Analysis Of ClPsy1 Promoter Activity And Detection Of CRISPR/Cas9 System Target Sites In Watermelon

Watermelon（Citrullus lanatus）is an important economic crop of Cucurbitaceae.The pulp contains a lot of antioxidant carotenoids,which can give watermelon pulp different colors.Psy（phytoene synthase）is the rate-limiting enzyme in the process of carotenoid synthesis and a key gene in the carotenoid metabolic pathway.The most important and widely studied of the carotenoid metabolic pathways isβ-carotene,the synthetic precursor of vitamin A.In our previous study,we used canary-yellow pulp COS and orange pulp PI 192938 to make generations of population,and concluded that ClPsy1 was a key candidate gene for the formation of orange watermelon flesh through fine mapping.The study speculated that the Cl MYB21（Cla97C10G196920）transcription factors might be ClPsy1 regulatory factors.This experiment was based on four different watermelon accessions:canary-yellow flesh COS（Cream of Saskatchewan）,light yellow flesh PI 635597,golden flesh PI 192938 and red-colored flesh LSW-177,according to the published RNA-seq and q RT-PCR analysis of the relative expression of Cl MYB21.Construction of VIGS（Virus induced gene silencing）vector for transient transformation of tomato fruits to verify the function of Cl MYB21.The vector of ClPsy1promoter-GUS deletion fragment was constructed for transient transformation into tomato fruits,and the expression of（β-glucuronidase）GUS enzyme activity in tomato fruits was determined to preliminarily predict the key reaction region of ClPsy1 promoter activity.Orange pulp PI 192938rich inβ-carotene was used as the material for optimizing regeneration system of watermelon.The CRISPR/Cas9 gene editing vector of ClPsy1 gene was constructed and transferred into Agrobacterium rhizogenes K599 to detect the knockout efficiency of target sites,which provided the vector basis for the subsequent genetic transformation experiment of watermelon.The main results in this study were as follows:（1）Combined with RNA-seq showed that Cl MYB21 showed the similar expression trend as ClPsy1,and compared with fruit rind,which showed higher expression in fruit flesh.Its expression level was related to carotenoid contents.（2）The VIGS results showed that no treatment tomatoes turned to red normally,and the silenced tomato with Sl MYB21 showed abnormal carotenoid accumulation.The expression of Sl MYB21 and Sl Psy1 was lower than CK,which showed that Sl MYB21 may be involved in the synthesis of carotenoid in tomato.It was speculated that Cl MYB21 transcription factor may affect the expression of ClPsy1 and participate in the synthesis of carotenoid in watermelon flesh.（3）ClPsy1 promoter was isolated from four different accessions,and compared with the sequence structures differences of them.The results showed that there were 6 SNPS(342^th,598^th,898^th,1,257^th,1,634^thand 1,694^th)in COS,which were different from the other three lines.The T→C mutation at 598^thposition leads to the mutation of the binding site of MYC transcription factor,and the T→C mutation at 1257^thposition leads to the mutation of the binding site of MYB transcription factor.（4）The GUS activity in tomato fruits showed that ClPsy1 promoter could drive GUS gene expression in tomato fruits.The activity of ClPsy1 promoter in orange pulp PI 192938 was stronger than that in canary-yellow pulp COS.GUS gene expression activity varied from 1521bp to 1043bp,suggesting that ClPsy1 promoter activity was relatively strong in the region of-1521 to-1043bp.The MYB binding-site exists in this region.（5）The optimum explants for the regeneration system of golden flesh PI 192938 were the base of cotyledon,and the optimum ratio of culture-medium for adventitious bud differentiation was MS+1.0mg/L 6-BA+2.0mg/L Ag NO₃+30g/L sucrose+8g/L Agar.Bud elongation medium:MS+0.2mg/L KT+30g/L sucrose+8g/L Agar;root medium:MS+0.5mg/L NAA+30g/L sucrose+8g/L Agar,fully regenerated plants were formed in about 60 days.（6）We get a CRISPR/Cas9 gene editing vector and infected golden pulp watermelon PI192938 to knock out the ClPsy1 gene,and the sequencing results showed that the 285 bp to 695 bp different deletions of them,which proved that inducing gene editing to detect the target site is an effective method to detect the target site of CRISPR/Cas9 system,which could be used in subsequent gene-editing trials.