荠菜CRABS CLAW的克隆与拟南芥遗传转化

Cloning of Capsella bursa-pastoris CRABS CLAW Gene and Transform into Arabidopsis thaliana

根据GenBank收录的CRC基因cDNA序列设计引物,以短角果荠菜(Capsella bursa-pastoris)为材料,通过RT-PCR扩增出与拟南芥CRC基因同源的全长cDNA,进行测序、比对及同源性分析。结果显示,克隆的该基因cDNA序列与报道的拟南芥CRC序列一致性达到93%,可以推断其为荠菜CRC基因的cDNA(cbCRC)。以Ti质粒pWM101为载体,构建了由CaMV35S启动子调控的cbCRC基因植物表达载体pWM101-cbCRC,采用根癌农杆菌滴注柱头法转化拟南芥,获得了转cbCRC基因的拟南芥植株。转基因拟南芥心皮果荚形态大小发生了一定的变化,说明荠菜cbCRC基因在拟南芥中的表达对拟南芥心皮形态和大小都产生了一定影响,但其并没有使拟南芥表现出荠菜短角果的形态。

CRABS CLAW（CRC） is a key gene controlling the carpel development in Arabidopsis thaliana.It encodes a transcription factor that is belongs to MADS family.A pair of primer was designed and synthesized according to the CRC cDNA sequence of A.thaliana and applied to amplify the homologous cDNA of Capsella bursa-pastoris,a typical silicle cruciferous weed.A predict fragment of cDNA was amplified from the total RNA of C.bursa-pastoris by RT-PCR then it was cloned and sequenced.The cDNA is homologous to CRC of A.thaliana with the homologous identity 93% and it is also predicted encoding a MADs transcription factor protein.The cloned cDNA is subsequently cloned into Ti plasmids pWM101 under the control of a consistent promoter CaMV35S.The recombinant was then transformed into A.thaliana by inflorescence direct dipping with the agrobacteria.The transgenic A.thaliana are screened out and the phenotype of carpel is observed.The carpel varies both in size and shape but all attribute to silique.It is conclude that the homologous CRC expression in A.thaliana will make the carpel varies in some extent but keep in the shape of silique.