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

doi:10.3864/j.issn.0578-1752.2014.17.003

摘要/Abstract

摘要： 【目的】克隆谷氨酰胺合成酶BnGS2等位基因，分别构建其超量表达载体，并探讨其在转基因烟草中对氮代谢的影响。【方法】依据苎麻转录组unigenes和RT-PCR技术克隆苎麻BnGS2等位基因，利用内切酶TaqⅠ对目的等位基因在中苎1号自交F1和亲本中进行酶切鉴定，并利用生物信息学对基因序列和结构特征进行分析；通过同源重组技术分别构建BnGS2等位基因的超量表达载体，并在农杆菌（Agrobacterium tumefaciens）LBA4404的介导下，通过叶盘法将超量表达载体转入烟草中，通过Kan筛选、转化植株基因组DNA PCR验证获得转基因T0植株；利用qRT-PCR分析BnGS2等位基因在转基因T1植株中的相对表达水平，并测定植株叶片中的GS活性、株高、鲜重、可溶蛋白及总氮含量。【结果】首次从苎麻中克隆了一对GS2等位基因，命名为BnGS2-1和BnGS2-2，等位基因序列全长1 340 bp，含有一个1 293 bp的ORF区，编码430个氨基酸残基多肽；等位基因核苷酸序列在11个位点上存在差异，导致编码的多肽在195、382位点上的氨基酸残基存在替换现象（BnGS2-1为脯氨酸和天冬酰胺，BnGS2-2为苏氨酸和丝氨酸）；NCBI BLASTP分析表明苎麻BnGS2与Pisum sativum、Vigna radiata、Glycine max、Phaseolus vulgaris、Medicago truncatula具有很近的亲缘关系；构建了能分别超量表达BnGS2-1和BnGS2-2的载体，并获得能分别超量表达BnGS2-1和BnGS2-2转基因烟草植株；与野生型烟草植株相比，超量表达BnGS2等位基因（BnGS2-1和BnGS2-2）都能显著性提高转基因植株叶片GS活性、鲜重和可溶性蛋白的含量，株高和总氮含量也有增加，但没有达到显著性水平。另外，超量表达不同BnGS2等位基因（BnGS2-1和BnGS2-2）的转基因烟草植株，在株高、鲜重、叶片可溶性蛋白及总氮含量上并没有显著性差异。【结论】在烟草中分别超量表达来源苎麻的BnGS2等位基因（BnGS2-1或BnGS2-2）均能显著提高转基因植株的生物产量和氮利用效率，并且所克隆的2个等位基因BnGS2-1和BnGS2-2在功能上并无显著差异。

关键词: 苎麻 , GS2等位基因克隆 , 超量表达 , 转基因 , 氮利用效率

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 isolate BnGS2 allele genes which further identified by TaqⅠdigestion in self-bred F1 and 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 through Agrobacterium tumefaciens LBA4404 using “leaf-disk” transformation method into Nicotiana 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 of BnGS2 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】 Two BnGS2 allele 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, named BnGS2-1 and BnGS2-2. The diversity of nucleotide in 11 sites among BnGS2 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 to Pisum sativum, Vigna radiate, Glycine max, Phaseolus vulgaris, and Medicago truncatula. In addition, the over-expression vectors of BnGS2-1 and BnGS2-2 were successfully constructed according to homologous recombination technology and the independent transgenic plants over-expressing BnGS2-1 and BnGS2-2 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 different BnGS2 allele gene (BnGS2-1 or BnGS2-2) did not exhibit significant difference. 【Conclusion】 Over-expression BnGS2 allele genes (BnGS2-1 and BnGS2-2) 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 isolated BnGS2-1 and BnGS2-2.

Key words: Boehmeria nivea L. , GS2 allele genes cloning , over-expression , transgenic , nitrogen use efficiency

郑建树，喻春明，陈平，王延周，谭龙涛，陈继康，朱涛涛，卢凌霄，朱娟娟，段叶辉，熊和平. 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, 47(17): 3348-3358.

ZHENG Jian-shu, YU Chun-ming, CHEN Ping, WANG Yan-zhou, TAN Long-tao, CHEN Ji-kang, ZHU Tao-tao, LU Ling-xiao, ZHU Juan-juan, DUAN Ye-hui, XIONG He-ping. Cloning of Glutamine Synthetase BnGS2 Allele Genes from Ramie (Boehmeria nivea L.) and Study on Gene-Transforming Tobacco[J]. Scientia Agricultura Sinica, 2014, 47(17): 3348-3358.

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