鹅掌楸LcPAT8基因的克隆及功能初步分析
发布时间：2018-10-31 17:47
【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。
[Abstract]:[objective] based on the results of early correlation analysis of growth traits and EST-SSR markers in Liriodendron chinense, the genes corresponding to SSR loci associated with growth traits of Liriodendron were cloned and isolated, and their sequence and structural characteristics were analyzed. The relationship and expression characteristics of the homologous gene with other species were studied to understand the role of the gene in the growth and development of Liriodendron. At the same time, the function of the isolated genes was studied by genetic transformation technique, in order to lay a foundation for the study of molecular mechanism and functional gene mining of Liriodendron chinense. [methods] the leaf buds of Liriodendron were used as materials. With the help of the leaf transcriptome database, RT-PCR and RACE techniques were used to clone and isolate the EST sequence associated with the growth traits of Liriodendron (Liriodendron mandshurica) at locus 642, and its bioinformatics analysis was carried out. The relative expression of the gene in flower bud, leaf, petal, stamen and pistil of Liriodendron was detected by real-time fluorescence quantitative PCR. The plant overexpression vector of this gene was constructed by Gateway technique, and transformed into Arabidopsis thaliana by Agrobacterium GV3101 mediated floss soaking, and T2 generation transgenic plants were obtained. The phenotype of transgenic plants was observed directly. [results] A gene corresponding to locus 642 associated with growth traits of Liriodendron chinense was obtained. The gene was 1 968 bp (GenBank accession number: KU883608) and the open reading frame was 1 269 bp,. Encode 422 amino acids. Bioinformatics analysis showed that the protein sequence encoded by the gene contained a typical DHHC-CRD domain and belonged to the DHHC zinc finger protein family, which was highly similar to the predicted protein palmityl transferase (PAT) from other plants. According to the comparison results of Arabidopsis thaliana PAT gene with NCBI, it was named LcPAT8.. Tissue expression analysis showed that LcPAT8 gene was expressed in flower bud and leaf bud of Liriodendron, and the highest expression was in pistil. The expression of LcPAT8 gene in petals and leaves was higher than that in leaf bud and flower bud, but lowest in stamen. The plant overexpression vector of LcPAT8 gene of Liriodendron was constructed by Gateway technique, and T2 generation transgenic Arabidopsis thaliana was obtained. Compared with wild-type Arabidopsis thaliana, the number of rosette leaves and bolting time of Arabidopsis thaliana plants with overexpression of LcPAT8 gene did not change significantly. The transgenic plants still grow vigorously, leaves are fresh green and there are a lot of flowers. After the wild-type plants withered and died, the transgenic plants still have a large number of lateral branches growing and flowering. [conclusion] the LcPAT8 gene of Liriodendron has the highest expression in pistil. The second is petal, which may be involved in the expansion of petals and the development of carpels and embryos. Overexpression of LcPAT8 gene in Arabidopsis thaliana significantly prolonged the plant growth period. Therefore, the LcPAT8 gene of Liriodendron mandshurica may play an important role in plant growth and development.
【作者单位】： 南京林业大学南方现代林业协同创新中心;贵州省林业科学研究院;
【基金】：国家自然科学基金项目(31470660) 江苏省高校优势学科(PAPD)
【分类号】：S792.21
本文编号：2303067
[Abstract]:[objective] based on the results of early correlation analysis of growth traits and EST-SSR markers in Liriodendron chinense, the genes corresponding to SSR loci associated with growth traits of Liriodendron were cloned and isolated, and their sequence and structural characteristics were analyzed. The relationship and expression characteristics of the homologous gene with other species were studied to understand the role of the gene in the growth and development of Liriodendron. At the same time, the function of the isolated genes was studied by genetic transformation technique, in order to lay a foundation for the study of molecular mechanism and functional gene mining of Liriodendron chinense. [methods] the leaf buds of Liriodendron were used as materials. With the help of the leaf transcriptome database, RT-PCR and RACE techniques were used to clone and isolate the EST sequence associated with the growth traits of Liriodendron (Liriodendron mandshurica) at locus 642, and its bioinformatics analysis was carried out. The relative expression of the gene in flower bud, leaf, petal, stamen and pistil of Liriodendron was detected by real-time fluorescence quantitative PCR. The plant overexpression vector of this gene was constructed by Gateway technique, and transformed into Arabidopsis thaliana by Agrobacterium GV3101 mediated floss soaking, and T2 generation transgenic plants were obtained. The phenotype of transgenic plants was observed directly. [results] A gene corresponding to locus 642 associated with growth traits of Liriodendron chinense was obtained. The gene was 1 968 bp (GenBank accession number: KU883608) and the open reading frame was 1 269 bp,. Encode 422 amino acids. Bioinformatics analysis showed that the protein sequence encoded by the gene contained a typical DHHC-CRD domain and belonged to the DHHC zinc finger protein family, which was highly similar to the predicted protein palmityl transferase (PAT) from other plants. According to the comparison results of Arabidopsis thaliana PAT gene with NCBI, it was named LcPAT8.. Tissue expression analysis showed that LcPAT8 gene was expressed in flower bud and leaf bud of Liriodendron, and the highest expression was in pistil. The expression of LcPAT8 gene in petals and leaves was higher than that in leaf bud and flower bud, but lowest in stamen. The plant overexpression vector of LcPAT8 gene of Liriodendron was constructed by Gateway technique, and T2 generation transgenic Arabidopsis thaliana was obtained. Compared with wild-type Arabidopsis thaliana, the number of rosette leaves and bolting time of Arabidopsis thaliana plants with overexpression of LcPAT8 gene did not change significantly. The transgenic plants still grow vigorously, leaves are fresh green and there are a lot of flowers. After the wild-type plants withered and died, the transgenic plants still have a large number of lateral branches growing and flowering. [conclusion] the LcPAT8 gene of Liriodendron has the highest expression in pistil. The second is petal, which may be involved in the expansion of petals and the development of carpels and embryos. Overexpression of LcPAT8 gene in Arabidopsis thaliana significantly prolonged the plant growth period. Therefore, the LcPAT8 gene of Liriodendron mandshurica may play an important role in plant growth and development.
【作者单位】： 南京林业大学南方现代林业协同创新中心;贵州省林业科学研究院;
【基金】：国家自然科学基金项目(31470660) 江苏省高校优势学科(PAPD)
【分类号】：S792.21
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