Molecular Cloning and Expression Analysis of Nitrite Reductase Gene CsNiR in Tea Plant
Teanitrite reductase (CsNiR) was cloned by RT-PCR from cDNA isolated from leaves of cultivar ‘Longjing 43’ The complete ORF of the CsNiR was 1 764 bpencoding 587 amino acids.Alignment of amino acid sequences showed that CsNiR sharing more than 76% similarities to NiR in Betula pendula,Arabidopsis thaliana,Spinacia oleracea and Oryza sativa.Bioinformatics analysis indicated that the CsNiR was a hydrophilic non-secretory protein with molecular weight 68.648 kD and theoretical pI 6.12.Prediction results by InterProScan showed the secondary structure of CsNiR protein comprised of a hemoprotein beta component and a 4Fe-4S region,and its 3D structure was also predicted by Swiss Model.qRT-PCR analysis revealed that the expression abundance of CsNiR in mature leaves was the highest among the three tested tissues (two leaves and a bud,mature leaves and roots).Meanwhile,the transcript changes of CsNiR responding to different nitrogen (N) levels were studied by qRT-PCR after resupplying normal N (1 mmol · L-1 NH4NO3) and low N (0.1 mmol · L-1 NH4NO3) on hydroponic seedlings of three tea varieties with treatment of two week N starvation.The CsNiR expression levels were increased significantly in roots at 2 h and 6 h under normal N treatment,but they were changed since 24 h after N supplied in leaves.Furthermore,the transcription of CsNiR was also different among varieties.The transcript abundance of CsNiR increased more greatly under the normal N condition compared to those under low N treatment.Thus,factors like genotypes,tissues,nitrogen levels should be taken into consideration for the role of CsNiR in nitrogen utilization in tea plants.