Research On Cold Induced Early Flowering And Homologous Cloning Of MADS-box Genes In Tobacco

Tobacco is an important leaf economic crop, and prone to early flowering. In the actual production, early flowering has serious impact on tobacco leaf yield and quality. Low-temperature is one important influential factor for early flowering, but so far, the related researches take different interpretation and its molecular mechanism has been unclear. In this study, using low-temperature sensitive tobacco variety NC82, induced by low-temperature treatment (12℃10 d) at different seedling stage to study the low-temperature sensitive properties. Meanwhile, homologous cloning techniques of RT-PCR and RACE are used for cloning, and using semi-quantitative RT-PCR and Real-Time PCR technique for expression analysis of FLC-like MADS-box gene existing in NC82. The study has practical significance in production, and provides a good functional gene in cultivating tobacco varieties of no sensitive to low-temperature. Specific results are as follows.(1) Induction results of low-temperature: 16 days after transplanting, tobacco seedlings of low-temperature treatment at eight-leaf firstly appeared floral differentiation, indicating changes from vegetative growth to reproductive growth. 21 days after transplanting, tobacco seedlings of low-temperature treatment at six-leaf and eight-leaf stage initiated floral differentiation, and their controls appeared floral primordial until 26 days after transplanting, low temperature promoted floral differentiation 5 days and 8 days in advance, respectively. 31 days after transplanting, seedlings of low-temperature treatment at six-leaf and eight-leaf stage appeared early budding, while the controls were not budding until 36 days after transplanting. The time of flowering was during the 45th to 49th day after transplanting, and there are 2～3 days difference between low-temperature treatment and controls, but seedlings of low-temperature treatment at four-leaf stage was vice versa, the floral differentiation, budding and flowering were delayed 11d, 18 d, 16 d, respectively. So six-leaf and eight-leaf stage was temperature sensitive period for NC82, low- temperature promoted flowering, but seedling of low-temperature treatment at four-leaf stage was susceptible to injury and delayed flowering. From initial of floral differentiation to budding, the time of low-temperature treatment was longer than controls, indicating that low-temperature elongated floral primordial differentiation. Days of budding to flowering were fluctuating, that might be subject to environmental influence.(2) Conserved fragment cloning of MADS-box gene family in tobacco: Degenerate primers were designed according to conserved domain of various plant MADS-box family genes, and 27 cDNA fragments were successfully isolated from the young shoot tip of tobacco variety NC82 using RT-PCR technology, fragment length was between 122 bp and 146 bp, and of which 16 cDNA fragments'deduced amino acid sequences contained MADS_MEF2_like region using Blast in NCBI. Multiple sequence alignment analysis indicated that the 16 deduced amino acid sequences had high homology with MADS-box genes of Arabidopsis and other species, the average identity could reach 76%. Meanwhile, the 16 fragments contained 15 conserved amino acid sites, of which 7 sites involved in the interaction between protein and DNA, 5 sites with protein dimerization function. Phylogenetic analysis showed that these 16 conserved fragments were classified into 9 different MADS-box subfamilies in Arabidopsis, FLC, STMADS11, TM3 and AGL6 subfamily had the function of controlling flowering time; AG, AGL2 and DEF / GLO subfamily belong to C / D-type, E-type and B-type floral homeotic genes; genes of AGL15 and AGL12 subfamily were also expressed in the stem.(3) Full-length cloning and expression analysis of tabacco NtFLC. Using fragment LyE05 with the highest similarity with Arabidopsis AtFLC to design primers for 3'RACE and 5' entire cDNA cloning. Selecting cloned fragment A5 (805 bp) and B1 (433 bp) in 5' full length cloning, 3'RACE of 845bp length, and the conserved sequence LyE05 (146 bp) for the FLC-like full-length genes of tobacco, and obtained 1174 bp full-length cDNA of tobacco. The applications of NCBI's ORF finder software, and found it's Open Reading Frame (ORF) located between nucleic acids 172 to 786 bp, which encoding 204 amino acids, belongs to the MADS-box family and has the conserved MADS-box and K-box domain. NtFLC was homologous gene of Arabidopsis AtFLC using Phylogenetic analysis ,Semi-quantitative RT-PCR analysis showed that the expression of NtFLC mainly in the stem and shoot tip, and low expression in flower and fruit. Real-Time PCR for accurate quantitative analysis showed that the expression of NtFLC in the shoot tip was reduced after low temperature treatment. Therefore assumed that NtFLC might relate to low temperature sensitive and easily early flowering characteristics of NC82, and with similar function of Arabidopsis AtFLC.