[1]杜林娜,李秀兰,魏东旭,等.细脚拟青霉腺苷酸琥珀酸合酶基因的克隆与表达分析[J].黑龙江农业科学,2018,(06):11-16.[doi:10.11942/j.issn1002-2767.2018.06.0011]
　DU Lin-na,LI Xiu-lan,WEI Dong-xu,et al.Cloning and Expression Analysis of Ptadss Gene from Paecilomyces tenuipes[J].HEILONGJIANG AGRICULTURAL SCIENCES,2018,(06):11-16.[doi:10.11942/j.issn1002-2767.2018.06.0011]
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细脚拟青霉腺苷酸琥珀酸合酶基因的克隆与表达分析

《黑龙江农业科学》[ISSN:1002-2767/CN:23-1204/S] 卷: 期数: 2018年06期 页码: 11-16 栏目: 出版日期: 2018-06-10
Title:
Cloning and Expression Analysis of Ptadss Gene from Paecilomyces tenuipes
作者:
杜林娜1; 2; 李秀兰1; 魏东旭1; 陈鸿兵1; 姚宇1; 杨晶1; 2
1．吉林农业大学 生命科学学院，吉林 长春 130118; 2.吉林农业大学 生物反应器与药物开发教育部工程研究中心，吉林 长春 130118
Author(s):
DU Lin-na1; 2; LI Xiu-lan1; WEI Dong-xu1; CHEN Hong-bing1; YAO Yu1; YANG Jing1; 2
1.College of Life Science,Jilin Agricultural University，Changchun 130118，China; 2.Engineering Research Center of the Chinese Ministry of Education for Bioreactor and Pharmaceutical Development,Jilin Agricultural University,Changchun 130118,China
关键词:
细脚拟青霉; 腺苷酸琥珀酸合酶; 基因克隆; 序列分析; 表达分析
Keywords:
Paecilomyces tenuipes;  adenylosuccinate synthetase;  cloning;  sequence analysis
DOI:
10.11942/j.issn1002-2767.2018.06.0011
文献标志码:
A
摘要:
腺苷酸琥珀酸合酶（ADSS）为细脚拟青霉(Paecilomyces tenuipes)有效成分腺苷合成代谢途径的关键酶，而细脚拟青霉中腺苷酸琥珀酸合酶基因Ptadss的克隆与序列分析鲜见相关报道。本文在细脚拟青霉转录组测序结果的基础上，利用PCR技术克隆得到该菌Ptadss基因的cDNA序列，对Ptadss基因及其编码的蛋白进行生物信息学分析（相关理化性质、结构域、亲水性-疏水性、亚细胞定位、信号肽和高级结构等），并构建PtADSS与相关微生物ADSS的系统进化树。同时，采用实时荧光定量PCR技术，分析Ptadss基因在不同发酵阶段的细脚拟青霉菌丝体中的表达量，以初步明确该基因的表达量对于细脚拟青霉腺苷含量的影响。结果表明：克隆得到的Ptadss基因的ORF序列为1 670 bp。该基因编码的蛋白的基本性质为：423个氨基酸残基、相对分子量为46.7 663 kDa、等电点（pI）为5.59，含58个磷酸化位点，且该蛋白为定位于细胞质中的非分泌性和非跨膜的疏水性蛋白。结构预测结果表明该蛋白是由α-螺旋、β-转角、延伸链和无规则卷曲组成的复杂结构。序列分析结果提示，细脚拟青霉Ptadss基因与Cordyceps confragosa腺苷酸琥珀酸合酶基因同源性最高，可达91%。结合其他微生物的ADSS氨基酸序列构建系统树，结果表明细脚拟青霉与Cordyceps confragosa亲缘关系较近。另经对不同发酵阶段的细脚拟青霉中Ptadss的表达量及腺苷含量分析结果表明：Ptadss基因的表达水平与菌丝体中腺苷含量存在正相关性。
Abstract:
Adenosine is one of most important active ingredients of Paecilomyces tenuipes.Adenylosuccinate synthetase is a key enzyme of adenosine biosynthesis.The cloning and sequene analysis of adenylosuccinate synthetase in P.tenuipes has not been reported.In this study,cloning and sequence analysis of adenylosuccinate synthetase gene was performed.Previously,based on the results of RNA-seq of P.tenuipes,Ptadss gene were identified.In this project,a pair of 䥺Symbol`@@primers was designed and the full-length cDNA fragments of Ptadss gene is to be amplificated using gene cloning technology.Furthermore,characters of Ptadss gene and encoded amino acid residue sequence were analyzed using bioinformatics method,including the physical and chemical properties,hydrophobic/hydrophilic,localiztion sites in cells,signal peptide,secondary structure and teritiary structure.Then,the phylogeny evolution tree was constructed with other microorganism.Additionally,The expression level of Ptadss gene in P.tenuipes cultured different time and the relation between the expression level and the yield of adenosine were also investigated.The cloned Ptadss gene of P.tenuipes had an ORF of 1,670 bp which was predicted to encode a protein of 423 amino acids residues,including 58 phosphorylation sites.There was no signal peptide in Ptadss.The relative molecular weight and theoretical pI of protein are 46.7663 kDa and 5.59,respectively.In addition,subcelluar localization indicated that the prediction of protein was located in cytoplasm.Tertiary structure prediction showed that the predicated protein is mainly composed of alpha helix,β-turns,extended strand and random coil.Sequence alignment showed that predicted Ptadss had high consistency with adenylosuccinate synthetase gene of Cordyceps confragosa.Phylogenetic tree analyses showed that P.tenuipes is closely related to Cordyceps confragosa.Real-time PCR results showed that the expression of Ptadss gene was positively related with adenosine production.The results of this study will provide a foundation for exploring the molecular function of Ptadss involved in adenosine biosynthesis in P.teuipes.

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备注/Memo

收稿日期：2018-03-06基金项目：吉林农业大学科研启动基金资助项目（2015014）；吉林省教育厅“十三五”科学技术研究资助项目吉教科合字\[2016\]-第180号；2016大学生创新计划资助项目（2016-10193042）；吉林省科技厅优秀青年人才基金资助项目（2017-0520035JH）。第一作者简介：杜林娜（1985-），女，博士，从事微生物与生化药学研究。E-mail：dulinna0918@163.com。通讯作者：杨晶（1981），女，博士，副研究员，从事药用植物研究。E-mail：2690338278@qq.com。

更新日期/Last Update: 2018-09-11