【摘要】 以裸子植物高山松(Pinus densata)为试验材料,利用实验室前期构建的高山松miRNA数据库,通过生物信息学方法筛选了与生长发育相关的miR482a,并通过RLM-5′RACE的方法验证了NBS-LRR抗性蛋白(Unigenes45569)是高山松miR482a的靶基因。通过PCR方法克隆得到高山松miR482a前体序列是130 bp,并可形成稳定的茎环发夹结构,但其成熟序列的碱基保守性较差。系统进化显示pdemiR482a与裸子植物欧洲云杉的进化关系较近。经qRT-PCR分析表明,pde-miR482a在根中表达量最高,其次是在茎中。而靶基因Unigenes45569在根和茎中的表达水平相对较低,暗示pde-miR482a可以通过调控靶基因NBS-LRR抗性蛋白(Unigenes45569)而参与高山松的生长发育。更多还原

【Abstract】 Taking the gymnosperm Pinus densata as the experimental material,using the Pinus densata miRNA database constructed in the early stage of the laboratory,mir482 a related to growth and development was screened by bioinformatics method,and NBSLRR resistance protein(Unigenes45569)was verified to be the target gene of Pinus densata mir482 a by RLM-5′ RACE method. The mir482 a precursor sequence of Pinus densata cloned by PCR is 130 bp and can form a stable stem-loop hairpin structure,but the base conservation of its mature sequence is poor. Phylogeny showed that pde-mir482 a had a close evolutionary relationship with the gymnosperm Picea europaea. qRT-PCR analysis showed that the expression of pde-mir482 a was the highest in roots,followed by in stems. The expression level of target gene Unigenes45569 in roots and stems is relatively low,suggesting that pde-mir482 a can participate in the growth and development of Pinus densata by regulating target gene NBS-LRR resistance protein(Unigenes45569).更多还原