板栗FLC基因的分离及其RNAi载体的构建  

Isolation and RNAi Expression Vector Construction of FLC Gene from Castanea mollissima

作　　者：程华[1,2] 陈小玲[1,2] 李琳玲[1,2] 廖志琴[1,2] 程水源[1,3] 

机构地区：[1]经济林木种质改良与资源综合利用湖北省重点实验室,湖北黄冈438000 [2]黄冈师范学院生命科学学院,湖北黄冈438000 [3]武汉轻工大学生物与制药工程学院,湖北武汉430023

出　　处：《贵州农业科学》2015年第1期1-7,共7页Guizhou Agricultural Sciences

基　　金：湖北省自然科学基金重点资助项目"湖北省板栗良种早熟丰产结实机理及栽培调控技术的研究"(2010CBB03901);湖北省教育厅高校产学研合作重点资助项目"北板栗良种绿色栽培技术集成研究与示范"(C2010060);中央财政林业科技推广示范资助项目"板栗良种绿色栽培技术集成示范与推广"(2011BH0032)

摘　　要：为板栗FLC同源基因控制开花的相关功能及春化作用的相关分子机理提供研究基础,以罗田板栗为研究材料,分离并鉴定板栗花芽调控相关基因FLC,依据NCBI数据库提供的EST序列片段,利用RACE技术,从板栗叶片总RNA中分离了与开花相关的基因-CmFLCcDNA的序列,通过在线分析预测其序列同源性及3D结构,以pBluescript SK plus作为过渡载体构建其干扰表达载体。结果表明,该序列全长为968bp,其中有1个编码含227个氨基酸蛋白序列的开放阅读框。CmFLC蛋白的理论等电点为5.80,分子质量为25.62kDa,N端含有M盒保守序列,该蛋白的二级结构大部分由无规则卷曲和α-螺旋构成。CmFLC存在M盒、K盒两个特征性序列区域。CmFLC蛋白与毛白杨FLC蛋白的三维结构存在高度相似性。板栗的FLC蛋白属于植物进化分支,且与山核桃的FLC蛋白同属一支。设计特异引物扩增得到FLC基因的正、反义基因干扰片段,酶切结果显示插入片段大小为320bp,板栗FLC的干扰载体pc1301-ubi-CMFLCRNAi构建成功。结论:板栗中存在FLC同源基因,并可能参与板栗开花相关功能及春化调控。To provide the research foundation for molecular mechanism of homologous gene FLC controlling flowering-related functions and vernalization of C.mollissima,based on the sequence of EST from NCBI,the authours isolated a flowering locus C gene from Luotia C.mollissims according to RACE technology.The sequence homology and three dimensional structure was analyzed on line,and the interference expression vector was constructed with transition carrier pBluescript SK plus.Results：CmFLCcDNA sequence was 968 bp containing an open reading frame（ORF）,which encoded 227 amino acids with a predicted molecular mass of 25.62 kDa and the theoretical isoelectric point（PI）of 5.80.CmFLC was an intro-free gene,and its deduced polypeptide contained a M box of 61 amino acids in the N terminal.The secondary structure of CmFLC was mainly composed of alpha helix and random coil.CmFLC had a high similarity to other plant FLC proteins,and contained all the M box and K box.The three dimensional structure of CmFLC protein had high similarity with Populus tomentosa FLC.CmFLC and CcFLC were assigned to the same clade.Sense and antisense gene interference fragments were gained through specific primer amplification,size 320 bp according to enzyme digestion results.FLCinterference vector was successfully constructed：pc1301-ubi-CMFLC-RNAi.Conclusion：homologous gene FLC existed in C.mollissima,and maybe participate in the regulation of flowering and vernalization.

关 键 词：板栗 开花 FLC基因 RNA干扰载体 

分 类 号：S-9[农业科学]