Cloning And Characterization Of NAC-Like Genes Related To Fruit Ripening In Tomato

NAC transcription factor family genes are plant specific transcriptional regulators, and this gene family is one of the biggest gene families in plant. The NAC domain was originally characterized from consensus sequences from petunia NAM and from Arabidopsis ATAF1, ATAF2, and CUC2. NAC transcription factor genes play very important roles in plant, it express in various developmental stages and tissues to regulate plant development and growth. It's involved in embryo development, the form of apical meristem, the development of lateral roots, signal transduction, response to biotic and abiotic stress and fruit ripening.A T-DNA insert mutant was obtained in our lab. It's fruits can't be ripening and the phenotype are similar to rin and nor mutants. Analysis of the side sequence indicated that T-DNA insert into a member of NAC gene family, we defined this gene Nur, and the mutant nur. Bioinformatics analysis suggested that Rin,Nur and Nor genes are all transcription factors, this result indicate that transcription factors play very important roles the fruit ripening and maybe regulate at the upstream of fruit ripening. Bioinformatics analysis also suggested that Nur and Nor genes are both NAC-like transcription factor genes with a identity of 65%。Utilizing bioinformatics tools and combining with Nur,Nor and other known NAC genes, we wanted to find some NAC genes which related to fruit ripening, and analysis the influence of them. It's not only important to understanding fruit ripening, but also benefit to the agriculture production.The major results are as follow:1. Twenty-one full length NAC genes were obtained from tomato nucleic acid database by the conserved NAC domain. A multiple alignments were made to analysis the structure of NAC genes in tomato: they have a conserved NAC domain at N-terminal and C-terminal were diversity; NAC domain is constituted by five subdomains: A,B,C,D,E。2. A phylogeny tree was constructed and combined with known functions of some NAC genes, we classified tomato NAC genes into five groups and predicted the function of every group; two NAC genes: SGN-U213215 (NAC15) and SGN-U213218 (NAC18 were screened which maybe related to fruit ripening. 3. Southern blot indicated that the Nur gene was of a single copy in the tomato genome; RFLP analysis suggested that the Nur gene shows the polymorphism in M82 and LA716.4. The Nur gene was bin-mapped in chromosome 2-A using 76 IL populations.5. Three full length NAC genes: NAC15, NAC18 and Nur, were cloned and have been proved by sequencing.6. The sense and anti-sense expression vectors of NAC18 gene, sense expression vector of Nur gene were constructed.7. NurR2, transformed by the full length of Nur cDNA RNAi vector were detected by molecular and physiology technology. The results indicated the NurR2 plants are transgenic plants, fruits of which can not be ripening with very small seeds compared with the control plants.8. A homology gene of Nur: AtNAC2 was found that maybe related to fruit ripening in Arabidopsis thaliana, imply the possibility that there are common fruit ripeing regulators in all type fruits.