Bxy-egl-30调控松材线虫早期个体发育的生物学功能研究

The biological function of Bxy-egl-30 in regulating early-stage development of Bursaphelenchus xylophilus

“发育快,交配效率高”是松材线虫种群扩张和病害流行的基础。通过生物信息学分析、原位杂交和RNAi等技术,本研究明确了Bxy-egl-30在松材线虫(Bursaphelenchus xylophilus)个体生长发育过程中的功能。生物信息学分析结果表明Bxy-egl-30编码区长度为1128 bp,编码375个氨基酸,属于EGL-30蛋白亚基家族。原位杂交结果显示Bxy-egl-30基因在松材线虫不同发育阶段的表达具有特异性,在胚胎期与幼虫期该基因广泛表达于性腺、肠道和附近的体壁细胞等部位;在成虫期,该基因在雌虫的阴户肌肉和雄虫的性腺部位集中表达。RT-qPCR结果显示Bxy-egl-30在二龄期表达量最高,胚胎期次之,三龄期、四龄期和成虫期表达量依次降低。浸泡Bxy-egl-30双链RNA后,松材线虫胚胎的孵化率下降了10.88%,二龄期、三龄期幼虫活力明显下降,每分钟头摆频率约下降4次。致病性接种实验结果显示松材线虫被Bxy-egl-30双链RNA干扰后致病性减弱,减缓了黑松的死亡时间,干扰组黑松的死亡时间比对照组晚9 d。本研究明确了Bxy-egl-30基因在松材线虫不同发育阶段的时空动态表达特性和生物学功能,揭示了该基因调控松材线虫早期个体发育和运动等方面的重要作用。

Rapid development and efficient mating are the basis of population expansion and disease epidemic of B.xylophilus.Bioinformatics analysis,in situ hybridization and RNA interference were used to investigate the potential role of Bxy-egl-30 in the growth and development of B.xylophilus.The cloned coding region of the Bxy-egl-30 gene has a length of 1128 bp,encoding 375 amino acids,and the phylogenetic tree analysis showed that Bxy-egl-30 belonged to the EGL-30 protein subunit family.The results of in situ hybridization showed that the expression of Bxy-egl-30 was specific in different developmental stages of B.xylophilus,and it was widely expressed in the gonads,gut and nearby body wall cells,during embryonic and larval stages,the gene is expressed in the vulva muscle of females and the gonad of males.The results of RT-qPCR showed that the expression level of Bxy-egl-30 reached the highest level at the second larval stage,followed by the embryonic stage,and in the third instar stage,the fourth instar stage,and the adult stage,the expression level of Bxy-egl-30 decreased in turn.Knockdown of Bxy-egl-30 decreased the hatching rate of pinewood nematode embryos by 10.88%,and the activity of the second and third instar larvae decreased obviously,which the head swing frequency decreased about 4 times per minute.The results of the inoculation experiment showed that the pathogenicity of nematodes was weakened by Bxy-egl-30 RNAi and the death time of trees was slowed down.The death time of pine trees in the interference group was 9 days later than that in the control group.This study investigated the spatiotemporal dynamic expression patterns and biological functions of the Bxy-egl-30 gene at different deve-lopmental stages of B.xylophilus,and revealed the important role of the gene in the movement and development of B.xylophilus.