中国农业科学Issue(17)：3348-3358,11.
苎麻谷氨酰胺合成酶BnGS2等位基因的克隆及其转基因烟草特性
Cloning of Glutamine Synthetase BnGS2 Allele Genes from Ramie (Boehmeria nivea L.) and Study on Gene-Transforming Tobacco
摘要
Abstract
Objective] The objective of this paper is to clone and construct the over-expression vector of two glutamine synthetase (BnGS2) allele genes to study their effects on nitrogen metabolism of transgenic tobacco plants.[Method] The ramie transcriptome unigenes and RT-PCR were used to isolateBnGS2 allele genes which further identified byTaqⅠdigestion in self-bred F1and parents. Sequence and structure were analyzed by bioinformatics. In addition,BnGS2 allele genes over-expression vector was constructed respectively according to homologous recombination technology and transformed throughAgrobacterium tumefaciensLBA4404 using “leaf-disk” transformation method intoNicotiana tabacum. The transgenic T0 plants were verified by Kan screening and DNA PCR determination. The qRT-PCR was used for determining the relative expression levels ofBnGS2 allele genes in T1 transgenic tobacco plants as well as the leaf GS activity, fresh weight, plant height, leaf soluble protein and total nitrogen content of transgenic plants were determined.[Result] TwoBnGS2allele genes with length of 1 340 bp containing 1 293 bp ORF region encoded polypeptide of 430 amino acids were isolated for the first time from ramie, namedBnGS2-1 and BnGS2-2. The diversity of nucleotide in 11 sites amongBnGS2 allele genes resulted in amino acid residues substitution at sites 195 and 382 (Pro-195 and Asp-382 in BnGS2-1, Thr-195 and Ser-382 in BnGS2-2). The NCBI Blastp analysis displayed that BnGS2 was close toPisum sativum,Vigna radiate,Glycine max,Phaseolus vulgaris,andMedicago truncatula. In addition, the over-expression vectors ofBnGS2-1 andBnGS2-2 were successfully constructed according to homologous recombination technology and the independent transgenic plants over-expressingBnGS2-1 andBnGS2-2 were obtained, respectively. Compared with wild type plants, the transgenic plants exhibited significant increase in leaf GS activity, fresh weight and leaf soluble protein content. The plant height and leaf total nitrogen content were slightly higher but not reached significant levels. However, these parameters between the transgenic plants with over-expression differentBnGS2 allele gene (BnGS2-1 orBnGS2-2) did not exhibit significant difference.[Conclusion] Over-expression BnGS2 allele genes (BnGS2-1 andBnGS2-2) in the transgenic plants respectively resulted in a higher biomass and enhanced nitrogen use efficiency. In addition, there was no obvious difference in function between isolatedBnGS2-1 andBnGS2-2.关键词
苎麻/GS2等位基因克隆/超量表达/转基因/氮利用效率Key words
Boehmeria nivea L./GS2 allele genes cloning/over-expression/transgenic/nitrogen use efficiency引用本文复制引用
郑建树,段叶辉,熊和平,喻春明,陈平,王延周,谭龙涛,陈继康,朱涛涛,卢凌霄,朱娟娟..苎麻谷氨酰胺合成酶BnGS2等位基因的克隆及其转基因烟草特性[J].中国农业科学,2014,(17):3348-3358,11.基金项目
国家“十一五”科技支撑计划 （）
