Identification of Candidate Constitutive Expressed Resistant Genes of Pine Wilt Disease in <i>Pinus massoniana</i> Based on High-throughput Transcriptome Sequencing

LIU bin; LIU Qing-hua; ZHOU Zhi-chun; XU Liu-yi; CHEN Xue-lian; LUO Ning

doi:10.13275/j.cnki.lykxyj.2019.05.001

Volume 32 Issue 5

Oct. 2019

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Identification of Candidate Constitutive Expressed Resistant Genes of Pine Wilt Disease in Pinus massoniana Based on High-throughput Transcriptome Sequencing

1.
Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Key Laboratory of Tree Breeding of Zhejiang Province, Hangzhou 311400, Zhejiang, China
2.
Anhui Academy of Forestry, Hefei 230031, Anhui, China
3.
Linhai Natural Resources and Planning Bureau, Linhai 317000, Zhejiang, China

Corresponding author: LIU Qing-hua, 876009290@qq.com ;
Received Date: 2018-11-26
Accepted Date: 2019-03-11

Abstract

Objective To study the genes related to the resistance of Masson pine ( Pinus massoniana ) to pine wood nematode ( Bursaphelenchus xylophilus , PWN). Method Based on two genotypes of resistant and susceptible P. massoniana for inoculating pine wood nematode, and sampling at the 1 dpi, 15 dpi and 30 dpi after inoculation, the authors performed a transcriptome analysis to identify differentially expressed constitutive genes associated with resistance to PWN infected. Result Comparing resistant and susceptible transcriptomes of P. massoniana inoculated with B. xylophilus , 2 866, 679 and 1 657 differentially expressed genes were obtained at 1, 15, or 30 days post-inoculation (dpi), and there were relatively few common differentially expressed genes at different time points. The GO enrichment analysis was performed on the differentially expressed genes, which indicated that the most significant biological process at the 1 dpi, 15 dpi and 30 dpi was oxidation-reduction process, while the stimulus response, transcriptional regulation and the geranyl diphosphate metabolic process in GO items were also significantly enriched. A further analysis of these related genes in the stimulus response GO term, 26 R genes were found at three time points after inoculation. The expression of the other R genes was constitutive except for two R genes, and no significant difference was observed between PWN and water inoculation. In the transcriptional regulation GO term, only two differentially expressed genes of ERF transcription factors were annotated by nr database, and the rest differentially expressed genes were unknown. After inoculation with PWN, the expression level of ERF transcription factors in resistant P. massoniana was always higher than that in susceptible ones, and there was insignificantly difference between trees inoculating PWN and the control. The result means that expression of the two ERF transcription factors is constitutive. In geranyl diphosphate metabolic process GO term, three GGPPS genes having higher expression level in high resistant P. massoniana , are constitutive expression genes. Here, the authors also found that the TIR-NBS-LRR gene (c65785.graph_c0), ERF transcription factor (c78073.graph_c0), and the three GGPPS genes, having higher expression in resistant P. massoniana , the expression levels were extremely low or even zero in susceptible P. massoniana , which suggested that these genes can be chosen as candidate genes for the development of molecular markers to identify resistant P. massoniana . Conclusion Most genes changed the expression level at 1 dpi between resistant and susceptible P. massoniana . The R genes, ERF transcription factors and GGPPS are related to the resistance of P. massoniana , some of which are expected to be developed as molecular markers for identifying PWN-resistance P. massoniana .
- Pinus massoniana,
- pine wilt disease,
- resistance,
- transcriptome sequencing,
- constitutive expressed genes

References

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马尾松( Pinus massoniana Lamb.)速生、耐干旱瘠薄、适应性强，是我国南方荒山造林的先锋树种，在一些立地条件较差的山地，马尾松甚至是唯一可造林的本土树种。由松材线虫( Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle，PWN)引起的松树萎蔫病是一种毁灭性森林病害，其中，马尾松为松材线虫主要危害对象之一。马尾松群体中存在抗病基因型，利用抗病品种防治松材线虫病为最经济、有效的途径之一，现已在安徽、浙江等地选育出一批抗性较高的马尾松家系和无性系^[1]，但对其抗性机理并不完全清楚。

植物抗性基因(R基因)是其对病虫害防御反应的主要基因, 其中, 富含亮氨酸重复序列(LRRs)是植物R基因编码蛋白最重要的结构特征之一， NBS-LRR 、 LRR-STK 类是R基因中的两类^[2]。在植物与线虫互作过程中， NBS-LRR 类是植物抗病基因中数量最多的一类^[3]。依据 NBS 在N端的不同结构，又主要分为 TIR-NBS-LRR 和 CC-NBS-LRR 两种类型。 Hs1^pro-1是从甜菜( Beta vulgaris L.)中获得的第1个R基因^[4]。之后，在马铃薯( Solanum tuberosum L.)和樱桃( Cerasus pseudocerasus Lindl.)中发现的R基因编码蛋白皆具有核苷酸结合位点和富亮氨酸重复序列(NBS-LRR)结构，其中, Gpa2、Mi-1.2、Mi9和Hero A编码蛋白属于N端为卷曲螺旋结构域(CC-NBS-LRR)结构类别的抗性蛋白，而Ma和Gro1-4编码蛋白属于N端为Toll/白细胞介素受体(TIR-NBS-LRR)结构类别的抗性蛋白。 CC-NBS-LRR 与 TIR-NBS-LRR 这两类抗性基因在应对病原菌侵染时发挥着不同的功能^[5]。基因不受时期、部位以及环境影响均可较稳定表达即为组成型基因。在这些植物抗性基因中，基因组成型表达对抗性起重要作用。如 CC-NBS-LRR 亚家族中组成型表达基因RGC260在向日葵( Helianthus annuus L.)抗锈病过程中发挥重要作用^[6]。转录因子 CBF1 的组成型表达可增强冷敏感植物的抗寒性^[7]。感染柑桔黄龙病菌( Candidatus Liberibacter spp.)后，组成型表达基因 PtCDR2 和 PtCDR8 在抗黄龙病柑桔( Poncirus trifoliate L.)中表达丰度较高^[8]。

目前, 在松树对松材线虫病的抗性机制研究上进展相对较慢，仅发现一些抗性相关物质或基因。如Menéndez-Gutiérrez等对不同抗性的几种松树进行研究，发现组成型氮、多酚和磷在对松材线虫病抗性较强的火炬松( P. taeda L.)和地中海松( P. halepensis L.)木质部中的含量高于易感病的赤松( P. sylvestris L.)^[9]，松类内酚类、单宁、黄酮和木质素含量高不利于松材线虫的繁殖生长^[10]。在分子水平上发现与活性氧、木质素、萜类和乙烯等合成相关的基因与抗松材线虫病关系密切^[11-14]。本研究组已利用转录组高通量测序技术发现编码扩展蛋白、萜类合酶和活性氧等基因在接种松材线虫前后表达量发生显著的变化^[15]，且在高抗与易感马尾松间表达量差异也达到显著水平，但这种表达量的差异为诱导型，在不接种情况下，无法利用这些基因对高抗马尾松进行早期鉴定。本试验在前期研究基础上，着重于研究和识别高抗和易感马尾松间与抗松材线虫病相关的组成型表达基因，为解析马尾松抗性机理和高抗马尾松早期选择提供理论依据。

1. 材料与方法

1.1. 材料来源

试验材料来自安徽省林业科学研究院4年生马尾松无性系试验林。根据前期该院对试验林内无性系测定的抗性指数，于2017年7月中旬，选择高抗无性系‘休宁5’和易感无性系‘黄山1’各18株。供试松材线虫为高致病性株系‘广株3B’和‘全4’混合株系，于灰葡萄孢菌( Botrytis cinerea )菌落上纯培养，保存于4℃冰箱中备用。

1.2. 实验方法

1.2.1. 松材线虫悬浊液制备

将灰葡萄孢菌接种于PDA平板上，于28℃培养箱中培养5 d。待菌落长成后将松材线虫接种于该菌落中，28℃培养直至菌落完全被松材线虫取食，进一步用贝尔曼漏斗法分离松材线虫，12 h后收集于10 mL离心管中，3 000 r·min^-1离心5 min, 去除上清液。将各离心管中的松材线虫汇集并混匀，最终制备成每微升含有50条松材线虫的悬浊液。

1.2.2. 松材线虫接种马尾松

在高抗无性系与易感无性系中，分别选取9株接种松材线虫悬浊液，9株接种蒸馏水作为对照。参考Liu等处理方法^[16]进行接种与样品采集。所取样品包括高抗马尾松和易感马尾松2个基因型，接种松材线虫和接种蒸馏水2个处理，接种后第1、15和30天3个取样时间点，3个生物学重复，共计36个样品。

1.3. 马尾松RNA提取和cDNA合成

采用植物RNA试剂盒RN38 EASYSpin plus(Aidlab Biotech，Beijing，China)分别提取接种后1、15、30 d各样本RNA。利用Ultraspec TM 2100 Pro UV检测RNA的浓度，1%琼脂糖凝胶电泳检测RNA纯度以及完整性。选择RNA浓度≥400 ng·μL^-1、 OD_{260 / 280}为1.8～2.2、 OD_{260 / 230}＞2.0、RIN≥8.0作为高质量RNA。

1.4. 马尾松转录组组装和测序

马尾松无参考基因组，为获得全面的转录组数据，首先将36份高质量RNA等质量混合，利用Illumina HiSeqTM 2000测序平台进行转录组测序。同时对36个样品进行表达谱测序，并使用Bowtie方法将36份样品中高质量读长(reads)比对到转录组上，获取各样本中所有基因的表达量。

1.5. 高抗与易感马尾松差异基因识别及GO富集分析

转录组和表达谱数据已提交至NCBI，登录号为SRP103562。利用Benjamini-Hochberg方法进行 P 值校正，获得差异基因。采用GOseq R对差异基因进行GO富集分析， P 值≤0.05的GO term为显著富集。

1.6. 实时定量PCR(qRT-PCR)分析

使用Primer3.0软件对候选基因设计引物，采用ABI7300实时荧光定量PCR系统(Applied Biosystems，CA，USA)对候选基因表达情况进行qRT-PCR检测，以验证转录组测序结果。反应总体系为20 μL，包括0.4 μL ROX染料，2 μL cDNA，0.4 μL引物和10 μL SYBR Premix ExTaq^TM。PCR程序为95℃预变性10 s，95℃变性5 s，58℃退火30 s，72℃延伸30 s，共40个循环。3次生物学重复，3次技术重复。利用 EF1-α 作为参考基因对候选基因表达量进行标准化，并根据2^-ΔΔCT方法计算候选基因的相对表达水平。

3. 讨论

本研究发现，编码NBS-LRR蛋白及TIR-NBS-LRR蛋白的组成型基因表达量在高抗马尾松与易感马尾松间差异显著。Yamaguchi等^[17]分离出一个 NBS-LRR 抗性功能基因 TYNBS1，在西红柿( Lycopersicon esculentum Mill.)防御黄叶卷叶病中至关重要。Grand等^[18]研究表明，组成型 HSF 23和 CaMBP 基因在水稻( Oryza sativa L.)抗稻瘟病中起正向调节作用。Wen等^[19]在拟南芥( Arabidopsis thaliana (L.) Heynh.)和烟草( Nicotiana tabacum L.)中构建野生华东葡萄( Vitis pseudoreticulata W. T. Wang) VIR-NBS-ARC-LRR 基因的组成型异源过表达增强了对植物病原性真菌和细菌的抗性。王猛^[20]利用抑制消减杂交技术分析了松材线虫诱导后马尾松针叶中基因的表达情况，也提出NBS-LRR蛋白等组成型基因在抗松材线虫病过程中发挥了作用。这些结果表明， NBS-LRR 组成型基因参与了马尾松对松材线虫病的防御过程；但本研究发现，接种后1、15和30 d，大部分 NBS-LRR 及 TIR-NBS-LRR 组成型基因在易感马尾松上表达量更高。Sagi等^[21]在研究鹰嘴豆( Cicer arietinum Linn.)抵御叶枯病过程中也发现，在早期大多数 NBS-LRR 基因在易感鹰嘴豆中的表达量高于高抗鹰嘴豆。关于LRR-RLKs的作用机制，目前普遍认为植物利用LRR-RLKs蛋白激活MAPK信号途径，从而启动下游的防御反应，如诱发PR、GST等蛋白表达^[22]。本研究发现，LRR-RLKs蛋白激酶基因与 NBS-LRR 组成型基因表达量的调节方向一致，皆表现出在易感马尾松上表达量更高。 NBS-LRR 及 TIR-NBS-LRR 组成型基因在易感马尾松上高表达反而使马尾松感病，具体原因有待进一步研究。

乙烯响应因子( ERF )是一个很大的转录因子家族，可参与植物对生物或非生物的胁迫响应。如 ERF 109的过量表达可增强植物对氧化胁迫的抗性，也可减缓细胞程序性死亡的速度和提高植物对盐的耐受力^[23]。Berrocal-Lobo等^[24]发现，拟南芥中 ERF1的组成型表达即可增强其对尖孢镰刀菌( Fusarium oxysporum Schl.)的抗性，且接种尖孢镰刀菌后 ERF1表达量将上调。Shin等报道，接种线虫后，日本红松( Pinus koraiensis L.)中001_06-B09( AP2/ ERF 转录因子)组成型表达量上调^[25]。本研究发现， ERF 在高抗马尾松上的表达量高于易感马尾松上的表达量， ERF 转录因子可能在马尾松抵御松材线虫病中起重要调节作用。

已有研究认为，针叶树种中二萜是主要的防御化合物，松脂对病虫害的防御主要由松节油的化学毒害和二萜的物理阻碍形成，即二萜通过氧化聚合封闭伤口，以防御虫和病菌继续侵入^[26-29]，其中，二萜由甲基赤藓醇磷酸途径(MEP pathway)合成，GGPPS为异戊基转移酶^[30]，而 GGPPS 表达量与松脂产量相关， GGPPS 表达量高的马尾松松脂产量也较高^[15]。松脂产量则与针叶树种对病虫害的抗性密切相关^[31]， GGPPS 可能参与了对病虫害胁迫的抗性反应。本研究也发现， GGPPS 在高抗马尾松上的表达量始终远高于易感马尾松上的表达量，由此推测， GGPPS 可能在马尾松抵御松材线虫病的进程中起正调节作用。本研究组后续将对 LRR 基因、乙烯响应因子( ERF )和 GGPPS 等基因进行深入研究。

4. 结论

本研究通过表达谱分析揭示，接种松材线虫后第1天高抗与易感马尾松间差异基因最多，这些差异基因主要富集在生物学进程中的氧化-还原过程、跨膜转运、刺激反应、转录调节以及香叶基二磷酸代谢过程等GO项上，其中， LRR 基因、 ERF 转录因子和 GGPPS 可能与马尾松的抗性相关， TIR-NBS-LRR 基因(c65785.graph_c0)、乙烯响应转录因子(c78073.graph_c0)、 GGPPS 基因(c33360.graph_c0、c33943.graph_c0和c58549.graph_c0)为组成型表达基因，具有被用于开发鉴定高抗马尾松分子标记的潜力。

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基因本体编号 GO ID	类别 Term	差异基因 GO 富集 Different genes GO enrichment
基因本体编号 GO ID	类别 Term	接种后1 d 1d After inoculation	接种后15 d 15 d After inoculation	接种后30 d 30 d After inoculation
GO:0055114	氧化-还原过程 Oxidation-reduction process	2.80E-11	4.80E-11	3.50E-10
GO:0055085	跨膜转运 Transmembrane transport	2.30E-05	4.80E-05	1.10E-05
GO:0008152	代谢过程 Metabolic process	8.90E-05	－	－
GO:0042026	蛋白质重折叠 Protein refolding	9.70E-05	－	－
GO:0006886	胞内蛋白转运 Intracellular protein transport	0.000 32	0.000 35	－
GO:0009725	激素应答 Response to hormone	0.000 46	0.000 49	－
GO:0050896	刺激反应 Response to stimulus	0.000 50	0.000 30	0.000 49
GO:0006950	胁迫应答 Response to stress	0.000 97	0.001 11	0.000 93
GO:0006355	转录调节 Regulation of transcription, DNA-templated	0.001 25	0.001 85	0.001 25
GO:0033383	香叶基二磷酸代谢过程 Geranyl diphosphate metabolic process	0.001 57	0.001 46	0.000 56
GO:0006468	蛋白质磷酸化 Protein phosphorylation	－	0.000 18	0.000 17
GO:0006749	谷胱甘肽代谢过程 Glutathione metabolic process	－	0.000 42	－
GO:0006097	乙醛酸循环 Glyoxylate cycle	－		－
GO:0030435	孢子形成 Sporulation resulting in formation of a cellular spore	－		－
GO:0006886	胞内蛋白转运 Intracellular protein transport	－	－	0.000 31
GO:0009266	温度刺激应答 Response to temperature stimulus	－	－	0.002 32
GO:0006464	细胞蛋白质修饰过程 Cellular protein modification process	－	－	0.002 79

基因本体编号 GO ID	类别 Term	差异基因 GO 富集 Different genes GO enrichment
基因本体编号 GO ID	类别 Term	接种后1 d 1d After inoculation	接种后15 d 15 d After inoculation	接种后30 d 30 d After inoculation
GO:0016021	膜整体构成 Integral component of membrane	3.60E-24	1.20E-21	3.50E-17
GO:0005886	质膜 Plasma membrane	5.90E-11	2.90E-11	3.60E-11
GO:0009507	叶绿体 Chloroplast	1.10E-09	3.60E-10	6.00E-10
GO:0005829	细胞质基质 Cytosol	3.30E-07	3.10E-07	2.90E-07
GO:0009570	叶绿体基质 Chloroplast stroma	2.20E-05	1.70E-05	1.80E-05
GO:0044464	细胞组分 Cell part	4.80E-05	0.000 12	9.90E-05
GO:0005773	液泡 Vacuole	6.40E-05	5.40E-05	5.80E-05
GO:0005768	核内体 Endosome	9.60E-05	6.10E-05	7.30E-05
GO:0009941	叶绿体被膜 Chloroplast envelope	0.000 16	3.10E-05	0.000 14
GO:0005840	核糖体 Ribosome	0.000 21	1.50E-06	2.00E-05

基因本体编号 GO ID	类别 Term	差异基因 GO 富集 Different genes GO enrichment
基因本体编号 GO ID	类别 Term	接种后1 d 1d After inoculation	接种后15 d 15 d After inoculation	接种后30 d 30 d After inoculation
GO:0016491	氧化还原酶活性 Oxidoreductase activity	2.10E-10	8.80E-09	5.50E-08
GO:0005515	蛋白结合 Protein binding	1.90E-07	5.00E-08	2.00E-07
GO:0000981	DNA特异序列结合 Sequence-specific DNA binding	1.80E-05	4.10E-06	1.40E-05
GO:0003824	催化活性 Catalytic activity	4.60E-05	0.000 14	0.000 19
GO:0005488	结合 Binding	5.00E-05	－	－
GO:0004721	磷蛋白质磷酸蛋白活性 Phosphoprotein phosphatase activity	0.000 14	－	7.60E-05
GO:0010181	FMN结合 FMN binding	0.000 26	5.60E-05	0.000 17
GO:0008874	葡萄糖酸5-脱氢酶活性 Gluconate 5-dehydrogenase activity	0.000 49	－	－
GO:0016832	醛裂解酶活性 Aldehyde-lyase activity	0.000 53	－	－
GO:0004591	酮戊二酸脱氢酶 Oxoglutarate dehydrogenase	0.000 62	0.000 18	0.000 3
GO:0004738	丙酮酸脱氢酶活性 Pyruvate dehydrogenase activity	－	0.000 14	0.000 29
GO:0004375	甘胺酸脱氢酶 Glycine dehydrogenase	－	0.000 23	0.000 37
GO:0051287	NAD结合 NAD binding	－	0.000 39
GO:0016758	转移酶活性 Transferase activity	－	0.000 55	0.000 58

类别 Term	基因编号 Gene_ID	基因表达量 Relative level of gene expression/RPKM
类别 Term	基因编号 Gene_ID	RI₁	SI₁	RI₁₅	SI₁₅	RI₃₀	SI₃₀
受体丝氨酸/苏氨酸蛋白激酶 LRR receptor-like serine/ threonine-protein kinase	c63612.graph_c0	11.14	0.78	10.80	0.67	15.65	0.38
	c75632.graph_c0	25.21	75.21	-	-	8.99	43.55
	c74934.graph_c1	9.49	31.04	-	-	11.00	43.81
	c78638.graph_c0	1.18	11.57	-	-	0.30	9.71
	c72330.graph_c0	2.55	10.92	-	-	1.34	5.37
	c52089.graph_c0	0.61	5.38	-	-	-	-
	c77321.graph_c0	15.01	34.64	-	-	-	-
	c77212.graph_c0	5.26	13.39	-	-	-	-
	c76446.graph_c0	7.21	22.56	-	-	-	-
	c64844.graph_c0	1.10	5.51	-	-	-	-
	c71845.graph_c0	5.21	40.05	-	-	-	-
	c72922.graph_c0	-	-	-	-	17.85	60.44
NBS-LRR蛋白 NBS-LRR protein	c79162.graph_c0	0.00	5.56	0.65	5.82	-	-
	c49318.graph_c0	0.40	5.25	0.90	4.83	0.19	5.22
	c66133.graph_c0	10.50	30.33	-	-	12.53	30.92
	c73896.graph_c0	2.48	8.31	-	-	3.32	9.25
	c76976.graph_c0	11.13	40.39	-	-	9.57	29.29
	c76662.graph_c0	2.63	7.31	-	-	-	-
	c62473.graph_c0	1.72	7.55	-	-	-	-
TIR-NBS-LRR蛋白 TIR-NBS-LRR protein	c65785.graph_c0	2.68	0.00	-	-	2.71	0.00
	c73334.graph_c0	0.60	51.29	4.19	16.98	0.60	22.60
	c53610.graph_c0	2.44	7.51	-	-	2.20	7.67
	c76602.graph_c0	1.95	4.46	-	-	1.93	3.21
	c65671.graph_c0	0.00	3.34	-	-	0.00	4.84
	c75804.graph_c0	2.88	8.01	-	-	-	-
	c66096.graph_c0	4.10	23.63	-	-	-	-
注：RI₁，高抗马尾松接种松材线虫后第1天的基因表达；S₁，易感马尾松接种松材线虫后第1天的基因表达；RI₁₅，高抗马尾松接种松材线虫后第15天的基因表达；SI₁₅，易感马尾松接种松材线虫后第15天的基因表达；RI₃₀，高抗马尾松接种松材线虫后第30天的基因表达；SI₃₀，易感马尾松接种松材线虫后第30天的基因表达。 Notes: RI₁, Genes expression of resistant P. massoniana at 1day after PWN inoculation; SI₁, Genes expression of susceptible P. massoniana at 1day after PWN inoculation; RI₁₅, Genes expression of resistant P. massoniana at 15day after PWN inoculation; SI₁₅, Genes expression of susceptible P. massoniana at 15 days after PWN inoculation; RI₃₀, Genes expression of resistant P. massoniana at 30 day after PWN inoculation; SI₃₀, Genes expression of susceptible P. massoniana at 30 days after PWN inoculation.

Identification of Candidate Constitutive Expressed Resistant Genes of Pine Wilt Disease in Pinus massoniana Based on High-throughput Transcriptome Sequencing