大豆AK基因的克隆与序列多样性分析

Molecular Cloning and Gene Diversity Analysis of Gene AK in Soybean

基于NCBI网站下载获得的大豆AK基因cDNA序列设计特异引物,以大豆品种东农42总RNA为模板,通过RT-PCR获得约1 690 bp cDNA片段,经序列比对认定为AK基因序列。以19个赖氨酸和蛋氨酸含量特殊的大豆品种为材料,采用相同方法在RNA中进行扩增,并对产物测序,利用软件对所得序列进行分析。结果显示：在不同的大豆品种中AK基因的核酸序列存在两种变异方式,第一种是在336 bp产生1个T碱基突变的基因序列,第二种是在1 369-1 374 bp处出现“CAAGTG”6个碱基插入的序列;在蛋白质水平上,主要是在456-457个氨基酸处存在A和S两个氨基酸插入突变。AK基因结构完整,其编码的蛋白产物具有AA_kinase、Carbamate kinase-like、Aspartate kinase和ACT保守结构域。本研究首次对大豆中AK基因多样性进行了分析,为大豆天冬氨酸代谢途径其它相关酶基因的研究提供了理论依据,也为大豆AK基因在植物基因工程等领域的研究和应用奠定了良好的基础。

AK gene was cloned from soybean, a cDNA product about 1 690 bp was amplified from the total RNA of soybean leaves by reverse transcription PCR（RT-PCR） with specific primers designed with the full length sequence of AK gene from NCBI. The cDNA of 19 varieties were amplified with the same way. The PCR products were cloned, sequenced, and analyzed with bioinformatics software. The results showed that there were two mutant ＇alleles of nucleotide sequences in different soybean varieties, the first was the sequence which had a nucleotide substitution, the second was the sequence which had an insertion of 6 nucleotides between 1 369 and 1 374 bp. There were only one mutant allele of amino acid sequences in protein level, which was an insertion of 2 amino acids at 456 - 457 amino acid residue. The structure of AK was completed, the conserved domains of AA_kinase, Carbamate kinase-like, Aspartate kinase, and ACT were found in protein of AK. The gene diversity analysis of gene AK in soybean was studied for the first time in this research. The results provided a theoretical basis for the study of other related enzymes in the aspartate pathway of soybean, and also laid a good foundation for the research and application of AK gene in plant genetic engineering and other fields.