松墨天牛NCOA7基因的克隆及分析

Cloning and Analysis of Nuclear Receptor Coactivator 7 Gene from Monochamus alternatus

【目的】本文探究了松墨天牛核受体共激活因子7基因的功能和作用方式。【方法】采用cDNA末端快速扩增(RACE)方法获得了松墨天牛NCOA7基因的cDNA全长序列,命名为MaNCOA7(GenBank登录号:KU240004);通过RT-qPCR分析得出松墨天牛MaNCOA7基因在卵、幼虫、蛹、成虫4个虫态、幼虫脂肪体等6个组织和成虫鞘翅等10个部位的表达模式。【结果】MaNCOA7基因的cDNA序列全长为3345 bp,其中开放阅读框(ORF)长2970 bp,编码989个氨基酸,由此推测的蛋白分子质量为111 220.4ku,等电点为5.11。MaNCOA7的氨基酸序列与赤拟谷盗相似性最高,为83.6%,与赤拟谷盗处在系统发育树的同一个分支上;没有信号肽和跨膜区,属于亲水性氨基酸,含有丝氨酸(Ser)、苏氨酸(Thr)、酪氨酸(Tyr)磷酸化位点数分别为62、21、11个。MaNCOA7基因在幼虫虫态表达量最高,在卵虫态表达量最低;在幼虫6个组织中均有表达,且有显著差异(P<0.05),其中在幼虫脂肪体中表达量最高,在幼虫马氏管中表达量最低;在成虫10个部位中均有表达,且有显著差异(P<0.05),其中在成虫鞘翅的表达量最高,在成虫马氏管的表达量最低。【结论】推测MaNCOA7基因在松墨天牛幼虫脂肪体中参与调控某些代谢过程或组织器官的生理功能,并可能参与了松墨天牛羽化过程中鞘翅和前胸背板的骨化过程。

【Objective】In this paper,the function and mode of nuclear receptor coactivator 7 gene in Monochamus alternates were studied.【Method】A full length sequence of NCOA7 designated as MaNCOA7（ GenBank： KU240004） from Monochamus alternatus was cloned by RACE（ rapid-amplification of cDNA ends） method,and the expression pattern of MaNCOA7 in different stages and different parts of both larvae and adults of Monochamus alternatus were detected by RT-qPCR.【Result】The full-length cDNA of MaNCOA7 was 3345 bp,which contained a 2970 bp open reading frame（ ORF）,encoding 989 amino acids,with a calculated molecular mass of 111 220. 4 ku and an isoelectric point of 5. 11. The MaNCOA7 indicated the highest homology with Tribolium castaneum（ 83. 6 %）,and M. alternatus and T. castaneum were on the same branch in the phylogenetic tree; The MaNCOA7 protein was hydrophilic,without the signal peptide and transmembrane helices,and there were serine,threonine and p-hydroxyphenylalanine phosphorylation at 62,21 and 11 sites in MaNCOA7,respectively. The expression pattern of MaNCOA7 detected by RT-qPCR indicated that the expression level of MaNCOA7 in larvae was the highest,and the lowest in eggs; MaNCOA7 was prominently expressed in all of the larvae and adults parts（ P 0. 05）. The expression level in larvae fat-body and adults elytra were the highest while the lowest were in both larvae and adults Malpighian tubes（ P 0. 05）.【Conclusion】It is speculated that the MaNCOA7 gene participate in the regulation of certain metabolic processes or physiological functions of tissues and organs in the fat bodies of Monochamus alternates larvae and may be involved in the process of the ossification of elytron and pronotum of Monochamus alternates.