Functional Validation Of Key Differential Genes In Black And White Fruits Of Lycium Ruthenicum Murr.

In this study,based on the previous results of the experimental group,the key difference genes in the two black and white fruits of Lycium barbarum were unearthed and the gene functions were verified by combining transgenic technology.After extensive analysis,all the genes involved in the anthocyanin synthesis pathway in black and white fruits were narrowed down to 65 differential genes.Twenty-seven of these differential genes were quantitatively analyzed,and it was found that regulatory genes were not the factors responsible for the color differences in black and white fruits.Among the five important differential genes,F3’5’H-c102345 had the largest difference in expression in black and white fruits,with 2391 times more expression in black fruits than in white fruits;meanwhile,F3’5’H,as one of the key enzymes in the anthocyanin synthesis pathway of black fruit berries,determines the structure and color of anthocyanins accumulated in black fruit berries.DFR is also one of five important differentially expressed genes and is a key enzyme downstream of F3’5’H in the flavonoid biosynthesis pathway,and its substrate specificity is related to which anthocyanins the plant accumulates.Therefore,two genes,F3’5’H and DFR,were identified for functional validation in this experiment,and the main results are as follows.1.Analysis of anthocyanin content in black and white fruits showed that anthocyanin content in black fruits began to accumulate in early development,was highest in mid development,and always remained at a very high level in late development,whereas anthocyanin content in white fruits was always at a very low level,unaffected by developmental stage.The variation in anthocyanin content of both was found to be large throughout the stages.The expression patterns of F3’5’H and DFR genes in different developmental stages and different tissue sites of Lycium barbarum were analyzed by q RT-PCR,indicating very low or almost no expression of F3’5’H and DFR genes in stems,old leaves and new leaves.The results of this study are as follows.However,there were large differences in expression in fruit,with expression of key structural genes for anthocyanin glucoside synthesis significantly elevated in almost all compared to stem and leaf sites,especially in S3 and S4 stages.This suggests that the F3’5’H and DFR genes of Lycium barbarum are expressed only in that particular plant organ(fruit),that there are significant differences in their expression activity in fruit,and that their expression patterns are spatially and temporally specific.2.Based on the existing sequences,the Gen Bank database was applied for the cloning of Lr F3’5’H and Lr DFR target genes,which was simple and convenient,and after the detection of the cloned fragments,it showed that the fragments of the target genes required for the subsequent experiments were successfully cloned.The use of existing sequences and databases in this study greatly reduced the number of experimental steps for target gene cloning,and the success rate was very high.3.The p CAMBIA2300-Lr F3’5’H-GFP and p CAMBIA2300-Lr DFR-GFP plant overexpression vectors were constructed by double digestion and genomic DNA of transgenic SR tobacco was extracted with the help of Agrobacterium-mediated transformation.As a result,the gene was successfully transfected into the model plant,tobacco.4.The relative anthocyanin content of wild-type and transgenic tobacco flowers was measured at each period,and the difference in anthocyanin content between S1 and S5 was significantly higher than that of wild-type,and the difference in content reached the maximum at the flowering stage.This indicates that the exogenous genes were successfully transformed and stably expressed in the transgenic plants,further demonstrating the regulatory role of F3’5’H and DFR genes on plant flower color.