辣椒锌指蛋白DnaJ-Like基因家族鉴定及对高温胁迫的表达响应

doi:10.13560/j.cnki.biotech.bull.1985.2022-1033

摘要/Abstract

摘要：

植物DnaJ-Like锌指蛋白（DNAJE）是近年来发现的一类具有重要功能的蛋白，在光合作用、质体发育与运动和植物抗逆及防御反应中发挥重要作用。基于辣椒全基因组数据，对辣椒DNAJE基因家族（CaDNAJE）进行鉴定，利用生物信息学方法对基因和蛋白特征、比较进化、共线性关系及高温胁迫下基因表达模式等进行分析。结果表明，CaDNAJE基因家族有28个成员，不均匀的分布在10条染色体和3个Scaffold上，氨基酸序列长度为99-470 aa，分子量为10.05-52.26 kD，理论等电点（pI）为4.75-9.64，其编码蛋白主要定位在叶绿体和细胞核中；辣椒、番茄和拟南芥的比较进化分析可将其分为GroupⅠ 和GroupⅡ 2个亚族，同一亚族具有类似的蛋白保守motif和基因结构；CaDNAJE基因启动子区包含大量光响应、激素响应和胁迫应答元件；辣椒与拟南芥DNAJE之间存在10对共线性基因，辣椒种内仅有1对；辣椒不同组织转录组分析发现，GroupⅠ 中多数成员在各组织中均有高表达，而GroupⅡ 中除CaDNAJE2、CaDNAJE9等基因存在高表达外，多数基因表达量都很低甚至不表达；高温胁迫下，辣椒叶片过氧化氢含量急速上升后降低，丙二醛含量持续升高，抗氧化物酶活性也有不同程度的提高。同时，高温诱导热激转录因子Hsf和热激蛋白HSP耐热相关基因及CaDNAJE基因高表达，在不同胁迫时期发挥调控作用。推测CaDNAJE基因家族可能参与辣椒响应高温胁迫，提高其耐受力，以降低细胞损伤。

关键词: 辣椒, DnaJ-Like锌指蛋白, 全基因组鉴定, 高温胁迫, 基因表达, 调控

Abstract:

Plant DanJ-Like zine finger protein（DNAJE）is one of the important protein found in recent years, which plays an important role in photosynthesis, development and motility of plastids and the responses of plants to stresses. Here, the DNAJE whole gene family（CaDNAJE）was identified by the whole genome data of pepper（Capsicum annuum）, and the characteristic of gene and protein, comparative evolution, collinearity and transcriptional expression under high temperature stress were analyzed. Totally, 28 members of CaDNAJE genes were identified, and they were distributed unevenly on 10 chromosomes and 3 Scaffolds. The amino acid length, molecular mass and isoelectric point were between 99-470 aa, 10.05-52.26 kD and 4.75-9.64, respectively. Moreover, these encoded proteins were mainly located in the chloroplast and nucleus. The comparative evolution of pepper, tomato（Solanum lycopersicum）and Arabidopsis analysis can be divided into 2 subgroups: GroupⅠ and GroupⅡ, and the CaDNAJE protein in the same subgroup had similar conserved motifs and gene structures. Cis-acting element analysis in the promoter region showed that CaDNAJEs contained a large numbers of functional elements related to light response, hormone response and stress. There were 10 pairs of collinearity genes between pepper and Arabidopsis, but only one pair within pepper species. The transcriptome analysis of different pepper tissues indicated that most members of GroupⅠ were highly expressed in all tissues, while most members of GroupⅡ had low or no expression except for CaDNAJE2 and CaDNAJE9. The content of hydrogen peroxide in pepper leaves increased rapidly and then decreased. The content of malondialdehyde kept increasing and peroxidase activity also had been improved to different extent. High temperature stress induced the high expression of the heat shock factor/heat shock protein（Hsf/HSP）and CaDNAJE genes, which played a regulation role in different stress time. It is suggested that CaDNAJE family genes are involved in response to high temperature stress and then increased tolerance to reduce cell damage.

Key words: pepper, DnaJ-Like zinc finer protein, genome-wide identification, high temperature stress, gene expression, regulation

段敏杰, 李怡斐, 杨小苗, 王春萍, 黄启中, 黄任中, 张世才. 辣椒锌指蛋白DnaJ-Like基因家族鉴定及对高温胁迫的表达响应[J]. 生物技术通报, 2023, 39(1): 187-198.

DUAN Min-jie, LI Yi-fei, YANG Xiao-miao, WANG Chun-ping, HUANG Qi-zhong, HUANG Ren-zhong, ZHANG Shi-cai. Identification of Zinc Finger Protein DnaJ-Like Gene Family in Capsicum annuum and Its Expression Analysis Responses to High Temperature Stress[J]. Biotechnology Bulletin, 2023, 39(1): 187-198.

图/表 9

表1 实时荧光定量PCR引物

Table 1 Primers used for quantitative real-time PCR

基因名称 Gene name	上游引物 Forward primer（5'-3'）	下游引物 Reverse primer（5'-3'）
CaDNAJE2	TACCCCGAAAAGTGTCCGC	TCGTCGAAAACCACCCTATGAG
CaDNAJE3	TTCTGCTCTATGGGGCTCTTAC	TTTTCCTCCACGGGTTCCT
CaDNAJE11	AACACTCAGCCCTGCTTCCC	GCTGAATGCCACTCCCTTGAC
CaDNAJE13	TGGTGGTGCCGATGAGATT	GCATTCGTCGCAGAGGATAA
CaDNAJE15	CACCAAAGTGAGGAGCATCG	AATCCACCAAGAAATCCAGCA
CaDNAJE17	ATTCCAATCCCAGCCACAA	CAACAGCAGCGATGACCAA
CaDNAJE19	GCTCAGCAAGCGGTAAAT	TGCCATCACCATCCCAGT
CaDNAJE21	CCCTTTCACTTCCAACCCC	TCAGTCCCATTTAGCCCATTTA
CaDNAJE23	CGGTGCCGCTCTGGTGAAATC	CGACAACCCACGTCCCCATCT
CaDNAJE24	TGGGAAGAGTCTATTGATGAACG	ATTGGTAGACACGCTTGGTTTT
CaDNAJE27	ACGCAAAGTAAGAAGAAAGCAGC	CGAGAACAGTATCCAAGCCCAC
CaHsfB5	AAAGTTCCAGAAAGGGTGC	TCCATACATTGAGTGAGGC
CaHSP22.0	CCCCACTCACCATCCCAAAA	CCCCACTGATTCTCAACACCCTA
CaHSP16.4	ATGTCAAAGATGATCAGCTTATTG	GGGTACTCAACACGGGACAC
CaUBI3	GGACCCAAACCCCGATGAT	CACCCCAAGCACAATAAGACA

表2 辣椒DNAJE蛋白氨基酸保守基序

Table 2 Conserved motif of the DNAJE proteins in pepper

基序 Motif	长度 Length/aa	氨基酸保守基序 Conserved sequence of amino acid
motif 1	20	CEGCGGSGFVPCSTCNGSGK
motif 2	33	EDKIVIYFTSLRGIRKTFEDCNTVRMILESFRV
motif 3	45	LQNVLEGKKVSLPRVFIKGRYIGGAEEIKQL- HEEGELKKLLEGJP
motif 4	15	CPECNENGLIRCPIC
motif 5	15	VDERDVSMDSGFREE

图1 辣椒DNAJE基因家族成员染色体定位染色体颜色由绿到红表示基因密度由小到大

Fig. 1 Chromosomal location of DNAJE gene family members in pepper（Capsicum annuum） Chromosomal color ranging from green to red indicates the density of gene

图2 辣椒、拟南芥和番茄DNAJE基因家族成员进化树图中蓝色实心圆代表辣椒DNAJE蛋白，黄色三角代表拟南芥DNAJE蛋白，红色五角星代表番茄DNAJE蛋白

Fig. 2 Phylogenetic tree of DNAJE gene family members in pepper, Arabidopsis and tomato（Solanum lyc-opersicum） The blue solid dot indicates pepper DNAJE protein, yellow triangle indicates tomato DNAJE protein, and red star indicates Arabidopsis DNAJE protein

图3 辣椒DNAJE基因家族保守基序、基因结构和顺式作用元件分析 A：CaDNAJE蛋白进化；B：保守基序分布；C：基因结构；D：顺式作用元件分布

Fig. 3 Analysis of conserved motifs, gene structure and cis-acting elements of DNAJE gene family in pepper A: CaDNAJE protein evolution. B: Conserved motif. C: Gene structure. D: cis-acting element distribution

图4 辣椒和拟南芥DNAJE基因家族共线性分析图中Chr和数字1-5分别表示辣椒和拟南芥的染色体。红线表示种间DNAJE共线性关系，紫线表示种内DNAJE共线性关系，灰线表示种间所有基因成员的共线性关系

Fig. 4 Collinearity analysis of DNAJE gene family between pepper and Arabidopsis Chr and the number 1-5 indicate the chromosomes of pepper and Arabidopsis, respectively. The red lines indicate DNAJE collinearity between interspecific. The purple lines indicate DNAJE collinearity between intraspecific. The Gray lines indicate represent collinearity of all gene membres between interspecific

图5 DNAJE基因在辣椒不同组织及果实发育过程中的表达分析 A、B、C、D代表不同表达模式聚类

Fig. 5 Expression profile analysis of pepper DNAJE genes in different tissues and fruit development A, B, C and D indicate the clustering of different expression patterns

图6 辣椒高温胁迫相关基因表达分析 * 表示在0.05水平上差异显著，** 表示在0.01水平上差异显著

Fig. 6 Expression analysis of genes related to high temperature stress in pepper * indicates significant differences at 0.05 level, and ** indicates significant differences at 0.01 level

图7 辣椒幼苗对高温胁迫的生理生化响应

Fig. 7 Physiological and biochemical responses of pepper seedlings to high temperature stress

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