| 摘要: |
| 【目的】克隆西瓜（Citrullus lanatus (Thunb.) Matsum. et Nakai）渗调蛋白基因ClOsm，明确该基因编码蛋白的抑菌活性，为进一步研究ClOsm基因功能并开展西瓜抗病分子育种奠定基础。【方法】采用同源克隆法，克隆西瓜ClOsm基因，分析其序列特征，并构建系统进化树。利用实时荧光定量PCR（RT-PCR）技术分析ClOsm基因的表达特征。利用原核表达系统表达并纯化His-ClOsm重组蛋白，检测其对枯萎病菌、蔓枯病菌、白粉病菌、茄病镰刀菌和轮枝镰刀菌的体外抑菌活性。【结果】从枯萎病菌侵染的西瓜根系组织中克隆到渗调蛋白基因ClOsm（GenBank登录号为MF445019），其长度为768 bp，编码255个氨基酸，预测蛋白质分子质量为27.52 ku，等电点为7.78。氨基酸结构分析显示，ClOsm含有TLP蛋白的保守结构域。系统进化分析表明，ClOsm与冬瓜BhOsm蛋白的亲缘关系最近，相似度达95%，且归为osmotin类型的类甜蛋白。实时荧光定量分析显示，ClOsm基因在西瓜根、茎、叶组织中均有表达，根中表达量最高；枯萎病菌侵染能够显著诱导ClOsm基因的表达，在侵染120 h时表达量最高。融合蛋白His ClOsm体外抑菌活性检测结果表明，ClOsm蛋白可以显著抑制枯萎病菌、蔓枯病菌、白粉病菌、茄病镰刀菌和轮枝镰刀菌等病原菌的生长，抑制率分别为70.2%,60.8%,65.3%,55.4%和46.6%。【结论】克隆了西瓜ClOsm基因，并纯化得到ClOsm重组蛋白，该蛋白能够显著抑制病原真菌的生长。 |
| 关键词: 西瓜 渗调蛋白 基因表达 抗病育种 抑菌活性 |
| DOI： |
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| 基金项目:江苏农林职业技术学院科技项目（2020kj064） |
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| Cloning and antifungal activity of osmotin like protein gene ClOsm from watermelon |
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HUANG Xiaozhong,WEI Xiang,XU Xiaoyan
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| Abstract: |
| 【Objective】This work cloned the osmotin like gene (ClOsm) from watermelon and analyzed its antifungal activity to provide basis for further functional research and disease resistant molecular breeding.【Method】Based on the proteomic data of watermelon roots under fusarium oxysporum infection,the complete coding sequence of ClOsm was cloned using PCR.The sequence properties were analyzed before constructing phylogenetic tree.The real-time fluorescence quantification technique was employed to analyze the expression pattern of ClOsm gene.The recombinant protein His ClOsm was prokaryotically expressed and purified,and its antifungal activity Fusarium oxysporum,Ascochyta citrullina,Podosphaera xanthii,Fusarium solani and Fusarium verticillium was determined in vitro.【Result】An osmotin like protein gene named ClOsm (accesstion No.MF445019) was cloned from watermelon in this study.It contained 768 nucleotides and encoded a putative polypeptide of 255 amino acids with a calculated molecular mass of 27.52 ku and a theoretical isoelectric point of 7.78.Sequence alignment showed that ClOsm contained the conserved sixteen cysteines motif.Phylogenetic analysis indicated that ClOsm belonged to the osmotin cluster,and was close to Benincasa hispida Osm gene with sequence identity of 95%.Real-time PCR analysis revealed that ClOsm expressed in all examined tissues,with the highest in roots.Expression profiles of ClOsm under Fusarium oxysporum inoculation revealed that transcriptional level of ClOsm was significantly up regulated at 120 h.ClOsm was further constructed to pET 28a(+) vector to obtain a recombined His-ClOsm protein.Antifungal activity analysis showed that the His ClOsm protein could significantly inhibited growth of Fusarium oxysporum,Ascochyta citrullina,Podosphaera xanthii,Fusarium solani and Fusarium verticillium.The inhibition ratio was 70.2%,60.8%,65.3%,55.4% and 46.6%,respectively.【Conclusion】The ClOsmgene was cloned from watermelon and the recombinant ClOsm protein could significantly inhibited growth of different fungal pathogens. |
| Key words: watermelon osmotin-like protein gene expression disease resistant and breeding antifungal activity |
