Molecular Cloning And Functional Analysis Of HbMADS4from Hevea Brasiliensis

The rubber tree self-rooting juvenile clones which derived from somatic embryos of anthers and internal integuments were promising newly planting materials, with a comparative trial, self-rooting JCs exhibited higher rubber production than donor clones (DCs). It maybe owing to the faster growth, laticifer number, etc. Though substantial research including physiological studies and morphology has been conducted. The molecular mechanism underlying the high production of self-rooting JCs still remains unknown.In our previous study, our research group found differentially expressed of gene and protein existed in the latex of rubber tree self-rooting JCs and their donor clones by the subtractive hybridization and two-dimensional electrophoresis, which may be directly involved in differentially production between self-rooting JCs and DCs. In order to futher study the function of differentially expressed of gene, we cloned the relative genes and got some relative reseach aimed at futher study on the molecular mechanism of high production of rubber tree self-rooting JCs.The full-length cDNA encoding a MADS-box transcription factor, designated as HbMADS4, was isolated from Hevea brasiliensis by RACE method. The sequence analysis suggested that HbMADS4was984bp containing633-bp open reading frame encoding a deduced protein of230amino acid residues with a typical conserved MADS-box motif at the C-terminus and a K-box domain. The results of subcellular localization indicated that the HbMADS4::GFP in the transformed onion epidermal cells was localized in the nucleus under a fluorescent microscope, demonstrating the nuclear localization of HbMADS4was consist with its predicted function as a transcription factor. The real-time RT-PCR results showed in both of JCs and DCs, the expression of HbMADS4gene was tissue-specific, with the highest transcription in latex. A prokaryotic expression vector of pET-28a(+)-HbMADS4was constructed, and then transferred into E. Coli Rosetta (DE3). The recombinant protein was produced then. And the result of Western blot demonstrated the induced protein was correct. Yeast one hybrid analysis indicated that HbMADS4was able to specifically recognize and interacted with the promoter of HbSRPP. Transient gene expression and the co-expression of pHbSRPP::GUS with35S::HbMADS4in transgenic tobacco indicated that HbMADS4significantly decreased the HbSRPP promoter. The result suggested that HbMADS4maybe a negative regulator in natural rubber biosynithesis.