陆地棉GhDAHP基因的克隆与表达分析

棉花黄萎病是制约棉花产业健康发展的一种重要病害,在生产上未发现有效的根治办法,使用现代基因工程技术,从基因库中挖掘抗黄萎病相关基因,为棉花抗黄萎病育种工作的提供理论基础。本研究根据在GenBank中检索到抗黄萎病基因SDAHP(GU479467,1,3-脱氧-阿拉伯庚酮糖-7-磷酸合成酶),从陆地棉基因组数据库中检索到3个同源基因(NM_016893351.1,XM_016849738.1和XM_0168151150.1),根据3个同源基因核苷酸序列设计引物,以陆地棉cDNA为模板进行克隆,获得3个目的基因,依次命名为GhDAHP-1、GhDAHP-2和GhDAHP-3。利用ProtParam和Prot Scale等在线软件对目的基因进行生物信息学分析;RT-PCR分析黄萎病菌诱导后目的基因在陆地棉叶片中的表达量。结果表明:GhDAHP-1序列全长1983 bp,氨基酸总数为539,分子质量约59.45378 kD,理论等电点pI为8.7;GhDAHP-2序列全长1832 bp,氨基酸总数516,分子质量约57.02998 kD,理论等电点pI为7.68;GhDAHP-3序列全长1652 bp,氨基酸总数517,分子质量约56.95491 kD,理论等电点pI为8.52。系统进化树分析,GhDAHP与雷蒙德氏棉(Gossypium raimindii)、木本棉(Gossypium arboreum)的亲缘关系最近。棉花叶片在黄萎病菌的处理下,GhDAHP基因表达量呈现先升高后降低的趋势,推测受到黄萎病诱导后,GhDAHP基因可能参与了黄萎病菌侵染的防卫过程。

Cloning and Expression Analysis of GhDAHP Gene in Gossypium hirsutum

Cotton Verticillium wilt was an important disease that restricts the healthy development of the cotton industry,no effective radical cure has been found in production.Using modern genetic engineering techniques,mining Verticillium wilt resistance related genes from GenBank,to provided theoretical basis for cotton breeding for Verticillium wilt resistance.In this study,according to the GenBank retrieved that the resistance Verticillium wilt gene was StDAHP(Gene accession number:GU479467,1,3-deoxy-D-arabino-heptulosonate 7-phosphate).It was retrieved 3 homologous genes in Gossypium hirsutum genetics database(NM_016893351.1,XM_016849738.1 and XM_0168151150.1).According to the 3 homologous genes nucleotide sequence for designed primer,used Gossypium hirsutum cDNA was template for cloning that got 3 target gene,they were named respectively as follows GhDAHP-1 GhDAHP-2 and GhDAHP-3.For bioinformatics analyzed of target gene by online software ProtParam and Prot Scale so on.When cotton was induced by Verticillium dahliae,RT-PCR analyzed the target gene's expression in leaves of Gossypium hirsutum L.The results showed that:GhDAHP-1,its sequence length was 1983 bp,total amino acids was 539,molecular weight was about 59.45378 kD,and theoretical isoelectric point(pI)was 8.7.GhDAHP-2,its sequence length was 1832 bp,total amino acids was 516,molecular weight was about 57.02998 kD,and theoretical isoelectric point(pI)was 7.68.GhDAHP-3,its sequence length was 1652 bp,total amino acids was 517,molecular weight was about 56.95491 kD,and theoretical isoelectric point(pI)was 8.52.Phylogenetic analyzed showed that GhDAHP was the closest to Gossypium raimindii and Gossypium arboreum.After vaccinated by Verticillium dahliae of cotton leaves,GhDAHP's expression was a tendency to go up first and then down.It was speculated that after being induced by Verticillium dahliae,GhDAHP may be involved in the defense process of Verticillium wilt infection.