茶树CsHIPP26.1互作蛋白的筛选与验证

doi:10.3864/j.issn.0578-1752.2022.08.013

摘要/Abstract

摘要：

【背景】‘黄金芽’属于光照敏感型黄化茶树（Camellia sinensis）品种,叶片色泽呈现强光黄化、弱光复绿的特点,但叶色响应光照的黄化机制并不明确。前期通过对黄化叶片、遮阴复绿叶片以及常绿品种叶片的蛋白组研究发现,重金属相关异戊二烯化植物蛋白CsHIPP26.1（TEA000549）的表达响应光照强度,表明CsHIPP26.1可能参与调控‘黄金芽’叶色黄化的光响应过程。【目的】通过筛选与CsHIPP26.1互作的光信号响应相关的蛋白,为叶片色泽响应光照信号变化提供科学依据。【方法】以‘黄金芽’茶树1芽2叶为材料进行CsHIPP26.1和互作基因的克隆,经酵母双杂交筛库,然后将筛选得到的目的蛋白进行酵母双杂交点对点验证、体内双分子荧光互补（BiFC）和体外pull-down等技术进行蛋白互作的进一步验证。【结果】通过酵母双杂交对茶树cDNA文库进行筛选,共筛选到26个候选互作蛋白,主要集中在细胞组分、结合以及催化活性方面发挥作用,其中生物素合成代谢过程富集程度较高,与光信号通路及叶绿素合成相关的蛋白只有编号为TEA026466.1的bHLH30转录因子。克隆bHLH30转录因子后发现,该转录因子与茶树光信号传导途径蛋白PIF4处于同一进化树分支,且含有与茶树PIF4蛋白相同的HLH和ACT结构域,将该bHLH30转录因子命名为CsPIF4.2,GenBank登记号为MW116834。进一步通过体外Pull-down蛋白互作和体内双分子荧光互补（BiFC）验证发现,CsHIPP26.1和CsPIF4.2蛋白能够发生互作,并且发生互作的部位在细胞核内。【结论】初步筛选出26个与CsHIPP26.1互作的蛋白,并验证发现CsHIPP26.1能够在细胞核内与其中一个光敏色素互作因子CsPIF4.2发生蛋白相互作用。

关键词: HIPP, PIF4, 蛋白互作, 黄化品种, 光响应

Abstract:

【Background】 Huangjinya is a light sensitive chlorotic tea (Camellia sinensis) variety, the leaf color of which presents yellow under strong light and presents green under weak light, but the chlorotic mechanism of leaf color in response to light is not clear. Previous proteomic studies on etiolated leaves, shaded green leaves and evergreen leaves found that the expression of heavy metal-associated isoprenylated plant protein CsHIPP26.1 (TEA000549) responded to light intensity, indicating that CsHIPP26.1 may be involved in regulating the light response process of leaf color etiolation of Huangjinya. 【Objective】The proteins related to the light signal response interacting with CsHIPP26.1 were screened to provide a scientific basis for leaf color response to light signal changes. 【Method】The gene were cloned from one bud and two leaves of Huangjinya. And the screened target protein was further verified by yeast two hybrid point-to-point verification, in vivo bimolecular fluorescence complementarity (BiFC), and in vitro pull-down techniques. 【Result】 The tea cDNA library was screened by yeast two hybrid, and a total of 26 candidate interaction proteins were screened, which mainly played a role in cell components, binding and catalytic activity. Among them, the enrichment degree of biotin anabolism process was high, and the proteins related to light signal pathway and chlorophyll synthesis were only the bHLH30 transcription factor, and its gene ID was TEA026466.1. After cloning the gene of bHLH30 transcription factor, it was found that the transcription factor was in the same evolutionary tree branch as tea light signal transduction pathway protein PIF4, and contained the same HLH and ACT domains as tea PIF4 protein. Therefore, the bHLH30 transcription factor was named CsPIF4.2, GenBank registration number: MW16834. And through the pull-down protein interaction in vitro and bimolecular fluorescence complementarity (BiFC) in vivo, it was found that CsHIPP26.1 and CsPIF4.2 proteins could indeed interact, and the site of interaction was in the nucleus. 【Conclusion】 26 proteins interacting with CsHIPP26.1 were preliminarily screened, and it was found that CsHIPP26.1 could interact with one of the phytochrome interacting factors CsPIF4.2 in the nucleus.

Key words: HIPP, PIF4, protein interaction, yellowing varieties, light response

范延艮,王域,刘富浩,赵秀秀,向勤锃,张丽霞. 茶树CsHIPP26.1互作蛋白的筛选与验证[J]. 中国农业科学, 2022, 55(8): 1630-1641.

FAN YanGen,WANG Yu,LIU FuHao,ZHAO XiuXiu,XIANG QinZeng,ZHANG LiXia. Screening and Verification of CsHIPP26.1 Interaction Protein in Tea Plant[J]. Scientia Agricultura Sinica, 2022, 55(8): 1630-1641.

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https://www.chinaagrisci.com/CN/Y2022/V55/I8/1630

图/表 6

表1

图1

表2

互作蛋白信息"

编号Number	蛋白 Protein	名称 Definition	KEGG通路注释 Annotation of KEGG pathway
1	TEA026466.1	转录因子bHLH30 Transcription factor bHLH30 (CsPIF4-like)	ko04010 MAPK信号通路 ko04010 MAPK signaling pathway
2	TEA017417.1	类Barwin内切葡聚糖酶 Barwin-like endoglucanase	ko04010 MAPK信号通路 ko04010 MAPK signaling pathway
3	TEA006996.1	BRI1激酶抑制剂1 BRI1 kinase inhibitor 1	ko04075植物激素信号转导 Ko04075 Plant hormone signal transduction
4	TEA027305.1	细胞周期蛋白 Cyclin-like protein	ko03022基础转录因子 ko03022 Basal transcription factors
5	TEA028934.1	生物素合成酶 Biotin synthase	ko00780生物素代谢 ko00780 Biotin metabolism
6	TEA032431.1	未表征的蛋白质 Uncharacterized protein	ko00790叶酸生物合成 ko00790 Folate biosynthesis
7	TEA006267.1	未表征的蛋白质 Uncharacterized protein	ko00790叶酸生物合成 ko00790 Folate biosynthesis
8	TEA016398.1	未表征的蛋白质 Uncharacterized protein	ko00941 黄酮类生物合成 ko00941 Flavonoid biosynthesis
9	TEA022238.1	UDP-糖基转移酶 UDP-glycosyltransferase	ko00942花青素生物合成 ko00942 Anthocyanin biosynthesis
10	TEA030717.1	HXXXD 型酰基转移酶家族蛋白 HXXXD-type acyl-transferase family protein	ko00940苯丙烷生物合成 ko00940 Phenylpropanoid biosynthesis ko00941黄酮类生物合成 ko00941 Flavonoid biosynthesis ko00945芪、二芳基庚烷和姜酚的生物合成 ko00945 Stilbenoid, diarylheptanoid and gingerol biosynthesis
11	TEA032735.1	咖啡酰莽草酸酯酶 Caffeoylshikimate esterase	ko00561甘油脂代谢 ko00561 Glycerolipid metabolism
12	TEA011538.1	未表征的蛋白质 Unnamed protein product	ko03010核糖体 ko03010 Ribosome
13	TEA019010.1	30S核糖体蛋白S17 30S ribosomal protein S17	ko03010核糖体 ko03010 Ribosome
14	TEA028503.1	U2小核核糖核蛋白B U2 small nuclear ribonucleoprotein B	ko03040剪接体 ko03040 Spliceosome
15	TEA019956.1	导入-5 Importin-5	ko03013核质运输 ko03013 Nucleocytoplasmic transport
16	TEA014840.1	圈套蛋白YKt Snare protein YKt	ko04130囊泡运输中的SNARE相互作用 ko04130 SNARE interactions in vesicular transport
17	TEA015767.1	蛋白质STAY-GREEN Protein STAY-GREEN	-
18	TEA033782.1	Poly(rC)结合蛋白 Poly(rC)-binding protein	-
19	TEA011052.1	胚花2 Embryonic flower 2	-
20	TEA012266.1	Phragmoplast定向驱动蛋白2 Phragmoplast orienting kinesin 2	-
21	TEA015624.1	质脂相关蛋白 Plastid-lipid-associated protein	-
22	TEA011412.1	肽基-脯氨酰顺反异构酶CYP95 Peptidyl-prolyl cis-trans isomerase CYP95	-
23	TEA005217.1	DnaJ同源物1 DnaJ homolog 1	-
24	TEA027912.1	UDP-糖基转移酶 UDP-glycosyltransferase	-
25	TEA012996.1	未表征的蛋白质 Uncharacterized protein	-
26	TEA010659.1	未命名的蛋白质产物 Unnamed protein product	-

表2

图2

图3

图4

参考文献 7

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引物名称 The Primer	序列 Sequence (5′-3′)	序列 Sequence (3′-5′)
BD-CsHIPP26.1	CATATGATGGGTGCTCTGGATCATCTC	GGATCCCATGACAACACAAGCAGCAG
AD-CsPIF4.2	GAATTCATGATGTGTGGGAAAAAGG	GGATCCAAGAGACCTGTGTTCAAGGAT
His-CsHIPP26.1	GGATCCATGGGTGCTCTGGATCATCTC	GTCGACCATGACAACACAAGCAGCAG
GST-CsPIF4.2	GAATTCATGATGTGTGGGAAAAAGG	GTCGACAAAAGAGACCTGTGTTCAAG
BiFC-N-CsHIPP26.1	GGATCCATGGGTGCTCTGGATCATCTC	GTCGACCATGACAACACAAGCAGCAG
BiFC-C-CsPIF4.2	GGATCCATGATGTGTGGGAAAAAGGA	GTCGACAAGAGACCTGTGTTCAAGGAT