植物抗菌蛋白GmPGIP3和BvGLP1转基因小麦的分子检测和抗病性鉴定
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摘要
GmPGIP3是大豆多聚半乳糖醛酸酶抑制蛋白3,能够抑制一些真菌内切多聚半乳糖醛酸活性,从而减弱真菌对植株的侵害。BvGLP1是甜菜中的一类萌发素蛋白,萌发素催化草酸氧化产生过氧化氢,过氧化氢能够诱导与抗性有关的基因表达,从而使植物产生相关抗性。本研究利用基因工程技术构建了GmPGIP3和BvGLP1基因的单子叶植物表达载体pA25-GmPGIP3和pA20-BvGLP1,通过基因枪介导法将其转入推广的小麦品种扬麦18和扬麦19中,成功的获得了转基因小麦植株。经过多代的鉴定和选育,最终挑选出了抗小麦纹枯病、赤霉病和根腐病的小麦新种质,主要研究工作如下:
1.抗小麦纹枯病扬麦19种质的获得。对转GmPGIP3基因扬麦19的T0至T4代植株,进行PCR、Southern blot、半定量RT-PCR、荧光定量Q-RT-PCR分析,人工接种致病菌禾谷丝核菌后进行抗性鉴定。分子检测结果表明,GmPGIP3已转入扬麦19,已验证在部分转基因小麦不同株系中以一至二个拷贝存在,并在转录水平上表达;不同转基因株系的抗病性对比受体材料有明显提高而且可以稳定遗传。同时调查主要农艺性状与受体材料没有明显差异,说明外源基因对小麦正常生长基本没有影响。
2.抗小麦赤霉病扬麦19种质的获得。对转GmPGIP3基因扬麦19的T0至T4代植株,进行上述分子检测,在江苏省农业科学院生物技术研究所网室内,采用人工接种致病菌禾谷镰刀菌后进行抗性鉴定。分子检测结果表明,GmPGIP3已转入扬麦19,已验证在部分转基因小麦不同株系中以一至二个拷贝存在,并在转录水平上表达;不同转基因株系的抗病性对比受体材料有明显提高而且可以稳定遗传。
3.抗小麦根腐病扬麦18种质的获得。对转GmPGIP3和BvGLP-1基因扬麦18的T0至T2代植株进行上述分子检测,人工接种混合致病菌后进行抗性鉴定。分子检测结果表明,GmPGIP3和BvGLP-1已分别转入不同株系的扬麦18。已验证在部分转基因小麦不同株系中以一至二个拷贝存在,并在转录水平上表达;不同转基因株系的抗病性对比受体材料有明显提高而且可以稳定遗传。同时调查主要农艺性状与受体材料没有明显差异,说明外源基因对小麦正常生长基本没有影响。
GmPGIP3 is soybean polygalacturonase-inhibiting protein 3, could inhibition of fungal endo-polygalacturonase acid activity, which weakened pathogen fungi to plant Infection. BvGLP1 is a kind of sugar beet Germin-like protein(GLP), GLP catalytic oxalic acid oxidation to produce hydrogen peroxide, hydrogen peroxide can induce resistance gene expression so that the plants associated with resistance. In this study, the use of genetic engineering technology to build the GmPGIP3 and BvGLP1 genes monocots expression vector pA25-GmPGIP3 and pA20-BvGLP1, by gene gun-mediated and related molecular detection techniques that GmPGIP3 and BvGLP1 into wheat varieties have been promoted Yangmai 18 and Yangmai 19. After several generations of identification and selection, finally picked Enhanced-resistance to Sharp Eyespot, Fusarium Head Blight and wheat root rot new species, the main research work are as follows:
1. Access of enhanced-resistance Yangmai 19 to Sharp Eyespot. We characterized the GmPGIP3 transgenic wheat plants in T0 to T4 generations by PCR, Southern blot, RT-PCR, and Q-RT-PCR analyses. We also evaluated the disease resistance in these GmPGIP3 transgenic plants through inoculating R. cerealis. The results show that, GmPGIP3 in different transgenic wheatlines with one to two copies, and the alien gene GmPGIP3 was over-expressed in seven transgenic wheat lines; different transgenic lines disease resistance contrast with Yangmai 19 showed significantly-enhanced resistance to R. cerealis.The ability of disease resistance can be stably inherited, the main agronomic index with no significant differences with receptor material Yangmai 19.
2. Access of enhanced-resistance Yangmai 19 to Fusarium Head Blight. We characterized the GmPGIP3 transgenic wheat plants in T0 to T4 generations by PCR, Southern blot, RT-PCR, and Q-RT-PCR analyses. We also evaluated the disease resistance in these GmPGIP3 transgenic plants through inoculating F. graminearum. The results show that, GmPGIP3 in different transgenic wheatlines with one to two copies, and the alien gene GmPGIP3 was over-expressed in seven transgenic wheat lines; different transgenic lines disease resistance contrast with Yangmai 19 showed significantly-enhanced resistance to F. graminearum.The ability of disease resistance can be stably inherited.
3. Access of enhanced-resistance Yangmai 18 to wheat root rot. We characterized the GmPGIP3 and BvGLP1 transgenic wheat plants in T0 to T2 generations by PCR, RT-PCR, and Q-RT-PCR analyses. We also evaluated the disease resistance in these GmPGIP3 and BvGLP1 transgenic plants through inoculating Bipolaris sorokiniana. The results show that, the alien gene GmPGIP3 and BvGLP1 was over-expressed in seven transgenic wheat lines; different transgenic lines disease resistance contrast with Yangmai 18 showed significantly-enhanced resistance to Bipolaris sorokiniana.The ability of disease resistance can be stably inherited, the main agronomic index with no significant differences with receptor material Yangmai 18.
1.抗小麦纹枯病扬麦19种质的获得。对转GmPGIP3基因扬麦19的T0至T4代植株,进行PCR、Southern blot、半定量RT-PCR、荧光定量Q-RT-PCR分析,人工接种致病菌禾谷丝核菌后进行抗性鉴定。分子检测结果表明,GmPGIP3已转入扬麦19,已验证在部分转基因小麦不同株系中以一至二个拷贝存在,并在转录水平上表达;不同转基因株系的抗病性对比受体材料有明显提高而且可以稳定遗传。同时调查主要农艺性状与受体材料没有明显差异,说明外源基因对小麦正常生长基本没有影响。
2.抗小麦赤霉病扬麦19种质的获得。对转GmPGIP3基因扬麦19的T0至T4代植株,进行上述分子检测,在江苏省农业科学院生物技术研究所网室内,采用人工接种致病菌禾谷镰刀菌后进行抗性鉴定。分子检测结果表明,GmPGIP3已转入扬麦19,已验证在部分转基因小麦不同株系中以一至二个拷贝存在,并在转录水平上表达;不同转基因株系的抗病性对比受体材料有明显提高而且可以稳定遗传。
3.抗小麦根腐病扬麦18种质的获得。对转GmPGIP3和BvGLP-1基因扬麦18的T0至T2代植株进行上述分子检测,人工接种混合致病菌后进行抗性鉴定。分子检测结果表明,GmPGIP3和BvGLP-1已分别转入不同株系的扬麦18。已验证在部分转基因小麦不同株系中以一至二个拷贝存在,并在转录水平上表达;不同转基因株系的抗病性对比受体材料有明显提高而且可以稳定遗传。同时调查主要农艺性状与受体材料没有明显差异,说明外源基因对小麦正常生长基本没有影响。
GmPGIP3 is soybean polygalacturonase-inhibiting protein 3, could inhibition of fungal endo-polygalacturonase acid activity, which weakened pathogen fungi to plant Infection. BvGLP1 is a kind of sugar beet Germin-like protein(GLP), GLP catalytic oxalic acid oxidation to produce hydrogen peroxide, hydrogen peroxide can induce resistance gene expression so that the plants associated with resistance. In this study, the use of genetic engineering technology to build the GmPGIP3 and BvGLP1 genes monocots expression vector pA25-GmPGIP3 and pA20-BvGLP1, by gene gun-mediated and related molecular detection techniques that GmPGIP3 and BvGLP1 into wheat varieties have been promoted Yangmai 18 and Yangmai 19. After several generations of identification and selection, finally picked Enhanced-resistance to Sharp Eyespot, Fusarium Head Blight and wheat root rot new species, the main research work are as follows:
1. Access of enhanced-resistance Yangmai 19 to Sharp Eyespot. We characterized the GmPGIP3 transgenic wheat plants in T0 to T4 generations by PCR, Southern blot, RT-PCR, and Q-RT-PCR analyses. We also evaluated the disease resistance in these GmPGIP3 transgenic plants through inoculating R. cerealis. The results show that, GmPGIP3 in different transgenic wheatlines with one to two copies, and the alien gene GmPGIP3 was over-expressed in seven transgenic wheat lines; different transgenic lines disease resistance contrast with Yangmai 19 showed significantly-enhanced resistance to R. cerealis.The ability of disease resistance can be stably inherited, the main agronomic index with no significant differences with receptor material Yangmai 19.
2. Access of enhanced-resistance Yangmai 19 to Fusarium Head Blight. We characterized the GmPGIP3 transgenic wheat plants in T0 to T4 generations by PCR, Southern blot, RT-PCR, and Q-RT-PCR analyses. We also evaluated the disease resistance in these GmPGIP3 transgenic plants through inoculating F. graminearum. The results show that, GmPGIP3 in different transgenic wheatlines with one to two copies, and the alien gene GmPGIP3 was over-expressed in seven transgenic wheat lines; different transgenic lines disease resistance contrast with Yangmai 19 showed significantly-enhanced resistance to F. graminearum.The ability of disease resistance can be stably inherited.
3. Access of enhanced-resistance Yangmai 18 to wheat root rot. We characterized the GmPGIP3 and BvGLP1 transgenic wheat plants in T0 to T2 generations by PCR, RT-PCR, and Q-RT-PCR analyses. We also evaluated the disease resistance in these GmPGIP3 and BvGLP1 transgenic plants through inoculating Bipolaris sorokiniana. The results show that, the alien gene GmPGIP3 and BvGLP1 was over-expressed in seven transgenic wheat lines; different transgenic lines disease resistance contrast with Yangmai 18 showed significantly-enhanced resistance to Bipolaris sorokiniana.The ability of disease resistance can be stably inherited, the main agronomic index with no significant differences with receptor material Yangmai 18.
引文
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