Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (17): 3348-3358.doi: 10.3864/j.issn.0578-1752.2014.17.003

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES • Previous Articles     Next Articles

Cloning of Glutamine Synthetase BnGS2 Allele Genes from Ramie (Boehmeria nivea L.) and Study on Gene-Transforming Tobacco

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   

  1. Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205
  • Received:2014-03-03 Online:2014-09-01 Published:2014-05-26

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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

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