甘蓝KAPP编码基因的克隆与序列分析

采用PCR、RT-PCR及其他分子生物学方法,以甘蓝基因组DNA、花蕾RNA和叶片RNA为模板,分别对甘蓝KAPP gDNA和KAPP cDNA进行扩增,分别获得3247bp的KAPP gDNA片段、1699bp的KAPP cDNA片段、1578bp的花蕾KAPP2 cDNA片段和1581bp的叶片KAPP2cDNA片段。对克隆的甘蓝KAPP gDNA和cDNA(结合报道的KAPPcDNA)进行比对表明,甘蓝KAPP基因包含11个内含子,均符合"GU-AG"剪接规则,并且克隆得到的KAPP cDNA序列与报道的KAPP cDNA序列有6处单个碱基的差异,但两者的氨基酸序列完全一致。然而花蕾KAPP2 cDNA、叶片KAPP2 cDNA片段与报道的KAPP cDNA序列相似性分别为85.2%和85.0%。这两个序列分别在590bp和593bp处较早出现一个无义突变引起的终止密码子。Blast分析表明,两基因编码的氨基酸序列与拟南芥KAPP氨基酸序列相似性最高,其次为甘蓝KAPP氨基酸序列。以已报道的8种植物KAPP的CDS及本实验所克隆的两个KAPP2序列构建分子进化树,获得序列与甘蓝KAPP序列聚为一支。结合比较作图及分子进化树,推测KAPP基因在甘蓝基因组上有两个拷贝,而笔者克隆到的KAPP2 cDNA序列是其中一个拷贝,是KAPP进化过程中突变失活的拟基因。

Cloning and Sequence Analysis of KAPP Gene in Brassica oleracea

The gDNA and cdna fragments of KAPP gene were amplified from genomic DNA, bud Rna and leaf Rna in Brassica oleracea by PCR and RT-PCR and other molecular biology methods. We initially obtained a fragment of KAPP gDNA with length of 3 247 bp, a fragment of KAPP cDNA with length of 1 699 bp, as well as a bud cdna with length of 1 578 bp and a leaf cdna with length of 1 581 bp which were called KAPP2 cDNA of bud and KAPP2 cDNA of leaf respectively. Compared gDNA with cDNA of KAPP, we found that there were 11 introns in KAPP gene, and these introns all followed typical GU-AG rule. Between KAPP cDNA which we cloned and KAPP cDNA released there are six nucleotide differences, but they encode a same amino acid sequence. Sequences analysis of the KAPP2 cDNA which we cloned from bud cdna and leaf cdna in Brassica oleracea showed that they share 85.2% and 85.0% identity with the reported KAPP cDNA respectively.We also found that the nonsense mutation in 590 bp of KAPP2 of bud cDNA and 593 bp of KAPP2 of leaf cDNA led to the early appearance of termination codon, who’s Blast indicated that both of them shared more identity in Arabidopsis thaliana than in Brassica oleracea. Based on KAPP cDNA sequence of eight species released by NCBI and two sequences of KAPP2 cDNA cloned in this study, we constructed a phylogenetic tree of KAPP gene, which showed that the two KAPP2 sequences were in the same group with released KAPP cDNA in Brassica oleracea. Based on all the above analysis, as well as some view of the comparative mapping research, we speculated that KAPP gene may have more than one copy in the genome of Brassica oleracea,the KAPP2 sequence cloned in this study may be the other copies of KAPP gene. Moreover, they are likely to be the pseudogene which was inactivated by mutation during evolution process. It has an important significance for the deep research between KAPP and Self-incompatibility. The results will provide some new insights into the research of molecular mechanism in SI and molecular evolution in Brassica oleracea.