基于 TMT和PRM 技术筛选番茄响应盐胁迫差异表达蛋白

doi:10.6048/j.issn.1001-4330.2022.06.013

摘要/Abstract

摘要：

【目的】 盐胁迫是造成番茄产量损失和品质严重下降的关键非生物胁迫之一,研究番茄耐盐的分子机制,为认识番茄幼苗应答盐胁迫分子调控机制奠定基础。【方法】 研究利用番茄耐盐渐渗系IL-7-5-5和番茄盐敏感M82为试材,采用同位素相对标记与绝对定量(TMT)技术结合定量蛋白质组平行反应监测(PRM)技术,对200 mM盐胁迫12 h的番茄幼苗叶片进行蛋白质组学研究,筛选出盐胁迫响应显著的潜在靶标蛋白。【结果】 (1)共鉴定出286个差异显著表达蛋白(DEPs)。盐胁迫下IL-7-5-5鉴定到191个DEPs,其中119个表达上调,72个表达下调。在M82中鉴定出157个DEPs,其中84个表达上调,73个表达下调。维恩图分析显示,有129和95个差异蛋白分别特异于IL-7-5-5和M82。有62个显著差异蛋白共表达,其中 28 个在IL-7-5-5和M82中均上调,15 个均为下调,表现出对盐胁迫的一致响应。19个显著差异蛋白在表达相反,有5个蛋白质在ST中下调,在SS中上调,14个差异蛋白在ST中上调,在SS中下调;(2)番茄盐反应蛋白种类诱导能力强,主要与代谢过程、单组织过程以及细胞过程有关,细胞组成主要涉及细胞、细胞器、分子复合物和膜,基因本体分子功能显示,这些差异性蛋白主要参与催化活性、绑定和分子功能调控。(3)选择11个显著差异蛋白PRM验证结果与TMT 定量表现出相同的趋势。差异蛋白包括 A0A3Q7E8T9、A0A3Q7EK65、A0A3Q7FY19、A0A3Q7G430、A0A3Q7ITH0、A0A3Q7J1Y7、P05116、Q43779、A0A3Q7F8W6、A0A3Q7GKU3、A0A3Q7J0Z4,可能是番茄幼苗耐盐的潜在靶标蛋白。【结论】 研究采用 TMT 结合 PRM 技术,筛选出番茄幼苗响应盐胁迫差异表达蛋白。

关键词: 番茄; 盐胁迫; 蛋白质组学; TMT; PRM

Abstract:

【Objective】 Salt stress is one of the key abiotic stresses that cause yield loss and quality decline of tomato.Therefore, it is of great significance to reveal the molecular mechanism of tomato salt tolerance. 【Methods】 In this study, tomato salt-tolerant introduction system IL-7-5-5 and tomato salt-sensitive M82 were used as test materials, and isotope relative labeling and absolute quantification (TMT) technology were combined with quantitative parallel reaction monitoring (PRM) technology.The proteomics of leaves of tomato seedlings under 200 mM salt stress for 12 h were studied to screen out potential target proteins with significant salt stress response.【Results】 (1)A total of 286 differentially expressed proteins (DEPs) were identified.Under salt stress, 191 DEPs were identified by IL-7-5-5, of which 119 were up-regulated and 72 down-regulated.157 DEPs were identified in M82, of which 84 were up-regulated and 73 down-regulated.Venn diagram analysis showed that 129 and 95 differential proteins were specific to IL-7-5-5 and M82, respectively.There were 62 differentially expressed proteins, among which 28 were up-regulated in IL-7-5-5 and M82, and 15 were down-regulated, showing consistent response to salt stress.Among the 19 differentially expressed proteins, 5 proteins were down-regulated in ST and up-regulated in SS, and 14 differentially expressed proteins were up-regulated in ST and down-regulated in SS; (2) The proteomic analysis showed that the GO enrichment tomato had strong salt reactive protein type induction ability, mainly related to metabolic process, single-tissue processes, and cellular processes and the cellular composition mainly involved cells, organelles, molecular compounds and membrane. And gene ontology revealed that these differential proteins were mainly involved in the regulation of catalytic activity, bound with molecular function,.(3) PRM validation results of 11 significantly different proteins showed the same trend as TMT quantitative results.Differential proteins included A0A3Q7E8T9, A0A3Q7EK65, A0A3Q7FY19, A0A3Q7G430, A0A3Q7ITH0, A0A3Q7J1Y7, P05116, Q43779, A0A3Q7F8W6,A0A3Q7GKU3 and A0A3Q7J0Z4, which might be potential target proteins for salt tolerance of tomato seedlings. 【Conclusion】 In this study, TMT combined with PRM technology was used to screen out differentially expressed proteins in tomato seedlings responding to salt stress, which could lay a foundation for further understanding of the molecular regulatory mechanism of tomato seedlings responding to salt stress.

Key words: tomato; salt stress; proteomics; TMT; PRM

中图分类号:

S188
S641.2

王强, 刘会芳, 韩宏伟, 庄红梅, 王柏柯, 王娟, 杨涛, 王浩, 秦勇. 基于 TMT和PRM 技术筛选番茄响应盐胁迫差异表达蛋白[J]. 新疆农业科学, 2022, 59(6): 1418-1428.

WANG Qiang, LIU Huifang, HAN Hongwei, ZHUANG Hongmei, WANG Baike, WANG Juan, YANG Tao, WANG Hao, QIN Yong. Screening Differentially Expressed Proteins in Response to Salt Stress of Tomato Leaves Based on TMT and PRM Techniques[J]. Xinjiang Agricultural Sciences, 2022, 59(6): 1418-1428.

图/表 5

图1 2种番茄品种差异表达蛋白比较注:(A)差异表达蛋白(DEPs)比较的数量。(B)两个番茄幼苗DEPs蛋白的维恩图

Fig.1 Comparative analysis of differentially expressed proteins in two tomato varieties. Note: (A) Number of up-regulated and down-regulated differentially expressed proteins (DEPs). (B) Venn diagram of tomato DEPs in two maize varieties.

图2 差异蛋白的 GO 功能分类注:A、D分别代表SS12 vs SS0、ST12 vs ST0生物学过程;图B、E分别代表SS12 vs SS0、ST12 vs ST0细胞成分;图C、F分别代表SS12 vs SS0、ST12 vs ST0分子功能

Fig.2 Gene ontology enrichment analysis of differentially expressed protein Note:A and D represent the biological processes of SS12 vs SS0 and ST12 vs ST0 respectively.Figure2- B and E represent SS12 vs SS0 and ST12 vs ST0 cell components respectively.Figure 2-C and F represent the molecular functions of SS12 vs SS0 and ST12 vs ST0 respectively

图3 ST12vsST0响应盐胁迫差异蛋白的KOG功能分类

Fig.3 KOG functional classification of ST12vsST0 differential proteins in response to salt stress

图4 SS12vsSS0响应盐胁迫差异蛋白的KOG功能分类

Fig.4 KOG functional classification of SS12vsSS0 differential proteins in response to salt stress

表1 PRM 和 TMT 的定量结果比较

Table 1 Comparison of PRM and TMT quantification result

蛋白号 Protein Accession	蛋白质的描述 Protein description	SS12/SS0 TMT PRM		ST12/ST0 TMT PRM
A0A3Q7E8T9	过氧化物酶^*	/	/	1.56	2.72
A0A3Q7EK65	精氨酸酶 2^*	/	/	1.97	3.98
A0A3Q7FY19	可能乙酰辅酶a乙酰转移酶^*	/	/	1.64	2.51
A0A3Q7G430	黄氧素脱氢酶	0.64	0.54	3.37	3.25
A0A3Q7ITH0	过氧化物酶	1.82	1.24	1.64	1.89
A0A3Q7J1Y7	可能多胺氧化酶 2	0.81	0.61	2.26	6.47
P05116	1-氨基环丙烷-1-羧酸氧化酶1	3.41	4.96	1.67	2.67
Q43779	超氧化物歧化酶^*	1.53	1.8	/	/
A0A3Q7F8W6	微管蛋白α链	0.6	0.48	1.57	1.63
A0A3Q7GKU3	40S核糖体蛋白S13样^*	/	/	1.62	1.44
A0A3Q7J0Z4	延长因子1α	0.57	0.3	2.22	3.48

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