慈菇蛋白酶抑制剂通过花粉管途径对小麦的导入及转基因植株分析  

Transformation of Wheat with Insecticide Gene of Arrowhead Proteinase Inhibitor by Pollen Tube Pathway and Analysis of Transgenic Plants

作　　者：牟红梅[1] 刘树俊[1] 周文娟[1] 文玉香[1] 张文俊[1] 魏荣瑄[1] 

机构地区：[1]中国科学院遗传所植物细胞与染色体工程国家重点实验室,北京100101

出　　处：《Acta Genetica Sinica》1999年第6期634-642,共9页

摘　　要：慈菇蛋白酶抑制剂（ArrowheadProteinaseInhbitor，API）是来源于慈菇（Sagittariatrifolia）储藏器官（根茎）的一种天然抗虫物质，属于丝氨酸蛋白酶抑制剂类，能抑制胰蛋白酶、胰凝乳蛋白酶和激肽释放酶，对某些鳞翅目、双翅目以及鞘翅目等昆虫有毒杀作用。通过花粉管途径，将构建的含抗虫基因API－A、API－B和选择性标记基因NPT－Ⅱ的质粒pBLAH－A（B）导入3个冬小麦品种（JD-1、8866、866554），通过卡那霉素抗性的筛选和聚合酶链反应（PCR）技术分析表明，Kmr绿苗中有3株为PCR阳性株（866554有2株，JD-1有1株），阳性率为0．29％。用API基因的片段为探针，对Kmr绿苗分别进行基因组DNA的Southern杂交分析，在3株PCR阳性株中都检测出了API基因片段的存在，说明了外源目的基因已整合到小麦基因组中。对转化阳性植株899554-3的自交后代进行了PCR分析和Southern杂交分析，部分植株表现为PCR和Southern杂交阳性，这表明整合到基因组中的外源基因能稳定遗传给后代。用ELISA的方法检测与API基因融合的NPT－Ⅱ基因的表达含量，发现在PCR和Southern杂交分析为阳性反应的3植株中都显示出NPT－Ⅱ的高含量，为杀虫基因API在转基因小麦中的整合提供了进一步证据。Arrowhead Proteinase Inhibitor(API), one kind of Pure natural material, was derived from storage organ of Sagittaria trifolia. It belongs to serine proteinase inhibitor, and can inhibit trypsin, chemotrypsin and kallikrein. Furthermore, API is toxical to some species of insectS such as lepidotera, Coleoptera and Diptrea etc. By means of pollen tube pathway, plasmid PBMH-A(B) containing insect-resistant genes of API-A, API-B and selective marker gene of NPT-Ⅱ were transferred into three lines of local winter wheat-JD-1, 8866, 866554. Then, Kanamycin-resistant screening and PCR analysis of genetic transformed Plants showed that three of Kmr green Plants (tWo from 866554, one from JD-1) were PCR positihve with the positive rate of 0.29%. When the fragment of API gene was used as probe to hybrid with genomic DNA of Kmr green plants separately, all of three PCR positive ones displayed a single strong hybridizing band. Such results demonstrated that foreign target gene had been integrated into wheat genome already. Simultaneously, PCR analysis and Southern hybridization were camed out among selfiedoffsprings of transformed positive Plant of the line 899554-3, some of them were PCR and Southern blotting positive, indicating that foreign gene integrated into wheat genome could stably transmitted into next generation. In addition, the expression level of NPT-Ⅱ gene was checked via ELISA in our study, all of three PCR and Southern blot positive Plants could yield high level of NPT-Ⅱ. This data provided a more powerful evidence for integration of insecticide gene into wheat genome.

关 键 词：慈菇 抗虫基因 小麦遗传转化 花粉管理管法 API 

分 类 号：S512.103.4[农业科学—作物学] Q785[生物学—分子生物学]