Expression Analysis And Functional Characteristics Of TFL1/CEN Homologous Gene Of Poncirus Trifoliata (L.) Raf.

For woody plants, especially those economic important forest and fruit trees, the mechnism of juvenile development has always been a major concern. How to effectively reduce the juvenile phase and accelerate the breeding process is a very important problem in molecular biological research. In 1976, a mutant of Poncirus trifoliata (L.)Raf with a short juvenile phase was found in Yichang, Hubei Province of China and was named as precocious trifoliate orange.20% of the seedlings germinated from the mutant seeds flowered the next year after germination, and 100% of the seedlings flowered the third year after germination. The mutant can flower 2-3 times every year. Compared with the wild type trifoliate orange, the mutant has a spontaneous short juvenile phase, so it is advantaged in being an ideal material for studying the juvenile development.The previous studies about the floral transition were mainly focused on physiological and biochemical basis, such as environmental factors and culture techniques, but less on the molecular mechanisms and genetic control.Our study mainly investigated the two critical genes PtTFL1 and PtTFL2 which regulate the juvenile development of the woody plants.We characterized the function of the two gene by bioinformatics analysis, real-time PCR after various environmental factors treatments and genetic transformation. The main results are as follows:1.After analysis of the genomic sequence and conservative sites, we found the two genes PtTFL1 and PtTFL2 are consisted of four exons and three introns, encoding a putative protein of 173 amino acids with a predicted molecular mass of 19.5kDa and 19.2kDa, and pI of 9.24 and 8.79, respectively. There are a PEBP motif in the Open Reading Frame of the two genes, and the two critical sites His88 and Asp144 are very conservative.The coding region exhibited 98% and 71% identity to that of the sweet orange CiTFLl at the nucleotide level.In addition, the coding region of PtTFLl exhibited 77% identity to that of PtTFL2 at the nucleotide and amino acid level.2.We investigated the expression of the PtTFL1 and PtTFL2 in precocious trifoliate orange after photoperiod, GA3 and temperature treatment, respectively. The results showed that the two genes had a downward trend under short-day and low temperature treatment, this is possibly related with the repression of short-day and low temperature on the vegetative growth. But the expression of the two genes appeared differences after the long-day, GA3 and cold treatment, their expression were opposite, although the function delaying flowering was similar. It's possibly that the two genes involve different regulatory pathway and result in different response to some environment factors.3.After the test about influence of HygB on the tobacco leaves and the stems of precocious trifoliate orange, we found that not only tobacco leaves but also the stems of precocious trifoliate orange were very sensitive to HygB.Combining the test results with the damage of antibiotics on explants,we confirmed that:the selection concentration of HygB on Nicotiana tabacum Linn.leaves was 5mg/L, Nicotiana nudicaulis leaves and the stems of precocious trifoliate orange were 2.5mg/L.This would provide the basis for selection of transgenic plants by Hygromycin B.4.We overexpressed the two genes PtTFL1 and PtTFL2 in two kinds of tobaccos, and we derived 27 and 38 positive plants in N. nudicaulis of 35::PtTFL1 and 35::PtTFL2, and the positive rate were 90% and 47.5%,respectively. The transgenic To-generation plants presented different degrees of late flowering except 2 tansformants, and flowered 20d later than the wild-type tobacco, and PtTFLl transformants flowered 2d earlier than PtTFL2 on average. We obtained 5 and 11 positive plants in N. tabacum Linn.of 35::PtTFLl and 35::PtTFL2, and the positive rate were 83.3% and 61.1%,respectively. The transgenic To-generation plants also showed late-flowering phenotypes when compared with wild type plants, and firstly the phenotypic difference was mainly in axillary buds and internodes length. The PtTFLl transformants had a large number of axillary buds, and the internodes were very short; But the PtTFL2 transformants had no axillary bud, and the internodes were longer than PtTFLl transformants and wild-type plants.This might imply that PtTFL1 gene is mainly correlated with the vegetative development.5.We made preliminary exploration about transformation of precocious trifoliate orange, and obtained eight resistant plants for 35S::PtTFL1 and 35S::PtTFL2, respectively. We selected six resistant plants for micro grafting, and two for rooting. Due to time constraints, we couldn't analyze the transformants, but all of these provide base for the later research about the function of the PtTFLl and PtTFL2 in precocious trifoliate orange.