龙眼胚性愈伤组织RNA结合蛋白LSm14的基因克隆、亚细胞定位及表达分析  

Cloning,subcellular localization and expression analysis of RNA binding protein LSm14 gene from embryogenic calli of Dimocarpus longan Lour.

作　　者：厉雪 苏立遥 陈燕 张舒婷[1] 徐小萍[1] 陈晓慧[1] 陈荣珠 赖钟雄[1] 林玉玲[1] LI Xue;SU Liyao;CHEN Yan;ZHANG Shuting;XU Xiaoping;CHEN Xiaohui;CHEN Rongzhu;LAI Zhongxiong;LIN Yuling(Institute of Horticultural Biotechnology,Fujian Agricultural and Forestry University,Fuzhou 350002,Fujian,China)

机构地区：[1]福建农林大学园艺植物生物工程研究所,福州350002

出　　处：《果树学报》2020年第6期808-818,共11页Journal of Fruit Science

基　　金：国家自然科学基金(31672127);福建省高原学科建设经费(102/71201801101);福建农林大学创新基金(CXZX2017187,CXZX2018078)。

摘　　要：【目的】探究LSm14对龙眼体胚发生的影响,克隆RNA结合蛋白DlLSm14e基因并观察其亚细胞定位,分析其表达特性。【方法】以龙眼胚性愈伤组织为材料,依据转录组数据和基因组数据,采用RT-PCR克隆了一个DlLSm14基因,将其命名为DlLSm14e。对该基因进行生物信息学分析、亚细胞定位观察、龙眼非胚性愈伤组织和不同胚性培养物以及不同激素处理下的FPKM值分析和不同浓度细胞分裂素(KT)处理下的qRT-PCR检测。【结果】该基因CDS全长1707 bp,编码568个氨基酸,该蛋白不含信号肽和跨膜结构,不属于分泌蛋白和跨膜蛋白;此外,该蛋白含有N端保守结构域以及延长的C端,并且含有74个磷酸化修饰位点;系统发育分析表明,DlLSm14e蛋白与漾濞槭(Acer yangbiense)相似度较高,与单子叶植物亲缘关系最远;顺式作用元件分析表明,该基因含有大量光响应元件和多种激素响应元件;亚细胞定位结果表明,该基因为核定位基因;表达谱与实时荧光定量分析显示,DlLSm14e的FPKM值在非胚性愈伤组织(NEC)阶段最低,在球形胚阶段达到最高,推测DlLSm14e大量累积可能有利于龙眼体胚早期形态建成;不同激素处理下,2,4-D抑制该基因表达,KT促进其表达,而KT的浓度能影响该基因的表达。【结论】龙眼LSm14e基因定位于细胞核,该基因FPKM值随龙眼体胚胚性增加而升高,并且响应多种与龙眼体胚发生相关的激素,推测该基因可能参与龙眼体胚早期的形态建成。【Objective】The LSm14 protein is an important RNA-binding protein with conserved N-terminal and extended C-terminal, which can participate in the metabolism of RNA and affect embryonic development. The LSm14 has been shown to be involved in cell division and post-transcriptional regulation in animals. Longan is an important tropical and subtropical woody fruit tree in China. Its embryogenic state significantly affects the quality and yield of the fruits. At present, the effect of the DlLSm14 on nonembryonic callus(NEC) and different embryogenic cultures of longan has not been reported. To investigate the effect of the LSm14 on somatic embryogenesis of longan, we cloned RNA-binding protein DlLSm14 e, observed its subcellular localization, predicted the physicochemical properties, structure and functional sites of the protein, and analyzed its expression characteristics under different concentrations of kinin(KT) treatments.【Methods】In this study, firstly, the five longan LSm14 genes(Dlo026784.1,Dlo006168.1, Dlo011575.1, Dlo021757.1, Dlo029021.1) were selected by using the amino acid sequence of Arabidopsis thaliana LSm14 as the probe sequences and local blast with the longan genome database(NCBI accession number: BioProject PRJNA305337). The Longan LSm14 genes were named as the DlLSm14 a, DlLSm14 b, DlLSm14 c, DlLSm14 d and DlLSm14 e according to the search annotation of the Arabidopsis LSm14 in the longan genome and its chromosomal location. Through the previous data analysis, we selected the DlLSm14 e for cDNA full-length validation and overexpression vector construction. DNAMAN was used to design specific primers DlLSm14 e-CF and DlLSm14 e-CR at both ends of the CDS sequence. RT-PCR was used toclone the full-length. The homologous sequences of 15 bp linear cloned vectors were introduced into the 5 and 3 ends of the target fragment by RT-PCR, respectively.Hieff CloneTMPlus One Step Cloning Kit was used to recombine the target fragment with the terminal homologous sequence of the linearized clone vector wi

关 键 词：龙眼 DlLSm14基因 克隆 亚细胞定位 表达 

分 类 号：S667.2[农业科学—果树学]