Acta Agriculturae Boreali-Sinica ›› 2022, Vol. 37 ›› Issue (2): 42-48. doi: 10.7668/hbnxb.20192675

Special Issue: Apple Biotechnology

• Crop Genetics & Breeding·Germplasm Resources·Biotechnology • Previous Articles     Next Articles

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

ZHAO Huiying1, YANG Guangkai1, GAO Yan1,2, ZHANG Xiaojun1,2, HAO Yanyan1,2   

  1. 1.College of Horticulture,Shanxi Agricultural University,Taigu 030801,China
    2.Shanxi Key Laboratory of Germplasm Improvement and Utilization in Pomology,Taiyuan 030031,China
  • Received:2021-12-13 Published:2022-04-28

苹果MdSGR2基因克隆及植物超表达载体构建

赵惠英1, 杨光凯1, 高燕1,2, 张小军1,2, 郝燕燕1,2   

  1. 1.山西农业大学 园艺学院,山西 太谷 030801
    2.果树种质创制和利用山西省重点实验室,山西 太原 030031
  • 作者简介:
    作者简介:赵惠英(1997—),女,河北邢台人,在读硕士,主要从事园艺植物栽培生理与品质调控研究。
  • 基金资助:
    山西省基础研究计划项目(20210302123396)

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

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