Cloning and Expression Analysis of Banana EARLY FLOWERING 3（ELF3）Genes

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

Abstract

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

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.

Figures/Tables 10

References 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