花生4CL基因家族鉴定及对干旱与盐胁迫响应分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-1266

摘要/Abstract

摘要：

目的分析花生4-香豆酸：CoA连接酶（4-coumarate：CoA ligase, 4CL）基因家族成员基本特性及其对干旱和盐胁迫的响应，为培育花生耐旱抗盐品种提供重要的目标基因。方法通过HMM文件及NCBI CDD和Pfam数据库在全基因组水平鉴定花生4CL基因家族成员；利用ExPASy-ProtParam工具进行蛋白理化性质分析；通过MEGA7及itol工具进行系统进化分析；通过MEME及NCBI中的CD-search工具进行蛋白保守基序和保守结构域分析；通过PlantCARE及TBtools进行启动子元件分析及可视化；通过RNA-seq数据及RT-qPCR分析花生4CLs转录水平变化。结果借助花生Tifrunner基因组参考数据，鉴定到56个花生4CL基因，氨基酸长度介于239‒1 208之间，pI介于5.5‒9.22之间，蛋白脂肪系数介于80.2‒103.13之间，不稳定性指数在25.51‒48.79之间，GRAVY值在-0.367‒0.139之间；花生A与B基因组的20条染色体上，Ah4CLs基因不均匀地分布，5和15号染色体Ah4CLs基因密度最高；同一个聚类分支，Ah4CLs具有相似的保守基序组成及相似的内含子‒外显子分布结构，外显子数量在1‒18之间；Ah4CLs启动子区域富含光、非生物胁迫、激素及生长发育响应元件；Ah4CLs表达具有组织特异性，在根、花及种子中表达量较高；在ABA、盐与干旱胁迫处理下，部分Ah4CLs转录水平显著增加，特别是Ah4CL28在ABA、干旱及盐处理下均明显转录上调，这些基因在花生应对非生物胁迫中可能发挥着重要作用。结论鉴定到的56个花生4CL基因家族成员有不同结构与特性，在部分基序及结构域上具有保守型，Ah4CLs在影响花生生长发育的同时，还参与非生物胁迫响应。

关键词: 花生, 4CL基因, 基因家族, 进化分析, 共线性分析, 启动子分析, 干旱胁迫, 盐胁迫

Abstract:

Objective To analyze the basic characteristics of peanut 4CL (4-coumarate:CoA ligase) gene family members and their responses to drought and salt stress in order to provide important target genes for breeding peanut varieties tolerant to drought and salt. Method The members of peanut 4CL gene family were identified by HMM file, NCBI CDD, and Pfam databases at the whole genome level. ExPASy-ProtParam was used to analyze the physicochemical properties of proteins. MEGA7 and itol tools were used for phylogenetic analysis. The conserved motifs and conserved domains of proteins were analyzed by MEME and CD-search tools in NCBI, respectively. The cis-acting elements were analyzed and visualized by PlantCARE and TBtools, respectively. The transcriptional changes of peanut 4CLs were analyzed by RNA-seq data and RT-qPCR. Result Based on the reference data of peanut Tifrunner genome, 56 peanut 4CL genes were identified, with amino acid length ranging from 239 to 1 208, pI ranging from 5.5 to 9.22, and aliphatic index ranging from 80.2 to 103.13. The instability index (II) was 25.51-48.79, and GRAVY was -0.367-0.139. The Ah4CLs were unevenly distributed on 20 chromosomes of peanut A and B genome, and the Ah4CLs gene density was the highest on chromosomes 5 and 15. In the same clustering branch, Ah4CLs had similar conserved motif composition and intron-exon distribution structure, with exon number ranging from 1 to 18. Ah4CLs promoter regions were rich in light, abiotic stress, hormone and growth response elements. The expression of Ah4CLs was tissue-specific and higher in the root, flower and seed. Under ABA, salt and drought stress, the transcription levels of some Ah4CLs significantly increased, especially Ah4CL28 was significantly up-regulated under ABA, drought, and salt stress. These genes may play an important role in peanut response to abiotic stress. Conclusion The 56 identified peanut 4CL gene family members have different structures and characteristics, and some motifs and domains are conserved. Ah4CLs not only affects plant growth and development but also participates in abiotic stress response.

Key words: peanut, 4CL genes, gene family, phylogenetic analysis, synteny analysis, promoter analysis, drought stress, salt stress

张泽, 杨秀丽, 宁东贤. 花生4CL基因家族鉴定及对干旱与盐胁迫响应分析[J]. 生物技术通报, 2025, 41(7): 117-127.

ZHANG Ze, YANG Xiu-li, NING Dong-xian. Identification of 4CL Gene Family in Arachis hypogaea L. and Expression Analysis in Response to Drought and Salt Stress[J]. Biotechnology Bulletin, 2025, 41(7): 117-127.

图/表 10

表1 RT-qPCR所用基因引物

Table 1 Genetic primers for RT-qPCR

基因名称 Gene name	正向引物 Forward primer （5′‒3′）	反向引物 Reverse primer （5′‒3′）
Ahactin7	TTGGAATGGGTCAGAAGGATGC	AGTGGTGCCTCAGTAAGAAGC
Ah4CL21	GAGAGAGCGAGGATGAAGGC	AGCTCCTCTGACCACGATCT
Ah4CL22	TCTTGGACAGAGCGGGGATA	GTGTCCTCTTCCAATGCCGA
Ah4CL28	GCTACACTTGGACCCTTGCT	GGCAGAGGAAGGATGGTGTC
Ah4CL37	AGCCCTTGTGCCTTTCTCTC	CATGGCCCTCAACTGAACCT
Ah4CL49	CGAGAATTTGAGCAGCGTGG	ACTCCACCATCTCCTCCTCC
Ah4CL53	CTCTGCACTCCCTCAACCTG	GCCTGGTTTACGCTCTCCTT

图1 花生与拟南芥4CL蛋白的系统进化分析不同区域颜色表示不同分类亚组分支，包括Clade 4CL与Clade A‒F

Fig. 1 Phylogenetic analysis of 4CL protein in peanut (Arachis hypogaea L.) and Arabidopsis thalianaDifferent regional colors indicate different classification subgroup branches, including Clade 4CL and Clade A‒F

图2 花生4CL基因染色体分布分析

Fig. 2 Analysis of chromosome distribution of 4CL gene in peanut

图3 花生4CL基因家族成员共线性分析粉红色框表示花生染色体，灰色线条指示花生4CL基因位置，蓝色线条连接的Ah4CL基因之间存在共线性

Fig. 3 Syntenic relationship of peanut 4CL gene family membersThe pink box indicates the peanut chromosomes, while the gray lines denote the positions of the 4CL genes in peanuts. The blue lines illustrate the collinearity observed between the Ah4CL genes

图4 花生4CL家族成员进化关系（A）、保守基序（B）、保守结构域（C）及基因结构（D）分析

Fig. 4 Phylogenetic relationship (A), conserved motif (B), conserved domain (C), and gene structure (D) analysis of peanut 4CL family members

表2 花生4CL家族蛋白保守基序序列

Table 2 Conserved motif sequences of peanut 4CL family proteins

基序 Motif	序列 Sequence	E值 E_value
Motif 1	GWLHTGDLGYIDEDGYJFIVDRLKELIKYKGEQVAPAELEAVLYSHP	3.8e-1 534
Motif 2	LLYSSGTTGLPKGVVLTHRGL	1.8e-653
Motif 3	GEICIRGPTIMKGYLKBPEAT	2.1e-581
Motif 4	DAAVVPRPDEEAGEVPCAFVV	4.8e-540
Motif 5	VVFIDSJPKTSTGKILRKDLR	2.8e-488
Motif 6	KSEDVYLWTLPMFHVNGLCFP	2.8e-448
Motif 7	SPAFYELHLAVPMAGAVLTTANP	7.2e-402
Motif 8	AVGGTNVCMRKFDAKAILEAIEKHKVT	5.4e-457
Motif 9	PGAIVSQGYGMTETG	6.9e-322
Motif 10	ITEEEIIEFCAKQVAPYKRPK	3.5e-402

图5 花生4CL基因启动子区顺式作用元件分析

Fig. 5 Analysis of cis-acting elements in the promoter regions of peanut 4CL genes

图6 花生4CL基因在不同组织（A）及在干旱、盐及ABA处理后（B）的表达分析

Fig. 6 Expression analysis of peanut 4CL genes in different tissues (A) and after drought, salt and ABA treatment (B)

图7 模拟干旱处理后Ah4CL28及Ah4CL37表达水平变化通过Student’s t检验进行显著性分析，*表示P<0.05，**表示P<0.01。下同

Fig. 7 Ah4CL28 and Ah4CL37 expression alterations after simulated drought treatmentSignificance analysis is conducted by Student's t-test, with * indicating P<0.05 and ** indicating P<0.01. The same below

图8 盐处理后Ah4CL21、 Ah4CL22、 Ah4CL28、 Ah4CL49及Ah4CL53表达水平变化

Fig. 8 Ah4CL21, Ah4CL22, Ah4CL28, Ah4CL49, and Ah4CL53 expression alterations after salt treatment

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