Cloning And Expression In Yeast Cells Of Teansciption Factor Gene MdHB-1from Apple

The regulation mechanism of ACC oxidase has been focused by scientists, which is thelast key biosynthetic enzyme in ethylene biosynthetic pathway. The gaseous hormoneethylene plays the more important role in ripening, senescence of apple, and apple is typicalclimacteric fruit. The study confirmed the existence of large number ACO1genes in matureapple, and it revealed that ACO1regulated indirectly fruit ripening and senescence.In the present report, in order to explore the molecular regulation mechanism of ACO1in apple fruit, especially, the specific binding about its promoter and the correspondingtranscription factor, using the DNA of Royal Gala as materials, the Md-ACO1promotersequence was obtained by PCR。Besides, A Blastn search of apple expressed sequence tag(EST) databases was performed using the LeHB-1cDNA sequence of tomato LeACO1, andthe EST sequences were identified as the closest match to LeHB-1We designed a pair ofspecific primers corresponding to the identified EST sequence, and extracted total RNA fromfloral organ of Royal Gala. A cDNA for was amplified by reverse transcription PCR(RT-PCR), named MdHB-1. MdHB-1was cloned into expression vector of pPICZA，andtransformed into Pichia pastoris of GS115by electroporation. Methanol induction werecarried out to obtain the high level expression transformants, and the expression ofMdHB-1was Detected by SDS-PAGE and Western blotting analysis. The main results were asfollows:1. Gene specific primers were derived from ACO1gene promoter sequences in GenBank,and a cDNA fragment which contains1650bp was cloned. Compared with ACO1genepromoter sequences in GenBank, the sequences cloned homologous rates of nucleotide was97.83%.We concluded that this gene fragment cloned was ACO1gene promoter.2. Sequence of“MdHB-1”（Its GenBank accession number is JQ678788）analysisshowed that the fragment for1051bp contains a full coding region of1011bp. The sequenceswere up to99%identical with the target gene sequences of “Royal Gala” apple in EST on thenucleotide sequence，and the conserved amino acid residues present more than85%homologous traits with HD-Zip domain of LeHB-1and AtHB-1. We concluded that this gene fragment was MdHB-1.3. The eukaryotic expression vector pPICZA–MdHB-1was constructed and identifiedcorrectly by PCR, restriction analysis and sequencing. It was transformed into Pichia pastorisof GS115and the transformants were obtained by antibiotic selection of the highconcentration.4. SDS-PAGE and Western blotting analysis about yeast total protein showed thatspecific bands didn’t appear and the ideal result still didn’t occur after several improvements,which made DNA-binding of downstream not proceed successfully. I give an explanationabout the test results by according to the practice and the literature summary.