华山松大小蠹β-肌动蛋白基因的克隆与组织表达

Cloning and tissue expression ofβ-actin gene from Dendroctonus armandi(Coleoptera:Scolytidae)

【目的】获得华山松大小蠹β-肌动蛋白基因序列,探讨其作为内参基因的可行性。【方法】通过比对其他昆虫β-肌动蛋白的氨基酸序列设计简并引物,用PCR的方法获得华山松大小蠹β-肌动蛋白基因片段,进一步通过RT-PCR和qRT-PCR的方法,研究β-肌动蛋白基因在华山松大小蠹不同发育阶段和成虫不同组织的表达情况。【结果】获得了1 461bp的华山松大小蠹β-肌动蛋白基因cDNA序列,与其他昆虫肌动蛋白基因核苷酸序列的相似性在86%以上,且该基因在华山松大小蠹幼虫、蛹、成虫3个发育阶段以及成虫的头、胸、腹、足、翅等组织中表达稳定,且表达量无明显差异。【结论】成功获得了华山松大小蠹β-肌动蛋白基因片段,且该基因片段可以作为基因表达差异性研究的内参基因。

【Objective】The aim of this study was to obtain the sequence of Dendroctonus armandiβ-actin gene and test its applicability of being a reference gene.【Method】Degenerate primers were designed based on the conserved nucleotide sequences of insectβ-actin genes.A fragment ofβ-actin gene was amplified from the cDNA of D.armandi.The expression levels of D.armandi were detected by RT-PCR and qRT-PCR.【Result】A fragment of 1 461bp was cloned fromD.armandi,which was>86%identical with the corresponding nucleotide sequences of other insectβ-actin genes.The D.armandiβ-actin gene stably expressed in different stages of larva,pupa,and adult,and different tissues of head,thorax,abdomen,foot and wing.Moreover,there was no significant difference in the expression levels of D.armandiβ-actin gene.【Conclusion】D.armandiβ-actin gene was cloned successfully and it was feasible to be used as a reference gene in expression studies.