摘要
目的分别克隆灰毡毛忍冬野生型与湘蕾型ACS3基因,并进行生物信息学和表达模式分析。方法筛选灰毡毛忍冬转录组数据库中与ACS蛋白同源性较高的Unigene序列,设计引物,利用实时荧光定量PCR(qRT-PCR)及RACE技术对该Unigene进行全长克隆;用生物信息学软件分析蛋白的理化性质、结构域和同源性等特性;借助qRT-PCR检测1-氨基环丙烷-1-羧酸合酶(ACS3)基因在不同品种、不同花期灰毡毛忍冬中表达模式。结果从灰毡毛忍冬野生品种及湘蕾品种中分别克隆得Lm-ACS3(GenBank:MH724196)及Lm-XL-ACS3(GenBank:MH724197),开放阅读框长度均为1452bp,编码483个氨基酸,包含保守的Aminotran_1_2结构域,与其他植物的ACC合酶具较高相似度;qRT-PCR结果显示,野生型ACS3基因不同花期间表达差异显著,从花期3开始呈整体明显上升趋势,湘蕾型中花期间表达差异相对较小。结论分别克隆得到灰毡毛忍冬野生型和湘蕾型ACS3基因,该基因在2品种中具不同表达模式;推测ACS3基因或为导致灰毡毛忍冬不同品种间差异表型的可能功能基因之一,为进一步验证该基因在调控蕾期长短及花表型的生物学功能提供实验基础。
Objective To clone ACS3 gene from wild type and ?Xianglei? cultivar of Lonicera macranthoides, respectively, followed by bioinformatics analysis and detection of spatio-temporal expression pattern. Methods Unigene sequence which is highly homologous with ACS protein from the transcriptome database of L. macranthoides was screened, the primers were designed based on it to amplify the full length of the Unigene by qRT-PCR and RACE techniques;Bioinformatics tools were used to analyze and identify the physicochemical property, conserved domain and gene homology of ACS3 proteins;Finally, qRT-PCR technique was used to detect the gene expression patterns of different species of L. macranthoides. Results Lm-ACS3(GenBank: MH724196) and Lm-XL-ACS3(GenBank: MH724197) were isolated from wild type and ?Xianglei? cultivar of L. macranthoides, respectively. The length of open reading frame(ORF) were all 1 452 bp, encoding 483 amino acids, containing the conserved Aminotran12 structural domain, which were highly similar to the ACC synthase of other plants;And qRT-PCR results showsed that the expression quantity of ACS3 gene in wild L. macranthoides changed significantly at different blossoming stages, the overall trend was upward from flowering stage 3, while the expression difference between the flowering stages was relatively small in "Xianglei"cultivar. Conclusion Lm-ACS3 and Lm-XL-ACS3 gene were separately obtained from L. macranthoides and L. macranthoides?Xianglei? cultivar, the expression patterns of ACS3 in this two varieties were different;It?s speculated that ACS3 gene might be a possible functional gene that causing different phenotypes of two strains of L. macranthoides, it provides theoretical basis for further verifying the biological function of ACS3 gene in regulating flower?s bud duration and phenotype.
作者
刘畅宇
陈勋
陈娅
龙雨青
童巧珍
刘湘丹
周日宝
LIU Chang-yu;CHEN Xun;CHEN Ya;LONG Yu-qing;TONG Qiao-zhen;LIU Xiang-dan;ZHOU Ri-bao(College of Pharmacy,Hunan University of Chinese Medicine,Changsha 41020& China;Hunan Traditional Chinese Medicine Piece Standardization and Function Technology Research Center,Changsha 410208,China)
出处
《中草药》
CAS
CSCD
北大核心
2019年第9期2154-2164,共11页
Chinese Traditional and Herbal Drugs
基金
国家自然科学基金资助项目(81673546)
国家自然科学基金资助项目(81203007)
中央引导地方科技发展专项资金项目(2017XF5044)
湖南省自然科学基金资助项目(2017JJ3237)
湖南省教育厅资助科研项目(16B193)
湖南省科技厅重点研发项目(2017SK2124)
国家中医药管理局中药标准化项目(ZYBZH-Y-HUN-24)
湖湘中药资源保护与利用2011协同创新中心项目(湘教通[2015] 351)
湖南中医药大学中药学一流学科项目(校行科字[2018] 3号)
