Clonging And Expression Analysis Of Cabernet Sauvignon(vitis Vinifera L.) Vvaos Gene

Jasmonic acid(JA)is an important plant secondary metabolites.As a new plant endogenous hormones,it improves the physical environment of plants species between adaptation and competitiveness of infringement against predators,enhances disease resistance.Allene oxide synthase(AOS)is a key enzyme in the synthesis of JA.AOS closely control the biosynthesis of JA and involved in the regulation of many physiological processes of plants.In this paper,wine grape variety Vitis vinifera L.cv.Carbernet Sauvignon was taken as test material.A full-length sequence of AOS was obtained with reverse transcription PCR method and named VvAOS,and expressed in prokaryotic cells.Also influences of wounding and Methyl jasmonate(MeJA)applying treatment on gene VvAOS expresses was studied.The results are as follows:1.According to the grape genome(PN40024),EST sequences spliced genes of grape plants and other AOS logged in NCBI,we forecasted VvAOS gene open reading frame and designed primers of grapes AOS sequences and clone it.The obtained open reading frame of VvAOS is 1452bp,encoding 483 amino acids.Sequence analysis shows that the protein encoded VvAOS is highly similar with other plants AOS,which belongs to the cytochrome P450 gene superfamily,CYP74A family member.2.Quantitative RT-PCR analysis showed that the expression levels of methyl jasmonate changes with wounding and MeJA.It increased rapidly after treatment and remained at a high level of expression.After the introduction VvAOS expression of wounding had a slight decline in a short time,and then a sharp increase at 12 hours as the highest level.And then it declined slightly.After spraying MeJA,gene expression of the grape AOS gradually increased,and after 24 hours in the highest position.3.Prokaryotic expression vector pMAL-c4x-VvAOS and pET-32a(+)-VvAOS were constructed successfully,which were transformed into the expression host strain E.coli BL21(DE3).After cultivating for 22 hours under being induced with 0.1 mM IPTG,22? at the speed of 150rpm,the product was subjected to SDS-PAGE.We obtained two proteins about 90KD and 78KD.According to the prokaryotic expression vector has protein size itself,the VvAOS gene encoding the right size of protein.This suggests that grape AOS gene highly expressed in E.coli expression system.