紫云英(Astragalus sinicus)LHY基因克隆及鉴定  

Cloning and Identification of LHY Gene in Chinese Milk Vetch(Astragalus sinicus)

作　　者：张贤[1] 庄俐[1] 曹卫东[2] 曹凯[1] 徐静[1] 斯林林 王建红[1] ZHANG Xian;ZHUANG Li;CAO Wei-Dong;CAO Kai;XU Jing;SI Lin-Lin;WANG Jian-Hong(Institute of Environment,Resource,Soil&Fertilizer,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China;Institute of Agricultural Resources and Regional Planning,Chinese Academy of Agricultural Sciences,Beijing 100081,China)

机构地区：[1]浙江省农业科学院环境资源与土壤肥料研究所,杭州310021 [2]中国农业科学院农业资源与农业区划研究所,北京100081

出　　处：《农业生物技术学报》2023年第11期2263-2271,共9页Journal of Agricultural Biotechnology

基　　金：财政部和农业部国家现代农业产业技术体系(CARS-22);浙江省自然科学基金(No.LY14D010004)。

摘　　要：紫云英(Astragalus sinicus)是我国南方稻区主要绿肥作物之一,对土壤资源可持续利用具有重要意义,花期调控是紫云英育种中的重要目标。生物钟基因响应昼夜节律变化,通过影响光周期途径中成花基因的表达参与植物开花调控,LHY(late elongated hypocoty l)基因是关键生物钟基因之一。为探究紫云英中LHY基因的功能,本研究利用Illumina Hiseq方法和RACE(rapid-amplification of cDNA ends)克隆技术分离了紫云英LHY基因全长序列;利用生物信息学分析软件进行LHY基因的生物信息学分析;q RTPCR分析LHY基因在不同组织中的表达模式,揭示基因表达与紫云英成花的关系;并通过拟南芥(Arabidopsis thaliana)遗传转化,进一步鉴定紫云英LHY基因的功能,明确其对成花的影响。结果表明,本文首次克隆了紫云英LHY基因(GenBank No.OP455117),cDNA全长为2865 bp,含开放阅读框长度为2259 bp,编码752个氨基酸,LHY基因的同源蛋白在不同植物间具有较高的相似性,尤其与蒺藜苜蓿(Medicago truncatula)、鹰嘴豆(Cicer arietinum)、野生大豆(Glycine soja)等豆科植物相似性达75%以上。qRT-PCR分析结果显示,在紫云英叶片、花和花芽中LHY基因的表达量最高。超表达紫云英LHY基因的转基因拟南芥株系,表现出了显著的晚花性状,抽薹天数平均比野生型晚18.36 d,开花天数平均比野生型晚20.72 d,表明紫云英LHY基因可能具有晚花功能,抑制植物开花。本研究为解析紫云英开花调控机理提供了科学依据。Astragalus sinicus has been identified as one of the most important green manure for paddy fields in the south of China.It is vital to the sustainable utilization of soil resources.Based on different farming systems,selecting flowering varieties is an important breeding target of A.sinicus.Flowering time is regulated by the circadian clock in response to the external environment.Late elongated hypocoty l(LHY)is a key biorhythm clock gene.In the present study,in order to explore the function of the LHY gene in A.sinicus,the Illumina and rapid-amplification of cDNA ends(RACE)technology were performed to obtain the sequence of LHY in A.sinicus;the expression profiles of the LHY gene in tissues of A.sinicus were subsequently analyzed by qRT-PCR;the function of LHY gene of A.sinicus is verified by the transformation of LHY gene into Arabidopsis.The full-length cDNA of the LHY gene(GenBank No.OP455117)was isolated in A.sinicus,which was 2865 bp in length and containing the ORF of 2259 bp,encoding 752 amino acid polypeptide.The encoded amino acid sequence is highly homologous to the LHY proteins of Medicago truncatula,Cicer arietinum,and Glycine soja with a more than 75%similarity.The qRT-PCR analysis showed that the LHY gene had the highest expression accumulation in leaves,flowers,and flower buds of Astragalus sinicus.The LHYoverexpression transgenic Arabidopsis lines showed a significant late flowering phenotype.The bolting days and flowering days of transgenic Arabidopsis lines were 18.36 and 20.72 d later than that of the wild-type respectively.This study provides important implications for genetic modification of flowering in A.sinicus.

关 键 词：紫云英 LHY 成花 生物信息学分析 表达分析 

分 类 号：S529[农业科学—作物学]