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园艺学报 ›› 2021, Vol. 48 ›› Issue (5): 908-920.doi: 10.16420/j.issn.0513-353x.2020-0579

• 研究论文 • 上一篇    下一篇

龙眼pri-miR319a编码短肽活性的研究

苏立遥1, 王培育2, 蒋梦琦1, 黄倏祺1, 薛晓东1, 刘梦雨1, 肖学宸1, 赖春旺1, 张梓浩1, 陈裕坤1, 赖钟雄1, 林玉玲1,*()   

  1. 1福建农林大学园艺植物生物工程研究所,福州 350002
    2三明农业科学研究院,福建三明 365000
  • 收稿日期:2020-09-15 修回日期:2021-04-06 出版日期:2021-05-25 发布日期:2021-06-07
  • 通讯作者: 林玉玲 E-mail:buliang84@163.com
  • 基金资助:
    国家自然科学基金项目(31672127);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);福建农林大学园艺学院优秀硕士学位论文资助项目(2018S02);福建农林大学科技创新专项基金项目(CXZX2017187)

The Activity Verification of pri-miR319a Encode Regulatory Peptide of Dimocarpus longan

SU Liyao1, WANG Peiyu2, JIANG Mengqi1, HUANG Shuqi1, XUE Xiaodong1, LIU Mengyu1, XIAO Xuechen1, LAI Chunwang1, ZHANG Zihao1, CHEN Yukun1, LAI Zhongxiong1, LIN Yuling1,*()   

  1. 1Institute of Horticultural Biotechnology,Fujian Agriculture and Forestry University,Fuzhou 350002,China
    2Sanming Academy of Agricultural Sciences,Sanming,Fujian 365000,China
  • Received:2020-09-15 Revised:2021-04-06 Online:2021-05-25 Published:2021-06-07
  • Contact: LIN Yuling E-mail:buliang84@163.com

摘要:

为研究龙眼miRNA编码短肽(miRNA encode regulatory peptide,miPEP)的潜在活性,以miR319为对象,从7个龙眼品种中克隆得到pri-miR319a 序列并预测其潜在编码miPEP,并通过遗传转化和人工合成miPEP的方法验证miPEP319a的活性。结果显示,7个龙眼品种中pri-miR319a序列存在10个核苷酸的差异,并具有3个潜在ORF可以编码miPEP,其中1个核苷酸突变位点导致miPEP319a氨基酸序列的改变。进一步通过原位转化法获得转化miPEP的过表达载体及相应突变表达载体的龙眼幼芽,并结合人工合成miPEP319处理龙眼胚性愈伤组织,进一步验证龙眼miPEP的生物学活性。结果显示,pri-miR319a编码的3个潜在miPEP仅miPEP319a-2具有生物学活性并促进下游miR319a的表达。最后采用烟草叶片的瞬时转化法验证龙眼miPEP319a在烟草当中的活性及对miR319a的影响。结果表明龙眼miPEP319a具有物种特异性,在烟草叶片中miR319a的表达模式无差异。该研究结果提示龙眼pri-miR319序列在不同品种中具有多态性,且其编码的3个潜在miPEP仅miPEP319a-2具有生物学活性,并具有物种特异性,同时也暗示龙眼miPEP319a-2可以参与到龙眼的生长发育过程中。

关键词: 龙眼, miR319a, miRNA编码短肽, 基因克隆, 遗传转化

Abstract:

To study the potential activity of pri-miR319a encoding regulatory peptide of Dimocarpus longan,the pri-miR319a full-length sequences were cloned from seven longan varieties and the positions of potential miPEP(miRNA encode regulatory peptide)in longan pri-miR319a sequences were further analyzed. Then,the activity of miPEP319a was verified by genetic transformation and artificial synthesis of miPEP319a. The results showed that among the seven longan varieties,pri-miR319a had 10 nucleotide differences and three potential ORFs that could encode miPEPs,and one nucleotide mutation site resulted in the change of miPEP319a amino acid sequence. The biological activity of longan miPEP was further verified by thein plantatransformation technology and the synthetic miPEP319 treated embryogenic callus of longan. The results indicated that only the miPEP319a-2 of the three potential miPEPs was biological activity and promoted the expression of miR319a. Finally,the vectors were transiently expressed in tobacco leaves by Agrobacterium-mediated transformation method to verify the activity of longan miPEP319a in tobacco. The results showed that there was no significant difference in the expression of miR319a in tobacco leaves,which indicated that miPEP319a was specie-specific. The results suggested that longan miPEP319a-2 had biological activity and species-specific,and also suggested that longan miPEP319a-2 might participate in the growth and development of longan.

Key words: Dimocarpus longan, miR319a, miRNA encode regulatory peptide, gene clone, genetic transformation

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