转基因小麦环境释放中基因漂移研究

基因漂移供体是转基因小麦，受体有转基因小麦的非转基因分离体、受体对照品系和地方小麦品种，以外源uidA基因表达作为基因漂移的分子标记，在供体开花期间，距供体5个不同距离上收集花粉，约80％的花粉分布在距供体中心15m以内，距离35m处只检测到约6％的花粉，在11个检测样点检测受体和供体间杂交率，供体与分离体混种同一小区时，基因漂移频率较高(1．08％)；与其受体对照播种于相邻小区(相距约0．5m)时，基因漂移频率居中(0．63％)；地方品种距供体边缘0．5～2．5m处，频率较低(0．25％)；3m以外没有检测到基因漂移，认为转基因小麦基因漂移距离应在3m以内。     

大穗小麦幼穗分化规律研究

对目前具有代表性的大穗小麦品系幼穗分化进行了２年系统观察，结果表明：大穗小麦幼穗分化速度快，比对照品种提高１９．４６％和２２．８２％；单棱分化时期短，比对照品种少４２．５ｄ，降低４０．９６％；进入二棱期早且二棱期长，比对照品种早５０～８６ｄ，增加２５．７ｄ；护颖分化期和小花原基分化期比对照品种增长５．５ｄ和５．７ｄ，雌雄蕊原基分化期和药隔分化期差异不大。其中，幼穗分化速度快是决定大穗小麦具有较多小穗数的主要原因。     

合系42号AGP转基因株系产量性状研究

粳稻品种合系42号经AGP基因导入后，得到性状稳定的转基因株系，经品比试验，有1个株系的产量高于对照且有显著差异，初步认为转基因水稻材料可能是通过提高结实率来提高产量。     

应用基因枪将外源基因导入小麦幼胚获可育的…

以小麦晋２１４８，７５０６，７６０６，５９２６四个春性品种的幼胚为材料，用ＪＱ７００型高速基因枪将ＢＰＩ１２１质粒导入小麦，研究并确立了轰击速度、钨粉直径、上方式、ＤＮＡ浓度等基因枪转化参数。在以上研究基础上进一步将导入外源基因的小麦幼胚质片细胞通过胚胎发生途径再生成植株。供试的４个品种中有３个（７５０６，７６０６，５９２６）获得了可育的转基因植株。ＧＵＳ基因的转化频率分别为０．６％，０．６％和１     

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。     

导入野生大豆DNA小麦后代的农艺性状研究

用花粉管通道法将野生大豆总DNA导入小麦，获得了转基因小麦，其后代的农艺性状有较大的变化。田间实验分析了变异系小麦植株与对照植株在叶片的光合速率、蒸腾速率、叶面积及株高、穗粒、穗重等性状上的差异。t检验结果显示，变异系小麦在叶面性状及产量性状上的差异是显著的，外源DNA的导入改善了植物的农艺性状，并在后代中表达，为选育高产、优质的新小麦品种提供了依据。     

多重PCR法快速鉴定转基因小麦植株及后代

根据转基因植物中常用的报告基因uidA、选择基因bar的序列,设计合成两对不同的引物,建立了在转基因小麦材料分子确证中,应用一次PCR反应同时扩增检测两种或多种外源基因的多重PCR方法．通过对质粒pAHC25以及30个转基因小麦样品进行PCR扩增分析,比较多重PCR的检测结果与单基因的PCR扩增结果,发现两者结果完全一致．     

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。     

陆地棉基因转移技术研究初报

应用农杆菌菌株Ａｔ４４０４携带抗卡那霉素基因（氨基葡萄糖磷酸转移酶）、Ｇｕｓ基因（葡萄糖苷酸酶）以及Ｂ．Ｔ毒蛋白基因，对陆地棉４个不同栽培品种鲁棉６号、鲁棉１０２４、中棉１２、中棉１９的无菌苗下胚轴和茎尖分生组织区作基因导入的研究，获得转基因的棉花试管苗．在以胚轴作为转化体系的培养过程中，只有鲁棉６号经过了愈伤组织发生、胚状体形成及植株再生途径．若以３～５ｄ的无菌苗芽顶端分生组织作为转化体系，经转化的茎尖分生组织培养在无激素或低浓度激素的ＭＳ培养基上，则可较容易地从多个品种中获得转基因的再生植株．以此方法进行基因转移，时间短、方法简便、不受品种来源及基因型的限制．这一技术将有利于提高棉花转基因植株的成活率     

转1Dx5基因小麦突变株系的研究

以转1Dx5基因小麦3个高分子量麦谷蛋白突变株系B73-6-1-1、B73-6-1-2和B73-6-1-3及转基因受体L88-6为材料,对其主要农艺性状、低分子量麦谷蛋白表达情况及染色体核型进行研究.结果表明:在田间栽培条件下,纯合稳定转基因突变株系之间及其与转基因受体之间主要农艺性状存在差异,其中株高和千粒重存在显著差异(P=0.05);同时,SDS-PAGE结果显示,在小麦低分子量麦谷蛋白区域,突变株系之间无明显差异,而突变株系与转基因受体之间部分区域表达量存在明显差异并产生一个突变条带;通过核型分析比较,得出突变株系之间及其与转基因受体之间,染色体无论是相对长度还是臂比值都没有显著差异.     

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 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…     

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.     

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。     

Introduction of rice chitinase gene into wheat via low energy Ar+ beam implantation

A chitinase gene (RCH8) in plasmid vector pCAMBIA1308 was delivered into 3 wheat cultivars (Yangmai 158, Wan 9210, Wanmai 32) by low energy Ar⁺ beam-mediated method. Preliminary calli from treated mature embryos were first selected on hygromycin (Hm, 20 or 30 mg/L) containing medium. After the resistant calli formed, they were transferred to the regeneration medium with 10 or 20 mg/L Hm. All the three wheat varieties obtained transgenic plants. PCR and PCR-Southern assays showed that most plants regenerated from the resistant calli were positive transgenic plants. Southern blot of the positive green plants confirmed stable integration of alien DNA into wheat genome. The plant transformation frequencies varied with the variety and ion dose implanted. Wanmai 32 possessed the highest transformation frequency, reaching 3.8% at a suitable implantation dose. The transformation frequency of Yangmai 158 and Wan 9210 varied from 0.5% to 2.5% and from 0.5% to 1.4%, respectively. Progeny test for resistance to wheat scab showed that the leaf extract of R₁ generation inhibited the growth of wheat scab strain R₀ and F₁₅.     

不同基因型冬小麦花后干物质的累积、转运及氮动态变化研究

采用田间小区试验,研究了2个不同基因型冬小麦(小偃22号和小偃6号)对花后不同营养器官干物质的累积与转运、收获指数及籽粒灌浆过程中茎、叶、颖壳、籽粒的全氮含量的动态变化。结果表明:茎、叶干物质的积累量,从开花期到成熟期一直呈下降的趋势;穗干物质的积累量,在开花期和灌浆期,随着施氮量的增加均呈升高的趋势;灌浆期到成熟期无明显趋势。开花期,不同干物质转运量在不同器官中的分配为"茎叶穗";冬小麦不同器官转移的干物质对子粒的贡献叶片最大,其次是茎,穗部最小;施氮处理下,收获指数显著高于不施氮处理。在籽粒灌浆过程中,全氮含量在茎、叶、籽粒中呈先降低后升高的趋势,而颖壳中的全氮含量呈"降低—升高—降低"的趋势;冬小麦不同器官全氮含量为茎颖壳叶籽粒,施氮量在150 kg/hm2处理下,冬小麦各器官全氮含量保持较高水平。试验中选用的2个基因型冬小麦品种,小偃22号干物质的积累量、收获指数显著高于小偃6号;施氮处理下,小偃22号不同器官的干物质转运量、干物质转运率和干物质对籽粒的贡献率都显著高于小偃6号。小偃22号具有较高的氮素吸收能力。     

As在小麦植株内吸收、分配和累积的研究

通过模拟As污染的田间试验,以郑州9023为供试小麦品种,研究了小麦不同生育期不同部位对As的吸收、分配和累积的动态变化规律.结果表明,在小麦整个生长过程中,不同部位的As含量随着时间的推移而变化,根、茎和籽粒3者的关系始终为:根>茎>籽粒;在灌浆末期,各部位As含量从大到小依次为:根>废弃物>叶>叶鞘>茎>穗轴>颖片>籽粒,地上各部位As累积量从大到小依次为:废弃物>茎>叶鞘>叶>籽粒>颖片>穗轴,因此小麦植株中较易富集As的部位是根和废弃物,其次是叶和叶鞘,而籽粒中As水平相对较低;从试验结果还可以看出,在小麦灌浆初-中期As的吸收量及吸收速率都显著高于其它时期.