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松材线虫病,即松树枯萎病(pine wilt disease, PWD),是一种以松材线虫(Bursaphelenchus xylophilus)为病原的国际检疫性林业病害[1-2]。松材线虫病具有发病部位隐秘、发病速度快、传播蔓延迅速、传播途径多、治理难度大等特点,因此该病害又被称为松树的“癌症”[3]。我国于1982年在南京中山陵首次发现松材线虫病[4],随后在我国逐渐蔓延,已经造成了重大的经济损失并严重威胁着我国众多名胜风景区和松林生态系统安全。细胞色素P450(cytochrome p450, P450),又称单加氧酶,是一类由数量众多、功能复杂的血红素结合蛋白组成的同工酶,广泛存在于细菌、真菌、动物和植物等生物体中[5]。细胞色素P450在内源性化合物(脂肪酸、胆汁酸、类固醇、蜕皮激素和保幼激素等)和外源性化合物(农药、植物毒素和环境致癌物等)代谢过程中十分重要[6-8]。大量研究表明细胞色素P450基因参与代谢过程和抗性机制主要通过上调来实现。Maja等[9]研究发现秀丽隐杆线虫受到毒物质侵害时,cyp-33D3基因表达量上调明显。松材线虫全基因组序列于2011年测序完成,为研究松材线虫致病相关基因的功能奠定了良好的基础[10]。我们前期研究发现松材线虫感染松树后,cytochrome P450 33D3 (cyp-33D3) 基因表达量上调显著,表明cyp-33D3基因在松材线虫致病过程中发挥了重要作用[11]。目前关于cyp-33D3基因的研究主要集中在过量表达方面,而关于该基因的功能研究鲜见报道。
RNA干扰(RNA interference, RNAi)现象广泛存在于真核生物细胞中,它通过小分子双链RNA诱导同源mRNA降解从而阻断体内特定基因表达[12]。随着RNA干扰机制被阐明,RNA干扰被认为是一种可以与基因敲除相媲美的技术,在基因功能、信号传导通路和基因治疗等研究领域得到了广泛的应用[13-16]。本研究采用RNA干扰技术研究cyp-33D3基因沉默后对松材线虫取食、繁殖和致病性的影响,阐明cyp-33D3基因在松材线虫致病过程中的功能,为进一步明确松材线虫分子致病机制奠定理论基础。
松材线虫cyp-33D3基因的RNA干扰及其功能研究
Study on the Function of cyp-33D3 Gene of Bursaphelenchus xylophilus with RNA Interference Method
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摘要:
目的 研究松材线虫cytochrome P450 33D3 (cyp-33D3) 基因功能,为揭示松材线虫分子致病机制及其生物防治提供理论依据。 方法 采用双链RNA对cyp-33D3基因的表达进行干扰,测定该基因沉默后对松材线虫取食速率、个体大小、产卵数量、孵化率及致病力的影响。 结果 ddH2O和gfp dsRNA处理的线虫取食速度明显较快,在第5天已经几乎将灰葡萄孢菌丝取食殆尽,而cyp-33D3 dsRNA处理松材线虫取食灰葡萄孢的面积较小;cyp-33D3基因RNA干扰对松材线虫雌、雄成虫的体长无显著影响(P > 0.05);与ddH2O和gfp dsRNA处理相比,cyp-33D3dsRNA处理每条雌虫平均产卵数量分别减低了12和11粒,虫卵孵化率分别减低了46%和43%;在接种40 d后,ddH2O处理的黑松苗发病率达到100%,cyp-33D3 dsRNA处理的黑松发病率为43.1%;而gfp dsRNA处理的黑松生长状况良好。 结论 cyp-33D3基因RNA干扰减缓了松材线虫的取食速度,减少了松材线虫的产卵数量,降低了松材线虫的虫卵孵化率和对黑松的致病力;松材线虫cyp-33D3基因RNA干扰对线虫的个体大小无明显影响。 -
关键词:
- 松材线虫
- / cyp-33D3基因
- / RNA干扰
- / 繁殖能力
- / 致病力
Abstract:Objective To study the function of cytochrome P450 33D3 (cyp-33D3) gene in Bursaphelenchus xylophilus in order to further reveal the molecular pathogenesis mechanism of B. xylophilus, and provide useful information for its biological control. Method Double strand RNA (dsRNA) interference was used to investigate the effect of silencing of cyp-33D3 on feeding speed, individual size, number of eggs, hatching rate and pathogenicity of B. xylophilus. Result It was found that the feeding speed of B. xylophilus under ddH2O and gfp dsRNA treatments were faster than that under cyp-33D3dsRNA interference treatment in the first 5 days. Almost all the hyphae of Botrytis cinerea were consumed by B. xylophilus soaked under ddH2O and gfp dsRNA treatments. In contrast, a large portion of the hyphae still existed under cyp-33D3 dsRNA treatment. No significant effect was found on the individual size of B. xylophilus when cyp-33D3 gene was interfered by dsRNA (P > 0.05). Compared to ddH2O and gfp dsRNA treatments, the average number of eggs per female decreased 12 and 11 under cyp-33D3 treatment, and the hatching rate of eggs decreased by 46% and 43%, respectively. 40 days after the soaked B. xylophilus were inoculated in Pinus thunbergii seedlings, the pathogenicity of B. xylophilus under ddH2O and cyp-33D3 treatments were 100% and 43.1%, respectively, while the P. thunbergii seedlings inoculated with B. xylophilus under gfp dsRNA treatment still grew well. Conclusion The RNA interference of cyp-33D3 gene decreases the feeding speed, the number of eggs, hatching rate and pathogenicity of B. xylophilus. However, RNA interference of cyp-33D3 gene exhibits no significant effect on individual size of B. xylophilus. -
Key words:
- Bursaphelenchus xylophilus
- / cyp-33D3 gene
- / RNA interference
- / reproduction ability
- / pathogenicity
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