| 摘要: |
| 【目的】从番木瓜4个不同成熟期果实的转录组中筛选并克隆得到1个转录因子CpMYB114L，构建番木瓜果实的cDNA文库，筛选与CpMYB114L互作的蛋白，探究CpMYB114L参与调控果实成熟的分子机理。【方法】以番木瓜‘GZBG’品种的果实为材料，设计特异引物，克隆CpMYB114L CDS区，利用生物信息学软件分析该基因序列及其编码的蛋白序列。提取番木瓜不同成熟期果实的总RNA，利用all-direct法建立酵母双杂交cDNA文库。构建pGBKT7-CpMYB114L诱饵载体，对其自激活检测后，通过共转化从文库中筛选CpMYB114L互作蛋白。通过在NCBI网站上进行序列比对，明确互作蛋白的功能分类，最后对各类蛋白基因在番木瓜不同成熟期果实中的表达进行分析。【结果】CpMYB114L基因的CDS序列编码227个氨基酸，预测其相对分子质量为26 304.3，理论等电点为6.23，亲水性总平均值为－0.893。CpMYB114L蛋白序列与甜樱桃（PaWERL）、蒺藜苜蓿（MtMYB1）、黧豆（MpMYB113）、大豆（GmMYB1）和苹果（MdMYB308L）的同源性较近。cDNA文库总库容为1.15×107 CFU，重组率100%，插入片段长度为750~2 000 bp。诱饵载体pGBKT7-CpMYB114L不存在自激活。从酵母双杂交文库中筛选到30个初始阳性克隆，经回转验证后最终获得26个与CpMYB114L互作的蛋白，共分为4类，包括乙烯响应受体1（CpERS1）、酰基辅酶A硫酯酶13（CpAcots13）、热休克蛋白42（CpHSP42）和叶绿体转运子159（CpTOC159）。在果实成熟过程中，CpERS1和CpHSP42表达量先上升后下降，CpAcots13表达量持续上升，CpTOC159表达量剧增后趋于平稳。【结论】推测CpMYB114L与CpERS1蛋白互作参与番木瓜果实成熟的调控。 |
| 关键词: 番木瓜 酵母双杂交 果实成熟 CpMYB114L 互作蛋白筛选 |
| DOI： |
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| 基金项目:国家自然科学基金项目（32102355）；广东省现代农业产业技术体系创新团队建设项目（优稀水果）（2021KJ116）；广州市科技计划项目（202102020038）；广东省基础与应用基础研究基金项目（2020A1515011166）；广东省农业科学院院长基金项目（202011） |
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| Construction of yeast two-hybrid library and screening of CpMYB114L interacting protein in papaya fruit |
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ZHOU Chenping,YANG Min,GUO Jinju,et al
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| Abstract: |
| 【Objective】A transcription factor related to fruit repining,named CpMYB114L,was screened from the transcriptomes of papaya fruit at four different mature stages.The cDNA library of papaya fruit was constructed and the proteins interacting with CpMYB114L were screened to reveal the molecular mechanism of CpMYB114L in regulating papaya fruit ripening.【Method】The full length CDS region of CpMYB114L was cloned from ‘GZBG’ papaya fruit with the designed specific primers,and its gene and coding protein sequence were analyzed using bioinformatics software.The total RNA was extracted from fruits at different mature stages,and the yeast two-hybrid cDNA library was established by the all-direct method.The pGBKT7-CpMYB114L bait vector was constructed and tested for self activation.The CpMYB114L interacting proteins were screened from the library by co-transformation.Through sequence alignment on the NCBI website,the functional classification of the interacting proteins was clarified and their gene expression in papaya fruit at different mature stages was analyzed.【Result】The CDS region of CpMYB114L gene encoded 227 amino acids with predicted relative molecular mass of 26 304.3,theoretical isoelectric point of 6.23 and average hydrophobic value of -0.893.The protein sequence of CpMYB114L was homologous to Prunus avium (PaWERL),Medicago truncatula (MtMYB1),Mucuna pruriens (MpMYB113),Glycine max (GmMYB1),and Malus domestica (MdMYB308L).The library capacity was 1.15×107 CFU,the recombination rate was 100%,and the length of inserted fragment was from 750-2 000 bp.The pGBKT7-CpMYB114L bait vector showed no self-activation in yeast.Thirty initial positive clones were screened from the yeast two-hybrid library.After identification of interacting proteins,26 potential interacting proteins were obtained and divided into four categories,including ERS1,Acots13,HSP42 and TOC159.During fruit ripening,the expression levels of CpERS1and CpHSP42 increased and then decreased,that of CpAcots13 continuously increased,while that of CpTOC159 increased and then stabilized.【Conclusion】It was speculated that CpMYB114L interacted with CpERS1 protein to regulate papaya fruit ripening. |
| Key words: papaya yeast two-hybrid fruit ripening CpMYB114L interaction protein screening |
