中国农业科学 ›› 2019, Vol. 52 ›› Issue (23): 4274-4284.doi: 10.3864/j.issn.0578-1752.2019.23.008
收稿日期:
2019-05-28
接受日期:
2019-08-14
出版日期:
2019-12-01
发布日期:
2019-12-01
通讯作者:
钱伟
作者简介:
远俊虎,E-mail:jhyuan1998@163.com|丁一娟,E-mail:dding1989@163.com
基金资助:
YUAN JunHu,DING YiJuan,YANG WenJing,YAN BaoQin,CHAI YaRu,MEI JiaQin,QIAN Wei()
Received:
2019-05-28
Accepted:
2019-08-14
Online:
2019-12-01
Published:
2019-12-01
Contact:
Wei QIAN
摘要:
【目的】由核盘菌(Sclerotinia sclerotiorum)引起的菌核病是我国油菜种植上的主要问题,严重威胁着菜籽产量及品质。分泌性蛋白在病原菌致病过程中起着重要作用,核盘菌基因组中包含大量编码分泌性蛋白的基因,本研究旨在鉴定并筛选与致病性相关的分泌蛋白基因,揭示核盘菌的致病机理,为菌核病防控提供重要靶标。【方法】采用SMART软件对核盘菌在侵染抗病、感病甘蓝过程中差异表达明显的8个具有信号肽的候选基因进行蛋白质结构域的分析,随后将SMART分析得到的结构域分别于SCOP、Pfam、PDB数据库进行功能注释。利用基因特异性引物进行目的基因特异片段的PCR扩增,构建pTRV2-Gene和pTRV2-GFP载体。随后等量混合含有pTRV1及pTRV2-Gene,pTRV1及pTRV2-GFP载体的重悬菌液。室温静置3 h后,利用针筒浸润法将pTRV2-Gene载体及对照(TRV-GFP)侵染5—6周龄的本氏烟(Nicotiana benthamiana)。侵染植株于黑暗环境中培养48 h后,再置于正常光照条件的环境中生长7 d。将直径6 mm的核盘菌PDA菌丝块接种于侵染9 d后的烟草叶片叶腹的中央,其中带菌面紧贴叶片,随后将接种植株培养48 h后统计病斑面积。提取接种48 h后的病斑及病斑周围组织叶片(距腐烂组织边缘1 cm左右)的RNA,利用特异引物进行目的基因的qRT-PCR,计算目的基因在携带TRV-HIGS载体的烟草植株中的相对表达量。【结果】SMART及结构域注释预测这8个候选基因可能参与了蛋白、核酸或多糖的水解,影响植物的免疫反应,参与核盘菌对药物耐受性的调节及生物素合成。核盘菌接种携带这8个基因的TRV-HIGS载体及对照载体的烟草,48 h后对照植株的病斑面积平均为3.44 cm 2,除SS1G_07655外,其余7个候选基因的TRV-HIGS植株上的病斑面积相比对照植株均显著减小(P≤0.05),其病斑面积介于1.63—2.61 cm 2。qRT-PCR结果显示,这7个致病相关的候选基因在核盘菌侵染烟草过程中的基因表达水平均显著低于对照(P≤0.05)。【结论】利用TRV介导的HIGS技术成功地对核盘菌中8个未知功能的分泌蛋白基因进行了功能鉴定,筛选到7个可能与核盘菌致病性相关的基因,其中对核盘菌致病性影响最大的SS1G_03146预测可能参与核盘菌生物素合成,同时SS1G_04343及SS1G_11912预测可能参与影响植物的免疫反应。
远俊虎,丁一娟,杨文静,闫宝琴,柴亚茹,梅家琴,钱伟. 利用TRV-HIGS技术鉴定核盘菌致病相关的分泌蛋白基因[J]. 中国农业科学, 2019, 52(23): 4274-4284.
YUAN JunHu,DING YiJuan,YANG WenJing,YAN BaoQin,CHAI YaRu,MEI JiaQin,QIAN Wei. Identification of Genes Encoding Secretory Proteins Related to the Pathogenicity of Sclerotinia sclerotiorum Using TRV-HIGS[J]. Scientia Agricultura Sinica, 2019, 52(23): 4274-4284.
表1
本试验所用引物信息"
基因 Gene | 引物序列 Primer sequence (5′-3′) | 产物大小 Product size (bp) | 用途 Usage |
---|---|---|---|
SS1G_00263 | F: CCGGAATTCCGGCAGCGCCTCAAGCTCGACTCAAATC R: CGAGCTCGCTGACGGTAGCGGAACCAACAACGG qF: TCTTTGAGGATGGAACTTGGAC qR: AGCCTGGCAAGCATAATCG | 273 | 基因扩增Gene amplification qRT-PCR |
SS1G_04945 | F: CCGGAATTCCGGTCTCCTTCCTTCTCGGC R: CGAGCTCGCTCGTAATCGGCACCAT qF: TCTCAACGGTGCTCTTTACTTC qR: TTGCTATCAGGGACCCATCC | 263 | 基因扩增Gene amplification qRT-PCR |
SS1G_03181 | F: CCGGAATTCCGGTGAGCGATGCTACCAACAGCGCCTAC R: CGAGCTCGCACCAGCACTTTGGAGAGCACCGTAA qF: TGCCGATTACGAGGGAACA qR: TGGAAACATCGACGGTGAAG | 210 | 基因扩增Gene amplification qRT-PCR |
SS1G_04343 | F: CCGGAATTCCGGTGGTCTTTACGCTGGGTATTTC R: CGAGCTCGCACCGTTGGTTGTGTTTTCATT qF: TGGTCTTTACGCTGGGTATTTC qR: CAGACACTTGCGAATGGAGC | 284 | 基因扩增Gene amplification qRT-PCR |
SS1G_03146 | F: CCGGAATTCCGGACATTTCCTCTTGAACCATCCCGTA R: CGAGCTCGTTTATGACACCCTTGTTTCCAGCGA qF: GCACATTTCCTCTTGAACCATC qR: GCAGTGTCACTTCCCACCATT | 309 | 基因扩增Gene amplification qRT-PCR |
SS1G_02250 | F: CCGGAATTCCGGTCACACTTTTGGCATT R: CGAGCTCGATCAGCACGTTTTTCT qF: AAGCCAACACCAACCTCATC qR: CACTGGAGCGTAGTTCTCGTAG | 272 | 基因扩增Gene amplification qRT-PCR |
SS1G_11912 | F: CCGGAATTCCGGTCAGCAGCTCCACCTCCACCACC R: CGAGCTCGATTCGCCTTTCCAAAATCCGTAT qF: TTCCCGAAACCGTTCCTAGT qR: TCACCATTGCTACTGCCACTT | 257 | 基因扩增Gene amplification qRT-PCR |
SS1G_07655 Sstub1 (内参基因Actin gene) | F: CCGGAATTCCGGGCTACTGTTCCTTTGGACTACGCT R: CGAGCTCGTTACTGAGGAGTGAGTCGTGTCGG qF: AATATGCCAGAGCCATCACA qR: CAGCGTAGTCCAAAGGAACAG qF: GTGAGGCTGAGGGCTGTGTGA qR: CCTTTGGCGATGGGACG | 322 | 基因扩增Gene amplification qRT-PCR qRT-PCR |
图1
候选基因在侵染抗、感病甘蓝过程中的表达热图 核盘菌候选基因在接种抗、感甘蓝茎秆(A)0、12、24 h和叶片(B) 0、6、12 h后的表达量Relative expression levels of candidate genes in the susceptible and resistant B. oleracea after inoculation with S. sclerotiorum in stem (A) post 0, 12, 24 h and leaf (B) post 0, 6, 12 h。Rs:抗病甘蓝材料的茎秆Resistant B. oleracea stem;Ss:感病甘蓝材料的茎秆Susceptible B. oleracea stem;Rl:抗病甘蓝材料的叶片Resistant B. oleracea leaf;Sl:感病甘蓝材料的叶片Susceptible B. oleracea leaf。以lg (FPKM+1)值绘制基因表达量的热图,红色表示高表达基因,蓝色表示低表达基因。颜色从红到蓝,表示lg (FPKM+1)从大到小。基因表达热图构建方法见文献[11] The lg (FPKM+1) value was used for the heatmap. Red indicated high expression genes and blue indicated low expression genes. The colors ranged from red to blue, indicating that lg (FPKM+1) from large to small. The heatmap was conducted according to reference [11]"
表2
候选基因的结构域功能分析"
基因 Gene | 结构域名称 Domain name | 结构域位置 Location (aa) | E期望值 E-value | 可能的功能或特性 Putative function or feature |
---|---|---|---|---|
SS1G_00263 | SCOP:d1hw1a2 | 14-79 | 0.77 | 调节药物的耐受性 Regulation of drug tolerance |
SS1G_04945 | Pfam:Glyco_hydro_7 | 21-446 | 2.6e-200 | O型糖苷水解酶 O-glycoside hydrolase |
PDB:3PL3|A | 19-446 | 0 | 纤维二糖水解酶 Cellobiohydrolase | |
SCOP:d1gpia_ | 19-450 | 0 | 伴刀豆球蛋白A样凝集素/葡聚糖酶 Concanavalin A/Glucanase | |
SS1G_03181 | Pfam:ASP | 91-399 | 3.2e-69 | 天冬氨酰蛋白酶(酸性蛋白酶)Aspartyl proteases (acid proteases) |
SCOP:d1bxoa_ | 79-399 | 1e-81 | 酸性蛋白酶 Acid proteases | |
PDB:3APP|A | 83-398 | 1e-90 | 酸性蛋白酶 Acid proteases | |
SS1G_04343 | SCOP:d1gh8a_ | 339-377 | 0.39 | 翻译延长因子 Translation elongation factor |
SCOP:d1fuia2 | 411-457 | 0.68 | 异构酶 Isomerase | |
SS1G_03146 | SCOP:d1h4vb2 | 18-62 | 1.8 | 生物素合成酶 Biotin synthetase |
SS1G_02250 | SCOP:d1ed8a_ | 43-90 | 5.7 | 碱性磷酸酶 Alkaline phosphatase |
SCOP:d1dofa_ | 215-241 | 0.86 | L型天冬氨酸 L-aspartic acid | |
SS1G_11912 | Pfam:NPP1 | 52-242 | 4.5e-61 | 坏死诱导蛋白 Necrosis inducing protein |
PDB:3GNZ|P | 32-242 | 4e-65 | 毒素 Toxin | |
SCOP:d1fuia2 | 188-240 | 0.58 | 异构酶 Isomerase | |
SS1G_07655 | Pro-kuma_activ | 35-181 | 3.25e-19 | 肽酶 Peptidase |
PDB:3EDY|A | 35-581 | 2e-67 | 肽酶 Peptidase | |
SCOP:d1gt91_ | 36-581 | 9e-19 | 枯草杆菌蛋白酶类 Subtilisins |
图3
TRV-HIGS转化烟草植株的鉴定 A:注射含有TRV-Gene重组质粒9 d后的烟草植株的PCR鉴定The identification of N. benthamiana at 9 days post TRV-Gene inoculation. M: Marker; 1: SS1G_00263; 2: SS1G_04945; 3: SS1G_03181; 4: SS1G_04343; 5: SS1G_03146; 6: SS1G_02250; 7: SS1G_11912; 8: SS1G_07655;B:注射不含重组质粒的重悬液9 d后的烟草植株的GFP信号观察GFP signal observation of N. benthamiana at 9 days after suspension without recombinant vectors inoculation;C:注射TRV-GFP载体9 d后的烟草植株的GFP信号观察GFP signal observation of N. benthamiana at 9 days post TRV-GFP inoculation。标尺Bar = 30 μm"
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