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作物学报

作物学报 ›› 2019, Vol. 45 ›› Issue (1): 55-69.doi: 10.3724/SP.J.1006.2019.84001

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

基于iTRAQ技术的甘蔗受黑穗病菌侵染蛋白组分析

宋奇琦1(),Pratiksha SINGH2,Rajesh Kumar SINGH2,宋修鹏1,李海碧1,农友业2,杨丽涛1,2,*(),李杨瑞1,2,*()   

  1. 1 中国农业科学院甘蔗研究中心 / 广西壮族自治区农业科学院 / 农业部广西甘蔗生物技术与遗传改良重点实验室 / 广西甘蔗遗传改良重点实验室, 广西南宁530007
    2 广西大学农学院 / 亚热带农业生物保护与利用国家重点实验室, 广西南宁530005
  • 收稿日期:2018-01-04 接受日期:2018-08-20 出版日期:2018-09-26 网络出版日期:2018-09-26
  • 通讯作者: 杨丽涛,李杨瑞
  • 基金资助:
    本研究由广西八桂学者和特聘专家专项经费项目(GKAD17195100);广西科技基地和人才专项(桂科AD17195100);国家现代农业产业技术体系广西甘蔗创新团队项目(gjnytxgxcxtd-03-01);广西自然科学基金项目(2015GXNSFBA139060);广西甘蔗遗传改良重点实验室项目资助(16-K-02-01)

Proteomic analysis of sugarcane-Sporisorium scitamineum interaction based on iTRAQ technique

Qi-Qi SONG1(),SINGH Pratiksha2,Kumar SINGH Rajesh2,Xiu-Peng SONG1,Hai-Bi LI1,You-Ye NONG2,Li-Tao YANG1,2,*(),Yang-Rui LI1,2,*()   

  1. 1 Sugarcane Research Center, Chinese Academy of Agricultural Sciences / Guangxi Academy of Agricultural Sciences / Key Laboratory of Sugarcane Biotechnology and Genetic Improvement (Guangxi), Ministry of Agriculture / Guangxi Key Laboratory of Sugarcane Genetic Improvement, Nanning 530007, Guangxi, China
    2 Agricultural College, Guangxi University / State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Nanning 530005, Guangxi, China
  • Received:2018-01-04 Accepted:2018-08-20 Published:2018-09-26 Published online:2018-09-26
  • Contact: Li-Tao YANG,Yang-Rui LI
  • Supported by:
    This study was supported by the Guangxi Special Fund for Scientific Base and Talent(GKAD17195100);the Special Funds for Bagui Scholars and Distinguished Experts in Guangxi(桂科AD17195100);Guangxi Sugarcane Innovation Team of National Agricultural Industry Technology System(gjnytxgxcxtd-03-01);Guangxi Natural Science Fundation(2015GXNSFBA139060);Guangxi Key Laboratory of Sugarcane Genetic Improvement(16-K-02-01)

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摘要：

黑穗病已成为影响甘蔗产量和含糖量的重要病害。为从蛋白质水平探讨甘蔗应答黑穗病菌的分子机制, 本实验选用抗黑穗病品种桂糖29号和感黑穗病品种崖城71-374, 处理组用浸渍法接种黑穗病菌, 对照组用无菌水模拟接菌, 在接种180 d后采取甘蔗叶片, 使用iTRAQ技术对蛋白质组进行研究。结果显示, 桂糖29号中有定量信息蛋白1429个, 差异表达蛋白290个, 其中上调表达蛋白153个, 下调表达蛋白137个; 崖城71-374中有定量信息蛋白1576个, 差异表达蛋白125个, 其中上调表达蛋白55个, 下调表达蛋白70个。抗病品种桂糖29号中差异表达蛋白数多于感病品种崖城71-374, 且桂糖29号在KEGG富集到的代谢通路也更多, 可能被侵染后抗病品种的免疫调节机制更为复杂, 涉及的调控通路网更广。经对光合作用、抗氧化系统、钙信号、苯丙烷类代谢、激素相关差异表达蛋白及共有差异表达蛋白分析, 发现光合作用通路、ROS、ABA、钙信号通路相关蛋白在2个品种中多为上调表达, 且桂糖29号的上调表达蛋白数多于崖城71-374, 可能参与甘蔗后期对黑穗病的应答。植物抗病是一个复杂的过程, 需要多种功能与途径参与调控。在本实验中没有发现苯丙烷类代谢途径及一些酶(谷胱甘肽过氧化物酶、抗坏血酸过氧化物酶、超氧化物歧化酶、谷胱甘肽硫转移酶、过氧化氢酶)、激素(生长素、乙烯、赤霉素)参与甘蔗的抗病过程, 可能与采样时间有关。

关键词: 甘蔗, 黑穗病, iTRAQ, 蛋白质组

Abstract:

Sugarcane smut has become an important disease affecting sugarcane yield and sugar content. In order to investigate the molecular mechanism of sugarcane responding to smut at protein level, the smut-resistant variety GT29 and the smut-susceptible variety Yacheng 71-374 were used in this study. Both varieties were inoculated with the teliospore suspension of smut pathogen by dipping method whereas the control was treated with sterile water. Leaf samples were collected and used for proteomic analysis by iTRAQ technique at 180 days after treatment. The that 1429 proteins presented in GT29 with quantitative information, including 290 differentially expressed proteins with 153 up-regulated and 137 down-regulated; while 1576 proteins in Yacheng 71-374 with quantitative information, including 125 differentially expressed proteins with 55 up-regulated and 70 down-regulated. The number of differentially expressed proteins in resistant variety was higher than that in susceptible variety, and GT29 enriched more metabolic pathways in KEGG, indicating that the immunomodulatory mechanism of the resistant variety may be more complicated, and the regulatory network involved in response was broader. Through the analysis of photosynthesis, antioxidant system, calcium signal, phenylpropane metabolism, hormone related differential expressed protein and co-owned differentially expressed protein, it was found that the photosynthesis pathway, ROS, ABA, calcium signal pathway related protein were up-regulated in both varieties. The up-regulated expressed proteins were more in GT29 than in Yacheng 71-374, which may be involved in resistance response in sugarcane against smut pathogen at later growth stage. Plant disease resistance is a complex process that requires multiple functions and pathways to participate in regulation. The phenylpropanoid metabolic pathway, enzymes GPX, APX, SOD, GST, and CAT, and hormones IAA, ETH, and GA were not found to be involved in the disease resistance process of sugarcane.

Key words: sugarcane, smut, iTRAQ, proteomics

图1

对照和接种黑穗病菌甘蔗DNA的PCR扩增产物电泳图 M: molecular size marker (100~5000 bp); P: 黑穗病孢子DNA正对照; N: 无菌水负对照; G(T): GT29处理; G(C): GT29 对照; Y(T): Yacheng 71-374处理; Y(C): Yacheng 71-374对照。"

表1

2个甘蔗品种中鉴定到的肽段和蛋白数"

品种
Variety		 总谱图数
Total profiles		 鉴定谱图数
Identified profiles		 鉴定肽段数
Identified peptides		 鉴定蛋白数
Identified proteins		 定量蛋白数
Quantified proteins
GT29 112 817 8009 3512 1452 1429
Yacheng 71-374 114 550 8832 3990 1594 1576

图2

GO注释分析"

"

分类
Category		 GO ID 功能类别
Functional group		 P-value
生物过程
Biological process		 GO:0019684 Photosynthesis, light reaction 0
GO:0009765 Photosynthesis, light harvesting 0.0002
GO:0015979 Photosynthesis 0.0002
GO:0009772 Photosynthetic electron transport in photosystem II 0.0156
GO:0009767 Photosynthetic electron transport chain 0.0167
GO:0006412 Translation 0.0233
GO:0006091 Generation of precursor metabolites and energy 0.0283
GO:0043043 Peptide biosynthetic process 0.0315
GO:0022900 Electron transport chain 0.0342
GO:0006518 Peptide metabolic process 0.0347
GO:0034645 Cellular macromolecule biosynthetic process 0.0375
GO:0009059 Macromolecule biosynthetic process 0.0375
GO:0043604 Amide biosynthetic process 0.0381
GO:0043603 Cellular amide metabolic process 0.0418
GO:0044267 Cellular protein metabolic process 0.0484
分子功能
Molecular function		 GO:0004478 Methionine adenosyltransferase activity 0.0140
GO:0004857 Enzyme inhibitor activity 0.0140
GO:0016859 Cis-trans isomerase activity 0.0145
GO:0003755 Peptidyl-prolyl cis-trans isomerase activity 0.0145
GO:0051536 Iron-sulfur cluster binding 0.0216
GO:0051540 Metal cluster binding 0.0216
GO:0030234 Enzyme regulator activity 0.0367
GO:0098772 Molecular function regulator 0.0465
细胞组分
Cellular component		 GO:0016020 Membrane 0.0024
GO:0043228 Non-membrane-bounded organelle 0.0386
GO:0043232 Intracellular non-membrane-bounded organelle 0.0386
GO:0031224 Intrinsic component of membrane 0.0410

"

分类
Category		 GO ID 功能类别
Functional group		 P-value
生物过程
Biological process		 GO:0006979 Response to oxidative stress 0.0086
GO:0019222 Regulation of metabolic process 0.0094
GO:0006560 Proline metabolic process 0.0151
GO:0006561 Proline biosynthetic process 0.0151
GO:0060255 Regulation of macromolecule metabolic process 0.0244
GO:0031323 Regulation of cellular metabolic process 0.0301
GO:0055114 Oxidation-reduction process 0.0465
分子功能
Molecular function		 GO:0004097 Catechol oxidase activity 0.0039
GO:0016841 Ammonia-lyase activity 0.0039
GO:0016682 Oxidoreductase activity, acting on diphenols and related
substances as donors, oxygen as acceptor		 0.0039
GO:0004089 Carbonate dehydratase activity 0.0042
GO:0016684 Oxidoreductase activity, acting on peroxide as acceptor 0.0057
GO:0004601 Peroxidase activity 0.0057
GO:0016829 Lyase activity 0.0083
GO:0016491 Oxidoreductase activity 0.0107
GO:0016840 Carbon-nitrogen lyase activity 0.0113
GO:0016209 Antioxidant activity 0.0154
GO:0020037 Heme binding 0.0174
GO:0046872 Metal ion binding 0.0187
GO:0046914 Transition metal ion binding 0.0208
GO:0043169 Cation binding 0.0242
GO:0046906 Tetrapyrrole binding 0.0244
GO:0045735 Nutrient reservoir activity 0.0347
GO:0016679 Oxidoreductase activity, acting on diphenols and related
substances as donors		 0.0347
GO:0004611 Phosphoenolpyruvate carboxykinase activity 0.0347
GO:0008964 Phosphoenolpyruvate carboxylase activity 0.0347
GO:0016835 Carbon-oxygen lyase activity 0.0385
细胞组分
Cellular component		 GO:0009521 Photosystem 0.0195
GO:0034357 Photosynthetic membrane 0.0266
GO:0044436 Thylakoid part 0.0266
GO:0009522 Photosystem I 0.0281
GO:0009579 Thylakoid 0.0307
GO:0009538 Photosystem I reaction center 0.0357

表2

光合作用相关差异表达蛋白"

蛋白ID
Protein ID		 蛋白名称
Protein name		 差异倍数 Fold change
GT29 Yacheng 71-374
c54318_g1;orf1 光系统I反应中心亚基II Photosystem I reaction center subunit II 1.2995↑ 1.2562↑
c69501_g1;orf1 光系统I PsaH, 反应中心亚基III Photosystem I PsaH, reaction centre subunit III 1.9614↑ 0.9303
c48673_g1;orf1 光系统I反应中心亚基IV A Photosystem I reaction center subunit IV A 1.1149 1.3041↑
c56023_g1;orf1 光系统I反应中心亚基V Photosystem I reaction center subunit V 1.5387↑ 1.0456
c43081_g1;orf1 光系统I PsaH, 反应中心亚基VI Photosystem I PsaH, reaction centre subunit VI 0.8432 1.2040↑
c129327_g1;orf1 光系统I亚基VII Subunit VII of photosystem I 1.1871 1.2700↑
c55121_g1;orf1 光系统I反应中心亚基N Photosystem I reaction center subunit N 1.0602 1.4252↑
c56941_g1;orf1 光系统I反应中心亚基XI Photosystem I reaction center subunit XI 1.3609↑ 1.1204
c32636_g1;orf1 铁氧还蛋白[2Fe-2S] Ferredoxin[2Fe-2S] 0.7977↓ 1.0785
c70387_g2;orf1 光系统I组装蛋白ycf4 Photosystem I assembly protein ycf4 1.2823↑ 0.7661↓
c73345_g1;orf1 叶绿素a-b结合蛋白8 Chlorophyll a-b binding protein 8 0.8225↓ 1.1336
c99995_g1;orf1 叶绿素a-b结合蛋白CP26 Chlorophyll a-b binding protein CP26 0.9317 0.7892↓
c52958_g1;orf1 叶绿素a-b结合蛋白 Chlorophyll a-b binding protein 1.3946↑ 1.2335↑
c66930_g1;orf1 叶绿素a-b结合蛋白 Chlorophyll a-b binding protein 1.5718↑ 1.1641
c69028_g1;orf1 叶绿素a-b结合蛋白 Chlorophyll a-b binding protein 2.4059↑ 1.1324
c51419_g1;orf1 叶绿素a-b结合蛋白 Chlorophyll a-b binding protein 1.0915 1.3043↑
c55759_g1;orf1 叶绿素a-b结合蛋白 Chlorophyll a-b binding protein 1.9421↑ 1.2397↑
c57415_g1;orf1 叶绿素a-b结合蛋白 Chlorophyll a-b binding protein 1.3181↑ 1.0380
c69028_g3;orf1 叶绿素a-b结合蛋白 Chlorophyll a-b binding protein 1.8089↑ 1.0351
c55792_g1;orf1 叶绿素a-b结合蛋白 Chlorophyll a-b binding protein 1.8236↑ 1.0311
c51241_g4;orf1 叶绿素a-b结合蛋白 Chlorophyll a-b binding protein 1.8423↑ 1.0049
c69028_g2;orf1 叶绿素a-b结合蛋白 Chlorophyll a-b binding protein 1.9144↑ 0.9817
c121256_g1;orf1 叶绿素a-b结合蛋白 Chlorophyll a-b binding protein 2.4187↑ 0.8355
c73247_g2;orf1 光系统II蛋白D1 Photosystem II protein D1 3.4261↑ 0.9206
c78076_g1;orf1 光系统II蛋白D2 Photosystem II D2 protein 1.3682↑ 0.9089
c54219_g1;orf2 细胞色素b559亚基α Cytochrome b559 subunit α 1.6695↑ 0.7424↓
c77580_g1;orf1 光系统II CP43脱辅基蛋白 Photosystem II CP43 chlorophyll apoprotein 1.4055↑ 0.9421
蛋白ID
Protein ID		 蛋白名称
Protein name		 差异倍数 Fold change
GT29 Yacheng 71-374
c126236_g1;orf1 放氧增强蛋白1 Oxygen-evolving enhancer protein 1 1.0064 1.2372↑
c59540_g1;orf1 放氧增强蛋白3 Oxygen-evolving enhancer protein 3 0.8754 1.8631↑
c100226_g1;orf1 放氧增强蛋白 Oxygen-evolving enhancer protein 1.5306↑ 0.9906
c55903_g1;orf1 叶绿体放氧增强蛋白1 (部分) Chloroplast oxygen-evolving enhancer protein 1 (partial) 1.0456 1.2517↑
c51741_g1;orf1 光系统II PsbR Photosystem II PsbR 1.9037↑ 1.0547
c22663_g1;orf1 光系统II 47kDa蛋白 Photosystem II 47 kDa protein 1.7295↑ 0.9404
c56158_g2;orf1 细胞色素b6 (部分) Cytochrome b6 (partial) 1.4786↑ 1.0816
c72546_g2;orf1 细胞色素f Cytochrome f 1.2540↑ 0.9964
c67723_g1;orf1 ATP合成酶CF1α亚基 ATP synthase CF1 α subunit 1.3837↑ 1.0172
c57360_g1;orf2 ATP合成酶 ATP synthase 0.6735↓ 1.0127
c61429_g2;orf1 叶绿体Ptr-ToxA结合蛋白 Chloroplast Ptr ToxA-binding protein 1.4116↑ 0.9843
c54911_g1;orf1 16 kDa膜蛋白 16 kDa membrane protein 1.6364↑ 0.7557↓
c47557_g1;orf1 铜蛋白 Blue (type 1) Copper protein Blue (type 1) 1.1170 1.5826↑

表3

抗氧化相关差异表达蛋白"

蛋白ID
Protein ID		 蛋白名称
Protein name		 差异倍数 Fold change
GT29 Yacheng 71-374
过氧化物酶POD
c50862_g1;orf1 过氧化物酶-5 Peroxiredoxin-5 0.6309↓ 0.9930
c67975_g2;orf2 过氧化物酶54 Peroxidase 54 1.1999 2.0388↑
c59322_g3;orf1 过氧化物酶54前体 Peroxidase 54 precursor 1.4571↑ 1.5671↑
c67975_g1;orf1 过氧化物酶54前体 Peroxidase 54 precursor 1.4967↑ 2.0387↑
c66988_g1;orf1 过氧化物酶12前体 Peroxidase 12 precursor 1.5653↑ 1.0408
c53549_g1;orf1 过氧化物酶-2E-1 Peroxiredoxin-2E-1 0.8258↓ 0.9233
c59322_g4;orf1 过氧化物酶 Peroxidase 1.4017↑ 0.9072
c59146_g1;orf1 过氧化物酶 Peroxidase 0.7301↓ 0.6919↓
c72776_g2;orf1 过氧化物酶 Peroxidase 1.1594 0.6714↓
蛋白ID
Protein ID		 蛋白名称
Protein name		 差异倍数 Fold change
GT29 Yacheng 71-374
c58718_g1;orf1 过氧化物酶 Peroxidase 0.4462↓ 0.4493↓
c67818_g4;orf1 过氧化物酶 Peroxidase 1.3312↑ 1.6457↑
c64648_g9;orf1 过氧化物酶 Peroxidase 2.7030↑ 1.4510↑
c70898_g1;orf2 过氧化物酶 Peroxidase 1.2262↑ 1.3997↑
c50276_g1;orf1 过氧化物酶 Peroxidase 1.2289↑ 1.3558↑
c58269_g2;orf1 过氧化物酶 Peroxidase 1.4748↑ 1.2872↑
c46626_g1;orf1 过氧化物酶 Peroxidase 1.4542↑ 1.2559↑
c70898_g1;orf1 过氧化物酶 Peroxidase 1.3312↑ 1.2195↑
谷胱甘肽过氧化物酶GPX
c73435_g2;orf1 谷胱甘肽过氧化物酶 Glutathione peroxidase 0.8064↓ 1.0474
c55250_g2;orf1 磷脂氢过氧化物谷胱甘肽过氧化物酶6
Probable phospholipid hydroperoxide glutathione peroxidase 6		 0.7848↓ 0.9799
抗坏血酸过氧化物酶APX
c79083_g1;orf1 L-抗坏血酸过氧化物酶 L-ascorbate peroxidase 0.7024↓ 0.9451
c113714_g1;orf1 APx1-细胞溶质抗坏血酸过氧化物酶 APx1-cytosolic Ascorbate Peroxidase 0.7028↓ 0.9186
c76142_g1;orf1 抗坏血酸过氧化物酶 Ascorbate peroxidase 0.7592↓ 0.9356
c64041_g1;orf1 抗坏血酸过氧化物酶 Ascorbate peroxidase 0.7935↓ 0.9019
c62865_g1;orf1 抗坏血酸过氧化物酶 Ascorbate peroxidase 0.9393 0.7810↓
超氧化物歧化酶SOD
c73325_g1;orf1 超氧化物歧化酶(Cu/Zn) Superoxide dismutase (Cu/Zn) 0.6040↓ 1.4209↑
c73325_g2;orf2 超氧化物歧化酶(Cu/Zn) Superoxide dismutase (Cu/Zn) 0.4810↓ 1.1765
c55722_g2;orf1 超氧化物歧化酶 Superoxide dismutase 0.5893↓ 1.0278
谷胱甘肽硫转移酶GST
c66937_g2;orf2 谷胱甘肽S-转移酶4 Glutathione S-transferase 4 0.4706↓ 1.0162
c71423_g2;orf2 谷胱甘肽S-转移酶F8 Glutathione S-transferase F8 0.7288↓ 1.3394↑
c57078_g1;orf1 谷胱甘肽S-转移酶GST 12 Glutathione S-transferase GST 12 1.0751 1.2122↑
c47481_g1;orf1 谷胱甘肽S-转移酶GST 14 Glutathione S-transferase GST 14 0.9491 1.3272↑
c49019_g1;orf1 谷胱甘肽S-转移酶 Glutathione S-transferase 0.9292 1.2484↑
c125211_g1;orf1 谷胱甘肽S-转移酶 Glutathione S-transferase 1.1719 1.2102↑
c67259_g1;orf1 谷胱甘肽S-转移酶 Glutathione S-transferase 0.6751↓ 1.0939
c66937_g2;orf1 谷胱甘肽S-转移酶 Glutathione S-transferase 0.5350↓ 0.9349
c65037_g3;orf1 谷胱甘肽S-转移酶 Glutathione S-transferase 1.2785↑ 0.9165
c66547_g1;orf2 蛋白IN2-1同系物B同型X2 Protein IN2-1 homolog B isoform X2 0.6158↓ 1.0739
过氧化氢酶CAT
c63190_g2;orf1 过氧化氢酶 Catalase-1-like 0.7984↓ 0.9734
c63190_g2;orf2 过氧化氢酶 Catalase-1-like 0.8116↓ 0.8982
c63190_g1;orf1 过氧化氢酶 Catalase 1.2415↑ 0.7038↓

图3

共有差异表达蛋白COG功能分类"

表4

在GT29中上调表达、在Yacheng 71-374中下调表达的共有差异表达蛋白"

蛋白ID
Protein ID		 蛋白名称
Annotation		 差异倍数 Fold change
GT29 Yacheng 71-374
c62874_g2;orf1 40S核糖体蛋白S5 40S ribosomal protein S5 1.4018↑ 0.8042↓
c60900_g1;orf1 40S核糖体蛋白S18 40S ribosomal protein S18 1.4850↑ 0.7965↓
c61922_g1;orf1 40S核糖体蛋白S10 40S ribosomal protein S10 2.3718↑ 0.7673↓
c53669_g1;orf1 60S核糖体蛋白L35 60S ribosomal protein L35 2.2003↑ 0.8276↓
c71804_g5;orf1 NAD(P)H-醌氧化还原酶亚基H NAD(P)H-quinone oxidoreductase subunit H 1.9719↑ 0.7819↓
c63190_g1;orf1 过氧化氢酶 Catalase 1.2415↑ 0.7038↓
c54219_g1;orf2 细胞色素b559亚基α Cytochrome b559 subunit α 1.6695↑ 0.7424↓
c70387_g2;orf1 光系统I组装蛋白ycf4 Photosystem I assembly protein ycf4 1.2823↑ 0.7661↓
c58104_g1;orf1 叶绿体光系统II 22kDa蛋白质 Chloroplast photosystem II 22 kDa protein 1.3253↑ 0.8263↓
c54911_g1;orf1 16 kDa膜蛋白 16 kDa membrane protein 1.6364↑ 0.7557↓
c42694_g1;orf1 C4磷酸烯醇式丙酮酸羧化酶 Putative C4 phosphoenolpyruvate carboxylase 1.5936↑ 0.7203↓
c42694_g2;orf1 C4磷酸烯醇式丙酮酸羧化 Putative C4 phosphoenolpyruvate carboxylase 1.4599↑ 0.7465↓
c53868_g3;orf1 C4磷酸烯醇式丙酮酸羧化酶 Putative C4 phosphoenolpyruvate carboxylase 1.3213↑ 0.7792↓
c107621_g1;orf1 2A型丝氨酸/苏氨酸蛋白磷酸酶65 kDa调节亚基Aβ亚型
Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A beta isoform		 1.3104↑ 0.8029↓
c68372_g4;orf1 γ-生育酚甲基转移酶 γ-tocopherol methyltransferase 1.2630↑ 0.7888↓
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