棉花<i>GaLMI1-like</i>基因功能及其启动子表达分析

doi:10.7668/hbnxb.20192873

摘要/Abstract

摘要：

LMI1基因是叶片锯齿状结构发育调控的关键基因。为了研究棉花鸡脚叶发育的机理,通过PCR扩增技术从A基因组棉花亚洲棉石溪亚1号中克隆出GaLMI1-like基因及其启动子序列,大小分别为681,1 439 bp。结构域分析发现,GaLMI1-like蛋白含有与陆地棉中同源基因一样的homeobox结构域,进一步构建了GaLMI1-like基因过表达载体p6MYC-GaLMI1-like,转化拟南芥后验证了GaLMI1-like基因具有调控叶片缺刻表型发育的功能。对启动子序列进行顺式作用元件分析,发现其除了具有CACA-box和TATA-box等基本作用元件外,还具有光响应及根、茎和叶肉特异性表达相关元件。构建了GaLMI1-like启动子的GUS融合表达载体并转化拟南芥,GUS染色结果显示,该启动子能够驱动GUS基因在根中柱、茎和叶片中表达,其中在叶片中染色较深。上述结果表明,GaLMI1-like基因具有调控缺刻叶形成的功能,且此调控棉花叶形发育的功能是通过GaLMI1-like启动子调控其在叶片中强表达实现的。

关键词: 棉花, GaLMI1-like基因, 叶形, 启动子, 顺式作用元件, 转基因拟南芥, GUS染色分析

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

杨小贝, 陈二永, 李成伟. 棉花GaLMI1-like基因功能及其启动子表达分析[J]. 华北农学报, 2022, 37(3): 27-36. doi: 10.7668/hbnxb.20192873.

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.

http://www.hbnxb.net/CN/Y2022/V37/I3/27

图/表 11

图1 GaLMI1-like蛋白结构域及进化分析 A.GaLMI1-like蛋白结构域分析;B.GaLMI1-like及其同源蛋白进化树分析。

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.

图2 GaLMI1-like基因序列扩增 M.DNA Marker;1,2.GaLMI1-like基因片段。

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

图3 p6MYC-GaLMI1-like重组表达载体酶切鉴定 M.DNA Marker;1-3.p6MYC-GaLMI1-like表达载体。

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

图4 植物表达载体p6MYC-GaLMI1-like的构建示意图

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

图5 GaLMI1-like 转基因拟南芥鉴定及表型分析 A.半定量鉴定GaLMI1-like转基因拟南芥阳性植株;B-D.GaLMI1-like转基因拟南芥阳性植株表型观察。

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.

图6 GaLMI1-like启动子序列扩增 M.DNA Marker;1,2.GaLMI1-like启动子片段。

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

表1 棉花GaLMI1-like启动子顺式作用元件分析

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

图7 pCAMBIA1391-pGaLMI1-like重组表达载体酶切鉴定 M.DNA Marker;1-3.pCAMBIA1391-pGaLMI1-like表达载体。

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

图8 植物表达载体pCAMBIA1391-pGaLMI1-like的构建示意图

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

图9 pCAMBIA1391-pGaLMI1-like转基因拟南芥T₁植株的PCR鉴定 M.DNA Marker;Col-0.野生型拟南芥(阴性对照);1-5.转基因拟南芥。

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.

图10 pCAMBIA1391-pGaLMI1-like转基因拟南芥不同生长时期的GUS染色分析 A.种子;B.刚萌发的种子;C.3 d的幼苗;D.12 d的幼苗; E、F.茎生叶;G.花;H.角果。

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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棉花GaLMI1-like基因功能及其启动子表达分析

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棉花GaLMI1-like基因功能及其启动子表达分析

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

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