Construction Of ACO ? Gene RNAi Expression Vector And Its Genetic Transformation To Jujube

Ziziphus jujuba Mill.is native to China and is one of the most characteristic and representative economic fruit trees in northern Shaanxi.However,the ripening period of jujube fruit is short and concentrated,and it coincides with the rainy season,which leads to severe fruit cracking in the producing areas.The jujube fruit is susceptible to aging and softening after harvest,resulting in dehydration,rot,decomposition of the flesh tissue,metabolic disorders,and accumulation of large amounts of harmful substances such as ethanol.The fruit's vitamin C is almost completely destroyed,which not only reduces the quality of jujube fruit,but also greatly shortens the shelf life of the fresh jujube market.Therefore,the cultivation of jujube varieties with ripening and storage characteristics is of great significance for promoting the development of the fresh jujube industry.With the continuous deepening of the research on the molecular mechanism of related enzymes in the ethylene biosynthesis pathway,it is possible to use genetic engineering technology to regulate the expression of ACC oxidase gene in the process of ethylene synthesis,thus making it possible to reduce the production of ethylene and improve the preservation and storage of jujube fruit..In this study,the ACO I gene was used as the target and RNAi technology was used to interfere with the endogenous ACO I gene in jujube,in order to reduce the expression of ACO gene in ethylene synthesis,and then obtain the jujube variety with preservation and storage.After research,the following results have been obtained:1.Select two gene fragments between 566~859(exon 3)and 1002~1267(exon 4)as target from the ORF of ACO I gene c DNA fragment of Gonodendron chinense,and use target PCR fragments to target the target gene fragment.Fusion was performed and the fusion gene ACO I3-4 with a size of 558 bp was obtained.The blast analysis of the homology of the fusion gene sequences showed that the homology of the ACO1 gene was 96.53%.2.The RNAi plant expression vector p ARTACO I3-4 containing the forward and reverse ACO I3-4 fragment driven by the Ca MV 35 S promoter was constructed and introduced into Agrobacterium tumefaciens LBA4404.3.Determine the selective pressure of Kan at the callus regeneration from the leaves of jujube(20 mg/L),the critical concentration of Kan(30 mg/L)for the normal growth of tissue culture seedlings,and the selection of 400 mg/L of Carb for the agrobacterial bactericides.Carb has the effect of promoting callus formation in the regeneration of recipient leaves.4.An efficient genetic transformation system was established for the leaves of the wood jujube: The leaves were cut into approximately 0.3 cm2 squares,precultured for 1 d,then inoculated in an Agrobacterium sp.broth with an OD600 of 0.6-0.8 for 10 min and secretly co-cultured for 3 d.The resistance callus induction rate was 19.00%.At the end of co-culture,after about 20 days of dark culture,callus induction rate and callus browning can be significantly reduced.5.In this experiment,94 transformant resistant shoots were obtained by total transformation.After screening by 30 mg/L Kan resistance,16 positive plants were detected by PCR,which preliminarily proved that the ACO I gene fragment was successfully integrated into the jujube genome.