Functional Analysis Of A Transcription Factor PCF7 In Rice(Oryza Sativa L)

Plant morphogenesis is mainly determined by regulating the growth and differentiation of organs,, the size and characteristic of the plant organs,depending on its continued growth in the number of cells, size and characteristic.It is the process of cell division, cell proliferation and cell differentiation.For generating the complex structure of a plant, the process of tight regulation is necessary. However, the main regulators of cell division, cell proliferation and cell differentiation are transcription factors, which combine with DNA or promoter to initiate the expression of the gene,eventually determining the characteristics of cells and the entire organization, so as to regulate the development of plants.The TCP gene family was first described in1999 as a new family of transcription factors, it has focused on the various research of the cell, organ, tissue and hormone level of plant developmental, which shows the TCPs are of great importance in plant growth and development.Many previous studies have shown that TCP transcription factor family is the main regulator of plant morphogenesis,and regulates cell proliferation and hormone responses of plant by various paths.TCP transcription factor is a kind of ancient plant specific transcription factor, which regulates cell division and development in vegetative and reproductive stage.Studies have reported that TCP involved in important evolution regulation of morphological traits, including cell division,meristematic activation,coordinated growth,plant architecture,organ boundaries,the structure of the plant organs,flower symmetry,the pollen development,circadian rhythms and morphogenesis and senescence of leaf. Os PCF7 gene was deteremined as the candidate gene. In this study, the expression pattern and function of Os PCF7 were analyzed.The followings are the main results of this study:① Cloning, expression pattern and bioinformatics analysis of Os PCF7 gene1) The m RNA of various tissues of rice in different growth stages were extracted, and was reverse transcribed into c DNA.We picked a c DNA as template and cloned Os PCF7 gene, using real-time fluorescent quantitative RT-PCR(q PCR).With wild type rice(Zhong hua 11) as contrast, Os PCF7 genes were analyzed in different tissues and organs for the relative quantitative analysis.The research results show that the Os PCF7 was mainly expressed in the roots, leaves and stems of rice tillering stage,which is the highest, followed by the filling stage, the expression of the seedlings and full stage is similar.2) Bioinformatics analysis of Os PCF7 indicated that it’s 813 bp in length, encoding 270 amino acids. It predominantly consists of α-helix and coils. The analysis of Os PCF7 protein showed that it was a hydrophilic protein.Protein phosphorylation showed Os PCF7 has three Tyr phosphorylation sites, five Thr phosphorylation sites and five Ser phosphorylation sites,and these modifications to Os PCF7 proteins may play an important role in rice.Phylogenetic analysis and multiple sequence alignment of Os PCF7 demonstrated that it possessed several conserved structure domains and fell into a branch with TB1, At TCP3 and At TCP4 genes.② The subcellular localization of Os PCF7 geneThe expression vector 35s::Os PCF7-GFP fusion protein was constructed, and the result by transient expression in onion epidermal cells indicated that Os PCF7 protein localized on the cell nucleus.③ The influence of higher expression of Os PCF7 gene on rice plant morphologicalBy Agrobacterium-mediated method,35s::Os PCF7 recombinant plasmid was transferred into rice,then obtained 35s::Os PCF7 transgenic plants.Using the 35s::Os PCF7 transgenic plants T1 generation research shows that: transgenic plants express higher, plant height is also higher, root growth is also better, root length is longer, the number is also more,compared with wild type plants. The transgenic plants and wild type plants are grown in rice field for about 40 days, the tiller number of the transgenic plants is more obviously than the wild type, the average tiller number of transgenic plants is 5 to 8, while the average for the wild type is two t0 three, and plant height is slightly higher than the wild type, the average height of the transgenic plants is 49-60 centimeters, average height of wild type is 40 cm.and the height of plants is slightly higher than the wild type.④ The effect of Os PCF7 on downstream genesWe tested downstream genes expression of the Os PCF7 by q PCR.The expression of STM, KNAT2 and KNAT6 gene associated leaf blade apex meristem genes are increased compared with the expression of wild type, the expression of LOX2, AS1 and IAA3 are increased compared with the expression of wild-type, which suggested that Os PCF7 may have certain regulation effect on downstream genes.⑤ The construction of p CAMBIA1301- Os PCF7 Ri vectorThe p CAMBIA1301- Os PCF7 Ri vector was constructed and transformed into rice callus by Agrobacterium-mediated method, callus is at the screening stage.