大蒜叶凝集素基因ASAL的克隆与表达载体的构建  

Cloning of ASAL Gene and Construction of Its Plant Expression Vector

作　　者：陈德西[1] 何忠全[1] 向运佳[1] 胡荣平[1] 

机构地区：[1]四川省农业科学院植物保护研究所,农业部西南作物有害生物综合治理重点实验室,四川成都610066

出　　处：《西南农业学报》2016年第10期2445-2449,共5页Southwest China Journal of Agricultural Sciences

基　　金：国家科技支撑计划西南稻区水稻重大病虫防控技术研究与集成示范(2012BAD19B03-09);四川省财政基因工程优秀论文基金项目(2011LWJJ-005)

摘　　要：大蒜叶凝集素基因(Allium sativum leaf agglutinin,ASAL)是一种高活性的凝集素基因,且具显著的抗虫性。克隆大蒜ASAL基因将为其在转基因作物中抗虫特性研究奠定基础。本文以GenBank公布的ASAL基因序列设计特异引物,采用RT-PCR法从当地大蒜品种二水早和新都软叶的幼苗中克隆ASAL基因。结果表明,克隆的两个ASAL基因与已知基因的碱基序列相似性分别为98.7%和98.9%,氨基酸序列相似性为98.3%。将其亚克隆至表达载体中,经菌落PCR和酶切鉴定,成功构建了重组质粒。将该载体导入农杆菌EHA5a进行保存。ASAL gene was a highly active gene in many plants and had high insect resistance. Cloning ASAL gene isolated from garlic would lay the foundation for transgenic crops characteristic research in insect resistance. In this paper, a pair of specific primers was designed according to the sequence of ASAL gene in GenBank, and using the total RNA of garlic seedlings of the local varieties Eershuizao and Xindu soft leaves as the templates, the complete cDNA sequences by RT-PCR were obtained. The results showed that the recombinant plasmids containing ASAL gene from Eershuizao and Xindu were similar to reach 98.7 % and 98.9 % respectively with ASAL gene in GenBank and the amino acid sequences were similar to reach 98.3 % . The recombinant plasmids were successfully constructed by PCR amplification and digestion with restriction endonucleases. The vectors were transformed into the Agrobactrium tumefaciens EHA50 for preservation.

关 键 词：ASAL基因 刺吸式害虫 基因克隆 表达载体 构建 

分 类 号：S633.4[农业科学—蔬菜学]