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

doi:10.12302/j.issn.1000-2006.202104004

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南京林业大学学报（自然科学版） ›› 2022, Vol. 46 ›› Issue (4): 15-20.doi: 10.12302/j.issn.1000-2006.202104004

所属专题：松材线虫病致病机理与病害防治

• 专题报道Ⅰ：松材线虫病致病机理与病害防治（执行主编　叶建仁） • 上一篇下一篇

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

韦鹏飞¹^,²(), 理永霞¹^,²^,^*(), 冯宇倩¹^,², 刘振凯¹^,², 张星耀¹^,²

1.南京林业大学,南方现代林业协同创新中心,江苏南京 210037
2.中国林业科学研究院森林生态环境与自然保护研究所,国家林业和草原局森林保护学重点实验室,北京 100091

收稿日期:2021-04-02 修回日期:2021-08-01 出版日期:2022-07-30 发布日期:2022-08-01
通讯作者: 理永霞
基金资助:
国家重点研发计划(2018YFC1200400)

Expression characteristics and biological functions of the sex determination gene Bx-sex-1 in Bursaphelenchus xylophilus

WEI Pengfei¹^,²(), LI Yongxia¹^,²^,^*(), FENG Yuqian¹^,², LIU Zhenkai¹^,², ZHANG Xingyao¹^,²

1. Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
2. Key Laboratory of Forest Protection of National Forestry and Grassland Administration, Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing 100091, China

Received:2021-04-02 Revised:2021-08-01 Online:2022-07-30 Published:2022-08-01
Contact: LI Yongxia

摘要/Abstract

摘要：

【目的】揭示松材线虫(Bursaphelenchus xylophilus)性别决定基因Bx-sex-1的表达特征和生物学功能。【方法】通过Mega 6中的Jones-Taylor-Thornton (JTT)模型,采用邻接法(neighbor joining,NJ)对Bx-sex-1和其他线虫的氨基酸序列进行系统发育构建。采用同步培养的方法分别收集不同龄期的松材线虫并提取总RNA,反转录获得cDNA并采用实时荧光定量PCR (qRT-PCR) 的方法,研究Bx-sex-1在松材线虫不同发育阶段的相对表达量。同时采用qRT-PCR并通过绝对定量法分析Bx-sex-1基因在雌虫和雄虫基因组的拷贝数。采用dsRNA浸泡法对目的基因进行干扰,观察松材线虫后代卵的孵化率和雌雄比的变化。【结果】系统进化树显示松材线虫Bx-sex-1和其他两个植物寄生线虫成为一支,与自由生线虫和寄生动物线虫分别进化为不同的分支。松材线虫Bx-sex-1在不同发育阶段相对表达量有差异,在卵期相对表达量最高,而在2龄幼虫时期最低,从2龄幼虫时期到成虫时期相对表达量逐渐升高。qRT-PCR结果显示Bx-sex-1基因在雌虫和雄虫基因组中均为单拷贝。Bx-sex-1被干扰后,卵的孵化率下降,雌雄比降低。【结论】松材线虫Bx-sex-1基因在卵的发育过程和性别决定中可能具有重要作用。

关键词: 松材线虫, 性别决定基因, RNA干扰, 雌雄比

Abstract:

【Objective】 This study aimed to determine the expression characteristics and biological functions of Bx-sex-1, a sex determination gene in Bursaphelenchus xylophilus. 【Method】 The phylogenetic analysis was performed using the neighbor joining (NJ) method, based on the Jones-Taylor-Thornton (JTT) model in the molecular evolutionary genetic analysis (Mega 6). After collecting pine wood nematodes at different developmental stages using synchronous culture, cDNA was synthesized from the extracted RNA, and the relative expression level of Bx-sex-1 was studied using real-time quantitative PCR (qRT-PCR), where the copy number of Bx-sex-1 in the female and male genomes was analyzed through the absolute quantitative analysis. B. xylophilus adults were socked with double-strand RNA (dsRNA), and the hatching rate and the female to male sex ratio of the progeny were observed. 【Result】 The phylogenetic tree showed that the pine wood nematode Bx-sex-1 was a clade with two other plant parasitic nematodes, and had evolved into a different clade from the free and animal parasitic nematodes, respectively. The Bx-sex-1 gene exhibited the highest relative expression level in the egg period, with the lowest expression in the L₂ period that increased gradually up to the adult period. Both female and male genomes had a single copy of the gene. Compared with untreated B. xylophilus, the hatching rate of the eggs and the sex ratio decreased in the experimental samples. 【Conclusion】 The Bx-sex-1 gene of B. xylophilus may play an important role in egg development and sex determination.

Key words: Bursaphelenchus xylophilus, sex determination gene, RNA interference(RNAi), sex ratio of female to male

中图分类号:

S763.11

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[J]. 南京林业大学学报（自然科学版）, 2022, 46(4): 15-20.

WEI Pengfei, LI Yongxia, FENG Yuqian, LIU Zhenkai, ZHANG Xingyao. Expression characteristics and biological functions of the sex determination gene Bx-sex-1 in Bursaphelenchus xylophilus[J].Journal of Nanjing Forestry University (Natural Science Edition), 2022, 46(4): 15-20.DOI: 10.12302/j.issn.1000-2006.202104004.

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参考文献 27

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引物名称 primer name	序列(5'—3') sequence(5'—3')
Bx-sex-1-5'-F	ATGGTCATGGATAATGTAGGC
Bx-sex-1-5'-R	ACAACTCTGACAACGAACT
Bx-sex-1-3'-F	AAACGGACCCTGAAGTCCCC
Bx-sex-1-3'-R	TCATGTCGCAGCCTGTCCTGATC
C-Bx-daf-12-F	TACGGTGGTCAACACGACTG
C-Bx-daf-12-R	TCGACCGGTTGACTCACAAG
C-Bx-sex-1-F	CAAAGGAGAACCGGACCGAA
C-Bx-sex-1-R	CTGGCCATTGGGGAGTGAAT
Bx-sex-1-T7-F	GGATCCTAATACGACTCACTATAGGGCTCCACAACCTCAACT
Bx-sex-1-R	GGCTCTGCTTGTTCATCA
Bx-sex-1-T7-R	GGATCCTAATACGACTCACTATAGGGGCTCTGCTTGTTCATCA
Bx-sex-1-F	GCTCCATCAACCTCAACT
q-Bx-sex-1-F	CTTCAAGGAGTCGGAATAATG
q-Bx-sex-1-R	ACTGGAGTCGTATGATTGG
β-actin-F	TCCGTACCCTGAAGTTGGCTAACC
β-actin-R	AAGTGGAGACGAGGGAATGGAACC

样本 sample	拷贝数/×10^-6 copy number		Bx-daf-12与Bx-sex-1 拷贝数之比 copy number ratio of Bx-daf-12 to Bx-sex-1
样本 sample	Bx-daf-12	Bx-sex-1
雌虫1 female 1	6.33	5.10	1.24
雌虫2 female 2	4.87	3.74	1.30
雄虫1 male 1	3.80	3.01	1.26
雄虫2 male 2	3.04	2.31	1.31

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

Expression characteristics and biological functions of the sex determination gene Bx-sex-1 in Bursaphelenchus xylophilus

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