Function and Promoter Expression Analysis of GaLMI1-like Gene in Cotton

doi:10.7668/hbnxb.20192873

Abstract

Abstract:

LMI1 is a key gene in the regulation of serrated leaf development.In order to study the mechanism of okra leaf development in cotton,we cloned GaLMI1-like gene and GaLMI1-like promoter by PCR amplification technology from Shixiya 1,which is an Asia cotton species of a genome.The sequence lengths were 681,1 439 bp,respectively.Domain analysis found that GaLMI1-like contained a homeobox domain.Then,we constructed a reorganization overexpression vector p6MYC-GaLMI1-like.The transgenic Arabidopsis showed incised leaf shape when GaLMI1-like was overexpressed in Arabidopsis.Moreover,we analyzed the cis-acting elements of the promoter sequence of GaLMI1-like.It was found that it had basic acting elements,such as CACA-box and TATA-box,and also had photoresponsive element,root,stem and mesophyll-specific expression elements.GUS fusion expression vector of GaLMI1-like promoter was constructed and transformed into Arabidopsis thaliana.GUS staining results showed that the promoter could drive GUS gene expression in root column,stem and leaf,and the expression was especially high in leaf.These results indicated that GaLMI1-like had the function of regulating the formation of incised leaves,and its function was realized by the strong expression of GaLMI1-like regulated by GaLMI1-like promoter in cotton leaves.

Key words: Cotton, GaLMI1-like gene, Leaf shape, Promoter, Cis-acting element, Transgenic Arabidopsis, GUS staining analysis

YANG Xiaobei, CHEN Eryong, LI Chengwei. Function and Promoter Expression Analysis of GaLMI1-like Gene in Cotton[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(3): 27-36. doi: 10.7668/hbnxb.20192873.

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Figures/Tables 11

Fig.1 Domain and evolutionary analysis of GaLMI1-like protein A.The domain analysis of GaLMI1-like;B.Phylogenetic tree analysis of GaLMI1-like and its homologous proteins.

Fig.2 The sequence amplification of GaLMI1-like gene M.DNA Marker;1,2.GaLMI1-like gene fragment.

Fig.3 Restriction enzyme digestion of recombinant expression vector p6MYC-GaLMI1-like M.DNA Marker;1-3.p6MYC-GaLMI1-like expression vector.

Fig.4 The construction diagram of plant expression vector p6MYC-GaLMI1-like

Fig.5 Identification and phenotypic analysis of GaLMI1-like transgenic Arabidopsis A.Identification of GaLMI1-like transgenic Arabidopsis by semi-quantitative PCR;B-D.The phenotypic analysis of GaLMI1-like transgenic Arabidopsis.

Fig.6 The sequence amplification of GaLMI1-like promoter M.DNA Marker;1,2.GaLMI1-like promoter fragment.

Tab.1 Analysis of cis-acting elements of GaLMI1-like promoter in cotton

元件 Component	序列 Sequence	数量/个 Number	功能 Function
Box 4	ATTAAT	1	部分光响应模块
G-Box	TACGTG	6	光反应中的顺式调节元件
GT1-motif	GGTTAA	6	光应答元件
MRE	AACCTAA	7	MYB结合位点参与光响应
MSA-like	(T/C)C(T/C)AACGG(T/C)(T/C)A	9	参与细胞周期调节的顺式作用元件
Circadian	CAAAGATATC	9	参与昼夜节律控制的顺式调节元件
ARR1AT	ARR1AT	10	细胞分裂素响应元件
INRNTPSADB	YTCANTYY	10	光调节基因结合位点
CAAT-box	GGGTCAATCT	22	启动子和增强子中常见的作用元件
CACTFTPPCA1	CACTFTPPCA1	15	叶肉特异表达相关元件
TATA-box	TATAATAAT	4	核心启动子元件
NODCON	CTCTT	4	茎特异表达相关元件
ROOTMOTIFTAPOX1	ATATT	4	根特异表达相关元件

Fig.7 Restriction enzyme digestion of recombinant expression vector pCAMBIA1391-pGaLMI1-like M.DNA Marker;1-3.pCAMBIA1391-pGaLMI1-like expression vector.

Fig.8 The construction diagram of plant expression vector pCAMBIA1391-pGaLMI1-like

Fig.9 PCR identification of T₁ transgenic Arabidopsis with pCAMBIA1391-pGaLMI1-like M.DNA Marker;Col-0.Wild Arabidopsis (Negative control); 1-5.Transgenic Arabidopsis.

Fig.10 GUS staining analysis of pCAMBIA1391-p GaLMI1-like transgenic Arabidopsis at different time A.Seed;B.Budding seed;C.Three days old seedling; D.Twelve days old seedling;E,F.Stem leaf;G.Flower;H.Silique.

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