农杆菌介导抗虫基因转化芥菜型油菜的研究

用农杆菌介导法对芥菜型油菜进行了转化，并对影响根癌农杆菌转化效率的因素进行了研究，建立了以油菜子叶柄为外植体的转化体系．用携带蝎毒素(BmKITs)和几丁质酶(chi)双价基因的农杆菌转化油菜，共获得了抗卡那霉素的再生苗153株，移栽成活21株．对成活植株进行了PCR分子检测，证实有外源基因的整合．转基因植株形态正常，开花、结实．     

褐飞虱喂养试验显示表达GNA的转基因水稻纯系抗褐飞虱

利用基因枪法将含有3个不同基因（hpt,gus和gna）的质粒pWRG1515和pRSSGNA1共同转化粳稻品种鄂晚5号成熟胚诱导的愈伤组织。共再生出35株独立转基因植株。PCR／Southern印迹法分析发现,83％的转基因植株含有所有3个外源基因。Western印迹法分析发现79％的含gna基因的转基因植株以不同水平表达GNA。遗传分析证实外源基因在转基因植株后代中以孟德尔方式遗传。从其R1代亲本为孟德尔3：1方式遗传的R2代中,鉴定出2个含有所有3个外源基因的独立转基因植株纯系。这些纯系具有相似的外源基因表达量。褐飞虱喂养试验表明,这些纯系对褐飞虱具有显著的抑制作用。这些褐飞虱抗性提高的转基因纯系将应用于水稻抗虫育种中。实验证明,通过遗传转化和筛选可获得含在农业上有应用价值基因的转基因水稻纯系。     

转基因水稻纯系对褐飞虱的抗性研究

利用基因枪法将含潮霉素抗性基因、ＧＵＳ报告基因和雪花莲凝集基因的２个质粒ｐＷＲＧ１５１５和ｐＲＳＳＧＡ１共同转化粳稻品种鄂宜１０５的成熟胚诱导的愈伤组织,从轰击的１５２块愈伤组织中共再生出２６株独立转基因植株,ＰＣＲ／Ｓｏｕｔｈｅｒｎ印迹法分析发现,７３％的转基因植株含有所有３个外源基因,遗传分析证实外泊基因在转基因植株后代中以孟德尔方式遗传,从其Ｒ１代亲本为孟德尔３：１方式遗传的Ｒ２代中,鉴定出     

通过遗传转化获得含多基因的水稻转基因纯合植株

利用基因枪法将含有4个不同基因的3个质粒共转化由粳稻品种鄂宜105号和鄂晚5号种子胚诱导的愈伤组织（5－10d龄）。从轰击的986块愈合组织中共再生出169株独立的转基因水稻植株（转化率为17％）。PCR／Southern blot分析显示70％以上的转基因植株含有所有4个基因。GUS组织化学分析、Western blot和或RT－PCR分析表明所有4个基因的共表达率为70％。未观察到任何质粒在整合中存在优势,转基因拷贝数也与基因表达量无关。遗传分析证实外源基因在后代植株中大多以孟德尔方式遗传。从其R1代为3：1孟德尔方式遗传的后代R2代植株中,鉴定含有3个或4个不同基因的转基因纯合植株系。PCR／Southern blot分析证实了这些转基因纯合植株系。这些系的植株具有相似的外源基因表达量。我们证实通过基因枪介导的共转化,结合常规育种方法筛选可以获得含多基因的转基因水稻纯合植株。这项技术为利用基因同时改良作用多个性状提供了一种途径。     

几种水稻籼型恢复系和不育系离体培养和遗传转化的研究

对二系杂交水稻亲本籼型恢复系 2 88,198,112 5和籼型不育系株 1- s进行了离体培养和遗传转化的研究 ,建立了这些籼稻新品系的胚性愈伤组织高频诱导与再生系统及其基因枪转化系统 .它们的胚性愈伤组织诱导率分别可达 87.4 % ,90 .6% ,78.9%和 88.0 % ;其分化率分别可达 60 .0 % ,4 5 .8% ,4 6.9%和 60 .6% ;其基因枪转化率分别可达 16.69% ,2 .65 % ,2 .0 4 %和 10 .78% ,并获得了一批可育的水稻转基因植株     

多基因双T-DNA植物表达载体的构建及拟南芥转化

多基因植物表达载体用于植物遗传转化是培育具有多种优良品质作物的有效策略. 双T-DNA系统是实现筛选完成后选择标记基因删除的一种简便可行的方式. 为培育高度抗逆或去除标记基因的农作物,构建了多基因双T-DNA植物表达载体2T-bbgdD,其中含有一个抗除草剂基因bar, 3个抗逆相关基因(DREB1A, Na+依赖性Pi转运体基因（d5）, betA)和一个报告基因gfp. 利用农杆菌介导法将该载体转入拟南芥,获得了多基因共转化及去除标记基因的转基因拟南芥. 可将此植物表达载体进一步用于作物的遗传转化.     

害虫对转基因植物抗性进化的计算机模拟

采用人工生命的方法，定量模拟了不同条件下害虫对转基因植物抗性性的进化速度。结果显示只转入植物一个毒性基因常使害虫在短时间内产生抗性，若转３－４个不会产生交互抗性的毒性基因则可大大推迟到或阻止抗性的产生。     

尖镰胞菌细胞色素P450 55a1基因超表达载体的构建和水稻日本晴遗传转化

要将细胞色素P450 55a1基因克隆到镰刀菌素植物超表达载体pCAMBIA1302中,构建了pCAMBIA1302-cyp55a1-gfp1植物超表达载体,以水稻日本晴为遗传转化的受体对象,通过农杆菌介导侵染方法进行了遗传转化.结果表明：成功构建了细胞色素P450 55a1基因超表达载体,获得了多个细胞色素P450 55a1基因超表达的阳性植株,并以RT-PCR技术分析了阳性植株中细胞色素P450 55a1基因的表达水平.     

转基因植物标记基因的研究进展

随着商业化植物转基因品种的不断出现,人们对转基因植物的安全问题谈论得越来越多,其中争论的焦点之一即筛选标记基因的安全性。科学工作者尝试培育具安全选择标记基因或无选择标记的转基因植物,目的是提高转基因植物安全性,使之更易为广大消费者所接受。本文就这方面的研究进展作一综述。     

含编码类铁氧还双亲性蛋白ap1基因的转基因水稻植株的获得

利用基因枪法将含有潮霉素抗性基因（hpt),gusA报告基因和ap1基因的2个质粒（pJIMB15和pBiSAP1)共同转化同转化由粳稻品种鄂宜105号种 子在胚诱导的愈伤组织（2－3周龄）。ap1基因编码一种双亲性的蛋白。该蛋白能延缓因假单孢菌感染所引起的非寄主植物中的过敏反应。经过2轮潮霉素（30mg/L)筛选,抗性愈伤组织被转入含30mg/L潮霉素的再生培养基中再生植株。从轰击的186块愈伤组织中共再生出32株独立的转基因水稻植株（转化率为17．2％）,PCR／Southern blot分析显示84％的转基因植株含有所有3个基因。     

Light-regulated and organ-specific expression of a wheat Cab gene in transgenic tobacco

Many of our most important crop plants are monocotyledons, including wheat, corn, rice and barley. No routine transformation system for monocotyledons has been reported, such as the Ti-mediated gene transfer system for dicotyledons facilitated by Agrobacterium tumefaciens. Indirect evidence suggests that Ti-plasmid DNA is transferred into and expressed in A. tumefaciens-infected wound tissues of plants from Liliaceae and Amaryllidaceae, but these observations have not been extended to monocotyledons of greatest agricultural importance. Regeneration of monocotyledons is usually blocked at the callus-stage, further complicating the possibility of exploring the regulated expression of their genes, and thus preventing identification of the regulatory domains of monocotyledonous genes in a homologous nuclear background. To circumvent these difficulties, we investigated whether monocotyledonous genes can be expressed and correctly regulated in dicotyledons. We have introduced a wheat gene (whAB1.6) encoding the major chlorophyll a/b binding protein (Cab) of the light-harvesting complex into the genomes of tobacco (Nicotiana tabacum SR1) and petunia (Petunia hybrida) via a Ti-DNA-mediated gene transfer system which allows the transformed cells to regenerate into whole plants. Here we report for the first time the light-regulated and organ-specific expression of a monocotyledonous gene in transgenic dicotyledonous plants.     

Inheritance and expression of multiple disease and insect resistance genes in transgenic rice

2—3 anti-fungal disease genes are coinserted with hygromycin phosphotransferase in the same vector. Two insecticidal genes and PPT acetyl transferase genes are placed in another one. The vectors are co-delivered to rice embryonic cellus tissue at a molar ratio of 1︰1 using the particle gun method. 55 independent regenerated lines have been obtained through screening for hygromycin resistance. Of these, 70% transgenic plants harbor 6—7 foreign genes. The genes on the same vectors are always co-delivered to rice plant. Northern blot analysis has indicated that the multiple foreign genes give stable expression. In the 6 transgenic plants carrying 6—7 foreign genes, multiple foreign genes tend to integrate in 1 or 2 genetic loci. Progeny segregation is consistent with Mendel’s 3︰1 segregation law. 8 homozygous R₁ transgenic plants harboring 2—3 anti-fungal and 2 insecticidal genes are selected from large number of transgenic progeny screening for hygromycin and Basta resistance.     

Functional characterization and mapping of two MADS box genes from peach (Prunus persica)

Previously an AGAMOUS gene homologue PpMADS4 and a FRUITFULL gene homologue PpMADS6 were isolated from peach (Prunus persica), and both genes were shown to express in the developing floral and fruits. To gain insight into their function, the two genes were constitutively expressed in Arabidopsis thaliana and their effects on plant growth and floral organ development were studied in this work. The transgenic plants all displayed early flowering and conversion of inflorescence to floral meristem. However, the two genes had different effects on the floral organ structures in A. thaliana. The transgenic plants overexpressing PpMADS4 displayed homeotic conversion of floral organs, and par- ticularly the perianth abscission was inhibited. The plants overexpressing PpMADS6 showed early flowering, produced higher number of carpels, petals, and stamens than nontransgenic plants, and pod shatter was prevented; significantly, the transgenic plants yielded more than one siliques from a single flower. A SSR molecular marker was developed for PpMADS4, and it was then assigned into the G5 linkage group of Prunus sp. Both PpMADS4 and PpMADS6 genes were located at the same region in the G5 linkage group. Our results showed the potential application of these two MADS box genes for crop and fruit tree improvement.     

植物外源基因拷贝数及插入位点的检测方法与技术

随着转基因技术的快速发展,转基因植物在世界范围内已经大量种植.近年来,转基因植物的安全性越来越受到公众关注.转基因植物安全性与有效性的关键因素是外源基因拷贝数与插入位点.本文综述了近年来植物外源基因拷贝数与插入位点的检测方法.     

Salt tolerance of transgenic rice (Oryza sativa L.) with mtlD gene and gutD gene

Southern blot analysis indicated that mtlD gene (encoding mannitol-1-phosphate dehydrogenase) and gutD gene (encoding glucitol-6-phosphate dehydrogenase) had been integrated into the rice genome mediated by Agrobacterium tumefaciens LBA4404(pBIGM). The expression of the above two genes in transgenic rice plants was demonstrated by Northern blot analysis and enzymatic activity assay. Analysis of sugar alcohol showed that transgenic rice plants could produce and accumulate mannitol and sorbitol. The salt tolerance of transgenic plants was much higher than that of their controls.     

Studies on introduction of arrowhead proteinase inhibitor gene intoN. tobacco protoplasts

The Arrowhead Proteinase Inhibitor (API) gene was introduced into the protoplasts of mesophyll cells ofN. tobacco by PEG-mediated. The transformed protoplasts underging differentiation of callus and regeneration of plantlet have been growing into transgenic plants. Restriction endonuclease analysis of products amplificated by PCR indicates the existence of the API gene in the transformed plantlet. The extract of the leaves from the transformed plants shows trypsin inhibitory activity, which indicates the expression on the introduced API gene and the transformed plants can accumulate the inhibitor. However, the variation of the inhibitory activity of the transformed plants reveals the importance of the integration site of the API gene in the genome. Wang Xin: born in Dec. 1965. Master of science. Present address     

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.     

农杆菌介导的双价抗虫基因对番茄遗传转化的研究

采用农杆菌介导法将含有苏云金芽孢杆菌毒蛋白基因(CryIAc)与半夏凝集素抗虫基因(Pta)的高效植物表达载体pCAMBIA3300转入番茄品系Micro Tom的子叶外植体中。经过共培养、除草剂筛选和分化再生,获得了24个具有除草剂抗性的株系。再将转化后的番茄植株经过PCR检测和Southern Blot检测,确定检测后呈阳性反应的株系为8个。通过小菜蛾幼虫初步抗性试验证明,转基因株系表现出较强的抗虫性。实验结果为进一步研究番茄抗虫性和培育抗虫番茄新品种奠定了重要基础。     

甘蓝型油菜3个AP3重复基因植物反义表达载体的构建及遗传转化

利用pFGC5941构建了甘蓝型油菜3个AP3重复基因的植物反义表达载体pFGC5941-BnAP3-2、pFGC5941-BnAP3-3和 pFGC5941-BnAP3-4.采用快速冻融法,将载体导入农杆菌EHA105,转化甘蓝型油菜下胚轴.在5mg/L PPT的MS培养基中筛选,获得反义BnAP3-2基因的抗性再生植株31株,反义BnAP3-3基因的抗性再生植株20株,反义BnAP3-4基因的抗性再生植株42株.PCR鉴定结果显示,获得BnAP3-2反义的基因植株12株,BnAP3-3反义的转基因植