木薯Ⅴ型几丁质酶MeCHITV基因生物信息学分析及其表达载体构建  

Bioinformatics analysis and expression vectors construction of a classⅤchitinase MeCHITV in Manihot esculenta

作　　者：刘沁雲 向春瑜 蔡杰 LIU Qinyun;XIANG Chunyu;CAI Jie(Tropical Crops Genetic Resources Institute,Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Ministry of Agriculture for Germplasm Resources Conservation and Utilization of Cassava,Haikou,Hainan 571101,China;Sanya Research Institute of Chinese Academy of Tropical Agricultural Sciences,Sanya,Hainan 572024,China;School of Life and Health,Hainan University,Haikou,Hainan 570228,China)

机构地区：[1]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,海南海口571101 [2]中国热带农业科学院三亚研究院,海南三亚572024 [3]海南大学生命健康学院,海南海口570228

出　　处：《福建农林大学学报(自然科学版)》2025年第3期289-298,共10页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：国家自然科学基金青年基金项目(32101840);中国热带农业科学院国家热带农业科学中心科技创新团队项目(CATASCXTD202301)。

摘　　要：【目的】研究木薯Ⅴ型几丁质酶基因MeCHITV,以为后续阐明其在共生中的功能奠定基础,并为木薯高效栽培与养分利用提供理论依据。【方法】通过RT-PCR技术成功克隆了SC8木薯中的Ⅴ型几丁质酶基因MeCHITV,并采用生物信息学方法对其蛋白结构、组织特异性表达模式和遗传进化关系进行了全面分析。构建了MeCHITV基因的原核表达载体、过表达载体和基因编辑载体。【结果】MeCHITV基因的编码序列(CDS)全长1137 bp,编码378个氨基酸。MeCHITV蛋白的分子质量为42.09 ku,等电点为6.92,不稳定系数为33.97,是稳定蛋白且具有亲水性。其N端含有一个由21个氨基酸组成的信号肽序列。二级结构由33.33%的α螺旋、4.76%的β折叠、41.01%无规则卷曲和20.90%延伸链构成。与截形苜蓿Ⅴ型几丁质酶MtNFH1的同源性最高,亲缘关系最近。MeCHITV基因在木薯须根中表达量最高。此外,成功构建了原核表达载体pET28a-MeCHIV、过表达载体pCAMBIA1300-35S-MeCHITV和基因编辑载体pYAO:hSpCas9-MeCHITV,并分别成功转化至原核表达菌株Rossetta(DE3)和根癌农杆菌LBA4404。【结论】根据MeCHITV基因在须根中的高表达及对其所编码蛋白的结构,推断MeCHITV在根际发挥功能。结合同源性蛋白功能的分析,推测MeCHITV参与了木薯与丛枝菌根真菌(AMF)共生信号的调控。【Objective】The study of the classⅤchitinase gene in Manihot esculenta lays the foundation for further studies on symbiotic function,and provides a theoretical basis for the efficient cultivation and utilization of nutrients in M.esculenta.【Method】The classⅤchitinase gene MeCHITV was successfully cloned from M.esculenta SC8 by RT-PCR.The structure,tissue-specific expression pattern and genetic evolutionary relationship of MeCHITV were then comprehensively analysed by bioinformatics methods.Molecular cloning techniqueswere employed to construct prokaryotic expression vectors,overexpression vectors,and gene editing vectors for MeCHITV.【Result】The coding sequence of this gene was 1137 bp in length and encodes 378 amino acids.The molecular weight of the MeCHITV protein was 42.09 ku,the isoelectric point was 6.92,and the instability coefficient was 33.97.These characteristics indicate that MeCHITV is stable and hydrophilic protein.Additionally,the protein contains a signal peptide sequence of 21 amino acids at the N-terminal.The secondary structure of MeCHITV protein was found to comprise 33.33%αhelices,4.76%βsheets,41.01%random coils,and 20.90%extended strands.MeCHITV exhibited evolutionary conservation with the Medicago truncatula classⅤchitinase MtNFH1.Furthermore,the expression level of MeCHITV was highly expressed in the fibrous roots.Besides,the prokaryotic expression vector pET28a-MeCHITV,overexpression vector pCAMBIA1300-35S-MeCHITV and gene editing vector pYAO:hSpCas9-MeCHITV were constructed,and successfully transformed into Escherichia coli Rossetta(DE3)or Agrobacterium rhizogenes LBA4404 respectively.【Conclusion】Given the high expression of the MeCHITV gene in the fibrous roots and the predicated structure of this protein,MeCHITV was recognized as a protein operates at the rhizosphere.Considering the homologous protein functions,it is postulated that MeCHITV plays a role in the regulation of symbiotic signalling between M.esculenta and the arbuscular mycorrhizal fungus(AMF).

关 键 词：木薯 丛枝菌根真菌 Ⅴ型几丁质酶 MeCHITV 表达载体 基因编辑 

分 类 号：S184[农业科学—农业基础科学]