苹果谷胱甘肽S-转移酶基因MdGSTF3的克隆及其表达分析

樊连梅1,2, 刘更森2, 李思琪1, 原永兵2,*
青岛农业大学1生命科学学院/山东省高校植物生物技术重点实验室; 2园艺学院/青岛市现代农业质量与安全工程重点实验室, 山东青岛266109

通信作者:原永兵;E-mail: yyb@qau.edu.cn;Tel: 0532-88030513

摘 要:

以‘富士’苹果果皮为试材, 利用RT-PCR和RACE-PCR克隆得到1个全长为868 bp的苹果谷胱甘肽S-转移酶基因MdGSTF3全长cDNA序列, GenBank的登录号为KP234026, 该基因5'非翻译区49 bp, 3'非翻译区151 bp, 含有26 bp的PolyA尾, 编码区为642 bp, 编码213个氨基酸, 编码的蛋白质含有19种氨基酸, 不含有半胱氨酸Cys, 相对分子质量为23.993 kDa, 等电点为6.17。氨基酸多重序列比对分析结果表明MdGST与梅的PmGST蛋白(XP_008235026)遗传相似性为83%。实时荧光定量PCR结果表明, MdGSTF3在条红‘富士’的茎、叶和花中表达量高于其突变体片红‘富士’; 而在果皮和果肉中, 片红‘富士’该基因的表达量高于条红‘富士’; 在表达量达到最大值时, 片红‘富士’比条红‘富士’分别高出46.6%和22.1%。实验结果表明, MdGSTF3的表达与苹果果实不同组织花青苷的积累和分布密切相关。

关键词:‘富士’苹果; 谷胱甘肽S-转移酶(GST); 花青苷; 克隆; 表达分析

收稿:2015-05-04   修定:2015-08-28

资助:青岛农业大学高层次人才科研基金(630929)、青岛农业大学大学生科技创新基金项目、山东省高等学校优秀中青年骨干教师国际合作培养计划、公益性行业(农业)科研专项项目(201203075-04)和山东省现代农业产业体系水果创新团队建设基金(SDAIT-03-022-10)。

Cloning and Expression Analysis of Glutathione S-Transferase Gene MdGSTF3 from Apple (Malus domestica)

FAN Lian-Mei1,2, LIU Geng-Sen2, LI Si-Qi1, YUAN Yong-Bing2,*
1College of Life Sciences/Key Lab of Plant Biotechnology in Universities of Shandong; 2College of Horticulture/Qingdao Key Lab of Modern Agriculture Quality and Safety Engineering, Qingdao Agricultural University, Qingdao, Shandong 266109, China

Corresponding author: YUAN Yong-Bing; E-mail: yyb@qau.edu.cn; Tel: 0532-88030513

Abstract:

A full-length glutathione S-transferase gene named MdGSTF3 was cloned with RT-PCR and RACEPCR from Malus domestica cv. ‘Fuji’ peel. The GenBank accession number was KP234026. MdGSTF3 was 868 bp in length including 49 bp of 5' untranslated region, 151 bp of 3' untranslated region and 26 bp polyA. It had a coding sequence (CDS) of 642 bp, encoding 213 amino acid residues. This predicted polypeptide included 19 kinds of amino acids except for cysteine, with the molecular mass of 23.993 kDa, isoelectric point of 6.17. Real-time fluorescent quantitative PCR analysis showed that the expression level of MdGSTF3 in stems, leaves, flowers of striped-red ‘Fuji’ was higher than that of the mutant blushed-red ‘Fuji’, but the expression level of MdGSTF3 in peel and pulp of blushed-red ‘Fuji’ was higher than that of striped-red ‘Fuji’. The most relative expression quantity of MdGSTF3 from peel and pulp of blushed-red ‘Fuji’ were 46.6% and 22.1% higher than that of striped-red ‘Fuji’ respectively. The results indicated that the expression level of MdGSTF3 was closely related with anthocyanin accumulation and distribution of apple peel.

Key words: Malus domestica cv. ‘Fuji’; glutathione S-transferase (GST); anthocyanin; cloning; expression analysis

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