四季秋海棠<i>BsMYB62</i>基因酵母双杂交诱饵载体的构建及互作蛋白的筛选

刘静; 王阳; 姚珂心; 屈莹; 王瑞博; 刘胜男; 李永华; 张开明

doi:10.19303/j.issn.1008-0384.2021.04.006

四季秋海棠BsMYB62基因酵母双杂交诱饵载体的构建及互作蛋白的筛选

Construction of Yeast Two-hybrid Bait Vector and Identification of Proteins Interacting with BsMYB62 in Begonia semperflorens

摘要

摘要:
目的低温和高光均能引起四季秋海棠（Begonia semperflorens）叶片因合成积累花色素苷而变红。本研究从低温和高光处理四季秋海棠的转录组中筛选并克隆得到1个 MYB 转录因子基因并命名为 BsMYB62 ，对其进行生物学信息分析，并从四季秋海棠cDNA文库中筛选出与其互作的蛋白。
方法设计BsMYB62引物，克隆 BsMYB62 CDS区，利用生物信息学软件对BsMYB62基因序列及其编码蛋白序列分析。构建 pGBKT7-BsMYB62 酵母双杂交诱饵载体，对该诱饵载体进行毒性和自激活检测后, 用 Mating 法筛选BsMYB62的互作蛋白。
结果 BsMYB62 基因的 CDS 区为 801 bp（NCBI 登录号：MT560845），编码266个氨基酸。 pGBKT7-BsMYB62 有自激活现象，对宿主酵母菌无毒性。在含 45mM 3-AT 的 SD/-Trp-Leu-His-Ade 培养基上能明显抑制 BsMYB62 蛋白的自激活。酵母双杂交结果显示与 BsMYB62 蛋白互作的蛋白有 9 类，分别是 XTH9、LHB1B2、EBS7、PSI-F、UBE2、FDH、RBCS1A、FP6 和 BCA。
结论推测四季秋海棠BsMYB62 蛋白与以上9类蛋白互作响应低温、高光胁迫。

Abstract:
Objective The selected transcriptomes of Begonia semperflorens that can trigger the anthocyanin biosynthesis in the plant was used to clone the MYB transcription factor, and the cDNA databank applied to identify the potential interacting proteins associated with the biological process.
Method Transcriptomes of B. semperflorens plant exposed to low temperature or intense light to induce the anthocyanin biosynthesis that turns the leaves red were screened for positive identification and cloning the full length cDNA of an MYB transcription factor, BsMYB62 (Gene ID: MT560845). Bioinformatics and potential interacting proteins of BsMYB62 were obtained with the designed primers and cloned cDNA. A pGBKT7-BsMYB62 bait vector was constructed and tested for toxicity and auto-activation. From the yeast two-hybrid library using the mating method, potential interacting proteins were named.
Result The full length cDNA of BsMYB62 was determined to be 801 bp. The pGBKT7-BsMYB62 was found not toxic to the host yeast and capable of significantly inhibiting auto-activating in SD/-Trp-Leu-His-Ade medium including 45 mM of 3-AT. Nine potential interacting proteins were identified to include XTH9, LHB1B2, EBS7, PSI-F, UBE2, FDH, RBCS1A, FP6, and BCA.
Conclusion It was speculated that BsMYB62 of B. semperflorens interacted with 9 proteins in response to the stress of low temperature or intense light.

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