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马尾松(Pinus massoniana Lamb.)是我国南方重要荒山造林树种,具有耐贫瘠,速生丰产等优点,被誉为“先锋树种”。松材线虫病被视为松树的“癌症”,其具有危害大、隐蔽性强、致使寄主快速死亡并可人为远距离传播等特点,致使防治该病害的工作存在较大困难,松材线虫病疫点数量和发生面积逐年增大。有鉴于此,明确马尾松抗松材线虫病的基因表达调控过程是当前利用现代生物学技术防控松材线虫病害研究中不可缺少的部分。
非编码微小RNA (microRNA, miRNA)由于具有高效沉默靶向mRNA表达的特性,成为当前生命科学研究中最受关注的对象之一[1-2]。这种长度为20~24 nt的单链非编码小RNA能够以RNA诱导沉默复合体的形式结合到靶基因上,对靶基因进行降解或抑制靶基因的翻译,从而在基因转录后对其进行表达沉默,降低基因的表达丰度。这一新奇的基因表达调控方式自从21世纪初发现以来已受到国内外研究团队的密切关注[3-5]。
近年来的诸多研究均表明,miRNA在调控植物生长发育和响应逆境胁迫的过程中起着十分重要的作用,这包括调控植物抗旱、耐冷、抗病等多种行为[6-11]。在林木研究中,Wan等[12]利用本地BLAST和MIREAP程序从高山松(Pinus densata Mast.)的转录组数据中鉴定得到了34个保守的miRNA,其中,25个miRNA家族共有72个靶基因,且大部分miRNA与mRNA具有靶向作用关系。Quinn等[13]在火炬松(Pinus taeda L.)的序列表达标签中发现12个未被报道的miRNA,这些miRNA作用的靶基因参与表达调控、代谢和信号转导。此外,Lu等[14]在对火炬松miRNA及其与松梭锈病病害发生的相关性研究中,从火炬松茎干的木质部克隆并鉴定了26个miRNA,其中,9个家族miRNA能够有效调控43个靶基因的表达。Xie等[15]测定了松材线虫侵染1、2、3 d及未受侵染的对照马尾松样本中miRNA表达谱,结果显示,侵染不同天数下的马尾松针叶中有10个miRNA均较对照样本发生差异表达,其对应的靶基因在植物激素信号通路等途径富集。与此同时,笔者前期也测定了松材线虫侵染1、2、3 d的马尾松针叶mRNA表达谱。本文在上述研究的基础上,进一步研究松材线虫侵染下,马尾松针叶的mRNA及miRNA表达关联情况,通过比较二者的表达变化模式,明确miRNA对重要mRNA的有效调控作用,为揭示马尾松响应松材线虫侵染胁迫下的转录调控过程提供参考。
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基于前期RNA-seq得到的松材线虫侵染1、2、3 d的马尾松及其对照样本针叶的miRNA表达谱数据,分析不同miRNA的表达变化模式,结果显示:侵染不同天数的马尾松针叶的miRNA表达量变化呈多种模式(profile),其中profile 1和3中的miRNA在侵染不同天数下的马尾松中较对照组表达变化差异显著,其p值均小于0.05,且这些miRNA在侵染第2天的马尾松针叶中的表达较对照样本变化程度最大,说明松材线虫侵染2 d的马尾松针叶中基因表达响应强烈。通过对各组不同表达变化模式的miRNA数量进行统计,结果显示:profile 1和3中分别有10个和14个miRNA (图 1)。
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进一步研究了RNA-seq得到的松材线虫侵染1、2、3 d的马尾松及其对照样本针叶的mRNA表达谱,对mRNA的表达变化模式进行分析,结果显示mRNA表达变化模式具有显著性的有8组,分别是profile 2 (119个基因),profile 4 (205个基因),profile 6 (321个基因),profile 7 (376个基因),profile 11 (374个基因),profile 14 (282个基因),profile 22 (88个基因)和profile 23 (123个基因) (图 2)。
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为研究松材线虫侵染的马尾松针叶中可能由miRNA的降解作用而引起表达变化的mRNA,进一步对表达变化模式相反的miRNA和mRNA进行关联性分析。结果在miRNA表达变化模式中获得15个具有统计学关联性的miRNA(表 1),与这些miRNA表达变化模式相反的靶标mRNA为12个,这些基因包括: (1)与病原识别相关的类CC-NBS-LRR抗性基因(CC-NBS-LRR resistance-like gene)、NBS、TIR/NBS以及ACRE (Avr9/Cf-9rapidly elicited defense-related gene, ACRE); (2)与转录调控相关的类MYB转录因子MBF1 (MYB-like transcriptional factor, MBF1); (3)与防御相关的,位于保守激酶2和P-Loop结构阈区间的抗性基因(resistance gene, region between conserved kinase-2 and P-Loop domains)、棉子糖合成酶家族蛋白质异构体1 (Raffinose synthase family protein isoform 1)基因、类乙烯不敏感蛋白3异构体1(ethylene insensitive 3-like isoform 1)基因; (4)与植物生长发育相关的,类copia反转录转座子(copia-like retrotransposable element)、1, 2-α-L-岩藻糖苷酶(1, 2-alpha-L-fucosidases)、5’-腺苷酰硫酸还原酶3(5’-adenylylsulfate reductase 3)、反转录酶(reverse transcriptase)等的编码基因。在这些表达关联的miRNA与mRNA中,novel-m0040-3p和novel-m0040-5p靶向作用的CC-NBS-LRR抗性基因,miR946、novel-m0136-3p、novel-m0051-3p靶向作用的ACRE基因,二者均是植物识别病原信号的直接基因。
表 1 松材线虫侵染下马尾松针叶关联表达的miRNA和mRNA
Table 1. The correlation between miRNA and mRNA expression pattern on needle leaf of P.massoniana after different days of B.xylophilus infestation comparison with the control group
miRNA 表达量(TPM)
miRNA expression预测勒标mRNA
Predicted target mRNAmRNA表达量
(RPKM) mRNA expressionmiRNACT TR1 TR2 TR3 CT TR1 TR2 TR3 病原识别功能(pathogen recognition) miR946 7 792.11 9 754.86 8 739.33 9 491.99 R gene, a homology of Avr9/Cf-9 rapidly elicited defense-related gene (ACRE) (Pinus sylvestris) 316.78 137.35 159.70 221.38 novel-m0136-3p 449.23 509.23 520.53 539.26 R gene, a homology of Avr9/Cf-9 rapidly elicited defense-related gene (ACRE) (Pinus sylvestris) 316.77 137.35 159.69 221.38 novel-m0051-3p 393.08 509.23 453.00 504.99 R gene, a homology of Avr9/Cf-9 rapidly elicited defense-related gene (ACRE) (Pinus sylvestris) 316.77 137.35 159.69 221.38 novel-m0040-5p 122.84 289.90 194.14 172.64 CC-NBS-LRR resistance-like gene(Pinus lambertiana) 2.27 0 0 0 novel-m0040-3p 66.68 153.60 136.93 78.24 CC-NBS-LRR resistance-like gene(Pinus lambertiana) 2.27 0 0 0 miR472 5.62 0.69 0.94 5.17 NBS (Pinus taeda) 3.67 5.31 8.40 2.54 miR3705 6.32 0.69 5.63 2.59 TIR/NBS (Pinus taeda) 0.53 1.33 0.50 3.68 转录调控功肯旨(transcriptional regulation) miR858 197.94 100.32 60.03 200.44 MYB-like transcriptional factor MBF1 (Picea mariana) 0.94 3.33 1.85 1.14 防御功能(defense) miR156 1 000.25 2 315.04 1 365.58 1 379.83 Raffinose synthase family protein isoform 1 (Theobroma cacao) 8.83 4.15 13.79 3.98 miR5658 4.91 1.38 1.88 0.01 resistance gene;region between conserved kinase-2 and P-Loop domains (Pinus radiate) 0.24 1.55 0.46 2.96 novel-m0012-5p 4.83 0.01 0.01 0.01 ethylene insensitive 3-like isoform 1 (Cucumis sativus) 5.47 6.57 6.20 6.43 植物生长发育相关(plant growth and development) novel-m0172-3p 155.13 69.19 69.40 105.39 copia-like retrotransposable element (Arabidopsis thaliana) 1.04 2.12 1.09 1.78 novel-m0071-5p 80.72 36.67 28.13 65.31 1, 2-alpha-L-fucosidases (Theobroma cacao) 2.11 3.10 2.38 0.95 novel-m0157-3p 25.27 13.15 51.58 33.62 5’adenylylsulfate reductase 3, chloroplastic-like isoform 1 (Vitis vinifera) 5.09 10.05 1.88 3.64 novel-m0019-3p 124.94 56.04 87.22 128.03 reverse transcriptase (Arabidopsis thaliana) 1.42 2.80 1.84 0.25 注:CT:对照; TR1:侵染1天; TR2:侵染2天; TR3:侵染3天。
Note:CT:Control;TR1:1 d after infestation;TR2:2 d after infestation;TR3:3 d after infestation.深入分析表达关联的miRNA与mRNA的基因信息可见,松材线虫侵染后,马尾松针叶中的类CC-NBS-LRR基因、ACRE基因受到其对应miRNA的沉默作用后,其在受侵染的马尾松样本中的表达都较对照样本中的表达量低,而相应的miRNA则在受侵染样本中的表达量均高于对照样本,miRNA与靶基因的表达变化模式相反体现了miRNA对靶标mRNA的负调控作用。此外,位于保守激酶2和P-Loop结构域之间的类似抗性基因与miRNA5658关联,其中,miRNA5658在处理样本中下调表达,而其对应的靶标基因即抗性基因则上调表达。逆转录酶基因在侵染1、2天的样本中上调表达并在第3天较对照样本表达下调,其相应的novel-m0019-3p则在侵染第1、2天的处理样本中下调表达,而在第3天表达上调。与光合作用相关的5’-腺苷酰硫酸还原酶-3的编码基因在处理的第1天表达上调,在随后的第2、3天表达下调,对应的novel-m0157-3p则是先下调表达后上调表达,符合其对靶标mRNA的调控特征。此外,编码棉子糖合成酶家族蛋白质异构体1的基因在侵染第1天的样本中的表达较对照样本下调,侵染第2天的处理样本中的表达则高于对照样本,侵染第3天的表达又低于对照样本,其相应的miRNA156则在处理第1天的样本中表达量显著增加,随后在侵染第2、3天时表达量降低(图 3)。
松材线虫侵染下马尾松针叶miRNA和mRNA的关联表达
Expression Correlation Between miRNA and mRNA From Needle Leaves of Pinus massoniana with Bursaphelenchus xylophilus Infestation
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摘要:
目的 微小RNA(microRNA,miRNA)具有靶向沉默信使RNA(mRNA)表达的功能,是基因表达的负调控因子;研究并明确马尾松在遭受松材线虫侵染下是否存类似的调控模式,对于未来探析寄主植物对病原侵染胁迫下的应激机制及获得调控马尾松抗松材线虫病的miRNA及其靶标mRNA都具有重要意义。 方法 以前期高通量测序获得的松材线虫侵染1、2、3 d的马尾松针叶mRNA和miRNA表达谱为研究对象,采用STEM软件分别分析mRNA和miRNA的表达变化模式,并运用斯皮尔曼等级相关法研究松材线虫侵染下的马尾松针叶中miRNA与mRNA的表达关联情况。 结果 松材线虫侵染下的马尾松针叶中的miRNA呈2种显著的表达变化模式,mRNA呈8种显著的表达变化模式,且15个miRNA与其12个靶标mRNA的表达变化模式相反,符合miRNA对靶标mRNA的负调控特点,这些靶标mRNA编码具有识别病原作用的ACRE、CC-NBS-LRR基因等。 结论 松材线虫侵染下的马尾松针叶中miRNA及mRNA均呈多种表达变化模式,且部分miRNA与靶标mRNA的表达变化模式相反,推测其中部分miRNA可能是病原识别基因的负调控因子。 Abstract:Objective MicroRNA (miRNA) has the function of silence target mRNA expression, which is considered as a negative role in the regulation of gene expression. This study aims to reveal the potential expression correlation between miRNA and mRNA from needle leaves of Pinus massoniana with Bursaphelenchus xylophilus infestation, which helps to further indicate the mechanism of host plant in response to the pathogen infestation, and to obtain the particular miRNA and the target mRNA that involved in the regulation of masson pine resistance to pine wilt disease. Method Expression pools of miRNA and mRNA from P. massoniana with B. xylophilus infestation for 1, 2, and 3 d, which were generated using RNA-seq in our previous studies, were used as materials. The different expression profiles of miRNA or mRNA from P. massoniana with B. xylophilus infestation for different days were figured using STEM (Short Time-series Expression Miner) software, and method of Spearman Rank Correlation was employed to comparatively analyze the miRNA and mRNA expression profiles of P. massoniana. Result Two significant expression profiles were generated from the miRNA pools of P. massoniana with B. xylophilus infestation for 1, 2, and 3 d, whilst 8 significant profiles were generated from that of mRNA pools. Fifteen miRNAs were considered as candidates that obviously represented correlation with twelve mRNAs from the expression pools. The reverse expression profiles between the mRNA and miRNA validate the bio-function of transcriptional silence from miRNA to the target mRNA. The miRNA-targeted mRNA encodes ACRE, CC-NBS-LRR resistance-like gene, etc. These genes play roles in plant pathogen recognition. Conclusion It was clear that the infestation of B. xylophilus resulted in various expression profiles of miRNA and mRNA from the P. massoniana, and numbers of miRNA presented a reverse expression pattern in comparison with target mRNA. Among these miRNAs, several of them were speculated that function in silenced expression of genes involved with pathogen recognition. -
Key words:
- Pinus massoniana
- / Bursaphelenchus xylophilus
- / microRNA
- / mRNA
- / expression correlation
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表 1 松材线虫侵染下马尾松针叶关联表达的miRNA和mRNA
Table 1. The correlation between miRNA and mRNA expression pattern on needle leaf of P.massoniana after different days of B.xylophilus infestation comparison with the control group
miRNA 表达量(TPM)
miRNA expression预测勒标mRNA
Predicted target mRNAmRNA表达量
(RPKM) mRNA expressionmiRNACT TR1 TR2 TR3 CT TR1 TR2 TR3 病原识别功能(pathogen recognition) miR946 7 792.11 9 754.86 8 739.33 9 491.99 R gene, a homology of Avr9/Cf-9 rapidly elicited defense-related gene (ACRE) (Pinus sylvestris) 316.78 137.35 159.70 221.38 novel-m0136-3p 449.23 509.23 520.53 539.26 R gene, a homology of Avr9/Cf-9 rapidly elicited defense-related gene (ACRE) (Pinus sylvestris) 316.77 137.35 159.69 221.38 novel-m0051-3p 393.08 509.23 453.00 504.99 R gene, a homology of Avr9/Cf-9 rapidly elicited defense-related gene (ACRE) (Pinus sylvestris) 316.77 137.35 159.69 221.38 novel-m0040-5p 122.84 289.90 194.14 172.64 CC-NBS-LRR resistance-like gene(Pinus lambertiana) 2.27 0 0 0 novel-m0040-3p 66.68 153.60 136.93 78.24 CC-NBS-LRR resistance-like gene(Pinus lambertiana) 2.27 0 0 0 miR472 5.62 0.69 0.94 5.17 NBS (Pinus taeda) 3.67 5.31 8.40 2.54 miR3705 6.32 0.69 5.63 2.59 TIR/NBS (Pinus taeda) 0.53 1.33 0.50 3.68 转录调控功肯旨(transcriptional regulation) miR858 197.94 100.32 60.03 200.44 MYB-like transcriptional factor MBF1 (Picea mariana) 0.94 3.33 1.85 1.14 防御功能(defense) miR156 1 000.25 2 315.04 1 365.58 1 379.83 Raffinose synthase family protein isoform 1 (Theobroma cacao) 8.83 4.15 13.79 3.98 miR5658 4.91 1.38 1.88 0.01 resistance gene;region between conserved kinase-2 and P-Loop domains (Pinus radiate) 0.24 1.55 0.46 2.96 novel-m0012-5p 4.83 0.01 0.01 0.01 ethylene insensitive 3-like isoform 1 (Cucumis sativus) 5.47 6.57 6.20 6.43 植物生长发育相关(plant growth and development) novel-m0172-3p 155.13 69.19 69.40 105.39 copia-like retrotransposable element (Arabidopsis thaliana) 1.04 2.12 1.09 1.78 novel-m0071-5p 80.72 36.67 28.13 65.31 1, 2-alpha-L-fucosidases (Theobroma cacao) 2.11 3.10 2.38 0.95 novel-m0157-3p 25.27 13.15 51.58 33.62 5’adenylylsulfate reductase 3, chloroplastic-like isoform 1 (Vitis vinifera) 5.09 10.05 1.88 3.64 novel-m0019-3p 124.94 56.04 87.22 128.03 reverse transcriptase (Arabidopsis thaliana) 1.42 2.80 1.84 0.25 注:CT:对照; TR1:侵染1天; TR2:侵染2天; TR3:侵染3天。
Note:CT:Control;TR1:1 d after infestation;TR2:2 d after infestation;TR3:3 d after infestation. -
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