| 摘要: |
| 【目的】克隆苦荞半胱氨酸蛋白酶基因（FtCP）并进行原核和真核表达分析，为探究FtCP与苦荞营养 物质积累与代谢的关系奠定基础。【 方法】提取苦荞灌浆期种子RNA，反转录合成cDNA。采用RT-PCR方法克隆获得FtCP基因，对其进行测序分析。采用半定量RT-PCR检测FtCP基因在苦荞不同组织器官（花、叶、茎、种子）中的表达情况。构建原核表达载体pET47b-FtCP，将其转化原核表达系统BL21（ DE3）中，用异丙基-β-D-硫代半乳糖苷 （IPTG）进行诱导表达，对表达产物进行亲和层析纯化；以纯化的FtCP为抗原，制备特异性多克隆抗体，利用Western blotting分析多克隆抗体的特异性。构建FtCP基因的真核表达载体pPICZ（alpha）A-FtCP，将其转化毕赤酵母表达系统进行诱导表达，对表达产物进行酶活性与酶学特性分析。【结果】FtCP基因开放阅读框（ORF）大小为1 059 bp，编码352个氨基酸，具有保守的序列结构特征。FtCP基因在4种组织中均有表达，但在花、叶中的表达水平高于茎和种 子，表明FtCP的表达具有组织特异性。原核表达获得FtCP蛋白的分子质量约为39.78 ku；所制备的FtCP多克隆抗体能特异性识别苦荞种子中FtCP及其同源蛋白。真核表达的FtCP酶对底物偶氮酪蛋白具有催化活性，酶活性为159 U/mL；FtCP酶最适pH为 6.5，最适温度为55 ℃；苯甲基磺酰氟会显著抑制酶活性，而Mg2+和1% Triton X-100对酶活性无影响。【结论】克隆了FtCP基因序列并进行了异源表达，制备了具有活性的FtCP酶，为阐明苦荞种子蛋 白质营养积累机理提供了试验材料。 |
| 关键词: 苦荞 半胱氨酸蛋白酶 异源表达 酶活性 |
| DOI：10. 13207/j. jnwafu. 2026. 12. 005 |
| 分类号: |
| 基金项目:西安市科技计划项目（25FWQY10）；陕西省教育厅自然科学专项（20JK0876）；西安市科技计划梧桐树科转行动创新团队项目（25WTCY03） |
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| Cloning and heterologous expression of cysteine protease gene from Fagopyrum tataricum |
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LI Meng1, CHEN Peng2, CHEN Peng1
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1.School of Biological and Environmental Engineering，Xi’an University，Xi’an，Shaanxi 710065，China;2.College of Life Sciences， Northwest A&F University，Yangling，Shaanxi 712100，China
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| Abstract: |
| 【Objective】This research aims to clone the cysteine protease gene from Fagopyrum tataricum（FtCP），which plays a crucial role in plant growth and development，and serves as a key enzyme in the regula-tion of protein accumulation in Fagopyrum tataricum seeds.【Method】The FtCP gene was cloned via RTPCR，using cDNA that was reverse-transcribed from RNA extracted from F. tataricum seeds during the filling stage as the template，and subsequently subjected to sequencing analysis. The expression levels of FtCP gene in different tissues and organs of F. tataricum（flower，leaf，stem，and seed） were detected by semi-quantitative RT-PCR. A prokaryotic expression vector pET47b-FtCP was constructed and transformed into E.coli BL21 （DE3） expression system. Isopropyl β-D-1-thiogalactopyranoside（IPTG） was used to induce gene expression，and the expressed product was purified through affinity chromatography.The purified FtCP was used as an antigen to generate specific polyclonal antibodies，and the tissue specificity of the FtCP protein was analyzed by using Western blotting. The eukaryotic expression vector pPICZ（alpha） A-FtCP was constructed，and trans‐formed into the expression system of Pichia pastoris to induce the expression of the gene. The enzyme activity and enzymatic characteristics of the expressed product were analyzed.【Result】The FtCP open reading frame （ORF） was 1 059 bp，encoding 352 amino acids with conserved structural motifs. The FtCP gene was expressed in all four tissues，but the expression levels in flower and leaf were higher than those in stem and seed，indicating that the expression of FtCP was tissue-specific.The prokaryotically expressed FtCP antigen exhibited a molecular mass of 39. 78 ku. The prepared polyclonal antibodies specifically recognized FtCP and its homolo‐gous proteins in F.tataricum seeds，with tissue-specific expression patterns observed. The eukaryotically expressed FtCP enzyme displayed catalytic activity towards the substrate azocasein，with a specific activity rate of 159 U/mL. The optimum pH of FtCP enzyme was 6.5 and optimum temperature was 55 ℃.Phenylmethylsulfo‐nyl fluoride（PMSF） notably inhibited the enzyme activity，whereas magnesium ions and 1% Triton X-100 showed no impact on the enzyme activity.【Conclusion】In this study，the FtCP gene was cloned and an active FtCP enzyme was obtained through heterologous expression，providing the experimental materials for elucidating the mechanism of nutritional accumulation at the protein level in F.tataricum seeds. |
| Key words: Fagogyrum tartaricum cysteine proteinase heterologous expression enzymatic activity |
