灰毡毛忍冬AP1同源基因的克隆及互作蛋白鉴定  

Cloning and interacted protein identification of AP1 homologous gene from Lonicera macranthoides

作　　者：俞亚欣 龙丽君 李昌珠[2] 曾慧杰[2] 乔中全[2] 刘思思[2] 马英姿[1] YU Ya-xin;LONG Li-jun;LI Chang-zhu;ZENG Hui-jie;QIAO Zhong-quan;LIU Si-si;MA Ying-zi(College of Life and Environmental Sciences,Central South University of Forestry and Technology,Changsha 410004,China;State Key Laboratory of Utilization of Woody Oil Resource,Hunan Academy of Forestry,Changsha 410004,China)

机构地区：[1]中南林业科技大学生命与环境科学学院,湖南长沙410004 [2]湖南省林业科学院省部共建木本油料资源利用国家重点实验室,湖南长沙410004

出　　处：《药学学报》2024年第10期2880-2888,共9页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金项目(82304683);湖南省自然科学基金项目(2022JJ30326);湖南省重大科技创新平台项目(2023PT1001)。

摘　　要：MADS-box家族基因是一类十分重要的转录调控基因,在植物的整个生长发育过程中发挥重要作用。其中家族成员APETALA1(AP1)基因不仅在植物开花转变过程中发挥调控作用,还控制着花器官的特征发育。灰毡毛忍冬以干燥花蕾和初开的花入药,因此研究AP1基因参与调控灰毡毛忍冬花器官发育的潜在机制可以为分子手段提高其药用价值提供基础。本研究采用逆转录PCR(RT-PCR)对AP1同源基因的cDNA全长进行扩增,并将其命名为LmMADS4。结果显示,LmMADS4基因CDS序列长729 bp,编码242个氨基酸,且LmMADS4蛋白不含信号肽,无跨膜结构,为不稳定的亲水性蛋白。通过序列比对及系统进化分析,LmMADS4与忍冬MADS27蛋白聚为一类,亲缘关系最近。最后利用实时荧光定量逆转录聚合酶链式反应(qRT-PCR)、酵母双杂交技术对LmMADS4基因的表达模式、蛋白间的相互作用进行了分析。qRT-PCR结果表明,LmMADS4基因在灰毡毛忍冬花蕾型龙花和野生型白云的茎、叶以及花蕾不同发育时期均有差异表达;其中LmMADS4基因主要在龙花和白云生殖器官花蕾中高表达,且随着灰毡毛忍冬花蕾的发育,LmMADS4基因在花蕾型品种龙花中呈现持续上调表达的趋势,而在白云花蕾末期的表达水平较花蕾晚期有所降低,但差异不显著。酵母双杂交结果表明,构建的诱饵载体pGBKT7-LmMADS4对酵母菌株无毒性,也无自激活活性,LmMADS4蛋白与LmSVP1蛋白、LmSVP3蛋白和LmSOC1s蛋白间均存在相互作用。该研究可为分子层面探索灰毡毛忍冬蕾期长和花冠不展开的作用机制以及品种改良提供理论依据。The MADS-box gene family is a very important transcriptional regulator gene,which plays a role in the whole growth and development process of plants.The APETALA1(AP1)gene is considered to play an important regulatory role in the transformation of plant flowering,but also to control the characteristic development of floral organs.Lonicera macranthoides is used as medicine with dry buds and early flowers.Therefore,studying the potential mechanism of AP1 gene in regulating flower organ development can provide a basis for improving its medicinal value by molecular means.To explore the potential mechanism of the AP1 gene in the regulation of floral organ development in L.macranthoides,the full-length cDNA of the AP1 was cloned by reverse transcription PCR(RT-PCR)and named LmMADS4.The results show that the CDS of the LmMADS4 gene is 729 bp and encodes 242 amino acids,and the LmMADS4 protein contains no signal peptide and no transmembrane structure,which is an unstable hydrophilic protein.Through homologous sequence alignment and phylogenetic analysis,LmMADS4 and L.japonica MADS27 protein cluster into one class and are closely related.Finally,the expression pattern and protein interaction pattern of LmMADS4 were analyzed by real-time reverse transcription-PCR(qRT-PCR)and yeast two-hybrid technology.The qRT-PCR showed that LmMADS4 gene was differentially expressed in the stems,leaves and flower bud at different developmental stages,including bud type variety Longhua and common variety Baiyun;and LmMADS4 gene was highly expressed in the flower buds,and with the development of flower buds,LmMADS4 gene was continuously up-regulated in the flower bud variety Longhua,however,the expression level of LmMADS4 in the Baiyun terminal flower bud was lower than that in the late flower bud,but the difference was not significant.The yeast two-hybrid results showed that the bait vector pGBKT7-LmMADS4 was not toxic to yeast strains and had no self-activating activity.LmMADS4 protein interacted with LmSVP1,LmSVP3 and LmSOC1s proteins.Thi

关 键 词：灰毡毛忍冬 MADS-BOX 基因克隆 花器官 蛋白互作 

分 类 号：R931[医药卫生—生药学]