中国农业科学 ›› 2019, Vol. 52 ›› Issue (21): 3794-3805.doi: 10.3864/j.issn.0578-1752.2019.21.008
收稿日期:
2019-05-22
接受日期:
2019-06-28
出版日期:
2019-11-01
发布日期:
2019-11-12
通讯作者:
杨秀芬
作者简介:
梁颖博,E-mail: 基金资助:
LIANG YingBo,LI Ze,QIU DeWen,ZENG HongMei,LI GuangYue,YANG XiuFen()
Received:
2019-05-22
Accepted:
2019-06-28
Online:
2019-11-01
Published:
2019-11-12
Contact:
XiuFen YANG
摘要:
目的 通过RNA-Seq筛选本生烟(Nicotiana benthamiana)响应大丽轮枝菌(Verticillium dahliae)蛋白激发子PevD1的差异表达基因(differentially expressed gene,DEG),分析PevD1诱导植物产生抗病性的潜在分子机制。方法 用10 μmol·L -1的PevD1蛋白液渗入4周龄的本生烟叶片,分别在处理后6、12和24 h取样提取RNA,构建mRNA文库后采用BGISEQ-500平台进行测序。筛选各时间点的差异表达基因进行GO和KEGG分析;重点分析与诱导抗病相关的富含亮氨酸重复序列类受体蛋白激酶(leucine-rich repeats RLKs,LRR-RLKs)、转录因子(transcription factor,TF)以及病程相关蛋白(pathogenesis related protein,PR蛋白)家族差异表达基因;采用qRT-PCR对差异表达基因进行定量验证。 结果 GO功能富集以及KEGG通路富集分析表明,PevD1诱导6 h后的差异表达基因主要与细胞识别、光合作用、光收割等功能相关,显著富集在光合作用-天线蛋白通路、萜类化合物合成通路、黄酮和黄酮醇等次生代谢产物合成相关通路中;12 h和24 h的差异表达基因主要与细胞识别和胞内激酶等生物学功能相关,显著富集在植物-病原互作通路、倍半萜和三萜生物合成通路、黄酮和黄酮醇生物合成通路、亚麻酸代谢等次生代谢产物合成相关通路中。与光合作用相关的差异表达基因主要呈下调趋势,与萜类、黄酮类等抗病相关次生代谢产物合成通路相关的差异表达基因主要呈上调趋势。PevD1诱导后大量的LRR-RLKs、TF以及PR蛋白家族基因显著上调表达,这些基因与激发子识别、基因转录调控和抗病性相关。经qRT-PCR验证后,所检测基因的表达趋势与转录组测序结果一致。结论 PevD1诱导本生烟中大量基因转录重排,大量LRR-RLKs、TF和PR蛋白家族基因上调表达,激活了植物免疫系统,使植物产生抗病性。研究结果可为今后深入探讨PevD1诱导植物免疫的机理提供依据。
梁颖博,李泽,邱德文,曾洪梅,李广悦,杨秀芬. 本生烟响应蛋白激发子PevD1的差异表达基因鉴定与分析[J]. 中国农业科学, 2019, 52(21): 3794-3805.
LIANG YingBo,LI Ze,QIU DeWen,ZENG HongMei,LI GuangYue,YANG XiuFen. Identification and Analysis of Differentially Expressed Genes Induced by Protein Elicitor PevD1 in Nicotiana benthamiana[J]. Scientia Agricultura Sinica, 2019, 52(21): 3794-3805.
表1
待测差异表达基因的定量验证引物"
基因ID Gene ID | 基因名称Gene name | 正向引物Forward primer | 反向引物Reverse primer |
---|---|---|---|
Niben101Scf00245g03004 | LHCA4 | CTCCTACTTATCTTGATGGAA | CACTGGTGAGAACCTCAG |
Niben101Scf01094g03014 | PTI5 | TTCCCAGAAATGTCCTTC | GCTTCTTCTGCTGTTTCA |
Niben101Scf01297g04006 | WRKY7 | GGTCTCCTATCAGTAAGTC | GCTTGTGGTTTGAACTAC |
Niben101Scf01426g00001 | ERF3 | CTTCGAGCTGGATCTCAC | GTCGCCACAAAGAATCAA |
Niben101Scf02430g03006 | WRKY8 | CCTACTGAACTCTTGGAC | CTGCTCTTCCAGTTAAAAG |
Niben101Scf03160g01004 | NAC90 | TGTCGAGTTTACGTCATA | TCAGCTGCTAATTCTTGA |
Niben101Scf03816g01001 | SOBIR1 | CACCAGAATACCATCAGA | GATCCTCAGAAGTCATTACA |
Niben101Scf04504g03008 | LHCB3 | CCAAGTAGTGCTAATGGG | AGTGGGTCAAAGTACTGG |
Niben101Scf06017g02002 | MYB4 | CACCCACTTGAAGAAGAG | CTTGGATGTTGTTAAAATTGTG |
Niben101Scf06509g02006 | RLK5 | GCTGTTAAGAGGATTTGG | GCAACAAAGCAGTTTTAC |
Niben101Scf10735g00018 | PR10 | CTGGAAATGGAGGATGTA | AACGGTCTTGAAAAGTTC |
Niben101Scf18667g01002 | MKS1 | GCTTCTCTTCCACCTATA | GGGCTAGATAAATAACTGTG |
表2
差异表达基因中LRR-RLKs的筛选"
Unigene ID | log2FC | 蛋白特性 Protein property | ||
---|---|---|---|---|
6 hpi | 12 hpi | 24 hpi | ||
Niben101Scf00160g06027 | 2.80 | 3.42 | 2.12 | Putative receptor-like protein kinase At3g47111 |
Niben101Scf00182g06001 | — | 5.47 | — | Pollen receptor-like kinase 2 |
Niben101Scf00245g00006 | 10.00 | 6.88 | 4.19 | Probable LRR receptor-like serine/threonine-protein kinase At1g74361 |
Niben101Scf00742g01037 | — | 2.35 | — | Protein STRUBBELIG-RECEPTOR FAMILY 3-like isoform X4 |
Niben101Scf00744g08015 | 2.10 | 1.82 | — | Receptor-like protein kinase HAIKU3 |
Niben101Scf00887g04011 | 1.80 | 2.26 | — | Putative receptor-like protein kinase At3g47111 |
Niben101Scf00953g00004 | 2.65 | 3.06 | 1.66 | Receptor-like protein kinase HAIKU3 |
Niben101Scf01203g01002 | 2.12 | 4.39 | 2.65 | Probable LRR receptor-like serine/threonine-protein kinase At3g47570 isoform X2 |
Niben101Scf01205g02008 | -2.00 | — | -2.10 | Probable LRR receptor-like serine/threonine-protein kinase At4g36181 |
Niben101Scf01225g04031 | 2.51 | 2.85 | 2.08 | Probably inactive leucine-rich repeat receptor-like protein kinase At5g48381 |
Niben101Scf01278g09008 | — | 2.04 | 1.54 | Probable LRR receptor-like serine/threonine-protein kinase At1g56130 isoform X3 |
Niben101Scf01673g02002 | — | 6.26 | — | Probable LRR receptor-like serine/threonine-protein kinase RLK |
Niben101Scf01819g01001 | 1.31 | 2.21 | — | Phytosulfokine receptor 2 |
Niben101Scf02217g05010 | 1.65 | 2.29 | — | Receptor-like protein kinase HSL2 |
Niben101Scf02531g00007 | — | 2.88 | — | LRR receptor-like serine/threonine-protein kinase EFR |
Niben101Scf02862g00007 | -1.90 | -2.80 | — | Probable LRR receptor-like serine/threonine-protein kinase At4g36181 |
Niben101Scf03374g08019 | 3.80 | 3.71 | 1.10 | Probable LRR receptor-like serine/threonine-protein kinase At3g47571 |
Niben101Scf03816g01001 | 2.03 | 2.07 | 1.39 | AHW85126.2//leucine-rich repeat receptor-like kinase |
Niben101Scf04053g03008 | 2.06 | 2.50 | 1.79 | Receptor-like protein kinase HAIKU3 |
Niben101Scf04099g05004 | 2.38 | 2.35 | 1.72 | Leucine-rich repeat receptor-like serine/threonine/tyrosine-protein kinase SOBIR2 |
Niben101Scf05349g01040 | 1.30 | 2.32 | 2.20 | Protein STRUBBELIG-RECEPTOR FAMILY 5 isoform X2 |
Niben101Scf05928g03007 | 5.32 | 6.75 | 3.32 | Probable LRR receptor-like serine/threonine-protein kinase At1g74361 |
Niben101Scf06509g02006 | 5.34 | 5.87 | 2.71 | Receptor-like protein kinase 6 |
Niben101Scf08137g01034 | 2.82 | 3.29 | — | Putative receptor-like protein kinase |
Niben101Scf08564g00001 | 1.15 | 3.10 | — | Probable LRR receptor-like serine/threonine-protein kinase At1g56141 |
Niben101Scf10381g03006 | — | 3.10 | 2.42 | Probable LRR receptor-like serine/threonine-protein kinase At4g08851 |
Niben101Scf20124g00014 | 2.27 | 2.20 | 1.73 | Leucine-rich repeat receptor-like tyrosine-protein kinase PXC4 |
表3
PevD1诱导病程相关蛋白家族差异表达基因的筛选"
PR 家族基因 PR family gene | Unigene ID | log2 Ratio (WT_PevD1/WT_CK) | 蛋白特性 Protein property | ||
---|---|---|---|---|---|
6 hpi | 12 hpi | 24 hpi | |||
PR1 | Niben101Scf00107g03008 | -2.10 | 2.38 | 4.32 | Pathogenesis-related protein 1A |
Niben101Scf00953g03008 | — | 5.98 | 3.86 | Basic form of pathogenesis-related protein 1 | |
Niben101Scf00953g03009 | 4.60 | 7.73 | 5.58 | Basic form of pathogenesis-related protein 1-like | |
Niben101Scf01999g07002 | — | 3.17 | 2.58 | Pathogenesis-related protein 1C-like | |
Niben101Scf03376g03004 | -6.50 | 2.87 | 6.24 | Pathogenesis-related protein 1A | |
Niben101Scf04053g02006 | 1.64 | 5.47 | 3.81 | Basic form of pathogenesis-related protein 1-like | |
Niben101Scf04053g02007 | 5.23 | 8.30 | 6.87 | Basic form of pathogenesis-related protein 1 | |
PR2 | Niben101Ctg13736g00004 | — | 3.09 | 4.15 | Glucan endo-1,3-beta-glucosidase |
Niben101Scf01001g00003 | — | 2.86 | 3.91 | Glucan endo-1,3-beta-glucosidase | |
Niben101Scf01001g00004 | — | 2.86 | 3.91 | Glucan endo-1,3-beta-glucosidase | |
Niben101Scf01001g00005 | — | 2.86 | 3.91 | Glucan endo-1,3-beta-glucosidase | |
Niben101Scf01001g00006 | — | — | 3.70 | Glucan endo-1,3-beta-glucosidase | |
Niben101Scf01934g02004 | — | 5.40 | 3.21 | Glucan endo-1,3-beta-glucosidase | |
Niben101Scf03905g02017 | 1.32 | 5.13 | 2.91 | Glucan endo-1,3-beta-glucosidase | |
Niben101Scf04869g03002 | — | 3.25 | 3.39 | Glucan endo-1,3-beta-glucosidase | |
PR3 | Niben101Scf02041g00002 | — | 1.57 | 3.85 | Acidic endochitinase Q |
Niben101Scf03200g01014 | — | 2.22 | 2.15 | Endochitinase A | |
Niben101Scf03949g00023 | — | 4.49 | 1.38 | Endochitinase 3 | |
Niben101Scf07491g00003 | — | 4.87 | 3.48 | Endochitinase A | |
PR4 | Niben101Scf01015g01002 | 1.84 | 4.45 | — | Wound-induced protein WIN1-like |
Niben101Scf02171g00007 | 2.01 | 5.84 | 3.06 | Endochitinase PR4-like | |
Niben101Scf12045g06025 | 1.41 | 4.73 | 6.29 | Pathogenesis-related protein PR-4B | |
PR5 | Niben101Scf03937g02019 | — | — | 1.69 | Pathogenesis-related protein 5-like |
Niben101Scf05732g04031 | — | — | 4.88 | Pathogenesis-related protein 5-like | |
PR6 | Niben101Scf04053g01004 | — | — | 6.66 | Pathogenesis-related leaf protein 6-like |
PR10 | Niben101Scf10735g00018 | 1.36 | 2.10 | 2.50 | Pathogenesis-related protein PR-10 |
PR11 | Niben101Scf01789g04010 | 2.80 | 3.62 | 2.77 | Chitotriosidase-1-like |
Niben101Scf06295g04023 | 1.91 | 2.85 | 2.19 | Chitotriosidase-1-like | |
PR17 | Niben101Scf03385g02011 | — | 2.02 | 3.33 | Uncharacterized protein LOC104232799 |
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