苹果<i>MdSGR2</i>基因克隆及植物超表达载体构建

doi:10.7668/hbnxb.20192675

摘要/Abstract

摘要：

STAYGREEN(SGR)基因在植物叶绿素降解代谢过程中起重要作用。为探究苹果SGR2基因的生物学功能,利用RT-PCR技术从澳洲青苹成熟果皮中克隆得到MdSGR2基因,利用生物信息学分析软件对其编码蛋白进行了同源性、理化性质、蛋白结构、启动子顺式作用元件等预测和分析,并构建其植物超表达载体。结果表明,MdSGR2基因完整开放阅读框(ORF)cDNA序列为840 bp,编码279个氨基酸,该蛋白属于Staygreen超家族。理化性质分析表明,该蛋白分子质量为31.27 ku,等电点pI为8.52,属不稳定亲水蛋白。同源性分析表明,MdSGR2蛋白与秋子梨SGR同源性最高,达84.12%。蛋白质结构分析表明,该蛋白二级结构和三级结构主要由α-螺旋、无规则卷曲构成,二者比例分别为40.50%,44.44%。启动子分析表明,该基因启动子区域包含低温顺式作用元件、光响应元件和脱落酸、水杨酸、茉莉酸甲酯响应元件等诱导型顺式作用元件,说明MdSGR2基因可能受环境和激素等多种因素的调控。同时研究成功构建了MdSGR2基因植物超表达载体pCAMBIA2301-MdSGR2。

关键词: 苹果, MdSGR2, 基因克隆, 生物信息学分析, 超表达载体

Abstract:

Gene STAYGREEN(SGR)plays an important role in the process of plant chlorophyll degradation and metabolism.To explore the biological function of gene SGR2,MdSGR2 gene sequence was cloned from the peel of Granny Smith by RT-PCR.The homology,physicochemical properties,protein structure and cis-acting elements of the promoter were predicted and analyzed by bioinformatics software.The plant overexpression vector was constructed by double enzyme digestion.The results showed that the complete open reading frame(ORF)cDNA of gene MdSGR2 was 840 bp.Encoding 279 amino acids in total,it belonged to the Staygreen superfamily.Physicochemical analysis showed that the total molecular weight of the protein was 31.27 ku,and the isoelectric point pI was 8.52,indicating that the protein was an unstable hydrophilic protein.Homology analysis showed that MdSGR2 encoded amino acid sequence was of the highest homology with Pyrus ussuriensis,reaching 84.12%.The protein structure analysis showed that the secondary structure and tertiary structure of MdSGR2 was determined structurally to be 40.55% α-helix and 44.44% random coil.Promoter analysis showed that the promoter region of the gene contained cryogenic cis-acting elements,photo-responsive elements and induced cis-acting elements such as abscisic acid,salicylic acid and methyl jasmonate response elements.These results suggest that gene MdSGR2 may be regulated by many factors such as environment and hormones.The plant overexpression vector pCAMBIA2301-MdSGR2 was successfully constructed.

Key words: Apple, MdSGR2, Gene cloning, Bioinformatics analysis, Overexpression vector

赵惠英, 杨光凯, 高燕, 张小军, 郝燕燕. 苹果MdSGR2基因克隆及植物超表达载体构建[J]. 华北农学报, 2022, 37(2): 42-48. doi: 10.7668/hbnxb.20192675.

ZHAO Huiying, YANG Guangkai, GAO Yan, ZHANG Xiaojun, HAO Yanyan. Cloning of Gene MdSGR2 from Apple and Construction of Plant Overexpression Vector[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(2): 42-48. doi: 10.7668/hbnxb.20192675.

http://www.hbnxb.net/CN/Y2022/V37/I2/42

图/表 6

图1 苹果MdSGR2基因PCR扩增
M.D2000 DNA Marker;1—4.MdSGR2基因PCR扩增产物。

Fig.1 PCR amplification of gene MdSGR2
M.D2000 DNA Marker;1—4.PCR products of MdSGR2.

图2 MdSGR2基因同源氨基酸序列比对
Md.苹果;Pu.秋子梨;Pp.桃;Pa.甜樱桃;Fa.草莓。

Fig.2 Homologous amino acid sequence alignment of gene MdSGR2
Md.Malus domestica;Pu.Pyrus ussuriensis;Pp.Prunus persica;Pa.Prunus avium;Fa.Fragaria vesca subsp.vesca.

图3 苹果MdSGR2与其他同源蛋白系统进化分析

Fig.3 Phylogenetic analysis of MdSGR2 protein and other homologous proteins

图4 MdSGR2蛋白三级结构模型

Fig.4 Tertiary structure model of MdSGR2 protein

表1 MdSGR2基因启动子区顺式作用元件及功能

Tab.1 Cis-active elements and functions in the promoter region of MdSGR2

顺式作用元件 Cis-acting element	序列 Sequence	功能 Function	数量 Number
ABRE	CACGTG/ACGTG	脱落酸响应顺式作用元件	5
ACE	CTAACGTATT	光响应顺式作用元件	1
CGTCA-motif/TGACG-motif	CGTCA/TGACG	茉莉酸甲酯响应顺式调节元件	4
G-Box	CACGTG/CACGTT/CACGAC/ACACGTGT	光响应顺式调节元件	7
LTR	CCGAAA	低温响应顺式作用元件	2
TCA-element	CCATCTTTTT	水杨酸响应顺式作用元件	1

图5 pCAMBIA2301-MdSGR2超表达载体构建
A.pMD-MdSGR2双酶切;B.pCAMBIA2301载体双酶切;C.超表达载体pCAMBIA2301-MdSGR2鉴定结果。M.D2000 DNA Marker;1.pMD-MdSGR2质粒双酶切产物;2.pCAMBIA2301载体双酶切产物;3.超表达载体pCAMBIA2301-MdSGR2双酶切产物。

Fig.5 Construction of pCAMBIA2301-MdSGR2 overexpression vector
A.Double enzyme-digested of pMD-MdSGR2;B.Double enzyme-digested of vector pCAMBIA2301;C.Results of identification of overexpression vector pCAMBIA2301-MdSGR2.M.D2000 DNA Marker;1.Double digestion product of pMD-MdSGR2 plasmid;2.The double digestion product of vector pCAMBIA2301;3.Double enzyme-digested product of pCAMBIA2301-MdSGR2.

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苹果MdSGR2基因克隆及植物超表达载体构建

Cloning of Gene MdSGR2 from Apple and Construction of Plant Overexpression Vector

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