miR395分子进化特性及其在龙眼早期体胚发生中的表达分析  

Analysis of the molecular evolution characteristics and the expression of miR395 at the early stage of somatic embryogenesis in Dimocarpus longan

作　　者：黄倏祺 苏立遥 蒋梦琦 赖钟雄[1] 林玉玲[1] HUANG Shuqi;SU Liyao;JIANG Mengqi;LAI Zhongxiong;LIN Yuling(Institute of Horticulture Biotechnology,Fujian Agriculture 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

出　　处：《果树学报》2020年第1期18-28,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);福建农林大学科技创新专项基金(CXZX2018078);福建农林大学科技创新专项基金(CXZX2017314)

摘　　要：【目的】研究miR395的分子进化特性及其在龙眼早期体胚发生中的表达分析。【方法】通过龙眼miRNA文库、转录组数据库和miRBase数据库,得到所有植物miR395家族成员成熟体和前体序列,对其进行序列比对及进化分析、保守性分析、前体二级结构预测、潜在靶基因预测,对龙眼miR395a在龙眼早期体胚发生(SE)阶段的表达模式进行分析。【结果】龙眼miR395家族包含5条前体序列和2条成熟体序列。Mfold预测分析发现,龙眼pre-miR395序列均能形成稳定的发卡结构,其最小折叠自由能ΔG在-44.70~64.80 kal·mol-1,提示与序列长度没有显著相关性。其次,保守性分析显示,植物pre-miR395两端序列较保守,而中间序列不保守;系统进化分析提示,龙眼pre-miR395家族与脐橙、葡萄和苹果的亲缘关系更近。psRNAtarget预测显示,龙眼miR395调控的潜在靶基因主要包含富含亮氨酸重复受体蛋白激酶LRR-RLKs、ATP硫酸化酶APS1、蛋白磷酸酶PP2C、前体RNA蛋白质TSR1同系物TSR1、酮酯酰CoA硫解酶KAT2和EH结构域蛋白EHD1等6个,且都是以裂解靶标的方式进行调控。qRT-PCR分析表明,在龙眼早期体胚发生阶段,miR395a呈上调趋势,且miR395a与APS1呈显著负相关。【结论】龙眼miR395与其他植物miR395功能具有高度保守性和物种特异性,同时龙眼miR395a上调表达可能促进龙眼球形胚的形态建成。【Objective】MiR395 is an ancient and highly conserved family of miRNAs,which plays an important role in nutrient absorption and assimilation,and is strongly induced by sulfur deficiency stress.Previous researches showed that miR395 guided cleavage APS genes and Sultr2;1 at post-transcriptional level,which played an important role in resisting abiotic stress,such as heavy metal,plant disease,chilling injury and sulfur nutrition.MiR395 family generally comprises multiple miR395 members in plants,which may exhibit functional redundancy and specificity.Longan(Dimocarpus longan Lour.)is a kind of subtropical and tropical fruit tree is economically valuable in Southeast Asia.In longan,embryo plays an important role,and the development of embryos is closely related to seed size,fruit quality and yield.Studies have shown that miRNAs are important to longan somatic embryogenesis(SE).However,miR395 evolutionary characteristics and its function of plants somatic embryos had not been reported yet.Therefore,we studied the molecular evolution characteristics of miR395 and its expression at the early SE in longan.【Methods】The matures and precursors of miR395 family members were extracted from longan miRNA library,transcriptional database and miRBase database.Then we analyzed the number of all the pre-miR395 and miR395 in plants.We carried out the longan sequence alignment using DNAMAN6.0.And the phylogenetic trees of mature and precursor miR395 sequences of plants were performed by MEGA7.0 through NJ(Neighbor-joining)method with 1000 bootstrap,then we made use of the iTOL online software to beautify.In addition,their conserved sequences were analyzed by Web Logo software,Mfold online software was used to predict longan pre-miR395 secondary structures.In order to obtain the longan miR395s’target genes,psRNATarget online software was uesed.Finally,SYBR Prumix EX TaqTMII kit,Tip-mix kit and Roche LightCycler 480 real-time quantitative PCR method were used to verify the relationship between the predicted target genes and l

关 键 词：龙眼 miRNA395 进化特性 早期体胚发生 实时荧光定量PCR 

分 类 号：S667.2[农业科学—果树学]