BADH基因转化番茄提高耐盐性的研究

甜菜碱是植物在盐、干旱或其它胁迫下在细胞中迅速积累的一种相容性有机小分子化合物，它在细胞中的积累与植物抗盐性的提高密切相关。甜菜碱醛脱氢酶（BADH）催化甜菜碱醛转化为甜菜碱。我们将来源于耐盐植物山菠菜（Atriplx hortensis L.）的BADH基因通过农杆菌介导法导入‘百丽春’番茄(Lycopersicon esculentum L. ‘Bailichun’)中，并获得15株转化植株，PCR、Southern和Northern检测表明，其中的6株有外源BADH基因的整合，5株中BADH基因能够正常表达，但不同植株间BADH基因的表达水平和BADH酶活力有较大差异。对叶片电导率的测定表明，转基因植株比野生型的耐盐性有较大提高。T1代分析表明，检测的两个转基因株系后代遵循孟德尔分离规律，90mmol/L NaCl胁迫下种子发芽率提高了2~4倍，幼苗的苗高、根长和须根数三个指标均明显优于对照。部分T1代植株在水培条件下能够耐受180mmol/L NaCl胁迫。 植物耐盐的另一机理就是利用液泡膜上存在的转运蛋白将细胞内的有毒离子区域化。我们将已转入编码转运蛋白基因AtNHX1的番茄品种‘Moneymaker’（L. esculentum‘Moneymaker’）株系X1OEA1通过农杆菌介导法转入山菠菜BADH基因，以期获得转双基因耐盐番茄。目前已获得转基因植株，PCR结果证明部分抗性幼苗中已整合了BADH基因，其它各项分子检测正在进行中。

农杆菌介导的双价抗盐基因转化番茄的研究

近年来，植物耐盐生物技术研究取得了可喜的进展，特别是通过抗盐基因转化在一定程度上使植物的耐盐性得到了提高。然而，植物的耐盐性是一个多基因控制的复杂性状，依赖于多个基因之间的相互作用。因此，只是将单个基因导入植物获得的抗逆性还是远不能达到满意的效果。一般认为，将多个与耐盐相关的基因转入到同一个植物（即所谓的“复合基因转化”）将会大大提高转基因植物的耐盐能力。 渗透调节是植物抵御盐胁迫的主要方式。植物渗透调节的方式分为两类：一是在细胞中吸收和积累无机盐，如通过离子通道、Na+/H+逆向运输蛋白和ATP酶/H+泵;二是在细胞中合成有机溶质，如脯氨酸和甘氨酸甜菜碱。 我们通过农杆菌介导法向转AtNHX1（拟南芥Na+/H+逆向运输蛋白编码基因）的番茄（Lycopersicum esculentum L. ‘Moneymaker’）株系X1OEA1自交二代植株（T2）中转入山菠菜甜菜碱醛脱氢酶基因（BADH）。PCR、Southern、RT-PCR和甜菜碱含量分析结果证明，BADH已经整合到目标植物基因组，并在转基因植株中转录和翻译表达。叶绿素荧光（Fv/Fm）、相对电导率（Rc/Rc’）、叶绿素含量（Chla+b）、叶绿素a/b比（Chla/b）和光合速率（Pn）测定结果表明，在200 mM NaCl 胁迫下，二次转化的番茄植株各项生理指标均优于转单基因AtNHX1的番茄。初步证明“复合基因转化”有助于进一步提高植物的耐盐性。同时对番茄的转化系统进行了优化，结果表明使用抗生素‘特美汀’作为抑制农杆菌的抗生素的转化效率明显高于使用头孢霉素。

SeNHX1和BADH基因提高植物耐盐性的研究

土壤的盐碱化问题已经严重影响到世界范围内许多重要作物的生产。培育耐盐作物是解决这一问题的最有效途经。利用耐盐相关基因的转化可以在不改变或很少改变植物其它性状的情况下提高植物的耐盐性，因此基因工程方法对于改良植物耐盐性及其机理的研究具有重要的意义。目前植物耐盐基因工程从调控渗透调节物质和盐离子区隔化两个方面开展了较多的研究。已经获得一些耐盐性提高的转基因植物。 本研究拟用耐盐性较强植物山菠菜中的甜菜碱合成关键基因BADH和盐生植物盐角草的液泡膜Na+/H+ anitiporter基因SeNHX1对模式植物烟草进行转化，以确定其各自在耐盐性方面所起的作用。同时，现有的研究表明植物的耐盐性是多基因控制的复杂性状，因此拟把SeNHX1和BADH 这两个涉及不同耐盐机理的基因构建到同一个植物表达载体上，以比较单基因转化和双基因转化在提高植物耐盐性方面的优劣。除此之外，并对已经转入BADH基因番茄的耐盐性和遗传稳定性分析进行了研究。 转BADH基因番茄已经稳定遗传到T4世代。通过对5个转BADH基因番茄株系在T0世代、T3世代和T4世代的分析，表明除了株系T4-3由T0世代的3个拷贝变为1个拷贝外，其余各株系拷贝数均没有发生变化。外源基因编码的酶活性和最终催化产物甜菜碱在盐分胁迫下都能较容易的检测到，说明外源基因在番茄基因组中的遗传是稳定的，没有发生丢失。在连续2个世代的耐盐性鉴定中，各转基因株系的耐盐性较为一致，均比野生型有了较大的提高。其中株系T4-5连续2年表现出了较低的减产率，株系T4-8也在连续的2年中表现出了最高的单株产量。盐分胁迫下转BADH基因各个株系比野生型有较高的K+和Ca2+含量，较低的Na+含量，转基因株系较野生型有较低的脐腐病果率。 通过SeNHX1、BADH单独转化以及构建双价载体共转化的方法获得了3种类型的转基因烟草。Southern和Northern 检测结果表明，外源基因已经整合到烟草基因组中，并得到了正确的表达。转BADH基因烟草在盐分胁迫下能检测到明显的BADH酶活性和甜菜碱含量。转基因烟草T0代对盐分胁迫、氧化胁迫的抗性均较野生型对照有较大的提高。转基因株系在200 mM NaCl胁迫下较野生型有较高的光合速度。百草枯处理过的野生型叶盘比转基因株系积累了更多的丙二醛，表明野生型受到了更大的氧化胁迫。 已经获得3种转不同基因烟草的T1代，且T1代具有较强的耐渗透胁迫能力。转基因烟草的T0种子均能在含100 mg/L 卡钠霉素培养基上发芽和正常生长，其中部分种子能够在含200 mM NaCl 培养基上发芽并能较好的生长，而野生型根本不能发芽。从200 mM甘露醇胁迫1周后，又转移到营养液中的生长1周的情况来看，转基因烟草能较快的恢复正常的生长，有新的叶子和根长出，而野生型却不能，同时转基因株系比野生型具有更大的单株鲜重。 转BADH基因番茄在遗传上是稳定的，并且其耐盐性有了较大的提高。双基因转化烟草的抗盐性要好于单基因转化，但SeNHX1基因转化要好于BADH基因转化。说明SeNHX1基因在提高植物耐盐性方面要比BADH基因有更强的功能，同时，也表明多基因转化在植物的耐盐改良方面可能是一个更为有效的方法。

两种完熟相关基因反义RNA在转因番茄中的表达及其对果实完熟的仰制作用

植物激素乙烯作为一种信使分子调节控制果实完熟。ACC合成酶是植物体内乙烯生物合成途径的限速酶，其反义RNA的表达将能有效地抑制乙烯的生物合成而延缓果实完熟，利用反转录PCR技术克隆获得了ACC合成酶多基因家族成员之一LE-ACC2阅读框架约1.7kb的cDNA，经酶切图谱和序列分析鉴定无误后，反向连入植物表达载体pBin437中构建成组成型表达ACC合成酶反义RNA的双元载体。经农杆菌途径转化番茄“丽春”品种，获得了60株抗卡那再生杭株，PCR检测证明有6株为转基因植株，Southern杂交和Northern杂交分析进一步确证了外源基因的插入及其转录活性。反义番茄果实的乙烯释放受到明显抑制，表现出更好的耐储保鲜特性，并且与对照相比，在果实品质上没有明显差别。大田培育Fl和F2代转化番茄植株，反义番茄纯合品系的筛选工作正在进行之中。 同时，本研究利用已经获得的ACC合成酶和PG的cDNA克隆，构建了两个嵌合转化基因载体pPGACC1、pPGACC10，它包括1300bp的ACC合成酶cDNA编码序列，并分别含有反向与正向的250bp的5’端PG基因片断。酶切图谱和序列分析鉴定无误后，以pBin437为植物表达载体构建了双元载体pBPGACC1和pBPGACC10，分别表达PG正义RNA和反义RNA，并均表达ACC合成酶反义RNA。经农杆菌转化番茄子叶，植株的再生培育有待进行。通过对转基因植物的分析，我们期望阐明用单一嵌合基因表达载体通过反义抑制与抑制作用实现对内源两同源基因——PG和ACC合成酶下降调节的可能性，并可望得到具有更好耐储效果且品质优良的番茄品系。

番茄种子萌发的高温耐性诱导及其生理效应

番茄（Lycopersicon esculentum Miller）原产于南美西部高原地带，适应原产地赤道附近高地干燥冷凉的气候特点，不耐高温多湿，种子萌发期间对高温非常敏感。本研究以佳粉17番茄种子为材料，试图寻找诱导番茄种子萌发期间高温耐性的方法，并通过研究高温耐性被诱导前后种子内部发生的生理生化变化，探索番茄种子萌发期间不耐高温以及高温耐性诱导的机理。试验结果显示： 25 ℃是种子萌发的最适温度，种子发芽率为97.5%。高温抑制种子的萌发，33 ℃和35 ℃条件下萌发率分别为58.5%和8.5%。 萌发适宜温度预吸胀、低温预吸胀、吸湿-回干预处理可提高番茄种子萌发期间的高温耐性，而水杨酸处理则没有明显效果。种子经25 ℃预吸胀30 h、0 ℃预吸胀10 h、吸湿-回干预处理后在33 ℃条件下的萌发率分别为81.5%、78.0%、90%，在35 ℃下的萌发率分别达到33.5%、42%、48.5%。经以上处理后，种子萌发速率提高，萌发高峰期提前，幼苗生长健壮，根干重增加，活力指数变大。 番茄种子萌发期间遭受高温危害时，电解质渗漏增加，相对电导率升高；脂膜过氧化作用加剧，其产物MDA的含量增加。经萌发适宜温度预吸胀、低温预吸胀、吸湿-回干等方法预处理后，高温伤害减轻，膜的完整性增强，电解质渗漏减缓，膜脂过氧化作用减弱，因而相对电导率降低，MDA含量减少。 高温抑制了抗氧化酶的活性，种子内部SOD、APX、CAT、GR等抗氧化酶活性降低。经萌发适宜温度预吸胀、低温预吸胀、吸湿-回干等方法预处理以后，抗氧化酶活性有不同程度的提高，清除细胞内超氧阴离子自由基的能力增强，因而使过氧化伤害减弱， 适宜温度预吸胀、低温预吸胀、吸湿-回干等预处理方法在保护生物膜的同时，增强抗氧化作用，抑制过氧化伤害，从而提高了番茄种子萌发的高温耐性，这是番茄种子高温耐性提高的生理机制之一。

Mitochondrial DNA variation in cattle of South China: origin and introgression

Ten restriction endonucleases were used to investigate the mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) of 11 native cattle breeds and one cultivated cattle breed in South China. Twenty-three restriction morphs were detected, which can be sorted into five haplotypes, A phylogenetic tree of the haplotypes was constructed by using the 'upgma' method. Our study showed that haplotype I and II are identical to the zebu (Bos indicus) and taurine (Bos taurus) haplotypes, respectively. Zebu and taurine were the two major origins of cattle populations in South China, and the zebu probably had more influence on the native cattle population than taurine did. Haplotype III is identical to haplotype I of yak (Bos grunniens), which was only detected in the Diqing cattle breed. Haplotype IV was detected for the first time. This haplotype, found only in Dehong cattle, might be from an independent domestication event, probably from another Bos indicus population. Divergence of haplotypes I and IV occurred about 268,000-535 000 years ago, much earlier than the 10,000-year history of cattle husbandry. Our results also suggest a secondary introgression of mtDNA from yak to Diqing cattle.

Deep RNA sequencing at single base-pair resolution reveals high complexity of the rice transcriptome

Understanding the dynamics of eukaryotic transcriptome is essential for studying the complexity of transcriptional regulation and its impact on phenotype. However, comprehensive studies of transcriptomes at single base resolution are rare, even for modern organisms, and lacking for rice. Here, we present the first transcriptome atlas for eight organs of cultivated rice. Using high-throughput paired-end RNA-seq, we unambiguously detected transcripts expressing at an extremely low level, as well as a substantial number of novel transcripts, exons, and untranslated regions. An analysis of alternative splicing in the rice transcriptome revealed that alternative cis-splicing occurred in similar to 33% of all rice genes. This is far more than previously reported. In addition, we also identified 234 putative chimeric transcripts that seem to be produced by trans-splicing, indicating that transcript fusion events are more common than expected. In-depth analysis revealed a multitude of fusion transcripts that might be by-products of alternative splicing. Validation and chimeric transcript structural analysis provided evidence that some of these transcripts are likely to be functional in the cell. Taken together, our data provide extensive evidence that transcriptional regulation in rice is vastly more complex than previously believed.

Aerobic biodegradation of hexachlorobenzene by an acclimated microbial community

The aerobic degradation of hexachlorobenzene (HCB) by an acclimated microbial community which isolated from a contaminated site and acclimated in our laboratory was investigated. The enriched microbial community was capable of biodegrading HCB when cultivated in minimal salts medium and supplied HCB as the sole carbon source. The efficiencies of microbial community in the degradation of HCB under different pH and temperatures were examined. The phylogenetic analysis for the nearly complete sequences of 16S rDNA demonstrated that the bacteria assemblage in the microbial community was dominated by Azospirillum and Alcaligenes groups.

Growth kinetics of 1-2 mm and 3-4 mm colonies of Nostoc sphaeroides (Cyanophyta) in outdoor culture

Nostoc sphaeroides Kutzing was cultivated in paddlewheel-driven raceway ponds and the growth kinetics of 1-2 mm and 3-4 mm colonies of N. sphaeroides was studied. The biomass productivities in 2.5 m(2) raceway ponds inoculated with 1-2 mm and 3-4 mm colonies were 5.2 and 0.25 g dry wt m(-2) stop d(-1), respectively. Furthermore, differently sized colonies showed different relative water content, total soluble carbohydrates, chlorophyll a content and density of filaments. This is the first report on mass culture of N. sphaeroides under outdoor conditions.

The accumulation and metabolism of astaxanthin in Scenedesmus obliquus (chlorophyceae)

The biosynthesis and metabolism of astaxanthin in coenobium alga Scenedesmus obliquus were investigated using a two-stage culture. The first stage was for the analysis of biosynthesis and accumulation of astaxanthin in alga cells which were cultured under induction conditions (incubation at 30 degrees C and illumination of 180 mu mol m(-2) s(-1)) for 48 h. The composition of the secondary carotenoids in algal cells was analyzed and seven ketocarotenoids were identified. The results implied that S. obliquus synthesized astaxanthin from beta-carotene through three possible pathways. In the second stage, the cultures were transferred to normal conditions (incubation at 25 C and illumination of 80 mu mol m(-2) s(-1)) for 72 h. Algal cells accumulated more chlorophyll and biosynthesis of secondary carotenoids terminated, the content of secondary carotenoids decreased from 59.48 to 6.57%. The results inferred that accumulation and metabolism of astaxanthin could be controlled by cultivated conditions which also could lead the mobilization of secondary carotenoids to support the algal cell growth. The results also implied that presumed conversions from astaxanthin to lutein or antheraxanthin could be modulated by culturing conditions. (C) 2008 Published by Elsevier Ltd.

Clonal diversity and genealogical relationships of gibel carp in four hatcheries

To conserve and utilize the genetic pool of gynogenetic gibel carp (Carassius auratus gibelio), the Fangzheng and Qihe stock hatcheries have been established in China. However, little information is available on the amount of genetic variation within and between these populations. In this study, clonal diversity in 101 fish from these two stock hatcheries and 35 fish from two other hatcheries in Wuhan and Pengze respectively was analysed for variation in serum transferrin. Thirteen clones were found in Fangzheng and Qihe, of which 12 were novel. Six clones were specific to Fangzheng and three specific to Qihe, whereas four were shared among the Fangzheng and Qihe fish. To obtain more knowledge on genetic diversity and genealogical relationships within gibel carp, the complete mitochondrial DNA (mtDNA) control region (similar to 920 bp) was sequenced in 64 individuals representing all 14 clones identified in the four hatcheries. Differences in the mtDNA sequences varied remarkably among hatcheries, with the Fangzheng and Qihe lines demonstrating high diversity and Wuhan and Pengze showing no variation. The Fangzheng and Qihe lines might represent two distinct matrilineal sources. One of the Qihe samples carried the haplotype shared by a most widely cultivated Fangzheng clone, indicating that a Fangzheng clone escaped from cultivated ponds and moved into the Qihe hatchery. Four Fangzheng samples clustered within the lineage formed mainly by Qihe samples, most likely reflecting historical gene flow from Qihe to Fangzheng. It is suggested that clones in Wuhan originated from Fangzheng, consistent with their introduction history, supporting the hypothesis that gibel carp in Pengze were domesticated from individuals in the Fangzheng hatchery.

Different tolerances and responses to low temperature and darkness between waterbloom forming cyanobacterium Microcystis and a green alga Scenedesmus

The dynamics of planktonic cyanobacteria in eutrophicated freshwaters play an important role in formation of annual summer blooms, yet overwintering mechanisms of these water bloom forming cyanobacteria remain unknown. The responses to darkness and low temperature of three strains (unicellular Microcystis aeruginosa FACHB-905, colonial M. aeruginosa FACHB-938, and a green alga Scenedesmus quadricauda FACHB-45) were investigated in the present study. After a 30-day incubation under darkness and low temperature, cell morphology, cell numbers, chlorophyll a, photosynthetic activity (ETRmax and I-k), and malodialdehyde (MDA) content exhibited significant changes in Scenedesmus. In contrast, Microcystis aeruginosa cells did not change markedly in morphology, chlorophyll a, photosynthetic activity, and MDA content. The stress caused by low temperature and darkness resulted in an increase of the antioxidative enzyme-catalase (CAT) in all three strains. When the three strains re-grew under routine cultivated condition subjected to darkness and low temperature, specific growth rate of Scenedesmus was lower than that of Microcystis. Flow cytometry (FCM) examination indicated that two distinct types of metabolic response to darkness and low temperature existed in the three strains. The results from the present study reveal that the cyanobacterium Microcystis, especially colonial Microcystis, has greater endurance and adaptation ability to the stress of darkness and low temperature than the green alga Scenedesmus.

Man-made desert algal crusts as affected by environmental factors in Inner Mongolia, China

Man-made desert algal crusts were constructed on a large scale (3000m(2)) in Inner Mongolia, China. Microcoleus vaginatus was mass cultivated and inoculated directly onto unconsolidated sand dune and irrigated by automatic sprinkling micro-irrigation facilities. The crusts were formed in a short time and could resist the erosion of winds and rainfalls 22 days after inoculation. The maximum biomass in the man-made algal crusts could also reach 35 mu g Chl a/cm(2) of soil. Effects of environmental factors such as temperature, irrigation, rainfall and soil nutrients on algal biomass of man-made algal crusts were also studied. It was found that rainfalls and lower light intensity had significantly positive effects on the biomass of man-made algal crusts. The preliminary results suggested that man-made algal crusts could be formed rapidly, and thus it might be a new feasible alternative method for fixing unconsolidated sand. (c) 2006 Elsevier Ltd. All rights reserved.

Growth promotion of vegetative gametophytes of Undaria pinnatifida by blue light

Through an acclimation period of 10 days, compared to white light, the maximal net photosynthetic rates were significantly higher for gametophytes of Undaria pinnatifida cultivated under blue light (400-500 nm), and were lower under red light (600-700 nm). Chlorophyll c and the carotenoid content of gametophytes were similar under blue light and red light but were much lower under white light. The growth rate of female gametophytes under blue light was higher than that under other lights, and the growth rate of male gametophytes showed little variation with respect to blue and white light. Male and female gametophytes were mixed together to form sporophytes under white, blue and red light. After approximately 5 days, 50% gametophytes became fertile under blue and white light, but remained vegetative under red light after 10 days.

Composition of volatile oil of Primula obconica in Central China

Primula obconica was introduced to Europe from Hubei, China in 1880, and has been cultivated worldwide as one of popular ornamental plants. The volatile oil of wild P. obconica collected from its original place, Yichang, Hubei was first investigated. A total of 43 compounds constituting 93.49% of the oil were identified by using GC and GC-MS. The major compounds were methyl 2,4-dihydroxy-5-methyl benzoate (30.41%), methyl 2,6-dihydroxy-4-methyl benzoate (29.27%), and hypnone (8.92%) etc. In comparison with the published data of some European cultivars, the native P. obconica seems to be allergen-free due to absence of primin and miconidin.