龙眼pri-miR319a编码短肽活性的研究

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

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mi R319a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mi PEP319a-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。

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 miPEP319 a 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 miPEP319 a amino acid sequence. The biological activity of longan miPEP was further verified by the in planta transformation 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.