聚乙烯／淀粉共混物的光、生物降解性能研究

目前，可降解塑料作为一种解决塑料废弃物污染环境的最有效的全新技术途径，是各国塑料领域研究的热门课题之一。降解塑料大体可分为光降解、生物降解、光／生物降解和水降解塑料，其中光／生物双降解塑料的发展最快，形成产业化的商品也比较多。本论文选用通用塑料中应用最广泛的聚乙烯和天然可降解高分子材料中最具有代表性的淀粉作为共混体系，应用硬脂酸铁作为光敏化剂，系统的研究了共混体系的光降解特性和生物降解性能，同时还对淀粉的改性加工进行了研究。1、天然淀粉不具有热加工性能，采用甘油为淀粉的增塑剂，经过适当的工艺处理，可使其转化为具有加工性能的热塑性淀粉。并通过差热分析、流变学研究，讨论了热塑性淀粉的稳定性和加工性能。2、采用硬脂酸铁作为聚乙烯／淀粉共混物的光敏化剂，随着光敏化剂用量和光照时间的增加，聚乙烯和聚乙烯／淀粉共混物的力学性能呈明显下降趋势，分子量降低，碳基指数上升。说明硬脂酸铁对聚乙烯及其与淀粉的共混物具有很好的光敏化效果，在降解过程中伴有交联反应发生。同时，淀粉的加入对聚乙烯光氧化降解的发生具有促进作用。3、聚乙烯／淀粉共混物在堆肥一段时间后，表面出现大量的空洞，淀粉含量越高空洞越多，当淀粉含量）30％时，聚乙烯的分子量开始下降，说明共混物具有明显的生物降解特性。

花椒水浸液及酚酸类化感物质对作物种子萌发和幼苗生长的影响效应

花椒（Zanthoxylum piperitum）是川西干旱河谷地区的重要经济作物，化感作用是花椒连作障碍的原因之一。系统研究花椒化感作用有助于深入理解并最终解决花椒连作障碍。本文通过研究花椒叶、林下土壤浸提液及单一纯化感物质对花椒幼苗生长、苜蓿种子萌发及幼苗生长的影响作用，从生理生化角度揭示浸提液及纯化感物质的作用方式。通过室外和室内模拟实验，对浸提液及纯化感物质的化感效应进行比较，为花椒连作障碍的解决和化感作用机制的深入理解提供依据。主要结果如下： 1.花椒叶及林下土壤浸提液对地下生物量影响作用强与对地上生物量的化感效应，两种浸提液的化感效应强度不同，叶浸提液作用表现更显著。其中在Y6、Y8 、T6和T8处理时，花椒幼苗地下生物量分别降低了31.2%、32.1%、31.6%和31.7%。 2.两种浸提液均能显著影响花椒幼苗体内的保护性酶活性，总体说来，在高浓度下抑制各种抗氧化物酶活性，幼苗体内丙二醛含量增加，幼苗受害严重；在较低浓度下，各种保护性酶活性有所增加，丙二醛含量减少，幼苗伤害减轻。同时，不同月份里，各种酶的活性高低显著不同，9月份的活性显著低于7月份的酶活性。对于养分含量的影响，Y8、T8的影响强度最大，分别使碳元素含量降低了27.8%和30.8%，使钾元素含量降低了34.7%和25.6%。 3.花椒叶及林下土壤浸提液对苜蓿种子萌发及幼苗生长有化感作用，表现在最终萌发率、不同物质代谢及保护性酶活性的差异上。两种浸提液对苜蓿种子萌发过程中蛋白质的含量均无显著性影响，对淀粉和可溶性糖的影响作用类似，高浓度处理无明显化感效应，较低浓度处理显著降低二者在萌发苜蓿种子中的含量。Y2、Y4与T4处理分别使可溶性糖含量减少了32.3%、29.1%和18.8%，Y2与T2处理分别使淀粉含量降低了29.3%和26.8%。 4.苜蓿种子在4种单一化感物质最高浓度即10-3 mol•L-1处理下，萌发率显著降低，半数萌发时间推迟，随着处理浓度降低，抑制作用逐渐减弱，当降低到10-6 mol•L-1时，又能够表现出对苜蓿种子萌发的促进作用。 5.纯化感物质在10-6 mol•L-1时使苜蓿幼苗叶片的保护性酶活性显著升高，丙二醛含量显著降低；在10-3 mol•L-1时使苜蓿叶片中保护性酶活性显著降低，丙二醛含量增加，膜脂过氧化程度加重。 Zanthoxylum piperitum is one of the most important cash crops and has been extensively cultivated in Eastern Tibetan Plateau, especially in the fragile dry valley areas. Allelopathic effects could be a reason for Z. piperitum’s continuous cropping impediment. Systemmatically research of the effect of Z. piperitum allelopathy could help to comprehend the continuous cropping impediment. The allelopathic effects on seedlings growth and seed germination of aqueous extracts of Zanthoxylum piperitum and phenolic allelochemicals were studied, and the action mechanism of the two substances was also discussed from physiology. Indoor and outdoor experiments were set to compare the difference between aqueous extracts and pure allelochemicals. The main results showed that: 1. The aqueous extracts of leaf and soil had significant allelopathic effects on aboveground and underground biomass, but the effect on underground biomass was stronger than the effect on underground evidently. Treated with Y6、Y8 、T6 and T8, the underground biomass was reduced 31.2%、32.1%、31.6% and 31.7% respectively. 2. The activity of activities of superoxide dismutase, catalase, peroxidase and ascorbate peroxidase were significantly reduced, while the content of MDA was increased and the seedlings were suffered stronger, when treated by the high concentration; but at the low concentration, these were reversed. And then, at the different month, the activities of antioxidant enzyme were significantly distinct. As for the contents of nutrient element, Y8、T8 had the more intensive effects than other treatments. 3. The results showed that the two types of aqueous extracts had significant allelopathic effects on seed germination, substances metabolize and the activity of antioxidant enzyme. But the aqueous extracts had no effects on the content of protein, while had the similar effects on the content of starch and soluble sugar. At Y2、Y4 and T4, the content of soluble sugar decreased 32.3%、29.1% and 18.8% respectively. 4. Treated with 10-3 mol•L-1 of the four allelochemicals, the seed germination of alfalfa was significantly inhibited. Ferulic acid, coumarin and vanillic acid at 10-3 mol•L-1 significantly reduced the activities of antioxidant enzyme, while the content of MDA in alfalfa seedling was significantly increased. The restrain effects became weakened with the treat concentration falled. However, ferulic acid, coumarin and vanillic acid could increase the activities of antioxidant enzyme at 10-6mol•L-1.

大气CO2浓度升高对红桦碳水化合物含量和分配的影响

碳水化合物按其存在的形式可分为结构性碳水化合物和非结构性碳水化合物两种。前者主要用于植物体的形态建成；后者是参与植物生命代谢的重要物质。迄今为止，有关CO2浓度升高对植物叶片中的碳水化合物含量的研究较多，而对其它器官中碳水化合物含量以及碳水化合物在植物体内的分配响应研究较少。碳水化合物含量在植物各器官中的变化能够反映光合同化产物在叶和茎、枝和根中的转运情况；碳水化合物的分配与植物的生长模式相关，它的变化会对植物的生长情况产生影响。因此，为全面认识植物生理生化与生长过程对大气CO2浓度升高响应情况，需要对CO2浓度升高条件下植物体内碳水化合物的含量及分配变化进行深入的研究与探讨。本文应用自控、独立、封闭的生长室系统，研究了红桦幼苗根、茎、叶和枝的碳水化合物含量以及分配格局对大气CO2浓度升高(环境CO2浓度+350 µmol·mol-1) 的响应。研究结果表明：1) CO2浓度升高使红桦幼苗叶片中的非结构性碳水化合物含量显著增加。这可能会对光合作用造成反馈抑制，降低光合速率。2) CO2浓度升高使红桦幼苗根、茎和枝中的还原糖、蔗糖、总可溶性糖、淀粉和总的非结构性碳水化合物(TNC) 含量显著增加。说明CO2浓度升高促进了碳水化合物由叶片向枝、茎和根中的运输转移，支持了Finn和Brun的假设。3) 在总的非结构性碳水化合物(TNC) 中，淀粉所占比例最大。同样地，CO2浓度升高使TNC含量增加的部分中，淀粉所占的比例也最大。在叶片、枝、茎和根中淀粉含量增加部分占TNC含量增加部分的91.45%、88.23%、83.23%和82.01%。4) CO2浓度升高使红桦幼苗根、茎、叶和枝内的纤维素含量有增加的趋势，但未达到显著水平。需要进一步研究长期CO2浓度升高下，纤维素含量的响应程度。5) CO2浓度升高使碳水化合物在红桦幼苗体内的分配发生了改变。红桦幼苗体内碳水化合物分配变化的一致趋势是由地上部分向地下部分分配转移。其中，测定的所有碳水化合物均向根中分配增多。同时，CO2浓度升高使红桦幼苗的根冠比显著增大；根系干重显著增加。这些结果支持了Gorissen 和Cotrufo的假设，即碳水化合物向根中分配增多是根冠比增大的主要原因。6) CO2浓度升高使红桦幼苗体内的氮含量显著下降。氮含量的下降可能主要是由生长的加快和TNC (主要是淀粉) 含量的增加对氮的稀释造成的。Carbohydrates found in plants are frequently grouped into two different classes:structural carbohydrates and non-structural carbohydrates. The former mainlyconstruct the plant basic framework, while the latter are essential for plant growth andmetabolism. As yet there is lack of information on the effects of elevated CO2concentration on carbohydrate contents in stem, branch and root of plant, and oncarbohydrate allocation in organs of plant although there have been many reports onthe responses of carbohydrate contents to elevated CO2 concentration in plant foliages.A shift of carbohydrate contents in plant reflects a change in transporting ofphotosynthetic production from leaf to stem, branch and root of plant. The allocationof carbohydrates that is correlated to plant growth patterns affects plant growth. Thus,in order to understand the influences of elevated CO2 on biochemical process,physiological change and plant growth well, the response of carbohydrate contentsand allocation in plant to elevated CO2 should be further investigated. In our study, theeffects of elevated CO2 on carbohydrate contents and their allocation between leaf,stem, branch and root tissue of Betula albosinensis seedlings were determined. Theseedlings were grown in independent and enclosed-top chambers. Chambers werecontrolled to reproduce ambient (CK) and ambient + 350 µmol·mol-1 CO2 (EC)concentration for 1 year. The results here showed that,1) Elevated CO2 significantly increased non-structural carbohydrate contents in leafof red birch seedlings. This will reduce photosynthetic rate.2) Elevated CO2 also significantly increased non-structural carbohydrate contentsin root, stem and branch of red birch seedlings. These findings supported thehypothesis that elevated CO2 accelerated carbohydrates from leaf to branch, stem androot.3) Starch makes up the largest parts of total non-structural carbohydrate. In thesame way, the increase of starch plays a main role in the increase of totalnon-structural carbohydrate under elevated CO2. In leaf, branch, stem and root, theincrements of starch contents comprised 91.45%, 88.23%, 83.23% and 82.01% of theincrements of total non-structural carbohydrate contents.4) Under elevated CO2 the cellulose contents have an increasing tendency in redbirch seedlings. It is needed to investigate the effects of long-term elevated CO2 oncellulose contents in plant.5) There are significant CO2 effects on the allocation of carbohydrate in organs ofred birch seedlings. Under elevated CO2 more carbohydrates were allocated to root.Moreover, CO2 enrichment significantly increased the root to shoot ratio of red birchseedlings and the dry weight of roots. These results supported Gorissen and Cotrufo ‘shypothesis that increase of carbohydrate allocation to root mostly contributed to theincrease of root to shoot ratio.6) Elevated CO2 brought about a reduction in the nitrogen contents of leaf, stem,branch and root. The decline of nitrogen contents under elevated CO2 is mainlycaused by the dilution effects of increasing starch level and growth of red birchseedlings.

青杨(Populus cathayana Rehd.)对逆境胁迫的反应及lea基因的表达分析

杨树具有分布广、适应性强，在生态环境治理和解决木材短缺方面均占有重要位置。青杨(Populus cathayana Rehd.)是青杨派树种的重要成员之一，也是我国的特有种。本研究通过对不同水分梯度的干旱胁迫下青杨形态和生理生化的反应，不同pH值盐碱胁迫下不同海拔和不同气候地区的四个青杨种群在生理生态上的反应差异，以及在干旱和低温胁迫下青杨lea2, lea3组基因表达差异的研究，从形态、生理、生化和分子生物学水平系统地研究了青杨在不同逆境胁迫下的反应和青杨不同种群在盐碱胁迫下的反应差异。主要研究结果如下： 1. 青杨在干旱胁迫下的反应机制：中度和重度干旱胁迫下植株的生长受到明显抑制。表现在光合系统上青杨的净光合同化速率(A)下降，主要原因是气孔导度(gs)，胞间二氧化碳浓度(Ci)下降。另外最大量子产量(Fv/Fm)、光化学猝灭效率(qP)降低反应了干旱胁迫下光合系统II(PSII)受到严重损伤, 而且非光化学猝灭效率(qN)上升，导致可利用化学能产量下降，叶绿体产生淀粉的量减少。qP降低qN上升导致产生的过量电子对光合系统的伤害造成活性氧以及丙二醛(MDA)的含量增加。超微解剖结构显示，干旱胁迫增强时，叶绿体内淀粉粒的数目减少，而且叶绿体、线粒体等细胞器中嗜锇颗粒的数目增加。为清除细胞内的活性氧，植物一般的反应是抗氧化系统酶活性增加，对青杨来讲超氧化物歧化酶(SOD), 抗坏血酸过氧化物酶(APx)活性的增加远大于过氧化物酶(POD)，这显示了在青杨中SOD、APx酶在清除活性氧的作用上大于POD。另外同工酶研究结果显示这些酶活性的升高主要是由于各条同工酶带表达量的增加，而不是诱导新酶带的产生。另外，75% FC水分处理下有些指标非但没有下降，像A和有效光量子产量(Y)的值都略有增加，而且gs同时增加。另外，100% FC比75% FC细胞内淀粉粒的数目少一些，但有少量的嗜锇颗粒。这证明100% FC土壤水分也许并非最适合青杨生长。 2. 盐碱胁迫对不同海拔地区青杨种群的反应差异：青杨高海拔和低海拔种群的各种生理特性随着pH值上升都受到了很大的影响。两种群叶和根中Na+、K+ 含量, Na+/K+比率随着pH值的上升影响显著。在pH值高于10.4时高海拔种群叶和根中Na+/K+比率急剧下降但是低海拔种群中却一直维持在较高水平。两种群中MDA、脯氨酸(Proline)的含量，抗氧化系统酶的活性都受到了严重的影响，证明两个种群都属于盐碱胁迫敏感类型但是高海拔的种群对盐碱胁迫的耐性要高于低海拔。这主要是由于高海拔种群一般具有耐干旱、低温胁迫的能力，而植物的抗逆机制一般都有共通之处。 3. 盐碱胁迫对不同气候地区青杨种群的反应差异：盐碱胁迫下两种群的光合作用受到明显的抑制，具体表现在叶绿素的含量和A 显著下降。净光合速率的下降主要是由于叶片gs，Ci 值降低引起的。与湿润地区的种群相比盐碱胁迫增强时，干旱地区的种群叶绿素含量和光合能力的升高与K+离子含量增加有关。植物维持细胞质高K+/Na+值对植物的抗盐性有很重要的作用。为清除盐碱胁迫产生的活性氧，抗氧化系统酶活性增加。盐碱胁迫下干旱地区的种群在SOD、CAT 和谷胱甘肽还原酶(GR)等酶的活性均显著上升，而湿润地区种群只有谷胱甘肽氧化酶(GST)的活性明显增加，说明干旱种群的抗氧化酶系统在较高盐碱胁迫下的保护作用要强于湿润种群。这主要是由于植物抗盐碱胁迫与抗干旱胁迫在一些方面的机制是一致的，抗旱种群一般也能抵抗一定程度的盐碱胁迫。 4. 青杨lea2、lea3 基因在干旱和低温胁迫下的表达差异：通过荧光定量PCR 分析，lea2、lea3 组基因在干旱和低温胁迫下在mRNA 水平的瞬时表达量明显升高，说明了两基因在青杨耐干旱和低温胁迫上都起显著的作用。而且两基因在干旱胁迫下，表达量的升高和降低的时间近乎同步，表明两基因在干旱胁迫下对植物应急保护机制的启动都发挥着重要的作用。低温胁迫下lea3 基因在mRNA 水平上表达量显著上升的时间要早于lea2，而且lea3 基因的持续作用时间明显长于lea2 组基因，说明了低温胁迫开始时lea3基因在植物应对逆境的作用上要大于lea2 基因。 Poplars play an important role in lumber supply, and are important components of ecosystems due to their wide distribution and well adaptation. Populus cathayana Rehd., which belongs to Populus Sect. Tacamahaca Spach, is one of the most important resources of poplars and is specialist to china. In this study, different altitudes and climates populations of P. cathayana were used as experiment materials to investigate the adaptability to drought and salt-alkali stresses. And the cultures of P. cathayana were used to analyze the lea2 and 3 group genes expression when exposed to drought and low temperature stresses. The results are as follows: 1. A large set of parallel responses to drought stress: Drought stress caused pronounced growth inhibition. A decreased significantly and was mainly the result of gs and Ci down. Besides, Fv/Fm, qP decreased and that reflected the harmful effects to PSII of drought stress. In accordance with qN increasing, decreased useful energy production caused the starch numbers reduction in chloroplast. The qP up and qN down improved the levels of ROS and MDA. Starch numbers in chloroplast reduced and plastoglobuli numbers increased when soil water content decreased. To reduce ROS, the activities of SOD, APX, CAT and PPO were activated. The isozymes results show that the rising activities of the antioxidant enzymes resulted from certain isoform content increased, and not from the new band produced. Interestingly, morphological results show 100%FC maybe wasn’t the favorite water content for P. cathayana growth. 2. Effect of salt-alkali stress on morphological and physiological changes in two different altitudes populations of P. cathayana: We compared the physiological responses of two populations of Populus cathayana Rehder, originating from altitudes 2,840 m and 1,450 m. Our results demonstated that Na+ and K+ contents, and Na+/K+ ratios in leaves and roots are greatly affected by pH values. At pH 10.4, the Na+/K+ ratios in both leaves and roots sharply dropped in the higher altitude population but were always maintained at higher levels in the lower altitude population. The pH values causing maximum malondialdehyde (MDA) level, free proline content and antioxidant enzyme activities were significantly different in two populations. These results indicated that the higher altitude population exhibits greater tolerance to alkalinity stress than does the lower altitude population. 3. Morphological and physiological changes in two different climates populations of P. cathayana when exposed to salt-alkali stress. Salt-alkali stress caused pronounced inhibition of the growth and especially in photosystem. Pigments content and A decreased significantly and at the same time gs and Ci decreased too. Compared with wet climate population, the Chlorophyll content and A increased in drought climate population as pH value rising was related to the K+ content increasing. It is important to resist salt-alkali stress that the K+/Na+ ratio matained at high level in cytoplasm. To reduce ROS content, the SOD, CAT and GR activities rised significantly in drought population but only GST increased in wet population. The drought population showed higher salt-alkali tolerance than the wet population mainly resulted from the fact that drought tolerance was in accordance with salt-alkali tolerance to some extent. 4. The different expressional model of lea2 and lea3 gene when P. cathayana was exposed to drought and cold stress. RT-PCR results show both lea2 and lea3 suddenly expressed significantly in mRNA level under drought and cold stress. The expression level of two genes reached optimal level at the same time. But under cold stress, the earlier significantly rising expressional time and the longer maintained higher level time in lea3 than lea2 elucidated that lea3 may be more important than lea2 in resisting cold stress in short time in P. cathayana.

糯小麦杂交后代的基因型鉴定及Wx基因的效应研究

糯小麦在食品加工业、淀粉加工业及其它工业上有着重要用途，是近年来许多国家小麦研究的重要课题。国外糯小麦选育尚未突破高产与糯性相结合的难点，国内目前还没有培育出高蛋白强筋型的糯小麦品种，这在一定程度上与缺乏合适的育种方法和高效、实用的糯小麦分子标记辅助育种技术有关。国内外对Wx基因效应的研究主要利用缺体－四体系、重组自交系或近等基因系，还未见有利用遗传背景相同的BC5F2代回交改良群体的报道。 糯性位点近等基因系是小麦淀粉品质育种的重要材料，而我国目前还没有一套中国栽培小麦遗传背景的糯性位点近等基因系。为了选育部分糯小麦、全糯小麦和中国栽培小麦遗传背景的糯性位点近等基因系，我们利用Wx蛋白电泳和高效实用的分子标记技术体系来鉴定糯小麦杂交后代的基因型，结果证明该体系能有效地用于糯小麦的分子标记辅助育种。以中国春糯性位点全套近等基因系为研究材料，对小麦Wx基因的6个STS标记和1个CAPS标记进行了筛选，改良PCR扩增条件以及产物检测方式后，从这些标记中筛选出3个标记，包括鉴定Wx-A1、Wx-D1位点的2个共显性STS标记和Wx-B1位点的1个显性STS标记。利用上述3个分子标记从BC5F2 代回交改良群体中筛选出了8种Wx基因型，经卡方检验，其分离比符合3对基因的分离比例，其中基因型为aabbdd的植株有2株，直链淀粉含量分别为1.81%和0.82%，为全糯小麦；基因型为AAbbdd，aabbDD的部分糯性植株各有1株，直链淀粉含量分别为15.24%和17.57%。以上4株植株的农艺性状和品质性状接近回交亲本“川育12”，并明显优于全糯材料“98Y1441”，表明采用回交法与Wx基因分子标记辅助选择相结合，有助于培育高产、优质的全糯和部分糯小麦。同时，本研究中建立的分子标记技术体系，也为选育具有中国栽培小麦遗传背景的糯性位点近等基因系奠定了基础。 在基因型鉴定的基础上，利用糯小麦杂交后代BC5F2代回交改良群体研究了各基因缺失降低直链淀粉含量的效果和各基因合成直链淀粉的能力，以及直链淀粉含量与农艺性状、品质性状、淀粉糊化特性等的相关性。缺失不同Wx基因的8种基因型，直链淀粉含量差异显著。研究单缺失基因型发现，减少效应最大的是Wx-B1b基因，Wx-B1b和Wx-D1b基因没有显著差异，减少效应最小的是Wx-A1b基因。研究双缺失基因型发现，Wx基因合成直链淀粉的能力，Wx-B1a基因最高，Wx-A1a基因最低，而Wx-B1a和Wx-D1a基因差异很小；直链淀粉含量与株高、穗长、小穗数、穗粒数、千粒重等农艺性状相关不显著，表明淀粉品质育种可以与高产育种实现有机结合；直链淀粉含量与SDS-沉降值呈显著负相关(r=-0.726)，说明直链淀粉含量降低在一定程度上有利于提高小麦营养与加工品质，这一结果至今未见有文献报道；全糯类型的淀粉糊化特性与其他类型显著不同，具有最高的峰值粘度和稀懈值，最低的低谷粘度、最终粘度、反弹值、峰值时间、糊化温度、起始糊化温度，表明糯小麦淀粉在食品和工业上具有特殊用途；稀懈值与直链淀粉含量呈极显著负相关(r=-0.969)，其他粘度参数与直链淀粉含量呈显著正相关(最终粘度r=0.797，低谷粘度r=0.910、反弹值r=0.954、峰值时间r=0.970、糊化温度r=0.962、起始糊化温度r=0.932)。以上结论可为糯小麦品种选育和淀粉品质改良提供理论依据。 Waxy wheat is very important in food processing industry, starch processing industry and the other industries, so it is a focus of wheat research in many countries these years. Foreign wheat breeders have not conquered the difficulty of high yield combined with waxy character, and there is no waxy wheat variety with high protein and strong gluten in China at present，all of which were caused by lacking proper breeding methods and effective, applied molecular markers-assisted selection technique at a certain extent. Until now, research about the effect of waxy genes mainly depended on nullisomic-tetrasomic lines, recombinant lines or near-isogenic lines, and it is lacking the reports of using improved BC5F2 backcross progenies under the common genetic background. Near-isogenic lines at the Wx loci are important materials for wheat starch quality breeding. However, there are no such lines under the Chinese cultivated wheat genetic background. To develop partial waxy wheats, waxy wheats and near-isogenic lines at the Wx loci under the Chinese cultivated wheat genetic background, we use SDS-PAGE of waxy proteins and effective, applied molecular marker-assisted selection technical system to identify the genotype of waxy wheat’s progenies. The results indicated that such a system is applicable in waxy wheat’s molecular marker-assisted selection effectively. A series of Chinese Spring Wx loci near-isogenic lines were used to identify the specific bands of six STS markers and one CAPS marker of Wx genes. After optimizing PCR amplification and separating of PCR products, three co-dominant and dominant STS-markers were identified at the Wx-A1, Wx-D1 and Wx-B1 loci, respectively, which were used to identify the genotype of waxy wheat’s progenies. Eight Wx genotypes were developed from the improved BC5F2 backcross progenies, which follows Mendelian segregation. Among them, there were two aabbdd waxy plants whose amylose content in the BC5F3 seeds were 1.81% and 0.82%, respectively. In addition, there were partial waxy plants (AAbbdd and aabbDD) whose amylose content in the BC5F3 seeds were 15.24 % and 17.57%, respectively. Most agronomic and quality traits of these four plants resembled those of the recurrent parent “Chuanyu 12”, and superior to waxy wheat parent “98Y1441”. This shows that backcross approach in combination with molecular marker-assisted selection of waxy genes is helpful to develop partial and full waxy wheat with good traits in the waxy wheat breeding program. At the same time, molecular marker-assisted selection technical system in this paper, also establish the base for breeding the near-isogenic lines at the Wx loci under the Chinese cultivated wheat genetic background. According to the results of genotype identification, we use waxy wheat’s improved BC5F2 backcross progenies to verify the effects of null alleles on reducing amylose content and determine the amylose synthesis capacity of each Wx gene independently, and investigate the relativity among amylose content with agonomic traits, quality traits, starch pasting properties respectively. There was significant difference in amylose content of the eight genotypes carrying different null Wx alleles. The reducing effect of the single null alleles was the most significant in Wx-B1b, followed by Wx-D1b and Wx-A1b, and there was no significant difference between Wx-B1b and Wx-D1b. The results of the double null lines further demonstrated that for the capacity of amylose synthesis, Wx-B1a was the highest, followed by Wx-D1a and Wx-A1a, and there was no significant difference between Wx-B1a and Wx-D1a. Amylose contents of the eight genotypes were not significantly correlated with plant height, spike length, spikelets per spike, grains per spike, 1000-grain weight, which showed that starch quality breeding could integrate with high yield breeding. Amylose contents of the eight genotypes were negatively significantly correlated with SDS-sedimentation value(r=-0.726), which suggested that reduction in amylose content is propitious to improve quality at a certain extent. Starch pasting properties of the full waxy type was significantly different from the other seven types, with the highest peak viscosity and breakdown, and lowest valley viscosity, final viscosity, setback, peak time, pasting temperature and starting pasting temperature. It indicated that waxy wheat starch has special use in food and industry. Breakdown was negatively significantly correlated with amylose contents (r=-0.969), and the other parameters were positively significantly correlated with amylose contents (r=0.797 for final viscosity, r=0.910 for trough viscosity, r =0.954 for setback, r =0.970 for peak time, r=0.962 for pasting temperature and r=0.932 for starting pasting temperature ). The results of the study are very useful for waxy wheat variety breeding and starch quality improvement.

青藏高原栽培青稞遗传多样性研究

青稞，是我国藏区居民对裸大麦的称谓，它不仅是藏民的主要食粮、燃料和牲畜饲料，而且也是啤酒、医药和保健品生产的原料；青稞不仅为藏区人民的健康和经济发展做出了很大的贡献，而且对人类健康和社会经济的可持续发展都有重要的意义。青藏高原是我国及世界上青稞分布和种植面积最大的地区，资源极其丰富。虽然从经典遗传直到分子标记对我国大麦遗传多样性都有研究，但研究手段、数量仍然不够深入，对我国大麦资源遗传多样性研究的信息非常有限，不能很好地满足大麦遗传研究和育种应用的需要，尤其是对西藏栽培大麦的遗传多样性的研究还只是刚刚开始，关于栽培青稞多态性的研究报道很少。本研究采用SSR标记和蛋白质电泳两类技术，从SSR标记位点、单体醇溶蛋白、B组醇溶蛋白和淀粉粒结合蛋白（SGP）等四个方面对我国青藏高原栽培青稞的遗传多样性进行了综合评价。 SSR标记具有基因组分布广泛、数量丰富、多态性高、容易检测、共显性、结果稳定可靠、实验重现性好、操作简单、经济、易于高通量分析等许多优点，被认为是用于遗传多样性、品种鉴定、物种的系统发育、亲缘关系及起源等研究的非常有效的分子标记。本研究采用SSR标记分析了64份青藏高原栽培青稞的遗传多样性，同时评估SSR标记在我国大麦育种和品种鉴定中的应用潜力。选择了30个已知作图位点SSR标记，其中25个标记与重要性状的控制位点连锁紧密。选择的30个SSR标记，5个未得到很好的扩增产物，3个无多态性。22个多态性SSR标记位点中，每位点检测出等位基因2~15个，共检测出等位基因132个，平均每位点6.0 个。各多态位点检测出基因型为2~11种，位点HVM33的基因型最多。各多态位点的多态信息指数为0.16~0.91, 平均为0.65。根据PIC值选择了13个SSR标记用于我国青藏高原栽培青稞基因型鉴定，这些标记的PIC值为0.6以上。结合PIC值和基因型差异，选择了8个多态信息含量高的SSR标记，构建了高效指纹图谱，此图谱能把64份材料完全区分。 贮藏蛋白电泳分析是研究相关编码蛋白基因多态性的非常有效的方法。大麦单体蛋白与小麦醇溶蛋白相对应，具有丰富的多态性，可用于大麦遗传多样性、品种鉴定和群体进化等研究。本研究通过A-PAGE电泳技术研究了84份青藏高原栽培青稞的单体醇溶蛋白多态性。大麦单体醇溶蛋白图谱与小麦醇溶蛋白电泳图谱类似，所分离的蛋白清晰地分为ω－，γ－，β－和α－四个部分。青藏高原栽培青稞单体醇溶蛋白具有丰富的多态性，84份青稞材料中存在43条不同的蛋白带，75种组合带谱；其中67种为单一材料所独有，另8种则分别包含了2-3份材料。每份材料中拥有醇溶蛋白带为6－16条，含有6-10条单体醇溶蛋白带材料较多。西藏和四川材料群体单体醇溶蛋白多态性不同，具有区域特异性。西藏材料中发现了40条不同蛋白带，3条特异带，46 种蛋白组合；四川材料中出现了40种不同蛋白带，26种条带组合, 3条特异带。基于单体蛋白多态性的聚类与材料的来源有一定的相关性。A-PAGE单体蛋白具有丰富的多态性，可作为遗传研究和品种鉴定的标记。 大麦醇溶蛋白（hordein）是大麦籽粒的主要贮藏蛋白，与大麦的营养品质和加工品质密切相关，而且具有丰富的多态性，广泛用于品种鉴定、种质筛选、遗传多样性和亲缘关系研究。B组醇溶蛋白是主要的醇溶蛋白组份，约占总醇溶蛋白的80%，而且具有丰富的多态性。本研究采用SDS-PAGE分析了72份青藏高原栽培青稞B组醇溶蛋白的遗传多样性。青藏高原栽培青稞B组醇溶蛋白具有丰富的多态性，72份青稞材料中存在15种蛋白带，30种组合带谱，其中15种为单一材料所独有，另15种则分别包含了2-10份材料。每份材料中B组醇溶蛋白条带数为4-8条，含5、6条的材料较常见。不同来源的群体材料间B组醇溶蛋白组成存在差异，西藏青稞含有26种蛋白组合带谱，其中有19种特异带谱；四川群体中共发现11种蛋白组合带型，其中有4种特有带谱。两群体中都存在稀有条带。聚类分析将材料分成三组，材料聚类与材料来源地没有明显的相关性。 淀粉粒蛋白（Starch granule proteins, SGPs）是一类与淀粉粒结合的微量蛋白，一些淀粉粒蛋白具有淀粉生化合成中主要的酶蛋白功能，其变异会影响淀粉含量和特性，从而影响淀粉的应用。关于我国大麦淀粉粒组成研究还未见报道。本实验首次开创了我国大麦淀粉粒结合蛋白的研究工作。采用SDS-PAGE电泳技术研究了青藏高原栽培青稞的SGP组成，并分析了不同SGP组合间淀粉含量的差异，初步探索了所分离的SGP蛋白与淀粉合成的关系。66份青稞材料中分离了10种主要的SGP，其表观分子量为40-100KD，低于60KD的SGP带有7条，共有16种组合带谱；各SGP蛋白和组合带谱出现的频率存在差异，青藏高原青稞的SGP组成存在多态性。西藏青稞和四川青稞的SGP组成有很大差异，SGP组成具有地域差异性，西藏青稞含有12种蛋白组合带谱，其中有9种特异带谱；四川群体中共发现7种蛋白组合带型，其中有4种特有带谱；两群体中仅有3种共同的蛋白组合带谱。SGP蛋白特性将66份青稞分为三组, 即Ⅰ、Ⅱ、Ⅲ，材料聚类与材料来源具有一定的相关性。不同组合带谱材料间淀粉含量差异显著性检验结果显示，不同带谱间材料的总淀粉含量、直链淀粉含量和支链淀粉含量有差异，带谱2（SGP1+3+7+9+10）和8（SGP1+2+4+6+8）的总淀粉含量及支链淀粉含量显著大于组合带谱3（SGP1+3+7+10）的总淀粉含量。组合带谱7（SGP1+2+6+8）的直链淀粉含量显著低于带谱11（SGP1+5+8）的直链淀粉。带谱SGP2、3、4、5、6、7、8、9、10可能参与淀粉合成，SGP9可能与高支链淀粉的合成相关。 SSR标记位点、单体醇溶蛋白、B组醇溶蛋白、淀粉结合蛋白等四个方面的研究结果表明青藏高原SSR标记多态性、单体醇溶蛋白多态性、B组醇溶蛋白多态性和SGP多态性都非常丰富，与青藏高原是栽培青稞的多样性分布中心的观点一致。 青藏高原栽培青稞的SSR标记、单体醇溶蛋白、B组醇溶蛋白和SGP多态性表现出很大差异。SSR标记覆盖了整个基因组，多态性非常高。单体蛋白、B组醇溶蛋白、SGP蛋白是育种中非常关注的性状，他们只是代表基因组中的某一区域或位点，多态性相对较低。但单体蛋白多态性很高，84份材料中检测出43条不同蛋白带，75种不同的组合带谱。SSR标记技术和单体蛋白技术都是遗传多样性研究的有力工具，但单体蛋白技术不仅多态性高，而且经济、操作简便，是种质鉴定的理想方法。 对不同标记的多态性材料数据进行聚类，聚类图能为我们提供各材料间的遗传相似信息，为材料选择提供参考。但材料聚类与材料来源的地理区域的相关性表现不一致。SSR聚类和B组醇溶蛋白聚类与材料的来源地无相关性，而单体醇溶蛋白和SGP聚类与材料来源地有一定相关性，即西藏群体和四川群体分别有集中类群，这可能是人为选择的附加效应。 不同来源的群体材料的遗传多样性不同，具有区域特异稀有基因，加强不同地区间资源的交换和配合使用，有利于增加群体遗传多样性和新品种培育。 青藏高原栽培青稞的麦芽浸提性状、淀粉性状、病虫及裸粒等重要农艺性状控制位点存在丰富的变异，遗传基础宽广，可能蕴藏着多种不同的等位基因，是研究重要性状遗传特性、基因资源挖掘和遗传育种的宝贵资源库。 Hulless barley, due to its favorable attributes such as high feed value, good human nutrition，rich dietary fiber and ease processing, attracts people,s attention . Hulless barley plays a very important role in Tibetan life, used as essential food crop, main animal feed and important fuel. In addition to tsampa (roasted barley flour), a main food for Tibetan, hulless barley is also made into cake, soup, porridge, recent naked barley liquor and cornmeal. Qinghai-Tibet Plateau is one of a few areas which plant naked barley widely in the world and also has a long growing history. Genetic diversity of the cultivated hulless barley in this region , however, has not been documented. The study of genetic diversity existing within this population is of particular interest in germplasm identification, preservation, and new cultivar development. This study analyzed the genetic diversity of the cultivated naked barley from Qinghai-Tibet plateau through the study of SSR marker loci and monomeric prolamins, B-horden and starch granule proteins. SSRs are present abundantly in genomes of higher organisms and have become a popular marker system in plant studies. SSRs offer a number of advantages, such as the high level of polymorphisms, locus specificity, co-dominance, reproducibility, ease of use through PCRand random distribution throughout the genome. In barley, several hundred SSRs have been developed and genetically mapped and can therefore be selected from specific genomic regions. The genetic diversity of 64 cultivated naked barley from Tibet and Sichuan was studied with 30 SSRs of known map location.Among the selected SSR markers, PCR products of 5 SSR markers were not obtained and 3 SSR marker loci were monomeric. A total of 132 alleles were identified at 22 polyomeric SSR loci. The number of alleles per locus ranged from 2 to 15, with an average of 6.0. The polymorphism information content values for the SSRs ranged from 0.08 to 0.94, with an average of 0.65. 13 SSR markers with the PIC value >0.6 have been selected for discrimination of Qinghai-Tibet naked barley genotypews. A finger Print map was developed through 7 SSR markers with the high PIC value. It could be used as an efficient tool for gene discovery and identification of gernplasm. Hordeins, the main storage proteins of the barley seed, are composed of momomeric and polymeric prolamins and divided into -A, B, C and D groups in order of decreasing electrophoretic mobility. Hordeins show high inter-genotypic variation and have been extensively used as markers for cultivar identification and analyzing the genetic diversity. This study analyzed the genetic diversity of B-hordein in 72 naked barley from Qinqhai-Tibet Plateau. Extensive diversity was observed. A total of 15 different bands and 30 distinct patterns were found. Jaccard's coefficient of similarity was calculated, and the accessions were divided into three　main groups by cluster analysis using UPGMA. Differentiation among the populations from different collecting regions based on the polymorphism of B-hordein was investigated. Monomeric prolamins show high inter-genotypic variation and have been used as molecular markers for cultivar identification, analyzing the genetic diversity in collections and investigating the evolution processes and structure of populations However, the cultivated hulless accessions from Qinghai-Tibet Pateau in China have never been examined with respect to monomeric prolamins. This study analyzed the genetic diversity of monomeric prolamins (protein fraction corresponding to wheat gliadins) using the Acid -PAGE technique in eighty-four cultivated hulless barley from Qinqhai-Tibet Plateau in China. Extensive diversity was observed. A total of 43 different bands were found, of which 21 different bands were in the region of ω group, 8 in the region of γ, 8 in the region of β, and 6 in the region of α group. Among the 86 accessions, 75 distinct patterns were identified. The number of bands ranged from 6 to 16, depending on the variety. Jaccard’s coefficient of similarity was calculated, and the lines were grouped by cluster analysis using UPGMA. A dendrogram was obtained from the analysis of the groups and five main clusters were identified. No relationship between the distribution in the dendrogram and growth habits and origins of the cultivars could be detected. Starch is the major constituent of the cereal endosperm, comprising approximately 65% of the dry weight of the mature wheat grain. The starch formed in all organs of plants is packaged into starch granules, which vary widely between species and cultivars in size and shape. Wheat endosperm starch granules contain about corresponding to the main biosynthase of starch. This report firstly dealed with intraspecific variation of the major SGPs in cultivated naked barley from Qinghai-Tibet plateau. A total of 10 major SGPs were observed in the range of 40KD-100KD and 16 types of patterns were found. Based on the variation of SGPs, accessions studied were classified into 3 groups. A geographical cline of electrophoregram was observed. In addition, significance test of the difference of starch content among groups and types of patterns were done, and the results indicated those SGPs could be related to the content of starch. Diagram obtained through cluster analysis exhibited a structuration of diversity and genetic relationship among cultivated hulless accessions. In breeding program, parents with genetically distant relationship for hybridization will increase genetic diversity of progenies. In conclusion, cultivated naked barley from Qinghai-Tibet Plateau in China presents a high variability with respect to monomeric prolamins，SSR markers , B- hordeins and SGPs. The result of this study supports Qinghai-Tibet Plateau is the center of cultivated hulless barley and the cultivated naked barley is considered to be a gene pool with large diversity and could be applied to breeding for cereal.

青藏高原青稞淀粉资源的评价

青稞（Hordeum vulgare L.var．nudum Hook．f.），即裸大麦，是兼食用、饲用和酿造于一体的作物，有着重要的利用价值。淀粉是青稞籽粒中含量最多、最重要的碳水化合物，淀粉含量、直支淀粉比将会直接影响淀粉的功能特性，进而影响淀粉的应用领域。我国青藏高原青稞的栽培和食用历史悠久，特色青稞资源极其丰富。目前关于青藏高原青稞淀粉特性的报道还不多见，筛选和培育特色淀粉青稞利于拓展青稞的应用领域, 从而提高其经济价值。 本研究以114份青藏高原青稞品种(系)为实验材料，通过SDS-PAGE对材料的胚乳淀粉颗粒结合蛋白（SGAPs）进行分离，确定各蛋白的分子量大小、组合类型和多态性等。然后按照国标法测试材料的籽粒总淀粉含量和直链淀粉含量，通过微型糊化粘度仪分析相应的淀粉糊化特性，最后使用显微镜观察比较了青稞的淀粉颗粒形态特征。主要结果如下： 1、114种青稞中共分离出20种不同的SGAP条带，条带分子量为35.00～112.39 KDa，分布频率为12.28～97.37%。材料含有的SGAPs条带数从10到14不等，超过一半的材料含11种SGAP条带。20种条带形成16种组合类型，其中西藏地区青稞包含所有16个组合类型，四川地区青稞包含其中12个组合类型。青藏高原青稞籽粒淀粉颗粒结合蛋白的差异很大，遗传多样性丰富。 2、114份青稞的总淀粉含量、直链淀粉含量、直支淀粉比、峰值粘度、糊化温度和峰值温度的变幅分别为51.26～66.70%、14.64～29.74%、0.17～0.42、194～1135BU、58.8～65.2℃和81.4～92.4℃，相应的平均值分别为59.82%、23.60%、0.31、722.30BU、62.1℃和88.8℃。群体在总淀粉含量、直链淀粉含量、直支淀粉比、峰值粘度、糊化温度和峰值温度上的分布具有明显的正态性；所有胚乳淀粉体的淀粉粒都呈复粒结构。对西藏和四川的材料进行了分组比较， 两地区的青稞在直链淀粉含量和直支淀粉比上的差异达到显著水平。 3、筛选出18份具有特殊淀粉特性的青稞品种，其中5份材料的总淀粉含量超过65%，包括NB63-1、NB67、甘孜白六棱、98221-1和NB63；3份材料的直链淀粉含量大于29%，包括藏青85、藏青3号和喜马拉6号；8份材料的直支淀粉比小于0.25，包括99033-6、春青稞、阿坝330、Jan-03、米麦114、396、NB63-1和92013；7份材料的糊化温度低于60℃，同时材料的峰值粘度大于1000BU,并且峰值温度低于90℃，包括足捉春、Jan-03、阿坝330、米麦114、春青稞、20003和阿青5号。 4、各淀粉特性间存在高度相关性。直链淀粉含量和直支淀粉比与糊化温度成极显著正相关，与峰值粘度成极显著负相关，与A型淀粉粒数量和大小呈负相关。不同SGAPs组合的品种之间，淀粉含量和淀粉糊化特性间差异均达显著水平。SGAP2、SGAP5、SGAP6和SGAP7可能对籽粒直链淀粉含量、直支淀粉比和糊化温度有正向效应；SGAP3、SGAP9∼SGAP20可能对峰值粘度有正向效应。 本研究对青藏高原青稞淀粉资源进行了较为全面的评价，对该区青稞淀粉特性有了系统的认识。研究筛选出的特殊青稞品种可作为青稞育种和青稞淀粉工业应用的潜在资源，淀粉特性差异巨大的众多青稞品种也为拓宽青稞应用领域提供了丰富的资源保障。本研究对部分SGAPs在性质上的鉴定和功能上的初步推断为青稞材料的筛选提供了指导，也为品质育种提供了理论参考。 Hulless barley (naked barley, Hordeum vulgare L.) is a short- season, early maturing crop with a wide range of adaptation. It has been attracting more and more attention due to its superior nutrition and extensive industrial applications. Starch is the main ingredient in hulless barley seeds which makes up 65 percent of hulless barley’s dry weight. The ratio of the amylose/amylopectin and the size, shape, distribution of starch granules can affect the physico-chemical and functional properties of starch, which may turn affect its utilizations. The Qinghai-Tibet Plateau, which is located in southwestern China, is a typical area of vertical agricultural ecosystem and one of the barley origin centers with abundant hulless barley resources. There are little reports about hulless barley in Qinghai-Tibet Plateau at present. To screen and cultivate some characteristic hulless barley can improve its value. An improved SDS-PAGE was used to identify SGAPs combination of 114 hulless barley varieties. Starch content (total starch and amylose starch) was determined according to the standard methods GB5006-85 and GB/T 15683 using PerkinElmer M341 Precision Automatic Polarimeter and UV spectrophotometer 755B respectively. The pasting properties were measured by BRABENDER Micrio Visco-Amylo- Graph 803201. The morphology of starch granules were observed and compared with Axioplan 2 Imaging light microscopy. The following were the results obtained: 1. There were 20 major SGAPs presented in 114 varieties, with the molecular weight ranged from 35.00 to 112.39 KDa, and the frequencies ranged from 12.28% to 97.37%. The number of SGAP bands in each accession varied from 10 to 14, more than half of the population had 11 bands. There were 16 distinct SGAP patterns in the 114 varieties, the Tibet hulless barley had all of the 16 types and the Sichuan hulless barley had 12 types. The results indicated the Qinghai-Tibet Plateau hulless barley had a polymorphism of the SGAPs. 2. The ranges of the total starch content, amylose content, Am/Ap, peak viscosity, pasting temperature and peak temperature of the 114 hulless barley were 51.26～66.70%,14.64～29.74%,0.17～0.42,194～1135BU,58.8～65.2 and 81.4℃～92.4, with an average of ℃59.82%, 23.60%, 0.31, 722.30BU, 62.1 and 88.8,℃℃ respectively. The distributions of the total starch content, amylose content, Am/Ap, peak viscosity, pasting temperature and peak temperature were visibly normal school. All of the amyloplasts in endosperm of varieties showed bimodal size distributions.The main starch properties of hulless barley from Tibet and Sichuan were separated and compared, the differences on amylose content and Am/Ap were obvious. 3. Eighteen accessions which had special starch properties were screened out. Five accessions with total starch content beyond 65%, including NB63-1, NB67, Ganzibailiuleng, 98221-1 and NB63; three accessions, Zangqing85, Zangqing3 and Ximala6, with the highest amylose content (>29%); five accessions with Am/Ap less than 0.25, including 99033-6, Chun Qingke, A Ba 330, Jan-03, Mi Mai114, 396, NB63-1 and 92013; seven accessions had a pasting temperature under 60, ℃meanwhile their peak viscosity beyond 1000BU and their peak temperature under 90℃，including Zu Cuochun, Jan-03, A Ba 330, Mi Mai 114, Chun Qingke, 20003 and A Qing 5. 4. There were high correlations between starch properties. Amylose content and Am/Ap were positively correlated to pasting temperature, negatively correlated to peak viscosity, negatively correlated to the number and granule size of A-type granule. Different SGAP combinations caused significant diversities in starch content and pasting properties. SGAP2, SGAP5, SGAP6 and SGAP7 may have positive effect on amylose content, Am/Ap and pasting temperature; SGAP3, SGAP9∼SGAP20 may have positive effect on peak viscosity. Our research made a comprehensive evaluation on the hulless barley starch from the Qinghai-Tibet Plateau, we can get a systemic understanding. Some special accessions were screened out can be used on the hulless barley breeding lines and industries utilization.The combination of the SGAPs may become a criterion to evaluate the hulless barley endosperm starch quality. Consequently, the results will be good information for further studies on the hulless barley.

毛壳菌产抗真菌活性物质菌株筛选及种间原生质体融合研究

毛壳菌属很多种类具有重要生防价值，其生防机理包括对植物病原真菌的重寄生作用、诱导植物产生抗病性、产生抗真菌活性的次生代谢产物等。迄今，学界对毛壳菌的研究主要集中在毛壳菌的生防机理，毛壳菌活性次生代谢产物的分离等方面。本研究致力于产抗生素的毛壳菌的种间原生质体融合，从产抗生素毛壳菌菌株的筛选开始，进而对产抗生素的角毛壳菌进行诱变选育，最终用产不同抗生素的角毛壳菌与球毛壳菌进行种间原生质体融合。主要有以下五方面研究结果。 1、毛壳菌抗真菌活性物质产生菌株的筛选：不同毛壳菌菌株发酵液采用琼脂扩散法对植物病原真菌进行抑菌活性试验，结果显示，菌株CH08和CH23的发酵液对芒果炭疽、苹果炭疽和马铃薯晚疫菌具有抑制作用。菌株CH16和CH17的发酵液对芒果炭疽菌、苹果炭疽菌有抑制作用。菌株CH21发酵液对辣椒炭疽菌和西瓜枯萎菌有抑制作用。经形态学研究，菌株CH08、CH16、CH17和CH23鉴定为球毛壳菌，菌株CH21鉴定为角毛壳菌。对角毛壳菌与球毛壳菌菌株发酵液抑菌谱比较，发现角毛壳菌与球毛壳菌发酵液具有明显不同的抑菌谱，表明角毛壳菌与球毛壳菌产生不同的抗真菌活性物质。 2、角毛壳菌（CH21）和球毛壳菌（CH08）原生质体制备和再生条件研究：考察了菌龄、酶浓度、稳渗剂及其浓度、酶解温度、酶解时间及再生培养基对原生质体制备和再生的影响。用菌龄为生长54 h的角毛壳菌菌丝，以0.06 M磷酸缓冲液（pH6.0）配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液，30℃酶解1.5 h，原生质体释放量2.02×107个/g；以PDA为再生培养基，0.7 mol/L的蔗糖再生稳渗剂，再生率可达51.45%。用菌龄为生长48 h的球毛壳菌菌丝，以0.06 M磷酸缓冲液（pH6.0）配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液，30℃酶解1 h，原生质体释放量达1.57×108个/g；以PDA为再生培养基，0.7 mol/L的蔗糖为再生稳渗剂，再生率可达41.48%。 3、角毛壳菌（CH21）原生质体紫外诱变选育：以CH21为出发菌株，制备原生质体进行紫外诱变，诱变条件为：15 w紫外灯，距离30 cm，照射90 s，致死率80%~85%。建立了诱变菌株初筛的双层平板筛选模型。经平板初筛和摇瓶复筛，获得一株突变菌株CH21-I-402，其发酵液抑菌活性较出发菌株提高18.3%。 4、抗性标记菌株的获得：菌株CH21-I-402和CH08抗生素药敏试验表明， CH21-I-402菌株对潮霉素有抗性、对G418（Geneticin）敏感，菌株CH08对潮霉素和G418都敏感。根癌农杆菌EHA105介导的新霉素磷酸转移酶基因转化球毛壳菌，经PCR检测，新霉素磷酸转移酶基因成功转化进菌株CH08-GR70，CH08-GR120。转化子对G418抗性提高3～4倍，对潮霉素仍然比较敏感。 5、以G418和潮霉素抗性为筛选标记的原生质体融合与融合菌株AFLP分析：制备角毛壳菌CH21-I-402和球毛壳菌CH08-GR70原生质体，以35％的PEG6000为助融剂进行原生质体融合，以65 μg/ml的潮霉素和60 μg/ml G418为抗性筛选标记，获得46个再生菌株。再生菌株连续传代5代后，再生菌株表现出多种形态类型。利用AFLP技术对再生菌株及亲本菌株基因组DNA分析表明，再生菌株PF1、PF26为融合菌株。抑菌活性测试表明，融合菌株PF26发酵液对芒果炭疽菌和苹果轮纹菌有强的抑制作用，且抑菌活性比亲本球毛壳菌明显提高。 Chaetomium spp. have great potentials as biocontrol agents against a range of plant pathogens on the basis of its mycoparasitism, induced plant disease resistance, production of antifungal metabolites, and so on. Previous researches on C. spp. mostly focused on the mechanisms of its biocontrol and the isolation of secondary metabolites. In this study, screening antifungal C. spp., mutation breeding of C. cupreum and interspecies protoplast fusion between C. cupreum and C. globosum were carried out, respectively. The corresponding results are as follows: Firstly, among more than 40 C. spp., the strains produced anti-fungal antibiotics were screened by agar diffusion experiments. Results showed that both CH08 and CH23 had inhibition against Colletotrichum gloeosporioides, Cladosporium fulvum, and Phytophthora infestans. Both CH16 and CH17 had inhibition against Colletotrichum gloeosporioides and Cladosporium fulvum. In addition, CH21 exhibited anti-fungal activity against Fusarium oxysporum f. sp niveum and Colletotrichum capsici. Furthermore, CH08, CH16, CH17 and CH23 were identified as C. globosum, CH21 was proved to be C. cupreum based on morphology. The comparison of the anti-fungal spectrum between C. cupreum and C. globosum, showed they could produce different antibiotics. Secondly, specified protocols for preparing and regenerating protoplasts from mycelia of C. cupreum CH21 and C. globosum CH08 were studied. The effects of the age mycelia, the concentration of enzyme, digestion temperature and time, kinds of osmotic stabilizer and regeneration medium on protoplasts preparation and regeneration were all optimized, respectively. In one protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1.5 h at 30 ºC, 2.02×107 protoplasts from each gram mycelia were obtained from cultures of C. cupreum CH21 grown in potato dextrose broth (PDB) medium for 54 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA (potato dextrose agar with osmotic stabilize), the regeneration efficiency of protoplasts was 51.45%. In another protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1 h at 30 ºC, 1.57×108 protoplasts from each gram mycelia were obtained from cultures of C. globosum CH08 grown in PDB for 48 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA, the regeneration efficiency of protoplasts was 41.48%. Thirdly, the mutagenesis conditions and secondary screening model of C. cupreum CH21 were explored. An 80% to 85% death rate could be achieved when the protoplasts of C. cupreum CH21 were irradiated by 15 w UV lamp from 30 cm distance for 90 s. In addition, the doublelayer plate’s method for the primary screening of high-producing antibiotics strains was established. A high yielding antibiotic mutant CH21-I-402 was obtained through the primary screening on plate and the secondary selection in Erlenmeyer flask, compared to the original CH21 strain, the antifungal activity of the mutant CH21-I-402 was increased by 18.3%. Fourth, the sensitivity to antibiotics of both C. cupreum CH21-I-402 and C. globusm CH08 was detected. Results showed C. cupreum CH21-I-402 was sensitive to G418 (Geneticin) (Gs) and resistant to Hygromycin B(Hr), and C. globusm CH08 was sensitive to both G418 (Geneticin) (Gs) and Hygromycin B(Hs). At the same time, neomycin phosphotransferase II (npt II) gene was transformed into C. globusm CH08(Gs, Hs) mediated by Agrobacterium tumefaciens EHA105, and the npt II gene was verified by polymerase chain reaction in resistance to G418 strains CH08-GR70 and CH08-GR120. The transformants still showed sensitive to Hygromycin B(Hs). Finally, a selection system for hybrids was set up by interspecies protoplast fusion between C. cupreum and C. globusm using dominant selective drug resistance markers. At first, protoplasts of C. cupreum CH21-I-402 (Hr, Gs) and C. globusm CH08-GR70 (Hs, Gr) were prepared, then the protoplasts were fused in the presence of 35% polyethylene glycol 6000 and regenerated on OPDA medium with 65 μg/ml Hygromycin B and 60μg/ml G418, at last 46 colonies with Hr and Gr were obtained. Even after 5 generations’ subculture, most of the colonies displayed significant difference in taxonomic characteristics with their parental strains. Regenerated strains PF1 and PF26 were confirmed as fusants by amplified fragment length polymorphisms analysis with the genomic DNA as the model. PF26 showed higher inhibitory activity against Colletotrichum gloeosporioides and Macrophoma kuwatsukai than that of the parental strain C. globusm.

PVA降解菌的产酶条件及特性研究

目前对PVA生物降解研究重点逐渐转移到对PVA降解菌和PVA降解酶的研究开发上，随着对PVA降解高效新菌株的不断发现和PVA降解酶作用机理和分泌机制的深入了解，利用高效微生物或酶法治理PVA这类高聚物的污染将具有较大的应用潜力。本论文研究工作正是基于这种客观条件下进行的，对本实验室前期分离的PVA降解菌株P1、共生菌B1+B2、Pa、Pb为研究对象，重点研究了菌株P1和共生菌B1+B2的产酶条件和产酶特性，验证找出了影响菌株P1产酶的生长因子，论证了菌株B1+B2的产酶特性，优化得出了菌株B1+B2的最佳产酶条件；然后对共生菌B1+B2的PVA降解酶的稳定性进行了研究；最后研究了最佳组合菌的产酶特性和最佳产酶条件。主要研究结果如下： 1 通过对菌株P1产酶因子的研究，找出了核黄素是菌株P1产酶的必须因子，在以淀粉为碳源时，核黄素只是产酶的必须因子，而不是菌体生长的必须因子；在以PVA为碳源时，核黄素既是生长的必须因子，也是产酶的必须因子，是菌株P1的生长因子。 2 对共生菌B1+B2的产酶条件和产酶特性进行了研究，并通过正交实验找出了影响菌株产酶的主要条件和菌株产酶的最佳条件。 3 对共生菌PVA降解酶的稳定性进行了研究，确定了影响酶稳定性的主要理化条件。 4 通过对菌株降解性能的比较，确定菌株Pa、Pb、共生菌、P1的作为组合菌的组成菌，然后通过复配实验确定出菌株的最佳组合为菌株Pa、P1、共生菌，最后通过正交实验确定最佳组合菌的最佳配比。 5对影响组合菌产酶的因素进行了研究，通过正交实验确定了影响组合菌产酶的主要因素和最佳产酶条件。 本文通过对PVA降解菌株产酶条件和特性的研究，旨在为PVA降解菌生产酶制剂及进一步优化PVA降解菌在PVA废水治理中的应用提供理论和应用依据。 Now the PVA-degrading bacteria and polyvinyl alcohol-degrading enzyme are the key studies on the PVA biological degradation. It has great application potential using special bacteria and enzyme to treat pollution of PVA, with some high efficient Strain and enzyme were found. The study of this paper was based on that objective condition. The stain P1, symbiotic bacteria B1+B2, stain Pa and strain Pb were studied .The conditions of enzyme production and enzyme production characteristic of stain P1, symbiotic bacteria B1+B2 were our key study, we tested and verified the growth factor which effected enzyme production of strain P1, demonstrated enzyme production characteristic of symbiotic bacteria B1+B2, optimized and obtained the optimum conditions of enzyme production; then we studied the stability of polyvinyl alcohol-degrading enzyme of strain B1+B2; last the enzyme characteristic and the optimum conditions of alcohol-degrading enzyme production of optimum combination stains were studied. The main study results are below: 1. Through the study of enzyme production factor we found that lactoflavin is the necessary factor in strain P1 enzyme production. When we used starch to be carbon energy, lactoflavin is only the necessary factor of enzyme production, but not growth factor. When we used PVA to be carbon energy, lactoflavin was not only the necessary growth factor ,but also the necessary enzyme production factor.So it was the growth factor of strain P1 2. The enzyme production conditions and enzyme production characteristic of symbiotic bacteria B1+B2 were studied. Through the orthogonal experimental design, the main conditions which effected the enzyme production and the optimum conditions of enzyme production were obtained 3. Through the study of the stability of polyvinyl alcohol-degrading enzyme, the main physical and chemical conditions which effected the enzyme stability were 4. The stain P1,symbiotic bacteria B1+B2, stain Pa and strain Pb were selected to form combination bacteria. The stain P1,symbiotic bacteria B1+B2,stain Pa were the optimum combination through duplication experiment. Then the optimum ratio was obtained through orthogonal experiment. 5. Studied the factors which effected the polyvinyl alcohol-degrading enzyme activity, then through orthogonal experiment, the main factors and condition of enzyme production which effected the combination bacteria were achieved. The result of the study was valuable for the ferment of the PVA-degrading enzyme and the optimization of the PVA-degrading performance in the PVA wastewater.

三孢布拉氏霉发酵产β-胡萝卜素的研究

本论文研究了利用三孢布拉氏霉(Blakeslea trispora)发酵产β-胡萝卜素的培养条件。主要包括：发酵培养基的确定，发酵条件的优化。还考察了发酵菌丝体中β-胡萝卜素的提取方法及薄层层析等。 首先研究了培养基成分对三孢布拉氏霉发酵产β-胡萝卜素的影响。确立了玉米淀粉作为碳源，黄豆粉（热榨）作为氮源，棉籽油作为植物油的发酵培养基配方，其成分为：玉米淀粉 3％，黄豆粉（热榨） 2％，棉籽油 3％，KH2PO4 0.2%，MgSO4·7H2O 0.2%，维生素B1 0.002％，pH值6.0。 其次，通过比较不同的发酵影响因子，分别得到最适的条件：如三孢布拉氏霉正负菌接种比例为1.3：0.7，培养基pH值为7.0（灭菌后），发酵促进因子为Triton X-100。并采用正交试验法，确定其最佳发酵条件为正负菌接种比例1.3/0.7，发酵培养基pH为7.0，在培养基中添加表面活性基Triton X-100 0.08％。使该菌株产β-胡萝卜素的量达到0.73g/L，较初始发酵条件提高了3.3倍。 研究中还找到一个简便有效的对β-胡萝卜素的提取方法，选用盐酸-热处理法进行细胞破壁，并选用沸程为60～90℃的石油醚进行萃取。 用三孢布拉霉菌丝体内类胡萝卜索的石油醚提取液点样于硅胶G板，以丙酮：石油醚（5：95）为展开剂能将β-胡萝卜素与其它类胡萝卜索分离。该方法简便快速，并有一定实用价值。 The fermentative conditions of β-carotene by Blakeslea trispora have been investigated. These conditions include fermentation medium, the optimization of some fermentation factor. The extracting methods and the TLC of carotenoids were also researched. Firstly, the effects of composition of fermentation medium on the yield of β-carotene were studied. the results showed that the best fermentation medium was corn starch 3％，soybean power 2％，cottonseed oil 3％，KH2PO4 0.2%，MgSO4·7H2O 0.2%，vitamin B1 0.002％，pH value 6.0. Secondly, through compared some factors, such as different proportion of plus and minus strains, pH value, nonionic surfactants, respective best values have been obtained. The best proportion of plus and minus strains is 1.3:0.7, pH value of fermentation medium (sterilized) is 7.0, fermentation accelerant which acts as surfactants is Triton x-100. Farther on, the fermentative conditions were optimized through orthogonal experiment, the optimization showed that proportion of plus and minus strains is 1.3:0.7，pH value is 7.0, content of Triton x-100 is 0.08％. And the yield of β-carotene reached 0.73g/L, which was up to 3.3 times through the fermentation. In the extracting study, it has showed hydrochloric acid-heat treatment is a simple, convenient and effective extracting methods is which was used to destroy the cell wall, and the extracting organic solvent is petroleum ether whose boiling range is 60~90 ℃. In the TLC experiments, extracting contents in the petroleum ether were spotted in the silicagel plate, and the mixed liquor of acetone and petroleum ether (5：95) is developping agent, which can distinguish β-carotene from other carotenoids. It is a simple and quick technique.

酿酒酵母和运动发酵单胞菌木薯乙醇发酵条件研究

本文结合我国燃料乙醇发展的方针政策，以酿酒酵母和运动发酵单胞菌为菌种研究其在非粮能源作物木薯中乙醇发酵的情况，为木薯原料更好地应用于生产中提供了理论依据。 酿酒酵母木薯高浓度乙醇发酵的研究。实验采用的木薯干淀粉含量约70-75%。以酿酒酵母为菌种进行高浓度乙醇发酵的工艺条件研究，最佳条件为：木薯干粉碎细度为35目，料水比1：2，α-淀粉酶用量0.09 KNU/g淀粉，蒸煮温度85 ℃，蒸煮时间15 min。采用30 ℃同步糖化发酵工艺，糖化酶用量为3.4 AGU/g淀粉，发酵时间30 h。在10 L发酵罐中，乙醇质量比达127.88 g/kg，发酵效率为88.28%，发酵强度4.263 g/kg/h，100 L中试研究中乙醇浓度为127.75 g/kg，发酵强度4.258 g/kg/h。利用高效液相色谱对发酵液中残糖进行了分析，证明葡萄糖、果糖等单糖已完全被菌体利用，剩余糖为二糖，三糖等不可发酵的低聚糖。 运动发酵单胞菌快速乙醇发酵的研究。对实验室保藏的8株运动发酵单胞菌进行比较，选择发酵速度最快的Zymomonas mobilis232B进行研究。该菌在纯葡萄糖中的最佳发酵条件为：葡萄糖浓度18%，起始pH 6-7，发酵温度30 ℃，发酵时间18 h，乙醇浓度88 g/kg。在以木薯为底物同步糖化快速乙醇发酵中，采用Full Factorial设计和最速上升实验确定了培养基成分中的2个显著性因子及其最适浓度：酵母粉4 g/kg，硫酸铵0.8 g/kg。在最适培养基条件下，对木薯料水比和糖化酶用量进行了优化，得到Z.mobilis232B木薯乙醇发酵最佳料水比1：3，糖化酶浓度4 AGU/g淀粉，乙醇发酵4.915 g/kg/h。利用高效液相色谱对发酵液中残糖进行了分析，剩余糖为二糖，三糖等，但成分较酵母发酵后复杂。 According to the fuel ethanol development plans and policies in our country, the ethanol production from cassava by Saccharomyces cerevisiae and Zymomonas mobilis was studied. It provided theoretical basis for ethanol fermentation by cassava in industry. Part 1 is the study of VHG (very high gravity) ethanol fermentation by Saccharomyces cerevisiae. The content of starch in cassava was 70-75%. Compared with the performances under different experimental conditions, the following optimal conditions for VHG fermentation were obtained: Granule size of dry cassava 35 mashes, hydromodulus of cassava to water at 1:2, α-amylase enzyme dosage 0.09 KNU/g starch, cooking temperature 85 ℃ for 15 min, using the SSF process (simultaneous saccharification and fermentation) and the amount of glucoamylase 3.4 AGU/g starch. Accordingly, the final ethanol concentration was up to 127.88 g/kg; the ethanol yield reached 88.28%, and ethanol productivity was 4.263 g/kg/h after 30 h. When the fermentation scale expanded to 100 L, the final ethanol concentration was 127.75 g/kg, and the ethanol productivity was 4.258 g/kg/h in 30 h. The residual sugar was analyzed by high performance liquid chromatography, and proved that there was no glucose and fructose. The residual reducing sugar was some unfermentable oligosaccharide Part 2 is the study of the rapid ethanol production by Zymomonas mobilis. Compare with other seven stains, Zymomonas mobilis 232B was selected for research. The optimum condition in glucose medium was as follow: glucose concentration 18%, initial pH 6-7, and fermentation temperature 30 ℃. The ethanol concentration was 88g/kg in 18 h. After that, rapid ethanol production from cassava in SSF by Zymomonas mobilis 232B was studied. Through a series of experiments aided by Full Factorial Design and steepest ascent search, the optimal concentration yeast extract and ammonium sulfate were determined: 4 g/kg and 0.8 g/kg, each. Under optimum medium conditions, the optimal hydromodulus of cassava to water and glucoamylase dosages were obtained: hydromodulus of cassava to water at 1:3 and glucoamylase dosages 4 AGU/g starch. The ethanol production reached 4.915 g/kg/h. The residual sugar was analyzed by HPLC, and proved that the residual reducing sugar was some unfermentable oligosaccharide，but the components were more complex than that fermentation by Saccharomyces cerevisiae.

非粮原料燃料乙醇发酵关键技术的研究

生物质燃料乙醇是一种高度清洁的交通液体燃料，是减少温室气体排放，缓解大气污染的最佳技术选择。以非粮原料生产燃料乙醇可以在进行能源生产的同时保证粮食安全，有利于产业的可持续发展。在众多的非粮原料中，甘薯是我国开发潜力最大的生物质能源作物之一。我国占世界甘薯种植总面积和产量的90％。同时，甘薯的单位面积燃料乙醇产量远大于玉米和小麦。其成本是目前酒精中最低廉的，因此利用甘薯生产乙醇是发展生物质燃料乙醇的首要选择。目前采用薯类全原料主要采用分批发酵生产乙醇，其技术水平低，发酵强度低，一般在0.7－2.5g/（L•h），乙醇浓度低，甘薯发酵乙醇为6－8％(v/v)，能耗高，环境负荷大，污染严重。针对上述问题，本文从菌株选育、原料预处理、中试放大、残糖成分分析等方面进行研究。 为了研究乙醇发酵生产规模扩大过程中，大型发酵罐底部高压条件下，CO2对酵母乙醇发酵的影响，我们通过CO2 加压的方法进行模拟试验，研究结果表明，发酵时间随压强的升高而逐渐延长，高压CO2 对乙醇发酵效率影响不大，在0.3 MPa 以下时，发酵效率均可达到90%以上。高压CO2 对发酵的抑制作用是高压和CO2 这两个因素联合作用的结果。高压CO2 条件下，酵母胞外酶和胞内重要酶类的酶活均表现出特征性。0.2 MPa 下，酶活性的变化趋势和0.1 MPa 条件下的较为一致。而0.3 MPa 下的酶活变化趋势与0.4 MPa 下的酶活更为接近。通过全基因表达分析发现在CO2 压力为0.3 MPa 下，乙醇发酵途径中多个基因表达量下调，同时海藻糖合成酶和热激蛋白基因表达量上调。 筛选耐高温的乙醇酵母菌株能够解决糖化温度和发酵温度不协调的矛盾，实现真正意义上的边糖化边发酵。高温发酵还能够降低发酵时的冷却成本，实现乙醇的周年生产。本研究筛选出一株高温发酵菌株Y-H1，进而我们对该菌株的胞外酶和胞内乙醇代谢重要酶类的酶活性进行了分析。结果表明Y-H1 能够在40 ℃条件下正常进行乙醇发酵，发酵33h，最终乙醇浓度达到10.7%（w/w），发酵效率达到90%以上。同时发酵液最终pH 在3.5 左右，显示菌株具有一定的耐酸性能力。同时观察到40 ℃下，菌株的胞外酶和胞内乙醇代谢重要酶类的酶活性发生了变化，乙醇发酵途径中关键酶基因表达下调，而海藻糖合成酶与热激蛋白基因表达量上调，这些结果为进一步研究酵母菌耐热调控机理提供了依据。 糖蜜是一种大规模工业生产乙醇的理想原料，本研究利用选育高浓度乙醇发酵菌株结合配套的发酵稳定剂，研究了糖蜜高浓度乙醇发酵情况。结果表明采用冷酸沉淀预处理糖蜜溶液，采用分批补料的发酵方式，乙醇浓度最高达到了10.26% (w/w)，发酵时间为42 h。同时观察到在糖蜜发酵中，乙醛含量与乙醇浓度存在一定的相关性。 快速乙醇发酵对于缩短乙醇生产周期、降低乙醇生产成本、减少原料腐烂损失具有重要意义。本研究诱变和筛选得到了一株快速乙醇发酵菌株10232B。在优化后的发酵条件下，采用10L 发酵罐进行分批乙醇发酵，经过18h,乙醇的最终浓度达到88.5g/L,发酵效率93.6%，平均乙醇生产速度达到4.92 g/L/h。此菌株在保持较高乙醇生产浓度的同时，拥有快速生产乙醇的能力，适合作为快速乙醇发酵生产菌种。 由于鲜甘薯具有粘度大的特点，传统液化糖化处理很难在短时间内充分糖化原料；高粘度的醪液也难以进行管道输送，容易堵塞管路；同时，也会降低后续的乙醇发酵效率。 本文采用了快速粘度分析法对鲜甘薯糊化粘度特性进行了分析，进而对预处理条件进行了研究，在最佳预处理条件下，糖化2h 后，醪液葡萄糖值最高可达99.3，粘度4.5×104 mPa.s，而采用传统糖化工艺，醪液DE 值仅为85.8，粘度大于1.0×105 mPa.s。 此预处理方法也可用于快速糖化不加水的醪液。后续的乙醇发酵试验表明，通过此预处理方法获得的糖化醪液对乙醇发酵无负面影响。 在前期已实现了实验室水平的鲜甘薯燃料乙醇快速乙醇发酵基础上，进一步将发酵规模扩大到500L，在中试水平上对甘薯乙醇发酵进行了研究。结果表明在500L 中试规模，采用边糖化边发酵（SSF）工艺，在料液比为3∶1，发酵醪液最高粘度为6×104mPa.s 条件下，发酵37h，乙醇浓度达到了12.7%（v/v），发酵效率91%，发酵强度为2.7 g/（L•h）。与目前国内的薯类乙醇发酵生产技术水平具有明显的优越性。 为研究甘薯、木薯乙醇发酵中残糖的组成，采用了高效液相色谱—蒸发光散射检测法，对乙醇发酵残糖进行了分析。结果表明，甘薯、木薯乙醇发酵残糖均为寡聚糖，主要由葡萄糖、木糖、半乳糖、阿拉伯糖和甘露糖构成。随着发酵时间延长，寡聚糖中的葡萄糖、半乳糖、甘露糖可被缓慢的水解释放。提高糖化酶量仅在一定程度上降低残糖，过量的糖化酶反而会导致残糖增加。同时发现3, 5-二硝基水杨酸法不能准确测定甘薯、木薯乙醇发酵中的残总糖含量。进一步筛选了两株残糖降解菌株，对甘薯乙醇发酵残糖的降解利用率均达到了40%以上，而且还能显著降低发酵醪液粘度。经形态学和rRNA ITS 序列分析，确定这两株菌分别属于为木霉属和曲霉属黑曲霉组。 通过对以甘薯原料为代表的非粮原料发酵技术研究开发，以期形成乙醇转化率高，能耗低，生产效率高、季节适应性好，原料适应性广，经济性强，符合清洁生产机制的燃料乙醇高效转化技术，为具有我国特色的燃料乙醇发展模式提供技术支持。 Sweet potato is one of the major feedstock for the fuel ethanol production in China. The planting area and the yield in China take 90% of the world. Sweet potato is an efficient kind of energy crops. The energy outcome per area is higher than corn or wheat. And the manufacture cost of ethanol is the lowest, compared with corn and wheat. So sweet potato is the favorable crop for the bioethanol production in China. However, the low-level fermentation technology restricts the development of ethanol production by sweet potato, including slow ethanol production rate, low ethanol concentration and high energy cost. To solve these problems, we conducted research on the strain breeding, pretreatment, pilot fermentation test and residual saccharides analysis. To study the impact of hyperbaric condition at bottom of the large fermentor on yeast fermentation, high pressure carbon dioxide (CO2) was adopted to simulate the situation. The results showed that the fermentation was prolonged with the increasing pressure. The pressure of CO2 had little impact on the ethanol yield which could reach 90% under the pressure below 0.3 MPa. The inhibition was combined by the high pressure and CO2. Under the high CO2 pressure, the extracellular and important intracellular enzyme activities were different from those under normal state. The changes under 0.1 MPa and 0.2 MPa were similar. The changes under 0.3 MPa were closer to those under 0.4 MPa. The application of thermotolerance yeast could solve the problem of the inconsistent temperature between fermentation and saccharificaton and fulfill the real simultaneous saccharification and fermentation. And it could reduce the cooling cost. A thermotolerance strain Y-H1 was isolated in our research. It gave high ethanol concentration of 10.7%（w/w）at 40 ℃ for 33 h. The ethanol yield efficiency was over 90%. At 40 ℃, the extracellular and important intracellular enzyme activities of Y-H1 showed the difference with normal state, which may indicate its physiological changes at the high temperature. Molasses is another feedstock for industrial ethanol production. By our ethanol-tolerance strain and the regulation reagents, the fermentation with high ethanol concentration was investigated. In fed-batch mode combined with cold acid deposition, the highest ethanol concentration was 10.26% (w/w) for 42h. The aldehyde concentration in fermentation was found to be related to ethanol concentration. The development of a rapid ethanol fermentation strain of Zymomonas mobilis is essential for reducing the cost of ethanol production and for the timely utilization of fresh material that is easily decayed in the Chinese bioethanol industry. A mutant Z. mobilis strain, 10232B, was generated by UV mutagenesis. Under these optimized conditions, fermentation of the mutant Z. mobilis 10232B strain was completed in just 18 h with a high ethanol production rate, at an average of 4.92 gL-1h-1 per batch. The final maximum ethanol concentration was 88.5 gL-1, with an ethanol yield efficiency of 93.6%. This result illustrated the potential use of the mutant Z. mobilis 10232B strain in rapid ethanol fermentation in order to help reduce the cost of industrial ethanol production. As fresh sweet potato syrup shows high viscosity, it is hard to be fully converted to glucose by enzymes in the traditional saccharification process. The high-viscosity syrup is difficult to be transmitted in pipes, which may be easily blocked. Meanwhile it could also reduce the later ethanol fermentation efficiency. To solve these problems, effects of the pretreatment conditions were investigated. The highest dextrose equivalent value of 99.3 and the lowest viscosity of 4.5×104 mPa.s were obtained by the most favorable pretreatment conditions, while those of 85.8 and over 1.0×105 mPa.s was produced by traditional treatment conditions. The pretreatment could also be applied on the material syrup without adding water. The later experiments showed that the pretreated syrup had no negative effect on the ethanol fermentation and exhibited lower viscosity. The fuel ethanol rapid production from fresh sweet potato was enlarged in the 500L pilot scale after its fulfillment on the laboratory level. The optimal ratio of material to water was 3 to 1 in 500L fermentor. With low-temperature-cooking (85 ℃) using SSF, the Saccharomyces cerevisiae was able to produce ethanol 97.44 g/kg for 37h, which reached 92% of theoretical yield. The average ethanol production rate was 4.06 g/kg/h. And the maximum viscosity of syrup reached 6×104mPa.s. The results showed its superiority over current industrial ethanol fermentation. The compositions of the residual saccharides in the ethanol fermentation by sweet potato and cassava were analyzed by high performance liquid chromatography coupled with evaporative light-scattering detector. The results showed that all the residual saccharides were oligosaccharides, mainly composed of glucose, xylose, galactose, arabinose and mannose. The glucose, galactose and mannose could be slowly hydrolyzed from oligosaccharides in syrup during a long period. To increase the glucoamylase dosage could lower the residual saccharides to a certain extent. However, excess glucoamylase dosage led to more residual saccharides. And the method of 3, 5-dinitrosalicylic acid could not accurately quantify the residual total saccharides content. Two residual saccharides degrading strains were isolated, which could utilize 40% of total residual saccharide and lower the syrup viscosity. With the analysis of morphology and internal transcribed spacer sequence, they were finally identified as species of Trichoderma and Aspergillus niger.

组合白腐真菌预处理木质纤维素原料的研究

木质纤维素原料种类多、分布广、数量巨大，通过燃料乙醇生产技术、厌氧沼气发酵技术将其转化成乙醇、沼气等二次能源，一定程度上可以缓解化石能源的不断消耗所带来的能源危机，也解决了农林废弃物引起的环境污染问题。其中以木质纤维素原料生产燃料乙醇，还可以避免以淀粉类和糖类原料生产燃料乙醇时带来的“与人争粮”等一系列问题。因此具有重要的经济效益、环境效益和社会效益。 然而，木质纤维素原料结构致密，木质素包裹在纤维素、半纤维素外围，导致其很难被降解利用，必须进行适当的预处理，去除木质素，打破原有的致密结构，利于原料的后续利用。因此，预处理成为木质纤维素原料能源化利用的关键。而目前预处理环节的费用过于昂贵，于是寻找一种高效、低成本的预处理方法是当今研究的热点。 本论文采用组合白腐真菌对木质纤维素原料进行生物预处理研究，与其他物理化学法相比，该法有着专一性较强、反应温和、不造成环境污染、成本低等优势。白腐真菌主要通过分泌木质素降解酶对木质素进行降解，从而破坏原料的致密结构，提高后续利用效率。所以木质素降解酶酶活的高低是影响原料预处理效果的一个关键因素。于是本论文首先通过将白腐真菌进行组合的方式提高木质素降解酶（漆酶，Lac）酶活；接着对组合菌的菌株相互作用机理进行研究，阐明组合菌Lac 酶活提高的原因，为菌株组合提高Lac 酶活这种方法的应用提供理论依据，同时也为后续组合白腐真菌预处理木质纤维素原料提供指导；进一步采用固态发酵和木质素降解酶两种方式对木质纤维素原料进行预处理研究，最大化去除木质素成分，破坏原料的致密结构；最终对预处理后原料的酶解糖化进行初步研究，为原料后续的能源化应用奠定基础。具体研究结果如下： （1） 以实验室保存的三株主要分泌Lac 的白腐真菌为出发菌株，筛选得到一组Lac 酶活明显提高的组合菌55+m-6，其中菌株55 为Trametes trogii sp.，m-6 为Trametes versicolor sp.，组合后Lac 酶活较单菌株分别提高24.13倍和4.07 倍。组合菌的最适产酶条件为pH 6.5、C/N 16:1、Tween 80 添加量为0.01%，在该条件下组合菌的Lac 酶活峰值比未优化时提高4.11倍。 （2） 对组合菌55+m-6 菌株间相互作用机理进行研究，发现菌株之间不存在抑制作用；平板培养时，菌丝交界处Lac 酶活最高并分泌棕色色素；液体培养时，菌株m-6 对组合后Lac 酶活的提高起着更为重要的作用：菌株m-6的菌块、过滤灭菌胞外物以及高温灭菌胞外物均能明显刺激菌株55 的Lac产生；菌株55、m-6 进行组合后，同工酶种类未发生增减，但有三种Lac同工酶浓度有所提高；对菌株胞外物进行薄层层析和质谱分析，结果表明组合前后菌株胞外物中各物质在浓度上存在较大的变化。推测组合菌Lac酶活的明显提高，主要是由于菌株m-6 胞外物中的一些物质能刺激菌株55 分泌大量Lac 进行代谢，且这些刺激物质并非菌株m-6 特有，菌株55自身也可以代谢生成，但是适当的浓度才能刺激Lac 的大量分泌。 （3） 将组合菌55+m-6 用于固态发酵预处理木质纤维素原料，发现其对玉米秆的降解程度最大，在粉碎度40 目、含水率65%的最优处理条件下，处理至第15d，秸秆失重率为41.24%，其中木质素、纤维素、半纤维素均有降解，且Lac 和纤维素酶（CMC）酶活以及还原糖量均达到峰值。 （4） 对玉米秆进行木质素降解酶预处理，发现Lac/1-羟基苯并三唑（HBT）系统对玉米秆木质素的降解效果最好，在最优处理条件时，即HBT 用量0.2%、处理时间1d、Lac 用量50U/g，木质素降解率可达12.60%。预处理后玉米秆的致密结构被破坏，比表面积增大，利于后续酶与纤维素、半纤维素成分的结合。 （5） 对预处理后的玉米秆进行酶解糖化，其中组合菌固态发酵预处理后玉米秆的糖化率比对照高4.33 倍；Lac/HBT 系统预处理后玉米秆的糖化率比对照高2.99％，糖化液中主要含有木糖、葡萄糖两种单糖。 There are many kinds and large quantities of lignocellulosic biomass widely distributed on the earth. They can be converted into secondary energy such as fuel ethanol, biogas, et al., which can relieve the energy crisis caused by consumption of fossil energy resources and solve the problem of environmental pollution caused by agriculture and forestry waste. Meanwhile, the production of fuel ethanol from lignocellulosic biomass can ensure food supply to human kind instead of starch- and sugar-containing raw materials. So the energy conversion of lignocellulosic biomass contributes considerable economic, environment and social benefits. However, lignocellulosic biomass has the compact structure, in which lignin surrounds cellulose and hemicellulose, so it must be pretreated before energy usage and pretreatment is one of the most critical steps in the energy conversion of lignocellulosic biomass. At present, the cost of pretreatment is too expensive, so looking for an efficient and low-cost pre-treatment method is one of recent research hot spots. In this research, combined white rot fungi pretreatment method was used, which had some advantages in low cost, high specificity, mild reacting conditions and friendly environmental effects compared with the other physical and chemical methods. White rot fungi secrete lignin degrading enzymes to degrade the content of lignin and damage the contact structure of lignocellulosic biomass, so the activity of the lignin degrading enzymes is the key factor to the degradation effect of raw materials. Firstly, the combined fungi with high laccase activity were screened; secondly, the interaction mechanism between strains was studied, and the cause of higher laccase activity after strains combination was also preliminary clarified; under the guidance of the mechanism, lignocellulosic biomass was pretreated by the combined fungi; lastly, the enzymatic hydrolysis of pretreated lignocellulosic biomass was also preliminary studied; all of the researches could lay the foundation for the energy application of lignocellulosic biomass. The specific research results were as follows: (1) The combined fungi 55+m-6 with significant higher laccase activity were screened from the three white rot fungi stored in our lab which mainly secreted laccase. Strain 55 and strain m-6 were Trametes trogii sp. and Trametes versicolor sp., respectively. The laccase activity of combined fungi was 24.13 and 4.07-fold than strain 55 and strain m-6, respectively. The optimized condition for laccase production of the combined fungi in liquid medium was pH 6.5, C/N 16:1 and Tween 80 0.01%. In this optimized condition, the laccase activity of combined fungi was 4.11-fold higher comparing with which in non-optimized medium. (2) The interaction mechanism between strain 55 and strain m-6 was further studied, and no inhibition effect was observed. Brown pigment was secreted on the junction of the two strains on the plate, where the highest laccase activity was detected. Strain m-6 was much important to boost laccase activity of combined fungi in liquid medium, and strain 55 was stimulated by fungal plug, filter sterilized extracellular substances and high temperature sterilized extracellular substances of strain m-6 to produce laccase. The types of laccase isozymes did not change after combining strain 55 and strain m-6, but the concentrations of three types increased. Mass Spectrometry and TLC analysis of extracellular substances of each strain showed that concentration of some substances considerably changed after strains were combined. It was supposed that the cause of higher laccase activity of combined fungi was mainly due to some extracellular substances of strain m-6 with the appropriate concentration which stimulated laccase secretion of strain 55 and generated not only by strain m-6 but also by strain 55. (3) Combined fungi 55+m-6 were used to lignocellulosic biomass pretreatment with the type of solid-state fermentation. The highest degree of degradation of corn straw was obtained, including the rate of weight loss was 41.24% and the lignin, cellulose and hemicellulose were degraded partially under the optimized condition of 40 mesh, 65% water content on 15th day. Laccase, CMCase activities and content of reducing sugar reached the maximum value on that day. (4) Lignin degrading enzymes from combined fungi 55+m-6 were used for corn straw pretreatment. The most remarkable degradation of lignin in corn straw with Lac/1-hydroxybenzotriazole (HBT) system was observed, and the 12.60% lignin degradation was obtained under the optimized condition of 0.2% HBT, 50 U/g laccase for 1 d. After pretreated by Lac/HBT, the tight structure of corn straw was demolished and specific surface area increased, which had advantages for accessible of enzyme to cellulose and hemicellulose. (5) The corn straws pretreated by combined fungi 55+m-6 with the type of solid-state fermentation and Lac/HBT were used for enzymatic hydrolysis, and the saccharification rates of each pretreatment type were 4.33 times and 2.99% higher than CK, respectively. The enzymatic hydrolysis liquid of corn straw pretreated by Lac/HBT mainly contained xylose and glucose.

Effect of gamma-ray radiation on physiological, morphological characters and chromosome aberrations of minitubers in Solanum tuberosum L.

Purpose: To investigate the effects of gamma-ray radiation on the physiological, morphological characters and chromosome aberrations of minitubers. Materials and methods: Minitubers of one potato cultivar, 'Shepody', were irradiated with 8 doses of gamma-rays (0, 10, 20, 30, 40, 50, 60, 70 and 80 Gy [Gray]) to investigate the effects of radiation on emergence ability, plant height and root length, morphological variations, chromosome aberrations, M-1 (first generation mutants) tuber number and size of minituber plants. Results: Compared with the non-irradiated controls, the whole period of emergence was prolonged by 10-15 days for minitubers treated with gamma-ray radiation, but low doses of radiation (10, 20 and 30 Gy) promoted the emergence percentage of minitubers. With an increase in radiation dose, the emergence percentage, plant height and root length of minituber plants were significantly inhibited at 40 and 50 Gy. No emergence occurred at 60 Gy and higher doses. After radiation, a series of morphological variations and chromosome aberrations appeared in minituber plants. Radiation with 20 Gy promoted tuber formation, and the average number and diameter of M-1 tubers per plant were significantly increased over the control by 71% and 34%, respectively. Conclusion: Low doses of radiation (10-30 Gy) might be used as a valuable parameter to study the improvement of minitubers by gamma-ray radiation treatment.

Fruit anatomy,seed germination and seedling development in the Japanese seagrass phyllospadix

Phyllospadix iwatensis Makino and phyllospadix japonicus Makino have similar frunt morphology and anatomy.The rhomboid fruit of Japanese phyllospadix is dark brown in colour and is characterized by two arms bearing stiff inflected bristles which can act as an anchoring system. The fruit covering consists of a thin cuticular seed coat and pericarp remains mainly fibrous endocarp. In the groove region of the fruit.the cuticular seed coat and endocarp are replaced by nucellus cells with wall in growths and crushed pigment strands with lignified walls.these tissues appera to control the transfer of nutrients to developing seed.the seed is oval with a small embryo and a large hypocotyl. the embryo is straight and simple,with the plumule containing three leaf primordia and a pair of root primordia surrounded by a cotyledon.the hypocotyl has large vontral lobe containing central provascular tissue and two small dorsal lobes.the hypocotyl contains starch.lipid and protein.and acts as a nutrient store.the seed of P.iwatensis has a dormancy period of 2-6 weeks and germination eventually reaches-65%.but is not synchronized.during germination the leaves emerge first.and then after at least three young leaves have formed and abseised.the roots emerge,usually?6 months after the commencement of germination.Utilizaton of the nutrient reserves is initially from the perihpery of the hypocotyl and then progressively towards its centre.