Persimmon ACC Synthase Gene Cloning And Its Plant Expression Vector Constrution

Persimmon fruit, a climacteric fruit, is very difficult to be storaged and transported because it is easy to be soften after harvested . The traditional storage, which not only need high charge but also have a limited fresh period, can not solute the softing problem of persimmon fruit ultimately. Ethylene, which is one of importment plant auxin and plays an very important regulatory role in various plant. Make use of trnasgene technique to repress or lower the ethylene biosynthesis of climacteric fruit, can lower the content of ethylene in fruit. ACC synthase and ACC oxidase are the key enzymes during the biosynthesis of ethylene. We can increase the storaging function of climacteric fruit by repressing the expression of the two enzymes with transgene method. In the present study, we have amplified the ACS gene fragment using total RNA extracted from the persimmon fruit, cloned it in a T-vector, identified by sequencing and further constructed in the plant expression vector. The majorresults of this study were followed:1. The quality RNA was extracted by Rneasy^? Plant Mini Kit. The value of OD₂₆₀/OD₂₈₀ is 2.0979.2. Reverse translation of mRNA was conducted with Omniscript^? RT Kit, which is a kind of a high quality mRNA extracted kit. Its reverse translation is much higher than some other buffer and kit. High quality RNA was obtainded by the kit, which was base for successful PCR.3. According to the amino acid sequences of Hiratanenashi' persimmon, several pair of primers were designed of which one pair primer was showed a repeatable result. The primers used for the PCR were 5'-TTYCARGAYTAYCAYGGCYTSCC-3'and 5'-AAAKACVCKRAACCARCCCGGYTC-3'.4. The stable RT-PCR reaction system for ACS of Fuyu persimmon fruit was established. The optimizing reaction system contained: each primer (10μM) 2μL, 10 × PCR Buffer 5μL dNTP Mixture (2.5mM each dNTP) 4μL, cDNA 4μL, ddH₂O 32.75μL, Ex Taq DNA Polymerase (5U/μL) 0.25μL. A sharp and repeatable PCR band was stably showed.5. One piece of ACS sequence from persimmon fruit was cloned. It was amplified by Reverse Transcription Polymerse Chain Reaction (RT-PCR) from ripening persimmon fruit. Sequence analysis showed that it was 1178bp which share high identity with DK-ACS1 registerd in GenBank. There was only one different nucleotide and amino acid from...