共转化CBF4和bar基因蒙农杂种冰草植株的分子检测

在获得转CBF4和bar基因蒙农杂种冰草植株的基础上,应用PCR和Southern杂交技术检测外源基因在转基因蒙农杂种冰草植株中的整合及拷贝数.确定外源基因CBF4和bar基因已经整合到冰草基因组中,并且是以多拷贝的整合方式插入受体细胞的染色体上.说明外源基因对蒙农杂种冰草成功地进行了转化,可以作为后期转基因冰草植株研究及培育转基因冰草新品种的材料.     

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.     

小鼠转基因及传代研究

综述了湖北省农科院生物技术研究所近年来有关转基因小鼠的主要研究进展。应用原核注射技术 ,将POMT PGH、hDAF、pBHSA、pSHSA、PT HBV 1 3、Bcl xL、hCD5 9、hCD5 9+hMCP等 8种外源基因 ,注入并移植 4 874枚小鼠的受精卵 ,得到 5 5 6只小鼠 ;经PCR和Southern杂交检测 ,确认原代转基因小鼠 10 8只。基因整合率平均 19.4 % ,转基因效率平均 2 .2 %。应用混合注射的方法得到了转双基因小鼠 ,双基因共整合率 2 2 .2 %。表达外源蛋白的转基因小鼠在 5 0 %～ 10 0 %之间。研究了小鼠的超数排卵和影响转基因效率的几个因素。通过转Bcl xL小鼠与非转基因小鼠连续五个世代的传代交配和检测 ,研究了转基因小鼠外源基因的遗传规律 ,表明四只原代小鼠中 ,只有一只能稳定地将外源基因传递给后代。并非所有的转基因小鼠都具有遗传的稳定性 ,欲建立小鼠的转基因品系 ,尚需对原代转基因小鼠进行筛选。     

转基因植物检测技术研究进展

随着植物基因工程技术的发展,转基因植物的研究和开发取得了令人瞩目的成绩,已培育了一批抗虫、抗病、抗除草剂和高产优质的转基因农作物新品种。与此同时,转基因植物检测技术不断丰富和完善,促进了转基因植物科学鉴定和评价,以及转基因植物商业化种植。目前报道的转基因植物检测方法主要有3类,一是在整合水平上进行的检测,包括PCR检测、Southern blot检测和染色体原位杂交检测等;二是在转录水平上进行的检测,包括RT-PCR检测、Northern blot杂交检测等;三是在表达水平上进行的检测,包括组织化学染色检测、荧光蛋白检测、Western blot检测、ELISA检测、叶片退绿检测和叶片涂抹除草剂检测等。     

转几丁质酶基因烟草后代的遗传分析

本论文采用分子检测(PCR扩增、PCR-Southern杂交)与表型检测(接真菌实验、酶活力测定)相结合的手段，对花粉管通道法及载体法导入几丁质酶基因烟草后代的遗传表现进行研究。结果证明通过不同方法导入受体细胞基因组中的外源基因均能遗传给后代，并能在转化受体当代及后代中高效表达。但采用花粉管通道法导入的外源基因虽然能够遗传给后代，但分离比复杂，遗传规律性较差。而载体法导入的外源基因T1代x^2检测符合3：1的遗传分离比，遗传稳定性要好于DNA直接导入。因此，建立良好的遗传转化系统是外源基因稳定遗传和表达的前提。     

利用基因枪法向高羊茅导入bar基因的研究

利用基因枪法,以bar基因转化高羊茅(Festuca arundinacea Schreb.)的下胚轴愈伤组织,对获得的再生植株进行的PCR检测和点杂交分析表明,外源bar基因片段已经整合至高羊茅基因组中.同时,对转基因植株进行的草丁膦涂抹实验发现,转基因植株对除草剂的耐性提高.     

乙型肝炎病毒转基因小鼠外源基因的复制和传代稳定性观察

目的研究乙型肝炎病毒(HBV)转基因小鼠外源基因的复制、传代稳定性.方法通过nest-PCR和Southern分子杂交等检测方法,对48#、87#、90#三个系列中各15只整合阳性小鼠定期进行血清中HBV DNA存在情况以及对各系列繁育F1-F5代小鼠外源基因整合传代进行检测.结果各系列转基因鼠血清都有HBV DNA存在,并以1月龄时血清中HBV DNA阳性率最高,但随月龄改变,其DNA阳性率也变化,并存在系列、个体及月龄上的差异.结论HBV基因可在转基因小鼠体内复制、传代,且目前已稳定传至第五代(F5).     

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.     

植物转基因沉默的原因及对策

转基因植物中转基因沉默已成为植物基因工程的一大障碍,转基因沉默的原因是多方面的,可能是由于转录前外源基因和内源基因的结构特性、伴置效应以及宿主植物的遗传调控;也可能是因为转录时启动子、转录因子和终止子的作用;还可能是由于转录后的修饰作用、外源基因表达特异性和环境等因素。为了克服转基因植物中转基因沉默,可以采取下列对策：选择适宜的外源基因和调控元件、采用适当的转化方法、使用更加简便快捷的筛选策略等。     

转基因稻是否会通过基因逃逸导致生态风险?

外源基因从转基因稻逃逸到非转基因稻品种或其野生近缘种有可能导致生态风险.通常转基因逃逸必须满足如下3个条件:(1)转基因稻和非转基因稻或野生近缘种在空间上分布重叠,相邻生长;(2)转基因稻和非转基因稻或野生近缘种在时间上花期相遇;(3)栽培稻与相关近缘野生种在生物学上有一定的杂交亲和性,且杂种后代能正常繁殖.总结分析了栽培稻及含AA基因组近缘种的地理分布、开花习性、种间杂交亲和性以及栽培稻与多年生普通野生稻之间基因流的研究结果,探讨转基因稻的外源基因是否可能逃逸到环境中而导致生态风险.     

Simple and effective detection method for gene expression product of transgenic plant

It is a crucial problem in gene engineering whether an exogenous gene could be correctly expressed in cells of transgenic animal or plant, and how the expression products could be detected quantitatively in translational level. It is very difficult to analyze some gene expression products, such as isopentenyl transferase (ipt), in transgenic investigation because they are trace in organisms. A convenient method is described to determine trace content gene expression product which is hard to purify. The method includes predicting and synthesizing the antigenic peptide according to the cDNA sequence, coupling the synthetic antigenic peptide with carrier protein, raising specific antibodies against the synthetic antigen, and detecting the gene expression product by ELISA and Western blot.     

转基因马铃薯植株中外源基因PCR扩增和拷贝数测定

本文报道一种简便、快速、可靠，同时又适合大量转基因植株中外源基因测定的ＰＣＲ检测技术，同时改进了转基因植物中总ＤＮＡ提取方法，并且用窄缝转移杂交测定了转基因植株中外源基因拷贝数。     

Salt tolerance of transgenic rice (Oryza sativa L.) withmtlD gene andgutD gene

Southern blot analysis indicated thatmtlD gene (encoding mannitol-1-phosphate dehydrogenase) andgutD gene (encoding glucitol-6-phosphate dehydrogenase) had been integrated into the rice genome mediated byAgrobacterium 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.     

抗除草剂旱稻转基因植株的获得

以旱稻丹粳旱５－５５、６－２４、６－３７的悬浮细胞及未成熟胚为受体材料，采用基因枪法将含ＢＡＲ基因的ｐＤＭ３０２质粒ＤＮＡ导入旱稻细胞中，经ＰＰＴ筛选获得抗性愈伤组织，筛选出的愈伤组织在分化培养基上再生出完整的旱稻转基因植株。Ｓｏｕｔｈｅｒｎ分子杂交分析表明，外源ＢＡＲ基因已整合到水稻基因组内，抗除草剂试验结果表明，转化植株对０．０１％Ｂａｓｔａ（有效成分为ＰＰＴ）有一定程度的抗性，说明外源ＢＡＲ     

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.     

Integration and inheritance stability of foreignBt toxin gene in the bivalent insect-resistant transgenic cotton plants

Genetic and expressional stability of Bt toxin gene is crucial for the breeding of insect-resistant transgenic cotton varieties and their commercialization. Genomic Southern blot analysis of R₃, R₄ and R₅ generations of bivalent transgenic insect-resistant cotton plants was done in order to determine the integration, the copy number and the inheritance stability of Bt toxin gene in the transgenic cotton plants. The results indicated that there was a 4.7 kb positive band in the Southern blot when the genomic DNA of the bivalent transgenic insect-resistant cotton plants and the positive control (the plasmid) were digested with HindⅢ respectively. This result proved that the Bt toxin gene had been integrated into the genome of the cotton in full length. There is only one XhoⅠ restriction site in the Bt toxin gene. Southern blot analysis indicated that many copies of Bt toxin gene had been integrated into the genome of the cotton when the genomic DNA of transgenic plants was digested with XhoⅠ. Among them, there were four copies (about 17.7, 8, 5.5 and 4.7 kb in size) existing in all the tested plants of ₃, R₄ and R₅ generations. The preliminary conclusion was that there were more than four copies of Bt toxin gene integrated into the genome of the cotton, among them, more than one copy can express and inherit steadily. This result provides a scientific basis for the breeding of the bivalent insect-resis- tant transgenic cotton plants and its commercialization.     

Transformation of the salt-tolerant gene of<Emphasis Type="Italic">Avicennia marina</Emphasis> into tobacco plants and cultivation of salt-tolerant lines

Salt-tolerant gene, CSRG1, which was isolated from a kind of salt-tolerant mangroves, Avicennia marina, constructed the transgenic plasmid, pGAM189/CSRG1. CSRG1, GUS, Kmr and Hyg^r could be transferred into tobacco genome by the ameliorated leaf discs method of agro-bacterium-mediate transformation. Thirteen stable resistant lines were obtained when fifty transgenic explants were selected through 50 mg/L hygromycin and 150 mg/L kanamycin. Assessments of PCR amplification, Southern blot analysis and GUS histochemical staining showed that CSRG1 has been integrated into the genome of the eleven transgenic lines (frequency of transformation was 22%). Northern bolt analysis revealed that CSRG1 had expressed in transgenic lines. The assessments of salt-tolerant ability and photosyn-thetic rates indicated that the survival rate of the transgenic lines is 80%—90% and the transgenic lines could increase by 30%—40% in plant height, even when they were cultivated in MS medium containing 2% NaCl and the total seawater (salinity 24). It is supposed that the special physiologic metabolic pathway formed by the products of CSRG1 can really endow the tobacco plants with the high salt-tolerant ability, not only to Na^ stress, but also to the comprehensive stress of various ions.     

丙型肝炎病毒E2基因在转基因番茄植株中的表达

以T-E1E2为模板扩增得到丙型肝炎病毒包膜蛋白基因E2,构建该基因的植物表达载体p35s-E2．通过农杆菌介导的叶盘法转化番茄子叶．转基因番茄植株叶片总DNA的PCR、Southern blot检测结果表明,E2基因已整合进了转基因番茄植株基因组中;RT-PCR,Western blot分析证实E2基因在转基因番茄植株叶片中表达．     

Function of resveratrol derived from transgenic plant expressing resveratrol synthase gene

Two genes from grapevine coding for resveratrol synthase, named RS1 and RS2, were cloned by RT-PCR. AnEscherichia coli expression vector was constructed by insertion of RS1 into pBV221. A specific protein with the same molecular weight (42 ku) as the resveratrol synthase was expressed and used to prepare the rabbit antiserum. A plant expression vector was constructed by inserting the RS1 gene into pBin438 downstream of the doubled CaMV 35S promoter and TMV-Ω fragment. PCR-positive transgenic tobacco plants were obtained after transformation withAgrobacterium tumefaciens LBA4404 harboring the plant expression vector. Southern blot analysis demonstrated that the foreign gene was integrated into the tobacco genome. The results of RT-PCR and Western blot indicated that the RS1 gene was transcribed and expressed. Formation of resveratrol in transgenic tobacco was further determined by thin-layer chromatography of silica gel and HPLC. Increased accumulation of human breast adenocarcinoma cells in G₀ and G₁ phases of cell cycle was observed in cells treated with resveratrol purified from transgenic tobacco as compared to the untreated cells.     

Overexpression of phospholipase <Emphasis Type="Italic">D</Emphasis>α gene enhances drought and salt tolerance of <Emphasis Type="Italic">Populus tomentosa</Emphasis>

The cDNA of AtPLDa （Arabidopsis thaliana Phospholipase Da） gene was introduced into P. tomentosa （Populus tomentosa） under the control of the Cauliflower mosaic virus 35S promoter. Southern and Northern blot analyses suggested that the AtPLDa gene has been transferred into the P. tomentosa genome. No obvious morphological or developmental difference was observed between the transgenic and wild-type （WT） plants. Drought and salt tolerance and gene expression of seedlings of several transgenic lines and WT plants （control） were studied. The results showed that the rhizogenesis rate and the average root-length of transgenic lines were significantly higher than WT plants after mannitol and NaCI treatment under the same growth conditions. Northern blot analysis indicated that the higher the PLDa expression in the transgenic plants, the more tolerant the transgenic plants are to drought and salt treatment. Meanwhile, another group of these transgenic lines and WT plants （control） were treated with PEG6000 and NaCI separately. The contents of chlorophylls and the activities of some anti- oxidant enzymes （superoxide dismutase, guaiacol peroxidase and catalase） as well as malondialdehyde and relative electrical conductivity were analyzed. Altogether, our results demonstrated that overexpression of the PLDa gene can enhance the drought and salt tolerance in transgenic P. tomentosa plants.