番茄4CL基因家族鉴定和氮素处理下的表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-1272

摘要/Abstract

摘要：

探究番茄4CL基因家族的基本理化性质、分子进化特性和其在不同浓度氮素下的表达模式。对番茄4CL基因家族成员进行全基因组鉴定,采用生物信息学方法对其进行分析,并在不同浓度氮素处理下进行qPCR验证。在番茄中共鉴定出6个4CL基因家族成员,全部成员均为稳定蛋白,有5个成员呈酸性,编码的氨基酸数在538-957之间,二级结构以无规则卷曲为主,三级结构除Sl4CL04以5bsw.1.A为模型,其他成员均以5bsr.1.A为模型。番茄4CL基因家族内含子数量在5-11之间,12个Motif在Sl4CL中排序及位置都非常保守,具有较高的同源性。启动子顺式作用元件分析发现MYB作用元件的分布最多,在不同浓度氮素处理下,6个基因表达量均有显著差异。对Sl4CL03构建过表达载体,亚细胞定位结果显示位于过氧化物酶体、叶绿体和细胞质。番茄4CL家族进化具有保守性和特异性,能够在氮素处理时进行相关机理调节。

关键词: 番茄, 4CL基因家族, 成员鉴定, 氮素, 表达分析

Abstract:

The objective is to explore the basic physical and chemical properties,molecular evolution characteristics of tomato 4CL gene family and its expression pattern under different concentrations of nitrogen. The whole genome of tomato 4CL gene family members were identified,analyzed by bioinformatics methods,and verified by qPCR under different concentrations of nitrogen treatment. As results,a total of 6 4CL gene family members were identified in tomato（Solanum lycopersicum）,all members were stable proteins,5 members belonged to acidic protein and the number of amino acids encoded by 4CL genes in tomato ranged from 538 aa to 957 aa. The secondary structure was mainly random coils,and the tertiary structure except for Sl4CL04 used 5bsw.1.A as the model,the other members all used 5bsr.1.A as the model. The number of introns in the tomato 4CL gene family was 5-11. The 12 Motifs were very conserved in order and position in Sl4CL,and had high homology. Cis-acting elements analysis indicated that MYB binding sites were the most widely distributed in the promoter region. The expression levels of the 6 genes were significantly different under different concentrations of nitrogen. The overexpression vector was constructed for Sl4CL03,and the subcellular localization results showed that it was located in peroxisome,chloroplast and cytoplasm. In conclusion,the evolution of the tomato 4CL family is conservative and specific,and can be regulated by related mechanisms during nitrogen treatment.

Key words: tomato, 4CL gene family, member identification, nitrogen, expression analysis

马馨馨, 许洋, 赵欢欢, 火兆燕, 王树彬, 钟凤林. 番茄4CL基因家族鉴定和氮素处理下的表达分析[J]. 生物技术通报, 2022, 38(4): 163-173.

MA Xin-xin, XU Yang, ZHAO Huan-huan, HUO Zhao-yan, WANG Shu-bin, ZHONG Feng-lin. Identification of Tomato 4CL Gene Family and Expression Analysis Under Nitrogen Treatment[J]. Biotechnology Bulletin, 2022, 38(4): 163-173.

图/表 11

表1 PCR所用到的引物

Table 1 Primers for PCR

类型 Type	基因ID Gene ID	基因名称Gene name	正向引物Forward（5'-3'）	反向引物Reverse（5'-3'）
RT-qPCR	Solyc03g097030	Sl4CL01	TTTGAGGAGAGGACATGTATTATCG	GACGATGCTAGAATGGTAAA
	Solyc03g117870	Sl4CL02	ATGTTGACACACAAAGGCT	ATTGCTGCTCCGACTCTC
	Solyc06g068650	Sl4CL03	TGTCTTCCTTGGTGCGTC	GCATAATCCTTCACCTTGTTC
	Solyc08g076300	Sl4CL04	TAAGTCAGCAAGATACAGCG	GAGAGAAAGCCCATACACAT
	Solyc11g069050	Sl4CL05	TCAACCTATGAGTATCTTTGCTGC	AAAACCGTTGTGCCGTGC
	Solyc12g042460	Sl4CL06	AGTAAGGATAGTCATTTCTGGG	GCTAAGCACATTGTTATAGGTG
		Actin	GTCCTCTTCCAGCCATCCAT	ACCACTGAGCACAATGTTACCG
RT-PCR	Solyc06g068650	Sl4CL03	ATGACAAATGCAACGATGGAG	ATTTGGA TATCCAGCAG CT
RT-PCR		pCAMBIA1302	GTGGATTGATGTGATATCTCC	CTGACAGAAAATTTGTGCCC

表2 番茄4CL基因家族蛋白理化性质

Table 2 Physico-chemical properties of 4CL family proteins in tomato（Solanum lycopersicum）

基因名称Gene name	氨基酸个数 Number of amino acids	分子量 Molecular weight/kD	等电点 Isoelectric point/pI	不稳定指数Instability index	总平均亲水系数Grand average of hydropathicity	原子总数Total number of atoms	亚细胞定位Subcellular localization
Sl4CL01	570	62080.54	5.34	34.92	0.106	8820	Peroxisome Chloroplast
Sl4CL02	699	77723.64	5.64	37.7	-0.126	10926	Peroxisome
Sl4CL03	560	61700.2	5.49	33.69	-0.028	8735	Peroxisome
Sl4CL04	957	102255.02	5.45	31.47	0.476	14550	Peroxisome
Sl4CL05	538	58930.46	8.49	30.69	0.07	8369	Peroxisome
Sl4CL06	548	60394.02	5.9	32.36	0.045	8590	Peroxisome

表3 番茄4CL基因家族二级结构

Table 3 Secondary structure of tomato 4CL gene family

基因名称 Gene name	α-螺旋α-helix/%	延伸链 Extended strand/%	β-转角 β-turn/%	无规则卷曲 Random coil/%
Sl4CL01	32.28	18.6	6.84	42.28
Sl4CL02	33.19	18.6	9.01	39.2
Sl4CL03	30.89	20.71	7.5	40.89
Sl4CL04	41.69	15.57	7.94	34.8
Sl4CL05	30.67	20.07	8.36	40.89
Sl4CL06	31.93	19.53	8.58	39.96

图1 番茄4CL基因家族三级结构

Fig.1 Tertiary structure of tomato 4CL gene family

图2 番茄、拟南芥和水稻4CL基因家族成员系统发育树

Fig.2 Phylogenetic tree of 4CL gene family in Solanum lycopersicum,Arabidopsis thaliana and Oryza sativa

图3 番茄4CL基因家族成员染色体定位

Fig.3 Localization of 4CL in chromosomes of tomato

图4 番茄4CL基因家族基因结构、保守结构域分布分析

Fig.4 Gene structure and conserved domain analysis of 4CL gene family in tomato

图5 番茄4CL基因家族启动子顺式调控元件类型和数量

Fig.5 Type and number of cis-regulatory elements in the promoter of tomato 4CL gene family

图6 番茄4CL基因在不同浓度氮素下的表达模式不同柱上不同字母表示显著差异（P<0.05）

Fig.6 Expression patterns of tomato 4CL genes under different concentrations of nitrogen Histograms with different letters indicate significant difference at the 0.05 level

图7 Sl4CL03基因的克隆（A）及Sl4CL03基因大肠杆菌菌液PCR（B）

Fig.7 Cloning of Sl4CL03 gene（A）and Escherichia coli bacteria liquid PCR of Sl4CL03 gene（B）

图8 Sl4CL03蛋白在烟草叶片细胞的亚细胞定位 A-D：分别为pCAMBIA1302空载的荧光通道、叶绿体荧光通道、明场、叠加图;E-H：分别为Sl4CL03蛋白的荧光通道、叶绿体荧光通道、明场、叠加图

Fig.8 Subcellular localization of Sl4CL03 in Nicotiana tabacum leaf cells A-D：pCAMBIA1302 empty vector fluorescence,chloroplast fluorescence,bright,and merged.E-H：Sl4CL03 protein fluorescence,chloroplast fluorescence,bright,and merged

参考文献 31

[1]	杜红豆. 氮素对飞机草(Eupatorium odoratum)4CL 酶活性和基因表达影响的研究[D]. 哈尔滨:哈尔滨师范大学, 2012.
	Du HD. Influence of nitrogen on activity of 4-coumarate:coenzyme a ligase(4CL)and its gene expression in Eupatorium odoratum[D]. Harbin:Harbin Normal University, 2012.
[2]	李晓瑞. 干旱胁迫下小麦苯丙烷类代谢及MYB转录因子TaMpc1-D4响应机制研究[D]. 杨凌:西北农林科技大学, 2020.
	Li XR. The mechanisms of the phenylpropanoid pathway and MYB transcription factor TaMpc1-D4 in responses to drought stress in wheat[D]. Yangling:Northwest A＆F University, 2020.
[3]	常胜合, 孙威, 许桂莺, 等. 利用4-香豆酸:CoA连接酶基因Mu4CL15提高香蕉植株的倒伏抗性[J]. 分子植物育种, 2017, 15(12):4905-4911.
	Chang SH, Sun W, Xu GY, et al. Enhancing lodging resistance of banana plant by transforming 4-coumarate:CoA ligase gene Mu4CL15[J]. Mol Plant Breed, 2017, 15(12):4905-4911.
[4]	Sun SC, Xiong XP, Zhang XL, et al. Correction to:Characterization of the Gh4CL gene family reveals a role of Gh4CL7 in drought tolerance[J]. BMC Plant Biol, 2021, 21(1):65. doi: 10.1186/s12870-021-02834-9 URL
[5]	Zhang MY, Wang DJ, Gao XX, et al. Exogenous caffeic acid and epicatechin enhance resistance against Botrytis cinerea through activation of the phenylpropanoid pathway in apples[J]. Sci Hortic, 2020, 268:109348.
[6]	Zhang CH, Ma T, Luo WC, et al. Identification of 4CL genes in desert poplars and their changes in expression in response to salt stress[J]. Genes:Basel, 2015, 6(3):901-917.
[7]	Wei HY, Rao GD, Wang YK, et al. Cloning and analysis of a new 4CL-like gene in Populus tomentosa[J]. For Sci Pract, 2013, 15(2):98-104. doi: 10.1007/s11632-013-0204-z URL
[8]	Liu XY, Cui XM, Ji DC, et al. Luteolin-induced activation of the phenylpropanoid metabolic pathway contributes to quality maintenance and disease resistance of sweet cherry[J]. Food Chem, 2021, 342:128309.
[9]	Dong NQ, Lin HX. Contribution of phenylpropanoid metabolism to plant development and plant-environment interactions[J]. J Integr Plant Biol, 2021, 63(1):180-209. doi: 10.1111/jipb.13054 URL
[10]	Lavhale SG, Kalunke RM, Giri AP. Structural, functional and evolutionary diversity of 4-coumarate-CoA ligase in plants[J]. Planta, 2018, 248(5):1063-1078. doi: 10.1007/s00425-018-2965-z URL
[11]	Li SS, Li Y, Sun LL, et al. Identification and expression analysis of 4-Coumarate:Coenzyme A ligase gene family in Dryopteris Fragrans[J]. Cell Mol Biol:Noisy-Le-Grand, 2015, 61(4):25-33.
[12]	Badhan S, Ball AS, Mantri N. First report of CRISPR/Cas9 mediated DNA-free editing of 4CL and RVE7 genes in chickpea protoplasts[J]. Int J Mol Sci, 2021, 22(1):396. doi: 10.3390/ijms22010396 URL
[13]	王惠, 张顺斌, 金贺, 等. 4-香豆酸辅酶A连接酶响应大豆孢囊线虫胁迫的潜在功能[J]. 生物技术通报, 2021, 37(7):71-80. doi: 10.13560/j.cnki.biotech.bull.1985.2021-0378
	Wang H, Zhang SB, Jin H, et al. Potential function of 4-coumaric acid-CoA ligase in response to soybean cyst nematode stress[J]. Biotechnol Bull, 2021, 37(7):71-80.
[14]	杨克彬, 单雪萌, 史晶晶, 等. 毛竹4-香豆酸辅酶A连接酶基因家族鉴定及表达分析[J]. 核农学报, 2021, 35(1):72-82.
	Yang KB, Shan XM, Shi JJ, et al. Identification and expression analysis of 4CL gene family in Phyllostachys edulis[J]. J Nucl Agric Sci, 2021, 35(1):72-82.
[15]	洪平静, 徐小萍, 王静宇, 等. 龙眼体胚发生早期4CL基因家族鉴定与功能分析[J]. 热带作物学报, 2021, 42(4):909-919.
	Hong PJ, Xu XP, Wang JY, et al. Identification and functional analysis of 4CL gene family during early somatic embryogenesis in Dimocarpus longan lour[J]. Chin J Trop Crop, 2021, 42(4):909-919.
[16]	唐映红. 苎麻4CL和CCR基因家族成员的克隆、表达及功能研究[D]. 长沙:湖南农业大学, 2018.
	Tang YH. Study on the cloning, expression and functional of Boehmeria nivea 4CL and CCR gene family members[D]. Changsha:Hunan Agricultural University, 2018.
[17]	申晚霞, 王志彬, 薛杨, 等. 柑橘4CL基因家族的结构及其功能分析[J]. 园艺学报, 2019, 46(6):1068-1078.
	Shen WX, Wang ZB, Xue Y, et al. Characterization of 4-coumarate:CoA ligase(4CL)gene family in Citrus[J]. Acta Hortic Sin, 2019, 46(6):1068-1078.
[18]	陈夏晔, 彭宣翔. 普通烟草Nt4CL基因家族的生物信息学分析[J]. 贵州农业科学, 2018, 46(8):11-15.
	Chen XY, Peng XX. Bioinformatics analysis on tobacco Nt4CL gene family[J]. Guizhou Agric Sci, 2018, 46(8):11-15.
[19]	曹运鹏, 方志, 李姝妹, 等. 砀山酥梨4CL基因家族的全基因组鉴定与分析[J]. 遗传, 2015, 37(7):711-719.
	Cao YP, Fang Z, Li SM, et al. Genome-wide identification and analyses of 4CL gene families in Pyrus bretschneideri Rehd[J]. Hereditas, 2015, 37(7):711-719.
[20]	Meng J, Li CN, Zhao ML, et al. Lignin biosynjournal regulated by the antisense 4CL gene in alfalfa[J]. Czech J Genet Plant Breed, 2018, 54(No. 1):26-29. doi: 10.17221/23/2017-CJGPB URL
[21]	孙士超. 绿色棉纤维转录组分析及4CL基因家族的鉴定[D]. 石河子:石河子大学, 2020.
	Sun SC. Transcriptome analysis of green cotton fiber and identification of 4CL gene family[D]. Shihezi:Shihezi University, 2020.
[22]	杨婷, 潘翔, 饶国栋, 等. 植物4CL基因家族结构功能与表达特性研究进展[J]. 成都大学学报:自然科学版, 2011, 30(1):4-7.
	Yang T, Pan X, Rao GD, et al. Research progress in structural function and expression characteristic of 4CL’s gene family in plants[J]. J Chengdu Univ:Nat Sci Ed, 2011, 30(1):4-7.
[23]	田晓明, 颜立红, 向光锋, 等. 植物4香豆酸:辅酶A连接酶研究进展[J]. 生物技术通报, 2017, 33(4):19-26. doi: 10.13560/j.cnki.biotech.bull.1985.2017.04.003
	Tian XM, Yan LH, Xiang GF, et al. Research progress on 4-coumarate:coenzyme A ligase(4CL)in plants[J]. Biotechnol Bull, 2017, 33(4):19-26.
[24]	乔中全, 王晓明, 曾慧杰, 等. 灰毡毛忍冬Lm4CL基因克隆及表达分析[J]. 中南林业科技大学学报, 2021, 41(5):122-132.
	Qiao ZQ, Wang XM, Zeng HJ, et al. Clone and expression analysis of Lm4CL in Lonicera macranthoides Hand-Mazz[J]. J Central South Univ For Technol, 2021, 41(5):122-132.
[25]	Sun H, Guo K, Feng S, et al. Positive selection drives adaptive diversification of the 4-coumarate:CoA ligase(4CL)gene in angiosperms[J]. Ecol Evol, 2015, 5(16):3413-3420. doi: 10.1002/ece3.1613 URL
[26]	汤崴, 白云赫, 胡雨晴, 等. 葡萄木质素相关基因PAL、4CL和CAD特征鉴定及其应答赤霉素在果实发育过程中的作用[J]. 南京农业大学学报, 2019, 42(3):430-439.
	Tang W, Bai YH, Hu YQ, et al. Characterization of grape lignin-related genes PAL, 4CL, CAD and the role of GA in the berry development[J]. J Nanjing Agric Univ, 2019, 42(3):430-439.
[27]	王星淇, 孙雪丽, 车婧如, 等. 两个非洲菊4CL基因的克隆及表达[J]. 应用与环境生物学报, 2020, 26(2):272-279.
	Wang XQ, Sun XL, Che JR, et al. Cloning and expression analysis of two 4CL genes in Gerbera[J]. Chin J Appl Environ Biol, 2020, 26(2):272-279.
[28]	Park JJ, Yoo CG, Flanagan A, et al. Defined Tetra-allelic gene disruption of the 4-coumarate:coenzyme A ligase 1(Pv4CL1)gene by CRISPR/Cas9 in switchgrass results in lignin reduction and improved sugar release[J]. Biotechnol Biofuels, 2017, 10:284. doi: 10.1186/s13068-017-0972-0 URL
[29]	Muna Alariqi. 陆地棉基因GhLDOX和Gh4CL3的功能解析[D]. 武汉:华中农业大学, 2020.
	Muna Alariqi. Functional studies of GhLDOX & Gh4CL3 genes in upland cotton Gossypium hirsutum[D]. Wuhan:Huazhong Agricultural University, 2020.
[30]	熊显鹏. Gh4CL30和GhWRKY70D13在棉花抗黄萎病中的功能研究[D]. 石河子:石河子大学, 2020.
	Xiong XP. Functional analysis of Gh4CL30 and GhWRKY70D13 in Cotton Resistance to Verticillium Wilt[D]. Shihezi:Shihezi University, 2020.
[31]	Kienow L, Schneider K, Bartsch M, et al. Jasmonates meet fatty acids:functional analysis of a new acyl-coenzyme A synthetase family from Arabidopsis thaliana[J]. J Exp Bot, 2008, 59(2):403-419. doi: 10.1093/jxb/erm325 pmid: 18267944