杉木天冬酰胺合成酶基因的克隆与表达分析

摘要

为了解天冬酰胺合成酶(asparagine synthetase,AS)基因在杉木生长发育及逆境中的作用机制,以杉木种子及其萌发的幼苗为材料,利用RT-PCR和RACE技术获得杉木ASN基因的cDNA序列(ClASN1)和基因组序列,并对该基因在不同生长发育阶段和逆境胁迫下的表达模式和Asn含量变化进行分析.结果表明:ClASN1基因cDNA全长1 609bp,编码443个氨基酸;生物信息学分析结果表明,该基因为含跨膜结构的不稳定亲水蛋白,无信号肽,定位于过氧化物酶体,具有多样的磷酸化位点;进化树分析结果显示,该基因与北美云杉和樟子松亲缘关系最近;ClASN1基因组序列全长3 210 bp,含10个外显子和9个内含子;qPCR结果表明,该基因从浸种24h到幼苗期表达量呈上升趋势,且在干旱胁迫、铝胁迫和光暗处理下均可诱导表达;除铝胁迫24h和持续光照24 h外,ClASN1表达模式与Asn含量的变化趋势相同,此结果表明ClASN1基因通过催化Asn的合成参与杉木幼苗的生长发育及胁迫响应.%In order to investigate the underlying mechanism of ASN gene of Cunninghamia lanceolata in the growth stages and under abiotic stresses,RT-PCR combined with RACE was employed to clone the complete eDNA sequence and DNA sequence from Cunninghamia lanceolata seedling,and the expression of ClASN1 gene and the Ash content under various growth stages and abiotic stresses were analyzed.The results showed that the complete cDNA sequence of ClASN1 was 1 609 bp,encoding 443 amino acids.The bioinformatics software predicted that ClASN1 protein was unstable and hydrophilic with transmembrane structure without signal peptide,located in peroxidase and with various phosphorylation sites.Anglicizing phylogenetic tree of ASN genes in different plants indicated that ClASN1 had the closest relative with Picea sitchensis and Pinus sylvestris.The full length of genomic ClASN1 was 3 210 bp,which contained 10 exons and 9 introns,qPCR results showed that the expression of ClASN1 increased with the time elapsed from seed germination to seedling stage,and was also induced under drought stress,aluminum stress and light dark stress,moreover,the general expression tendencies of ClASN1gene accorded with the content of Asn under various stages from seed germination to seedling growth and different stresses expect for aluminum stress 24 h and light stress 24 h.In conclusion,the above results suggested that ClASN1 may play a role in the various stages from seed germination to seedling growth and stress responses.