节瓜CqCHP1的低温响应分析及互作蛋白的筛选与验证

doi:10.16420/j.issn.0513-353x.2022-0854

摘要/Abstract

摘要：

以‘粤广节瓜’为材料，克隆得到基因CqCHP1（编码富含半胱氨酸和组氨酸C1结构域）。CqCHP1的开放阅读框为816 bp，编码271个氨基酸残基，蛋白二级结构主要由无规则卷曲构成，含23个丝氨酸磷酸化位点。进化树与氨基酸序列比对分析发现CqCHP1和黄瓜Csa_4G297435同源性最高。qRT-PCR分析表明，CqCHP1在节瓜不同组织中均有表达，并受低温显著诱导；苗期和结果期时，CqCHP1在耐冷材料AW66中的表达量显著高于冷敏材料AW9，但在营养生长期和开花期冷敏材料AW9的表达显著高于耐冷材料AW66。亚细胞定位结果显示该蛋白定位于细胞核中。利用酵母文库进行互作蛋白筛选发现CqCHP1可以与bHLH92在细胞核中互作，且双分子荧光互补实验和荧光素酶互补实验验证了其互作的准确性。

关键词: 节瓜, 低温, CHP1蛋白, 蛋白互作, bHLH92转录因子

Abstract:

In this study，CqCHP1 was cloned from‘Yueguang Chieh-qua’，which encoded cysteine/ histidine-rich C1 domain protein. The gene’s open reading frame was 816 bp with 271 amino acids. The secondary structure indicated that CqCHP1 contained main random curl and 23 serine phosphorylation sites. Phylogenetic tree and amino acid sequence alignment analysis showed that CqCHP1 had the highest homology with cucumber Csa_4G297435. CqCHP1 was expressed in different tissues and specifically induced by low temperature. And at seedling and fruiting stages，CqCHP1 showed higher expression in resistance materials than sensitive materials，while at vegetative growth and flowering periods，it’s the opposite. The protein was located in the nucleus. Analysis of interaction proteins obtained by yeast screening showed that CqCHP1 could interact with bHLH92 in nucleus. And the inteaction was verified by Bimolecular fluorescence complementation（BIFC）and Luciferase complementation imaging assay（LCI）.

Key words: chieh-qua, low temperature, CHP1 protein, protein-interation, bHLH92 transcription factor

王敏, 杨松光, 刘微, 何晓明, 史绍琪, 刘文睿, 陈林, 江彪, 彭庆务. 节瓜CqCHP1的低温响应分析及互作蛋白的筛选与验证[J]. 园艺学报, 2023, 50(11): 2376-2386.

WANG Min, YANG Songguang, LIU Wei, HE Xiaoming, SHI Shaoqi, LIU Wenrui, CHEN Lin, JIANG Biao, PENG Qingwu. Response to Low Temperature Stress and Screening and Validation of Interaction Protein of CqCHP1 in Chieh-qua[J]. Acta Horticulturae Sinica, 2023, 50(11): 2376-2386.

图/表 9

表1 本研究中使用的引物序列

Table 1 Primer sequences used in this study

用途	名称	引物序列（5′-3′）
Use	Name	Primer sequence
基因克隆Gene cloning	CqCHP1-F	ATGGCGCTGGTACCCGGAAA
	CqCHP1-R	CTAAGCACCAACAGAACCGA
实时荧光定量PCR	CqCHP1-QF	ACATACTGCTCCGATTCCCC
Quantitative real-time PCR	CqCHP1-QR	TTGGGGTGAATATTGACTCC
亚细胞定位 Sublocation	1305-GFP-CqCHP1-F	CGAGCTGTACAGATCTATGGCGCTGGTACCCGGAAA
	1305-GFP-CqCHP1-R	GGCCGCTTTAAGATCTCTACTAAGCACCAACAGAACCGA
酵母双杂 Yeast two-hybrid	BD-CqCHP1-F	GAATTCCCGGGGATCCGTATGGCGCTGGTACCCGGAAA
	BD-CqCHP1-R	GCAGGTCGACGGATCCCTAAGCACCAACAGAACCGA
	AD-bHLH92-F	CATCGATACGGGATCCATGGATGATAGCTTTTCTGT
	AD-bHLH92-R	CGAGCTCGATGGATCCTTAGCTTTCAAATTCTGGAC
双分子荧光互补	CqCHP1-BIFC-N-F	CCCTAGGAACTAGTATGGCGCTGGTACCCGGAAA
Bimolecular fluorescence	CqCHP1-BIFC-N-R	GATCCTCCACTAGTCTAAGCACCAACAGAACCGA
complementation	bHLH92-BIFC-C-F	TACGTAGTCACGTGATGGATGATAGCTTTTCTGT
	bHLH92-BIFC-C-R	CCGGACGTCACGTGTTAGCTTTCAAATTCTGGAC
荧光素酶互补	bHLH92-nLUC-F	ACGGGGGACGAGCTCGGTACCATGGATGATAGCTTTTCTGTG
Luciferase complementation	bHLH92-nLUC-R	CGCGTACGAGATCTGGTCGACGCTTTCAAATTCTGGACCCACA
imaging assay	CqCHP1-cLUC-F	TACGCGTCCCGGGGCGGTACCATGGCGCTGGTACCCGGAAA
	CqCHP1-cLUC-R	ACGAAAGCTCTGCAGGTCGACTCAAGCACCAACAGAACCGAAAAT

图1 CqCHP1的克隆及低温胁迫下的表达分析

Fig. 1 Cloning and expression analysis under low temperature of CqCHP1 ** P < 0.01.

图2 节瓜CqCHP1蛋白的进化分析 Os：水稻；AT：拟南芥；Csa：黄瓜；Solyc：番茄；A4A：烟草。

Fig. 2 Analysis of phylogenetic of CqCHP1 Os：Oryza sativa L.；AT：Arabidopsis thaliana（L.）Heynh；Csa：Cucumis sativus；Solyc：Solanum lycopersicum；A4A：Nicotiana attenuata.

图3 CqCHP1在‘粤广节瓜’不同组织中的表达分析

Fig. 3 Expression analysis of CqCHP1 in different tissues of‘Yueguang Chieh-qua’

图4 CqCHP1在耐冷节瓜（AW66）和冷敏节瓜（AW9）不同组织中的表达分析

Fig. 4 Expression analysis of CqCHP1 in different tissues of cold resistant（AW66）and cold-sensitive（AW9）materials ** P < 0.01.

图5 CqCHP1在正常和低温胁迫条件下的亚细胞定位

Fig. 5 Sublocation of CqCHP1 under normal and low temperature stress

图6 CqCHP1的自激活活性验证与互作蛋白的筛选

Fig. 6 Yeast self-activation and interaction identification of CqCHP1 in yeast -LW：-Leu/-Trp；-ALWH：-Ade/-His/-Leu/-Trp；-MEL1：-Ade/-His/-Leu/-Trp/X-α-gal.

图7 荧光素酶互补实验验证CqCHP1和bHLH92的互作

Fig. 7 Interaction identification of CqCHP1 and bHLH92 using LCI assay

图8 CqCHP1与bHLH92的BIFC验证

Fig. 8 Interaction identification of CqCHP1 and bHLH92 using BiFC assay

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