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作物学报

作物学报 ›› 2021, Vol. 47 ›› Issue (7): 1275-1296.doi: 10.3724/SP.J.1006.2021.04192

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蔗割手密种类甜蛋白家族鉴定及栽培种同源基因功能分析

苏亚春1,2, 李聪娜1, 苏炜华1, 尤垂淮3, 岑光莉1, 张畅1, 任永娟1, 阙友雄1,2,*()   

  1. 1福建农林大学农业农村部福建甘蔗生物学与遗传育种重点实验室, 福建福州 350002
    2福建农林大学教育部作物遗传育种与综合利用重点实验室, 福建福州 350002
    3福建农林大学生命科学学院, 福建福州 350002
  • 收稿日期:2020-08-21 接受日期:2020-12-01 出版日期:2021-07-12 网络出版日期:2021-01-04
  • 通讯作者: 阙友雄
  • 作者简介:E-mail: syc2009mail@163.com
  • 基金资助:
    本研究由国家重点研发计划项目(2019YFD1000500);国家自然科学基金项目(31671752);国家现代农业产业技术体系建设专项资助(CARS-17)

Identification of thaumatin-like protein family in Saccharum spontaneum and functional analysis of its homologous gene in sugarcane cultivar

SU Ya-Chun1,2, LI Cong-Na1, SU Wei-Hua1, YOU Chui-Huai3, CEN Guang-Li1, ZHANG Chang1, REN Yong-Juan1, QUE You-Xiong1,2,*()   

  1. 1Key Laboratory of Sugarcane Biology and Genetic Breeding (Fujian), Ministry of Agriculture and Rural Affairs, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
    2Key Laboratory of Crop Genetics and Breeding and Comprehensive Utilization, Ministry of Education, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
    3College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China;
  • Received:2020-08-21 Accepted:2020-12-01 Published:2021-07-12 Published online:2021-01-04
  • Contact: QUE You-Xiong
  • Supported by:
    This study was supported by the National Key Research and Development Program of China(2019YFD1000500);the National Natural Science Foundation of China(31671752);the China Agriculture Research System(CARS-17)

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摘要：

类甜蛋白(thaumatin-like protein, TLP)在植物生长发育和抵抗逆境胁迫中发挥重要作用。为全面了解甘蔗TLP家族基因的结构和功能特性, 本研究利用甘蔗野生种割手密基因组数据库和生物信息学方法, 首先筛选获得122个SsTLP家族基因并进行蛋白理化性质、基因染色体分布、基因结构和系统进化等分析; 其次, 从我国大陆主栽甘蔗品种ROC22中克隆到1条SsTLP87的同源基因ScTLP1, 并对其基因表达模式、亚细胞定位、瞬时表达和转录自激活活性进行鉴定。结果表明, 122个SsTLP家族基因分布在28条割手密染色体上, 且存在多基因聚集现象, 每条染色体上有1~13个SsTLP基因, 多数SsTLP蛋白被预测为定位于细胞质的酸性疏水性不稳定蛋白。SsTLP家族基因结构类型多样, 其内含子数目(1~30)、内含子相位(0~2)和Motif (1~5)存在多种类型和分布。系统进化树分析显示, SsTLP基因编码蛋白分别聚类在TLP家族的I~X分支上, 其中具有抑菌潜力的第V类SsTLP数量最多(36个)。从甘蔗栽培种ROC22克隆获得的ScTLP1基因编码227个氨基酸残基, 属于第V类进化聚类组, 定位于细胞膜, 不具有转录自激活活性, 其与割手密SsTLP87蛋白的氨基酸序列相似性高达99.56%。qRT-PCR分析表明, ScTLP1基因在甘蔗不同组织部位组成型表达, 且在蔗芽部位的表达量最高。ScTLP1基因在脱落酸(abscisic acid, ABA)和水杨酸(salicylic acid, SA)处理下的表达量无显著变化, 但受甘蔗黑穗病菌(Sporisorium scitaminea)、茉莉酸甲酯(methyl jasmonate, MeJA)、氯化钠(sodium chloride, NaCl)、聚乙二醇(polyethylene glycol, PEG)和4℃低温诱导上调表达。瞬时过表达ScTLP1基因后, 本氏烟(Nicotiana benthamiana)叶片中的DAB染色加深, 乙烯合成相关基因NtEFE26和茉莉酸代谢途径相关基因NtPR3上调表达, 且接种烟草青枯菌(Pseudomonas solanacearum)和烟草茄病镰刀菌蓝色变种(Fusarium solani var. coeruleum)的ScTLP1基因瞬时过表达叶片发病情况较对照组轻, 表明ScTLP1基因可以诱发本氏烟的过敏反应, 参与乙烯和茉莉酸信号传导途径, 增强本氏烟对病原菌的防御效应。研究结果为深入探究甘蔗TLP家族基因的结构和功能奠定了良好的基础。

关键词: 甘蔗, 类甜蛋白, 全基因组分析, 功能分析

Abstract:

Thaumatin like protein (TLP) plays an important role in plant growth and development and resistance to environmental stress. In order to fully understand the structure and functional characteristics of sugarcane TLP gene family, 122 SsTLP family genes were screened and analyzed for their physicochemical properties, gene chromosomal distribution, gene structure, and phylogenetic evolution using the genomic database of Saccharum spontaneum and bioinformatics methods. In addition, one homologous gene of SsTLP87, ScTLP1, was cloned from a major sugarcane cultivar ROC22 in mainland China. Gene expression pattern, subcellular localization, transient expression, and transcriptional self-activation activity of ScTLP1 were identified. The results showed that 122 SsTLP family genes were distributed on 28 chromosomes, and there was a gene clustering with 1-13 SsTLP genes on each chromosome. Most SsTLP proteins were predicted to be acidic, hydrophobic, and unstable proteins which located in cytoplasm. There were many types and distributions of intron number (1-30), intron phase (0-2), and motif (1-5) in SsTLP family genes. Phylogenetic tree analysis indicated that the coding proteins of SsTLP genes were clustered in the I-X branches of TLP family, among which, the number of the class V SsTLPs with antibacterial potential was the most (36). ScTLP1 gene obtained from sugarcane cultivar ROC22 encoded 227 amino acid residues, belonging to the class V member. ScTLP1 was located in the cell membrane and had no transcriptional self-activation activity. The similarity of amino acid sequence between ScTLP1 and SsTLP87 was as high as 99.56%. qRT-PCR demonstrated that ScTLP1 gene was constitutively expressed in different tissues of sugarcane with the highest expression level in the bud. The expression level of ScTLP1 gene had no significant change under the treatments of abscisic acid (ABA) and salicylic acid (SA), but it was up-regulated by Sporisorium scitaminea, methyl jasmonate (MeJA), sodium chloride (NaCl), polyethylene glycol (PEG), and low temperature of 4℃. After transient overexpression of ScTLP1 gene, DAB staining in Nicotiana benthamiana leaves was deepened, and the expression level of NtEFE26 and NtPR3 was up-regulated. Furthermore, the incidence of transient overexpression of ScTLP1 gene in N. benthamiana leaves inoculated with Pseudomonas solanacearum and Fusarium solani var. coeruleum was lighter than that of the control, suggesting that ScTLP1 gene could induce an allergic reaction of N. benthamiana, participate in the ethylene and jasmonic acid signal transduction pathways, and enhance the defense effect of N. benthamiana against pathogens. These results laid a good foundation for further study on the structure and function of the TLP gene family in sugarcane.

Key words: sugarcane, thaumatin-like protein, genome-wide analysis, functional analysis

表1

SsTLP家族蛋白的理化性质分析及亚细胞定位和信号肽预测"

基因名称 基因ID 氨基酸数目 分子量 等电点 不稳定系数 平均疏水性 亚细胞定位 信号肽
Gene name Gene ID Amino acids number Molecular weight (kD) Isoelectric point Instability index Hydrophobicity Subcellular localization Signal peptide
SsTLP1 Sspon.01G0006490-1A 294 29.00 4.81 27.25 0.13 细胞质Cytoplasm 无No
SsTLP2 Sspon.01G0006490-2C 324 32.03 5.59 29.88 0.24 细胞质Cytoplasm 有Yes
SsTLP3 Sspon.01G0006490-3D 325 32.05 5.23 30.42 0.26 细胞质Cytoplasm 有Yes
SsTLP4 Sspon.01G0016190-1A 625 64.44 5.20 56.53 -0.10 细胞质Cytoplasm 无No
SsTLP5 Sspon.01G0016190-2B 464 46.16 5.02 59.63 0.10 细胞膜, 细胞质Cell membrane, Cytoplasm 无No
SsTLP6 Sspon.01G0016190-3C 479 47.69 4.96 59.17 0.10 细胞膜, 细胞质Cell membrane, Cytoplasm 无No
SsTLP7 Sspon.01G0016190-4D 275 27.67 4.70 54.56 0.16 细胞质Cytoplasm 有Yes
SsTLP8 Sspon.01G0016220-1A 331 32.82 5.40 41.00 0.24 细胞质Cytoplasm 有Yes
SsTLP9 Sspon.01G0016220-2C 305 31.16 8.33 59.42 0.08 细胞质Cytoplasm 有Yes
SsTLP10 Sspon.01G0016220-3D 331 32.82 5.37 40.32 0.25 细胞质Cytoplasm 有Yes
SsTLP11 Sspon.01G0018320-1A 236 23.36 4.30 32.97 0.18 细胞质Cytoplasm 有Yes
SsTLP12 Sspon.01G0018320-2B 236 23.40 4.30 31.18 0.20 细胞质Cytoplasm 有Yes
SsTLP13 Sspon.01G0018320-3C 236 23.41 4.30 34.15 0.15 细胞质Cytoplasm 有Yes
SsTLP14 Sspon.01G0020410-2C 1053 113.01 6.79 45.32 -0.14 细胞核Nucleus 无No
SsTLP15 Sspon.01G0020420-1A 297 30.21 5.60 47.32 0.08 细胞质Cytoplasm 无No
SsTLP16 Sspon.01G0020420-2B 298 30.25 5.40 45.49 0.10 细胞质Cytoplasm 无No
SsTLP17 Sspon.01G0037110-1B 334 33.68 5.03 46.39 0.06 细胞质Cytoplasm 有Yes
SsTLP18 Sspon.01G0037110-1T 334 33.68 5.03 46.39 0.06 细胞质Cytoplasm 有Yes
SsTLP19 Sspon.01G0037110-2C 334 33.68 5.03 46.39 0.06 细胞质Cytoplasm 有Yes
SsTLP20 Sspon.01G0043210-1B 289 29.55 7.44 40.68 0.12 细胞质Cytoplasm 有Yes
SsTLP21 Sspon.01G0043210-2C 424 44.00 9.26 51.46 -0.17 细胞质Cytoplasm 有Yes
SsTLP22 Sspon.01G0043210-3D 292 29.81 7.44 41.03 0.12 细胞质Cytoplasm 有Yes
SsTLP23 Sspon.01G0044270-1B 109 11.37 8.28 71.74 -0.05 细胞质Cytoplasm 无No
SsTLP24 Sspon.01G0056200-1C 95 10.02 8.89 73.11 -0.62 细胞质Cytoplasm 无No
SsTLP25 Sspon.01G0056200-2D 144 14.95 6.51 44.39 -0.24 细胞质Cytoplasm 无No
SsTLP26 Sspon.02G0009140-1A 246 24.96 4.87 43.80 -0.16 细胞质Cytoplasm 无No
SsTLP27 Sspon.02G0009140-1P 326 34.51 6.21 46.96 -0.22 细胞质Cytoplasm 无No
SsTLP28 Sspon.02G0009140-2B 236 23.98 4.87 43.62 -0.12 细胞质Cytoplasm 无No
SsTLP29 Sspon.02G0009140-2P 308 32.49 4.99 43.67 -0.10 细胞质Cytoplasm 无No
SsTLP30 Sspon.02G0009140-3C 236 23.97 4.87 44.87 -0.13 细胞质Cytoplasm 无No
SsTLP31 Sspon.02G0009140-3P 316 33.34 5.36 45.83 -0.15 细胞质Cytoplasm 有Yes
SsTLP32 Sspon.02G0009140-4D 319 33.65 5.36 45.49 -0.13 细胞质Cytoplasm 有Yes
SsTLP33 Sspon.02G0009150-1A 328 32.84 4.91 47.87 0.16 细胞质Cytoplasm 有Yes
SsTLP34 Sspon.02G0009150-2D 318 31.83 4.79 46.30 0.10 细胞质Cytoplasm 无No
SsTLP35 Sspon.02G0011230-1A 323 34.14 6.06 43.42 0.00 细胞质Cytoplasm 有Yes
SsTLP36 Sspon.02G0011230-1P 220 22.52 4.44 61.16 -0.17 细胞质Cytoplasm 无No
SsTLP37 Sspon.02G0011230-2B 289 30.43 7.36 44.32 -0.11 细胞质Cytoplasm 无No
SsTLP38 Sspon.02G0011230-2P 231 23.89 4.87 43.93 0.03 细胞质Cytoplasm 有Yes
SsTLP39 Sspon.02G0011230-3C 382 38.73 4.45 51.65 -0.12 细胞质Cytoplasm 有Yes
SsTLP40 Sspon.02G0011230-4D 323 34.23 6.18 42.60 0.00 细胞质Cytoplasm 有Yes
SsTLP41 Sspon.02G0020000-1A 137 14.35 8.32 61.73 -0.36 细胞质Cytoplasm 无No
SsTLP42 Sspon.02G0020000-1P 288 29.63 7.39 48.63 -0.12 细胞质Cytoplasm 有Yes
SsTLP43 Sspon.02G0029050-1A 135 13.93 6.52 46.66 0.35 细胞质Cytoplasm 有Yes
SsTLP44 Sspon.02G0029050-2B 227 23.17 4.74 39.91 0.25 细胞质Cytoplasm 有Yes
SsTLP45 Sspon.02G0029050-3C 227 23.23 4.78 39.64 0.26 细胞质Cytoplasm 有Yes
SsTLP46 Sspon.02G0029050-4D 215 21.88 4.96 40.74 0.39 细胞质Cytoplasm 有Yes
SsTLP47 Sspon.02G0029070-1A 190 19.81 11.44 56.10 -0.22 细胞壁, 细胞质Cell wall, Cytoplasm 无No
SsTLP48 Sspon.02G0029080-1A 459 47.73 8.61 60.12 -0.25 细胞质Cytoplasm 无No
SsTLP49 Sspon.02G0032190-1A 163 17.00 5.76 46.52 -0.29 细胞壁, 细胞质Cell wall, Cytoplasm 无No
SsTLP50 Sspon.02G0032190-1P 170 17.33 4.94 40.68 0.02 细胞质Cytoplasm 有Yes
SsTLP51 Sspon.02G0032190-2B 168 17.20 5.54 42.99 0.04 细胞质Cytoplasm 有Yes
SsTLP52 Sspon.02G0032190-3C 163 17.02 5.52 45.34 -0.34 细胞壁, 细胞质Cell wall, Cytoplasm 无No
SsTLP53 Sspon.02G0032190-4D 168 17.20 5.54 42.99 0.04 细胞质Cytoplasm 有Yes
SsTLP54 Sspon.02G0032200-1A 179 18.78 9.85 50.54 0.04 细胞质Cytoplasm 有Yes
SsTLP55 Sspon.02G0032220-1A 171 17.61 4.68 29.57 -0.06 细胞质Cytoplasm 有Yes
SsTLP56 Sspon.02G0032220-2B 171 17.38 4.35 44.70 0.22 细胞质Cytoplasm 有Yes
SsTLP57 Sspon.02G0032220-3C 132 13.56 4.12 39.94 0.09 细胞质Cytoplasm 有Yes
SsTLP58 Sspon.02G0032230-1A 168 17.20 5.54 42.99 0.04 细胞质Cytoplasm 有Yes
SsTLP59 Sspon.02G0032230-2C 409 43.36 8.98 48.87 -0.31 细胞质, 细胞核Cytoplasm, Nucleus 有Yes
SsTLP60 Sspon.02G0032240-1A 201 20.71 6.38 26.29 -0.09 细胞质Cytoplasm 有Yes
SsTLP61 Sspon.02G0032240-2C 175 17.72 4.60 43.06 0.09 细胞质Cytoplasm 有Yes
SsTLP62 Sspon.02G0034650-1B 230 24.19 7.31 24.08 -0.01 细胞质Cytoplasm 有Yes
SsTLP63 Sspon.02G0034650-2D 224 23.68 7.30 22.53 0.02 细胞质Cytoplasm 有Yes
SsTLP64 Sspon.02G0036340-1B 317 32.21 4.51 39.86 0.12 细胞质Cytoplasm 无No
SsTLP65 Sspon.02G0036340-2C 322 32.67 4.51 38.40 0.13 细胞质Cytoplasm 有Yes
SsTLP66 Sspon.02G0036340-3D 316 32.12 4.51 39.95 0.12 细胞质Cytoplasm 无No
SsTLP67 Sspon.02G0041150-1B 291 30.58 8.03 51.00 0.04 细胞质Cytoplasm 有Yes
SsTLP68 Sspon.02G0041150-2C 383 40.61 8.39 48.90 -0.01 细胞质Cytoplasm 有Yes
SsTLP69 Sspon.02G0045690-2D 171 17.51 4.47 29.08 -0.03 细胞质Cytoplasm 有Yes
SsTLP70 Sspon.02G0045700-1B 173 17.67 4.26 31.34 0.10 细胞质Cytoplasm 有Yes
SsTLP71 Sspon.02G0045700-1P 416 42.99 9.33 35.28 -0.38 细胞质Cytoplasm 有Yes
SsTLP72 Sspon.02G0045700-2C 173 17.68 4.43 31.87 0.14 细胞质Cytoplasm 有Yes
SsTLP73 Sspon.02G0045700-3D 173 17.73 4.43 33.23 0.09 细胞质Cytoplasm 有Yes
SsTLP74 Sspon.02G0053220-1C 245 25.68 6.27 27.29 -0.32 细胞质Cytoplasm 有Yes
SsTLP75 Sspon.02G0053220-2D 271 28.16 5.87 38.49 -0.29 细胞质Cytoplasm 有Yes
SsTLP76 Sspon.02G0053240-1C 166 17.35 6.66 30.90 -0.17 细胞质Cytoplasm 有Yes
SsTLP77 Sspon.02G0053250-2D 233 24.46 8.92 60.54 -0.45 细胞质, 细胞核Cytoplasm, Nucleus 无No
SsTLP78 Sspon.02G0059040-1D 124 12.61 6.50 44.52 0.23 细胞膜, 细胞质Cell membrane, Cytoplasm 有Yes
SsTLP79 Sspon.02G0059050-1D 127 12.95 4.27 27.47 0.47 细胞质Cytoplasm 有Yes
SsTLP80 Sspon.03G0003060-1A 252 26.62 7.86 47.70 -0.05 细胞质Cytoplasm 有Yes
SsTLP81 Sspon.03G0003060-1P 253 26.73 7.86 47.55 -0.04 细胞质Cytoplasm 有Yes
SsTLP82 Sspon.03G0003060-2C 251 26.48 7.86 46.89 -0.06 细胞质Cytoplasm 有Yes
SsTLP83 Sspon.03G0014170-1A 633 69.61 6.63 48.05 -0.21 细胞核Nucleus 有Yes
SsTLP84 Sspon.03G0018890-1A 1028 111.78 9.42 52.82 -0.72 细胞核Nucleus 无No
SsTLP85 Sspon.03G0018890-2C 516 57.24 6.15 38.71 -0.30 细胞核Nucleus 无No
SsTLP86 Sspon.03G0018890-3D 727 80.10 6.23 49.20 -0.33 细胞核Nucleus 有Yes
SsTLP87 Sspon.03G0040280-1C 227 23.49 7.33 31.85 -0.08 细胞质Cytoplasm 有Yes
SsTLP88 Sspon.03G0040280-1P 227 23.51 7.33 31.48 -0.07 细胞质Cytoplasm 有Yes
SsTLP89 Sspon.04G0003480-1A 186 18.49 4.09 48.17 0.20 细胞质Cytoplasm 无No
SsTLP90 Sspon.04G0003480-2C 186 18.49 4.09 48.17 0.20 细胞质Cytoplasm 无No
SsTLP91 Sspon.04G0003480-3D 191 18.97 4.41 47.57 0.21 细胞质Cytoplasm 无No
SsTLP92 Sspon.05G0007890-1A 1136 124.24 5.26 52.74 -0.44 细胞核Nucleus 无No
SsTLP93 Sspon.05G0014480-1A 280 27.85 5.42 47.70 0.28 细胞质Cytoplasm 有Yes
SsTLP94 Sspon.05G0014480-1P 267 26.55 5.68 46.13 0.23 细胞质Cytoplasm 有Yes
SsTLP95 Sspon.05G0014480-2B 280 27.78 5.35 45.12 0.34 细胞质Cytoplasm 有Yes
SsTLP96 Sspon.05G0014480-3D 277 27.62 5.35 44.31 0.32 细胞质Cytoplasm 有Yes
SsTLP97 Sspon.05G0029070-1B 550 60.87 6.45 43.06 -0.32 细胞核Nucleus 有Yes
SsTLP98 Sspon.05G0029070-2C 514 56.87 6.33 45.88 -0.35 细胞核Nucleus 有Yes
SsTLP99 Sspon.05G0032300-1C 244 24.70 4.62 36.67 0.09 细胞质Cytoplasm 有Yes
SsTLP100 Sspon.05G0032300-2D 392 40.73 7.03 46.75 -0.08 细胞质Cytoplasm 无No
SsTLP101 Sspon.05G0032310-1C 242 25.22 4.53 47.73 -0.18 细胞质Cytoplasm 无No
SsTLP102 Sspon.05G0032310-2D 242 25.19 4.53 47.73 -0.17 细胞质Cytoplasm 无No
SsTLP103 Sspon.06G0027890-1B 241 25.13 6.50 41.80 -0.14 细胞质Cytoplasm 无No
SsTLP104 Sspon.06G0027890-1T 217 22.57 8.36 44.97 -0.11 细胞质Cytoplasm 无No
SsTLP105 Sspon.06G0027890-2C 251 26.21 8.10 39.51 -0.11 细胞质Cytoplasm 无No
SsTLP106 Sspon.07G0015550-1A 318 32.05 4.60 49.86 0.08 细胞质Cytoplasm 有Yes
SsTLP107 Sspon.07G0015550-2B 326 33.01 7.35 38.08 0.30 细胞质Cytoplasm 有Yes
SsTLP108 Sspon.07G0015560-1A 517 54.47 4.77 46.95 -0.11 细胞质, 细胞核, 液泡Cytoplasm, Nucleus, Vacuole 有Yes
SsTLP109 Sspon.07G0017710-1A 267 26.60 4.10 43.87 0.22 细胞质Cytoplasm 有Yes
SsTLP110 Sspon.07G0017710-2B 329 32.60 4.21 47.78 0.28 细胞膜, 细胞质, 液泡Cell membrane, Cytoplasm, Vacuole 有Yes
SsTLP111 Sspon.08G0001280-1A 260 25.63 4.82 46.65 0.35 细胞质Cytoplasm 有Yes
SsTLP112 Sspon.08G0001280-2C 260 25.58 4.65 46.15 0.36 细胞质Cytoplasm 有Yes
SsTLP113 Sspon.08G0001280-3D 279 27.61 4.92 47.72 0.10 细胞质Cytoplasm 无No
SsTLP114 Sspon.08G0003010-1A 248 25.25 8.59 38.06 0.13 细胞质Cytoplasm 有Yes
SsTLP115 Sspon.08G0003010-2B 254 25.87 8.59 36.97 0.14 细胞质Cytoplasm 有Yes
SsTLP116 Sspon.08G0003010-3C 248 25.26 8.59 37.63 0.15 细胞质Cytoplasm 有Yes
SsTLP117 Sspon.08G0007410-1A 313 31.28 4.74 33.53 0.25 细胞质Cytoplasm 有Yes
SsTLP118 Sspon.08G0007410-2C 313 31.25 4.74 34.28 0.24 细胞质Cytoplasm 有Yes
SsTLP119 Sspon.08G0015670-1A 309 31.95 6.13 49.19 0.07 细胞质Cytoplasm 有Yes
SsTLP120 Sspon.08G0015670-2B 309 31.92 6.20 46.42 0.07 细胞质Cytoplasm 有Yes
SsTLP121 Sspon.08G0015670-3D 251 26.04 9.12 55.78 -0.23 细胞质Cytoplasm 有Yes
SsTLP122 Sspon.08G0030460-1D 275 28.40 6.12 54.61 0.10 细胞质Cytoplasm 有Yes

图1

SsTLP家族基因的染色体定位"

图2

SsTLP家族基因结构 绿色方框代表编码序列(CDS); 黄色方框代表非编码序列(UTR); 方框中间的横线代表内含子; 0、1和2代表内含子相位。"

图3

SsTLP家族保守基序分析 不同颜色的方框代表基序种类与位置。"

图4

SsTLP家族系统进化树 红色圆点代表割手密SsTLP蛋白; 绿色圆点代表拟南芥AtTLP蛋白; 黑色圆点代表水稻OsTLP蛋白。"

图5

ScTLP1基因的核酸序列及其编码的氨基酸序列 蓝色字体为半胱氨酸位点; 方框序列为THN保守结构域; 黄色序列为信号肽; *为终止密码子。"

图6

甘蔗ScTLP1蛋白与其他近缘物种TLP蛋白的氨基酸序列多重比对"

图7

ScTLP1基因的组织特异性表达及在不同逆境胁迫下的表达 A: ScTLP1基因在甘蔗ROC22中的组织特异性表达。R: 蔗根; SP: 蔗肉; L: 蔗叶; SE: 蔗皮; B: 蔗芽。B: ScTLP1基因在甘蔗黑穗病菌侵染下的表达。C~H: ScTLP1基因在外源激素和非生物胁迫下的表达。ABA: 脱落酸; SA: 水杨酸; MeJA: 茉莉酸甲酯; NaCl: 氯化钠; PEG: 聚乙二醇; Low temperature: 4℃低温处理。误差线代表每组处理的标准误差(n = 3)。柱上标以不同小写字母表示在0.05水平显著差异。"

图8

ScTLP1基因在本氏烟叶片中的瞬时表达效应 A: ScTLP1在瞬时表达1 d后的本氏烟中的基因表达量。B: 烟草免疫相关基因在瞬时表达1 d后的对照组和实验组的基因表达量。C: 瞬时表达2 d后的本氏烟叶片的DAB染色结果。D和E: 本氏烟接种烟草青枯菌和烟草茄病镰刀菌蓝色变种7 d的叶片表型。实验数据以NtEF-1α基因的表达水平进行归一化处理。误差线代表每组处理的标准误差(n = 3)。柱上标以不同小写字母表示在0.05水平显著差异。"

图9

ScTLP1蛋白在本氏烟叶片表皮的亚细胞定位 该结果包含明场、绿色荧光、叠加场3个视野下拍摄的照片。箭头a、b和c分别表示细胞核、细胞膜和细胞质。35S::GFP为含有亚细胞定位空载的GV3101菌液注射本氏烟的结果; 35S::ScTLP1::GFP为含有目的基因ScTLP1的重组亚细胞定位载体的GV3101菌液注射本氏烟的结果。标尺为50 μm。"

图10

甘蔗ScTLP1蛋白的转录自激活活性验证 A: 不同浓度的阳性对照、阴性对照和pGBKT7-ScTLP1的Y2H Gold酵母菌液涂布在SD-Trp固体培养基上的生长情况。B: 利用X-α-gal染色液对SD-Trp固体培养基上的菌落的染色结果。C: X-α-gal染色液对添加了金担子素A抑制剂SD-Trp固体培养基上生长的菌落的染色结果。pGADT7-T+pGBKT7-p53: 阳性对照; pGBKT7: 阴性对照。"

表1

附SsTLP家族蛋白的二级结构预测"

蛋白名称
Protein name		 α-螺旋
Alpha helix		 延伸链
Extended strand		 不规则卷曲
Random coil		 蛋白名称
Protein
name		 α-螺旋
Alpha helix		 延伸链
Extended strand		 不规则卷曲
Random coil
SsTLP1 19.05 15.99 64.97 SsTLP62 18.70 15.65 65.65
SsTLP2 22.84 16.05 61.11 SsTLP63 19.20 16.52 64.29
SsTLP3 23.08 16.00 60.92 SsTLP64 24.92 14.20 60.88
SsTLP4 20.80 15.36 63.84 SsTLP65 23.91 14.91 61.18
SsTLP5 20.47 10.13 69.40 SsTLP66 24.68 14.24 61.08
SsTLP6 22.55 9.39 68.06 SsTLP67 14.78 20.27 64.95
SsTLP7 22.91 11.64 65.45 SsTLP68 18.28 15.93 65.80
SsTLP8 27.79 14.20 58.01 SsTLP69 9.36 22.81 67.84
SsTLP9 26.56 13.11 60.33 SsTLP70 9.25 18.50 72.25
SsTLP10 28.70 13.90 57.40 SsTLP71 14.90 18.51 66.59
SsTLP11 12.71 16.95 70.34 SsTLP72 9.25 20.23 70.52
SsTLP12 12.71 19.07 68.22 SsTLP73 9.25 18.50 72.25
SsTLP13 9.75 16.95 73.31 SsTLP74 7.76 19.59 72.65
SsTLP14 31.15 15.10 53.75 SsTLP75 10.33 13.65 76.01
SsTLP15 12.79 19.19 68.01 SsTLP76 7.83 21.08 71.08
SsTLP16 11.07 19.13 69.80 SsTLP77 19.74 12.45 67.81
SsTLP17 11.98 15.27 72.75 SsTLP78 26.61 20.97 52.42
SsTLP18 11.98 15.27 72.75 SsTLP79 18.11 22.83 59.06
SsTLP19 11.98 15.27 72.75 SsTLP80 17.86 15.87 66.27
SsTLP20 15.22 17.99 66.78 SsTLP81 18.18 15.81 66.01
SsTLP21 17.92 16.75 65.33 SsTLP82 17.93 16.33 65.74
SsTLP22 15.75 17.81 66.44 SsTLP83 29.07 17.85 53.08
SsTLP23 36.70 0.92 62.39 SsTLP84 27.04 9.92 63.04
SsTLP24 3.16 9.47 87.37 SsTLP85 40.50 13.76 45.74
SsTLP25 9.72 14.58 75.69 SsTLP86 30.54 17.06 52.41
SsTLP26 8.13 18.70 73.17 SsTLP87 24.23 11.01 64.76
SsTLP27 19.02 13.50 67.48 SsTLP88 24.23 11.45 64.32
SsTLP28 11.02 19.07 69.92 SsTLP89 9.68 21.51 68.82
SsTLP29 19.81 15.91 64.29 SsTLP90 9.68 21.51 68.82
SsTLP30 8.90 19.07 72.03 SsTLP91 9.95 23.56 66.49
SsTLP31 22.78 10.76 66.46 SsTLP92 40.49 10.56 48.94
SsTLP32 22.57 11.29 66.14 SsTLP93 13.21 22.86 63.93
SsTLP33 13.41 21.34 65.24 SsTLP94 11.99 20.97 67.04
SsTLP34 11.32 22.96 65.72 SsTLP95 13.93 22.14 63.93
SsTLP35 17.65 16.41 65.94 SsTLP96 15.88 21.30 62.82
SsTLP36 11.82 15.45 72.73 SsTLP97 29.09 18.18 52.73
SsTLP37 12.46 15.57 71.97 SsTLP98 29.77 16.54 53.70
SsTLP38 9.52 25.54 64.94 SsTLP99 12.30 21.72 65.98
SsTLP39 12.57 14.40 73.04 SsTLP100 15.31 20.66 64.03
SsTLP40 17.65 16.10 66.25 SsTLP101 12.40 24.79 62.81
SsTLP41 13.14 12.41 74.45 SsTLP102 12.40 24.79 62.81
SsTLP42 13.19 15.62 71.18 SsTLP103 13.69 12.03 74.27
SsTLP43 17.78 20.00 62.22 SsTLP104 16.59 14.75 68.66
SsTLP44 12.78 20.26 66.96 SsTLP105 16.33 11.16 72.51
SsTLP45 12.33 19.82 67.84 SsTLP106 17.30 16.98 65.72
SsTLP46 16.28 20.93 62.79 SsTLP107 26.99 21.17 51.84
SsTLP47 13.68 13.68 72.63 SsTLP108 31.53 12.77 55.71
SsTLP48 15.25 19.17 65.58 SsTLP109 16.48 19.48 64.04
SsTLP49 0.00 23.93 76.07 SsTLP110 19.45 17.33 63.22
SsTLP50 9.41 21.18 69.41 SsTLP111 29.23 12.69 58.08
SsTLP51 11.31 19.64 69.05 SsTLP112 28.46 13.46 58.08
SsTLP52 0.00 22.09 77.91 SsTLP113 22.94 13.98 63.08
SsTLP53 11.31 19.64 69.05 SsTLP114 17.74 20.97 61.29
SsTLP54 29.61 20.11 50.28 SsTLP115 21.26 20.47 58.27
SsTLP55 9.36 22.81 67.84 SsTLP116 18.95 21.37 59.68
SsTLP56 11.70 22.22 66.08 SsTLP117 22.68 18.85 58.47
SsTLP57 21.97 18.18 59.85 SsTLP118 22.36 18.85 58.79
SsTLP58 11.31 19.64 69.05 SsTLP119 20.39 16.50 63.11
SsTLP59 20.29 11.49 68.22 SsTLP120 19.09 16.50 64.40
SsTLP60 15.42 14.93 69.65 SsTLP121 17.13 10.36 72.51
SsTLP61 10.29 21.14 68.57 SsTLP122 21.45 14.55 64.00
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