Clonging And Functional Analysis Of Homologous Structural Gene And Their Regulator Of Anthocyanin's Biosynthesis In Rubber Tree

At present, long sexual generation cycle and low variability are the reasons why conventional breeding of rubber tree have low breeding efficiency. Without a appropriate report gene to identify the positive plant, limited transgenic technology play a significant role in the rubber tree breeding greatly, yet. Anthocyanin, as a novel report gene, can solve the low choose effenciency of transgenic rubber tree. Therefore, a regulator which can accumulate anthocyanin in rubber tree makes sense in choosing the positive transgenic rubber tree. In this study, some homologous structural genes and their transcription factors were cloned and identified the functions by tobacco transformation. At the same time, this study explored the mechanism of anthocyanin accumulation preliminary. The main results are as follows:1. Thirteen homologous structural genes in the anthocyanin synthesis pathway from rubber tree were obtained by cloning. 5 homologous structural genes which could accumulate anthocyanin were identified by 3'RACE technology and RT-PCR analysis.There were the HbLDOX1,HbF3H1, HbF3'H1, HbF3'H2 and HbCHSl, respectively. After transforming these genes into tobacco, HbF3'H1 can make tobacco accumulate anthocyanin. The qPCR results showed that the correlation between the accumulation degree of anthocyanin and the expression of HbF3'H1 in tobacco petals are positive.2. Three MYB transcription factors related to anthocyanin accumulation were cloned,named by Hb16MYB, Hb65MYB and Hb98MYB. After determining ORF region by 3'RACE amplification technology, we put these sequences on NCBI to get some homologous sequences and construct a phylogenetic tree, results showed that they are similar with the grape and apple MYB transcription factors which have been identified can accumulate anthocyanin. Then, three contructs with the transcription factors were made to overexpression in the tobacco, finally we get a transgenic tobacco which was dark red in the whole tobacco because of Hb16MYB. Analysis the expression level relationship of Hb16MYB?An2?An1?structural genes and anthocyanin accumulation by qPCR,the results showed that Hb16MYB could upregulate the expression of the downstream genes in the anthocyanin pathway of tobacco and affect the expression of An2 and An1 in some tissues.3. Cloned a bHLH transcription factor of rubber tree.4. Analyzed 5 homologous genes' promoter sequences, in addition to the common TATA-box and CAAT-box,they have light responsive elements?abscisic acid responsive elements,?ethylene responsive elements?salicylic acid responsive elements?MeJA responsive elements?heat stress responsive elements?fungal responsive elements and so on,the elements evident that these genes are high sensitivity to the light?the abscisic acid?the ethylene?the salicylic acid? the heat and the other stresses. In addition,endosperm-specific expression elements were found from these promoters, indicating that the expression of endosperm in the embryo of the rubber tree could stimulate the expression of the structural gene. At the same time, MYB binding site have been find from the HBLDOX1, HbF3'H2 and HbCHS1 genes' promoters. It is suggested that the MYB transcription factor of rubber tree play a regulating role in anthocyanin synthesis pathway,and it also lay the foundation for exploring the mechanism of MYB and structural gene's regulation.