| 胡国豪1, 杨子平2, 3, 刘 铜1, 张荣意1, 侯巨梅1*.橡胶树棒孢霉落叶病菌酵母单杂交cDNA文库及CcCas5启动子诱饵载体的构建[J].植物保护,2024,50(5):205-212. |
| 橡胶树棒孢霉落叶病菌酵母单杂交cDNA文库及CcCas5启动子诱饵载体的构建 |
| Construction of a yeast one-hybrid cDNA library and CcCas5 promoter bait vector of Corynespora cassiicola causing leaf fall disease of Hevea brasiliensis |
| 投稿时间：2023-10-09 修订日期：2023-12-05 |
| DOI：10.16688/j.zwbh.2023515 |
| 中文关键词: 橡胶树 多主棒孢 CcCas5 启动子 酵母单杂交 cDNA文库 |
| 英文关键词:Hevea brasiliensis Corynespora cassiicola CcCas5 promoter yeast one-hybrid cDNA library |
| 基金项目:国家自然科学基金 (32060589) |
| 作者 | 单位 | E-mail | | 胡国豪1, 杨子平2, 3, 刘 铜1, 张荣意1, 侯巨梅1* | 1. 海南大学热带农林学院, 海南省农用生物制剂创制工程研究中心, 海口 570228
2. 中国热带农业科学院南亚热带作物研究所, 湛江市热带作物遗传改良重点实验室, 湛江 524091
3. 中国热带农业科学院三亚研究院, 三亚 572025 | amyliutong@163.com |
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| 中文摘要: |
| 为了筛选出与CcCas5启动子结合的转录因子, 本研究构建了橡胶树棒孢霉落叶病菌的酵母单杂交cDNA表达文库和CcCas5启动子诱饵载体, 并对3-氨基-1, 2, 4-三唑 (3-amino-1, 2, 4-triazole, 3-AT)的最佳抑制浓度进行了筛选。结果显示, 构建的酵母单杂交cDNA表达文库总容量为3.49×106 cfu, 插入片段主要分布于750~2 000 bp之间, 重组率为95.8%; 克隆了CcCas5基因2 600 bp的启动子序列, 预测获得真菌主要的10大类转录因子的结合位点165个; 构建了分别携带1 500 bp和1 000 bp启动子序列的酵母单杂交启动子诱饵载体, 10 mmol/L的3-AT能抑制pHis2.1-pCcCas5-1000和pHis2.1-pCcCas5-1500的背景表达。试验结果为通过酵母单杂交筛选与CcCas5启动子结合的转录因子和进一步研究转录因子对CcCas5表达的调控作用奠定基础。 |
| 英文摘要: |
| In order to screen the transcription factors that bind to the CcCas5 promoter, a yeast one-hybrid cDNA expression library of Corynespora cassiicola and a CcCas5 promoter bait vector were constructed, and the optimal inhibitory concentration of 3-amino-1, 2, 4-triazole (3-AT) was screened in this study. The results showed that the total capacity of the constructed yeast one-hybrid cDNA expression library was 3.49×106 cfu, the inserted fragments mainly distributed between 750 to 2 000 bp, and the recombination rate was 95.8%. The 2 600 bp promoter sequence of CcCas5 was cloned and the binding sites of 165 main fungal transcription factors in 10 families were predicted. Yeast one-hybrid promoter bait vectors carrying 1 500 bp and 1 000 bp promoter sequences were constructed, respectively, and 10 mmol/L of 3-AT could inhibit the background expression of pHis2.1-pCcCas5-1000 and pHis2.1-pCcCas5-1500. The above results lay the foundation for screening transcription factors that bind to the CcCas5 promoter through yeast one-hybrid screening and further studying the regulatory effects of transcription factors on CcCas5 expression. |
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