松材线虫cul-1基因的表达特性与功能  

Expression Characteristics and Function of cul-1 Gene in Bursaphelenchus xylophilus

作　　者：杨雪晴 刘文义 陈静[1] 孙士淼 周立峰 胡加付[1] Yang Xueqing;Liu Wenyi;Chen Jing;Sun Shimiao;Zhou Lifeng;Hu Jiafu(Zhejiang Agriculture and Forestry University,Hangzhou 311300)

机构地区：[1]浙江农林大学,杭州311300

出　　处：《林业科学》2023年第9期95-105,共11页Scientia Silvae Sinicae

基　　金：国家自然科学基金项目(31870633,31670652)。

摘　　要：【目的】研究松材线虫Bxy-cul-1基因的表达特性和生物学功能,明确该基因在松材线虫生长发育中的作用,为从生长发育角度探索特异性的线虫种群增长控制措施提供理论基础。【方法】根据松材线虫基因组数据设计引物、克隆Bxy-cul-1基因,对Bxy-cul-1进行序列、系统发育和蛋白结构预测等生物信息学分析。利用实时荧光定量PCR技术和原位杂交技术探究Bxy-cul-1基因在松材线虫各龄期的表达水平和表达部位,明确其时空动态表达特性,采用RNA干扰技术探究该基因在松材线虫生长发育中的作用。【结果】生物信息学分析结果显示,Bxy-cul-1基因CDS全长2292 bp,编码763个氨基酸,属于Cullin蛋白家族。原位杂交结果表明,Bxy-cul-1基因在松材线虫各发育阶段均有表达,胚胎期全胚胎表达,2龄期广泛表达,3龄期和4龄期主要在肠道、体壁肌肉和尾部表达;成虫期,Bxy-cul-1基因在雌虫的卵母细胞和阴户、雄虫的腹部、交合刺和尾部表达。实时荧光定量PCR结果显示,Bxy-cul-1基因在松材线虫2龄期表达量最高,胚胎期次之,3龄期、4龄期、成虫期表达量依次递减。对松材线虫胚胎干扰后发现,松材线虫胚胎孵化率下降10.44%,未孵化出的胚胎大多停留在1龄末期,虫体腹部形成畸形肿大,不能突破卵壳;孵化出的部分2龄期幼虫也出现畸形性状,畸形虫无法正常伸展,中部食道腺异常膨大,尾部蜷缩。干扰2龄期幼虫,虫体活力下降明显,每30 s头摆频率约下降5次,部分2龄期幼虫也出现畸形性状,畸形虫体形短小,头部、食道腺异常膨大,尾部和腹部扭转在一起,虫体无法正常运动。发育进度试验结果表明,沉默Bxy-cul-1基因后,松材线虫发育进度减慢,干扰组2龄虫发育至成虫比对照组晚4天。【结论】松材线虫Bxy-cul-1基因是Cullin蛋白家族中的一员,基因表达水平及表达部位在不同发育阶段具有特异性。沉默该基因�【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 200000 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 2292 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 i

关 键 词：松材线虫 Bxy-cul-1 发育进度 RNAI 原位杂交 

分 类 号：S76[农业科学—森林保护学]