龙眼miR408与DlLAC12克隆及其在球形胚发生和非生物胁迫下的表达分析  

Gene Cloning and Expression Analysis of miR408 and Its Target DlLAC12 in Globular Embryo Development and Abiotic Stress in Dimocarpus longan

作　　者：徐小萍 曹清影 蔡柔荻 官庆栩 张梓浩[1] 陈裕坤[1] 徐涵[1,2] 林玉玲 赖钟雄[1] XU Xiaoping;CAO Qingying;CAI Roudi;GUAN Qingxu;ZHANG Zihao;CHEN Yukun;XU HAN;LIN Yuling;LAI Zhongxiong(College of Horticulture,Fujian Agriculture and Forestry University,Fuzhou 350002,China;Institut de la Recherche Interdisciplinaire de Toulouse,IRIT-ARI,Toulouse 31300,France)

机构地区：[1]福建农林大学园艺学院,福州350002 [2]法国图卢兹综合科学研究所(IRIT-ARI),图卢兹31300

出　　处：《园艺学报》2022年第9期1866-1882,共17页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(31572088);福建省高原学科建设经费项目(102/71201801101);福建农林大学创新基金项目(CXZX2017189,CXZX2017314,CXZX2018076,CXZX2019037S)。

摘　　要：为探讨miR408及靶基因DlLAC12在龙眼球形体细胞胚诱导及不同非生物胁迫下的表达模式,采用miR-RACE PCR和Tail-PCR克隆获得pri-miR408 cDNA和转录起始位点、dlo-miR408 gDNA、靶基因DlLAC12 cDNA及启动子pro-MIR408序列。研究结果显示:dlo-pri-miR408 cDNA、gDNA全长均为706 bp,5′端转录起始位点为胞嘧啶(C)。DlLAC12 cDNA全长为1725 bp,pro-MIR408全长为1532 bp。pro-MIR408序列上存在ABA、GA_(3)、JA等激素信号传导顺式作用元件和响应光、低温胁迫等相关元件。qRT-PCR结果显示,dlo-miR408-3p随外源添加的蔗糖浓度、Cu^(2+)浓度及培养温度的变化呈现动态表达;而dlo-miR408-5p1对蔗糖不敏感,在铜离子失衡和低温时下调表达。dlo-miR408-3p与DlLAC12负调控模式能响应高浓度ABA(≥500μmol•L^(-1))处理,而不同浓度GA_(3)处理下二者表达模式一致。dlo-miR408-3p与DlLAC12在球形胚诱导不同天数呈现负调控,说明在球形胚诱导过程发挥作用。试验表明,dlo-miR408与靶基因DlLAC12可参与龙眼体胚发生与非生物胁迫响应。To investigate the expression patterns of miR408 and its target DlLAC12 during longan globular embryo(GE)development and different abiotic stresses.The miR-RACE PCR and Tail-PCR were used to clone the cDNA and gDNA of dlo-miR408 and cDNA of DlLAC12,and the transcriptional start site(TSS),and promoter of MIR408(pro-MIR408)were ascertained.Characteristics of dlo-miR408 sequence,homologous and cis-acting elements of the promoter were bioinformatically analyzed.The transcriptional expression levels of the members of dlo-miR408 family at various abio-stress conditions were verified with qRT-PCR,and the expression patterns of dlo-miR408-3p with its target gene DlLAC12 were further analyzed at early somatic embryogenesis stages with various plant growth regulator treatments.pro-MIR408 sequence contains hormone signal transducing cis-acting elements such as ABA,GA_(3)and JA,and light and low temperature stress cis-elements.The results showed that the length of pri-miR408 cDNA,gDNA were 706 bp.The TSS was“C”.The length of DlLAC12 cDNA was 1725 bp.The length of pro-MIR408 was 1532 bp.dlo-miR408-3p showed dynamic expressions in responding to sucrose,Cu^(2+)and temperature treatments by means of qRT-PCR,whereas dlo-miR408-5p1 showed downregulation in the imbalanced Cu^(2+)and low temperature conditions,and maintained stable expression to sucrose changes.Moreover,dlo-miR408-3p and DlLAC12 responded to high concentration of ABA(≥500μmol•L^(-1))with negative regulations,and GA_(3)treatment made them same in the expression pattern.The negative regulation at the late globular embryo(GE)developmental stage played an important role in the GE morphogenesis.The present work showed that dlo-miR408 and DlLAC12 were involved in the longan somatic embryogenesis and abiotic stress.

关 键 词：龙眼 体胚发生 miR408 DlLAC12 非生物胁迫 

分 类 号：S667.2[农业科学—果树学]