茶花CjAPL1基因干扰表达载体的构建与遗传转化  

RNA Interference Expression Vector Construction of CjAPL1 Gene from Camellia japonica and Genetic Transformation

作　　者：孙迎坤[1,2] 李纪元[1] 殷恒福[1] 

机构地区：[1]中国林业科学研究院亚热带林业研究所,浙江富阳311400 [2]青岛农业大学园林与林学院

出　　处：《青岛农业大学学报（自然科学版）》2013年第2期114-117,123,共5页Journal of Qingdao Agricultural University(Natural Science)

基　　金：国家国际科技合作计划项目(2011DFA30490);国家十二五科技支撑课题(2012BAD01B07);中央级公益性科研院所基本科研业务费专向资金(RISF6141)

摘　　要：茶花CjAPL1基因是MADS-box基因家族的AP1同源基因,AP1基因对于植物花器官的发育具有重要调控功能。本实验选取CjAPL1基因中长度为292bp的基因干扰片段,采用双酶切方法构建了有反向重复序列的RNA干扰中间载体pUCC-CjAPL1。分别用Pst I单酶切pUCC-CjAPL1中间载体和pCAMBIA1300植物表达载体,将有反向重复序列的小片段与pCAMBIA1300大片段连接,得到最终植物干扰表达载体pCAMRNAi-CjAPL1。采用热激法转化农杆菌GV3101,花序浸染法转化野生型拟南芥植株。利用潮霉素筛选阳性植株,进一步进行PCR扩增验证和表型观察统计。结果表明,pCAMRNAi-CjAPL1表达载体构建完整,且干扰载体成功地导入拟南芥基因组中。转基因植株表现出雄蕊缺失1～2枚的花型变化,证明CjAPL1基因对于雄蕊等花器官的发育起到了调控作用。CjAPL 1 gene cloned from Camellia japonica is the AP 1 homologous gene belonged to MADS-- box gene family. And AP 1 could have important regulatory function to floral organs development. The fragment length of 292bp in CjAPL 1 gene was selected and used to construct RNA interference intermedi- ate vector with inverted repeat sequence pUCC- CjAPL1 by double enzyme digestion. The pUCC -- CjAPL 1 vector and PCAMBIA1300 expression vector were respectively digested by Pst I. Then small fragment with inverted repeat sequence and larger fragment in pCAMBIA1300 achieved were connected to construct pCAM--RNAiCjAPL1 expression vector finally. The vector was transformed to Agrobacterium tumefaciens GV3101 and then guided into Arabidopsis thaliana wild type by inflorescence impregnation. The positive plants with hygromycin resistance were dealt with PCR amplification and phenotypic observa- tion statistics. The result showed that pCAMRNAi-- CjAPL1 expression vector was completely construc- ted and successfully guided into genome of Arabidopsis thaliana. The positive plants showed floral organs change of 1 or 2 stamens deletion. This indicated that CjAPL1 gene could regulate and control the floral organs （such as stamen, and so on） development.

关 键 词：茶花 CjAPL1基因 干扰表达 载体构建 遗传转化 

分 类 号：S685.14[农业科学—观赏园艺]