文献类型：期刊文献

中文题名：日本落叶松LaSPL2和LaSPL3在体细胞胚发育中的表达分析

英文题名：Expression of SPL-like Gene LaSPL2 and LaSPL3 in Japanese Larch(Larix leptolepis)During Somatic Embryogenesis

作者：范艳如[1] 兰倩[2] 韩素英[3] 齐力旺[1] 张立峰[1]

机构：[1]中国林业科学研究院林业研究所,林木遗传育种国家重点实验室,国家林业和草原局林木育种与培育重点实验室,北京100091;[2]中国林业科学研究院林业科技信息研究所,北京100091;[3]中国林业科学研究院森林生态环境与保护研究所,北京100091

年份：2021

卷号：34

期号：5

起止页码：79-87

中文期刊名：林业科学研究

外文期刊名：Forest Research

收录：CSTPCD;;Scopus;北大核心:【北大核心2020】；CSCD:【CSCD2021_2022】；

基金：国家自然基金项目(31330017、31600544);中央级公益性科研院所基本科研业务费专项资金项目(CAFYBB2019SY011);国家科技重大专项课题(2018ZX08020-003)。

语种：中文

中文关键词：日本落叶松;SPL;体细胞胚;基因克隆;表达分析

外文关键词：Larix leptolepis;SPL;somatic embryogenesis;gene cloning;expression analysis

分类号：S791.223

摘要：[目的]通过研究LaSPL2和LaSPL3的分子特征和表达模式,揭示其在落叶松体细胞胚发育中的功能。[方法]利用同源克隆及RACE技术,获得LaSPL2和LaSPL3的全长cDNA序列,通过生物信息学分析其编码蛋白保守结构域、进化关系等,利用烟草瞬时表达体系进行亚细胞定位研究,采用qRT-PCR检测2个基因在落叶松体细胞胚发育过程的表达模式。[结果]本研究获得了2个落叶松SPL同源基因LaSPL2和LaSPL3,分别编码532个氨基酸和191个氨基酸,其cDNA序列均存在miR156识别位点。多序列比对分析发现:它们均具有保守的SBP结构域。亚细胞定位结果显示:LaSPL2和LaSPL3定位于细胞核中。qRT-PCR结果表明:在落叶松体细胞胚发育早期,ABA下调LaSPL2和LaSPL3表达;随着体细胞胚的进一步发育,LaSPL2和LaSPL3的表达水平分别在10 d和14 d达到峰值;之后随着体细胞胚发育成熟,它们的转录水平逐渐下降,并在42 d达到最低值。[结论]通过分析ABA缺失对LaSPL2和LaSPL3表达的影响,表明ABA可能是落叶松体细胞胚发育早期LaSPL2和LaSPL3下调表达的主要因子;LaSPL2和LaSPL3的表达量在体细胞胚发育的早期阶段均达到最高峰,表明它们可能是早期胚胎形成的一个重要调控因子;LaSPL2和LaSPL3序列分析及表达模式,表明它们在体细胞胚发育中可能受miR156调控,并在体细胞胚的成熟过程中具有重要意义。
[Objective]To reveal LaSPL2 and LaSPL3 function during somatic embryogenesis,based on their molecular characteristics and expression patterns.[Method]The full-length cDNA sequences of LaSPL2 and LaSPL3 were obtained by homologous cloning and RACE techniques.The amino acid sequences for known SPL family proteins were aligned using the ClustalX 2.1 software.The full-length protein sequence alignment was used to construct a phylogenetic tree according to the neighbor-joining method of the MEGAX program.Subcellular localization was performed by transient expression system of tobacco.The expression patterns of LaSPL2 and LaSPL3 during somatic embryogenesis were detected by qRT-PCR.[Result]In this study,two SPL homologous genes,LaSPL2 and LaSPL3,were isolated from Japanese larch,which encoded 532 amino acids and 191 amino acids respectively.Further analyses revealed that LaSPL2 and LaSPL3 mRNA were targeted by miR156.The deduced amino acid sequences of the LaSPL2 and LaSPL3 harbored a highly conserved SBP domain.A transient expression analysis of the LaSPLs-GFP fusion proteins in tobacco leaf epidermal cells revealed that the LaSPL2 and LaSPL3 proteins were localized in the nucleus.Moreover,the expression of LaSPL2 and LaSPL3 was down regulated by ABA during the early stage of somatic embryogenesis.With the further development of somatic embryos,the expression levels of LaSPL2 and LaSPL3 peaked at 10 and 14 days,respectively.Subsequently,with the maturation of somatic embryos,their transcription levels decreased gradually to the lowest level at 42 days.[Conclusion]The effects of ABA removal on the expression of LaSPL2 and LaSPL3 indicate that ABA might be the main factor in their down-regulation during the early stage of somatic embryogenesis.Furthermore,the expression of LaSPL2 and LaSPL3 will reach the peak at the early stage of somatic embryogenesis,implying it might encode an important regulator of early embryonic pattern formation.The sequence analysis and expression patterns of LaSPL2 and LaSPL3 indicated that they may be regulated by miR156 during somatic embryogenesis and play an important role in the maturation of somatic embryos.