Genome-wide Analysis Of GRAS Family And Functional Analysis Of PeSCR And PeSCL3 From Phyllostachys Edulis

GRAS belongs to a transcription factor(TF) family specific to plants, which plays diverse roles in plant growth and development, such as root radial patterning and growth, shoot meristem maintenance, axillary meristem development, gibberellin signal transduction,phytochrome A signal transduction and so on. Bamboo is one of potential green resources with fast growth and wide usage, which has been payed much more attention. In this study, moso bamboo(Phyllostachys edulis) was selected for research, a genome-wide analysis of GRAS family in Ph. edulis was carried out, including the number of family member, gene structure and function prediction. Based on the analysis, the genes of PeSCR and PeSCL3 were further studied for revealing gene functions in root growth and development of Ph. edulis. The main results were as follows:1. Based on bioinformatics analysis, 59 GRAS genes encoding the GRAS TFs were obtained in Ph. edulis from the Bamboo GDB(www.bamboogdb.org). A comprehensive analysis of gene structure, protein motif organization, conserved motifs and phylogenetic analysis was conducted. The results showed that the GRAS TFs in Ph. edulis were distributed in nine subfamilies, which were AtSCL4/7(1), HAM(13), AtSCR(4), DLT(2), AtSCL3(4),DELLA(2), AtPAT1(13), AtSHR(4) and LISCL(10), respectively. The conserved motifs were existed in each subfamily, indicating that they might have specific functions. However, the members of At LAS subfamily were not found in Ph. edulis.2. Expression patterns of GRAS genes were detected using qPCR method. The results indicated that 57 genes had various expression patterns, except that PeGRAS-41 and PeGRAS-55 were not detected. The expression of PeGRAS-10 and PeGRAS-12 belonged to HAM subfamily had a significant difference compared to the others members(p<0.01), which expressed in stem with the highest level indicating that they might play critical roles in the development of bamboo stem. PeGRAS-18 of At SCR subfamily expressed in root with theleast level, indicating that it might be involved in both root and other tissues development.PeGRAS-39 of AtPAT1 subfamily was only expressed in stem and sheath, and it was much higher in sheath than that in stem, inferring that PeGRAS-39 might have vital roles in sheath development. PeGRAS-43 of At PAT1 subfamily expressed in leaf with the highest level, which was similar to the previous studies on AtPAT1 in regulating phytochrome signal transduction pathway. However, PeGRAS-32~PeGRAS-37 all expressed highest in root, which meant they had a close relationship with the development of root.3. In order to make the further study on the regulation of root growth and development in Ph. edulis, PeSCR and PeSCL3 genes were isolated from Ph. edulis. The full length cDNA of PeSCR gene was 2298 bp, which included 5′UTR of 238 bp, 3′UTR of 134 bp and ORF of1926 bp encoding 641 aa. The genome sequence corresponding to ORF of Pe SCR was 2598 bp,containing one intron. The full length cDNA of PeSCL3 gene was 1978 bp, comprised 5′UTR of 340 bp, 3′UTR of 303 bp and ORF of 1335 bp encoding 444 aa. The genome sequence corresponding to ORF of PeSCL3 was 1335 bp without introns. Amino acid structures showed that PeSCR and PeSCL3 had specific conversed motifs of motif I, motif II and motif III besides the five conversed motifs of GRAS family.4. The analysis of gene expression patterns demonstrated that PeSCR was mainly expressed in leaf and root, and PeSCL3 was constitutively expressed in leaf, root and stem. The expressions of PeSCR and PeSCL3 had a notable difference under different treatments of hormones and abiotic stresses. PeSCL3 was suppressed under GA3 treatment, while it was induced by ABA,. PeSCL3 was induced by NaCl and drought treatments. The expression of PeSCR in root was inhibited by GA3 in short time(1 h), but it was induced with the treatment time. Overall, PeSCR was suppressed exogenous ABA and NaCl treatment, while it was induced firstly and then suppressed by drought.5. Functional analysis of PeSCR and PeSCL3 were carried out using transgenic technology. The sense and antisense expression vectors of PeSCR and PeSCL3 were constructed and transferred to model plant Arabidopsis thaliana by dipping flower method,respectively. Compared with wild type, the antisense transgenic plants were short with smallleaves and undeveloped roots, while that of sense transgenic plants were similar to wild type,or a bit stronger than wild type, the development of roots were synchronous or faster than wild type, and the number of root was a little more than that of wild type. However, the antisense plants of PeSCL3 had no difference in rosette leaves with wild type, and had shorter roots after treated with PAC, which indicated that PeSCL3 might regulate GA biosynthesis to control the root development.6. The promoter activities of PeSCR and PeSCL3 were studied. The promoter regions of PeSCR and PeSCL3 were isolated and subcloned into the multiple cloning sites of pBI101,respectively. The expression vectors containing promoter region fused with GUS were transferred into A. thaliana, followed by the selection of Kan resistance. Histochemical staining showed that blue color was observed in root, leaf, petiole and position of top in transfergenic plants with PeSCL3 promoter, but no blue tissues were found in those of PeSCR promoter.Root is the most major organ of plants to absorb water and nutrients, and its growth and development has an important impact on the biomass yield of the entire plant. These results of PeSCR and PeSCL3 belonging to GRAS family provided a vital reference for further study on root growth and development of Ph. edulis, which will be helpful to regulate bamboo characters at molecular level through genetic engineering.