Construction Of Co-Knockout Vector Of LeETR3/Lcyb And Preliminary Analysis Of MDHAR Related To Maturity And Quality In Fruit

Tomato is a popular world economic crop,which contains abundant nutrients(such as lycopene).However,it is easily perishable after maturity and is not easy to transport and storage,resulting in significant economic losses.Thus how to extend the shelf life of fruit and ensure its quality has become an important research direction in the food field.Ethylene is an important hormone that promotes tomato maturity,while the ethylene receptor gene LeETR3 is related to fruit ripening.On the other hand,lycopene beta-cyclase(Lcyb)is a key enzyme that metabolizes lycopene to form carotenoids.Therefore,I attempts to mutate LeETR3 and Lcyb to delay fruits’ maturation and ensure its lycopene via CRISPR/Cas9 system.CRISPR/Cas9Pubi::AtU::LeETR3/Lcyb co-knockout vector which containing hygromycin(Hyg)resistance was successfully constructed.However,due to extremely sensitivity of tomato AC to Hyg,I fail to select positive callus under low concentration of Hyg which is below to 5 mg/L Then,I try to constructed the other vetors containing kanamycin resistance(CRISPR/Cas9P35s::AtU::LeETR3/Lcyb,CRISPR/Cas9::AtU::LeETR3)instead of Hyg resistance gene.Finally,I obtained Lcyb and LeETR3 double mutants through application of CRISPR/CAS9 multi-gene co-knockout approach.These genetic materials would contribute to further studying the role of LeETR3 and Lcyb in tomato.It is well known that kiwi fruit has rich Ascorbate(AsA),and it is also many people’s favorite fruit.AsA is important for maintaining the normal growth and health for people.Dehydroascorbic acid reductase(MDHAR)can reduce monohydrohydroascorbic acid(MDHA)to AsA.The sequencing results showed that there are 7 genes encoding MDHAR in kiwi,but the function of MDHARs in kiwi is not clear.Therefore,the second part of my thesis would explore its function through generating transgenic lines which overexpressed 7 kiwi’s MDHAR individually.In this study,I analyzed homozygous OE MDHAR1,OE MDHAR2 and OE MDHAR6 transgenic lines.And I found that transcript levels of MDHAR1 and MDHAR2 were accumulated in transgenic lines and they possess MDHAR enzyme activity and higher AsA level,especially for the OE MDHAR2-1 line.Thus they provide an important genetic material for further analysis of the function of MDHAR in kiwi.