林业科学 ›› 2017, Vol. 53 ›› Issue (9): 45-54.doi: 10.11707/j.1001-7488.20170906

• 论文与研究报告 • 上一篇    下一篇


徐嘉娟1,2, 李火根1   

  1. 1. 南京林业大学南方现代林业协同创新中心 南京 210037;
    2. 贵州省林业科学研究院 贵阳 550005
  • 收稿日期:2016-12-19 修回日期:2017-03-29 出版日期:2017-09-25 发布日期:2017-10-24
  • 基金资助:

Cloning and Primary Functional Analysis of LcPAT8 Gene from Liriodendron chinense

Xu Jiajuan1,2, Li Huogen1   

  1. 1. Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University Nanjing 210037;
    2. Guizhou Academy of Forestry Guiyang 550005
  • Received:2016-12-19 Revised:2017-03-29 Online:2017-09-25 Published:2017-10-24

摘要: [目的]基于前期鹅掌楸生长性状与EST-SSR分子标记关联分析结果,克隆分离与鹅掌楸生长性状相关联的SSR位点相对应的基因,通过分析其序列特征、结构特征、与其他物种同源基因的亲缘关系和表达特性,初步了解该基因在鹅掌楸生长发育过程中的作用。同时,利用遗传转化技术对分离的基因进行功能研究,以期为鹅掌楸生长发育的分子机制研究及功能基因挖掘奠定基础。[方法]以鹅掌楸叶芽为材料,借助其叶片转录组数据库,采用RT-PCR和RACE技术克隆分离与鹅掌楸生长性状相关联的642号位点对应的EST序列相关基因,对其进行生物信息学分析。通过实时荧光定量PCR检测该基因在鹅掌楸花芽、盛花期的叶芽、叶片、花瓣、雄蕊、雌蕊中的相对表达量。利用Gateway技术构建该基因的植物过表达载体,使用农杆菌GV3101介导的花絮浸染法转化拟南芥,获得T2代转基因植株,直接观察转基因植株表型。[结果]克隆获得与鹅掌楸生长性状相关联的642号位点相对应的基因,该基因全长1 968 bp (GenBank登录号:KU883608),开放阅读框为1 269 bp,编码422个氨基酸。生物信息学分析表明该基因编码的蛋白序列含有1个典型的DHHC-CRD结构域,属于DHHC型锌指蛋白家族成员,与其他植物预测的蛋白质棕榈酰基转移酶(PAT)高度相似,并根据与NCBI中拟南芥PAT基因的比对结果,将其命名为LcPAT8。组织表达分析表明,LcPAT8基因在鹅掌楸花芽、叶芽等6个组织中均有表达,在雌蕊中表达量最高,花瓣和叶片中的表达量高于叶芽和花芽,而在雄蕊中的表达量最低。利用Gateway技术,成功构建了鹅掌楸LcPAT8基因的植物过量表达载体,获得T2代转基因拟南芥。与野生型拟南芥相比,过表达LcPAT8基因的拟南芥植株的莲座叶片数目以及抽薹时间没有发生明显变化,而野生型植株大部分角果成熟、叶片枯黄掉落时,转基因植株依然生长旺盛,叶片鲜绿并且还有大量花絮,在野生型植株干枯死亡后,转基因植株还有大量侧枝生长、开花。[结论]鹅掌楸LcPAT8基因在雌蕊中表达量最高,其次是花瓣,可能参与花瓣的扩展、心皮及胚的发育;在拟南芥中过表达LcPAT8基因,明显延长了植株的生长期。因此,鹅掌楸LcPAT8基因可能在植物生长发育过程中起着重要的调控作用。

关键词: 鹅掌楸, 基因克隆, 组织表达特征, 异源表达, 功能分析

Abstract: [Objective] Based on the previous results of association analysis between growth traits and EST-SSR markers in Liriodendron, a gene corresponding to the marker locus associated with growth traits was cloned from Liriodendron chinense, the function of this gene was studied by analyzing its sequence characteristics, structural features, genetic relationship with other species homologous genes and expression characteristics. Finally, the gene function was validated through over-expression in Arabidopsis thaliana, the aim of this study was to understand the molecular mechanism of its growth and development and contribute to the further utilization of functional genes.[Method] Based on the transcriptome database of L. chinense leaf, RT-PCR and RACE were used to clone and separate the EST related gene corresponding to 642 locus that associated with growth traits of hybrid Liriodendron for analyzing bioinformatics of the sequences. Quantitative real-time PCR was used for the analysis of gene expression in different tissues such as floral bud, flowering leaf buds, leafs, petals, stamens and pistil of L. chinense. Using the Gateway Recombination Cloning Technology, the over-expression vector of LcPAT8 was constructed, and transformed into A. thaliana with the floral dipping method of Agrobacterium-mediated transformation, the T2 transgenic plants were obtained and then the phenotype was analyzed.[Result] The full-length cDNA of 1 968 bp was cloned(GenBank accession number:KU883608), containing 1 269 bp ORF and encoding 422 aa protein. Bioinformatics analysis showed that the protein encoded by this gene had a typical DHHC-CRD domain, and it belonged to the DHHC-type zinc finger protein family. BLAST analysis indicated its high similarity to predicted protein S-acyltransferase(PAT) of other plants, and according to the alignmentresult with AtPAT genes in NCBI, we named it LcPAT8. Gene expression suggested that the LcPAT8 was expressed in various tissues, it had the highest expression in pistil but the least in stamen, and the expression in petal and leaf were higher than that in flower bud and leaf bud. The over-expression vector of the LcPAT8 was successfully constructed by the Gateway technology, the T2 transgenic A. thaliana were obtained. The rosette leaf number and bolting time of transgenic plants over-expressed LcPAT8 had no significant difference from the wild type, but when most pods of the wild type ripen, and leaves turned yellow and fell, the transgenic plants were still vigorous in growth, their leaves were fresh and green,and a large number of flower wads were present; when the wild types withered and died, the transgenic plants still had a large number of branches growing and flowering.[Conclusion] The experimental data confirmed that the LcPAT8, a DHHC-type zinc finger protein gene from L. chinense, was highly-expressing in the pistil and petal, it might have been involved in petal expansion and the development of carpel and embryo. Functional analysis showed that the over-expression of LcPAT8 in A. thaliana led to obvious extension of growth cycle in the transgenic plants. This result indicated that the LcPAT8 played a vital role on the regulation of plant growth and development.

Key words: Liriodendron chinense, gene clone, tissue expression characteristics, heterologous expression, functional analysis