木薯V型几丁质酶MeCHITVb基因克隆及其表达载体构建  

Cloning of a Class V Chitinase MeCHITVb Gene of Cassava and Construction of Eukaryotic Expression Vector

作　　者：丁云 陈松笔[2] 李开绵[2] 蔡杰 Ding Yu;Chen Songbi;Li Kaimian;Cai Jie(College of Horticulture,Hainan University,Haikou,570228;Key Laboratory of Ministry of Agriculture for Germplasm Resources Conservation and Utilization of Cassava,Tropical Crops Genetic Resources Institute,Chinese Academy of Tropical Agricultural Sciences,Haikou,571101)

机构地区：[1]海南大学园艺学院,海口570228 [2]中国热带农业科学院热带作物品种资源研究所,农业农村部木薯种质资源保护与利用重点实验室,海口571101

出　　处：《分子植物育种》2021年第21期7113-7121,共9页Molecular Plant Breeding

基　　金：国家重点研发计划项目(2019YFD1001100);海南省自然科学基金项目(320RC730);中央公益性科研院所基本科研业务费(1630032019046)共同资助。

摘　　要：几丁质酶在生物逆境环境和非生物逆境环境中发挥着重要作用。为研究木薯几丁质酶在响应低温胁迫下的分子作用机制,利用RT-PCR技术,从木薯耐低温品种SC124中克隆到CHITVb基因,利用平末端连接技术构建pEASY-MeCHITVb克隆载体,用于测序确定目标基因序列,并对其序列进行分析,构建植物表达载体。结果表明,成功克隆木薯CHITVb基因,命名为MeCHITVb。通过生物信息学分析,该基因全长1050 bp,编码349个氨基酸,理论相对分子量为38.08 kD,理论等电点为4.53,是一种稳定蛋白。经序列比对和系统进化树分析表明,MeCHITVb基因与烟草、拟南芥V型几丁质酶基因亲缘关系最近。用pEASYMeCHITVb与植物表达载体pISV2678构建重组子pISV-MeCHITVb,利用"Golden Gate"克隆方法构建基因编辑载体CRISPR/Cas9-MeCHITVb。将植物过表达的pISV-MeCHITVb和CRISPR/Cas9-MeCHITVb载体成功转化为根状杆菌LBA4404。本研究为后续MeCHITVb基因功能研究、木薯抗寒、抗病品种的选育及木薯北移栽培提供理论依据。Chitinase play an important role in biotic and abiotic stress. In order to investigate the molecular mechanism of cassava chitinases in response to cold stress, a class V chitinase gene was amplified from cold-resistant cassava variety SC124 by RT-PCR method. Constructed pEASY-MeCHITVb cloning vector by using blunt-end ligation to sequencing and determining the correct target gene sequence. And then analyzed its sequence,constructed eukaryotic expression vectors. The results showed that a class V chitinase gene was successfully cloned and named MeCHITVb. Bioinformatics analysis indicated that the gene is 1 050 bp in length and encodes349 amino acides which theoretical relative molecular mass is 38.08 kD, theoretical isoelectric point is 4.53 and is a stable protein. Sequence alignment and phylogenetic analysis performed that MeCHITVb gene was closely related to tobacco NtChiV gene and Arabidopsis AtCh i C gene. pEASY-MeCHITVb and expression vector pISV2678 were used to construct recombinant pISV-MeCHITVb. At the same time, CRISPR/Cas9-MeCHITVb vector was constructed by using ’ Golden Gate’’ cloning. Finally, the plant over-expression pISV-MeCHITVb and CRISPR/Cas9-MeCHITVb vectors were successfully transformed into Agrobacterium tumefaciens LBA4404. This study provided theoretical basis for studying the function of MeCHITVb gene, selecting the cold-resistance and diseaseresistant cassava varieties, and transplanting to north China.

关 键 词：木薯(Manihot esculenta Crantz) V型几丁质酶 植物表达载体 抗寒性 

分 类 号：S533[农业科学—作物学]