| GRAS protein family is one kind of plant-specific transcription factor family. It includes eight subfamilies:HAM, DELLA, PAT1, SCR, SHR, LISCL, LS and SCL3. These proteins are generally made up of 400-700 amino acids and consist of conserved C-terminus and variable N-terminus. GRAS protein family play important roles in growth and development, signal transduction, biotic and abiotic stresses. In this study, combining with transcriptome data of salt-tolerant birch (Betula kirghisorum, abbreviated as BK) and the characteristics of GRAS protein family, we selected 19 BkGRAS members and performed bioinformatics analysis. We successfully cloned the BkGRAS2 gene and performed the verification of expression pattern under salt stress, the subcellular localization and transformed wild-type Arabidopsis thaliana. The results were as followed:In the transcriptome analyses of Betula kirghisorum, we screened out 13 transcription factor families related to abiotic stress:GRAS, WRKY, Zinc finger protein, NAC, MYB, bHLH, HSF, MADS, ERF, bZIP, AP2, ARF and HD-ZIP. This study mainly studied on the GRAS family members.Bioinformatics analysis of Betula kirghisorum GRAS family,19 members were identified. The 19 BkGRAS proteins included typical LHR â… , VHIID, LHR â…¡, PFYRE and SAW motif. Secondary structure prediction of proteins was mainly on the alpha helix and random coil.19 BkGRAS proteins were mainly localized in nucleus, cytoplasm or chloroplast. Interestingly, the BkGRAS2 protein was localized in both nucleus and cytoplasm. Phylogenetic anaysis of Betula kirghisorum, Populus and Arabidopsis thaliana revealed that 19 BkGRAS proteins were mainly distributed in six subfamilies:HAM, LISCL, AtPAT1, AtSCL3, DELLA and AtSCR.We had successfully cloned BkGRAS2 gene, and found that the ORF of BkGRAS2 gene was 1614bp at length and coded 537 amino acids. In the BkGRAS2 protein, the P-N-H-D-Q-L residues of VHIID motif were absolutely conserved, and SAW motif included three absolutely conserved residues:R-E, W-G and W-W. There were 7 base differences and 4 amino acid variances between BkGRAS2 gene and already published BkGRASl gene, and the two genes had higher similarity. Phylogenetic tree analysis showed that BkGRAS2 protein belonged the AtPAT1 subfamily. Like the other dicotyledons, U-ended or A-ended codons were preferentially used in BkGRAS2 gene.RT-PCR and qRT-PCR results indicated that BkGRAS2 gene were expressed in root, stem and leaf, and the expression level increased in turn. The expression level of BkGRAS2 gene was significantly increased in the root when the seedling was stressed 12h, implying that the root was more sensitive to 0.6% NaHCO3 stress. Compared with the control group (0h), the expression level of BkGRAS2 gene decreased in root, stem and leaf under stress 24h, indicating that it was an adaptive process to 0.6% NaHCO3 stress. The expression level of BkGRAS2 gene reached the maximum in stem and leaf when the seedling was stressed 48h.The subcellular localization result pointed out that both nucleus and cytoplasm of inonion epidermal cells transformed pBI121-BkGRAS2-GFP protein were detected green fluorescence, indicating that BkGRAS2 protein was localized in the nucleus and cytoplasm.Under lmmol/L and 4mmol/L NaHCO3 stress, seed germination rate of transgenic lines was higher than WT and the phenotype of transgenic lines was better than WT, indicating that BkGRAS2 gene could make response to NaHCO3 stress and improve tolerance of transgenic Arabidopsis to NaHCO3. NaHCO3 resistance experiments of seedling stage shown that the root length of transgenic lines was longer than WT under low concentration NaHCO3 (1mmol/L) and high concentration NaHCO3 (4mmol/L) stress, indicating that BkGRAS2 gene could improve tolerance of transgenic Arabidopsis to NaHCO3 and reduce the toxic effects of NaHCO3 on transgenic Arabidopsis. |
