《植物生理学报》 2015, 51(10): 1697-1705

番荔枝GA20氧化酶基因的克隆与表达分析

刘锴栋¹, 袁长春^1,*, 黎海利¹, 刘金祥¹, 陈燕¹, 莫亿伟², 李华富¹

¹岭南师范学院生命科学与技术学院, 广东湛江524048; ²绍兴文理学院生命科学学院, 浙江绍兴312000

通信作者：袁长春；E-mail: yuanchangchun@163.com；Tel: 0759-3183271

摘　要：

本研究利用同源克隆和RACE-PCR的方法获得番荔枝GA20ox基因的全长cDNA序列, 命名为AsGA20ox, GenBank登录号为KR676623。序列分析表明, 克隆获得的番荔枝AsGA20ox基因编码区长为1 257 bp, 编码418个氨基酸。序列比对显示与油棕和海枣等GA20ox的相似度分别为71%和70%。构建类似蛋白系统进化树显示, 番荔枝AsGA20ox与簇毛麦、香蕉等分子进化距离较近。预测AsGA20ox蛋白定位在细胞外基质和过氧化物酶体中, 不具备信号肽。蛋白质分子量为47.2 kDa, 等电点为6.21。实时定量RT-PCR结果表明, 在不同的组织、不同的器官中, AsGA20ox的表达量存在差异, 其中在花蕾期I、未成熟的种子、结果枝的幼嫩茎段和雌蕊中的表达量相对较高。

关键词：番荔枝; GA20ox; 基因克隆; 表达模式

收稿：2015-06-10   修定：2015-09-06

资助：广东省科技计划项目(2013B020304008)、国家自然科学基金项目(31201586)、广东省教育厅科技创新项目(2013KJCX0124)、湛江市热带特色资源植物技术开发重点实验室项目(2014A06008)和湛江师范学院科研创新团队资助项目(2013CXTD05)。

Cloning and Expression Analysis of GA20-Oxidase Gene from Sugar Apple (Annona squamosa)

LIU Kai-Dong¹, YUAN Chang-Chun^1,*, LI Hai-Li¹, LIU Jin-Xiang¹, CHEN Yan¹, MO Yi-Wei², LI Hua-Fu¹

¹Life Science and Technology School, Lingnan Normal University, Zhanjiang, Guangdong 524048, China; ²College of Life Science, Shaoxing University, Shaoxing, Zhejiang 312000, China

Corresponding author: YUAN Chang-Chun; E-mail: yuanchangchun@163.com; Tel: 0759-3183271

Abstract:

A full-length cDNA sequence of homologous GA20ox<.i> gene was cloned by employing homology gene cloning and RACE-PCR from sugar apple (Annona squamosa L.), which was named as AsGA20ox (Gen-Bank accession KR676623). Sequence analysis showed that the AsGA20ox gene has a 1 257 bp open reading frame (ORF) encoding 418 amino acids. Sequence alignment displayed that it was a similarity of 71% and 70% with GA20ox of Elaeis guineensis and Phoenix dactylifera, respectively. The phylogenetic tree showed that sugar apple AsGA20ox and Dasypyrum villosum GA20ox are closest in molecular evolution distance, followed by Musa acuminata subsp. Malaccensis GA20ox. It was speculated that AsGA20ox protein is located in the extracellular matrix and peroxisomal. It do not have a signal peptide. The putative protein molecular weight was 47.2 kDa and its theoretical isoelectric point was 6.21. The real-time PCR results suggested that AsGA20ox gene showed a high transcription level in flower buds I, immature seeds, young stems of fruiting branches and pistils. The results laid a solid foundation for further research on AsGA20ox protein structure and the relationship between the gene and plant flower development.

Key words: sugar apple (Annona squamosa); GA20ox; gene cloning; expression pattern

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