青花菜BoCHS2基因的克隆、表达和反义载体的构建

CLONING,EXPRESSION AND ANTISENSE VECTOR CONSTRUCTION OF A CHALCONE SYNTHASE GENE BoCHS2 FROM Brassica oleracea var. italica

根据前期的研究结果设计PCR引物,从青花菜(Brassica oleracea var.italica)叶片基因组DNA和cDNA中克隆到了查尔酮合成酶基因,定名为BoCHS2。BoCHS2的基因组DNA全长为1267bp,具一个长度为85bp的内含子,基因编码区全长为1182bp,编码393个氨基酸。基因序列已提交至NCBI,登录号为HQ189776。与BoCHS相比,在核苷酸和氨基酸组成上的差异分别为27bp和8个氨基酸。RT-PCR结果表明,在霜霉菌诱导下,叶片和子叶中BoCHS2的表达量增加。将BoCHS2片段反向插入到带有绿色荧光蛋白基因的pCAMBIA1300载体中,成功构建了植物反义表达载体。

PCR primers were designed according to our previous research results.A chalcone synthase gene,designated BoCHS2,was isolated from leaf genomic DNA and cDNA.Genomic DNA of BoCHS2 was 1267bp in length with an intron of 85bp.The complete coding sequence was 1182bp in length encoding 393 amino acids.The gene sequence was submitted to NCBI with an accession number of HQ189776.There were 27bp and 8 amino acids differences between BoCHS2 and BoCHS at nucleotide and protein level,respectively.Expression analysis results showed an increase of BoCHS2 transcription both in leaves and cotyledons while infected with Hyaloperonospora parasitica.Antisense segment of BoCHS2 was inserted into a pCAMBIA1300 vector with green fluorescent protein gene and plant expression vectors were successfully constructed.