Cloning and Functional Analysis of GhFUL1 Gene and Its Promoter in Cotton

doi:10.7668/hbnxb.20193992

Abstract

Abstract:

To investigate the function of GhFUL1 gene in branching development of cotton,the GhFUL1 gene in the MADS-box family and its promoter were cloned from cotton,and the function and promoter activity of the gene were analyzed.The results indicated that the open reading frame (ORF) of GhFUL1 gene was 726 bp and encoded 241 amino acids.Phylogenetic tree analysis showed that the amino acid sequence of FUL subclade was conserved,and GhFUL1 owned the highest similarity to Cacao TcAGL8-1 except for the homologous proteins in cotton.Subcellular localization result showed that GhFUL1 was localized in nucleus and membrane.The expression level of different cotton tissues was examined,and the result showed that GhFUL1 was highly expressed in shoot apical meristem.Overexpressing GhFUL1 in Arabidopsis thaliana increased the number of lateral branches by decreasing the expression of cytokinin oxidation/dehydrogenase genes AtCTK1 and AtCTK6.Compared with the wild type,transgenic plants did not result in significant changes of the flowering time and floral organ development.The present results suggested that GhFUL1 might affect the number of branches by regulating the cytokinin synthesis pathway.The promoter of GhFUL1 was predicted to include the TATA-box and CAAT-box elements,and cis-acting elements related to light response,auxin response and stress response by PlantCARE website.Then the promoter of GhFUL1 was cloned and constructed into expression vector pBI121 to detect promoter activity.The GUS staining of transgenic Arabidopsis lines showed that GhFUL1 promoter was specifically expressed in shoot apical meristem at seedling stage and flower organ at maturity.This study revealed that the gene and promoter of GhFUL1 played an important role in the Arabidopsis branching development process after transformation.

Key words: Cotton, GhFUL1, Subcellular localization, Function analysis, Promoter activity, GUS staining

ZHANG Xiaohong, XU Peiyang, YAN Xu, ZHANG Beibei, YU Chanchan. Cloning and Functional Analysis of GhFUL1 Gene and Its Promoter in Cotton[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(5): 60-68. doi: 10.7668/hbnxb.20193992.

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References 31

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引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')	用途 Purpose
GhFUL1-F	ATGGGGAGGGGTAGGGTTCAG	基因克隆
GhFUL1-R	CTCAATACGAGGAATCATCCA	基因克隆
pGhFUL1-F	CAAAAGGCTAATTCAAGCTCA	启动子克隆
pGhFUL1-R	TTTTCTTCCAAATTTCGACC	启动子克隆
qGhFUL1-F	CAATTGCCAAGATTCATCCTC	荧光定量PCR
qGhFUL1-R	CCCTCTTCTCTTCCATTGCT	荧光定量PCR
GhACTIN4-F	ATCCTCCGTCTTGACCTTG	荧光定量PCR
GhACTIN4-R	TGTCCGTCAGGCAACTCAT	荧光定量PCR
AtUBQ5-F	CTCGCCGACTACAACATCCAG	荧光定量PCR
AtUBQ5-R	CCTTCCTCAAACGCTGAACC	荧光定量PCR
AtCKX7-F	GGTTTTGCTACAGCCAGCTCC	荧光定量PCR
AtCKX7-R	GGTCAGCACCGTTGACAAAC	荧光定量PCR
AtCKX1-F	TACCTTCATTTGACCGTTGGAG	荧光定量PCR
AtCKX1-R	CCTGTAACAATCTCTAGCTGG	荧光定量PCR
AtCKX6-F	TATCAGAGCTAAGCTACGTGAC	荧光定量PCR
AtCKX6-R	GATCGGAGTTTTACCTCAGACAC	荧光定量PCR

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')	用途 Purpose
GhFUL1-F	ATGGGGAGGGGTAGGGTTCAG	基因克隆
GhFUL1-R	CTCAATACGAGGAATCATCCA	基因克隆
pGhFUL1-F	CAAAAGGCTAATTCAAGCTCA	启动子克隆
pGhFUL1-R	TTTTCTTCCAAATTTCGACC	启动子克隆
qGhFUL1-F	CAATTGCCAAGATTCATCCTC	荧光定量PCR
qGhFUL1-R	CCCTCTTCTCTTCCATTGCT	荧光定量PCR
GhACTIN4-F	ATCCTCCGTCTTGACCTTG	荧光定量PCR
GhACTIN4-R	TGTCCGTCAGGCAACTCAT	荧光定量PCR
AtUBQ5-F	CTCGCCGACTACAACATCCAG	荧光定量PCR
AtUBQ5-R	CCTTCCTCAAACGCTGAACC	荧光定量PCR
AtCKX7-F	GGTTTTGCTACAGCCAGCTCC	荧光定量PCR
AtCKX7-R	GGTCAGCACCGTTGACAAAC	荧光定量PCR
AtCKX1-F	TACCTTCATTTGACCGTTGGAG	荧光定量PCR
AtCKX1-R	CCTGTAACAATCTCTAGCTGG	荧光定量PCR
AtCKX6-F	TATCAGAGCTAAGCTACGTGAC	荧光定量PCR
AtCKX6-R	GATCGGAGTTTTACCTCAGACAC	荧光定量PCR

基因名 Gene name	基因ID Gene ID	染色体ID Chromosome ID	起始位点 Start position	终止位点 End position	链 Strand	氨基酸相似度/% Amino acid identity
GhFUL1-D	Gh_D07G0671	D07	7915954	7933806	-	95.47
GhFUL1-A	Gh_A07G0605	A07	8448050	8466601	-	100.00
GhFUL2-D	Gh_D03G0922	D03	31494711	31500162	+	82.26
GhFUL2-A	Gh_A03G0634	A03	17989506	17995387	+	81.88

基因名 Gene name	基因ID Gene ID	染色体ID Chromosome ID	起始位点 Start position	终止位点 End position	链 Strand	氨基酸相似度/% Amino acid identity
GhFUL1-D	Gh_D07G0671	D07	7915954	7933806	-	95.47
GhFUL1-A	Gh_A07G0605	A07	8448050	8466601	-	100.00
GhFUL2-D	Gh_D03G0922	D03	31494711	31500162	+	82.26
GhFUL2-A	Gh_A03G0634	A03	17989506	17995387	+	81.88

元件 Element	链 Strand	数量 Number	序列 Sequence	功能注释 Function annotation
TATA-box	+/-	51	TATATA	核心启动子元件
CAAT-box	+/-	22	CCAAT	启动子和增强子区域中常见的顺式作用元件
TGACG-motif/CGTCA-motif	+/-	2	TGACG/CGTCA	参与MeJA反应的顺式作用元件
ATCT-motif/Box4	+	2	AATCTAATCC/ATTAAT	参与光响应的保守DNA模块的部分元件
AT-rich element	-	1	ATAGAAATCAA	AT-rich DNA结合蛋白结合位点
TGA-element	+	1	AACGAC	生长素响应元件
TCA-element	+	1	CCATCTTTTT	参与SA响应的顺式作用元件
ARE	+/-	2	AAACCA	厌氧诱导必须元件
GT1-motif	+	4	GGTTAA	光响应元件
CCAAT-box	+	1	CAACGG	MYBHv1结合位点

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