| 摘要: |
| 为探究GbHCT14基因CDS区及启动子序列在棉花纤维发育中的作用，通过中国农业科学院棉花研究所数据库检索到GbHCT14基因CDS区及上游非编码区-2 000 bp片段序列，克隆CDS区及启动子，并检测启动子活性。构建cDNA文库，利用酵母单杂交技术筛选阳性克隆，获得候选转录因子，并进行生物信息学分析。结果表明:1)GbHCT14基因的CDS区全长1 144 bp，其翻译产物为亲水性稳定蛋白，预测定位在叶绿体，没有信号肽以及跨膜结构域。2)GbHCT14基因启动子预测到5个MYB结合位点参与植物苯丙烷代谢途径的调节，1个TCA-element参与水杨酸调节，6个ABA响应元件。3)GbHCT14启动子能够驱动GUS蛋白表达，具有启动活性。4)酵母单杂交初步筛选出16个候选基因，包括Gbar_D01G020710、Gbar_A09G017360、Gbar_A09G009680、Gbar_A11G003660、Gbar_A12G004430、Gbar_D04G021210、GbM_D09G1212、GbM_A11G0186、GbM_A03G0171、GbM_A09G1247、GbM_D10G0411、GbM_D10G1024、GbM_D02G1379、GbM_A11G1190、GbM_D12G0471和genome_Gbar-ZJU_D08。利用生物信息学分析预测表明，上述候选基因参与棉花纤维细胞壁的伸长、泛素化反应、细胞分裂、花发育以及果实成熟的过程，其中GbM_A09G1247与WAK2为相似基因，WAK2功能蛋白与果胶结合后促进细胞壁的伸长，而HCT家族基因则是影响果胶合成的关键因素之一。5)GbHCT14和WAK2基因在棉花开花后25 d内高表达，两者在棉花纤维发育调控存在相关性。综上，GbHCT14基因CDS区及启动子序列为首次获得，GbHCT14基因启动子具有启动活性，并且筛选到与裂合酶、转移酶、植物激素、质子泵和功能蛋白相关的16个上游调控的候选转录因子。 |
| 关键词: 海岛棉 HCT基因 启动子 转录因子 |
| DOI：10.11841/j.issn.1007-4333.2023.09.03 |
| 投稿时间：2022-11-03 |
| 基金项目:新疆维吾尔自治区重大科技专项(2021A02001-3)；国家育种攻关项目(2022MH-01) |
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| Cloning of GbHCT14 gene promoter and screening of upstream regulators in Gossypium barbadense |
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ZHU Yuting1,YANG Wenjun1,WANG Chaoyue1,LIU Chenxi1,CHEN Quanjia1*,ZHENG Kai2*
|
| (1.College of Agronomy/Xinjiang Key Laboratory of Crop Genetic Improvement and Germplasm Innovation, Xinjiang University of Agriculture, Urumqi 830000, China;2.Hainan Yazhou Bay Seed Laboratory, Sanya 572025, China) |
| Abstract: |
| To investigate the role of the CDS region and promoter sequence of the GbHCT14 gene in cotton fiber development, the CDS region and the upstream non-coding region of the GbHCT14 gene -2 000 bp were retrieved from the database of the Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Cotton. fragment, clone the CDS region and promoter, and detect the promoter activity. The cDNA library was constructed, positive clones were screened using yeast single hybridization technique to obtain candidate transcription factors, and bioinformatics analysis was performed. The results showed that: 1)The CDS region of GbHCT14 gene is 1 144 bp long, and its translation product is a hydrophilic stable protein predicted to be localized on chloroplasts without signal peptide and transmembrane structural domains. 2)The promoter of GbHCT14 gene predicts five MYB binding sites involved in the regulation of plant phenylpropane metabolic pathway, one TCA-element involved in salicylic acid regulation, and six ABA response elements. 3)The GbHCT14 promoter is able to drive GUS protein expression with initiation activity. 4)Yeast single hybridization initially screened 16 candidate genes, including Gbar_D01G020710, Gbar_A09G017360, Gbar_A09G009680, Gbar_A11G003660, Gbar_A12G004430, Gbar_D04G021210, GbM_D09G1212, GbM_A11G0186, GbM_A03G0171, GbM_A09G1247, GbM_D10G0411, GbM_D10G1024, GbM_D02G1379, GbM_A11G1190, GbM_D12G0471 and genome_Gbar-ZJU_D08. Using bioinformatics analysis, these candidate genes were predicted to be involved in fiber cell wall elongation, ubiquitination response, cell division, flower development, and fruit ripening, with GbM_A09G1247 being a similar gene to WAK2, and the WAK2 functional protein binding to GbM_A09G1247 and WAK2 are similar genes, and WAK2 functional protein binds to pectin to promote cell wall elongation, while HCT family genes are one of the key factors affecting pectin synthesis. 5)GbHCT14 and WAK2 genes are highly expressed in cotton within 25 d post-anthesis, and there is a correlation between them for the regulation of cotton fiber development. In summary, the CDS region and promoter sequence of GbHCT14 gene were obtained for the first time, and the promoter of GbHCT14 gene was identified to have driver activity, and 16 candidate transcription factors related to lytic enzymes, transferases, phytohormones, proton pumps and functional proteins were screened. |
| Key words: Gossypium barbadense HCT gene promoter transcription factor |
