烟草安全抗青枯病转基因研究
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摘要
我国烟叶生产每年因病虫害造成的损失近10亿元。烟草青枯病是烟草的重要病害,迄今尚无特效药可治。常规育种受抗源与不良基因连锁和育种年限长等影响,进展慢。随着功能基因组学和生物技术研究的深入研究,转基因烟草将成为选育烟草抗病虫害的有效手段。同时,在转基因安全性得到了普遍的关注后,寻找一条安全有效的转基因育种手段成为当前生物技术育种的热门。本研究在已得到烟草根特异基因和启动子序列,以及受细菌诱导启动子的基础上,构建了特异启动子和诱导启动子驱动抗病基因的表达载体,转入烟草进行功能验证;并对实验室所构建的抗生素自我删除载体进行了验证,结果表明转基因烟草是可能通过基因工程手段来解决其安全性的担忧的。
1.根据本实验已获得烟草根特异表达基因NtR12片段,通过合成下游3' RACE引物,扩增得到3’目的片段,完整了NtR12基因的全长序列;同时对已经得到的NtR12 5’上游的启动子区域进行了生物信息学分析,构建了从启始密码子ATG之前带不同长度启动子片段的表达载体,用于进一步鉴定启动子的结构和功能。
2.利用农杆菌叶盘法,将本实验室已构建的抗生素自我删除载体p35S-loxp-Gus转化烟草翠碧一号,在不定芽分化前后,用定量的地塞米松重诱导,培养出转基因烟草。通过对转基因烟草植株进行GUS染色,证实载体在一定浓度的地塞米松诱导下,重组酶Cre-GRB能正确识别loxP位点,删除潮霉素标记基因和重组酶基因表达元件,并使重组DNA,使35S启动子能驱动GUS基因表达,产生蓝色反应。因此该载体可用于将潮霉素基因删除,获得无标记基因的转基因烟草植株。
3.同源克隆苦瓜种子的核糖体失活蛋白基因,将已克隆的烟草根特异启动子与核糖体失活蛋白基因融合,构建植物表达载体pSC-NtR12-RIP,转化烟草翠碧一号,对转基因植株进行分子检测和青枯菌接种鉴定,结果表明转基因烟草抗烟草青枯病能力显著提高,以至表达高抗至免疫的水平,并且该基因只在烟草的根和茎部特异表达,不在烟草叶片表达,为解决转基因安全性问题奠定了基础。
4.将克隆得到的烟草几丁质酶基因,与受细胞诱导的诱导型启动子结合,构建了植物表达载体pSC-PP1-Chi,通过农杆菌叶盘法侵染烟草翠碧一号,获得了100多株转基因植株,对转基因后代进行PCR检测鉴定得到了上百株阳性转基因植株,对转基因阳性烟草植株进行青枯菌接种鉴定,结果表明在青枯菌诱导下,转基因烟草对青枯病不同程度的提高,至表达高抗水平。
Chinese tobacco production losses caused by pests and diseases reaches nearly 10 million every year. Bacterial wilt is an importance disease in tobacco, and so far, there is no specific drugs to cure it. As germplasm with high resistance genes often linked with the bad characters and the long period of breeding, progress for breeding high resistance varieties is slow by conventional method. With functional genomics and biotechnology devoping so swiftly, the breeding of transgenic tobacco will become an effective means of desease-resistant tobacco. Meanwhile, as transgene security become widespread concerns, finding a safe and effective means of transgenic breeding with biotechnology become popular. Based on available root-specific genes and its promoter sequences, and promoter specifically induced by bacteria, expression vectors harboring disease resistance gene driven by specific promoters were constructed and were transformed into tobacco for functional verification. Construction of laboratory self-deleted of antibiotics gene was verified. The results are showed as follows.
1. According to tobacco root-specific genes NtR12 fragment, downstream fragment was got through 3' RACE primers, amplified 3' fragments of the NtR12 completed the full length of NtR12 gene. At the same time, the available NtR12 5 'upstream promoter has been analyzed by bioinformatics method, and series of expression vectors were constructed with various lengths of promoter sequences starting before ATG initiation codon, which are being used to identify the functional elements of the promoter.
2.An antibiotics self-deleted vector, p35S-loxp-Gus, built in this laboratory before has been transformed into transgenic tobacco CuiBiyihao, mediated via Agrobacterium tumefaciens, and explants in bud differentiation stage were induced to remove antibiotics genes by dexamethasone and produce buds and grow into transgenic tobacco without antibiotics gene. Transgenic tobacco plants tested by GUS staining confirmed that under certain concentration of dexamethasone in the induction, the recombinant enzyme Cre-GRB can correctly identify the loxP sites and delete the expression units of both hygromycin marker gene and recombinant gene, and then recombine DNA so that 35S promoter can drive expression of GUS gene to produce a blue reaction. So the expression vector can be used to remove the hygromycin gene, producing unmarked transgenic tobacco plants.
3. Ribosome inactivating protein gene of balsam pear was homologous cloned, and the function of the genes was analysed. The vector was constructed by binding root specific promoter with ribosome inactivating protein gene,which was transformed to tabacco CuiBiyihao. Then the transgenic tabacco was molecular detected and identified by inoculation with Rostonia solanacearum. The result showed that transgenic tabacco increase their resistance significantly to bacterial wilt,some plants showed immune rection to bacterial wilt. It was characterized that RIP expressed specifically in root and stem, not in leaves,, which provided basic explanation for safety of transgenics.
4.Employing tobacco chitinase gene in associated with bacterium-induced promoter available in the lab, a plant expression vector Psc-PP1-Chi, was constructed and transformed into CuiBiyihao, mediated by Agrobacterium infection. More than 100 transgenic plants were received, and transgenic offspring were identified by PCR assay and nearly 100 positive transgenic plants were undergone R. solanacearum inoculation tests. And the results indicate that Ralstonia solanacearum infection could induce Chi gene expression that the resistance to bacterial wilt in transgenic plants were all seen to increase by different degrees, some showed high resistance.
1.根据本实验已获得烟草根特异表达基因NtR12片段,通过合成下游3' RACE引物,扩增得到3’目的片段,完整了NtR12基因的全长序列;同时对已经得到的NtR12 5’上游的启动子区域进行了生物信息学分析,构建了从启始密码子ATG之前带不同长度启动子片段的表达载体,用于进一步鉴定启动子的结构和功能。
2.利用农杆菌叶盘法,将本实验室已构建的抗生素自我删除载体p35S-loxp-Gus转化烟草翠碧一号,在不定芽分化前后,用定量的地塞米松重诱导,培养出转基因烟草。通过对转基因烟草植株进行GUS染色,证实载体在一定浓度的地塞米松诱导下,重组酶Cre-GRB能正确识别loxP位点,删除潮霉素标记基因和重组酶基因表达元件,并使重组DNA,使35S启动子能驱动GUS基因表达,产生蓝色反应。因此该载体可用于将潮霉素基因删除,获得无标记基因的转基因烟草植株。
3.同源克隆苦瓜种子的核糖体失活蛋白基因,将已克隆的烟草根特异启动子与核糖体失活蛋白基因融合,构建植物表达载体pSC-NtR12-RIP,转化烟草翠碧一号,对转基因植株进行分子检测和青枯菌接种鉴定,结果表明转基因烟草抗烟草青枯病能力显著提高,以至表达高抗至免疫的水平,并且该基因只在烟草的根和茎部特异表达,不在烟草叶片表达,为解决转基因安全性问题奠定了基础。
4.将克隆得到的烟草几丁质酶基因,与受细胞诱导的诱导型启动子结合,构建了植物表达载体pSC-PP1-Chi,通过农杆菌叶盘法侵染烟草翠碧一号,获得了100多株转基因植株,对转基因后代进行PCR检测鉴定得到了上百株阳性转基因植株,对转基因阳性烟草植株进行青枯菌接种鉴定,结果表明在青枯菌诱导下,转基因烟草对青枯病不同程度的提高,至表达高抗水平。
Chinese tobacco production losses caused by pests and diseases reaches nearly 10 million every year. Bacterial wilt is an importance disease in tobacco, and so far, there is no specific drugs to cure it. As germplasm with high resistance genes often linked with the bad characters and the long period of breeding, progress for breeding high resistance varieties is slow by conventional method. With functional genomics and biotechnology devoping so swiftly, the breeding of transgenic tobacco will become an effective means of desease-resistant tobacco. Meanwhile, as transgene security become widespread concerns, finding a safe and effective means of transgenic breeding with biotechnology become popular. Based on available root-specific genes and its promoter sequences, and promoter specifically induced by bacteria, expression vectors harboring disease resistance gene driven by specific promoters were constructed and were transformed into tobacco for functional verification. Construction of laboratory self-deleted of antibiotics gene was verified. The results are showed as follows.
1. According to tobacco root-specific genes NtR12 fragment, downstream fragment was got through 3' RACE primers, amplified 3' fragments of the NtR12 completed the full length of NtR12 gene. At the same time, the available NtR12 5 'upstream promoter has been analyzed by bioinformatics method, and series of expression vectors were constructed with various lengths of promoter sequences starting before ATG initiation codon, which are being used to identify the functional elements of the promoter.
2.An antibiotics self-deleted vector, p35S-loxp-Gus, built in this laboratory before has been transformed into transgenic tobacco CuiBiyihao, mediated via Agrobacterium tumefaciens, and explants in bud differentiation stage were induced to remove antibiotics genes by dexamethasone and produce buds and grow into transgenic tobacco without antibiotics gene. Transgenic tobacco plants tested by GUS staining confirmed that under certain concentration of dexamethasone in the induction, the recombinant enzyme Cre-GRB can correctly identify the loxP sites and delete the expression units of both hygromycin marker gene and recombinant gene, and then recombine DNA so that 35S promoter can drive expression of GUS gene to produce a blue reaction. So the expression vector can be used to remove the hygromycin gene, producing unmarked transgenic tobacco plants.
3. Ribosome inactivating protein gene of balsam pear was homologous cloned, and the function of the genes was analysed. The vector was constructed by binding root specific promoter with ribosome inactivating protein gene,which was transformed to tabacco CuiBiyihao. Then the transgenic tabacco was molecular detected and identified by inoculation with Rostonia solanacearum. The result showed that transgenic tabacco increase their resistance significantly to bacterial wilt,some plants showed immune rection to bacterial wilt. It was characterized that RIP expressed specifically in root and stem, not in leaves,, which provided basic explanation for safety of transgenics.
4.Employing tobacco chitinase gene in associated with bacterium-induced promoter available in the lab, a plant expression vector Psc-PP1-Chi, was constructed and transformed into CuiBiyihao, mediated by Agrobacterium infection. More than 100 transgenic plants were received, and transgenic offspring were identified by PCR assay and nearly 100 positive transgenic plants were undergone R. solanacearum inoculation tests. And the results indicate that Ralstonia solanacearum infection could induce Chi gene expression that the resistance to bacterial wilt in transgenic plants were all seen to increase by different degrees, some showed high resistance.
引文
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