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

Abstract

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

CLC Number:

S763

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.

Figures/Tables 7

Table 1

Fig.1

Fig.2

Fig.3

Fig.4

Fig.5

Fig.6

References 0

	陈凤毛, 汤　坚, 叶建仁. 松材线虫病鉴定方法与评价(综述). 安徽农业大学学报, 2005, 32 (1): 22- 25.
	Chen F M, Tang J, Ye J R. Identification and evaluation of pine wood nematode disease (review). Journal of Anhui Agricultural University, 2005, 32 (1): 22- 25.
	陈　茜, 刘文义, 周立峰, 等. Bxy-egl-30调控松材线虫早期个体发育的生物学功能研究. 植物病理学报, 2023, 53 (2): 267- 276.
	Chen X, Liu W Y, Zhou L F, et al. The biological function of Bxy-egl-30 in regulating early-stage development of Bursaphelenchus xylophilus. Acta Phytopathologica Sinica, 2023, 53 (2): 267- 276.
	陈怡帆, 周艳涛, 孙　红. 全国主要林业有害生物2022年发生情况及2023年趋势预测. 中国森林病虫, 2023, 42 (2): 51- 54.
	Chen Y F, Zhou Y T, Sun H. National occurrence of major forestry pests in 2022 and trend forecast in 2023. China Forest Pests and Diseases, 2023, 42 (2): 51- 54.
	马　芮, 刘文义, 余红仕, 等. 拟松材线虫G蛋白α亚基Bmu-gpa-1表达特性及功能. 林业科学, 2022, 58 (4): 95- 103. doi: 10.11707/j.1001-7488.20220410
	Ma R, Liu W Y, Yu H S, et al. Expression characteristics and function of G protein alpha subunit Bmu-gpa-1 in Bursaphelenchus mucronatus. Scientia Silvae Sinicae, 2022, 58 (4): 95- 103. doi: 10.11707/j.1001-7488.20220410
	马欣欣. 2020. 松材线虫Bxy-mab-3基因的表达特性与功能验证研究. 杭州: 浙江农林大学.
	Ma X X. 2020. Expression and regulation of Bxy-mab-3 genes in pinewood nematodes, Bursaphelenchus xylophilus. Hangzhou: Zhejiang A&F University. ［in Chinese］
	孟　和, 陈学辉, 王起山, 等. 短双链RNA对鸡胚盘细胞外源绿荧光蛋白基因表达的影响. 动物学报, 2004, 50 (2): 302- 307. doi: 10.3969/j.issn.1674-5507.2004.02.022
	Meng H, Chen X H, Wang Q S, et al. Effects of short double-stranded RNA on the expression of exogenous green fluorescent protein gene in chick blastoderm cells. Acta Zoologica Sinica, 2004, 50 (2): 302- 307. doi: 10.3969/j.issn.1674-5507.2004.02.022
	孙永春. 南京中山陵发现松材线虫. 江苏林业科技, 1982, 4 (12): 47.
	Sun Y C. Pine wood nematode found in Sun Yat-sen Mausoleum, Nanjing. Journal of Jiangsu Forestry Science & Technology, 1982, 4 (12): 47.
	韦鹏飞, 理永霞, 冯宇倩, 等. 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. 南京林业大学学报(自然科学版), 2022, 46 (4): 5- 20.
	Wei P F, Li Y X, Feng Y Q, et al. Expression characteristics and biological function of the sex-determining gene Bx-sex-1 in Bursaphelenchus xylophilus. Journal of Nanjing Forestry University (Natural Sciences Edition), 2022, 46 (4): 5- 20.
	叶为民, 冯志新. 松材线虫个体发育研究. 华南农业大学学报, 1993, 14 (2): 78- 83.
	Ye W M, Feng Z X. Ontogenesis of pine parasitic nematode Bursaphelenchus xylophilus. Journal of South China Agricultural University, 1993, 14 (2): 78- 83.
	周天鸿, 李月琴, 朱嘉明, 等. 真核细胞转录系统启动T7启动子起始的研究. 暨南大学学报, 2000, 21 (3): 95- 99.
	Zhou T H, Li Y Q, Zhu J M, et al. Study on the initiation of T7 promoter by eukaryotic transcription system. Journal of Jinan University, 2000, 21 (3): 95- 99.
	Barton M K, Schedl T B, Kimble J. Gain-of-function mutations of fem-3, a sex-determination gene in Caenorhabditis elegans. Genetics, 1987, 115 (1): 107- 119. doi: 10.1093/genetics/115.1.107
	Bolla R I, Jordan W. Cultivation of the pine wilt nematode, Bursaphelenchus xylophilus, in axenic culture media. Journal of nematology, 1982, 14 (3): 377- 381.
	Cheng X Y, Xie P Z, Cheng F X, et al. Competitive displacement of the native species Bursaphelenchus mucronatus by an alien species Bursaphelenchus xylophilus (Nematoda: Aphelenchida: Aphelenchoididae): a case of successful invasion. Biological Invasions, 2008, 11 (2): 205- 213.
	Chuang P T, Albertson D G, Meyer B J. DPY-27: a chromosome condensation protein homolog that regulates C. elegans dosage compensation through association with the X chromosome. Cell, 1994, 79 (3): 459- 474. doi: 10.1016/0092-8674(94)90255-0
	Cui J, Li Y X, Zhang W, et al 2020. The population structure and sex ratios of Bursaphelenchus xylophilus under α-pinene stress. Journal of Forestry Research, 31(1): 921–926.
	Farboud B, Novak C S, Nicoll M, et al. Dose-dependent action of the RNA binding protein FOX-1 to relay X-chromosome number and determine C. elegans sex. Elife, 2020, 29 (9): e62963.
	Hagstrom K A, Holmes V F, Cozzarelli N R, et al. C. elegans condensin promotes mitotic chromosome architecture, centromere organization, and sister chromatid segregation during mitosis and meiosis. Genes & Development, 2002, 16 (6): 729- 742..
	Hasegawa K, Mota M, Futai K, et al. Chromosome structure and behaviour in Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae) germ cells and early embryo. Nematology, 2006, 8 (3): 425- 434. doi: 10.1163/156854106778493475
	Hodgkin J. A genetic analysis of the sex-determining gene, tra-1, in the nematode Caenorhabditis elegans. Genes & Development, 1987, 1 (7): 731- 745.
	Hodgkin J A, Brenner S. Mutations causing transformation of sexual phenotype in the nematode Caenorhabditis elegans. Genetics, 1977, 86 (2): 275- 287. doi: 10.1093/genetics/86.2.275
	Hunter C P, Wood W B. The tra-1 gene determines sexual phenotype cell-autonomously in C. elegans. Cell, 1990, 63 (6): 1193- 1204. doi: 10.1016/0092-8674(90)90415-B
	Jikumaru S, Togashi K. Resistance of an indigenous biological system against expansion of the invasive nematode, Bursaphelenchus xylophilus, in cool areas of Japan. Nematology, 2008, 10 (5): 679- 687. doi: 10.1163/156854108785787172
	Kimble J, Hirsh D. The postembryonic cell lineages of the hermaphrodite and male gonads in Caenorhabditis elegans. Developmental Biology, 1979, 70 (2): 396- 417. doi: 10.1016/0012-1606(79)90035-6
	Liao S M Kasuga S, Togashi K. Suppressive effects of Bursaphelenchus mucronatus on pine wilt disease development and mortality of B. xylophilus-inoculated pine seedlings. Nematology, 2014, 16 (2): 219- 227. doi: 10.1163/15685411-00002760
	Lieb D J, Albrecht R M, Chuang P, et al. MIX-1: an essential component of the C. elegans mitotic machinery executes X chromosome dosage compensation. Cell, 1998, 92 (2): 265- 277. doi: 10.1016/S0092-8674(00)80920-4
	Lilley C J, Bakhetia M, Charlton W L, et al. Recent progress in the development of RNA interference for plant parasitic nematodes. Molecular Plant Pathology, 2007, 8 (5): 701- 711. doi: 10.1111/j.1364-3703.2007.00422.x
	Lyon M F. Gene action in the X-chromosome of the mouse (Mus musculus L.). Nature, 1961, 190 (1): 372- 373.
	Motohashi T, Tabara H, Kohara Y. [ 2006−07−24]. Protocols for large scale in situ hybridization on C. elegans larvae, in WormBook edited by The C. elegans Research Community, 1–8. https://doi.org/10.1895/wormbook.1.103.1.
	Ohno S, Kaplan W D, Kinosita R. Formation of the sex chromatin by a single X-chromosome in liver cells of Rattus norvegicus. Experimental Cell Research, 1959, 18 (2): 415- 418. doi: 10.1016/0014-4827(59)90031-X
	Perry M D, Trent C, Robertson B, et al. Sequenced alleles of the Caenorhabditis elegans sex-determining gene her-1 include a novel class of conditional promoter mutations. Genetics, 1994, 138 (2): 317- 327. doi: 10.1093/genetics/138.2.317
	Rahul S, Peter M. Dosage compensation and nuclear organization: cluster to control chromosome-wide gene expression. Current Opinion in Genetics & Development, 2016, 37 (1): 9- 16.
	Shinya R, Sun S M, Dayi M, et al. Possible stochastic sex determination in Bursaphelenchus nematodes. Nature Communications, 2022, 13, 2574. doi: 10.1038/s41467-022-30173-2
	Shpargel K B, Praveen K, Rajendra T K, et al. Gemin3 is an essential gene required for larval motor function and pupation in Drosophila. Molecular Biology of the Cell, 2009, 20 (1): 90- 101. doi: 10.1091/mbc.e08-01-0024
	Starostina N G, Liu J M, Schvarzstein M, et al. A CUL-2 ubiquitin ligase containing three FEM proteins degrades TRA-1 to regulate C. elegans sex determination. Developmental Cell, 2007, 13 (1): 127- 139. doi: 10.1016/j.devcel.2007.05.008
	Strunnikov V A. SMC proteins and chromosome structure. Trends in Cell Biology, 1998, 8 (11): 454- 459. doi: 10.1016/S0962-8924(98)01370-1
	Sun S M, Wang J H, Liu W Y, et al. Regulatory roles of Bxy-laf-1 in reproductive behaviour of Bursaphelenchus xylophilus. Frontiers in Physiology, 2022, 13 (1): 1024409.
	Wei P F, Li Y X, Zhang W, et al. Factors affecting the ratio of female to male of pinewood nematodes Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle. Forests, 2021, 12 (6): 763. doi: 10.3390/f12060763
	Zhou L F, Ji J J, Zhu N J, et al. Molecular characterization and functional analysis of akt-1 in pinewood nematode, Bursaphelenchus xylophilus. Forest Pathology, 2021, 51 (1): 1- 10.
	Zhou L F, Liu W Y, Bai L, et al. Female's war: a story of the invasion and competitive displacement between two xylophilus group nematode species. Journal of Pest Science, 2023, 3 (2): 1- 11.
	Zhou L F, Ma X X, Zhu N J, et al. The role of mab-3 in spermatogenesis and ontogenesis of pinewood nematode, Bursaphelenchus xylophilus. Pest Management Science, 2020, 77 (1): 138- 147.

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

[1]	Zhihong Li,Wei Zhang,Xinkang Zhao,Jinping Shu,Haojie Wang. Phenotypic Variation of Bursaphelenchus xylophilus in Different Occurrence Areas of Zhejiang Province [J]. Scientia Silvae Sinicae, 2025, 61(1): 137-149.
[2]	Runxin Ni,Yihang Ning,Ziyue Wang,Guangxin Liu,Yan Zhen,Mengli Xi. High Quality Metaphase Chromosome Preparation and rDNA Physical Localization of Juglans mandshurica [J]. Scientia Silvae Sinicae, 2024, 60(10): 50-55.
[3]	Jianan Wang,Shengwei Jiang,Ruizhi Zhang,Debin Li,Hao Wu,Zhao Xu,Yizhen Zhang,Jiaqing Wang,Danlei Li,Feng Wang. Prolonged Incubation Period of Bursaphelenchus xylophilus Results in the Over-year Death of Pinus koraiensis [J]. Scientia Silvae Sinicae, 2024, 60(10): 67-75.
[4]	Yang Xueqing, Liu Wenyi, Chen Jing, Sun Shimiao, Zhou Lifeng, Hu Jiafu. Expression Characteristics and Function of cul-1 Gene in Bursaphelenchus xylophilus [J]. Scientia Silvae Sinicae, 2023, 59(9): 95-105.
[5]	Junnan Li,Runkai Chen,Yu Fu,Mengling Cai,Bingrong Huang,Yun Xu,Songqing Wu,Feiping Zhang. Research on Paracarophenax alternatus Ⅲ. Phoretic Characteristics by Monochamus alternatus Adults [J]. Scientia Silvae Sinicae, 2022, 58(9): 128-137.
[6]	Xiaolei Ding,Qingtong Wang,Sixi Lin,Ruiwen Zhao,Yue Zhang,Jianren Ye. Analysis of Genetic Variations of Bursaphelenchus xylophilus Populations between Guangdong and Jiangsu Provinces with SNP Marker [J]. Scientia Silvae Sinicae, 2022, 58(8): 1-9.
[7]	Yefan Cao,Xizhuo Wang,Laifa Wang,Xiang Wang,Ming Xu,Shengrong Su,Wei Guo. Effects of Different Virulence Isolates of Bursaphelenchus xylophilus on Aetivitres Early Enzymes in Pinus koraiensis [J]. Scientia Silvae Sinicae, 2022, 58(8): 10-17.
[8]	Junnan Li,Runkai Chen,Yu Fu,Mengling Cai,Bingrong Huang,Yun Xu,Songqing Wu,Feiping Zhang. Research on Paracarophenax alternatusⅡ. Mobility and Tropism of Female Adults [J]. Scientia Silvae Sinicae, 2022, 58(8): 149-156.
[9]	Chuanmei Xu,Zihan Wang,Feng Xie,Xuyin Jin,Xinyan Wu,Rong Chen,Jianqin Huang. Comparison of 45S rDNA and 5S rDNA Distribution Characteristics of 12 Bamboo Species [J]. Scientia Silvae Sinicae, 2022, 58(7): 93-102.
[10]	Junnan Li,Yuzhen Zhu,Runkai Chen,Yu Fu,Mengling Cai,Bingrong Huang,Yun Xu,Songqing Wu,Feiping Zhang. Research on Paracarophenax alternatus Ⅰ. Biological Characteristics [J]. Scientia Silvae Sinicae, 2022, 58(6): 79-87.
[11]	Minjia Wang,Jianren Ye,Yusheng Tu,Fangping Du. Screening of Wood Rot Fungi for Treating the Infected Wood by Bursaphelenchus xylophilus [J]. Scientia Silvae Sinicae, 2021, 57(6): 93-102.
[12]	Lihua Zhu,Xinyue Zhang,Xinrui Xia,Yu Wan,Shanjun Dai,Jianren Ye. Pathogenicity of Aseptic Bursaphelenchus xylophilus on Pinus massoniana [J]. Scientia Silvae Sinicae, 2020, 56(7): 63-69.
[13]	Yefan Cao,Laifa Wang,Xizhuo Wang,Jiehong Fan. Pathogenicity of Bursaphelenchus xylophilus to Larix olgensis Seedlings [J]. Scientia Silvae Sinicae, 2020, 56(11): 108-115.
[14]	Yuting Mao,Tao Ma,Laijiao Lan,Xiujun Wen. Advances in Esteya vermicola, A Potential Biocontrol Fungus for Pine Wood Nematode [J]. Scientia Silvae Sinicae, 2020, 56(1): 180-190.
[15]	Ye Jianren. Epidemic Status of Pine Wilt Disease in China and Its Prevention and Control Techniques and Counter Measures [J]. Scientia Silvae Sinicae, 2019, 55(9): 1-10.

Expression Characteristics and Biological Function of mix-1 Gene in Bursaphelenchus xylophilus

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 7

References 0

Related Articles 15

Recommended Articles

Metrics

Comments