转基因小麦小花开放特性与基因漂移关系研究

选用3个转基因小麦、2个受体对照与170个地方品种对照，在武汉进行环境释放试验．约20％的小麦地方品种与转基因小麦花期相遇，所有地方品种开花时柱头都部分外露，且小花开放度和开放时间品种间有差异，表明存在着转基因小麦以花粉为媒介向地方品种发生基因漂移的可能性，且不同地方品种作为接受者，在不考虑其他影响因素时，因小花开放度的差异发生基因漂移的可能性也应存在差异．     

导入高梁DNA选育丰产、抗逆小麦新品系及其RAPD分子验证

通过花粉管通道法,将高粱ＤＮＡ导入小麦,结果在其后代产生了广泛的变异,并从中选育出高产、抗逆、耐盐碱小麦新品系８９１２２．在省级区试中,连续两年表现优良,比统一对照高原６０２增产２４．０８％,比原受体陇春１３号增产２１．０６％．在盐碱地,比当地主栽品种Ｖ２８增产１０．５％,比原受体增产２２．５％．光合效率明显提高．ＲＡＰＤ分析结果表明,新品系基因组出现多态性,并具有供体特异性ＤＮＡ带,表明该小麦新品系为转基因后代     

青海本地山羊血清运铁蛋白多态性的研究

采用聚丙烯酰胺凝胶垂直平板电泳法对298只青海本地山羊的血清运铁蛋白多态性进行了研究。结果发现：被检山羊有，IFAA、TFAB、邢B三种基因型，以TFAA为优势基因型(74．5％)；TF^A和TF^B等位基因频率分别为0．8624和0．1376，基因杂合度和有效等位基因数分别为0．2373和1．3111；经遗传距离分析，青海本地山羊与四川藏山羊有较近的亲缘关系。     

花粉管通道法转基因技术在甜瓜品种河套蜜瓜上的应用

为建立甜瓜(Cucumis melo L.)简便易行转化频率高的转基因技术,以甜瓜品种河套蜜瓜为受体材料,以含gus基因的植物双元表达载体pPZP221为外源基因供体,进行了花粉管通道法转基因研究.自交授粉后分别于1、2、3、4、5、6、7、8、9、10 h,切去柱头上端1/3部分,立即滴加质粒DNA溶液,果实成熟后收获转化种子.对T0代植株进行PCR检测,结果表明不同时间处理所得到的T0代植株均具有较高的转化频率,其中授粉后7 h滴加DNA溶液所获得的转化率为28.3%.对一部分PCR阳性的T0代植株基因组DNA进行Southern杂交分析,结果表明所有样品均出现特异性杂交条带,证明外源基因已整合到受体植物基因组中.     

转betA/als基因棉花生存竞争力和基因漂流的调查

对导入乙酰乳酸合成酶突变基因als及来自大肠杆菌的胆碱脱氢酶基因betA的棉花纯合株系T3、T4代植株的田间农艺学性状和基因漂流进行了研究。 结果表明,与野生型对照鲁棉研19相比,转betA/als基因棉花涉及生存竞争力的一些农艺学性状,如种子繁殖能力、贮存后活力以及盐碱地条件下的经济产量等显著提高,转基因棉花的棉花纤维马克隆值下降,其它农艺学性状无明显差别;获得的转基因性状之一除草剂抗性遗传稳定,并在田间表达良好;在转基因棉花释放区面积为6?m×6?m的条件下,als基因通过花粉介导进入野生型棉花的频率随着非转基因棉花种植区与释放区之间的距离增大而迅速降低,采取适当的隔离距离（大于200?m）,可以避免外源基因逃逸事件的发生。但设置隔离区时应考虑昆虫对花粉传播等不确定因素的影响,适当加大隔离距离。     

转Bt基因杨树(NL-80106)对杨小舟蛾抗虫性研究

以转Bt抗虫基因杨树(NL-80106)的大田移植苗为材料，在室内控制和自然条件下测定其对畅小舟蛾幼虫的抗虫性。结果表明：转Bt基因杨树对杨小舟蛾1龄幼虫12d校正死亡率高达84．3％；其杀虫活性存在明显的时间动态，3个抗虫转基因杨树无性系FIN5、FB51和FB56对1龄幼虫12d的抗性由6月的56．9％～84．3％下降至7～8月的35．6％～45．1％，9月又回升至59．4％～80．8％；不同龄期幼虫对转基因杨树敏感性不同，1～3龄幼虫6d校正死亡率为19．8％～25．4％，与对照存在显差异，而4～5龄幼虫6d校正死亡率与对照差异不显；转Bt基因杨树对大部分杨小舟蛾幼虫的生长具有明显抑制作用，取食量、体重增长速率显低于对照，并导致幼虫最终死亡。     

应用地高辛标记的PCR-ELISA技术快速检测转基因水稻

应用地高辛标记的PcR—ELISA(Dig-PcR—ELISA)技术进行转基因水稻检测研究。针对转基因水稻中普遍存在的花椰菜花叶病毒(CaMV)35S启动子(p35S)、胭脂碱合成酶(NOS)终止子(Thos)，筛选标记潮霉素磷酸转移酶基因(hpt,hph)、β—葡萄糖苷酸酶基因(gus)、抗草丁膦除草剂基因(bar)，建立Dig—PcR—ELISA检测方法；能进行半定量检测，敏感性试验表明，Dig—ELISA检测比常规电泳检测可提高敏感性达1000倍，可检测含量达0．1％的GM0样品。全过程可在24h内完成。     

根癌农杆菌介导大麦Mlo反义基因转化小麦

应用含有大麦Mlo反义基因表达载体的根癌农杆菌菌株Agl Ⅰ，对3种基因型小麦(核生3号、烟优361、扬麦158)的愈伤组织进行了转化．从其中2种基因型获得23株转基因植株，有2株是白化苗，转化频率分别为1．9％(核生3号)和2．8％(扬麦158)．PCR及Southem分析表明，外源基因已整合进植物基因组．实验结果表明，筛选压力的存在对转基因植株的获得至关重要，在无筛选压力下未获得转基因植株．7棵转基因植株移栽至大田后正常结实．     

转基因小麦T1代中外源基因的检测和分析

该研究采用Multiplex PCR和SDS-PAGE技术对转1 Dx5基因(ORF)T1代小麦进行了检测和分析.结果显示,Multiplex PCR能够扩增出转基因T1代材料中1 Dx2基因和1 Dx5基因的特征片段,表明外源基因已整合到受体基因组中;SDS-PAGE检测到一新的蛋白质亚基X,表明外源基因的插入引起了转基因T1代籽粒中HMW-GS组成的变化.该研究不仅验证了线性基因片段遗传转化策略的可行性,而且印证了普通小麦Glu-1D等位基因的多重PCR分子标记体系的有效性,为培育安全型的转基因作物新种质打下坚实的基础,加快小麦分子育种进程.     

超声波对烟草花粉的生物学效应及其诱导外源基因向烟草花粉的转移

用超声波法处理烟草花粉的生物学效应及它诱导ＧＵＳ基因转化烟草花粉,获得了短暂表达的研究结果．单核晚期和双核早中期的烟草游离花粉,置于含０．４ｍ ｏｌ／Ｌ甘露醇的缓冲液中,随着超声强度提高或／和处理时间的延长,对烟草花粉的活力和萌发率具有显著影响．声强０．５Ｗ／ｃｍ ２、时间１０～２０ｍ ｉｎ 是比较理想的参数．将游离花粉与质粒ｐＢⅠ１２１ 混合,以声强为０．５Ｗ／ｃｍ ２ 的脉冲超声波处理１０ｍ ｉｎ, 检测ＧＵＳ基因的平均短暂表达频率（ＴＴＦ％ ）,单核晚期花粉为１８．６％ ,双核早中期花粉为３５．７％ ．对超声转化的不同发育时期的烟草花粉,分别以两条不同途径操作和筛选转基因植株：（１） 单核晚期的花粉：直接进行体外培养、植株再生和筛选,以期获得单倍体转基因植株．在卡那霉素选择培养基上,花粉细胞进行两次或多次分裂后即行停止;（２） 双核早中期的花粉,在体外培养成熟后授粉,收获种子,检测种子中外源基因是否存在．在所收３４１ 粒种子中,没能筛选到卡那霉素抗性植株．就如何筛选超声波转化花粉、获得转基因植株的技术途径进行了讨论．     

Expression of lysine-rich protein gene and analysis of lysine content in transgenic wheat

Expression vector pBPC102, which carries winged bean lysine-rich protein (wblrp) gene and dihydropicolinate synthase (DHDPS) gene, was transferred into hexaploid winter wheat cv. Jinghua No.l, Jing411, You899 and Yangnongl5 explants of immature inflorescence and immature embryos by particle bombardment. More than 100 transgenic plants were obtained under the selection of s-(2-aminoethyl)-L-cysteine (AEC). Confirmed transgenic plants of To and TI generation by PCR and PCR-Southern blotting analyses showed successful integration of wblrp gene into wheat genome. Analysis of transgenic plant lines of T2 by Northern dot-blotting showed good expression of wblrp gene in offspring seed. The content of free lysine in leaves, contents of bound lysine and total proteins in seeds of T2 transgenie wheat lines were determined and analyzed. Among 34 tested transgenic lines, levels of free lysine content in leaves of 9 transgenic lines are 2~3times higher than un-trans-formed wild-type cultivars. Among 17 analyzed transgenic lines, bound lysine content of 4 transgenic lines is more than 10% higher than that of wild-type cultivars. Our research suggests that introducing wblrp gene into wheat is an effective way to improve its nutrition quality.     

Generation of transgenic wheat resistant to wheat yellow mosaic virus and identification of gene silence induced by virus infection

The plasmid containing the promoter Act1, the coat protein (cp) gene of wheat yellow mosaic virus (WYMV) and the selectable bar gene, was delivered via particle bombardment, directly into immature embryos of a wheat cultivars. PCR and PCR-RFLP were employed to screen the existence of the cp gene in T0 and T1 generations. Seeds from the positive T1 plants were sowed in fields heavily contaminated with WYMV to detect their resistance. In field trial of virus infection, one of the transgenic wheat lines, P8-T2, exhibited highly disease-resistance. Western blot and RT-PCR analysis showed that the expression level of cp gene in the resistant transgenic line was reduced greatly compared to those susceptible to WYMV infection. This provided evidence to presume that the resistance obtained by the transgenic wheat line was stimulated by the mechanism of the virus induced gene silencing.     

Transgene inheritance and quality improvement by expressing novel HMW glutenin subunit （HMW-GS）genes in winter wheat

The expression vector pBPC30, which carries the high molecular weight glutenin subunit (HMW-GS) 1Dx5 and 1Dy10 genes, was transferred into hexaploid winter wheat cv. Jinghua No. 1, Jing411 and Jingdong No. 6 explants of immature embryos and immature inflorescence by particle bombardment. A large number of resistant transgenic plants were obtained under the selection of herbicide bialaphos or phosphinothricin (PPT). Confirmed transgenic plants of To generation showed successful integration of HMW-GS genes and bar gene into the wheat genome. T1 generation of transgenic plants can resist 20--150 mg/L PPT.Protein analysis of T2 seed by SDS-PAGE showed that HMW-GS 1Dx5 and 1DylO genes were well expressed in offspring seed of transgenic lines by co-expression with or substitution of endogenous 1Dx2 or 1DylO. In one transgenic line, TG3-74, a new protein band between endogenous protein subunits 7 and 8 (marked as 8*) of glutenin appeared,but endogenous subunit 8 (encoded by 1By8 gene) was absent. Analysis of gluten rheological quality on seed proteins of 102 T3 plants showed that the sedimentation value of 5 transgenic lines (44.2149.0 mL) was remarkably improved,59.6%---64.3% higher than that of wild type Jinghua No. 1 and Jingdong No. 6, similar to bread wheat Cheyenne (48.0 mL). Analysis of dough rheological properties of transgenic lines showed that the dough stable time of 5 transgenic lines range from 16 to 30 min, whereas the dough stable time of wild type was only between 3--7 min. Our research suggests that introducing novel HMW-GS genes into wheat is an efficient way to improve its bread-making quality.     

基因枪法获得可育抗除草剂转基因小麦

用基因枪法对两个春小麦品种进行了遗传转化，获得１９株独立转化的，抗除草剂Ｂａｓｔａ的小麦植株，其中１５株自交可育，ＰＣＲ和ＤＮＡ印迹检测证实了该基因在转化植株中的表达，转基因及其表达已遗传到子三代，在已检测的７个转基因后代中，有４个植株其抗除草剂性状以单位点，显性性状的孟德尔方式遗传。     

From pollen actin to crop male sterility

Actin plays an important role in the life activity of animal and plant cells. Pollen cells have plenty of actin whose structure and characteristics are very similar to the animal actin. The nucleotide sequence and amino acid sequence of plant actin gene are very similar to those of the animal gene. The content of pollen actin from male sterile plants is much more lower than that from its maintainer plants. The expression of actin gene is organ-specific during the plant development. The expression quantity of actin gene in pollen is much more higher than those from root, stem and leaf. The expression plasmid of the anti-sense actin gene was constructed, transferred to the protoplasts of wheat and tomato to inhibit the expression of actin gene in pollen and thus the male sterile plants of wheat and tomato were obtained. The actin in pollens from the transgenic plants was reduced significantly, whereas the pistil was not affected. This study might pave a new way to breeding male sterile lines for the application of hybrid vigor of wheat and tomato.     

Transgene directionally integrated into C-genome of Brassica napus

Transgenic Brassica napus has been widely planted in Canada, the United States, and some other countries. In China, although the policy for genetically modified foods has not yet opened, genetically modified rape- seed oil as raw material for biodiesel of…     

Expression of the intact C<Subscript>4</Subscript> type<Emphasis Type="Italic">pepc</Emphasis> gene cloned from maize in transgenic winter wheat

Maize intact C4-pepc gene was amplified through LA-PCR and successfully sub-cloned into modified vector pGreen0029 to form a stable expression construct named as pBAC214 (12 kb), which contains CaMV 35S promoter driven bar gene as selection marker. Comparing the cloned DNA sequences (6.7 kb) with published maize C4-pepc gene (GenBank accession E17154) sequences, the identity of DNA sequence alignment is 98.96%. There are only 49 differences between these two intact DNA sequences, of which 13 occur in the region of promoter, 18 in introns, and 18 in exons. The homology of mRNA sequence alignment is 99.38%, and the putative amino acids sequence identity is 99.38%. There are only 15 differences between these two mRNA, and these differences bring 4 sites mutant on the putative amino acids of PEPC protein. Through biolistic bombardment of PDS1000/He system, expression vector pBAC214 has been transformed into winter wheat. Southern blotting results show that the intact C4-pepc gene has been integrated into genome of winter wheat. SDS-PAGE analysis of leaf soluble protein in transgenic wheat showed that the intact C4opepc gene was well transcribed, spliced and translated as in maize. The enzyme activity of leaf PEPC in transgenic wheat has been detected. The activities of leaf PEPC increased over 3-5 times in some transgenic plants. The data of photosynthesis rate and transpiration rate of transgenic wheat flag leaves showed that the C4-pepc gene can increase the photosynthesis rate and transpiration rate of transgenic wheat.     

拟南芥AtPLC4基因RNAi载体的构建及遗传转化

利用RNAi技术,构建了AtPLC4基因的RNAi双元载体,并进行了拟南芥的遗传转化,通过抗性筛选及PCR鉴定,获得20株遗传转化株系.进一步通过RT-PCR检测,得到3株AtPLC4基因表达降低的转基因植株,为运用反向遗传学的方法深入研究AtPLC基因家族的生物学功能提供基础.