Preliminary Study On Increasing The Content Of Glutathione In Rice And Vitamin C In Kiwifruit Through Genetic Modification

Glutathione(GSH)is an antioxidant that is beneficial to human health.It is also widely used in the food industry,such as health product and food additive.GSH also can be used to treat some diseases.Rice is a worldwide economic crop and a staple food in most areas.Improving the nutrients in rice is the focus in the food field.GSH is a stress metabolite of plants or animals.The GSH level of plants under normal conditions is not high.This article intends to increase the GSH content in rice through genetic modification.In the second part of this paper,two targets were designed for CATB and CATC.The promoter,g RNA and the linker target were linked together by enzyme digestion and ligation.The correct ligation product was amplified by nested PCR,which is also known as g RNA expression cassette.Finally,the g RNA expression cassette was connected to PYLCRISPR / Cas9 through the steps of enzyme digestion and ligation,and the CRISPR / Cas9 Pubi :: Os U :: CATC / CATB knockout vector was successfully constructed.six T0 positive seedlings containing Cas9 were screened.The sequencing results of the positive seedlings showed that the plants showed a variety of mutations such as single base deletion,insertion,replacement,and partial fragment deletion.The CAT enzymes activity of six T0 positive seedlings decreased significantly.These showed that this article successfully constructed a CAT knockout vector and obtained positive seedlings whose CAT were successfully targeted.Like GSH,ascorbic acid(ASC/ASA,also known as Vc)is also an important nutrient which is necessary for human health,and plays an important role in the pharmaceutical,food,cosmetics industries.The level of ASC is also closely related to the plants’ ability to resist stress.Monodehydroascorbate reductase(MDHAR)is an important enzyme in the regeneration pathway of ASC,which can reduce MDHA to ASC.The kiwifruit is known as the king of Vc,and the kiwifruit’s genome sequencing results show that there are seven potential genes that encode MDHAR whose function is unclear.Therefore,the third chapter of this paper intends to analyze the function of kiwi MDHAR in order to further increase the antioxidant ASC content in plants through genetic modification.The model plant Arabidopsis thaliana has a short life cycle and easy access to genetic material.Therefore,the transgenic Arabidopsis overexpressed MDHAR1,MDHAR2,MDHAR3,MDHAR5,MDHAR6,and MDHAR7 were used as research objects.The results show that MDHAR1,MDHAR2,MDHAR5,and MDHAR7 all have MDHAR enzyme activity and promote the synthesis of ASC.Among them,M2-1 and M2-3 transgenic lines overexpressing MDHAR2 have the most significant increase in enzyme activity and ASC,Overexpression of MDHAR2 also enhanced the salt resistance of the transgenic lines.These provide a theoretical basis for improving the ASC content in kiwifruit or fruits and vegetables through genetic modification.