棉花精氨琥珀酸合成酶基因GhASS1的克隆及表达分析

doi:10.3864/j.issn.0578-1752.2016.07.003

摘要/Abstract

摘要： 【目的】克隆棉花精氨琥珀酸合成酶基因GhASS1的cDNA序列，并利用原核表达系统高效表达融合蛋白，检测融合蛋白的精氨琥珀酸合成酶活性及其表达对工程菌生长、耐盐能力及与游离L-瓜氨酸（L-Cit）和L-精氨酸（L-Arg）含量的影响，为解析该基因的功能及作用机制奠定基础。【方法】以拟南芥推断的精氨琥珀酸合成酶cDNA序列作为探针，利用电子克隆和RT-PCR技术，从棉花幼叶中获得cDNA片段，T/A克隆测序后得其序列信息，利用生物信息学软件分析其DNA结构、蛋白质结构、功能域和同源性，并构建系统进化树。利用qRT-PCR检测其在盐胁迫下的表达模式；将GhASS1的开放阅读框连接到原核表达载体pCold-TF上，构建融合表达载体pCold-GhASS1，转化到大肠杆菌Rosetta(DE3)plysS中进行IPTG诱导表达，利用SDS-PAGE法测定表达产物分子量，焦磷酸荧光法测定酶活性和比活力，HPLC法测定工程菌中L-Cit和L-Arg的含量，对比不同温度和不同NaCl浓度下工程菌与对照菌的生长速度及其体内L-Cit、L-Arg含量及L-Arg/L-Cit。【结果】棉花GhASS1存在于D基因组的第1染色体上，由10个外显子和9个内含子构成，其cDNA全长序列共1 584 bp，其中5′非翻译区22 bp，开放阅读框1 485 bp，3′端非翻译区77 bp，编码产物由494个氨基酸组成，推断分子量为54 kD，等电点6.74；序列比对分析表明GhASS1与大肠杆菌、酵母和拟南芥中同源蛋白序列一致性分别为21.81%、41.1%和78.5%，且具有典型的精氨琥珀酸合成酶结构模序；系统进化分析显示GhASS1与番茄、马铃薯和烟草中同源蛋白亲缘关系最近，而与云杉、卷柏中同源蛋白亲缘关系最远；盐胁迫可上调叶片中GhASS1的表达，1 d时达到峰值，随后逐渐下降，推测GhASS1参与棉花对盐胁迫的早期响应。表达载体pCold-GhASS1在工程菌中表达的融合蛋白分子量约108 kD，与预期相符，在体外具有精氨琥珀酸合成酶活性；与对照菌相比，pCold-GhASS1工程菌中游离L-Cit含量下降，L-Arg含量上升，且在生长周期中较快进入对数生长期；在含高浓度NaCl的培养基中pCold-GhASS1工程菌表现出更强的生长活力和较高的L-Arg/L-Cit值，显示了其具有较强的L-Cit向L-Arg的代谢流。【结论】从棉花中克隆了植物精氨琥珀酸合成酶基因cDNA序列GhASS1，推测其在植物体内可参与植物的生长和耐盐能力的调控。

关键词: 棉花, 精氨琥珀酸合成酶基因, 耐盐胁迫, real-time PCR

Abstract: 【Objective】This research was conducted to clone argininosuccinate synthetase gene from Gossypium hirsutum, obtain the fusion protein by prokaryotic expression system, detect the argininosuccinate synthetase activity of the fusion protein, and identify its effects on the growth, salt tolerance, L-citrulline (L-Cit) and L-arginine (L-Arg) content of engineering bacteria, aiming to lay a foundation for this gene’s function and mechanism. 【Method】The homologous cDNA fragment, named as GhASS1, was obtained from young leaves of cotton by the querying probe, a putative argininosuccinate synthetase cDNA sequence from Arabidopsis thaliana, in silico cloning and RT-PCR reaction. This fragment’s information was acquired by T/A cloning and sequencing. The genomic DNA and putative protein structure, function domain and homology were analyzed by bioinformatics software, and the phylogenetic tree was built. Its expression responses to salt tolerance were investigated by qRT-PCR. After the open reading frame of GhASS1 was linked to pCold-TF, the fusion expression vector, pCold-GhASS1, was constructed, and then transformed into the bacterial strain of Rosetta (DE3) plysS for expressing the recombination protein under the induction of IPTG. The molecular weight of recombination protein was tested by SDS-PAGE. The enzyme activities and specific activities were determinated by the method of pyrophosphoric fluorescence. The contents of free L-Arg and L-Cit in pCold-GhASS1 engineering bacteria were assayed by HPLC. The growth status of engineering bacteria was monitored under different NaCl concentrations in culture medium and different growth temperatures. The contents of free L-Arg and L-Cit, L-Arg/L-Cit ratio were compared. 【Result】 Sequencing analysis showed the genomic DNA of GhASS1 had 10 exons and 9 introns which was located on chromosome 1 of D genome in cotton, its cDNA fragment length was 1 584 bp containing 5′-untranslated region of 22 bp, the open reading frame of 1 485 bp and 3′- untranslated region of 77 bp, encoding 494 amino acid residues with a molecular mass of 54 kD and isoelectric point of 6.74. Sequence alignment analysis showed that GhASS1 shared 21.81%, 41.1% and 78.5% identity with the homologues from E. coli, yeast and Arabidopsis thaliana, respectively, and had the typical function domains of argininosuccinate synthetase. Phylogenetic analysis showed that GhASS1 was most closely related to the homologues from tomato, potato and tobacco, while was farthest to those from Picea sitchensis and Selaginellae moellendorfii. The expression of GhASS1 was up-regulated to the highest after 1 d under salt stress, then gradually declined in the leaves, indicating GhASS1 mRNA took part in the early response to salt tolerance. The SDS-PAGE analysis revealed that the fusion recombinant protein of GhASS1 was a molecular weight of 108 kD as expected. The fusion protein had argininosuccinate synthase activity in vitro. Compared with the control bacteria with pCold-TF, the contents of free L-Cit were decreased, while those of free L-Arg were increased in the engineering bacteria with pCold-GhASS1. In addition, the engineering bacteria with pCold-GhASS1 could be earlier into the logarithmic growth than the control, also showed stronger growth vigor and higher free L-Arg/L-Cit ratio under high NaCl contents in LB medium, revealing the engineering bacteria had stronger metabolic flow from L-Cit to L-Arg than the control. 【Conclusion】 It was the first report on the identification of argininosuccinate synthetase gene from cotton in plant. Its catalysis and physiology function were analyzed by the prokaryotic expression system, suggesting that it could be involved in the regulation of growth and ability of salt resistance in plant.

Key words: cotton (Gossypium hirsutum), argininosuccinate synthetase gene, salt tolerance, real-time PCR

王慧飞，孙艳香，冯雪，张一名，杨江涛，马梅芳，陈光. 棉花精氨琥珀酸合成酶基因GhASS1的克隆及表达分析[J]. 中国农业科学, 2016, 49(7): 1242-1253.

WANG Hui-fei, SUN Yan-xiang, FENG Xue, ZHANG Yi-ming, YANG Jiang-tao, MA Mei-fang, CHEN Guang. Cloning and Expression Analysis of Argininosuccinate Synthetase Gene GhASS1 from Gossypium hirsutum[J]. Scientia Agricultura Sinica, 2016, 49(7): 1242-1253.

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