小麦miR395a前体克隆、遗传转化及组织表达模式分析  

Cloning,Genetic Transformation and Tissue Expression Pattern Analysis of Wheat miR395a Precursor

作　　者：丁超杰 关园园 李成伟 DING Chaojie;GUAN Yuanyuan;LI Chengwei(Henan Engineering Research Center of Crop Genome Editing,Xinxiang 453003,China;College of Life Science and Technology,Henan Institute of Science and Technology,Xinxiang 453003,China)

机构地区：[1]河南省粮食作物基因组编辑工程技术研究中心,河南新乡453003 [2]河南科技学院生命科技学院,河南新乡453003

出　　处：《华北农学报》2020年第4期52-56,共5页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金面上项目(31872129);河南省高等学校重点科研项目(20B210005);河南省农业科技攻关项目(182102110259,202102110013);河南科技学院百农英才项目(BNYC2018-2-16)。

摘　　要：miRNA是一类在调控植物发育和生物及非生物逆境胁迫响应过程中发挥重要功能的非编码小RNA,为了探究miR395a在小麦抗病中的功能和作用机制,比较了不同物种中的miR395a成熟序列,分析发现miR395a在不同物种中具有较高的保守性,其中小麦与拟南芥、番茄以及大豆中的miR395a成熟序列具有高度一致性。并从小麦叶片中克隆了miR395a的前体序列,并将其连接到植物表达载体pCambia1301上,构建了miR395a前体的过量表达载体,之后通过农杆菌介导的遗传转化法,转化至小麦栽培品种(百农207)中,获得56株转基因植株,并通过PCR鉴定出11株阳性转基因植株。用RT-qPCR技术对不同组织中miR395a的表达量进行分析,结果显示,miR395a为组成型表达,但在不同组织中的表达量有差异,其中在三叶期和抽穗期叶片中的表达量较高,叶鞘中次之,旗叶中表达量最低。综上,获得了过量表达miR395a前体的小麦转基因植株,为进一步研究miR395a在小麦抗病中的功能及作用机制提供了材料来源。miRNA are a group of small non-coding RNAs that play important roles in the process of developmental regulation and biotic and abiotic stress responses in plant.In order to explore the function and mechanism of miR395a in resistance to pathogen in wheat,the mature sequence of miR395a from different species was compared.It was found that miR395a was highly conserved in different species.The mature miR395a in wheat was highly consistent with that in Arabidopsis,tomato and soybean.The pre-miR395a was cloned from wheat leaves and connected to pCambia1301 to construct over-expression vector of pre-miR395a.pre-miR395a was transformed into wheat variety Bainong 207 through Agrobacterium-mediated method.56 transgenic plants were obtained and 11 positive transformants were determined by using PCR analysis.The tissue expression profiles detected by RT-qPCR indicated that miR395a was differently expressed during the process of grain development among the tested tissue.The expression level of miR395a was highest in leaves at three-leaf and heading stages,followed by that in leaf sheath and the expression level was lowest in the flag leaf.This study obtains wheat transgenic plants overexpressing the precursor of miR395a,and provides genetic resources for further study of its function in wheat resistance to pathogens.

关 键 词：小麦 miR395a前体 过量表达载体构建 遗传转化 组织表达模式分析 

分 类 号：S512.03[农业科学—作物学] Q78[生物学—分子生物学]