松材线虫mix-1基因的表达特性与生物学功能

doi:10.11707/j.1001-7488.LYKX20230572

摘要/Abstract

摘要：

目的: 研究松材线虫中与性别决定相关基因Bxy-mix-1的表达特性和生物学重要功能，明确该基因在松材线虫性别分化和繁殖交配中的作用，为从雌雄比例调节角度探索以该基因为靶标设计分子药物提供理论依据。方法: 根据松材线虫基因组设计引物、克隆Bxy-mix-1基因。对Bxy-mix-1进行生物信息学分析。利用RT-qPCR技术和原位杂交技术探究Bxy-mix-1基因在松材线虫各龄期的表达水平和表达部位，以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: 生物信息学结果显示，松材线虫Bxy-mix-1 基因的全长CDS为 2 163 bp，共编码1171个氨基酸，属于SMC蛋白家族。Bxy-mix-1基因在松材线虫各发育阶段均有表达，胚胎期表达量最低，2龄、3龄、4龄表达量逐渐升高，成虫期表达水平最高，且基因表达水平具有雌、雄虫差异性，即雌虫的基因表达水平显著高于雄虫。原位杂交结果表明Bxy-mix-1基因在松材线虫胚胎期呈现全胚胎表达，2龄期、3龄期表达集中在体中后段，4龄和成虫期出现雌、雄差异性表达，即在雌、雄虫的性腺以及雄虫的交合刺表达。Bxy-mix-1基因沉默后，雌雄比例统计结果显示，松材线虫的雌雄比率显著下降。交配结果显示，松材线虫雄虫错误定位率显著升高，雌虫被干扰后的平均产卵量显著下降。结论: 松材线虫Bxy-mix-1基因属SMC蛋白家族，基因表达水平及表达部位皆具有雌、雄虫显著差异性。研究表明Bxy-mix-1基因对松材线虫的性别决定和生殖交配行为具有重要的调控功能。

关键词: 松材线虫, Bxy-mix-1, 性别决定, RNAi, 原位杂交

Abstract:

Objective: This study aims to elucidate the role of Bxy-mix-1 gene in the growth and development of Bursaphelenchus xylophilus by analyzing the expression characteristics and biological functions of the gene associated with sex determination in B. xylophilus, so as to provide a theoretical foundation for exploring specific nematode population growth control measures from the perspective of sex ratio regulation. Method: Based on the genome data of B. xylophilus, primers were designed, by which the Bxy-mix-1 gene was cloned. Bioinformatics analysis of Bxy-mix-1 was performed. To elucidate the spatio-temporal dynamic expression characteristics of the Bxy-mix-1 gene, RT-qPCR and in situ hybridization techniques were employed to investigate its expression level and expression site in each age of B. xylophilus. Additionally, RNA interference technique was utilized to explore the role of this gene in the growth and development of B. xylophilus. Result: The results of bioinformatics analysis showed that the full-length CDS of Bxy-mix-1 gene was 2 163 bp, encoding 1 171 amino acids, belonging to the SMC protein family. Bxy-mix-1 gene was expressed in different developmental stages of B. xylophilus, with the lowest expression level in embryo stage, gradually increasing expression level in J2, J3 and J4 stages, and reaching the highest expression level in adult stage. Meanwhile, there was difference in gene expression level between female and male, showing a significantly higher expression level in the males than that in the females. The results of in situ hybridization showed that Bxy-mix-1 gene exhibited whole-embryo expression at embryonic stage, with expression concentrated in the middle and posterior parts of the body at J2 and J3 instars. At the J4 and adult stage, there were differential expressions of female and male in the gonads of female and male and in the mating spines of male. After silencing the Bxy-mix-1 gene, the statistical results showed a significant decrease in the male to female ratio of B. xylophilus. The results of mating experiment showed that the mislocalization rate of males increased significantly, and the average number of offspring of females decreased significantly. Conclusion: Bxy-mix-1 is a member of the SMC protein family which has sexual dimorphism in the expression level and location. This study indicates that Bxy-mix-1 plays an important role in regulating sex determination and reproductive behavior of B. xylophilus.

Key words: Bursaphelenchus xylophilus, Bxy-mix-1, sex determination, RNAi, in situ hybridization

中图分类号:

S763

钟伟良,庞凯飞,鲁建斌,范忠轰,孙士淼,邵蝴蝶,胡加付. 松材线虫mix-1基因的表达特性与生物学功能[J]. 林业科学, 2025, 61(1): 150-158.

Weiliang Zhong,Kaifei Pang,Jianbin Lu,Zhonghong Fan,Shimiao Sun,Hudie Shao,Jiafu Hu. Expression Characteristics and Biological Function of mix-1 Gene in Bursaphelenchus xylophilus[J]. Scientia Silvae Sinicae, 2025, 61(1): 150-158.

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引物 Primer	基因符号 Gene symbol	序列 Sequence (5' to 3')
原位杂交 In situ hybridization	Bxy-mix-1	F: GACGGAAAAACAACGTGCGA
	Bxy-mix-1	R: CGCTGATTCTGGGCGATTTG
	M13	F: CGCCAGGGTTTTCCCAGTCACGAC
	M13	R: CACACAGGAAACAGCTATGAC
荧光定量PCR Quantitative real-time PCR (RT-qPCR)	Bxy-mix-1	F: TGGTACAGCAAGACGAGGTG
	Bxy-mix-1	R: TTGTAGCGCACAATCACGGA
	tbb-2	F: GAATCCAACATGAACGATCT
	tbb-2	R: CTTCGTATTCACCATCATCT
	β-actin	F: CGCAAATACTCCGTCTGGATTGG
	β-actin	R: TTCGTCGTACTCTTGCTTGGAGA
dsRNA	Bxy-mix-1	F: TAATACGACTCACTATAGGGGACGG AAAAACAACGTGCGA
	Bxy-mix-1	R: TAATACGACTCACTATAGGGCGCT GATTCTGGGCGATTTG
	gfp	F: TAATACGACTCACTATAGGGAGAAT GAGTAAAGGAGAAG
	gfp	R: TAATACGACTCACTATAGGGAGAT TTGTATAGTTCATCCATG

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