香蕉ELF3的克隆与表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-1163

摘要/Abstract

摘要：

【目的】早花基因3（EARLY FLOWERING3, ELF3）是生物钟系统核心振荡器的重要组成成员，在植物生物钟和开花调控及逆境胁迫等过程扮演重要角色。目前尚无香蕉ELF3的相关报道，为揭示ELF3在香蕉抵御逆境胁迫中的功能对其进行了鉴定、克隆和表达分析。【方法】克隆了从香蕉基因组鉴定获得的4个MaELF3成员（MaELF3-1-MaELF3-4），利用生物信息学手段分析了它们的序列特征，基于转录组数据和实时荧光定量PCR研究了它们在高低温胁迫、香蕉枯萎病菌FocTR4侵染及茉莉酸甲酯（MeJA）和脱落酸（ABA）处理后的表达模式。【结果】4个MaELF3的CDS长度介于2 058-2 301 bp，可编码685-766 aa。除MaELF3-4含3个外显子外，其余MaELF3s均含4个外显子。4个MaELF3s均为定位于细胞核的不稳定碱性蛋白；与温带植物ELF3s不同，MaELF3s和多种热带植物的ELF3均无朊病毒样结构域（PrD）。系统进化结果显示，MaELF3-2和MaELF3-4与拟南芥ELF3（At2g25930）亲缘关系最近；MaELF3-1和MaELF3-3分别与粗柄象腿蕉（Ensete ventricosum）和野蕉（Musa balbisiana）ELF3亲缘关系最近。MaELF3s启动子上存在一些光、激素（MeJA、ABA等）和逆境胁迫（干旱、低温等）响应相关元件。基因表达分析结果显示，所有4个MaELF3s在香蕉叶片中的表达均受ABA和JA影响，且它们在香蕉根系中的表达受FocTR4显著抑制；除MaELF3-4外其他成员的表达均受高低温显著抑制。【结论】MaELF3s参与了香蕉对不同逆境胁迫的响应。

关键词: 早花基因3, 香蕉, 基因克隆, 序列和表达分析, 逆境响应

Abstract:

【Objective】EARLY FLOWERING 3（ELF3）, an important component of the core circadian oscillator, has been identified to function greatly in regulating circadian and flowering, and stress responses of plants. Up to now, however, there has been no report on banana ELF3. Our present study is aimed to explore the functions of ELF in banana responses to stresses.【Method】Four ELF3 genes（MaELF3-1-MaELF3-4）identified from banana（Musa acuminata）genome were cloned using reverse transcription PCR and subjected to a series of bioinformatic analysis. Their expression patterns under treatments of high and low temperature, banana wilt disease pathogen Fusarium oxysporum f. sp. cubense Tropical Race 4（FocTR4）, MeJA and ABA were analyzed using transcriptome data and quantitative real time PCR（RT-qPCR）. 【Result】The coding sequences of the four MaELF3s ranged from 2 058 bp to 2 301 bp, encoding proteins consisted of 685-766 amino acids. Except that MaELF3-4 had three exons, all the other three MaELF3s had four exons. Protein analysis revealed that all MaELF3s were nucleus-located unstable basic proteins; unlike the ELF3s from temperate plants, all ELF3s from banana and some other tropical plants did not contain the prion-like domain（PrD）. Phylogenetic analysis results showed that MaELF3-2 and MaELF3-4 shared the closest relationship with Arabidopsis ELF3（At2g25930）, while MaELF3-1 and MaELF3-3 was the closest to ELF3 from Ensete ventricosum and Musa balbisiana, respectively. The promoter regions of MaELF3s contained many light-, phytohormone（such as MeJA and ABA）- and stress（such as drought and low temperature）-responsive elements. Gene expression analysis revealed that, the expressions of all the four MaELF3s in the leaves were greatly influenced by JA and ABA treatments, and their expressions in the banana roots was significantly downregulated by FocTR4. Moreover, the expressions of all MaELF3s except MaELF3-4 were downregulated by both high and low temperature treatments. 【Conclusion】The results obtained in this study indicate that MaELF3s participate in the banana responses to different stresses.

Key words: EARLY FOLWERING 3, Musa acuminata, gene cloning, sequence and expression characterization, stress response

郝思怡, 张君珂, 王斌, 曲朋燕, 李瑞得, 程春振. 香蕉ELF3的克隆与表达分析[J]. 生物技术通报, 2024, 40(5): 131-140.

HAO Si-yi, ZHANG Jun-ke, WANG Bin, QU Peng-yan, LI Rui-de, CHENG Chun-zhen. Cloning and Expression Analysis of Banana EARLY FLOWERING 3（ELF3）Genes[J]. Biotechnology Bulletin, 2024, 40(5): 131-140.

图/表 10

参考文献 35

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基因Gene	引物名称Primer name	引物序列Primer sequence（5'-3'）	退火温度Annealing temperature/℃	用途Application
MaELF3-1	MaELF3-1-F	ATGAAAGGGGGAAAGGAGGA	62	基因克隆 Gene cloning
	MaELF3-1-R	TCAGTCATGTTGCAGCCTCTC	62	基因克隆 Gene cloning
	MaELF3-1-qF	ATCTACAGACGGAGAACATTGCT	60	实时荧光定量PCR RT-qPCR
	MaELF3-1-qR	GCCCACTTTCTAAAACCTGACGATG	60	实时荧光定量PCR RT-qPCR
MaELF3-2	MaELF3-2-F	ATGAAAGGGGCGGAGGAG	62	基因克隆 Gene cloning
	MaELF3-2-R	TCATGAGTCATGTTGTTGCCTC	62	基因克隆 Gene cloning
	MaELF3-2-qF	ATGCAATATGTCCCATCATCCGAA	60	实时荧光定量PCR RT-qPCR
	MaELF3-2-qR	TTGCTGTGCTTCCTTGTAACTCG	60	实时荧光定量PCR RT-qPCR
MaELF3-3	MaELF3-3-F	ATGAAAGGGGAGAAGGATGA	58	基因克隆 Gene cloning
	MaELF3-3-R	TCATGAATCATGCTGTTGTCTT	58	基因克隆 Gene cloning
	MaELF3-3-qF	ATGCAATATGTCCCATCATCCGAA	60	实时荧光定量PCR RT-qPCR
	MaELF3-3-qR	TTGCTGTGCTTCCTTGTAACTCG	60	实时荧光定量PCR RT-qPCR
MaELF3-4	MaELF3-4-F	ATGCTTTCTCCATTGTACATTTCTC	60	基因克隆 Gene cloning
	MaELF3-4-R	TCATGAGTCATGTTGTTGCCT	60	基因克隆 Gene cloning
	MaELF3-4-qF	ATCAACGAATGGCGATACAGT	60	实时荧光定量PCR RT-qPCR
	MaELF3-4-qR	GCCTCACCATCATTAATAACAGC	60	实时荧光定量PCR RT-qPCR

基因Gene	引物名称Primer name	引物序列Primer sequence（5'-3'）	退火温度Annealing temperature/℃	用途Application
MaELF3-1	MaELF3-1-F	ATGAAAGGGGGAAAGGAGGA	62	基因克隆 Gene cloning
	MaELF3-1-R	TCAGTCATGTTGCAGCCTCTC	62	基因克隆 Gene cloning
	MaELF3-1-qF	ATCTACAGACGGAGAACATTGCT	60	实时荧光定量PCR RT-qPCR
	MaELF3-1-qR	GCCCACTTTCTAAAACCTGACGATG	60	实时荧光定量PCR RT-qPCR
MaELF3-2	MaELF3-2-F	ATGAAAGGGGCGGAGGAG	62	基因克隆 Gene cloning
	MaELF3-2-R	TCATGAGTCATGTTGTTGCCTC	62	基因克隆 Gene cloning
	MaELF3-2-qF	ATGCAATATGTCCCATCATCCGAA	60	实时荧光定量PCR RT-qPCR
	MaELF3-2-qR	TTGCTGTGCTTCCTTGTAACTCG	60	实时荧光定量PCR RT-qPCR
MaELF3-3	MaELF3-3-F	ATGAAAGGGGAGAAGGATGA	58	基因克隆 Gene cloning
	MaELF3-3-R	TCATGAATCATGCTGTTGTCTT	58	基因克隆 Gene cloning
	MaELF3-3-qF	ATGCAATATGTCCCATCATCCGAA	60	实时荧光定量PCR RT-qPCR
	MaELF3-3-qR	TTGCTGTGCTTCCTTGTAACTCG	60	实时荧光定量PCR RT-qPCR
MaELF3-4	MaELF3-4-F	ATGCTTTCTCCATTGTACATTTCTC	60	基因克隆 Gene cloning
	MaELF3-4-R	TCATGAGTCATGTTGTTGCCT	60	基因克隆 Gene cloning
	MaELF3-4-qF	ATCAACGAATGGCGATACAGT	60	实时荧光定量PCR RT-qPCR
	MaELF3-4-qR	GCCTCACCATCATTAATAACAGC	60	实时荧光定量PCR RT-qPCR

蛋白 Protein	基因ID Gene ID	编码区长度 CDS length/bp	蛋白长度 Protein size/aa	分子量 Molecular weight/Da	等电点 pI	不稳定系数 Instability index	最大平均亲水性 GRAVY	亚细胞定位 Subcellular location
MaELF3-1	Macma4_01_g08270	2 211	736	80 267.16	8.52	65.72	-0.652	细胞核Nucleus
MaELF3-2	Macma4_04_g26880	2 301	766	82 762.16	8.94	66.59	-0.628	细胞核Nucleus
MaELF3-3	Macma4_05_g09710	2 265	754	81 749.59	8.66	63.25	-0.693	细胞核Nucleus
MaELF3-4	Macma4_06_g37150	2 058	685	74 061.53	8.96	69.77	-0.61	细胞核Nucleus

蛋白 Protein	基因ID Gene ID	编码区长度 CDS length/bp	蛋白长度 Protein size/aa	分子量 Molecular weight/Da	等电点 pI	不稳定系数 Instability index	最大平均亲水性 GRAVY	亚细胞定位 Subcellular location
MaELF3-1	Macma4_01_g08270	2 211	736	80 267.16	8.52	65.72	-0.652	细胞核Nucleus
MaELF3-2	Macma4_04_g26880	2 301	766	82 762.16	8.94	66.59	-0.628	细胞核Nucleus
MaELF3-3	Macma4_05_g09710	2 265	754	81 749.59	8.66	63.25	-0.693	细胞核Nucleus
MaELF3-4	Macma4_06_g37150	2 058	685	74 061.53	8.96	69.77	-0.61	细胞核Nucleus

功能Function		元件Element	MaELF3-1	MaELF3-2	MaELF3-3	MaELF3-4
光响应 Light responsive	光 Light	ACE		1
		AE-box			1	1
		Box 4		1	8
		Gap-box			1
		GATA-motif			3	2
		G-Box	3
		GT1-motif	1	1		1
		GTGGC-motif				1
		I-box	1	1	2
		Sp1		2		1
		TCCC-motif		1
		TCT-motif	2		1
生长发育 Growth and development	分生组织表达Meristem expression	CAT-box	1	1	2	1
生长发育 Growth and development	玉米蛋白代谢Zein metabolism	O2-site		1
激素响应 Phytohormone responsive	脱落酸Abscisic acid	ABRE	7	1	1
	生长素Auxin	AuxRR-core		1
	生长素Auxin	TGA-element				1
	茉莉酸甲酯Methyl jasmonate	CGTCA-motif	2	4	2	1
	茉莉酸甲酯Methyl jasmonate	TGACG-motif	2	4	2	1
	赤霉素Gibberellin	GARE-motif		1		1
		P-box				1
		TATC-box				1
	水杨酸Salicylic acid	TCA-element	1			1
胁迫响应 Stress responsive	厌氧诱导Anaerobic induction	ARE			1
	缺氧特异性诱导Anoxic specific induction	GC-motif	1	1	1	2
	低温Low temperature	LTR		1
	干旱诱导Drought induction	MBS	3	1