辣椒UGT基因家族的鉴定及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0269

摘要/Abstract

摘要：

【目的】鉴定分析辣椒UGT基因家族成员的结构和功能，为解析辣椒多糖糖基化分子机制奠定基础。【方法】利用BioEdit、BLASTP和Pfam比对搜索辣椒UGT成员；利用CDD和HMMER数据库验证保守结构域；利用ExPASy、Cell PLOC、MAGA X、MG2C、GSDS、STRING等分析预测蛋白理化性质、系统发育、染色体定位、基因结构和蛋白互作；利用转录组数据和实时荧光定量PCR分析辣椒UGT基因在各器官发育过程中的表达模式。【结果】从辣椒基因家族中鉴定到的140个辣椒UGT家族成员，编码的氨基酸数量介于253-534之间，等电点在4.7-8.45之间；CaUGTs蛋白大多数定位于细胞外，少部分定位于质膜和叶绿体中；分布在17个组中，定位于12条染色体上，其中25个成员定位在第12条染色体（Chromosome 12，chr.12）；所有成员均包含保守结构域motif 1、motif 3；响应与植物生长发育、胁迫有关的作用元件。辣椒UGT基因在不同组织、逆境胁迫及激素应答下的表达模式分析表明，UGT基因具有组织特异性表达差异，在花、果实发育阶段表达相对较高；在ABA、GA3、高温以及低温胁迫条件下，表达被显著诱导增加或者降低，在发育的特定阶段CaUGTs成员可能发挥不同的作用，很可能参与了调控辣椒逆境胁迫条件下的防御应答反应。【结论】140个辣椒UGT成员在分布及结构上存在多样性，而组内成员之间高度相似，CaUGTs基因可能在辣椒植株生长发育过程中响应非生物胁迫。

关键词: 辣椒, UGT基因家族, 生物信息分析, 成员鉴定, 功能预测, 表达分析

Abstract:

【Objective】The structure and function of UGT gene family members in pepper were identified and analyzed, laying the foundation for subsequent research.【Method】BioEdit, BLASTP and Pfam were used to align and search for UGT members in pepper. CDD and HMMER databases were used to verify conserved domains. ExPASy, Cell PLOC, MAGA X, MG2C, GSDS, and STRING were used to analyze and predict protein physicochemical properties, phylogeny, chromosome localization, gene structure and protein interaction. Transcriptome data and real-time PCR were used to analyze the expression pattern of UGT gene in various organs during the development of various organs.【Result】The prediction results showed that the 140 CaUGTs family members identified from the pepper gene family encoded amino acids in the range of 253-534 and the isoelectric point in the range of 4.7-8.45, most of the CaUGTs were located outside the cell, and a few were located in the plasma membrane and chloroplasts, and were distributed in 17 groups and mapped on 13 chromosomes, of which 25 members were located in chrom12, and all members contained the conserved domains motif 1 and motif 3. They responded to the action elements related to plant growth and development, stress. Real-time quantitative PCR analysis of the expression patterns of UGT gene in pepper under different tissues, stress and hormone response showed that UGT gene was of tissue-specific expression differences, and the expressionof UGT gene significantly increased or decreased under the conditions of ABA, GA3, high and low temperature stress, and CaUGTs members might play different roles in specific stages of development, and may be involved in regulating the defense response under stress in pepper. 【Conclusion】The distribution and structure of the 140 pepper UGT members are diverse, while the members within the group are highly similar, indicating that the CaUGTs gene may respond to abiotic stress during the growth and development of pepper plants.

Key words: pepper, UGT gene family, bioinformatics analysis, membership identification, functional prediction, expression analysis

吴慧琴, 王延宏, 刘涵, 司政, 刘雪晴, 王静, 阳宜, 成妍. 辣椒UGT基因家族的鉴定及表达分析[J]. 生物技术通报, 2024, 40(9): 198-211.

WU Hui-qin, WANG Yan-hong, LIU Han, SI Zheng, LIU Xue-qing, WANG Jing, YANG Yi, CHENG Yan. Identification and Expression Analysis of UGT Gene Family in Pepper[J]. Biotechnology Bulletin, 2024, 40(9): 198-211.

图/表 11

表1 CaUGTs家族的RT-qPCR引物序列

Table 1 RT-qPCR primer sequences of CaUGTs

基因 Gene	正向引物Forward primer（5'-3'）	正向引物Forward primer（5'-3'）
CaUGT31	GCTACAAAGCTGATGCAGGC	GCGAAGACACTCGTACCGAA
CaUGT39	AAGGAGGCCATCGACACAAG	TAGGCAGGATCAAGCTGTGC
CaUGT47	GGGCCTTCTTCCACGAATTT	CGATCCCAAGTCCTCCAAGTT
CaUGT53	GGTTCTTGACTCACAGCGGA	GCGAAAAATGGCCAGCAGAT
CaUGT63	TTAGCTTTGGGAGTGTCGCC	ATCCGCGGGAATCTTGGTTT
CaUGT74	GCTCCTCTTCCCTCTTCCAA	CAAACCGGCGAGACAAAAGA
CaUGT82	CTGAGATGGCATGGGGACTT	GGCAAATTTACAACGTCGTCC
CaUGT92	TGCCGTGTCAATTCGAAAGG	GCAACTTCAACAGCCCATGA
CaUGT127	ATGTCCAAACCCAACTTCACA	CACGGCAGCACCAAAAGTTA
CaUGT128	CCAATGAATGCCAGGTTGGTAG	TGGCCCTCAAATTTTCCCCCAT
CaUGT132	GCCCCTCAAGCAACAATTCT	CACTGGTTGGTCAATGTGCA
CaActin	GGTGACGAGGCTCAATCCAA	CTCTGGAGCCACACGAAGTT

表2 CaUGTs蛋白理化性质

Table 2 Physical and chemical properties of CaUGTs protein

项目 Item	蛋白长度 Protein length/aa	分子量 Molecular weight/kD	等电点 pI	不稳定指数 Instability index	疏水指数 GRAVY	带负电残基数 Asp + Glu	带正电残基数 Arg + Lys
分布范围	253-534	20.71×10³-59.65×10³	4.7-8.45	26.52-59.99	-0.334-0.054	29-73	20-64
平均值	450	50.61×10³	5.79	43.35	-0.139	55.53	45.86
亲水蛋白数	—	—	—	—	131	—	—
疏水蛋白数	—	—	—	—	9	—	—
酸性蛋白数	—	—	134	—	—	—	—
碱性蛋白数	—	—	6	—	—	—	—
稳定蛋白	—	—	—	40	—	—	—
不稳定蛋白	—	—	—	100	—	—	—
最小值成员	CaUGT43	CaUGT43	CaUGT101	CaUGT102	CaUGT67	CaUGT15	CaUGT137
最大值成员	CaUGT99	CaUGT99	CaUGT137	CaUGT61	CaUGT49	CaUGT91	CaUGT49

图1 拟南芥（112）、辣椒（140）、番茄（14）UGTs系统发育进化树蓝色圆点表示辣椒UGT成员

Fig. 1 Phylogenetic tree of Arabidopsis thaliana（112）, pepper（140）, tomato（14）UGTs The blue dots indicate pepper UGTS

图2 辣椒CaUGTs家族成员系统进化树、基因结构及motif分布 A：系统进化树；B：保守结构域；C：基因结构（外显子/内含子）

Fig. 2 Phylogngntic tree, gene structure and motif distribution of the pepper CaUGTs family member A: Phylogenetic tree; B: conserved domain composition; C: gene structure（exon/intron）

图3 CaUGTs家族染色体分布图

Fig. 3 Chromosome distribution of CaUGTs family

图4 CaUGTs启动子顺式作用元件分布

Fig. 4 Distribution of cis-acting elements of CaUGTs promoters

图5 CaUGTs家族成员蛋白互作网络

Fig. 5 Protein interactions network among members of the CaUGTs family

图6 辣椒UGT在不同胁迫下的表达分析

Fig. 6 Expression analysis of UGT under different stresses in pepper

图7 辣椒UGT家族在不同器官中的表达分析 L：叶；F：花；G：果实；ST：种子和胎座；S：种子；T：胎座

Fig. 7 Expression analysis of UGT genes in different organs L: Leaf; F: flower; G: fruit; ST: seed and placenta; S: seed; T: placenta

图8 CaUGTs在花、果实不同发育时期的表达分析 F1、F2、F3、F4、F5、F5、F6分别代表现蕾后1、3、5、7、8、9 d

Fig. 8 Expression analysis of CaUGTs at different developmental stages of flowers and fruits F1, F2, F3, F4, F5, F5, and F6 indicate 1, 3, 5, 7, 8 and 9 d respectively

图9 CaUGTs 在非生物胁迫处理下的表达热图

Fig. 9 Hotmap of CaUGTs expression under abiotic stress treatment

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