棉花叶绿体Cu/Zn-SOD基因植物表达载体的构建及转化
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摘要
短季棉的早熟早衰问题严重影响棉花的产量和品质；目前短季棉早熟早衰问题在生理生化水平已经作了比较详细的研究，已有的结果显示：早熟不早衰品种和早熟早衰品种相比，早熟不早衰品种的抗氧化酶活性后期表现高、稳定，在抗氧化酶系统中主要起作用的关键酶是SOD，因此，进一步从分子水平揭示早熟不早衰的分子机制，克隆相关基因并进行遗传转化研究是非常必要的，对于提高短季棉产量、品质具有重要的意义。
(1)棉花叶绿体Cu-ZnSOD基因植物表达载体的构建：以质粒PBI121作为植物遗传转化的载体，对所克隆的棉花叶绿体Cu-ZnSOD基因和PBI121作Bam HI／SacI双酶切，电泳回收目的基因片段和载体片段，再用连接酶将二者连接起来，并转化到农杆菌LBA4404感受态细胞中，即成为棉花叶绿体Cu-ZnSOD基因植物表达载体PBI-YSOD。对所构建的植物表达载体PBI-YSOD进行相关的质粒提取检测和PCR检测，结果显示：植物表达载体PCR结果和目的基因大小相同，转化质粒和空质粒相比，略有增大，表明目的基因片段已经转化到了农杆菌中，可以对植物进行遗传转化。
(2)烟草遗传转化与转化植株的检测：利用农杆菌介导法将所构建的植物表达载体导入NC89烟草，卡那霉素筛选得到23株阳性植株，经PCR、Southern blotting检测结果显示有四个烟草株系中整合了棉花叶绿体Cu／Zn-SOD基因，SOD酶活性测定结果显示四株转基因烟草植株SOD酶活性都明显高于非转基因烟草，离体叶片暗处理试验结果也证明四株转基因烟草比非转基因烟草SOD酶活下降速度明显减慢，百草枯喷施试验表明，转基因烟草都比非转基因烟草对百草枯的耐性增强，这一系列试验间接地说明外源基因的导入增强了烟草的耐衰老能力。
(3)棉花的遗传转化：以中棉所24号和早熟早衰材料652583为材料，利用农杆菌介导法将所构建的植物表达载体导入中棉所24号和早熟早衰材料652583，经过愈伤化，胚性愈伤，抗性芽三个阶段的卡那霉素筛选，目前得到9株中棉所24号卡那霉素抗性植株，早熟早衰材料652583抗性芽还在进一步的筛选中。
The problem of earliness with decrepitude has worse effect on the yield in theshort-season cotton in our country. Up to now, about the problem of earliness withdecrepitude in short-season cotton, careful research has been done on the physiologicallevel, the results of the formers: comparing the varieties of earliness with not-decrepitudewith the varieties of earliness with decrepitude, the antioxidant enzyme activity ofearliness with not-decrepitude are higher and steadier in the growth retrial period, the keyenzymes in system of antioxidant enzyme are SOD. If we want to understand themolecular mechanism about earliness with decrepitude, it is very important of cloningthe correlative genes and studying its function. These genes can help us to improve theyield and quality.
(1) plant binary vector construction of cotton chloroplast Cu-ZnSOD gene: PlasmidpBI 121 was used as the genetic transformation carrier. The cotton chloroplastCu-ZnSOD genes and pBI 121 was digested by BamHI/SacI, then extracted the genefragments and vector fragment by electrophoresis, linked up the two fragments andtransformed it into Agrobacterium tumefaciens LBA4404 competent cells, which is plantexpression vector, pBI-YSOD. The result of extraction plasmid of pBI-YSOD and PCRshowed that fragment of plant expression vector the same to the target gene, plasmidpBI-YSOD is bigger than plasmid pBI121, which showed that cotton chloroplastCu-ZnSOD gene fragments has be transferred into the Agrobacterium tumefaciens, andcan be used as carriers for genetic transformation.
(2) Tobacco Genetic transformation and detection of transgenic plant: Cotton chloroplastCu-ZnSOD gene plant expression vector was constructed and introduced into tobacco NC89 viaAgrobacterium tumefaciens. There 23 plant display positive reaction in kanamycin selection. The resultshowed that 4 plantlets display positive reaction in PCR analysis、Southern blotting and SOD enzymeactivity detection, also indicated that SOD enzyme activity in aparted cotyledon of 4 transgenic plantsis higher than of non transgenic plant, and the sensitivity to paraquat in non transgenic plant is higherthan in 4 transgenic plants. Totally, the integration of foreign gene increased tobacco resisted- caducityactivity.
(3) Cotton genetic transformation: CCRI 24 and breeding material 652583 were used as materials for Agrobacterium infection, Cotton chloroplast Cu-ZnSOD gene plantexpression vector was constructed and introduced into CCRI 24 and breeding material 652583via Agrobacterium tumefaciens. There are 9 plantlet CCRI 24 display positive reaction inkanamycin selection, but resist seeding of breeding material 652583 is in selection still.
(1)棉花叶绿体Cu-ZnSOD基因植物表达载体的构建：以质粒PBI121作为植物遗传转化的载体，对所克隆的棉花叶绿体Cu-ZnSOD基因和PBI121作Bam HI／SacI双酶切，电泳回收目的基因片段和载体片段，再用连接酶将二者连接起来，并转化到农杆菌LBA4404感受态细胞中，即成为棉花叶绿体Cu-ZnSOD基因植物表达载体PBI-YSOD。对所构建的植物表达载体PBI-YSOD进行相关的质粒提取检测和PCR检测，结果显示：植物表达载体PCR结果和目的基因大小相同，转化质粒和空质粒相比，略有增大，表明目的基因片段已经转化到了农杆菌中，可以对植物进行遗传转化。
(2)烟草遗传转化与转化植株的检测：利用农杆菌介导法将所构建的植物表达载体导入NC89烟草，卡那霉素筛选得到23株阳性植株，经PCR、Southern blotting检测结果显示有四个烟草株系中整合了棉花叶绿体Cu／Zn-SOD基因，SOD酶活性测定结果显示四株转基因烟草植株SOD酶活性都明显高于非转基因烟草，离体叶片暗处理试验结果也证明四株转基因烟草比非转基因烟草SOD酶活下降速度明显减慢，百草枯喷施试验表明，转基因烟草都比非转基因烟草对百草枯的耐性增强，这一系列试验间接地说明外源基因的导入增强了烟草的耐衰老能力。
(3)棉花的遗传转化：以中棉所24号和早熟早衰材料652583为材料，利用农杆菌介导法将所构建的植物表达载体导入中棉所24号和早熟早衰材料652583，经过愈伤化，胚性愈伤，抗性芽三个阶段的卡那霉素筛选，目前得到9株中棉所24号卡那霉素抗性植株，早熟早衰材料652583抗性芽还在进一步的筛选中。
The problem of earliness with decrepitude has worse effect on the yield in theshort-season cotton in our country. Up to now, about the problem of earliness withdecrepitude in short-season cotton, careful research has been done on the physiologicallevel, the results of the formers: comparing the varieties of earliness with not-decrepitudewith the varieties of earliness with decrepitude, the antioxidant enzyme activity ofearliness with not-decrepitude are higher and steadier in the growth retrial period, the keyenzymes in system of antioxidant enzyme are SOD. If we want to understand themolecular mechanism about earliness with decrepitude, it is very important of cloningthe correlative genes and studying its function. These genes can help us to improve theyield and quality.
(1) plant binary vector construction of cotton chloroplast Cu-ZnSOD gene: PlasmidpBI 121 was used as the genetic transformation carrier. The cotton chloroplastCu-ZnSOD genes and pBI 121 was digested by BamHI/SacI, then extracted the genefragments and vector fragment by electrophoresis, linked up the two fragments andtransformed it into Agrobacterium tumefaciens LBA4404 competent cells, which is plantexpression vector, pBI-YSOD. The result of extraction plasmid of pBI-YSOD and PCRshowed that fragment of plant expression vector the same to the target gene, plasmidpBI-YSOD is bigger than plasmid pBI121, which showed that cotton chloroplastCu-ZnSOD gene fragments has be transferred into the Agrobacterium tumefaciens, andcan be used as carriers for genetic transformation.
(2) Tobacco Genetic transformation and detection of transgenic plant: Cotton chloroplastCu-ZnSOD gene plant expression vector was constructed and introduced into tobacco NC89 viaAgrobacterium tumefaciens. There 23 plant display positive reaction in kanamycin selection. The resultshowed that 4 plantlets display positive reaction in PCR analysis、Southern blotting and SOD enzymeactivity detection, also indicated that SOD enzyme activity in aparted cotyledon of 4 transgenic plantsis higher than of non transgenic plant, and the sensitivity to paraquat in non transgenic plant is higherthan in 4 transgenic plants. Totally, the integration of foreign gene increased tobacco resisted- caducityactivity.
(3) Cotton genetic transformation: CCRI 24 and breeding material 652583 were used as materials for Agrobacterium infection, Cotton chloroplast Cu-ZnSOD gene plantexpression vector was constructed and introduced into CCRI 24 and breeding material 652583via Agrobacterium tumefaciens. There are 9 plantlet CCRI 24 display positive reaction inkanamycin selection, but resist seeding of breeding material 652583 is in selection still.
引文
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2．蔡艳俊．自求恩医科大学学报，1994：20(6)：591
3．曹仪植，宋占午．植物生理学．兰州：兰州大学出版社，1998，380-381
4．陈锦云，林祥永，兰志斌等．超氧化物歧化酶防治烟草气候斑点病效果初报．福建农业科技，1996，(5)：13
5．陈忠宁，毛宗万，唐雯霞．铜锌超氧化物歧化酶和结构、机理及其模拟研究进展．化学通报，1993，6：1-8
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