慈竹LEAFY基因和启动子的克隆及序列分析  

The Cloning and Sequence Analysis of LEAFY Gene and Promoter of Neosinocalamus affinis

作　　者：白琼[1] 孙晓娇[1] 于丽霞[1] 鄢波[1] 

机构地区：[1]西南林业大学园林学院,云南昆明650224

出　　处：《竹子研究汇刊》2014年第2期16-24,共9页Journal of Bamboo Research

基　　金：国家自然科学基金项目(31060042);国家自然科学基金项目(31160177)

摘　　要：研究以开花慈竹叶片为材料,采用RT-PCR、染色体步移和RACE相结合的方法克隆得到慈竹LFY基因的完整片段,该基因DNA全长为3 116 bp,包含5'UTR和3'UTR,3个外显子和2个内含子序列,有一个1 161 bp的完整开放阅读框,编码386个氨基酸,LFY蛋白相对分子质量为42.29 kDa,等电点为8.85。蛋白质二级结构预测发现α-螺旋结构和无规卷曲为主导。通过Tail-PCR技术克隆得到1 003 bp的慈竹LFY基因的启动子序列,包含花粉特异表达元件、种子特异表达元件、激活StDof1蛋白在保卫细胞特异表达元件、控制大豆胁迫下的应答元件MYC识别位点、光调节相关元件及AGL15结合位点保守元件等。利用MAGA5.1软件构建慈竹与44种已知的不同植物来源的LFY基因氨基酸序列的系统发育树,结果显示系统发育树中分为2个大的分支,其中裸子植物和蕨类植物遗传距离较近,禾本科植物中慈竹与玉米遗传距离较近,其次为小麦。该研究从慈竹中克隆得到与花分生组织形式相关基因LFY基因,该研究为今后从分子水平上研究竹类植物开花机理打下基础。A 3 116 bp DNA fragment of Neosinocalamus affinis was isolated and cloned by RTPCR,Genome Walking and RACE method. The DNA clone contained a complete sequence of3 116 bp LFY homologous gene which included 5'UTR,3'UTR,2 intron and 3 exons and 386amino acids. The molecular weight of the predicted protein was 4. 29 kDa,its theoretical PI was 8. 85,the gene's secondary structure mainly consists of α-helix and random coil. A 1 003bp fragment of LFY promoter was cloned by Thermal Asymmetric Interlaced PCR( Tail-PCR).Promoter. Sequence analysis showed that this promoter fragment had some regulatory elements,such as Pollen-specific expression elements,Seed-specific expression elements,active StDof1protein in guard cell specific expression elements,controlled soybean Stress response elements MYC's recognition sites,light adjustment related components and AGL15 conservative element binding sites. The LFY gene sequences were compared with other 44 plants by MAGA5. 1program to construct the phylogenetic tree. Two big branches were identified. The genetic distance of gymnosperms and ferns was relatively close. Among the grasses,the genetic distance of Neosinocalamus affinis was firstly close to corn,and secondly close to wheat. This may provide a basis at the molecular level for further study of bamboo flowering mechanism.

关 键 词：慈竹 LFY基因 克隆 启动子 系统进化树 

分 类 号：S795.5[农业科学—林木遗传育种]