Abstract: 【Objective】 The objective of this study is to investigate the relationship between nitrate reductase (NR) and growth and regeneration of mulberry. A full length cDNA named MaNR was cloned from mulberry cultivar Guiyou No.62 and the sequence characteristics of MaNR were analyzed. Furthermore, the expression mode and influence factors during regeneration of mulberry hypocotyl regeneration in vitro was studied.【Method】 Specific primer sequences were designed according to the annotated gene scaffold 570 released in Morus notabilis genome database http://morus.swu.edu.cn/morusdb. Total RNA and DNA were extracted from mulberry leaf treated by KNO3, using RNAiso Plus (TaKaRa) and a modified CTAB method. cDNA and DNA were used as templates, RT-PCR was used to amplify MaNRcDNA sequence from RNA pools generated from KNO3-treated mulberry leaves. Bioinformatical tools were employed to analyze its deduced aimino acid sequence. Mulberry hypocotyls used as explants were inoculated in different nitrogen sources and growth regulating substances of the medium under aseptic conditions. Real-time PCR was used to investigate its relative expression level during hypocotyls regeneration in vitro and effects of different nitrogen sources and growth regulating substances. 【Result】 The obtained cDNA sequence of NR was 2 730 bp in length, encoding a polypeptide of 909 amino acid residues consisting of five classic conserved domain, protein molecular weight was 102.84 kD and the isoelectric point was 6.76. It showed that the sequence of NR has 95% identity compared with Morus notabilis and 78% indentity with other Rosaceae trees by NCBI BLAST. The obtained DNA sequence of NR was 5 142 bp including five exons and four introns. Cluster analysis showed that monocotyledons were clustered to one group, Mulberry and Rosaceae were clustered to one group, and the GenBank number was KF992020.1 and KF992021.1 respectively. Quantitative RT-PCR showed that this gene was highly expressed in roots, low expressed in stems. Glutamic acid significantly raised the expression of MaNR in leaves and GA3 significantly raised the expression of MaNR in stems. Hypocotyls could not be induced callus and multiple shoots in the single NH4+-N media but could be in the single NO3--N media. These nitrogen sources played a less part in subculture of shoot clumps. The shoots could grow in the single NH4+-N media and in the single NO3--N media. Real-time PCR exhibited that MaNR was highly abundant in cytoledons and hypocotyls than radicals. The relative expression level increased during hypocotyls regeneration in vitro and reduced to stable after shoot clumps formed. As time went on, the relative expression level of MaNR was reduced in shoot clumps with single NO3--N media or single NH4+-N. Glutamic acid showed a little inhibition of NR in the shoots subculture. 【Conclusion】 The full length of MaNR was cloned from mulberry trees. NO3--N was necessary for hypocotyls regeneration in vitro and the expression of NR affected by mulberry cell dedifferentiation and differentiation.

Key words: mulberry , nitrate reductase , MaNR gene , regeneration , real-time PCR