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

doi: 10.19303/j.issn.1008-0384.2021.04.006

河南农业大学风景园林与艺术学院，河南　郑州　450002

基金项目: 河南省自然科学基金培育项目（182300410091）

详细信息

作者简介:
刘静（1997−），女，硕士研究生，研究方向：园林植物生长发育与调控（E-mail：liujing9223@126.com）

并列第一作者：王阳（1995−），男，硕士研究生，研究方向：园林植物生长发育与调控（E-mail：wangyang1995@stu.henau.edu.cn）

通讯作者:
张开明（1978−），女，博士，教授，研究方向：园林植物生长发育与调控（E-mail：miss1199@126.com）

中图分类号: S 682.19
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出版历程
- 收稿日期: 2020-09-04
- 修回日期: 2020-12-18
- 网络出版日期: 2021-03-27
- 刊出日期: 2021-04-30

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

College of Landscape Architecture and Arts, Henan Agriculture University, Zhengzhou, Henan　450002, China

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。
- 四季秋海棠 /
- BsMYB62 /
- 酵母双杂交 /
- 互作蛋白
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.
- Begonia semperflorens /
- BsMYB62 /
- yeast two-hybrid /
- interaction protein

HTML全文

图 1 BsMYB62 CDS区的克隆及SMART分析

注：A：BsMYB62扩增产物琼脂糖凝胶电泳检测（M：DL2000 DNA Marker，1～2：阳性克隆）；B：BsMYB62编码蛋白保守结构的预测

Figure 1. Cloning and SMART analysis of CDS region in BsMYB62

Note: A: Agarose gel electrophoresis of PCR products of BsMYB62 （M: DL2000 DNA Marker; 1～2: positive clone ）; B: Prediction of conserved structure of BsMYB62 encoding protein

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图 2 诱饵载体pGBKT7-BsMYB62 的分子鉴定

注：A: 带酶切位点片段的鉴定（M: DL2000 DNA Marker）; B: 质粒菌液PCR鉴定

Figure 2. Molecular identification of bait vector pGBKT7-BsMYB62

Note: A: Identification of fragments with restriction sites （M: DL2000 DNA Marker） ; B: PCR identification of plasmid bacteria

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图 3 pGBKT7-BsMYB62 在SD/-Trp培养基上的毒性鉴定

注：A: pGBKT7载体 B: pGBKT7-BsMYB62载体

Figure 3. Determination of pGBKT7-BsMYB62 toxicity on SD/-Trp medium

Note: A: pGBKT7 vector B: pGBKT7-BsMYB62 vector

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图 4 诱饵蛋白pGBKT7-BsMYB62 的自激活检测

Figure 4. Auto-activation detection of bait protein pGBKT7-BsMYB62

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图 5 抑制pGBKT7-BsMYB62 载体自激活的3-AT浓度筛选

注：A: 3-AT浓度的初步筛选；B; 3-AT浓度的优化

Figure 5. Selection of 3-AT concentration for testing auto-activation inhibition of pGBKT7-BsMYB62

Note: A: Preliminary screening of 3-At concentration; B: 3-AT concentration optimization

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图 6 阳性酵母菌液PCR产物电泳图结果

Figure 6. Electrophoresis of PCR product from positive yeast liquid

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表 1 引物序列

Table 1. Primer sequences

引物名称 Primer name	引物序列（5＇-3＇）　　 Primer sequence（5＇-3＇）	用途 purpose
BsMYB62-1F	ATGGCTGCGTCATCTTCATCTA	BsMYB62基因CDS区克隆 Cloning of BsMYB62 CDS region
BsMYB62-1R	TCACCATAGCTCATAACTGTTCCAC	BsMYB62基因CDS区克隆 Cloning of BsMYB62 CDS region
BsMYB62-in-F	CATGGAGGCCGAATTCATGGCTGCGTCATCTTCATCTA	引入酶切位点 Add restriction site
BsMYB62-in-R	GCAGGTCGACGGATCCTCACCATAGCTCATAACTGTTCCAC	引入酶切位点 Add restriction site

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表 2 阳性互作蛋白比对结果

Table 2. Alignment of positive interacting proteins

编号 Number	类型 Description	长度 Length/ bp	生物进程 Biological Process	分子功能 Molecular Function
1	木葡聚糖内转糖酶/水解酶9 Xyloglucan endotransglucosylase/hydrolase 9（XTH9）	1090	细胞壁生物发生，木葡聚糖代谢过程 cell wall biogenesis, xyloglucan metabolic process	水解酶活性，水解O -糖基化合物，木葡聚糖：木糖基转移酶活性 hydrolase activity, hydrolyzing O-glycosyl compounds, xyloglucan:xyloglucosyl transferase activity
2	光合系统Ⅱ Photosystem II lightharvesting complex geneB1B2（LHB1B2）	972	光合作用，光系统I中的光收集，光合作用，光系统II中的光收集，对光刺激的响应 photosynthesis, light harvesting in photosystem I, photosynthesis, light harvesting in photosystem II, response to light stimulus	叶绿素结合 chlorophyll binding
3		949
4		952
5	甲烷磺酸乙酯诱变的油菜素甾体不敏感1抑制剂 Ethylmethanesulfonate-mutagenized brassinosteroid-insensitive 1 supperssor 7（EBS7）	1037	ERAD途径 ERAD pathway	蛋白结合 protein binding
6	光系统I亚基 FPhotosystem I subunit F（PSI-F）	941	—	编码光系统Ⅰ的F亚基 encodes subunit F of photosystem I
7	泛素结合酶E2 Ubiquitin conjugating enzyme E2（UBE2）	876	蛋白多泛素化，蛋白泛素化，泛素依赖的蛋白分解代谢过程 protein polyubiquitination, protein ubiquitination, ubiquitin-dependent protein catabolic process	泛素结合酶活性，泛素-蛋白转移酶活性 ubiquitin conjugating enzyme activity, ubiquitin-protein transferase activity
8	甲酸脱氢酶 Formate dehydrogenase（FDH）	1057	甲酸分解过程，对镉离子的反应，对创伤的反应 formate catabolic process, response to cadmium ion, response to wounding	NAD结合，甲酸脱氢酶（NAD +）活性，氧化还原酶活性，作用于CH-OH基团的供体，NAD或NADP作为受体 NAD binding, formate dehydrogenase （NAD+） activity, oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor
9	核糖二磷酸羧化酶小链1A Ribulose bisphosphate carboxylase small chain 1A（RBCS1A）	914	叶绿体核酮糖二磷酸羧化酶复合体组装，光合作用，响应冷害 chloroplast ribulose bisphosphate carboxylasecomplex assembly, photosynthesis,response to cold	核酮糖-二磷酸羧化酶活性 ribulose-bisphosphate carboxylase activity
10		856		核酮糖-二磷酸羧化酶活性 ribulose-bisphosphate carboxylase activity
11	法尼基化蛋白 6Farnesylated protein 6（FP6）	711 bp	热适应，金属离子迁移 heat acclimation, metal ion transport	镉离子结合，铜离子结合，铅离子结合，蛋白质结合 cadmium ion binding, copper ion binding, lead ion binding, protein binding
12	β-碳酸酐酶4 Beta carbonic anhydrase 4（BCA4）	1063 bp	碳利用，氰酸盐分解代谢过程，气孔复合体发育的负调控，气孔运动的调控，对二氧化碳的响应 carbon utilization, cyanate catabolic process, negative regulation of stomatal complex development, regulation of stomatal movement, response to carbon dioxide	碳酸盐脱水酶活性，蛋白质结合，锌离子结合 carbonate dehydratase activity, protein binding, zinc ion binding

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