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园艺学报 ›› 2022, Vol. 49 ›› Issue (9): 1866-1882.doi: 10.16420/j.issn.0513-353x.2021-0573

• 研究论文 • 上一篇    下一篇

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

徐小萍1, 曹清影1, 蔡柔荻1, 官庆栩1, 张梓浩1, 陈裕坤1, 徐涵1,2, 林玉玲1, 赖钟雄1,*()   

  1. 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
  • 收稿日期:2021-10-01 修回日期:2022-06-16 出版日期:2022-09-25 发布日期:2022-10-08
  • 通讯作者: 赖钟雄 E-mail:laizx01@163.com
  • 基金资助:
    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);福建农林大学创新基金项目(CXZX2017314);福建农林大学创新基金项目(CXZX2018076);福建农林大学创新基金项目(CXZX2019037S)

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

XU Xiaoping1, CAO Qingying1, CAI Roudi1, GUAN Qingxu1, ZHANG Zihao1, CHEN Yukun1, XU HAN1,2, LIN Yuling1, LAI Zhongxiong1,*()   

  1. 1College of Horticulture,Fujian Agriculture and Forestry University,Fuzhou 350002,China
    2Institut de la Recherche Interdisciplinaire de Toulouse,IRIT-ARI,Toulouse 31300,France
  • Received:2021-10-01 Revised:2022-06-16 Online:2022-09-25 Published:2022-10-08
  • Contact: LAI Zhongxiong E-mail:laizx01@163.com

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1 725 bp,pro-MIR408全长为1 532 bp。pro-MIR408序列上存在ABA、GA3、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、Cu2+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(≥ 500 μmol · L-1)处理,而不同浓度GA3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。

关键词: 龙眼, 体胚发生, miR408, DlLAC12, 非生物胁迫

Abstract:

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,GA3 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 1 725 bp. The length of pro-MIR408 was 1 532 bp. dlo-miR408-3p showed dynamic expressions in responding to sucrose,Cu2+ and temperature treatments by means of qRT-PCR,whereas dlo-miR408-5p1 showed downregulation in the imbalanced Cu2+ and low temperature conditions,and maintained stable expression to sucrose changes. Moreover,dlo-miR408-3p and DlLAC12 responded to high concentration of ABA(≥ 500 μmol · L-1)with negative regulations,and GA3 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.

Key words: longan, somatic embryogenesis, miR408, DlLAC12, abiotic stress

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