关键词: 松材线虫, Bxy-cul-1, 发育进度, RNAi, 原位杂交

Abstract: Objective Bursaphelenchus xylophilus (pine wood nematode) has a short generation, large egg production, and strong population reproductive ability. At 25 ℃, it only takes 5 days for pine wood nematodes to complete a generation, and a pair of nematodes can reproduce 200 000 offsprings within 20 days, showing that the growth and development speed is very fast. In this paper, by studying the spatiotemporal expression characteristics and biological functions of B. xylophilus Bxy-cul-1, the role of this gene in the growth and development of B. xylophilus is clarified, so as to provide a theoretical basis for exploring specific measures for controlling nematode population growth from the perspective of growth and development.Method Based on the genome data of B. xylophilus, primers were designed and the Bxy-cul-1 gene was cloned. Bioinformatics analysis such as sequence, phylogenetic analysis and protein structure prediction of Bxy-cul-1 was performed. Real-time quantitative PCR technology and in situ hybridization technology were used to explore the expression level and expression site of Bxy-cul-1 gene in various instars of B. xylophilus, to clarify its spatiotemporal dynamic expression characteristics. RNA interference technology was used to explore the role of this gene in nematode growth and development. Result The results of bioinformatics analysis showed that the CDS of Bxy-cul-1 gene was 2 292 bp in full length, encoding 763 amino acids, belonging to the Cullin protein family. The results of in situ hybridization showed that Bxy-cul-1 gene was expressed in different developmental stages of B. xylophilus, expressed in the whole embryo in the embryonic stage, widely expressed in the second instar, and mainly in the intestine and body wall muscles and tail in the third and fourth instars. In the adult stage, the gene was expressed in oocytes and vulva of females, and abdomen, copulatory spines and tail of males. The results of real-time quantitative PCR showed that the expression level of Bxy-cul-1 gene was the highest in the second instar stage of B. xylophilus, followed by the embryonic stage, and decreased in turn in the third instar, fourth instar and adult stages. After interference of B. xylophilus embryos, it was found that the hatching rate of B. xylophilus embryos decreased by 10.44%, and most of the unhatched embryos stayed at the end of the first instar, and the abdomen of the worm was deformed and swollen, and the worm was not able to break through the egg shell. The instar larvae also appeared deformed, the deformed worms was not able to stretch normally, the middle esophagus glands were abnormally enlarged, and the tail curled up. Interference of the 2nd instar larvae resulted in a significant decrease in the viability of the worm body, with the head swing frequency dropped about 5 times every 30 seconds. Some 2nd instar larvae also appeared deformed, such as short and small body shape, abnormal enlargement of the head and esophageal glands, twisting of the tail and abdomen, and inability to normally move, and hence the worms could not move normally. The results of the developmental progress experiment showed that after silencing the Bxy-cul-1 gene, the developmental progress of B. xylophilus slowed down, and the second instar in the interference group developed to adult 4 days later than the control group.Conclusion Bxy-cul-1 gene of B. xylophilus is a member of the Cullin protein family, and its expression level and expression site are specific at different developmental stages. Silencing this gene can reduce the hatching rate of B. xylophilus embryos and cause deformities, and also reduce the movement ability and developmental progress of B. xylophilus, indicating that the Bxy-cul-1 gene plays an important role in the growth and development of B. xylophilus.

Key words: Bursaphelenchus xylophilus, Bxy-cul-1, developmental progress, RNAi, in situ hybridization