The Cloning、expression Analysis And Functional Study Of YABBY Gene Family Transcription Factors In Tomato

YABBY protein family is a classific of plant-specific transcription factors, with atypically N-terminal C2C2-type zinc finger domain and C-terminal helix-loop-helixdomain, YABBY family play an important role in process of higher plants development.Currently, the studying of YABBY family genes function in the model organismArabidopsis more clearly, but the molecular mechanisms are still not very clearly intomato.It is more importent to discuss the function of YABBY family genes in tomatodevelopmental processes. This study screened several closely related tomato YABBYfamily transcription factors, were named as SlYABBY5, SlYABBY1b and SlYABBY2arespectively, qRT-PCR technology was used for a few of these tissue-specific geneexpression pattern analysis; analysis of the expression under ABA, ASA, IAA, GA3endogenous hormone treatment,and expression analysis of SlYABBY5, SlYABBY1b andSlYABBY2a as well as low temperature, high salt and wound. SlYABBY1b andSlYABBY2a gene were used for constructed RNAi-mediated silencing vector, and theoverexpression vectors of SlYABBY2a gene. And constructed SlYABBY1b andSlYABBY2a RNAi interference expression vector by the method of Agrobacterium,transformed into the tomato genome. antibiotic selection and PCR identification oftransgenic tomato lines respectively, observed the phenotypic of the transgenic tomatoduring the tomato growth and development.analysisi of the results and molecularmechanisms of transgenic tomato,and analysis of its function. The main results are asfollows:1We were obtained SlYABBY5(GenBank accession number: AK246138),SlYABBY1b (GenBank accession number: AK326840) and SlYABBY2a (GenBankaccession number: AK328263) gene sequence and its encoded amino acid residues fromthe NCBI database, and using the RT-PCR technology cloned SlYABBY5gene whichcontained a complete ORF, cloning of SlYABBY1b and SlYABBY2a silence genefragment used for constract RNAi silencing vectors, cloning of SlYABBY2a gene whichcontained the complete ORF used for constract overexpression vector. All of clonedfragments were obtained for pGEM-T-easy-vector cloning vector, verified by PCRand sequencing to verify the correct sequence was obtained.2Extracting the total RNA of wild-type tomato AC++tissue materials and rinand Nr fruit ripening mutant materials, using RT-PCR method for the synthesis of the first strand cDNA.Using the qRT-PCR method analysis of wild-type tomato tissueexpression patterns, and using of Nr and rin fruit ripening mutants for the studying offruit mature. This results showed that, the expressing level of SlYABBY5, SlYABBY1band SlYABBY2a genes were in tomato roots and stems very lowly, but in the tissue ofleaf, fruit, sepals and flower in tomato showed constitutive expression, the expression indifferent tissues have significant differences; the Nr and rin fruit ripening mutantcompared with the wild-type tomato was no significant difference, had a similarexpression trend.3Choosing four weeks wild-type tomato leaf materials,using of IAA, ABA, GA3and ASA treated tomato seedlings, qRT-PCR technology showed that the expression ofSlYABBY5, SlYABBY1b and SlYABBY2a genes are subject to inhibition; underinglow-temperature, high salt and mechanical damage treatment, this results showed thatthe expression SlYABBY1b gene also been inhibited; the expression of SlYABBY5andSlYABBY2a genes was inhibited by cold stress, whereas the expression the expression ofSlYABBY5and SlYABBY2a genes induced by high salt and leaf damage stress.4Using the cloned specific sequences of SlYABBY1b gene constructedRNAi-mediated silencing vector pBIN19:: SlYABBY1b, PCR and restriction enzymedigestion to verify it was corrected,then using of Agrobacterium-mediated method tothe recombinant the silencing vector into the tomato genome, screening and verificationpositive transgenic lines by RCR and Kan resistent, five positive transgenic lines werechoosed; using of qRT-PCR technology to detect the expression of SlYABBY1b gene intransgene lines, all of the results showed that the expression level of the gene issignificantly reduced.5Using the cloned specific sequences of SlYABBY2a gene constructedRNAi-mediated silencing vector pBIN19:: SlYABBY2a, and cloning of SlYABBY2agene which contained the complete ORF used for constract overexpression vector.PCR and restriction enzyme digestion to verify it was corrected. Then using ofAgrobacterium-mediated method to recombinant the silencing vector into the tomatogenome, screening and verification positive transgenic lines by PCR and Kan resistent,two overexpression of SlYABBY2a gene and five RNAi silencing transgenic lines werechoosed. Using of the qRT-PCR technology to detect the expression of SlYABBY2agene in transgene lines, in SlYABBY2a overexpression transgene lines,SlYABBY2a genewas upregulated;all of the results showed that the expression level of the gene issignificantly reduced in RNAi transgene lines. 6Using part of SlYABBY1b transgene materials to detect the expression of auxinand gibberellin biosynthesis pathway related genes, the results showed that, theexpression of IAA3, IAA9, ARF7, ARF8and PIN4genes in RNAi transgenic linescompared with wild-type tomato were reduced. And the expression of IAA3, IAA9andARF8genes in RNAi2#transgenic line was significantly lower than RNAi4#transgenic lines. The expression of the Oxidase gene GA20ox1in RNAi transgene wereupregulated, whereas the expression of GA20ox2gene no significant difference in bothRNAi2#and RNAi4#tansgene lines, although the expressionof GA20ox3, GA3ox1and GA3ox2in RNAi transgenic lines were downregulated.