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付学森,刘紫璇,王玲,龙雨青,曾娟,周日宝,刘湘丹.灰毡毛忍冬MADS-box基因家族鉴定与CMB1基因克隆[J].湖南中医药大学学报,2024,44(3):383-394[点击复制] |
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| 灰毡毛忍冬MADS-box基因家族鉴定与CMB1基因克隆 |
| 付学森,刘紫璇,王玲,龙雨青,曾娟,周日宝,刘湘丹 |
| (湖南中医药大学药学院, 湖南 长沙 410208;湖南中医药大学药学院, 湖南 长沙 410208;湘产大宗道地药材种质资源及规范化种植重点研究室, 湖南 长沙 410208;湖南省普通高等学校中药现代化研究重点实验室, 湖南 长沙 410208;湖南省中药饮片标准化及功能工程技术研究中心, 湖南 长沙 410208) |
| 摘要: |
| 目的 鉴定灰毡毛忍冬Lonicera macranthoides MADS-box家族基因并进行生物信息学分析与表达模式验证，克隆湘蕾型和野生型灰毡毛忍冬MADS-box家族成员CMB1全长。方法 基于转录组数据，利用在线工具对灰毡毛忍冬MADS-box进行生物信息学分析，并利用qRT-PCR验证MADS-box基因在不同品种中的表达模式，通过RT-PCR、RACE技术克隆湘蕾型和野生型灰毡毛忍冬CMB1基因全长。结果 28个灰毡毛忍冬MADS-box蛋白长度为89～359 aa，碱性蛋白占比85.7%，不稳定蛋白占比96.4%，亲水性蛋白占比96.4%，均定位于细胞核，均为无跨膜结构的非分泌蛋白。转录组显示，与野生型比较，湘蕾型中28个MADS-box基因有17.86%表达下调，MIKCC型基因中有18.75%表达下调。克隆得到在两个品种的灰毡毛忍冬花中高度特异性表达的CMB1基因全长，均包含一个738 bp的ORF，编码245个氨基酸。CMB1基因在两个品种的花中表达量存在显著差异（P<0.01），且与茎、叶相比，在花中高度特异性表达（P<0.01）。结论 基于灰毡毛忍冬转录组数据，鉴定了28个MADS-box家族基因，克隆得到湘蕾型与野生型灰毡毛忍冬CMB1基因全长，为进一步研究灰毡毛忍冬花发育、优良表型形成的分子机制提供研究基础与理论依据。 |
| 关键词: 灰毡毛忍冬 MADS-box基因家族 表达分析 CMB1基因 基因克隆 表型变异 |
| DOI：10.3969/j.issn.1674-070X.2024.03.006 |
| 投稿时间：2023-11-27 |
| 基金项目:国家自然科学基金项目（82373992）；湖南省自然科学基金项目（2021JJ30497，2021JJ30515）；国家现代农业产业技术体系（CARS-21）；湖南中医药大学研究生创新训练项目（2022CX79，2023CX152）；2020年湖南省一流专业建设点：中药资源与开发；湖南中医药大学重点学科中药学科（校行发规字〔2023〕2号）。 |
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| Identification of the MADS-box gene family and cloning of the CMB1 gene in Lonicera macranthoides |
| FU Xuesen,LIU Zixuan,WANG Ling,LONG Yuqing,ZENG Juan,ZHOU Ribao,LIU Xiangdan |
| (School of Pharmacy, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;School of Pharmacy, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;Key Research Laboratory of Germplasm Resources and Standardized Planting of Genuine Regional Medicinal Materials Produced in Hunan Province, Changsha, Hunan 410208, China;Key Laboratory of Modern Research of Chinese Medicine, Education Department of Hunan Province, Changsha, Hunan 410208, China;Hunan Engineering Technology Research Center for Standardization and Function of Chinese Herbal Decoction Pieces, Changsha, Hunan 410208, China) |
| Abstract: |
| Objective To identify the MADS-box gene family of Lonicera macranthoides, analyze its bioinformatics, validate the expression patterns, and clone the full-length sequences of CMB1, a member of the MADS-box family of Lonicera macranthoides of Xianglei and wild types. Methods Based on the transcriptome data, the bioinformatics of Lonicera macranthoides MADS-box was analyzed with online tools, the expression patterns of MADS-box genes in different cultivars were verified using qRT-PCR, and the full-length sequences of Lonicera macranthoides CMB1 gene of the Xianglei and wild types were cloned by RT-PCR and RACE techniques. Results The sequence lengths of 28 MADS-box proteins of Lonicera macranthoides ranged from 89 aa to 359 aa, with alkali proteins accounting for 85.7%, unstable proteins accounting for 96.4%, and hydrophilic proteins accounting for 96.4%, which were localized in the nucleus, and were non-secretory proteins without transmembrane structure. The transcriptome showed that compared with the wild type, 17.86% of the 28 MADS-box genes in the Xianglei type and 18.75% of the genes of MIKCC type were downregulated. The full-length sequences of CMB1 genes, which were highly specifically expressed in two varieties of flowers of Lonicera macranthoides, were cloned. Both of them contained a 738 bp ORF, encoding 245 amino acids. There was a significant difference in expression level of CMB1 gene between the two varieties of flowers (P<0.01), and it was highly and specifically expressed in flowers compared to that in stems and leaves (P<0.01). Conclusion Based on the transcriptome data of Lonicera macranthoides, 28 MADS-box family genes were identified, and the full-length sequences of CMB1 gene of Xianglei and wild types were cloned, which provides the research foundation and theoretical basis for the further study of molecular mechanism of development and superior phenotype formation of Lonicera macranthoides. |
| Key words: Lonicera macranthoides MADS-box gene family expression analysis CMB1 gene gene clone phenotypic variation |
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