贵州地方稻种‘平塘黑糯’低温应答类糖基转移酶基因(OsCUGT1)的克隆及突变体创建  

Cloning and Mutant Construction of Cold-upregulated Glycosyltransferase-like Gene(OsCUGT1) from Guizhou Landrace Rice ’Pingtang Heinuo’(Oryza sativa ssp.japonica)

作　　者：蔡明亮 陈蓉 黄小贞 赵德刚[1,3] CAI Ming-Liang;CHEN Rong;HUANG Xiao-Zhen;ZHAO De-Gang(The Key Laboratory of Plant Resources Conservation and Germplasm Innovationin Mountainous Region(Ministry of Education),College of Life Sciences,Guizhou University,Guiyang 550025,China;College of Tea Sciences,Guizhou University,Guiyang 550025,China;Guizhou Academy of Agricultural Sciences,Guiyang Branch of DUS Center,Ministry of Agriculture and Rural Sciences,Guiyang 550006,China)

机构地区：[1]贵州大学生命科学学院,山地植物资源保护与种质创新省部共建教育部重点实验室,贵阳550025 [2]贵州大学茶学院贵阳,550025 [3]贵州省农业科学研究院,国家农业农村部DUS中心贵阳分中心,贵阳550006

出　　处：《农业生物技术学报》2021年第1期12-22,共11页Journal of Agricultural Biotechnology

基　　金：国家转基因生物新品种培育重大专项(2016ZX08010003);国家自然科学基金(31960615)。

摘　　要：糖基转移酶(glycosyltransferase, GTs)在植物的生长发育和逆境胁迫中有重要作用。为了进一步挖掘和研究水稻(Oryza sativa)中新的糖基转移酶的生物学功能,本研究从前期构建的贵州地方稻种‘平塘黑糯’(O. sativa ssp. japonica)低温响应转录组文库中,筛选并克隆了1个低温应答类糖基转移酶基因(cold-upregulated glycosyltransferase-like gene 1, OsCUGT1)。荧光定量PCR分析发现,OsCUGT1基因受到低温诱导表达。亚细胞定位结果显示其定位于叶绿体。序列比对结果表明,’平塘黑糯’中OsCUGT1基因与‘日本晴’(O. sativa ssp. Nipponbare)相比,存在着多个碱基的差异。其中第511 bp的碱基发生了改变,由C变为A,碱基的改变造成了第170位氨基酸由赖氨酸(lysine, Q)变成了谷氨酰胺(glutamine, K)。对该基因序列进行多样性分析,发现该基因序列具有丰富的单核苷酸多态性,并在基因的外显子区域找到12个SNP位点。系统遗传进化树分析显示,OsCUGT1氨基酸序列在单子叶植物中较为保守。进一步利用CRISPR/Cas9基因组编辑系统,构建了OsCUGT1基因敲除载体。通过农杆菌(Agrobacterium tumefaciens)介导的愈伤转化、潮霉素筛选及测序分析,成功获得了OsCUGT1基因的定点编辑突变体。该研究结果为进一步明确OsCUGT1在水稻生长发育以及抗逆过程中的作用提供了研究材料和理论依据。Plant glycosyltransferases(GTs) play an important role in regulating growth and stress adaptation.To further explore and study the biological functions of new glycosyltransferases in rice(Oryza sativa), in this study, a cold-upregulated glycosyltransferase-like gene 1(OsCUGT1) was isolated and cloned, which was based on the cold treatment transcriptome library of Guizhou landrace rice ’Pingtang Heinuo’(O. sativa ssp.japonica). The results of qRT-PCR showed that the expression of OsCUGT1 gene was significantly induced by cold treatment. The subcellular localization results showed that it was localized in the chloroplasts.Phylogenetic analysis showed that OsCUGT1 was conserved in monocot plant species. Furthermore, sequence alignment revealed that there were several SNP sites between ’Pingtang Heinuo’ and O. sativa ssp. Nipponbare in the OsCUGT1 gene. Among them, the site of 511 bp was changed from C to A, which consequently caused the 170 th amino acid to change from lysine(Q) to glutamine(K). Diversity analysis of the OsCUGT1 gene sequence revealed that there were 12 SNP sites of them were found in the exon region. To further explore the biological function of OsCUGT1, the OsCUGT1 gene was edited by CRISPR/Cas9 technique. The targeted editing mutants of oscugt1 were successfully obtained by optimizing Agrobacterium-mediated transformation method, screening of hygromycin selection and sequencing analysis. The results of this study can provide research materials and theoretical basis for further clarifying the role of OsCUGT1 in rice growth development and stress resistance.

关 键 词：水稻 糖基转移酶 亚细胞定位 CRISPR/Cas9 潮霉素 

分 类 号：S511[农业科学—作物学] Q812[生物学—生物工程]