紫云英LEAFY基因的克隆与鉴定  

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

作　　者：张贤[1] 王建红[1] 曹凯[1] 庄俐[1] 曹卫东[2] ZHANG Xian;WANG Jian-Hong;CAO Kai;ZHUANG Li;CAO Wei-Dong(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》2018年第8期1328-1341,共14页Journal of Agricultural Biotechnology

基　　金：国家绿肥产业技术体系(No.CARS-22);浙江省自然科学基金(No.LY14D010004)

摘　　要：紫云英(Astragalus sinicus)作为重要的蜜源植物和绿肥作物,对促进作物增产具有重要作用。根据当地的耕作制度,选育花期适当的品种是紫云英重要的育种目标。LEAFY(LFY)是植物花分生组织的特异基因,而目前关于紫云英LEAFY基因的研究尚无报道。为探究紫云英中LEAFY基因的功能,本研究采用Illumina Hiseq和c DNA末端快速克隆(rapid-amplification of c DNA ends,RACE)技术对紫云英LEAFY基因序列进行克隆;采用生物信息学分析软件对LEAFY基因进行生物信息学分析;采用q RT-PCR揭示该基因在不同组织中的表达特性;采用表达载体构建、侵染以及转基因T2代纯合株系的生长发育表型特征统计分析鉴定紫云英LEAFY基因功能。本研究获得的LEAFY基因全长c DNA长度为1 400 bp(Gen Bank No.MH352146),含有1个1 191 bp长的开放阅读框;编码的蛋白质含有396个氨基酸,含22个α螺旋和5个β折叠,其分子量为44.72 k D,理论等电点为6.41;编码的氨基酸序列与蒺藜苜蓿(Medicago truncatula)、紫花苜蓿(Medicago sativa)、鹰嘴豆(Cicer arietinum)等物种的LEAFY蛋白相似性均在80%以上,具有高度的同源性;q RT-PCR分析结果表明,紫云英LEAFY基因在各组织中的表达量由高到低依次为花芽、花、叶、叶芽、根、茎和荚;超表达LEAFY基因拟南芥(Arabidopsis thaliana)实验结果发现,超表达LEAFY基因拟南芥株系的出苗至抽薹天数比野生型平均早3 d,出苗至开花天数比野生型平均早2 d,开花数比野生型平均多1.79个。本研究表明紫云英LEAFY基因与豆科物种均有较高的同源性,在其花芽中表达量最高,并证实LEAFY基因可能调控其开花机制,为通过分子技术改良该物种成花提供理论依据,对农业生产具有重要意义。Astragalus sinicus is an important nectariferous plant and green manure crop. It plays an important role in promoting crop yield. Basing on the local farming system, selecting flowering varieties is an important breeding target of Astragalus sinicus. LEAFY （LFY） is a plant-specific flower meristem specific-gene. However, the study on LEAFY gene of Astragalus sinicus is not reported yet. In this study, in order to explore the function of LEAFY gene in Astragalus sinicus, the sequence of LEAFY in Astragalus sinicus was cloned by Illumina Hiseq method and rapid-amplification of cDNA ends （RACE） technology, and the bioinformatics analysis of LEAFY gene was performed by bioinformatics analysis software; The expression characteristics of the LEAFY gene in different tissues were analyzed by qRT-PCR; The function of LEAFY gene of Astragalus sinicus was identified after expression vector construction, infection and statistical analysis of phenotypic characteristics of T2 transgenic homozygous lines were performed. The results showed that the full-length cDNA of the LEAFY gene （GenBank No. MH352146） in Astragalus sinicus was 1 400 bp in length and contained the open reading frame of 1 191 bp; The encoded protein contains 396 amino acids, including 22 α-helices and 5 β-sheets, which has a molecular weight of 44.72 kD and a theoretical isoelectric point of 6.41; The similarity of the encoded amino acid sequences to the LEAFY proteins of Medicago truncatula, Medicago sativa, Cicer arietinum, and other species were all above 80%, which was highly homologous. The qRT-PCR analysis of LEAFY gene showed that the LEAFY gene was expressed in various organs, and the order from high to low of the expression levels were flower buds, flowers, leaves, leaf buds, roots, stems and pods. The results of the experiments with Arabidopsis thaliana over expressing LEAFY showed that the number of bolting days of transgenic Arabidopsis thaliana was 3 days earlier than that of wild type, and the time of transgenic A. thaliana flowerin

关 键 词：紫云英 LEAFY(LFY) 成花 生物信息学分析 表达分析 

分 类 号：S529[农业科学—作物学]