Knocking Out ACC Synthase Gene By CRISPR-Cas System And Verifying The Function In “Laohan” Melon

Melon(Cucumis melo L.)is a genus of Cucurbitaceae in cucumber,is a popular fruit,and”Lao Han melon”Uighur called "Bixiekeji",is specialty of xinjiang.With soft and tasty,moisture and high sugar,welcomed by the consumers.But after the melon mature,the softening speed is very fast,it is difficult to store and transport,resulting in great economic losses.Therefore,the improvement of Fruit Storability has been an important goal of melon breeding,but also a difficult problem to be solved on the production.With the development of science and technology,people found that ethylene can control fruit mature,while the ACC synthase is a key enzyme of ethylene production,this study provides a theoretical basis for improving the Storability of melon.CRISPR/Cas system is a new genome editing technology.Targeted genes can be targeting knock-out and knock-in by the technology of CRISPRs.The experiment is based on the ACC synthase gene of melon,and construced a vector which knockout ACC synthase gene by CRISPR-Cas9 system,and then the high efficient genetic transformation system is obtained which can knockout Arabidopsis thaliana genes by using the agrobacterium-mediated genetic transformation of CRISPR-Cas9 system.1.In the ACS gene sequence design a target site.Then the designed primers were connected to the vector p P1 C.4 which can knockout plant gene,to construct the expression vector p P1 C.4-ACS of melon.2.In this paper,the tissue culture system of Melon Variety "Lao Han melon" was explored and optimized.By Agrobacterium-mediated method,we got 3 transgenic melon seedlings.3.To observe the morphological changes of melon seedlings before and after transformation.After knocking out the ACS gene,it inhibited the elongation of roots and delayed the growth of plants.Extraction of crude enzyme in transgenic melon leaves,and detection of SOD,POD and CAT enzyme activity in transgenic plants,found that three kinds of enzymeactivity were higher than that of the wild type.The results indicated that ACC synthase plays an important role in plant senescence and stress tolerance.At the same time,it laid the foundation for further research on the effects of ACC synthase on the ripening of melon fruit.