| Shoot branching is one of the most important components of plant architecture, which are influenced by genetic factors, environmental conditions and so on. A large number of genes that significantly regulate the shoot branching belong to GRAS, TCP or R2R3-MYB gene family. The three of them all encode plant transcription factors, of which GRAS gene family play important roles in various aspects of plant growth and development. TCP gene family are associated with three-dimensional morphology development of plant organ and cell cycle regulation. R2R3-MYB transcription factors are widely involved in plant primary and secondary metabolism, biotic and abiotic stresses responses, and so on. In this study, the GRAS and TCP gene family were identified from tobacco genome. On this basis, we cloned Nt LS, Nt BRC1-Like and Nt RAX2 genes from tobacco, the orthologous genes of whom are all associated with the shoot branching. The three genes respectively belong to GRAS family, TCP family and R2R3-MYB family. For Nt LS and Nt RAX2 genes, we performed the over-expression analysis, gene silencing and Arabidopsis mutant complementary studies, to explore their biological functions and lay the foundation for breeding new varieties of tobacco which have no branching or less branching. The main results obtained in this study are as follows:(1) A total of 95 GRAS genes were identified in the tobacco genome, which were devided into ten subfamilies according to the phylogenetic analysis. All members of Nt GRAS contained five highly conserved motifs, that are LHR I, VHIID, LHR II, PFYRE and SAW. About 88.42% members of the family had no introns. 84 Nt GRASs which had location information were distributed on 22 chromosomes of tobacco. Tobacco GRAS genes were involved in the regulation of many biolodical processes. About 74% Nt GRAS proteins were nuclear localization. Most of 22 selected genes had the highest expression levels in root and leaf.(2) A total of 61 TCP genes were identified in the tobacco genome, which were devided into two subfamilies, Class I and Class II. The Class II could be further divided into CIN and CYC/TB1 subclusters. All Nt TCP members contained highly conserved TCP-domian. Only 12 members had another R-domian, ten of which belonged to CYC/TB1 subcluster. About 67.2% members of the family had no introns. 54 Nt TCPs that had location information were distributed on 20 chromosomes of tobacco. The majority of 22 selected genes had the highest expression levels in leaf.(3) The two copies of Nt LS gene were cloned from Nicotiana tabacum, one of them was derived from N. sylvestris, named Nt LS-S, another one was from N. tomentosiformis, named Nt LS-T. Having highly similar sequence and no introns, the two gene copies repectively corresponded to Nt LS and Nt LSa genes in the GRAS family. The expression patterns of two gene copies are similar to that of Le LS, At LAS and Dg LSL. We constructed the over-expression vector and CRES-T vector for Nt LS to perform transgenic experiment. The transgenic tobacco of T0 generation we obtained sent forth axillary buds earlier, and the Nt LS-S gene can restored the phenotype of Arabidopsis las mutant.(4) Four Nt BRC1-Like genes were cloned from N. tabacum, named Nt BRC1-Like1, 2, 3 and 4, which respectively corresponded to Nt TCP18 a, b, c and d in TCP family. These four genes belonged to CYC/TB1 subfamily that is associated with the development of shoot branching. The genetic distance between Nt BRC1-Like1 and 4 is nearer, as well as Nt BRC1-Like2 and 3. Four proteins encoded by Nt BRC1-Like genes possessed two domains, the core TCP-domain and R-domain, the latter one is specific to CYC/TB1 proteins. Nt BRC1-Like genes showed specific expression in different tissues, and their expression patterns can be divided into two types: Nt BRC1-Like 1/4 highly expressing in axillary bud and Nt BRC1-Like 2/3 highly expressing in leaf and axillary bud. Nt BRC1-Like2 and 3 were confirmed to be two copies of Nt BRC1 b gene, Nt BRC1b-S and Nt BRC1b-T, which were respectively derived from N. sylvestris and N. tomentosiformis. We already constructed the over-expression vector of Nt BRC1 b gene and studyed its function recently.(5) The two copies of Nt RAX2 gene were cloned in Nicotiana tabacum, Nt RAX2-S and Nt RAX2-T, respectively derived from N. sylvestris and N. tomentosiformis. The sequences of two gene copies are highly conversed. Nt RAX2-S and Nt RAX2-T highly expressed in axillary bud and flower, besides flower bud and shoot tip. We constructed the over-expression vector and CRES-T vector for Nt RAX2 to perform transgenic experiment. The transgenic tobacco of T0 generation we obtained sent forth axillary buds earlier, and the Nt RAX2-T gene can restored the phenotype of Arabidopsis rax2 mutant. |
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