松材线虫Bx-HSF-1转录激活活性研究

doi:10.12302/j.issn.1000-2006.202102019

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南京林业大学学报（自然科学版） ›› 2022, Vol. 46 ›› Issue (4): 8-14.doi: 10.12302/j.issn.1000-2006.202102019

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Bx-HSF-1转录激活活性研究

张瑞芝¹(), 姜生伟²^,³, 吴昊²^,³, 陈俏丽¹^,⁴, 李丹蕾¹^,³^,⁴, 王峰¹^,³^,⁴^,^*()

1.黑龙江省外来林木病虫害监测与防控重点实验室,东北林业大学林学院,黑龙江哈尔滨 150040
2.辽宁省林业和草原局有害生物防治检疫工作站,辽宁沈阳 110001
3.辽宁省危险性林业有害生物防控重点实验室,沈阳工学院, 辽宁沈阳 113122
4.森林生态系统可持续经营教育部重点实验室, 东北林业大学林学院,黑龙江哈尔滨 150040

收稿日期:2021-02-24 修回日期:2022-09-18 出版日期:2022-07-30 发布日期:2022-08-01
通讯作者: 王峰
基金资助:
国家自然科学基金项目(31971656);辽宁省重点研发计划(2019JH2/10200001)

Transcriptional activity of the Bx-HSF-1 in Bursaphelenchus xylophilus (Aphelenchida: Aphelenchoididae)

ZHANG Ruizhi¹(), JIANG Shengwei²^,³, WU Hao²^,³, CHEN Qiaoli¹^,⁴, LI Danlei¹^,³^,⁴, WANG Feng¹^,³^,⁴^,^*()

1. Key Laboratory of Alien Forest Pest Detection and Control-Heilongjiang Province, School of Forestry, Northeast Forestry University, Harbin 150040, China
2. Station of Forest and Grassland Pest Control and Quarantine, Liaoning Province Forestry and Grassland Bureau, Shenyang 110001, China
3. Liaoning Provincial Key Laboratory of Dangerous Forest Pest Management and Control, Shenyang Institute of Technology, Shenyang 113122, China
4. Key Laboratory of Sustainable Forest Ecosystem Management of Ministry of Education,School of Forestry, Northeast Forestry University,Harbin 150040,China

Received:2021-02-24 Revised:2022-09-18 Online:2022-07-30 Published:2022-08-01
Contact: WANG Feng

摘要/Abstract

摘要：

【目的】通过研究松材线虫热激转录因子Bx-HSF-1转录激活活性及其靶基因在松材线虫7个虫态(龄)的表达量,揭示Bx-HSF-1在松材线虫生长发育过程中的具体功能。【方法】构建pGBKT7-Bx-hsf-1重组质粒,转化酵母AH109菌株,验证Bx-HSF-1的转录激活活性。应用RNA干扰(RNAi)和转录组技术鉴定Bx-hsf-1基因沉默后松材线虫差异表达基因。对差异表达基因进行基因互作分析,筛选出受Bx-HSF-1调控的靶基因。应用RT-qPCR鉴定5条靶基因在J₂、DJ₃、DJ₄、J₃、J₄、雌成虫和雄成虫共7种虫态(龄)中的表达量。【结果】转入pGBKT7-Bx-hsf-1重组质粒的酵母AH109菌株能够在SD/-Trp和SD/-Trp/-His/-Ade培养基上正常生长,并且在SD/-Trp/-His/-Ade +X-α-gal培养基中显蓝色,表明Bx-HSF-1能够激活下游报告基因表达,具有转录激活活性。通过荧光显微观察及RT-qPCR验证,RNAi处理12 h后靶向siRNA可顺利导入松材线虫细胞,且有效抑制Bx-hsf-1的表达。RNAi处理后转录组数据分析共鉴定到110条差异表达基因,其中86条下调表达。基因互作分析筛选到5条靶基因(Bx-daf-21、Bx-hsp-1、Bx-hsp-70、Bx-sti和Bx-cdc),通过RT-qPCR对这5条靶基因在7种虫态(龄)中的表达量进行分析。在7种虫态(龄)中,Bx-daf-21与Bx-hsf-1的表达量变化趋势一致,Bx-hsp-70与Bx-hsf-1表达量变化趋势相反,Bx-sti和Bx-hsp-1与Bx-hsf-1表达量变化趋势无相关性。在DJ₃中,Bx-cdc与Bx-hsf-1表达量变化趋势相反。【结论】松材线虫Bx-HSF-1具有转录激活功能,通过转录调控参与松材线虫各虫态(龄)发育过程。

关键词: 松材线虫, 热激转录因子, 转录激活活性, 虫龄

Abstract:

【Objective】 The transcriptional activity of Bx-HSF-1 and the expression of its target genes at the seven life stages of Bursaphelenchus xylophilus were studied to reveal its role in the growth and development of B. xylophilus. 【Method】 The CDS (coding sequence) of Bx-hsf-1 gene was constructed into plasmid pGBKT7 to obtain the yeast (Saccharomyces cerevisiae) bait expression vector pGBKT7-Bx-hsf-1. The self-activation of pGBKT7-Bx-hsf-1 was detected. RNA interference and transcriptome sequencing techniques were used to identify the differentially expressed genes of B. xylophilus after Bx-hsf-1 gene was knocked down. The gene interaction analysis of differentially expressed genes was analyzed, and the target genes that were directly regulated by Bx-HSF-1 were screened out. RT-qPCR (quantitative real-time PCR) was used to identify the expression levels of five genes in J₂, DJ₃, DJ₄, J₃, J₄, female, and male stages of B. xylophilus. 【Result】 The pGBKT7-Bx-hsf-1 yeast turned blue on SD/-Trp/-His/-Ade+X-α-gal medium and could grow on SD/-Trp and SD/-Trp/-His/-Ade mediums. A yeast transactivation activity assay suggested that Bx-HSF-1 had transcriptional activity. RT-qPCR results revealed the significant suppression of Bx-hsf-1 after RNAi treatment for 12 h. The total 110 differentially expressed genes were identified using a transcriptome analysis, of which 86 were down regulated. Total five target genes (Bx-daf-21, Bx-hsp-1, Bx-hsp-70, Bx-sti and Bx-cdc) were filtered using a gene interaction analysis. The expression of Bx-daf-21 was consistent with the expression of Bx-hsf-1 in the development of seven life stages of B. xylophilus, whereas the expression of Bx-cdc in DJ₃ and the expression of Bx-hsp-70 were inconsistent with the expression of Bx-hsf-1. The expression of Bx-hsp-1 and Bx-sti was not correlated with the expression of Bx-hsf-1 in the seven life stages. 【Conclusion】 Bx-HSF-1 had a transcriptional activation function and participated in the development process of B. xylophilus through transcriptional regulations.

Key words: Bursaphelenchus xylophilus, heat shock transcription factor, transcriptional activation, life stage

中图分类号:

S763.11

张瑞芝,姜生伟,吴昊,等. 松材线虫Bx-HSF-1转录激活活性研究[J]. 南京林业大学学报（自然科学版）, 2022, 46(4): 8-14.

ZHANG Ruizhi, JIANG Shengwei, WU Hao, CHEN Qiaoli, LI Danlei, WANG Feng. Transcriptional activity of the Bx-HSF-1 in Bursaphelenchus xylophilus (Aphelenchida: Aphelenchoididae)[J].Journal of Nanjing Forestry University (Natural Science Edition), 2022, 46(4): 8-14.DOI: 10.12302/j.issn.1000-2006.202102019.

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松材线虫Bx-HSF-1转录激活活性研究

Transcriptional activity of the Bx-HSF-1 in Bursaphelenchus xylophilus (Aphelenchida: Aphelenchoididae)

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