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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编码序列长度均为1917bp,相似性97.1%,它们的gDNA序列均为单一序列,长度分别为3797、3752和3770bp,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。
The EXO70A1 gene was isolated from Brassica campestris, Chinese cabbage and Brassica napus. The sequence of the gene was analyzed by bioinformatics, and then transformed into yeast Y187. The expression of BoEXO70A1 gene was detected by semi-quantitative RT-PCR. The results showed that the sequences of EXO70A1 in three Brassica genotypes were 1917 bp in length and 97.1% in similarity, respectively. All of their gDNA sequences were of single sequence with lengths of 3797, 3752 and 3770 bp, respectively, with a consistency of 91.0% Exon and 11 introns. Except for the 4th, 5th, 6th and 8th introns, the other introns were less conservative than the exons. The deduced 3 kinds of protein sequences (BnEXO70A1 , BrEXO70A1 and BoEXO70A1) were 99.8% and 98.1%, respectively, with similar secondary structure, three-dimensional structure and physicochemical properties. The 11 introns of the EXO70A1 gene all have the “GU-AG” rule. The conserved sequence of the first 20 to 50 bases of the excision receptor (AG) is “CU (A / G) A (C / U) ”. The three exons of Brassica and the Arabidopsis EXO70A1 gene had the same length of corresponding exons and the sequence identity of coding region was 90.1% . The molecular evolution analysis showed that EXO70A1 showed high conservation in the entire EXO70 protein family and in different plants. The expression of BoEXO70A1 in yeast Y187 was weakly expressed ; EXO70A1 could express in stamens, young stems, young petals, pistils, young roots and leaves of cabbage, which may belong to the constitutive expression gene, but its expression was different in different organs at different developmental stages. The highest, the lowest in the stamens. Therefore, EXO70A1 is highly conserved in Brassica plants as a whole. However, constitutive expression of EXO70A1 in yeast and cabbage organs is different, suggesting that EXO70A1 has multiple important functions in plant cells.