农业生物技术学报2018，Vol.26Issue(2)：213-221,9.DOI:10.3969/j.issn.1674-7968.2018.02.004
枸杞Lb14-3-3a基因过表达载体的构建及转化马铃薯研究
Construction of Overexpression Vector of Lb14-3-3a Gene from Wolfberry (Lycium barbarum) and Study of Genetic Transformation to Potato (Solanum tuberosum)
摘要
Abstract
14-3-3 proteins play important regulated roles in plant growth,development,stress physiology and starch metabolism.Prerious studies have found a differentially expressed 14-3-3 protein in development anther of wolfberry (Lycium barbarum).In order to investigate the regulation of 14-3-3 protein on the starch metabolism during the anther development of L.barbarum,A differentially expressed 14-3-3 protein gene was isolated from the anther of wolfberry cultivar ‘Ning qi No.1’,named as Lb14-3-3a (GenBank accession No.MG189703) and the protein structure was predicted.Furthermore,The plant overexpression vectors of the gene were constructed,and the functions of Lb14-3-3a in potato (Solanum tuberosum) were studied.The full-length cDNA of Lb14-3-3a was determined by RT-PCR technique.The phylogenetic tree of 14-3-3 proteins was constructed using a neighber-joining method with the MEGA5.1 program (http://www.megasoftware.net/).The image of phylogenetic tree of 14-3-3 proteins which was drawn in MEGA5.1 Homology researches of the NCBI (National Center of Biotechnology Information) databases were performed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST) with default parameters.The plant overexpression vector pMDC83-Lb14-3-3a was constructed using gateway technology.The pMDC83-Lb14-3-3a was transformed into the potato cultivar ‘Zihuabai’ by Agrobacterium mediated method.The positive seedling was selected by PCR and the starch content in leaves at different growth stages was determined by spectrophotometric method.Biological information analysis indicated that the gene belonged to the 14-3-3 protein gene family.The opened reading frame (ORF) of Lb14-3-3a gene was 768 bp long and its encoded protein contained 256 amino acid residues,the molecular weight was 61.9 kD and the isoelectric point was 4.99,which had a conserved domain of 14-3-3 protein family.Lb14-3-3a was associated with tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum) on the same branch of the phylogenetic tree.Positive transgenic plant was obtained by resistance screening and PCR identification.Under the strong promoter of CaMV35s (35S promoter of cauliflower mosaic virus),the exogenous gene Lb14-3-3a stably expressed in potato.Determination of starch content showed that the starch content of wild type plants had little difference with genetically modified plant,but starch content of tuber stage and mature stage of the transgenic plant leaf were higher than that of wild type.It is speculated that the Lb14-3-3a gene may affect the synthesis of potato starch.The result provides a reference for further studies on gene function of Lb14-3-3a on the accumulation and hydrolysis of starch during anther development of wolfberry.关键词
枸杞/Lb14-3-3a/过表达载体/转基因/马铃薯/淀粉含量Key words
Lycium barbarum/Lb14-3-3a/Overexpression vector/Transgene/Potato/Starch content分类
农业科技引用本文复制引用
周丽,岳思君,徐蕊,徐惠娟,丁向真,罗惠霞,姚新灵,郑蕊..枸杞Lb14-3-3a基因过表达载体的构建及转化马铃薯研究[J].农业生物技术学报,2018,26(2):213-221,9.基金项目
国家自然科学基金(No.31360361,No.31560418和No.31360025) （No.31360361,No.31560418和No.31360025）
