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刺激植物响应蛋白基因Epl1克隆、原核表达及功能初探

遇文婧 宋小双 邓勋 平晓帆 周琦 刘志华

遇文婧, 宋小双, 邓勋, 平晓帆, 周琦, 刘志华. 刺激植物响应蛋白基因Epl1克隆、原核表达及功能初探[J]. 北京林业大学学报, 2018, 40(1): 17-26. doi: 10.13332/j.1000-1522.20170249
引用本文: 遇文婧, 宋小双, 邓勋, 平晓帆, 周琦, 刘志华. 刺激植物响应蛋白基因Epl1克隆、原核表达及功能初探[J]. 北京林业大学学报, 2018, 40(1): 17-26. doi: 10.13332/j.1000-1522.20170249
Yu Wen-jing, Song Xiao-shuang, Deng Xun, Ping Xiao-fan, Zhou Qi, Liu Zhi-hua. Cloning, prokaryotic expression and function of the eliciting plant response protein of Trichoderma asperellum[J]. Journal of Beijing Forestry University, 2018, 40(1): 17-26. doi: 10.13332/j.1000-1522.20170249
Citation: Yu Wen-jing, Song Xiao-shuang, Deng Xun, Ping Xiao-fan, Zhou Qi, Liu Zhi-hua. Cloning, prokaryotic expression and function of the eliciting plant response protein of Trichoderma asperellum[J]. Journal of Beijing Forestry University, 2018, 40(1): 17-26. doi: 10.13332/j.1000-1522.20170249

刺激植物响应蛋白基因Epl1克隆、原核表达及功能初探

doi: 10.13332/j.1000-1522.20170249
基金项目: 

国家自然科学基金项目 31670649

国家自然科学基金项目 31700564

哈尔滨市应用技术研究与开发项目科技创新人才 2016RQQXJ235

黑龙江省林科院青年基金项目 2015Q01

详细信息
    作者简介:

    遇文婧,博士,副研究员。主要研究方向:森林保护。Email:ywjlinda2008@163.com 地址:150040黑龙江省哈尔滨市南岗区哈平路134号

    责任作者:

    刘志华,副教授。主要研究方向:森林保护。Email:lzhnefu@126.com 地址:150040黑龙江省哈尔滨市和兴路26号东北林业大学林学院

  • 中图分类号: S763.1

Cloning, prokaryotic expression and function of the eliciting plant response protein of Trichoderma asperellum

  • 摘要: 目的研究刺激植物响应蛋白Epl1对木本植物山新杨的刺激响应功能, 为开发新一代提高植物免疫的新型诱导剂奠定基础。方法从棘孢木霉ACCC30153菌株中克隆获得一个刺激植物响应蛋白基因Epl1, 并进行序列分析和原核表达, 将获得的原核重组蛋白rEpl1与山新杨组培苗进行互作, 初步探讨rEpl1蛋白对山新杨生长、生理指标、以及生长和防御相关基因转录水平的影响。结果刺激植物响应蛋白基因Epl1的cDNA序列全长为417 bp, 预测蛋白分子量12.6 kDa, 属于Cerato-platanin家族; 通过构建原核表达载体, 成功获得重组蛋白rEpl1;在1 μg/mL rEpl1诱导下, 山新杨组培苗生长较快、根系旺盛, 生物量明显增加, CAT活性在诱导第1 d时为对照的3.51倍, 可溶性糖含量和脯氨酸含量在诱导初期急剧积累; 山新杨生长素基因LAX2/AUX和水杨酸防御基因JAR2的转录水平分别在蛋白诱导2 d和12 h时达到峰值, 分别为对照的873.10和388.02倍。结论Epl1基因的克隆、序列分析和原核表达为进一步研究Epl1蛋白诱导植物系统抗病性提供了理论基础; 重组蛋白rEpl1与山新杨组培苗互作实验, 初步Epl1蛋白能够刺激木本植物在生理及分子水平上的响应, 从而促进生长, 提高抗性。

     

  • 图  1  Epl1基因的cDNA及DNA扩增结果

    M.DNA;1~4. cDNA扩增产物;5~8. DNA扩增产物。M, DNA marker DL2000; 1-4, PCR product of cDNA; 5-8, PCR product of DNA.

    Figure  1.  PCR analysis of DNA and cDNA of Epl1

    图  2  Epl1的cDNA序列及预测氨基酸序列

    粗体. cDNA序列;斜体. cDNA编码的预测氨基酸序列。Bold, cDNA sequence; Italic, predicted amino acid sequence of cDNA.

    Figure  2.  cDNA sequence and predicted amino acid sequence of Epl1

    图  3  Epl1基因的DNA序列

    Figure  3.  DNA sequence of gene Epl1

    图  4  Epl1基因的氨基酸组成

    Figure  4.  Amino acid composition of Epl1

    图  5  刺激植物响应蛋白Epl1保守区、信号肽及三级结构预测图

    A.Epl1的保守区预测;B.人工神经网络模型(NN)信号肽预测;C.隐马可夫模型(HMM)信号肽预测;D~F.Glu1的三级结构。

    Figure  5.  Prediction image of Epl1 protein conserved region, signal peptide site, phosphorylation site and tertiary structure

    A, prediction of Epl1 protein conserved region; B, signal peptide site predicted by NN; C, signal peptide site predicted by HMM; D-F, tertiary structure of Glu1.

    图  6  重组质粒pGEX-Epl1的检测

    A.PCR检测;B.双酶切检测;M.DNA marker;1~4.转化子质粒的PCR产物;5.转化子质粒的双酶切产物。

    Figure  6.  Detection of recombinant plasmid pGEX-Epl1

    A, PCR detection; B, double enzyme detection; M, DNA marker; 1-4, PCR product of transformant; 5, double enzyme product of transformant.

    图  7  重组蛋白rEpl1的SDS-PAGE检测

    A.不同诱导时间下重组蛋白检测;B.纯化蛋白检测;M.Protein Marker;1和2. IPTG诱导2 h的重组转化子BL21-Epl1上清和沉淀;3和4. IPTG诱导4 h的重组转化子BL21-Epl1上清和沉淀;5和6. IPTG诱导2 h的对照转化子BL21-pGEX上清和沉淀;7和8. IPTG诱导4 h的对照转化子BL21-pGEX上清和沉淀;9.纯化后的重组蛋白。

    Figure  7.  SDS-PAGE detection of recombinant protein rEpl1

    A, detection of recombinant protein under different inducing time; B, detection of purified protein; M, Protein Marker; 1 and 2, the supernatant and precipitate of recombinant BL21-Epl1 by IPTG induced for 2 hours; 3 and 4, the supernatant and precipitate of recombinant BL21-Epl1 by IPTG induced for 4 hours; 5 and 6, the supernatant and precipitate of control BL21-pGEX by IPTG induced for 2 hours; 7 and 8, the supernatant and precipitate of control BL21-pGEX by IPTG induced for 4 hours; 9, purified recombinant protein.

    图  8  rEpl1诱导后山新杨组培苗生长状态

    A为对照;B、C、D分别表示rEpl1诱导质量浓度为0.2、0.5和1 μg/mL。

    Figure  8.  Growth of poplar tissue culture seedlings induced by rEpl1

    A, the control; B-D, the inducing concentration of rEpl1 was 0.2, 0.5 and 1 μg/mL, respectively.

    图  9  rEpl1对山新杨生理指标的影响

    Figure  9.  Effects of rEpl1 on physiology of poplar tissue culture seedlings

    图  10  rEpl1对山新杨生长和防御相关基因的影响

    Figure  10.  Effects of rEpl1 on growth related gene and disease-resistance gene of poplar tissue culture seedlings

    表  1  RT-qPCR引物

    Table  1.   RT-qPCR primers

    基因
    Gene
    基因全称
    Total gene name
    引物
    Primer
    序列(5′-3′)
    Sequence (5′-3′)
    产物大小
    Size of product/bp
    LAX2/AUX Auxin gene LAX2-L TCATTCGGCCTCTTCACCAAGT 213
    LAX2-R GCCATGATTGCCATCACACCTT
    JAR2 Jasmonic acid JAR1-L AGTGGTGAACCAAGCGAGGAG 241
    resistant gene JAR1-R GGATTTGCTGCACCTTGCTGTT
    Tublin β-Tublin gene tu-1 TACCGAGGCTGAGAGTAACAT 245
    tu-2 GGACCCACAACTCATTCACAT
    Ef Elongation factor gene ef-1 TCACACCTGCCACATTGCTGT 230
    ef-2 TCTTGATGACACCAACCGCCAC
    Actin Action gene actin-1 TTCCGTTGCCCTGAGGTCCTAT 239
    actin-2 TCAGGAGGAGCAACCACCTTGA
    下载: 导出CSV

    表  2  Epl1基因的氨基酸组成

    Table  2.   Amino acid composition of Epl1

    氨基酸Amino acid 比例Proportion/%
    Ala (A) 13.0
    Gly (G) 10.1
    Thr (T) 9.4
    Ser (S) 8.7
    Leu (L) 8.7
    Val (V) 7.2
    Asn (N) 6.5
    Asp (D) 5.1
    Gln (Q) 4.3
    Tyr (Y) 4.3
    Arg (R) 2.9
    Cys (C) 2.9
    Phe (F) 2.9
    Trp (W) 2.2
    Lys (K) 2.2
    Pro (P) 2.2
    Met (M) 1.4
    His (H) 1.4
    Glu (E) 0.0
    Sec (U) 0.0
    Pyl (O) 0.0
    Ile (I) 0.0
    Asx (B) 0.0
    Glx (Z) 0.0
    Xaa(X) 0.0
    下载: 导出CSV

    表  3  不同质量浓度rEpl1对山新杨组培苗生物量的影响

    Table  3.   Effects of varied concentration rEpl1 on biomass of poplar tissue culture seedlings

    生物量Biomass 对照Control 0.2 μg/mL 0.5 μg/mL 1 μg/mL
    株高Plant height 5.17±0.309 5.39±0.353 8.03±0.473 10.1±0.396
    根长Root length 1.27±0.866 1.54±0.649 3.23±0.588 4.08±0.411
    叶片数Leaf number 6.7±0.597 7.1±0.563 7.7±0.649 9.1±0.440
    鲜质量Fresh mass 0.988 5±0.708 1.142 5±0.893 1.397 3±0.686 1.616 4±0.459
    干质量Dry mass 0.223 7±2.213 0.184 04±1.103 0.383 5±1.531 0.560 8±0.678
    下载: 导出CSV
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出版历程
  • 收稿日期:  2017-07-19
  • 修回日期:  2017-10-11
  • 刊出日期:  2018-01-01

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