农杆菌介导遗传转化获得转β-甘露糖苷酶正义、反义基因的棉花
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摘要
棉纤维是纺织工业的重要原料，在国民经济中占有重要地位。因此，从分子水平阐明棉纤维发育的机理，具有重要的理论价值和现实意义。
为了研究β-甘露糖苷酶(GhManA2)在棉纤维发育中的确切功能，本研究将其基因序列以正向，反向构建到由纤维特异启动子E6驱动的植物表达载体pBI121中，通过农杆菌介导法将其转入棉花，试图通过该基因在纤维发育中的过量表达和抑制表达对棉纤维发育的影响来研究其在纤维发育中的确切功能。本研究包括以下2个方面：
利用DNA重组技术将基因GhManA2序列以正向，反向构建到由纤维特异启动子E6驱动的植物表达载体pBI121-E6中，经过SmaI，NotI酶切鉴定和特异引物的PCR扩增及测序证明载体构建成功，可用于棉花的遗传转化。分别以泗棉3号和W0品系为基因转化受体，将正义、反义GhManA2载体利用农杆菌介导法转化棉花，共得到53个愈伤系的500株转化的再生植株。
用NPTⅡ和启动子-目的基因引物对再生植株DNA进行PCR扩增，初步得到T_0代转化的阳性植株230株。正义载体的再生植株中，NPTⅡ基因引物阳性率为97.92％，启动子-目的基因引物的阳性率为87.23％，反义载体的再生植株中，NPTⅡ基因引物阳性率为95.19％，启动子-目的基因引物的阳性率为64.65％。只有用NPTⅡ基因引物和启动子-目的基因引物扩增结果都为阳性的植株才被初步认为是阳性植株。本实验中，一共嫁接了230株阳性植株，成活了196株。随机取T_0代8株PCR阳性再生植株做Southern杂交。结果证明NPTⅡ基因已经整合到植物基因组中。进一步以E6启动子为探针进行Southern杂交，有部分植株有特异的杂交信号说明T-DNA区的E6启动子已整合到棉花基因组中。对T_1、T_2代转基因植株提取叶片DNA，用启动子-基因特异引物对其进行PCR扩增，在检测的7个T_1转基因株系中，目的基因的分离比例都符合孟德尔一对基因的分离比例(3:1)，结合Southern杂交结果，推测多拷贝插入的目的基因可能整合到了同一条染色体上。
Cotton fiber is the most important textile material, plays an important role in ournational economy. So, it is important to elucidate fiber development process by molecularbiology.
To characterize the precise function ofβ-mannosidase in cotton fiber, in this study,the sense and antisense coding sequence of theβ-mannosidase gene were fused withfiber-specific E6 promoter to build the constructs of sense gene and antisense RNA gene,and with which to transform into cotton via Agrobacterium-mediated transformation, and tomake them over expression, or suppression the expression of the endogenesis gene in thecotton genome in the purpose of studying the real function of this gene in fiberdevelopment.
By DNA recombination technology, the sense and antisense coding sequence of theβ-mannosidase gene droved by fiber-specific E6 promoter were constructed using the binaryvector pBI121 as backbone vector. PCR with the special primer and sequencing of the PCRproduct and digestions with SmaI and NotI proved the two vectors were constructedsuccessfully and can be used for transformation. By Agrobacterium mediatedtransformation, the above-mentioned twoβ-marmosidase constructs were successfullytransferred to the cotton cultivars, Simian3 and W0, Around 500 T_0 regeneration plantsfrom 53 different calli lines were obtained.
PCR amplification and Southern blot analysis of the transgenic cotton plants wereused to confirm the integration of the transgenes. The primers were designed according tothe sequences of NPTⅡand E6-β-mannosidase gene and applied to PCR analyses of T_0transgenic plants. For the sense vector, the percentage of the plants with positive signals inNPTⅡprimer reaction is 97.92%, that of the plants in E6-β-mannosidase primer reactionis 87.23%; and for the antisense vector, the percentage of the plants with positive signals inNPTⅡprimer reaction is 95.19%, that of the plants in E6-β-marmosidase primer reactionis 64.65%. The plants showing both positive signals of NPTⅡand E6-β-mannosidaseprimers were regarded as positive transgenic plants primarily and were regenerated by grafting. In this research, 230 positive plants were grafted and 196 of them were survived. 8To plants which were regarded as positive transgenic plants primarily by PCR reaction werefurther analyzed with Southern blotting. Using NPTⅡgene as the probe, the results showedthat the NPTⅡgene had been inserted to the cotton genome, using E6 promoter as theprobe, the results showed that the E6 promoter had been integrated into the cotton genomein some T_0 plants. PCR analysis of T_1 and T_2 transgenic plants using the E6-β-mannosidase gene special primers showed that the segregation ratio of ManA2 gene inseven transgenic lines were inherited in a classical Mendelian manner (3:1). But Southernblot showed that there was more than one copy of ManA2 gene in some transgenic lines,which perhaps was because of mutiple-copy integration in one site of the cottonchromosome.
为了研究β-甘露糖苷酶(GhManA2)在棉纤维发育中的确切功能，本研究将其基因序列以正向，反向构建到由纤维特异启动子E6驱动的植物表达载体pBI121中，通过农杆菌介导法将其转入棉花，试图通过该基因在纤维发育中的过量表达和抑制表达对棉纤维发育的影响来研究其在纤维发育中的确切功能。本研究包括以下2个方面：
利用DNA重组技术将基因GhManA2序列以正向，反向构建到由纤维特异启动子E6驱动的植物表达载体pBI121-E6中，经过SmaI，NotI酶切鉴定和特异引物的PCR扩增及测序证明载体构建成功，可用于棉花的遗传转化。分别以泗棉3号和W0品系为基因转化受体，将正义、反义GhManA2载体利用农杆菌介导法转化棉花，共得到53个愈伤系的500株转化的再生植株。
用NPTⅡ和启动子-目的基因引物对再生植株DNA进行PCR扩增，初步得到T_0代转化的阳性植株230株。正义载体的再生植株中，NPTⅡ基因引物阳性率为97.92％，启动子-目的基因引物的阳性率为87.23％，反义载体的再生植株中，NPTⅡ基因引物阳性率为95.19％，启动子-目的基因引物的阳性率为64.65％。只有用NPTⅡ基因引物和启动子-目的基因引物扩增结果都为阳性的植株才被初步认为是阳性植株。本实验中，一共嫁接了230株阳性植株，成活了196株。随机取T_0代8株PCR阳性再生植株做Southern杂交。结果证明NPTⅡ基因已经整合到植物基因组中。进一步以E6启动子为探针进行Southern杂交，有部分植株有特异的杂交信号说明T-DNA区的E6启动子已整合到棉花基因组中。对T_1、T_2代转基因植株提取叶片DNA，用启动子-基因特异引物对其进行PCR扩增，在检测的7个T_1转基因株系中，目的基因的分离比例都符合孟德尔一对基因的分离比例(3:1)，结合Southern杂交结果，推测多拷贝插入的目的基因可能整合到了同一条染色体上。
Cotton fiber is the most important textile material, plays an important role in ournational economy. So, it is important to elucidate fiber development process by molecularbiology.
To characterize the precise function ofβ-mannosidase in cotton fiber, in this study,the sense and antisense coding sequence of theβ-mannosidase gene were fused withfiber-specific E6 promoter to build the constructs of sense gene and antisense RNA gene,and with which to transform into cotton via Agrobacterium-mediated transformation, and tomake them over expression, or suppression the expression of the endogenesis gene in thecotton genome in the purpose of studying the real function of this gene in fiberdevelopment.
By DNA recombination technology, the sense and antisense coding sequence of theβ-mannosidase gene droved by fiber-specific E6 promoter were constructed using the binaryvector pBI121 as backbone vector. PCR with the special primer and sequencing of the PCRproduct and digestions with SmaI and NotI proved the two vectors were constructedsuccessfully and can be used for transformation. By Agrobacterium mediatedtransformation, the above-mentioned twoβ-marmosidase constructs were successfullytransferred to the cotton cultivars, Simian3 and W0, Around 500 T_0 regeneration plantsfrom 53 different calli lines were obtained.
PCR amplification and Southern blot analysis of the transgenic cotton plants wereused to confirm the integration of the transgenes. The primers were designed according tothe sequences of NPTⅡand E6-β-mannosidase gene and applied to PCR analyses of T_0transgenic plants. For the sense vector, the percentage of the plants with positive signals inNPTⅡprimer reaction is 97.92%, that of the plants in E6-β-mannosidase primer reactionis 87.23%; and for the antisense vector, the percentage of the plants with positive signals inNPTⅡprimer reaction is 95.19%, that of the plants in E6-β-marmosidase primer reactionis 64.65%. The plants showing both positive signals of NPTⅡand E6-β-mannosidaseprimers were regarded as positive transgenic plants primarily and were regenerated by grafting. In this research, 230 positive plants were grafted and 196 of them were survived. 8To plants which were regarded as positive transgenic plants primarily by PCR reaction werefurther analyzed with Southern blotting. Using NPTⅡgene as the probe, the results showedthat the NPTⅡgene had been inserted to the cotton genome, using E6 promoter as theprobe, the results showed that the E6 promoter had been integrated into the cotton genomein some T_0 plants. PCR analysis of T_1 and T_2 transgenic plants using the E6-β-mannosidase gene special primers showed that the segregation ratio of ManA2 gene inseven transgenic lines were inherited in a classical Mendelian manner (3:1). But Southernblot showed that there was more than one copy of ManA2 gene in some transgenic lines,which perhaps was because of mutiple-copy integration in one site of the cottonchromosome.
引文
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