Introduction of pokeweed antiviral protein cDNA intoBrassica napus and acquisition of transgenic plants resistant to viruses

Using cotyledonary petioles as explants, the PAP cDNA controlled by wound-inducible promoter has been introduced intoBrassica napus by coculture withAgrobacterium tumefaciens and laser microbeam puncture respectively. Regenerated plants resistant to gentamycin have been selected out. PCR amplification and Southern blotting analysis indicated that PAP cDNA together with wound-inducible promoter had been integrated intoBrassica genome with transformation frequencies of 2.0% and 1.7% for two transformation methods respectively. The test of virus challenge showed that these transgenicBrassica plants were resistant in different degrees to mechanically inoculated TuMV.     

甘蓝磷酯酶D HKD2功能区基因片段的克隆与反义表达载体的构建

以甘蓝(Brassiaca oleracea)为材料，取幼叶分离mRNA，反转录合成cDNA，以cDNA第一链为模板，通过PCR扩增，获得甘蓝磷酯酶D(Phosphorate　Lipid　Dehydrolase，PLD)HKD2功能区的基因片段．对其进行Blast分析，结果表明，分离的目的片段核苷酸序列与Genbank中报道的甘蓝PLD基因相比同源率为99．7％，只有2个碱基发生改变．将得到的PLD基因片段插入植物表达载体pAT940，构建了PLD基因反义表达载体pATC—rPLD，为下一步进行抗逆转基因作物选育打下基础．     

Construction of plant seed-specific expression vectors pSCB and pSCAB and the obtainment of transgenic Brassica napus H165 expressing poly-3-hydroxybutyrate synthetic genes

The seed-specific promoter and transit peptide were amplified and fused to the three genes phbA, phbB and phbC encoding PHB synthetic enzymes, respectively. Seed-specific expression vectors pSCB containing phbC and phbB, and pSCAB containing phbC, phbB and phbA, were constructed by introducing the genes with promoter and peptide into the binary vector pBI101. Transgenic Brassica napus H165 were obtained by Agrobacterium-mediated transformation with these vectors. They were confirmed by PCR, Southern and RT-PCR analyses.     

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．     

转EPSPS基因抗草苷膦油菜基因漂移的初步研究

草苷膦是一种广谱内吸传导型除草剂[1].草苷膦通过竞争性抑制植物莽草酸代谢过程中磷酸烯醇式丙酮酸莽草酸-3-磷酸合酶(简称为EPSPS)活性,阻断芳香族氨基酸的生物合成导致植物死亡[2].因此,转EPSPS基因(来自微生物的基因)作物对草苷膦类型的除草剂具有抗性.在1996~2004年的9年间,抗除草剂生物技术产品一直占市场的主导地位.2004年,全球种植的抗除草剂的转基因作物有油菜、大豆、玉米及棉花等,种植面积共计有5,860万公顷,占全球转基因作物总种植面积(8,100万公顷)的72%.抗除草剂油菜品种在国外已广泛种植,据国际农业生物技术应用服务组…     

拟南芥AtGluRS相互作用蛋白质VDAC及其转基因植物表型分析

电压依赖性阴离子通道蛋白质(voltage-dependent anion channel,VDAC)是拟南芥谷氨酰tRNA合成酶(AtGluRS)相互作用的蛋白质．为了研究VDAC蛋白的功能和作用,通过构建VDAC稳定表达载体,农杆菌介导的方法转化拟南芥,筛选和鉴定出VDAC过量和抑制表达植株．研究结果证实VDAC的过量和抑制表达植株影响气孔关闭和种子萌发．VDAC的过量表达导致植株对ABA敏感,VDAC的抑制表达降低了植株对ABA的敏感性．推测VDAC蛋白可能参与ABA信号途径．     

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     

农杆菌介导外源基因进入油菜的研究

用甘蓝型油菜子叶为外植体，以农杆菌共培养法将苏云金杆菌δ－内毒素基因和ＮＰＴＩ基因导入油菜．所用的农杆菌为ＬＢＡ４４０４－δ（ｄｉｓａｒｍｅｄｐＡＬ４４０４，ｐＧＡ６４３－δ）．ＰＧＡ６４３－δ为表达载体，其中克隆有ＮＰＴＩ基因和δ－内毒素基因．共培养４ｄ后，转化的子叶被转入含有卡那霉素（Ｋｍ）的分化培养基［１］上，分化出芽．被切下的芽在含有Ｋｍ的生根培养基［２］上生根，移栽后成活的小抗Ｋｍ油菜苗生长良好．用ＤＮＡ分子杂交、ＥＬＩＳＡ分析和生物测定等方法证明外源基因被转移到油菜中．     

柑橘CsPRP4基因RNAi载体的构建

富含脯氨酸蛋白4(PRP4,proline-rich protein 4)是一类响应胁迫并在细胞发育过程中起作用一种细胞壁蛋白。用RT-PCR克隆柑橘CsPRP4基因的正、反义cDNA序列,分别连接到T载体pMD–19T上;经限制性核酸内切酶2次双酶切,将CsPRP4基因的正、反义片段定向连接至双元质粒pFGC5941查耳酮合成酶A(CHSA)内含子的两端,构成反向重复序列;经菌液PCR和测序验证,成功构建了CsPRP4基因的RNA干涉载体;经农杆菌介导法转化柑橘,转基因植株具有明显不同于对照的表现型,研究为进一步阐明CsPRP4的功能奠定了基础。     

农杆菌介导的DREB1A基因转化多花黑麦草及其转化体系的优化

采用携带卡那霉素抗性基因nptII和GUS基因的ubiquitin启动子驱动的表达载体pBI121／DREB1A的根癌农杆菌AGL1, 对多花黑麦草幼胚来源的胚性愈伤组织进行了遗传转化,并优化了各种影响因素。胚性愈伤组织经根癌农杆菌感染和共培养后,用50mg／L巴龙霉素筛选抗性愈伤组织,待抗性愈伤组织在IB分化培养基上分化成苗后用25mg／L卡那霉素进一步筛选再生植株, 获得了部分抗性植株。抗性植株的总DNA用DREB1A基因的特异引物进行PCR检测,转化频率为2.14％,PCR-Southern blot进一步验证了转化植株基因组中含有该外源基因。各种影响转化效率因素的优化实验表明,当转化时菌液浓度的OD600为2.0、侵染时间为1h、共培养时间为2d、共培养温度为21℃及在共培养期间使用乙酰丁香酮等,均可明显提高转化频率。     

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.     

Obtaining High Pest-resistant Tobacco Plants Carrying B.t. insecticidal Gene

To increase the expression level of CryIA(c) gene in transgenic plants, a plant expression vector pBinMoBc carrying the CryIA(c) gene under control of chimeric OM promoter and Ω factor was constructed. As a control, pBinoBc carrying the CryIA(c) gene with the CaMV 35S promoter was also constructed. The vectors were transferred into tobacco plants respectively via Agrobacterium-mediated transformation. ELISA assay showed that the expression level of the CryIA(c) gene in pBinMoBc transgenic tobacco plants was 2.44-times that in pBinoBc transgenic tobacco plants, and it could be up to 0.255% of total soluble proteins. Bioassay showed that pBinMoBc transgenic tobacco plants had more notable insecticidal effect than pBinoBc transgenic tobacco plants. The above results showed that the chimeric OM promoter was a stronger promoter than CaMV 35S promoter that was widely used in plant genetic engineering, and this is very useful in pest-resistant plant genetic engineering.     

甘蓝型油菜FAE1基因启动子的克隆和瞬时表达载体的构建及功能分析

油菜脂肪酸延长酶基因FAE1是广泛存在于植物中并定位于内质网上的一种能催化脂肪酸碳链延长的酮脂酰CoA合成酶,根据GenBank上已知的植物FAE1基因启动子序列设计引物,以从甘蓝型油菜(Braccica napus.L)叶片中提取的总DNA为模板,进行PCR扩增,获得了1 328 bp的片段.回收该片段,并连接到pMD18-T载体测序.序列在NCBI进行blast比对,同源性为99.78%,说明该片段与植物中已知的FAE1基因的启动子序列有极高的相似性.将该片段替换掉改造后的pBI221上面的35S启动子从而构建了原生质体瞬时表达载体.然后通过PEG介导的方式将含有FAE1启动子驱动的荧光素酶报告基因(LUC)表达载体导入油菜原生质体中,研究报告基因表达量,证明出脱落酸(ABA)影响了报告基因的表达,随着ABA浓度的不断增加,LUC的表达量呈现逐步下降的趋势.     

长叶车前花叶病毒上海株(RMVsh)外壳蛋白基因的克隆、序列分析及原核表达

长叶车前花叶病毒上海分离株（RMVsh)是从上海郊区的青菜（Brassica chinensis)上分离鉴定的，以提纯的病毒为材料，SDS－酚法纯化的基因组RNA作为模板，通过RT－PCR方法克服了该病毒的外壳蛋白基因（CP），DNA序列测定结果表明，外壳蛋白基因全长474个碱基，编码157个氨基酸，将CP基因插入原核表达载体pBAD/His-C中，转化E.coli Top10后，诱导表达，经聚丙烯酰胺凝胶电泳分析呈－特异性的蛋白条带，Western blot检测表明，表达产物与RMVsh抗血清呈阳性反应，RMVsh CP基因的核苷酸序列及氨基酸序列与烟草花叶病毒属中其他能侵染十字花科作物的成员相比，同源率分别为83．5％－98．9％和87．9％－99．4％，并讨论了RMVsh的分类地位为烟草花叶病毒属侵染十字花科植物2个亚组中的第一亚组的代表。     

Construction of plant seed-specific expression vectors pSCB and pSCAB and the obtainment of transgenicBrassica napus H165 expressing poly-3-hydroxybutyrate synthetic genes

The seed-specific promoter and transit peptide were amplified and fused to the three genesphbA, phbB andphbC encoding PHB synthetic enzymes, respectively. Seed-specific expression vectors pSCB containingphbC andphbB, and pSCAB containingphbC, phbB andphbA, were constructed by introducing the genes with promoter and peptide into the binary vector pBI101. TransgenicBrassica napus H165 were obtained byAgrobacterium-mediated transformation with these vectors. They were confirmed by PCR, Southern and RT-PCR analyses.     

甘蔗蔗糖磷酸合成酶基因启动子遗传转化烟草的研究

蔗糖磷酸合成酶(Sucrose Phosphate Synthase,SPS)是调节甘蔗蔗糖合成的关键酶之一.根据课题组已经构建的甘蔗SPS5侧翼序列驱动GUS表达的载体,通过根癌农杆菌介导的叶盘转化法,转化模式植物烟草,获得26棵抗性植株.抗性植株的PCR结果表明,获得3株转基因植株.GUS染色分析说明,SPS的5侧翼序列可以驱动GUS基因表达,SPS的5侧翼序列具有启动子活性.     

利用番茄U3snRNA基因上游启动区构建植物表达载体及对烟草的转化

利用番茄U3snRNA基因上游启动区和ACC合成酶反义RNA-核酶嵌合基因DNA片段,构建含U3snRNA基因上游启动区-ACC合成酶的反义RNA-核酶嵌合序列的表达载体,重组于植物双元表达载体pGA643中,得到pGU3R.用三亲融合法导入农杆菌LBA4404中,采用叶盘法转化烟草,诱导再生小植株,获得了卡那霉素的抗性植株.提取抗性植株总DNA,通过PCR、PCRSouthern杂交检测并分别用启动区序列和ACC合成酶的反义RNA-核酶嵌合序列作探针,通过Southern杂交检测,已筛选出整合有外源基因的转化植株.为进一步研究U3snRNA上游启动区增强反义RNA-核酶基因的表达奠定了基础.     

Transgenic tobacco plants harboring tomato proteinase inhibitor II gene and their insect resistance

The plant expression vectors pBCT2 and pBT2 were constructed with the cDNA sequence (tin2) and genomic DNA sequence (tin2i) of tomato proteinase inhibitor II gene respectively. Then the two expression vectors were transferred into tobacco via the Agrobacterium tumefaciens strain LBA4404, and transgenic tobacco plants were generated. Molecular analysis and trypsin activity assay showed that both cDNA and genomic DNA were expressed properly in the transgenic plants. Insecticidal activities in these transgenic plants indicated that transgenic tobacco plants carrying tin2i sequence were more resistant to 2-instar larvae of Heliothis armigera Hubner than those carrying tin2 sequence. Therefore the intron of tin2i sequence might be a contributor to insecticidal activity of the transgenic tobacco.     

商陆抗病毒蛋白cDNA的植物表达载体构建及转化烟草的研究

以商陆抗病毒蛋白的cDNA序列为依据,设计特异性引物,应用PCR技术扩增出5′端含有BamHI,3′端含有KpnI限制性内切酶酶切位点的目的片段,且在序列的开始和结尾分别加入起始密码子ATG和终止密码子TAA,将该目的片段定向克隆于载体pROKII中与CaMV35S启动子和Nos末端构成植物表达载体pRPAP,通过菌落直接PCR法和酶切分析鉴定出阳性重组子,用直接导入法将其导入农杆菌EHA105,以此作为工程菌进行烟草转化,对筛选到的阳性植株随机取样进行Southern印迹分析,结果表明,PAP基因已经整合到烟草基因组中,证明载体构建成功。     

表达长喙田菁SrPCS4烟草的获得及Cd抗性研究

植物螯合肽(phytochelatins,PCs)在植物解除重金属的毒性方面具有重要作用,是以谷胱甘肽为底物,在植物螯肽合成酶(phytochelatin synthase,PCS)催化下合成的.作者已经克隆得到的长喙田菁(Sesba-nia rostrata)植物螯合肽合成酶SrPCS4 cDNA长为1035 bp,其ORF编码177个氨基酸,以pHANNIBAL及pART27为基础,构建了CaMV35S启动子驱动的SrPCS4基因植物表达载体pAM25,采用电击转化方法将pAM25导入根癌农杆菌EHA105,并通过改良叶盘转化方法用该菌株对烟草进行了转化,对转基因烟草进行了PCR与northern-blot检测,研究结果表明得到了表达该基因的烟草,但表达该基因的烟草不能够提高对Cd的抗性.