【作者】 曹传增；

【导师】 蔡民华； 刘凡；

【作者基本信息】 首都师范大学 ， 遗传学， 2003， 硕士

【摘要】 蜘蛛杀虫蛋白(Spider insecticidal protein,SIP)基因可编码一种只有37个氨基酸的小肽，该多肽能杀死多种有害昆虫。质粒pBBBast-PinⅡ含有马铃薯蛋白酶抑制剂基因PinⅡ(Potato proteinase inhibitor Ⅱ)，PinⅡ基因编码的蛋白酶抑制剂具有广谱抗虫性。实验以蜘蛛杀虫肽基因为目的基因，将其转入质粒pBBBast-PinⅡ中，从而构建含有PinⅡ和SIP两个抗虫基因的双价植物表达载体——pBBBast-PinⅡ-SIP。同时，还将SIP基因转入pBBBast中，构建单价抗虫载体pBBBast-SIP。经过限制性内切酶酶切以及PCR目的片段扩增证明两个载体构建正确。 通过真空渗入和花序浸渍法，将上述双抗和单抗基因分别导入白菜不结球类型——49菜心中，获得了7棵抗性株。通过对标记性状——除草剂抗性的活体及离体检验(氯酚红法)、目的基因PCR扩增、PCR-Southern杂交等方法证实目的基因已整合到小白菜基因组中。其中2棵抗性株自交后代的遗传分析表明，真空渗入法所得的转化株为杂合体，且其分离比符合3：1的单基因分离规律。 对小白菜真空渗入转化法的转化条件进行了初步探索。结果表明植株生长阶段和真空渗入处理时间，所用菌株类型均对转化率有影响。其中以开花初期的植株，利用胭脂碱型农杆菌介导(如C58)，真空渗入5分钟的处理方法转化效率最高。更多还原

【Abstract】 Spider insecticidal protein (SIP) gene can encode a small polypeptide containing 37 amino acids. The polypeptide has strong toxicity to many insects. Plasmid pBBBast-PinII has the Potato proteinase inhibitor II (Pin II ) gene which encodes potato proteinase inhibitor. The SIP gene was digested by restriction enzyme Hindlll and inserted into the vector pBBBast-Pin II . So a plant expression vector with two insect-resistant genes -pBBBast-Pin II -SIP was constructed. A plant expression vector - pBBBast-SIP with one insect-resistant gene was also constructed by inserting the SIP gene into the plasmid pBBBast. Restriction enzyme digestion analysis and PCR analysis showed that these two plant expression vectors constructed were correct.49Caixin, one cultivar of the Brassica campestris L. ssp. chinensis , was transformed by vacuum infiltration method. 7 herbicide resistant plants were obtained. PCR, PCR-Southern blotting analysis showed that the target genes were integrated into the plant genome. The segregation ratio in T1 of two examed transformants was 3:1. It revealed that these two transformants were heterozygote, and the foreign gene inserted just to on locus in the plant genome. As the observed ratio of progeny of two transformants which on selfing was 3:1, it was proved to be heterozygote for two transformants.Several factors influencing the vacuum infiltration transformation efficiency in Brassica campestris L. ssp. chinensis were also studied. The developmental status of seedlings, vacuum infiltration time, type of Agrobacterium tumefaciens used, all influence the transformation frequency. It was concluded that using the nopaline type of Agrobacterium (e.g. C58), 5min vacuum infiltration, and using the plants with just several blooming flowers would give the best results.更多还原