Cloning And Analysis On SHR, SCR And Homologous Genes Form Pinus Massoniana GRAS Family

The Masson pine(Pinus massoniana), an indigenous pine species in China, is widely spread in southern China. Masson pine is widely utilized in the timber and paper pulp industry because of its fast growth and fine wood quality. However, it is hindered to effectively achieve maximum genetic gains because Masson pine is hard to root in asexual reproduction. This dissertation studied 3 genes related to rooting in P.massoniana: SHORT-ROOT(SHR), SCARECROW(SCR) and SCARECROW-like1(SCL1). The results are as follows:1)SHR, SCR and SCL1, members of the GRAS family transcription factors, are keys regulator in root formation. By homological cloning and RACE(rapid-amplification of cDNA ends), Three complete cDNA sequences were cloned from Masson pine named PmSHR, PmSCR, PmSCL1. The length of PmSHR, PmSCR, PmSCL1 complete cDNA are 2399 bp, 2827 bp, 3289 bp respectively. The length of ORF are 1509 bp, 2529 bp, 2415 bp respectively, by ORFFinder analysis; encoding 502, 842 and 804 amino acids respectively. Physical and chemical analysis showed PmSHR molecular formulais C2533H3889N699O770S28 and molecular weightis 57351.6 Da; PmSCR molecular formulais C4038H6336N1182O1280S31 and molecular weightis 92915.7 Da; PmSCL1 molecular formulais C3958H6200N1110O1237S27 and molecular weightis 89993.0 Da. Phylogenetic analysis showed PmSHR, PmSCR, PmSCL1 protein belongs to SHR, SCR and LISCL subfamily.2)qPCR result showed root had highest PmSHR and PmSCR expression from 50d-old seedlings, and the others from high to low as cotyledon, hypocotyl, epicotyl; cotyledon had highest PmSCL1 expression from 50d-old seedings, and the others from high to low as root, hypocotyl, epicotyl. Higher expression of the 3 GRAS genes suggested PmSHR, PmSCR, PmSCL1 could play important roles in root development of P. massoniana.3)PmSHR-GFP, PmSCR-GFP, PmSCL1-GFP fusion expressing vector were built and transient expressed in onion epidermal cells. Fluorescent images of fusion protein give evidences that PmSHR was expressed in cytoplasm, PmSCR and PmSCl1 were expressed in nucleus.4)PmSCR-GFP over-expression vector was transformed to tobacco mediated by agrobacterium tumefaciens. Transgenic plants were selected by PCR and fluorescent methods. Phenotype observation showed that transgenic tobacco had more and longer root than wild type comparatively, Hydroponic stem apex result showed transgenic tobacco has more adventitious root than wild type.