摘要
虎杖(Polygonum cuspidatum)是蓼科蓼属的多年生草本植物,具有重要的药用价值,作为传统中药,在临床上有广泛应用。O-甲基转移酶在植物次生代谢产物合成过程中发挥重要作用,通过对虎杖转录组数据库的分析,获得O-甲基转移酶基因的序列信息。提取生长8周的虎杖叶片的mRNA,经反转录获得cDNA。设计特异性引物进行PCR扩增,获得虎杖O-甲基转移酶基因全长序列,命名为PcOMT。PcOMT基因全长1116 bp,编码371个氨基酸,蛋白分子量为40.95 KDa。序列分析显示,该蛋白具有SAM结合位点等O-甲基转移酶特有的保守结构域。系统发育树证实,其与葡萄等植物亲缘关系较近。利用重叠延伸PCR将PcOMT基因序列与eGFP基因连为融合蛋白,并通过同源重组将该序列连接至植物表达载体。将PcOMT基因转入拟南芥并进行烟草的瞬时表达,亚细胞定位结果表明PcOMT-eGFP融合蛋白定位于烟草和拟南芥细胞质中,组织表达模式显示,PcOMT为组成型表达基因,在各组织中均有表达,在茎中表达水平最高。非生物胁迫情况下,其表达水平均有增加,显示其与植物抗逆有关。为深入研究虎杖-O-甲基转移酶的功能奠定基础,为改造虎杖品质提供基因资源。
Polygonum cuspidatum was an herbaceous perennial plant of the Polygonaceae family and has significant medicinal values.,widely used in clinical practice as Traditional Chinese Medicine.O-methyltransferase(OMT)played important roles in the synthesis of plant secondary metabolites in plants.The sequence information of the O-methyltransferase gene was obtained by analyzing the transcriptome database of Polygonum cuspidatum.The mRNA of the leaves of 8 weeks old Polygonum cuspidatum was extracted and then cDNA was obtained by reverse transcription PCR.Specific primers of OMT were designed and used for PCR amplification to obtain the full-length sequence of Polygonum cuspidatum O-methyltransferase gene(PcOMT).The PcOMT gene had 1116 bp and encoding 371 amino acids.The protein molecular weight of PcOMT was 40.95KDa.Sequence analysis showed that the protein had conserved O-methyltransferase-specific domain and including SAM binding sites.The phylogenetic tree confirmed that it was closely related to grapes and other plants.The gene sequence of PcOMT and eGFP gene were connected as a fusion gene by overlap extension PCR.Then,the fused sequence was ligated to the plant expression vector PBI121 by homologous recombination.The PcOMT gene was transferred into Arabidopsis and transiently expressed in tobacco.The subcellular localization results showed that PcOMT-eGFP fusion protein was localized in the cytoplasm of tobacco and Arabidopsis.The tissue expression pattern showed that PcOMT was a constitutively expressed gene in various tissues.The highest expression level was found in stem.Under the condition of abiotic stress,the expression level increased significantly,showed that it was related with plant resistance to environment.,which gave some suggestions for future study of functions of Polygonum cuspidatum O-methyltransferase and provided genetic resources for improving the plant quality of Polygonum cuspidatum.
作者
吴志军
王晓伟
陈默
胡红艳
陶玲玲
徐淑艳
张华
曹宏伟
王红
Wu Zhijun;Wang Xiaowei;Chen Mo;Hu Hongyan;Tao Lingling;Xu Shuyan;Zhang Hua;Cao Hongwei;Wang Hong(College of Life Science and Biotechnology,Heilongjiang Bayi Agricultural University,Daqing,163319;College of Life Sciences,University of Chinese Academy of Sciences;Research Center of Academic Theory,Heilongjiang Bayi Agricultural University;College of Science,Heilongjiang Bayi Agricultural University)
出处
《黑龙江八一农垦大学学报》
2021年第3期65-71,78,共8页
journal of heilongjiang bayi agricultural university
基金
国家自然科学基金面上项目(NSFC:61672489和NSFC:61972374)。
关键词
虎杖
中药
O-甲基转移酶
克隆
表达水平
Polygonum cuspidatum
Traditional Chinese Medicine
O-Methyltransferase
clone
expression level.
