棉花精氨酸酶基因GhARG1 cDNA的克隆与表达分析

Cloning and Expression Analysis of Arginase Gene GhARG1 cDNA from Gossypium hirsutum L.

为了深入研究棉花精氨酸酶基因对非生物胁迫的响应以及生理功能,利用电子克隆和RT-PCR技术从棉花中获得了精氨酸酶ORF序列Gh ARG1,盐渍、PEG6000胁迫及ABA、SA和MeJA处理棉花幼苗,并原核表达棉花精氨酸酶基因。生物信息学分析表明,其ORF序列长度为1 023 bp,编码340个氨基酸,具有典型的精氨酸酶保守结构域。q RT-PCR技术分析表明,在叶片中该基因转录水平上受盐渍、PEG6000胁迫及ABA、SA处理的诱导,而对MeJA下调其表达。将Gh ARG1完整的编码序列融合到原核表达载体p Cold-TF中,经过IPTG诱导及SDS-PAGE检测表明,Gh ARG1编码产物的分子量约为37 ku,与预期相符。p Cold-Gh ARG1重组菌粗酶液中精氨酸酶活性是对照菌的6倍。研究表明,棉花内源性精氨酸酶基因Gh ARG1可能通过ABA、SA信号途径参与棉花对盐渍和干旱胁迫的响应,且其克隆表达产物具有精氨酸酶活性,为开展其生理功能研究奠定了基础。

It is of great significance to investigate the response of arginase gene to abiotic stress and its physiological function in cotton. Bioinformatics analysis showed that the ORF sequence of GhARG1 was obtained by In silico cloning and RT-PCR technique. Salt stress,PEG6000 stress and ABA, SA and MeJA treatment cotton seedlings, and GhARG1 was expressed in E. coli prokaryotic. The ORF sequence contained 1 023 bp,and encoded 340 amino acids with typical conserved domains of arginase. Quantitative RT-PCR analysis showed that the expression of GhARG1 in leaves was up-regulated under stress condition of NaCl, PEG6000, and signal molecule of ABA,SA,respectively, but down-regulated by MeJA. The coding sequence of GhARG1 was fused to prokaroytic expression vector pCold-TF,and the combinant vectors were induced by IPTG and detected by SDS-PAGE. The molecule weight of protein GhARG1 was detected to be the expected 37 ku. The activity of arginase in the recombinant strain was 6 times that of the control. The results suggested that GhARG1 ,which coding product has arginase activity,might be involved in the response to salinity and drought stress through ABA and SA signaling pathway in cotton,and could lay foundation for further physiological functional identification.