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Acta Agron Sin ›› 2011, Vol. 37 ›› Issue (01): 28-39.doi: 10.3724/SP.J.1006.2011.00028

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

Isolation, Chromosome Assignment, and Expression Assay of Nitrite Reductase Gene and Regulatory Sequence in Wheat

SHE Mao-Yun1,CHEN Duo-Duo2,FENG Chen3,DU Li-Pu1,YE Xing-Guo1,*   

  1. 1 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences / National Key Facility of Crop Gene Resources and Genetic Improvement / Key Laboratory of Crop Genetic and Breeding, Ministry of Agriculture, Beijing 100081, China; 2 Institute of Life Science, Wuhan University, Wuhan 430072, China; 3 Beijing City University, Beijing 100083, China
  • Received:2010-07-19 Revised:2010-10-05 Online:2011-01-12 Published:2010-11-12
  • Contact: YE Xing-Guo, E-mail: yexg@mail.caas.net.cn, Tel: 010-82109765

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Abstract: Nitrite reductase is wildly involved in N metabolism in plants and has shown excellent relevance to regeneration potential in the tissue culture of rice (Oryza sativa L.). In this study, in silico and inverse PCR techniques were employed to isolate nitrite reductase encoded gene and its regulatory sequences from wheat (Triticum aestivum L.). This gene was predicted to contain four exons and three introns. The gDNA and cDNA sequences were 2 881 bp and 1 830 bp in length, respectively, and they were both submitted to GenBank under the accession numbers FJ555239 and FJ527909. Its deduced encoding protein product was approximately 65.7 kD, sharing high (more than 60%) identity with other nitrite reductase genes deposited in the NCBI database, especially with those from other monocot cereal crops (more than 80%). The 5′ flanking region was isolated and extended to -2 924 bp (counting from the start code: ATG) through inverse PCR. After induced by 1 mmol L-1 IPTG, a protein of ca. 70 kD was obtained in prokaryotic expression vector pET-28a, including a histidine tag of ca. 3.8 kD. The highest expression of gene in wheat seedlings was induced by 30 mmol L-1 KNO3 for 1 h. Measurement on nitrite reductase activity showed the enzyme activity increased following the treatment time extension at 5-d intervals under the above same condition. According to the result of AS-PCR analysis, at least one copy of the gene existed on chromosome 6A and 6B each in common wheat. The study provides a basis for functional determination of wheat nitrite reductase gene in subsequent research.

Key words: Wheat, Nitrite reductase, Expression assay, Promoter, Chromosome assignment

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