Isolation And Function Characterization Of The Brasssinosteroids Synthesis Genes Of Chinese Cherry Flower Bud

Chinese cherry (Prunns pseudocerasus) belongs to the Prunoideae subfamily of Rosaceae. There are many different cherry cultivars in China. P. pseudocerasus ’Duanbing’was one of the main landraces cultivated in Zhej iang Province because of its low amounts of cold requirement, early fruiting, large fruit, high yield and strong adaptability. It is also become an important material for scientific research.For deciduous fruit trees, blooming will not be happen until sufficient accumulation of chilling time. Shortage of chilling time will lead to affecting the output of the fruits quality and yield. Nowadays the dormancy and regulatory mechanismt of deciduous fruit trees are becoming a hot spot with the rapid development of fruit cultivation. Natural dormancy regulation also become an issue to be solved in the process of deciduous fruit cultivation. With the continuous development of molecular biology, dormancy study of deciduous fruit trees come deep into the molecular level. Plenty of evidence suggested that different stages of dormancy are regulated by endogenous hormones, including ABA, IAA, GA. Brasssinosteroid, as a steroidal hormones, plays many great important roles to promote germination in seed dormancy, cell division, cell elongation, the formation of the reproductive organs and building in the form of light. However, few studies were reported the regulatory mechanism in the process of flower bud dormancy breaking. So the role of the brasssinosteroids synthesis genes in flower bud dormancy breaking were revealed in the current study.This study gained four brasssinosteroid synthesis genes from Chinese cherry transcriptome database (GeneBank ID SRX695147). The expression and function were studied using bioinformatics analysis, qRT-PCR, instantaneous expression and genetic transformation technology. The main results were as follows:(1) Four brasssinosteroid synthesis genes, Unigene 12256, Unigene2085, Unigene2065 and Unigene5028 were revealed by browsering the transcriptome database of Chinese cherry. The high homology with DET2, BR6ox, DWF4 and CPD in p. mume and Malus domestica was found. So they were named Unigene12256 (PpcDET2), Unigene2085 (PpcBR6ox), Unigene2065 (PpcDWF4), Unigene5028 (PpcCPD) through the bioinformatics analysis of the protein secondary structure, hydrophobicity and homology comparison.(2) Total RNA were extracted from naturally dormant shoots and leaves treated with different methods. The qRT-PCR results showed that expression level of PpcDWF4 changed irregularly, expression level of PpcCPD was down regulated, the expression level of PpcDET andPpcBR6ox gene ware rised with the accumulation of low temperature. The expression level of PpcBR6ox gene was increased 39 times and upregulated significantly. The results indicated that PpcBR6ox was associated with cherry flower bud dormancy state regulation.(3) In order to clearly explore PpcBR6ox gene regulation expression and the relationship between the low temperature, promoter of PpcBR6ox was isolated from cherry flower bud genomic DNA by adopting the conventional PCR technology. The promoter contained many functional elements was predicted by PlantCare online software forecast analysis, including low temperature response and light response elements. The experiment on the interaction with CBF transcription factors was done showed that LUC and REN ratio is 3.5 times higher than the negative control after building pGreenII0800-luc transient expression vector, this stated that the expression of PpcBR6ox gene was switched on though CBF signal pathway, which was thought to be another response pathway to low temperature.(4) In order to further understand the function of PpcBR6ox gene’s, PpcBR6ox gene was cloned and PpcBR6ox gene over expression vector was built. PpcBR6ox over-expression transgenic arabidopsis strain were gained by agrobacterium mediated genetic transformation using arabidopsis inflorescence impregnation method. The results of over-expression strains and wild-type arabidopsis seed germination show that the germination rate of over-expression strain of Arabidopsis seeds was close to 90%, the wild type was about 10%, was significantly higher than the wild type in the next 48h. This shows that PpcBR6ox gene may be helpful in dormancy breaking of arabidopsis seeds and may promote seed germination. It can be hypothesized that PpcBR6ox genes may also promote the cherry flower bud dormancy breaking and promote germination.(5) In order to study the relevance between the sxpression of PpcBR6ox gene and bud dormancy breaking, PpcBR6ox gene was transformated to P. tomentosa by Agrobacterium mediated genetic variations and gained 48strains of transgenic Populus tomentosa after resistance selection and PCR identification. And laid a foundation for observing the influence of growth and development and bud dormancy of the PpcBR6ox over-expression transgenosis strains and wild-type P. tomentosa for the next step research.