番木瓜CpTIFY10A-like基因克隆及表达分析  

Cloning and expression analysis of CpTIFY10A-like gene in Carica papaya L.

作　　者：吕金慧[1] 王府润 陈萍[1] LYU Jin-hui;WANG Fu-run;CHEN Ping(College of Horticulture,Hainan University,Haikou 570228,China)

机构地区：[1]海南大学园艺学院,海口570228

出　　处：《南方农业学报》2020年第6期1308-1315,共8页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31560544);海南省自然科学基金项目(317009)。

摘　　要：【目的】克隆番木瓜CpTIFY10A-like基因序列,并分析其表达特性,为探索该基因功能和抗番木瓜环斑病毒(PRSV)分子机制提供理论依据。【方法】结合前期研究的抗PRSV转录组数据,利用RACE对上调表达基因Cp-TIFY10A-like进行克隆,获得该基因cDNA全长序列,分析其编码区序列(CDS)及进行生物信息学分析。利用已鉴定的PRSV对3月龄番木瓜植株进行不同处理,以感染PRSV且出现病症的植株(CK+)、1.0 mL/L禾甲安(CTS-N)灌施感染PRSV且出现病症的植株(B)为处理组,以清水灌施(不添加禾甲安)不感染PRSV的植株(CK-)为对照组,实时荧光定量PCR(qRT-PCR)检测不同处理下PRSV基因和CpTIFY10A-like基因在番木瓜叶片中的表达情况。最后将CpTIFY10A-like基因CDS序列连接至pET28a-sumo载体上构建原核表达载体,转化Rosetta(DE3)感受态细胞,通过不同浓度IPTG诱导蛋白表达,利用SDS-PAGE电泳检测原核表达情况。【结果】CpTIFY10A-like基因全长为1352 bp,CDS序列为822 bp,编码273个氨基酸残基;其编码蛋白理论等电点(pI)9.23,不稳定系数62.97,亲水性平均系数-0.458,属于不稳定的亲水性碱性蛋白,定位在细胞核上,其二级结构以无规则卷曲和α-螺旋为主,同时含有少量的β-折叠和延伸链。CpTIFY10A-like蛋白与酸枣和白梨TIFY蛋白的氨基酸序列相似性较高,均含有特定的TIFY结构域,属于TIFY蛋白家族,三者在系统发育进化树上聚在同一分支,表明亲缘关系较近。不同处理的番木瓜叶片中,PRSV基因的相对表达量排序为CK+(1.02)>B(0.39)>CK-(0),CpTIFY10A-like基因的相对表达量排序为B(53.12)>CK+(1.15)>CK-(1.02),且CpTIFY10A-like基因在经禾甲安处理的感染PRSV番木瓜叶片中相对表达量显著升高(P<0.05,下同),而PRSV基因的相对表达量显著降低。CpTIFY10A-like基因在原核细胞中表达蛋白的分子量与理论分子量(29.36 kD)相符,且不同浓度IPTG诱导下,IPTG浓度越高,蛋白表达量越多,表明�【Objective】This study cloned the papaya CpTIFY10A-like gene sequence and analyzed its expression characteristics,in order to provide a theoretical reference for exploring the function of this gene and the molecular mechanism of resistance to papaya ring spot virus(PRSV).【Method】Combined with the anti-PRSV transcriptome data of the previous research of the research group,the up-regulated gene CpTIFY10A-like was cloned using RACE technology.The fulllength cDNA was obtained and the coding sequence(CDS)was analyzed,then bioinformatic analysis of this gene was conducted.Three-month-old papaya plants were treated differently with the identified PRSV.With plants infected with PRSV and showing symptoms(CK+),plants infected with 1.0 mL/L CTS-N infected with PRSV and showing symptoms(B)were the treatment groups.The plants irrigated with clear water(without the addition of CTS-N)and not infected with PRSV(CK-)were as the control group.qRT-PCR was used to detect the expression of PRSV gene and CpTIFY10A-like gene in papaya leaves under different treatments.At last,connected the CDS sequence of CpTIFY10A-like gene to pET28asumo vector to construct a prokaryotic expression vector.The reconstruction vector was transformed into competent cell Rosetta(DE3)and induced protein expression by different concentrations of IPTG.The prokaryotic expression of this gene was detected by SDS-PAGE electrophoresis.【Result】The results demonstrated that the gene was 1352 bp in fulllength,of which the CDS sequence was 822 bp,and it encoded 273 amino acid residues.The theoretical isoelectric point(pI)of the encoded protein was 9.23,the instability coefficient was 62.97,and the average hydrophilicity coefficient was 0.458.It belonged to unstable hydrophilic basic protein.Subcellular localization predicted that the gene was located on the nucleus.The secondary structure of the protein expressed by this gene was mainly random curl andα-helix,and also contained a small amount ofβ-turn fold and extended chain.Amino acid sequence of CpTIFY

关 键 词：番木瓜 CpTIFY10A-like基因 生物信息学 原核表达 禾甲安(CTS-N) 番木瓜环斑病毒(PRSV) 

分 类 号：S667.9[农业科学—果树学]