甘蓝、大白菜和甘蓝型油菜EXO70A1基因的克隆与表达特性

从甘蓝、大白菜与甘蓝型油菜中分离出EXO70A1基因，对该基因的序列进行生物信息学分析, 然后转化酵母Y187, 应用半定量RT-PCR检测BoEXO70A1基因的表达特性。结果表明, 3种芸薹属植物EXO70A1序列长度均为1 917 bp，相似性97.1%, 它们的gDNA序列均为单一序列，长度分别为3 797、3 752和3 770 bp，一致度达91.0%，均由12个外显子及11个内含子组成，除了第4、第5、第6、第8个内含子外，其余内含子的保守性低于外显子; 推导的3种蛋白质序列(BnEXO70A1、BrEXO70A1和BoEXO70A1)的相似度与一致性分别达99.8%与98.1%，其二级结构、三维结构及理化特性高度相似。EXO70A1基因的11个内含子的剪切位点均符合“GU-AG”法则，剪切受体(AG)的前20~50个碱基存在一段保守的序列“CU(A/G)A(C/U)”; 3种芸薹属植物与拟南芥EXO70A1基因的12个外显子的对应序列长度完全相同，所构成的编码区的序列一致性达90.1%，相应的蛋白质序列的相似度与一致性分别达99.8%与93.7%; 分子进化分析表明, EXO70A1在整个EXO70蛋白家族中及不同的植物间表现出较高的保守性; BoEXO70A1在酵母细胞Y187呈现弱表达; EXO70A1在甘蓝的雄蕊、幼茎、幼嫩花瓣、雌蕊、幼根及叶片中均能表达，可能属于组成型表达基因，但是其表达量在不同发育时期的不同器官中存在差异，授粉前雌蕊中最高，雄蕊中最低。由此可知，EXO70A1在芸薹属植物中整体高度保守, 但在酵母转化株和甘蓝各器官中的组成型表达有所差异，推测EXO70A1在植物细胞中具有多种重要的功能。

Cloning and Expression Characteristics of EXO70A1 from Brassica oleracea,Brassica campestris,and Brassica napus

EXO70A1 is an important signal element of self-incompatibility in Brassica. In this study, coding sequences (CDS) of EXO70A1 and the corresponding gDNA were cloned by PCR. Sequence analysis was carried out by means of bioinformatics. RT-PCR was used to analyze expression characteristics of EXO70A1 in Brassica oleracea. And BoEXO70A1 was transformed into yeast Y187 to explore its expression. The results showed: That the gene lengths of BnEXO70A1, BrEXO70A1 and BoEXO70A1 were 3 797, 3 752, and 3 770 bp, respectively, and all consisted of 12 exons and 11 introns; their identity positions reached 91%. Sequence conservation was higher in exons than in introns excluding 4th, 5th, 6th, and 8th intron. CDS of three EXO70A1 genes consisted of
1 917 base pairs and sequence similarity was 97.1%. Three proteins of EXO70A1 conduced all consisted of 638 amino acids; the similarity and identity of EXO70A1 in the three species were 99.8% and 98.1%, respectively. BnEXO70A1, BrEXO70A1, and BoEXO70A1 shared similar secondary structures and three-dimensional structures. All introns started from the sequence GU and ended with the sequence AG (in the 5'' to 3'' direction). They are referred to as the splice donor and splice acceptor site, respectively. Another important sequence “CU(A/G)A(C/U)” in all introns of EXO70A1 genes was located at 20–50 bases upstream of the acceptor site. The corresponding sequence lengths of all 12 exons of EXO70A1 were identical among three Brassica species and Arabidopsis thaliana; and similarity of four coding sequences was 90.1%. The similarity and identity of EXO70A1 proteins in the four species reached 99.8% and 93.7%, respectively. EXO70A1 was a subunit of EXO70 family which showed high conservative in Magnoliophyta. The results showed that BoEXO70A1 presented weak expression in Y187, while expressed in stamens, young stems, petals, pistils, young roots and leaves. However, its expression quantity was different in different tissues, with strong expression in pistils and weak in stamens. Thus conclusions can be drawn that: EXO70A1, in Brassica, is high conservative; EXO70A1 perhaps plays very important roles in plant cells because of its different expression characteristics in different plant tissues.