LI Lin-ling,LIAO Zhi-qin,CHEN Xiao-ling,et al.Molecular Cloning and RNAi Expression Vector Construction of MADS-box Gene from Castanea mollissima [J].Northern Horticulture,2014,38(24):92-98.
板栗MADS-box基因的分离及RNAi表达载体构建
- Title:
- Molecular Cloning and RNAi Expression Vector Construction of MADS-box Gene from Castanea mollissima
- 文章编号:
- 1001-0009（2014）24-0092-07
- 关键词:
- 板栗; MADS-box基因; 开花; 成熟期
- Keywords:
- Castanea mollissima; MADS-box gene; flowering period; maturation stage
- 分类号:
- S 664.203.6
- 文献标志码:
- A
- 摘要:
- 以中国罗田板栗品种“玫瑰红”的幼叶和花为试材，采用EST数据库分析，结合RACE技术从板栗中分离到MADS基因的cDNA全长序列并构建了其RNA干扰载体，研究开花关键基因对板栗花芽分化的影响。结果表明：CmMADS基因全长为922 bp的CmMADS的cDNA序列，该序列含有1个681 bp的可读框，编码227个氨基酸序列。生物信息学预测CmMADS蛋白的分子质量为25.87 kDa，理论等电点为6.27，N端具有M盒保守序列，其二级结构主要由α-螺旋和无规则卷曲组成。蛋白质同源分析表明，CmMADS含有M盒和K盒2个特征性序列区域。同源建模分析显示CmMADS序列与苹果MADS蛋白的三维结构及活性位点高度相似。系统进化分析表明，板栗MADS蛋白归属植物进化分支，且与太行花的MADS蛋白归为一支。将CmMADS基因2段相同长度(283 bp)的反向互补片段RMADS和FMADS连入载体pBluescript SK plus，构成中间载体pBluescript SK plus-FR。用BanH I和Kpn I同时酶切中间载体pBluescript SK plus-FR和植物表达载体pCl301-ubi，回收pBluescript SK plus-FR的酶切小片段，连入pC1301-ubi大片段中，构成植物表达载体pC1301-ubi-CmMADS-RNAi。下一步拟用构建好的RNA干扰载体转化农杆菌并由其介导将重组质粒转入烟草，为深入研究该干扰载体的功能及CmMADS基因的功能提供参考。
- Abstract:
- Taking the flowers and leaves of chestnut ‘Meiguihong’ as materials,the cDNA sequence of MADS gene was isolated from the flowers and leaves according to RACE technology and EST database from NCBI,plant expression vector of RNA interference pCl301-ubi-CmMADS-dsRNAi had also constructed by the MADS ene to study effect of blossom key gene on flower bud differentiation of Chinese chestnut.The results showed that,CmMADS was 922 bp containing an open reading frame(ORF) of 681 bp,which encoded 227 amino acids with a predicted molecular mass of 25.87 kDa and the theoretical isoelectric point(PI) of 6.27,CmMADS was an intro-free gene,and its deduced polypeptide contained a M box of 58 amino acids in the N terminal.The secondary structure of CmMADS was mainly composed of alpha helix and random coil.Comparative analysis showed that CmMADS had a high similarity to other plant MADS proteins,and contained all the M box and K box.The homology based structural modeling showed that CmMADS had the typical structure of Malus domestica MADS.Phylogenetic tree revealed that CmMADS and TrMADS3 were assigned to the same clade.Use BanH I and Kpn I and enzyme intermediate carrier pBluescript SK plus-FR and plant expression vector pcl301-ubi,recycling pBluescript SK plus- FR enzyme cut into small pieces,connected pcl301-ubi in larger pieces,a plant expression vector pCl301-ubi-CmMADS-dsRNAi.Next build good carrier of RNA interference and conversion of agrobacterium mediated by its putting the recombinant plasmid into tobacco,the function of the carrier for further study of the interference and CmMADS gene function.
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备注/Memo
第一作者简介：李琳玲(1981-)，女，湖北十堰人，博士，讲师，现主要从事板栗种质资源评价与改良等研究工作。E-mail：lilinling1437@126.com.