中国青藏高原青稞抗穗发芽资源评价与α-淀粉酶基因的克隆及表达

穗发芽（PHS，preharvest sprouting）是影响禾本科作物生产的重要的灾害之一。收获时期如遇潮湿天气容易导致穗发芽发生。发生穗发芽的种子内部水解酶（主要是α-淀粉酶）活性急剧升高，胚乳贮藏物质开始降解，造成作物产量和品质严重降低。因此，选育低穗发芽风险的品种是当前作物育种工作中面临的重要任务。 青稞(Hordeum vulgare ssp. vulgare)主要分布于青藏高原，自古以来就是青藏高原人民的主要粮食。近年来，由于青稞丰富的营养成分和特有的保健品质、在燃料工业中的潜力以及在啤酒酿造工业中的利用前景，在发达国家日趋受到重视，掀起综合研究利用的热潮。我国拥有占全世界2/3 以上的青稞资源，具有发展青稞产业的得天独厚的条件。然而，由于青稞收获期间恰逢青藏高原雨季来临，常有穗发芽灾害发生，使青稞生产损失巨大。目前对青稞穗发芽研究很少，适用于育种的穗发芽抗性材料相对缺乏，不能很好的满足青稞穗发芽抗性育种的需要。本研究以青藏高原青稞为材料，对其穗发芽抗性的评价指标和体系进行构建，同时筛选青稞抗穗发芽品种并对其抗性进行评价，还利用分子生物学手段对青稞穗发芽抗性的分子机理进行了初步探讨。主要研究结果如下： 1. 本试验以来自于我国青藏高原地区的青稞为材料，对休眠性测定的温度范围进行探讨，并对各种穗发芽抗性测定方法的对青稞的适用性进行评测。通过探讨温度对13 个不同基因型的青稞籽粒发芽和休眠性表达的影响，对筛选青稞抗穗发芽资源的温度条件进行探索，并初步分析了其休眠性表达的机理。在10，15，20，25，30℃的黑暗条件下，选用新收获的13 个青稞品种为材料进行籽粒发芽实验，以发芽指数（GI）评价其休眠性。结果发现，不同品种对温度敏感性不同，其中温度不敏感品种，在各温度条件下均表现很低的休眠性；而温度敏感品种，其休眠性表达受低温抑制，受高温诱导。15℃至25℃是进行青稞休眠性鉴定的较适宜的温度范围。通过对供试材料发芽后的α-淀粉酶活性，发现温度对青稞种子的休眠性表达的影响至少在一定程度上表现在对α-淀粉酶活性的调控上。随后，对分别在马尔康和成都进行种植的34 份青稞穗发芽指数（SI），穗发芽率（SR），籽粒发芽指数（GI）和α-淀粉酶活性（AA）进行了测定和分析，发现它们均受基因型×栽培地点的极显著影响，且四个参数之间具有一定相关性。GI 参数由于其变异系数较低，在不同栽培地点稳定性好，且操作简便，是较可靠和理想的穗发芽评价参数。SI 参数可作为辅助，区别籽粒休眠性相似的材料（基因型）或全面评价材料（基因型）的穗发芽抗性特征。AA 参数稳定性较差，并且检测方法复杂，因此不建议在育种及大量材料筛选和评价时使用。此外，青稞穗发芽抗性受环境影响较大，评价时应考虑到尽可能多的抗性影响因素及其在不同栽培条件下的变异。 2. 对来自青藏高原的青稞穗发芽抗性特征及其与其它农艺性状间的关系进行研究。通过测定穗发芽指数（SI）、籽粒发芽指数（GI）和α-淀粉酶活性（AA），表明113 份青稞材料的穗发芽抗性具有显著差异。SI、GI 和AA 参数的变幅分别为1.00~8.86、0.01~0.97 和0.00~2.76，其均值分别为4.72、0.63 和1.22。根据SI 参数，六个基因型，包括‘XQ9-5’，‘XQ33-9’，‘XQ37-5’，‘XQ42-9’，‘XQ45-7’和‘JCL’被鉴定为抗性品种。综合SI、GI 和AA 参数，可以发现青稞的穗发芽抗性机制包含颖壳等穗部结构的抗性和种子自身的抗性（即种子休眠性），且供试材料中未发现较强的胚休眠品种，除‘XQ45-7’外，所有品种在发芽第四天均能检测出α-淀粉酶活性。穗部结构和种子休眠的抗性机制因基因型不同而不同，在穗发芽抗性中可单独作用或共同作用。农家品种和西藏群体分别比栽培品种和四川群体的穗发芽抗性强，而在不同籽粒颜色的青稞中未发现明显差异。相关性检验发现，青稞的穗发芽抗性，主要是种子休眠性，与百粒重、开花期、成熟期、穗长、芒长和剑叶长呈显著负相关关系，与株高相关性不显著。农艺性状可以作为穗发芽抗性材料选育中的辅助指标。本试验为青稞穗发芽抗性育种研究提供了必要的理论基础和可供使用的亲本材料。 3. α-淀粉酶是由多基因家族编码的蛋白质，在植物种子萌发时高度表达，与植物种子的萌发能力密切相关。在大麦种子发芽时，高等电点α-淀粉酶的活性远大于低等电点的α-淀粉酶。为了研究不同穗发芽抗性青稞品种中编码高等电点α-淀粉酶Amy1 基因结构与抗性间的关系，我们以筛选得到的抗性品种‘XQ32-5’（TR1）、‘XQ37-5’（TR2）、‘XQ45-7’（TR3），易感品种‘97-15’（TS1）、‘9657’（TS2）以及强休眠大麦品种‘SAMSON’（SAM）为材料，对其Amy1 基因的编码区序列进行克隆和结构分析，并对它们推导的氨基酸序列进行比较。结果显示，青稞Amy1 基因具有三个外显子、两个内含子，编码区中有13 个核苷酸变异位点，均位于2、3 号外显子，2 个变异位点位于2 号外显子。SAM 和TS1 分别在2 号外显子相应位置有5 个相同的碱基(GAACT)的插入片段。相应α-淀粉酶氨基酸序列推导发现，所有核苷酸变异中有8 个导致相应氨基酸残基的改变，其余位点为同义突变。青稞Amy1 基因编码区序列品种间相似度高达99%以上，部分序列变异可能与其穗发芽抗性有关。随后，我们又通过SYBR Green 荧光定量技术对该基因在不同发芽时间（1d~7d）的相对表达水平进行了差异性检测。结果发现，7 天内不能检测到SAM 的Amy1 基因表达，5 个青稞品种间的Amy1 基因的相对表达量均随着发芽时间延长而上升，但上升方式有所不同。弱抗品种该基因表达更早，转录本增加速率更大，且在4~5 天可达到平台期。发芽7 天中，抗性品种总转录水平明显低于易感品种。本研究结果表明，青稞Amy1 基因的转录水平是与其穗发芽抗性高度相关。 我国青藏高原青稞，尤其是农家品种的穗发芽抗性具有丰富的变异，蕴藏着穗发芽抗性育种的宝贵资源。本研究为青稞穗发芽抗性育种建立了合理抗性评价体系，筛选出可供育种使用的特殊材料，阐明了农艺性状可辅助穗发芽抗性育种，同时还对穗发芽抗性与α-淀粉酶基因的结构和表达关系进行分析，为青稞穗发芽抗性资源筛选奠定了基础。 Preharvest sprouting (PHS) is a serious problem in crop production. It often takes place when encountering damp, cold conditions at harvest time and results in the decrease of grain quality and great loss of yield by triggering the synthesis of endosperm degrading enzymes (mostly the α-amylase). Therefore, PHS is regarded as an important criterion for crop breeding. In order to minimize the risk of PHS, resistant genotypes are highly required. Hulless barley (Hordeum vulgare ssp. vulgare) is the staple food crop in Qinghai-Tibetan Plateau from of old, where is one of the origin and genetic diversity centers of hulless barley. Recently, interest in hulless barley has been sparked throughout the world due to the demonstrations of its great potential in health food industry and fuel alcohol production. Indeed, hulless barley can also be utilized to produce good quality malt if the appropriate malting conditions are used. In China, overcast and rainy conditions often occur at maturity of hulless barley and cause an adverse on its production and application. PHS resistant genotypes, therefore, are highly required for the hulless barley breeding programs. However, few investigations have been made so far on this issue. The objectives of this study were: 1) to assessment of methods used in testing preharvest sprouting resistance in hulless barley; 2) to evaluate the variability and characteristics of PHS resistance of hulless barley from Qinghai-Tibet Plateau in China; 3) to select potential parents for PHS resistance breeding; 4) to primarily study on the molecular mechanism of PHS resistance of hulless barley. Our results are as followed: 1. We investigated the temperature effects on seed germination and seed dormancy expression of hulless barley, discussed appropriate temperature range for screening of PHS resistant varieties, and analyzed the mechanism of seed dormancy expression of hulless barley. The dormancy level of 13 hulless barley were evaluated by GI (germination index) values calculating by seed germination tests at temperature of 10，15，20，25，30℃ in darkness. There were great differences in temperature sensitivity among these accessions. The insensitive accessions showed low dormancy at any temperature while the dormancy expression of sensitive accessions could be restrained by low temperature and induced by high temperature. The temperature range of 15℃ to 25℃ was workable for estimating of dormancy level of hulless barley according to our data. Analysis of α-amylase activity showed that the temperature effects on seed germination and the expression of seed dormancy be achieved probable via regulating of α-amylase activity. Furthermore, we evaluated the differences in sprouting index (SI), sprouting rate (SR), germination index (GI) and α-amylase activity (AA) between Maerkang and Chengdu among 34 accessions of hulless barley from Qinghai-Tibetan Plateau in China. These PHS sprouting parameters were significantly affected by accession×location, and they had correlation between each other. GI was the most reliable parameter because of its low CV value, good repeatability and simple operation. SI could assist in differentiating between accessions of similar dormancy or overall evaluation of the resistance. AA was bad in repeatability and had relatively complex testing method, therefore, not appropriate for breeding and evaluation and screening of PHS resistant materials. Besides, since PHS resistance of hulless barley was greatly influenced by its growth environment, possibly much influencing factors and variations between cultivated conditions should be considered. 2. In this study, large variation was found among 113 genotypes of hulless barley (Hordeum vulgare ssp.vulgare) from Qinghai-Tibetan Plateau in China, based on the sprouting index (SI), germination index (GI) and α-amylase activity (AA) which derived from sprouting test of intact spikes, germination test of threshed seeds and determination of α-amylase activity, respectively. The range of SI, GI and AA was 1.00~8.86, 0.01~0.97 and 0.00~2.76，the mean was 4.72, 0.63 and 1.22 espectively. Six resistant genotypes, including ‘XQ9-5’, ‘XQ33-9’, ‘XQ37-5’, ‘XQ42-9’, ‘XQ45-7’ and ‘JCL’, were identified based on SI. Integrating the three parameters, it was clear that both hulls and seeds involved in PHS resistance in intact spikes of hulless barley and there was no long-existent embryo dormancy found among the test genotypes. All the genotypes, except ‘XQ45-7’, had detectable α-amylase activity on the 4th day after germination. There was PHS resistance imposed by the hull and seed per se and the two factors can act together or independent of each other. Besides, landraces or Tibet hulless barley had a wider variation and relatively more PHS resistance when compared with cultivars or Sichuan hulless barley. No significant difference was found among hulless barley of different seed colors. The correlation analysis showed PHS resistance was negatively related to hundred grain weight, days to flowering, days to maturity, spike length, awn length and flag length but not related to plant height. This study provides essential information and several donor parents for breeding of resistance to PHS. 3. Alpha-amylase isozymes are encoded by a family of multigenes. They highly express in germinating seeds and is closely related to seed germination ability. In barley germinating seeds, the activity of high pI α-amylase is much higher than low pI α-amylase. The aim of this study was to determine the relationship between preharvest sprouting resistance of hulless barley and the gene structure of Amy1 gene which encodes high pI α-amylase. The coding region and cDNA of Amy1 gene of three resistant accessions, including ‘XQ32-5’ (TR1), ‘XQ37-5’ (TR2), ‘XQ45-7’ (TR3), two susceptible accessions ‘97-15’ (TS1), ‘9657’ (TS2) and one highly dormant barley accession ‘SAMSON’ (SAM) was cloned. Analysis of their DNA sequences revealed there were three exons and two introns in Amy1 gene. Thirteen variable sites were in exon2 and exon3, 2 variable sites were in intron2. SAM and TS1 had a GAACT insert segment in the same site in intron2. Only 8 variable sites caused the change of amino acid residues. There were 99% of similarity between the tested hulless barley and some of the variable sites might be related with preharvest sprouting resistance. Then, we investigated the expression level of Amy1 gene in the 7-day germination test. Results of quantitative real-time PCR indicated that the relative expression trends of Amy1 gene were the same but had significant differences in the increase fashion between hulless barleys and no detectable expression was found in SAM. Susceptible accessions had earlier expression and faster increase and reached the maximum on day 4 ~ day 5. Besides, total transcripts level was found lower in resistant accessions than susceptible accessions. This study indicated that α-amylase activity was highly related to the transcription level of Amy1 gene which not correlated to missense mutation sites. In conclusion, hulless barley, especially the landraces from Qinghai-Tibetan Plateau in China possesses high degree of variation in PHS performance, which indicates the potential of Tibetan hulless barley as a good source for breeding of resistance to PHS. This study provides several donor parents for breeding of resistance to PHS. Our results also demonstrate that agronomic traits may be used as assistants for PHS resistance selection in hulless barley. Besides, analysis of high pI α-amylase coding gene Amy1 revealed the relative high expression of was Amy1 one of the mainly reason of different PHS resistance level in hulless barley.

Antioxidant capacity and lipophilic content of seaweeds collected from the Qingdao coastline

Lipophilic extracts from 16 species of seaweeds collected along the Qingdao coastline were screened and evaluated for their antioxidant activities (AA) using the beta-carotene-linoleate assay system. The diethyl ether soluble extracts of all selected seaweeds exhibited various degrees of antioxidative efficacy in each screen. The highest antioxidant capacities among the tested samples were observed for Rhodomela confervoides and Symphyocladia latiuscula and were comparable with that of the well-known antioxidant butylated hydroxytoluene and greater than that of propyl gallate. The lipophilic content of all 16 samples and the chemical composition of 4 selected seaweeds, R. confervoides and S. latiuscula, which had higher AA, Laminaria japonica, which had intermediate AA, and Plocamium telfairiae, which had lower AA, were analyzed by gas chromatography and gas chromatography-mass spectrometry, respectively. Fatty acids and alkanes were found. The present data indicated an increase in antioxidative property with increasing content of unsaturated fatty acid. The result of this study suggests that seaweeds can be considered as a potential source for the extraction of lipophilic antioxidants, which might be used as dietary supplements or in production in the food industry. This is the first report on the antioxidant activities of lipophilic extracts from seaweeds.

Differentiation of the rainbow trout (Oncorhynchus mykiss) from Atlantic salmon (Salmon salar) by the AFLP-derived SCAR

A species-specific SCAR marker for rainbow trout, which was used to detect adulteration and fraudulent labeling in Atlantic salmon products, has been developed based on the AFLP analysis and evaluated in this study. The SCAR marker could be amplified and visualized in 1% agarose gel in all tested rainbow trout samples and absent in all salmon samples. Using DNA admixtures, the detection of 1% (0.5 ng), 10% (5 ng) rainbow trout DNA in Atlantic salmon DNA for fresh and processed samples, respectively was readily achieved. The molecular approach was sensitive and demonstrated to be a rapid and reliable method for identifying frauds in salmon products and could be extended for applications of species identification in food industry.

羟基自由基降解琼胶及其产物的活性研究

琼胶是一种从石花菜等红藻中提取的，目前生产工艺和结构等方面研究比较成熟的海藻多糖，广泛应用于医药、仪器等行业。但是，海藻多糖因为具有分子量大，粘度大，溶解度较小的等特点，而使其应用范围受到限制。利用降解的手段对其进行修饰，降低分子量和粘度，改善溶解性，可以拓展其应用范围。并且根据文献报道，琼 胶寡糖具有一些特殊的生物活性，如抗氧化性，抗炎症等。因此，对琼胶降解的研究具有生要意义。本研究中，为了选择一种合适的降解方法，进行了几种水解方法的尝试，其中包括在不同湿度和酸度下盐酸水解，过氧化氢和醋酸催化水解，Fenton体系羟基自由基降解。对于酸水解和Fenton体系氧化还原降解方法，通过粘度法对反应的速度进行了比较，表明氧化还原降解反应中琼胶的粘度降低比较快，并且具有代表性和新意，确定为本实验的降解琼胶的方法并对氧化还原降解所得的产物进行了活性实验。通过模仿自然界普遍存在的氧化还原降解反应，利用Vc诱导的Fenton体系产生的羟基自由基氧化还原降解琼胶得到低分子量的琼胶。降解产物经过高速离心、60％乙醇沉淀，除去分子量比较大的降解产物和磷酸盐，得到可溶于60％乙醇的分子量估计小于3000的降争产物，其产率为85％。利用经Sephadex-G25凝胶色谱分离所香的不同分子量的级分进行分子量和α－葡萄糖苷酶抑制活性关系的实验。降解产物对α－葡萄糖苷酶的抑制率和各级分的浓度呈线性正相关，并且各级分的IC_(50)则随着分子量的降低而降低。另外，对所得的降解产物混合物进行了红外吸收光谱、质子去偶核磁共震碳谱和负离子基质辅助激光诱导－飞行时间质谱结构分析。结果表明，氧化还原降解反应的专一性差，在得到寡糖的同时，在光谱图中出现一些比较复杂的副产物的结构信息。最后，根据MTT法的原理，以有体皮肤成纤维细胞为材料，通过紫外线辐射产生自由基造成氧化损伤，研究降解产物对成纤维细胞的保护作用。当无紫外线辐射时，降解产物对成纤维细胞具有显著的促进生长增殖作用：当经UVa、UBb辐射时则可以显著地表现出对损伤的保护作用，并且这种促进生长和保护作用呈显著的量效关系，表明降解产物具有清除基自由基的作用。但是，因为氧化还原降解以应的机理尚不十分明的以及琼羟胶的特殊结构，使得反应的副产物很难预测，也就使得分离工作难以进行，所以，根据目前所得的信息，尚不能确定是降解产物的什么级分产生的以上两种生物活性。