Cloning Ethylene Biosynthase Key Enzyme Upstream Regulate Factors In Banana Fruit

The aim of this study was to isolate some regulation factors of the fruit ripening specific expression gene ACOl. First mensurated the ethylene production and Respiration rate during the process of postharvest banana fruit ripening. And applied the technique of real-time PCR to analyzing the mRNA level expression of ACOl. The results showed that the ethylene production of postharvest banana fruit was obvious rising from the 18 day. And reach the peak at 25 day. So as the respiration rate. The mRNA level expression of ACOl was rising during process of postharvest, and reach the peak at 18 day. and then descend. Those results farther proved banana is climacteric fruit; ACOl is a fruit ripening specific expression gene, it may startup may startup releasing the ethylene of system 2.Using two methoes, I constructing two reporter vector for one-hybrid analysis. One is insert a kb sequence of the promoter of ACOl into the multiple cloning site of pHIS2,geted a report vector pACOHIS2. Another is according to wet site of plant care and gene regulator ect al analyze the reported promoter of banana's ACS and ACO by NCBI, then selected some elements .and artifical synthesized three 50-60bp DNA. And geted a 170bp DNA using the technology of overlapping PCR. Then insert this DNA into the multiple cloning site of pHIS2, and attained a report vector pMmakeAC0HIS2. As the result of real-time PCR showed the mRNA level expression of ACOl was higtest at 18 day. So we extracted the total RNA of this day. And constructed cDNA-GAL4 AD fusion lrbraries.As a result there are 60 clones appear when pACOHIS2 library screening on selecting culture medium SD/-His-Leu-Trp+0mM3-AT. But only 8 clones appear while inscreasethe 3-AT concentration to 60mM. And there are 156 clones appear when pMmake HIS2 library screening on selecting culture medium SD/-His-Leu-Trp+60mM3-AT. But only 46 clones appear while increase the 3-AT concentration to 200mM.This study set up a method to analyzing gene transcriptional expression base on real-time PCR; And isolated some genes that maybe regulate ACOl.