松材线虫性别决定基因Bx⁃sex⁃1表达特征和生物学功能分析

Expression characteristics and biological functions of the sex determination gene Bx⁃sex⁃1 in Bursaphelenchus xylophilus

【目的】揭示松材线虫(Bursaphelenchus xylophilus)性别决定基因Bx⁃sex⁃1的表达特征和生物学功能。【方法】通过Mega 6中的Jones⁃Taylor⁃Thornton(JTT)模型,采用邻接法(neighbor joining,NJ)对Bx⁃sex⁃1和其他线虫的氨基酸序列进行系统发育构建。采用同步培养的方法分别收集不同龄期的松材线虫并提取总RNA,反转录获得cDNA并采用实时荧光定量PCR(qRT⁃PCR)的方法,研究Bx⁃sex⁃1在松材线虫不同发育阶段的相对表达量。同时采用qRT⁃PCR并通过绝对定量法分析Bx⁃sex⁃1基因在雌虫和雄虫基因组的拷贝数。采用dsRNA浸泡法对目的基因进行干扰,观察松材线虫后代卵的孵化率和雌雄比的变化。【结果】系统进化树显示松材线虫Bx⁃sex⁃1和其他两个植物寄生线虫成为一支,与自由生线虫和寄生动物线虫分别进化为不同的分支。松材线虫Bx⁃sex⁃1在不同发育阶段相对表达量有差异,在卵期相对表达量最高,而在2龄幼虫时期最低,从2龄幼虫时期到成虫时期相对表达量逐渐升高。qRT⁃PCR结果显示Bx⁃sex⁃1基因在雌虫和雄虫基因组中均为单拷贝。Bx⁃sex⁃1被干扰后,卵的孵化率下降,雌雄比降低。【结论】松材线虫Bx⁃sex⁃1基因在卵的发育过程和性别决定中可能具有重要作用。

【Objective】This study aimed to determine the expression characteristics and biological functions of Bx⁃sex⁃1,a sex determination gene in Bursaphelenchus xylophilus.【Method】The phylogenetic analysis was performed using the neighbor joining(NJ)method,based on the Jones⁃Taylor⁃Thornton(JTT)model in the molecular evolutionary genetic analysis(Mega 6).After collecting pine wood nematodes at different developmental stages using synchronous culture,cDNA was synthesized from the extracted RNA,and the relative expression level of Bx⁃sex⁃1 was studied using real⁃time quantitative PCR(qRT⁃PCR),where the copy number of Bx⁃sex⁃1 in the female and male genomes was analyzed through the absolute quantitative analysis.B.xylophilus adults were socked with double⁃strand RNA(dsRNA),and the hatching rate and the female to male sex ratio of the progeny were observed.【Result】The phylogenetic tree showed that the pine wood nematode Bx⁃sex⁃1 was a clade with two other plant parasitic nematodes,and had evolved into a different clade from the free and animal parasitic nematodes,respectively.The Bx⁃sex⁃1 gene exhibited the highest relative expression level in the egg period,with the lowest expression in the L_(2) period that increased gradually up to the adult period.Both female and male genomes had a single copy of the gene.Compared with untreated B.xylophilus,the hatching rate of the eggs and the sex ratio decreased in the experimental samples.【Conclusion】The Bx⁃sex⁃1 gene of B.xylophilus may play an important role in egg development and sex determination.