摘要
【目的】克隆棉花精氨琥珀酸合成酶基因Gh ASS1的c DNA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c DNA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c DNA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q RT-PCR检测其在盐胁迫下的表达模式;将Gh ASS1的开放阅读框连接到原核表达载体p Cold-TF上,构建融合表达载体p Cold-Gh ASS1,转化到大肠杆菌Rosetta(DE3)plys S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Na Cl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Gh ASS1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c DNA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 k D,等电点6.74;序列比对分析表明Gh ASS1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Gh ASS1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Gh ASS1的表达,1 d时达到峰值,随后逐渐下降,推测Gh ASS1参与棉花对盐胁迫的早期响应。表达载体p Cold-Gh ASS1在工程菌中表达的融合蛋白分子量约108 k D,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,p Cold-Gh ASS1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Na Cl的培养基中p Cold-Gh ASS1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c DNA序列Gh ASS1,推测其在植物体内可参与植物的生长和耐盐能力的调控。
【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 c DNA fragment, named as Gh ASS1, was obtained from young leaves of cotton by the querying probe, a putative argininosuccinate synthetase c DNA 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 q RT-PCR. After the open reading frame of Gh ASS1 was linked to p Cold-TF, the fusion expression vector, p Cold-Gh ASS1, was constructed, and then transformed into the bacterial strain of Rosetta（DE3）plys S 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 p Cold-Gh ASS1 engineering bacteria were assayed by HPLC. The growth status of engineering bacteria was monitored under different Na Cl 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 Gh ASS1 had 10 exons and 9 introns which was located on chromosome 1 of D genome in cotton, its c DNA 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 k D and isoelectric point of 6.74. Sequence alignment analysis showed that Gh ASS1 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 Gh ASS1 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 Gh ASS1 was up-regulated to the highest after 1 d under salt stress, then gradually declined in the leaves, indicating Gh ASS1 m RNA took part in the early response to salt tolerance. The SDS-PAGE analysis revealed that the fusion recombinant protein of Gh ASS1 was a molecular weight of 108 k D as expected. The fusion protein had argininosuccinate synthase activity in vitro. Compared with the control bacteria with p Cold-TF, the contents of free L-Cit were decreased, while those of free L-Arg were increased in the engineering bacteria with p Cold-Gh ASS1. In addition, the engineering bacteria with p Cold-Gh ASS1 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 Na Cl 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.
出处
《中国农业科学》
CAS
CSCD
北大核心
2016年第7期1242-1253,共12页
Scientia Agricultura Sinica
基金
河北省自然科学基金(C2010001804)
河北省遗传学重点发展学科(201221)
