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林业科学 ›› 2019, Vol. 55 ›› Issue (2): 50-60.doi: 10.11707/j.1001-7488.20190206

• 论文与研究报告 • 上一篇    下一篇

核桃JrGA2ox基因的克隆、亚细胞定位及功能验证

张佳琦1, 胡恒康1, 徐川梅1, 胡渊渊1, 黄有军1, 夏国华1, 黄坚钦1, 常英英2, 叶磊1, 娄和强1, 张启香1   

  1. 1. 省部共建亚热带森林培育国家重点实验室 浙江农林大学林业与生物技术学院 杭州 311300;
    2. 林木遗传育种国家重点实验室 国家林业和草原局林木培育重点实验室 中国林业科学研究院林业研究所 北京 100091
  • 收稿日期:2018-03-12 修回日期:2018-05-10 出版日期:2019-02-25 发布日期:2019-03-20
  • 基金资助:
    国家自然科学基金项目(31470682;31800563;31670682;31600547);浙江省自然科学基金项目(LY18C150002;LY15C160003);浙江省农业(果品)新品种选育重大科技专项(2016C02052-13);浙江农林大学大学生科研训练项目(2013200044)。

Cloning, Subcellular Localization and Function Verification of Gibberellin 2-Oxidase Gene in Walnut (Juglans regia)

Zhang Jiaqi1, Hu Hengkang1, Xu Chuanmei1, Hu Yuanyuan1, Huang Youjun1, Xia Guohua1, Huang Jianqin1, Chang Yingying2, Ye Lei1, Lou Heqiang1, Zhang Qixiang1   

  1. 1. State Key Laboratory of Subtropical Silviculture School of Forestry and Biotechnology, Zhejiang A & F University Hangzhou 311300;
    2. State Key Laboratory of Tree Genetics and Breeding Key Laboratory of Tree Breeding and Cultivation of National Forestry and Grassland Administration Research Institute of Forestry, Chinese Academy of Forestry Beijing 100091
  • Received:2018-03-12 Revised:2018-05-10 Online:2019-02-25 Published:2019-03-20

摘要: [目的]GA2-oxidase (GA2ox)是赤霉素合成过程中起负调控作用的一种关键酶,能够催化有活性的赤霉素为无活性的赤霉素,从而对植物生长起到一定的抑制作用。本研究主要对核桃中赤霉素氧化酶基因JrGA2ox进行克隆及功能验证,有利于进一步探究核桃JrGA2ox基因在植物生长和发育过程中,尤其是在植物株高调控中的作用,从而助力于挖掘与利用核桃中更多的优质基因,培育出更多优良的核桃品种。[方法]采用PCR扩增技术,克隆获得核桃JrGA2ox基因的全长编码序列,进一步通过In-Fusion克隆技术构建具有强启动子的35S::JrGA2ox::GFP过表达载体;利用BLAST网络在线工具得到其他植物中的JrGA2ox同源氨基酸序列,并对其进行氨基酸同源序列比对和系统进化分析;其后,通过亚细胞定位揭示其发挥功能的场所,进一步利用农杆菌介导法将构建好的过表达载体转化到核桃体细胞胚中,获得JrGA2ox超表达的阳性转化植株,深入分析JrGA2ox基因的生物学特性。[结果]通过基因克隆,得到1条JrGA2ox开放阅读框,其全长为1 056 bp,共编码351个氨基酸,分子量为39.25 kDa。通过比对发现,该基因编码的蛋白序列含有保守2OG-FeⅡ-Oxy蛋白结构域,具有GA2-氧化酶蛋白家族共同的结构特点,表明JrGA2ox属于GA2-氧化酶基因家族。氨基酸进化树比对分析结果显示,核桃JrGA2ox与毛白杨PtGA2ox聚为一个分支。且JrGA2ox蛋白与木本植物川桑MnGA2ox1、西洋梨PcGA2ox、桃PpGA2ox1及苹果MdGA2ox1蛋白序列同源性较高。烟草叶片表皮细胞亚细胞定位分析表明,JrGA2ox是定位于细胞核与细胞膜中的蛋白。对核桃体细胞胚进行基因遗传转化后经荧光检测及PCR验证表明,35S::JrGA2ox::GFP过表达载体被成功转入核桃体细胞胚中。阳性再生植株株高与对照苗相比具显著性差异,其平均株高为对照植株的1/2;且其株高与JrGA2ox基因表达量呈负相关。[结论]核桃JrGA2ox蛋白亚细胞定位于细胞核与细胞膜中。JrGA2ox基因调控核桃株高,主要起到负调节作用。阳性基因转化再生植株中JrGA2ox基因的表达量升高,并表现出明显的矮化特征。本研究结果可为进一步分析该基因在核桃生长发育过程中的作用提供技术参考,且为优良的矮化核桃品种选育奠定一定的基础。

关键词: 核桃, GA2ox基因, 过表达, 亚细胞定位, 同源转化

Abstract: [Objective] GA2-oxidase (GA2ox) is a key enzyme which plays a negative role in regulating the gibberellins.GA2ox could convert bioactive gibberellins to inactive gibberellins during gibberellin biosynthesis.Accordingly,it has a certain inhibitory effect on plant growth.This experiment was aimed at cloning and verifying function of GA2ox encoding gene (JrGA2ox) in walnut (Juglans regia).It helps to further explore the role of JrGA2ox gene in the process of plant growth and development,especially in regulating the plant height.This further assists in discovering and utilizing more high-qualified genes in walnut and cultivating more improved walnut varieties.[Method] The full length of JrGA2ox gene was cloned and verified based on PCR technique.Furthermore,the 35S::JrGA2ox::GFP overexpression vector with a strong promoter was constructed by In-Fusion cloning technology.The JrGA2ox homologous amino acid sequences were obtained by the online tool of BLAST,and their comparative analysis and phylogenetic tree analysis were carried out.Thereafter subcellular localization assay was carried out to reveal where it worked.The overexpressed vector was transformed into walnut somatic embryos using the Agrobacterium-mediated method and the positive transformation plants of JrGA2ox overexpression types were obtained.Furthermore,the biological properties of JrGA2ox gene were further analyzed.[Result] The 1 056 bp open reading frame (ORF) of JrGA2ox was gained by cloning method,encoding 351 amino acids and its molecular weight was 39.25 kDa.Blast analysis showed that JrGA2ox had a common structural feature of GA2-oxidase family which was a conserved protein domain named 20G-FeⅡ-Oxy.It indicated that JrGA2ox belongs to GA2-oxidase gene family.The results of phylogenetic tree analysis showed that JrGA2ox and Populous tomentosa GA2ox were clustered into the same branch.And the protein encoded by JrGA2ox gene had a high homology with those proteins of woody plants,Morus notabilis GA2ox1,Pyrus communis GA2ox,Prunus persica GA2ox1,and Malus domestica GA2ox1.The analysis of subcellular localization assay in epidermal cells of tobacco (Nicotiana benthamiana)-leaves showed that JrGA2ox was mainly localized in the nucleus and plasma membrane.The results of GFP fluorescence detection and PCR inspection in the transformed somatic embryos showed that 35S::JrGA2ox::GFP had been transformed into walnut somatic embryos successfully.Comparing with the regenerated wild type walnut plants,there was a significant difference in plant height.The average height of the transformed plants was 1/2 of that of the control and it was negatively correlated with the expression of JrGA2ox gene.[Conclusion] The results indicated that JrGA2ox was located in the nucleus and plasma membrane.In addition,JrGA2ox gene regulated the height of walnut plants negatively.When the relative expression level raised in the transgenic walnut plants with 35S::JrGA2ox::GFP,these plants showed obvious dwarfing characteristics.This study provides a technical basis for further analysis of the functions of JrGA2ox gene in growth and development and for the breeding of dwarf walnut varieties.

Key words: Juglans regia, GA2ox gene, over-expression, subcellular localization, transformation

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