Cry2Aa2 和PamPAP 双价表达载体的构建及其对辣椒的遗传转化

 为了获得既抗虫又抗病毒病，又对人体和环境安全的转基因辣椒（Capsicum annuum L．）材料，将启动子CaMV35S、终止子Nos-ter 及缺失无毒型商陆抗病毒蛋白基因PamPAP 一起插入到植物表达载体pCAMBIA1301-Cry2Aa2-PMI 的多克隆位点上，构建双价表达载体，采用农杆菌介导法将其转入辣椒中，并用PMI/甘露糖筛选体系对转化植株筛选。经PCR 扩增和Southern 杂交检测，结果表明Cry2Aa2和PamPAP 已经整合到辣椒基因组中。进一步经RT-PCR 分析，证实Cry2Aa2 和PamPAP 在T₀ 代转基因辣椒植株中共表达。经抗虫性、抗病毒病表型鉴定：T₀ 代转基因辣椒植株对黄瓜花叶病毒（CMV）和斜纹夜蛾幼虫的抗性均显著高于非转基因植株。     

基因枪法和农杆菌介导的Bt抗虫基因转化芥蓝

以芥蓝品种小香菇为试材,分别采用基因枪法和农杆菌介导转化法将Bt抗虫基因Cry2Aa2转化到芥蓝中,并对两种转化方法进行了优化和比较。结果表明:基因枪法在轰击距离为6cm、轰击次数为1次时转化效率最高,为0.43%;农杆菌介导转化法以带子叶下胚轴为外植体、预培养2d、侵染10min、共培养2d、抑菌培养5d后转入筛选培养基为最佳参数,转化效率为0.37%。经PCR和PCR-Southern鉴定,基因枪法和农杆菌介导转化法均成功将目的基因导入芥蓝基因组中。经抗虫性表型鉴定,T_0代转基因芥蓝植株对甘蓝夜蛾幼虫的抗性高于非转基因植株。     

双抗虫基因转化欧美杨107的研究

以欧美杨107为试材,采用农杆介导法进行双抗虫基因(Bt Cry1Ac基因+API慈姑蛋白酶抑制剂基因)的遗传转化研究,探讨了影响欧美杨107的遗传转化的不同因素,初步建立了欧美杨107品种高效组织培养再生体系及遗传转化体系。结果表明:经PCR检测和虫试效果,证明了目的基因已经整合到欧美杨107的基因组中,从转基因株系中筛选出生长发育正常并抗虫能力强的优良株系。     

转Bt 基因早熟春甘蓝抗虫材料的获得

采用农杆菌介导法，将Bt cry1Ia8 抗虫基因转入早熟春甘蓝自交系F2011 中，共获得37 株卡那霉素抗性植株，其中23 株经PCR 检测呈阳性；Southern blot 检测结果表明，cry1Ia8 基因已成功整合到甘蓝基因组中；RT-PCR 和Western blot 检测结果表明，cry1Ia8 基因在RNA 水平和蛋白质水平均得到表达；对转基因植株进行ELISA 检测，其Bt 毒蛋白含量在201.9～241.3 ng·g-1（FW）之间；离体饲虫试验结果表明，转基因植株对敏感小菜蛾和Cry1Ac 抗性小菜蛾均具有较好的抗
性，且Bt 毒蛋白表达量越大，植株抗性越强。     

农杆菌介导Cry1Ac基因转化菊花

 用农杆菌介导法, 以茎段(节间) 为外植体, 将含有苏云金芽孢杆菌(Bt) 毒蛋白Cry1Ac基因的植物遗传载体导入菊花(切花菊) 品种‘日本黄’中, 得到126株抗性植株, PCR检测及基因组DNA的Southern杂交分析表明Cry1Ac基因已整合到菊花总DNA中, 共获得6株转基因植株。利用棉铃虫做转基因植株的盆栽和叶片平皿试验, 发现其中2株对棉铃虫具有很高抗性, 校正死亡率达90%以上; 1株对棉铃虫具有高抗性, 校正死亡率达80%以上; 3株对棉铃虫抗性较明显, 校正死亡率44.7%～60.9%。     

毒杀天牛基因mBt886cry3a转化小黄菊研究

毒蛋白编码基因 Bt886cry3Aa 对小黄菊进行了遗传转化,转化植株经过PCR和总DNA狭缝杂交验证,得到转基因小黄菊4个样品株.对菊天牛2龄幼虫的生物测定表明:4个样品株毒杀活性明显高于对照,校正死亡率分别为:45.89%、39.12%、35.42%和22.41%.mBt886cry3a转基因植株表现出较强的天牛毒杀活性,为天牛等钻蛀性害虫的防治提供了研究基础.     

农杆菌介导P2300-pa-8e抗虫基因转化马铃薯的研究

以马铃薯品种早大白和克新18号微型薯为外植体,利用根癌农杆菌介导法,将来源于Bt菌株Bt185的P2300-pa-8e抗虫基因转入马铃薯,并对其遗传转化的影响因素进行研究。获得抗性植株早大白19株、克新18号21株|经PCR检测,5株早大白、9株克新18号呈阳性|经Southern-blot杂交分析,4株早大白、6株克新18号呈阳性,证明P2300-pa-8e基因已整合到马铃薯基因组中。在网室内对转基因植株进行抗虫试验,转基因材料对鞘翅目害虫暗黑鳃金龟和华北大黑鳃金龟幼虫表现出抗性。     

转Cry1Ac青花菜回交后代鉴定方法研究

转Bt基因青花菜在遗传分离后代中的鉴定方法,对于准确筛选阳性单株和抗虫材料具有重要影响。利用5份青花菜材料、1份阳性对照（转Cry1Ac基因纯合材料）和5份回交父本（阴性对照）为试验对象,在基因水平上对Cry1Ac进行普通PCR扩增,蛋白水平上采用Bt-Cry1Ab/1Ac转基因检测试纸条测定,表观上采用小菜蛾离体饲虫试验进行抗性鉴定,比较阐明了3种方法的鉴定效果和优缺点。结果表明,3种方法分别从5份青花菜回交材料的159个分离单株中检测到82、77和75份阳性株,经卡方显著性检验,各材料阳性单株与非阳性单株分离后代均符合孟德尔遗传规律。分析表明,小菜蛾离体饲虫试验对于鉴定转Cry1Ac后代抗虫效果准确可靠,但需要很好地控制实验条件;Bt-Cry1Ab/1Ac转基因检测试纸条灵敏度高,可快速观察到Bt毒蛋白的实际表达量,基本不受条件限制,但必须控制人为误差;普通PCR扩增能够鉴定阳性株,但不能反映实际抗虫效果,必须结合小菜蛾饲虫试验或Bt毒蛋白水平上的检测才能实现精准鉴定。     

农杆菌介导PSAG12-IPT基因转化辣椒的研究

异戊烯基转移酶(Isopentenyl-Transferasas,IPT),也称作细胞分裂素合成酶,是植物中细胞分裂素合成的限速酶。IPT在转基因植株中超表达后,会使叶片中的细胞分裂素含量增加,从而延缓叶片衰老。但过高对植物的生长和育性都是有害的。如果将衰老特异性启动子(PSAG)与IPT基因融合,在它的驱动下表达,只有在叶片衰老时才表达合成分裂素,既能延缓衰老又不影响植物的生长发育。以p CAMBIA1301-PMI表达载体为基础载体,用衰老特异性启动子驱动目的基因IPT,用辣椒Flamingobill外植体作受体,采用农杆菌介导的方法转化辣椒,并利用甘露糖筛选体系对辣椒转化体进行筛选,对转基因植株进行分子生物学检测和抗衰老检测。结果表明,共获得82株抗性植株,PCR检测的阳性率约为50%。而在对T1代的PCR检测表明该基因能稳定遗传给下一代。这些经转化的植株具有叶片衰老延缓及植株生命周期延长等现象,花的衰老也有所延缓。T1的株高和侧芽萌发与对照相比无明显差异。     

转拟南芥AtNHX5基因烟草的耐盐性研究

采用农杆菌介导的叶盘法将来源于拟南芥的Na+/H+逆向转运蛋白基因AtNHX5导入烟草,经潮霉素筛选、PCR和RT-PCR检测,证明AtNHX5已导入烟草基因组并在转基因植株中表达。将转基因植株和非转基因(野生型)植株培养在含有300 mM NaCl的1/2MS培养基上,发现转基因植株的生长明显优于野生型。将在含有300 mM NaCl的1/2MS培养基中生长4周后的幼苗转移到不含NaCl的1/2MS培养基后,转基因植株快速恢复生长,而野生型植株则生长停滞。结果表明:超表达AtNHX5基因可以提高烟草的耐盐性。     

中国商陆抗病毒蛋白基因的克隆及其转化辣椒的研究

本研究的目的是获得抗病毒病的辣椒材料,为选育抗病毒病辣椒品种奠定基础。根据美洲商陆抗病毒蛋白基因序列设计引物,从中国商陆（Phytolacca acinosa）叶片基因组DNA扩增克隆缺失无毒型中国商陆抗病毒蛋白基因（PacPAP1,构建该基因的植物表达载体,采用农杆菌介导的叶盘法转化辣椒,对转基因植株进行分子检测、TMV及CMV的人工接种鉴定,接种25d后,统计发病情况。结果表明：克隆得到缺失无毒型PAP基因PacPAP,大小为714 pb,与美洲商陆抗病毒蛋白基因（PamPAP）的同源性为99.7%;得到了PacPAP1整合入辣椒基因组中的阳性植株;接种TMV、CMV阴性对照植株都明显发病,转PacPAP1基因植株未出现症状,说明转PacPAP1辣椒植株可获得抗TMV和CMV材料。     

乙肝表面抗原大蛋白(S1S2S)基因在转基因苹果中表达

构建了乙肝表面抗原大蛋白基因PRS-S1S2S的表达载体pYF9616,通过根癌农杆菌介导法首次将该基因导入苹果品种红爱佳中,得到了抗卡那霉素的GUS阳性转化植株。随机取3株GUS染色阳性的转基因苹果植株经PCR扩增及RT-PCR检测证实该基因已整合入转基因苹果的基因组,并在转录水平得到了表达,ELISA检测证明在苹果植株中正确表达了乙肝表面抗原大蛋白基因。     

Development and bioassay of Cry1Ac-transgenic eggplant (Solanum melongena L.) resistant to shoot and fruit borer

Summary

Eggplant (Solanum melongena L.) is an important fruit vegetable, commercially cultivated in the tropics and subtropics. However, the most serious constraint on eggplant production is damage caused by eggplant shoot and fruit borer (ESFB). The development of resistant transgenic plants, using an insecticidal crystal protein gene, is an available crop protection option. A Cry1Ac gene obtained from the National Research Center for Plant Biotechnology (NRCPB), New Delhi, India, was transformed via Agrobacterium-mediated gene transfer into an improved inbred line of eggplant (IVBL-9). Hypocotyls proved to be the most effective and suitable explants, with a transformation frequency of 17.3% and 2.9 shoots per explant. PCR and Southern blot analyses confirmed the presence of a single copy insertion of the Cry1Ac gene in seven independent transgenic plants. The insertion of a single copy of the gene was also confirmed by segregation analysis of T₁ seed from T₀ plants. ELISA analyses revealed the presence of the Cry1Ac protein, and quantitative estimates confirmed significant levels of Cry1Ac protein (2.46 – 4.33 ng ml^–1 leaf extract) expressed in all seven transformed plants. High levels of expression of this insecticidal protein resulted in significant larval mortality and in the reduced growth of any surviving larva on transformed eggplant tissue.     

黄瓜花叶病毒反义2b 基因构建及其转基因初步研究

 研究构建了CMV 反义2b 基因的植物表达载体pZM 612 , 通过农杆菌侵染的叶盘转化法导入烟草, 分子检测证实获得了转反义2b 基因的SR1 烟草转基因植株, 初步的CMV 攻毒试验表明反义2b 基因可介导对CMV 的抗性。在此基础上, 将pZM 612 转化番茄品种‘红玛213’, 获得了卡那霉素抗性苗, PCR检测表明得到了PCR 阳性转化株。     

Towards crop improvement in bell pepper (Capsicum annuum L.): Transgenics (uid A::hpt II) by a tissue-culture-independent Agrobacterium-mediated in planta approach

Agrobacterium tumefaciens-mediated transformation has been one of the methods used to generate transgenic plants in bell pepper. An alternate transformation method that avoids/minimizes tissue culture would be beneficial for the improvement of bell pepper due to its recalcitrant nature. In this report, transgenic bell pepper plants have been developed by a tissue-culture-independent A. tumefaciens-mediated in planta transformation procedure. In the present study, two open pollinated varieties viz., Arka Gaurav and Arka Mohini were used for transformation. Agrobacterium strain EHA105 harboring the binary vector pCAMBIA1301 that carries the genes for β-glucuronidase (uid A) and hygromycin phosphotransferase II (hpt II) was used for transformation. GUS histochemical analysis of T₀ and T₁ plants at various stages of growth followed by molecular analysis using PCR, Southern analysis and RT-PCR allowed selection of transgenics. The method resulted in 17.8% and 11.4% of the T₀ plants in Arka Gaurav and Arka Mohini being selected as chimeric and 35.0% and 29.7%, respectively, were identified as stable transformants in the T₁ generation based on PCR analysis.     

辣椒CaSn 基因超表达增强转基因烟草对南方根结线虫的抗性

 CaSn 基因是在辣椒（Capsicum annuum）‘Santaka’中发现的新型抗菌肽Snakin 家族基因。 通过RT-PCR,将CaSn 基因从辣椒中分离并构建到pBI-121 植物表达载体,通过农杆菌EHA105 介导转 化白肋烟（Nicotiana tabacum‘White burley’）,筛选到10 个独立的转基因株系,并对T1 代植株进行抗 南方根结线虫（Meloidogyne incognita）鉴定及靶标基因表达分析。结果表明CaSn 已转入白肋烟,能够进 行超量表达,并且转CaSn 基因白肋烟上的根结数量比转空载体对照和非转基因对照减少70%,同时不同 转基因烟草株系间对南方根结线虫的抗性也存在着一定差别。通过试验证明了辣椒中的抗菌肽基因CaSn 具有抗南方根结线虫作用,对于植物抗线虫资源的挖掘及利用提供了重要理论依据。     

Efficient production of transgenic plants using the bar gene for herbicide resistance in sweetpotato

Efficient production of transgenic sweetpotato (Ipomoea batatas (L.) Lam.) plants using the bar gene for herbicide resistance was achieved through the use of embryogenic suspension cultures and Agrobacterium tumefaciens-mediated transformation. Cell aggregates from embryogenic suspension cultures of sweetpotato cv. Lizixiang were cocultivated with A. tumefaciens strain EHA 105 harboring a binary vector pCAMBIA3300 with the bar gene and uidA gene. Selection culture was conducted using 0.5 mg/l PPT. A total of 1431 plants were produced from the inoculated 870 cell aggregates via somatic embryogenesis. GUS assay and PCR analysis of the regenerated plants randomly sampled showed that 86.5% of the regenerated plants were transgenic plants. Stable integration of the bar gene into the genome of transgenic plants was confirmed by Southern blot analysis and transgene expression was demonstrated by Northern blot analysis. The copy number of integrated bar gene ranged from 1 to 3. Transgenic plants exhibited functional expression of the bar gene by in vivo assay for herbicide resistance. This study also provides a simple and efficient transformation system of sweetpotato based on the use of bar gene as a selectable marker gene, which can be combined with other agronomically important genes for the improvement of sweetpotato.     

农杆菌介导Ferritin基因转化苹果的研究

以皇家嘎拉苹果的叶片为转化受体,通过根癌农杆菌介导将铁结合蛋白(Ferritin)基因导入受体中,诱导获得了抗性芽.经PCR检测从抗Km植株中选择到4株阳性植株,进一步经PCR-Southern杂交证实外源基因已整合到4株苹果基因组中,RT-PCR检测表明,Ferritin基因在4株转基因植株中得到了表达.