芸薹属栽培种ALS1-3的克隆及序列分析

Cloning and sequence analysis of ALS1-3 genes from cultivated Brassica species

【目的】揭示6种芸薹属植物乙酰乳酸合成酶（ALS）基因的结构特征,为ALS酶的遗传操作和抗除草剂种质创制奠定基础。【方法】利用特异PCR方法,对甘蓝型油菜（Brassica napus L.）、甘蓝（B.oleracea）、芥菜型油菜（B.juncea）、白菜型油菜（B.rapa）、埃塞俄比亚芥（B.carinata）、黑芥（B.nigra）等6个芸薹属16份材料的ALS基因进行扩增、克隆测序及生物信息学分析。【结果】除1个B.nigra材料未扩增出任何产物外,其余参试材料中均有PCR扩增产物,共成功克隆了30个ALS基因（GenBank登录号为KM816807-KM816836）,且克隆的基因ALS1、ALS2、ALS3均不含内含子,只有一个开放阅读框。ALS1基因开放阅读框长为1 968bp,编码655个氨基酸;ALS2基因开放阅读框长为1 914bp,编码637个氨基酸;ALS3基因开放阅读框长为1 959bp,编码652个氨基酸。参试材料中,在ALS1基因中检测到7个SNP,其中4个导致氨基酸突变;在ALS2基因中检测到3个SNP,其中2个导致氨基酸突变;在ALS3基因中检测到25个SNP,其中2个导致氨基酸突变。在3类ALS蛋白中,ALS1和ALS3遗传距离较近,与ALS2的遗传距离相对较远。ALS1基因定位于C01染色体上,ALS2和ALS3基因定位于A01染色体上。【结论】参试的6个芸薹属不同材料间ALS1、ALS2、ALS3基因序列及其编码的蛋白序列均存在差异。

[Objective] This study revealed the acetolactate synthetase （ALS） genes from six Brassica species to lay foundation for genetic manipulation of ALS and creation of herbicide resistant materials. [Method] Specific PCR method was used to isolate ALS genes from 16 accessions of six Brassica species, including Brassica napus L. , B. oleracea, B. juncea, B. rapa, B. carinata, and B. nigra. Bioinformatics and the isolated ALS genes and their encoding products were also analyzed. [Result] Except for one B. nigra accession,ALS genes from other 15 accessions were successfully amplified. In total,30 ALS genes were cloned and their GenBank numbers （from KM816807 to KM816836） were obtained. ALS1,ALS2 and ALS3 had no introns and only one open reading frame （ORF）. ORF of ALS1 was 1968 bp in length,encoding a peptide of 655 amino acids. ORF of ALS2 was 1 914 bp in length,encoding a peptide of 637 amino acids. ORF of ALS3 was 1 959 bp in length,encoding a peptide of 652 amino acids. Seven single nucleotide polymorphisms （SNP） were detected among ALSl,and four of them resulted in amino acid change. Three SNPs were detected among ALS2,and two of them resulted in amino acid change. Twenty-five SNPs were detected among ALS3,and two of them resulted in amino acid change. Genetic distance between ALS1 and ALS3 was relatively closer than that with ALS2. ALS1 was located in C01 chromosome,while ALS2 and ALS3 were located in A01 chromosome. [Conclusion] Nucleic acid differences in ALS1,ALS2, and ALS3 were detected among six Brassica species, resulting in changes of several amino acids of the encoded proteins.