Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (6): 1041-1050.doi: 10.3864/j.issn.0578-1752.2014.06.001

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

Cloning and Functional Analysis of a Cotton Nicotinamide Synthetase Gene GbNocotin and Its Promoter

 YANG  Yu-Wen-1, ZHOU  Jian-Wu-1, GAO  Yuan-Yuan-2, CHEN  Tian-Zi-1, ZHANG  Bao-Long-1, NI  Wan-Chao-2   

  1. 1、Institute of Biotechnology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014;
    2、Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014
  • Received:2013-09-05 Online:2014-03-15 Published:2013-12-03

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Abstract: 【Objective】Cloning and characterizing the expression pattern of a cotton nicotinamide synthase gene and its promoter to analyze it’s application in transgenic crop breeding.【Method】 According to the sequencing results of a clone (78L16) of Maxxa BAC, the promoter of a cotton nicotinamide synthase gene GbNocotin was obtained firstly. The online database PLACE was utilized to predict regulatory elements on the promoter. Its recombinant vector pGbNocotin::GUS was constructed and was transformed into Arabidopsis by flower dip method and the transgenic plants was obtained, which were used for GUS staining analysis at the seedling and mature stages. Then the open reading frame (ORF) of GbNocotin was obtained by RT-PCR. Phylogenetic tree was made by mega5.0 and expression analysis in different organs and by stress treatments was conducted by real time-PCR. 【Result】 A specific Fe-deficiency-related element IRO2OS, several stress-responsive such as dehydration, heavy metal and pathogen related elements and phytohormone responsive elements are present in the 1.8Kb promoter region of GbNocotin. GUS staining analysis of transgenic Arabidopsis plants by pGbNocotin::GUS showed that GUS gene was strongly expressed in the root and hypocotyls at the seedling stage, while it was also detected at the base of pods, inflorescence and petioles as well as root in the flowering Arabidopsis plants. GbNocotin has an ORF with 864 nucleotides and encodes a protein of 287 amino acids, its isoelectric point is 6.76 and the predicted molecular weight is 32.7 kD. Although the conserved NAS domain (PFAM accession number: PF03059) was located at amino acid of 11-286, but the similarity of GbNocotin with homogenous genes from other plant is very low, with the highest similarity of ATNAS3 in Arabidopsis is only 43%. GbNocotin exhibits an organ differently expressed pattern. It was strongly expressed in root and stem, moderately expressed in cotton fibers and flowers, in contrast to weakly expression levels in leaves and ovule. In addition, Fe deficiency induced the expression of GbNocotin significantly, as the expression level increased as 9 fold compared to the control plants after one week with the treatment of Fe deficiency. Although the treatments of CuSO4, PEG, abscisic acid (ABA) and gibberellin (GA) all depressed the expression of GbNocotin, but the content varied largely, as the expression all decreased dominantly at 24h by the 4 treatments but the inhibitory effects of ABA and CuSO4 were most significantly. And after 48 h, the expression level by PEG and GA treatments has been restored, while that of the CuSO4 and ABA treatments was still very low.【Conclusion】GbNocotin was differentially expressed in various tissues and organs. It was induced by Fe deficiency and depressed by CuSO4, PEG, ABA and GA treatments. The GbNocotin gene may participate in the absorption and utilization of Fe in cotton and may have potential applications in breeding.

Key words: cotton , nicotinamide synthetase gene , promoter , differences in organ , Fe deficiency induced

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