铁皮石斛中性/碱性转化酶(DoNI2)基因的克隆和表达分析  

Cloning and expression analysis of a novel alkaline/neutral invertase(DoNI2) gene in Dendrobium officinale

作　　者：苗小荣 牛俊奇 莫昭展 何龙飞[1] 王爱勤[1] MIAO Xiao-rong;NIU Jun-qi;MO Zhao-zhan;HE Long-fei;WANG Ai-qin(Agricultural College,Guangxi University,Nanning 530005,China;College of Biology and Pharmacy,Yulin Normal University,Yulin 537000,China)

机构地区：[1]广西大学农学院,广西南宁530005 [2]玉林师范学院生物与制药学院,广西玉林537000

出　　处：《中草药》2018年第15期3659-3666,共8页Chinese Traditional and Herbal Drugs

基　　金：国家高新技术研究发展计划("863"计划)(2013AA102604);广西科学研究与技术开发计划项目(桂科重1298001-3-1);广西省南宁市重点研发项目(20162094)

摘　　要：目的克隆珍稀濒危兰科药用植物铁皮石斛中性/碱性转化酶(alkaline/neutral invertase,NI)家族基因成员,并对其进行定量表达以及生物信息学分析。方法采用转录组测序和RACE技术相结合克隆NI基因的cDNA序列,利用生物信息学工具对其进行分析,并采用实时荧光定量PCR方法检测NI基因在铁皮石斛根、茎和叶中的表达情况。结果克隆获得1个新的铁皮石斛NI基因,命名为DoNI2(Gen Bank登录号KY794404),全长2 397 bp,开放阅读框(ORF)为1 836 bp,编码611个氨基酸。DoNI2蛋白预测的理论相对分子质量和等电点分别为69 050和6.38,不稳定系数为42.95,疏水性系数为-0.232。DoNI2基因在铁皮石斛根、茎和叶中均有表达,其中在茎中表达量最高,根中最低。不同生长年限茎中DoNI2基因表达量与NI酶活性呈显著正相关。结论克隆了线粒体型的DoNI2基因的全长c DNA序列,为进一步阐明该基因在铁皮石斛蔗糖代谢途径中的重要作用奠定基础。Objective To clone novel member of alkaline/neutral invertase（NI） gene in a rare and endangered medicinal plant of Dendrobium officinale, conduct bioinformatic analysis and detect the quantitative expression in different organs. Methods Primers were designed according to NI gene segment which was selected from leaf transcriptome sequencing results of D. officinale. The full-length c DNA of NI gene was cloned via homology-based cloning and rapid amplification of c DNA ends（RACE） approach. The physical and chemical properties, secondary structure and tertiary structure of NI protein were forecasted and analyzed using related software. The expression levels of NI gene in roots, stems, and leaves of D. officinale were detected using real-time PCR. Results A novel gene encoding a NI protein was cloned from D. officinale. This gene（named as DoNI2, Gen Bank accession number： KY794404） had a total length of 2 397 bp with an open reading frame of 1 836 bp, and encoded a predicted polypeptide of 611 amino acids with a molecular weight of 69 050. Bioinformatics predicted that the isoelectric point of DoNI2 gene encoding protein was 6.38, the instability coefficient was 44.95, and the hydrophobic coefficient was-0.232. RT-PCR showed that DoNI2 gene expressed in all organs with highest expression level in stems and the lowest in roots. DoNI2 gene expression was significantly positively correlation with NI enzymatic activities at different growth years of D. officinale. Conclusion The full length c DNA sequence in a mitochondrial DoNI2 gene was identified, facilitating future functional analysis of the gene involving in the regulation of sugar metabolism in D. officinale.

关 键 词：铁皮石斛 中性/碱性转化酶 基因克隆 基因表达 实时荧光定量PCR 

分 类 号：R282.12[医药卫生—中药学]