甘蓝型油菜CRABS CLAW基因克隆及其RNA干扰载体的构建  

Cloning of CRABS CLAW gene from Brassica napus and construction of its RNA interference vector

作　　者：周晓婴[1,2,3] 付三雄[1,2,3] 陈松[1,2,3] 张超[3] 戚存扣[1,2,3] 

机构地区：[1]国家油菜改良中心南京分中心,江苏南京210014 [2]农业部长江下游棉花与油菜重点实验室,江苏南京210014 [3]江苏省农业科学院经济作物研究所,江苏南京210014

出　　处：《江苏农业学报》2015年第4期737-742,共6页Jiangsu Journal of Agricultural Sciences

基　　金：江苏省农业科技自主创新基金项目[CX(11)4011];江苏省自然科学基金项目(BK2011668);国家"948"项目(2011-G23)

摘　　要：CRABS CLAW(CRC)转录因子是YABBY基因家族成员,在植物花器官发育过程中起重要作用。为进一步研究CRC转录因子在调控油菜花器官发育过程中的作用,本研究以甘蓝型油菜品种宁油10号花蕾RNA为材料,通过反转录PCR克隆到1个580 bp长度的CRC基因,并利用p Hurricane中间载体构建CRC基因的反向重复序列表达框,将CRC基因片段以正向的方式连接在1个可剪切的内含子的5'末端,以反向的方式连接到该内含子的3'末端;然后将Ca MV35S启动子序列和CRC基因的反向重复表达框再克隆到植物双元载体p CAMBIAI1390的p UC18多克隆位点,构建了干扰表达载体p A6-CRCi,经过酶切鉴定和测序分析,所构建的载体正确。CRABS CLAW ( CRC) is a member of the YABBYA transcription factor gene family and plays an impor-tant role in the development of plant flower organ. In order to study the CRC transcription factor’ s function in regulating the rape flower organ development, a 580-bp segment was cloned by RT-PCR from the total RNA of rapeseed flower organ. An inverted repeated expression cassette was firstly constructed by cloning the 580-bp segment of CRC gene into an intermediate pHurrican vector. The fragment was then ligated to a spliceable fad2 intron sequence. Finally, both the inverted repeat cas-sette and CaMV35S promoter were inserted into the multiple clone site of binary vector pCAMBIA1390. The ihpRNA ex-pression vector was named pA6-CRCi, and the construction of which was further confirmed by the digestion of restriction enzymes and sequencing.

关 键 词：油菜 CRABSCLAW基因 RNA干扰载体 

分 类 号：S565.4[农业科学—作物学]