甘薯几丁质酶基因IbChi的克隆及其耐盐性分析  

Cloning and Salt Tolerance Analysis of Chitinase Gene IbChi from Sweet-potato

作　　者：周桦楠 孙思凡 杨颖慧 聂楠 何绍贞[1] 刘庆昌[1] 翟红[1] Zhou Huanan;Sun Sifan;Yang Yinghui;Nie Nan;He Shaozhen;Liu Qingchang;Zhai Hong(Laboralory of Crop Heterosis and Uilization(Minisiry of Educaion),Key Lubratory of Sweet Poato Biology and Biotechmology(Ministry of Agri-culure and Rural Afair),China Agricultural University,Beijing,100193;Crop Research Institute,Liaoning Academy of Agricultural Sciences,Shenyang,110161)

机构地区：[1]中国农业大学农业农村部甘薯生物学与生物技术重点实验室教育部作物杂种优势研究与利用重点实验室,北京100193 [2]辽宁省农业科学院作物研究所,沈阳110161

出　　处：《分子植物育种》2022年第9期2812-2820,共9页Molecular Plant Breeding

基　　金：国家重点研发计划项目(2019YFD1001300,2019YFD1001301);财政部和农业农村部:国家现代农业产业技术体系项目(CARS-10,甘薯);辽宁省农业科学院院长基金项目(2021QN2004)共同资助。

摘　　要：几丁质酶基因在植物发育、抗病、抗冻、耐盐等方面都发挥着重要的作用。为了探究几丁质酶在甘薯抵御盐胁迫中的作用,本研究根据前期获得的差异表达EST序列,利用RACE方法从‘鲁薯3号’中克隆得到甘薯几丁质酶基因IbChi。该基因c DNA全长1226 bp,ORF为804 bp,编码267个氨基酸。该基因编码的蛋白质含有1条信号肽序列,并在5~27 aa位点推测有跨膜结构。生物信息学分析显示,IbChi蛋白是第Ⅰ类几丁质酶,属于糖基水解酶第19家族。氨基酸序列比对结果表明,IbChi与甘薯近缘野生种Ipomoea trilo ba(2x)同源蛋白ItlChi序列一致性为98.50%,同源性较高;进化树结果进一步显示IbChi与ItlChi亲缘关系最近。利用根癌农杆菌介导法将IbChi基因导入甘薯品种栗子香,IbChi基因的过表达显著提高了转基因甘薯植株的耐盐性。在86 mmol/L Na Cl的胁迫下,转基因植株的SOD活性、脯氨酸含量和叶绿素含量显著增加,丙二醛(MDA)含量显著降低。这些结果首次揭示了IbChi在植物耐盐性中的作用。本研究为后续甘薯IbChi耐盐分子机制研究奠定基础。Chitinase gene plays an important role in plant development,disease resistance,frost resistance and salt tolerance.In order to explore the role of chitinase in sweetpotato resistance to salt stress,we cloned IbChi from’Lushu3’by RACE method according to the sweetpotato EST sequences obtained in previous studies.The IbChi cDNA of 1226 bp in length,had an open reading frame(ORF)of 804 bp,encoded a 267 amino acids polypeptide,with one signal peptide,and a transmembrane structure at the site of 5~27 aa.Bioinformatics analysis showed that IbChi protein was a classⅠchitinase,belonging to the 19;family of glycohydrolases(GH19).Sequence alignment results showed that Ib Chi and Ipomoea triloba(2x)homologous protein ItlChi sequence consistency was 98.50%,had high homology.Phylogenetic tree results further showed that IbChi and ItlChi were most closely related.IbChi was transformed into sweetpotato cultivar Lizixiang via Agrobacterium tumefaciens-mediated transformation,and improved the salt tolerance of overexpression transgenic sweetpotato plants significantly.Under 86 mmol/L NaCl stress,the SOD activity,proline content and chlorophyll content were significantly increased whereas the MDA content was significantly decreased in transgenic plants compared to those of wild type plants.These results reveal for the first time the important role of IbChi in plant salt tolerance.This study laid a foundation for the following research on salt tolerance mechanism of Ibchi in sweetpotato.

关 键 词：甘薯 IbChi 转基因甘薯 耐盐性 

分 类 号：S531[农业科学—作物学]