利用酵母双杂交系统筛选水稻中与OsCRK5互作蛋白

doi:10.13560/j.cnki.biotech.bull.1985.2023-0090

摘要/Abstract

摘要：

水稻是重要的粮食作物，研究水稻生长发育调控机制可为水稻品种改良奠定理论基础。钙依赖蛋白激酶（calcium dependent protein kinases, CDPKs）是植物中重要的蛋白激酶，参与植物生长发育以及对环境反应的应答。水稻中的CRK5（CDPK-related kinase 5）在蛋白序列和结构上与CDPK高度同源。为进一步研究OsCRK5在水稻干旱反应中的功能，本研究利用酵母双杂交筛库技术筛选了OsCRK5的互作蛋白。首先将OsCRK5的1-1 332 bp片段克隆至pGBKT7载体中，获得诱饵载体pGBKT7-OsCRK5，经测序无误后，转化至酵母菌株Y2H Gold中。在营养缺陷培养基中观察到重组蛋白不具有毒性作用及自激活活性，同时利用Western blot分析重组蛋白的表达。进一步利用水稻cDNA文库筛选OsCRK5的互作蛋白，共得到77个阳性克隆。功能预测结果显示，互作蛋白涉及蛋白质合成、贮存和降解过程、转录调控、植物细胞生长和分裂过程、能量代谢和细胞代谢等方面的功能。最后，从阳性克隆中选取参与植物干旱胁迫应答的两个蛋白OsWR1和OsDi19-1，利用酵母双杂交和双分子荧光互补的方法验证其与OsCRK5的相互作用。研究结果为水稻抗旱的遗传改良奠定了基础。

关键词: 水稻, OsCRK5, 酵母双杂交, cDNA文库, 干旱胁迫

Abstract:

Rice（Oryza sativa L.）is an important food crop, research on the regulation mechanism of rice growth and development can lay a theoretical foundation for the improvement of rice varieties. Calcium dependent protein kinases（CDPKs）are important protein kinases in plants which participate in plant growth and development, as well as in response to environmental reactions. CRK5（CDPK related kinase 5）in rice is highly homologous to CDPK in protein sequence and structure. Yeast two-hybrid screening library technology was used to screen OsCRK5 interacting proteins related to plant drought resistance in order to explore the molecular mechanism of OsCRK5 participating in drought response in rice plants. Firstly, the 1-1 332 bp fragment of OsCRK5 was cloned into the plastmid pGBKT7, and the bait vector pGBKT7-OsCRK5 was obtained. After verification by sequencing, the bait vector was transformed into the yeast strain Y2H Gold.It was observed that the recombinant protein didn't demonstrate the toxic effects and self-activation in selective nutrition medium, and the expression of the recombinant protein was analyzed by Western blot. Furthermore, the interacting proteins of OsCRK5 were screened from the rice cDNA library and 77 positive clones were obtained. The functional prediction showed that the interacting proteins were involved in protein synthesis, degradation and storage process, transcriptional regulation, cell growth and division process, energy metabolism and cell metabolic processes. Finally, OsWR1 and OsDi19-1 related to plant drought stress response were selected from positive clones. The interaction between OsCRK5 and OsWR1 and OsDi19-1 was verified by yeast two-hybrid and biomolecular fluorescence complementation experiments. The results lay a foundation for the genetic improvement of drought tolerance in rice.

Key words: rice, OsCRK5, yeast two-hybrid system, cDNA library, drought stress

王子颖, 龙晨洁, 范兆宇, 张蕾. 利用酵母双杂交系统筛选水稻中与OsCRK5互作蛋白[J]. 生物技术通报, 2023, 39(9): 117-125.

WANG Zi-ying, LONG Chen-jie, FAN Zhao-yu, ZHANG Lei. Screening of OsCRK5-interacted Proteins in Rice Using Yeast Two-hybrid System[J]. Biotechnology Bulletin, 2023, 39(9): 117-125.

图/表 8

图1 OsCRK5参与水稻干旱胁迫反应 A：定量PCR检测PEG处理后水稻叶片中OsCRK5的表达情况；B：20% PEG处理20 d水稻幼苗的表型；C：20% PEG处理水稻幼苗后叶片的表型；D：定量PCR检测干旱诱导表达基因在野生型和OsCRK5-RNAi植株中的表达情况

Fig. 1 OsCRK5 participates in the response of rice to drought stress A: The expression of OsCRK5 in the leaves after PEG treatment by qPCR detection. B: Phenotype of 20 d rice seedlings treated with 20% PEG. C: Leaf phenotypes of rice seedlings treated with 20% PEG. D: Quantitative PCR to detect the expressions of drought-induced genes in wild-type and OsCRK5-RNAi plants

图2 pGBKT7-OsCRK51-444质粒在酵母细胞中的自激活检测 A：pGBKT7-OsCRK51-444质粒转化的酵母细胞在SD/Trp+X-α-gal培养基上的生长状况；B：pGBKT7-OsCRK51-444质粒转化的酵母细胞在单缺及三缺固体培养基上的生长状况。以pGBKT7空载质粒转化的酵母细胞生长状况为对照

Fig. 2 Detection of self-activation of plasmid pGBKT7-OsCRK51-444 in yeast cells A: Growth condition of the yeast cells transformed with pGBKT7-OsCRK51-444 plasmid on SD/Trp+X-α-gal plate. B: Growth condition of the yeast cells transformed with pGBKT7-OsCRK51-444 plasmid on single and triple deficiency plates. The growth of yeast transformed with pGBKT7 plasmid was used as control

图3 不同酵母菌株中诱导蛋白表达水平的检测 1-5为不同酵母菌株中提取的总蛋白

Fig. 3 Detection of expressions of trait protein in different yeast strains 1-5 were total proteins isolated from different yeast strains

图4 筛选获得的部分阳性克隆 A：40倍物镜下观察到的酵母合子；B：SD/-Trp/-Leu/-His/-Ade+X-α-gal+AbA培养基上二次筛选得到的部分阳性菌斑

Fig. 4 Partial positive clones from screened A: Yeast zygote observed under 40× objective lens. B: Partial positive clones screened from SD/-Trp/- Leu/- His/- Ade+X- α-gal+AbA plate

表1 酵母文库筛选部分互作蛋白信息

Table 1 Information of part interacting proteins screened by yeast library

NCBI号 Accession number of NCBI	基因名称 Gene name	基因注释 Gene annotation	参考文献 Reference
LOC4347054	ZHD9	锌指同源结构域蛋白9 Zinc-finger homeodomain protein 9-like	[27]
LOC107275681	ARP5	肌动蛋白相关蛋白5 Actin-related protein 5	[28]
LOC4339608	Di19-1	脱水诱导蛋白19 DEHYDRATION-INDUCED 19-like protein	[29]
LOC4336195	ATP23	线粒体内膜蛋白酶 Mitochondrial inner membrane protease	[30]
LOC4329772	OsFWL2	细胞数目调节因子2 Cell number regulator 2	[31]
LOC4337552	OseIF3f	真核翻译起始因子3 Eukaryotic translation initiation factor 3 subunit F	[32]
LOC4329963	OsCBP20	核帽结合蛋白亚基2 Nuclear cap-binding protein subunit 2-like	[33]
LOC4349349	OsEXPB3	类膨胀素B3 Expansin-B3-like	[34]
LOC4334101	rcs3	类半胱氨酸合成酶 Cysteine synthase-like	[35]
LOC4345839	OsMtATPd2	线粒体中d亚单位ATP合成酶 ATP synthase subunit d, mitochondrial	[36]
LOC4325989	BBTI8	胰蛋白酶抑制剂 Bowman-Birk type bran trypsin inhibitor	[37]
LOC4331740	OsCCR4b	碳分解代谢抑制蛋白4同源物1 Carbon catabolite repressor protein 4 homolog 1	[38]
LOC4349455	BSK2	丝氨酸/苏氨酸蛋白激酶 Serine/threonine-protein kinase	[39]
LOC4336306	OsHCT1	羟基肉桂酰转移酶 Hydroxycinnamoyltransferase 1-like	[40]
LOC4330573	BSH	染色体结构重塑蛋白 Chromatin structure-remodeling complex protein
LOC4340286	HPGT3	羟脯氨酸O-半乳糖基转移酶 Hydroxyproline O-galactosyltransferase	[41]
LOC9269857	OsWRKY29	WRKY转录因子29 Probable WRKY transcription factor 29	[42]
LOC4349610	OsWRKY70	WRKY转录因子70 Probable WRKY transcription factor 70	[43]
LOC4337733	OsSAMS1	类S-腺苷甲硫氨酸合酶1 S-adenosylmethionine synthase 1-like	[44]
LOC4339460	OsNAP1;2	核小体组装蛋白1;2 Nucleosome assembly protein 1;2-like
LOC4328653	OsWR1	乙烯应答转录因子WIN1 Ethylene-responsive transcription factor WIN1	[45]

表1 酵母文库筛选部分互作蛋白信息

Table 1 Information of part interacting proteins screened by yeast library

NCBI号 Accession number of NCBI	基因名称 Gene name	基因注释 Gene annotation	参考文献 Reference
LOC4347054	ZHD9	锌指同源结构域蛋白9 Zinc-finger homeodomain protein 9-like	[27]
LOC107275681	ARP5	肌动蛋白相关蛋白5 Actin-related protein 5	[28]
LOC4339608	Di19-1	脱水诱导蛋白19 DEHYDRATION-INDUCED 19-like protein	[29]
LOC4336195	ATP23	线粒体内膜蛋白酶 Mitochondrial inner membrane protease	[30]
LOC4329772	OsFWL2	细胞数目调节因子2 Cell number regulator 2	[31]
LOC4337552	OseIF3f	真核翻译起始因子3 Eukaryotic translation initiation factor 3 subunit F	[32]
LOC4329963	OsCBP20	核帽结合蛋白亚基2 Nuclear cap-binding protein subunit 2-like	[33]
LOC4349349	OsEXPB3	类膨胀素B3 Expansin-B3-like	[34]
LOC4334101	rcs3	类半胱氨酸合成酶 Cysteine synthase-like	[35]
LOC4345839	OsMtATPd2	线粒体中d亚单位ATP合成酶 ATP synthase subunit d, mitochondrial	[36]
LOC4325989	BBTI8	胰蛋白酶抑制剂 Bowman-Birk type bran trypsin inhibitor	[37]
LOC4331740	OsCCR4b	碳分解代谢抑制蛋白4同源物1 Carbon catabolite repressor protein 4 homolog 1	[38]
LOC4349455	BSK2	丝氨酸/苏氨酸蛋白激酶 Serine/threonine-protein kinase	[39]
LOC4336306	OsHCT1	羟基肉桂酰转移酶 Hydroxycinnamoyltransferase 1-like	[40]
LOC4330573	BSH	染色体结构重塑蛋白 Chromatin structure-remodeling complex protein
LOC4340286	HPGT3	羟脯氨酸O-半乳糖基转移酶 Hydroxyproline O-galactosyltransferase	[41]
LOC9269857	OsWRKY29	WRKY转录因子29 Probable WRKY transcription factor 29	[42]
LOC4349610	OsWRKY70	WRKY转录因子70 Probable WRKY transcription factor 70	[43]
LOC4337733	OsSAMS1	类S-腺苷甲硫氨酸合酶1 S-adenosylmethionine synthase 1-like	[44]
LOC4339460	OsNAP1;2	核小体组装蛋白1;2 Nucleosome assembly protein 1;2-like
LOC4328653	OsWR1	乙烯应答转录因子WIN1 Ethylene-responsive transcription factor WIN1	[45]

图5 筛选基因的功能分类

Fig. 5 Functional classification of screening genes

图6 酵母双杂交验证OsCRK5与OsDi19-1和OsWR1的互作

Fig. 6 Yeast two-hybrid confirmed the interaction between OsCRK5 and OsDi19-1 or OsWR1

图7 BiFC验证OsCRK5与OsDi19-1和OsWR1的互作

Fig. 7 BiFC confirmed the interaction between OsCRK5 and OsDi19-1 or OsWR1

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