Molecular Mechanism Of BZIP Transcription Factor CPRF2 Regulating Tomato Fruit Ripening

Fruit maturation refers to the process of a series of physiological and biochemical changes that occur after the fruit stops growing and development,including the accumulation of sugar,the softening of the fruit,the accumulation of pigments and the synthesis of aromatic substances,etc.,and finally reach an edible state.Tomato（Solamum lycopersicum）is one of the most widely planted fruits and vegetables in the world.It is also a model plant for genetic engineering research.During its maturation,it is often affected by various factors,resulting in changes in tomato appearance,nutritional quality,and maturity.Therefore,using tomato as the material to reveal the regulation mechanism of fruit ripening is of great significance to improve fruit quality,advance or delay the market,and extend shelf life.The b ZIP（basic region/leucine zipper motif,b ZIP）family genes play an important role in almost all tissues in the plant and the entire plant life cycle.At present,research on the b ZIP family genes is mainly focused on participating in the process of plant stress response,and with the in-depth study of the family genes,it has been discovered that the b ZIP family genes also play a vital role in regulating the formation of fruit ripening.However,such research is still relatively scarce.Therefore,studying the regulation mechanism of b ZIP family transcription factors on fruit ripening in tomato is of great significance for exploring and improving the function of this family gene.This thesis takes the Sl CPRF2 gene as the research object,amplifies the gene fragment and conducts bioinformatics analysis,analyzes the inducible factors and expression pattern of the gene by q PCR,uses genetic transformation to obtain transgenic plants and observes the phenotypes of their progeny plants,Cytological observation,transcriptomics analysis,screening tomato c DNA library through yeast two-hybrid to find the protein that interacts with Sl CPRF2,and preliminarily clarifying the mechanism of Sl CPRF2 gene in the regulation of tomato ripening.The main results are as follows:1.Using the leaves c DNA of wild-type tomato AC⁺⁺as a template to clone two different transcripts of tomato Sl CPRF2 gene,Sl CPRF2-X1（NCBI accession number:XM＿004244808.4）and Sl CPRF2-X2（NCBI accession number:XM＿010326484.2）.Sl CPRF2-X1 encodes 416 amino acids,and Sl CPRF2-X2 encodes 360 amino acids.Sl CPRF2 is located in the nucleus,has a conserved b ZIP domain,and is a member of the b ZIP gene family.2.Using the RNA from the roots,stems,young leaves,mature leaves,senescence leaf,flowers,sepals and fruits of wild-type tomato AC⁺⁺as materials,the expression of the Sl CPRF2 gene was analyzed by q PCR and it was found that the Sl CPRF2 gene was expressed in the flowers.The expression level of the Sl CPRF2 gene in the fruit gradually increased during the growth of the fruit,and reached the highest at the 4 days after breaker.And then decreased at the 7th day after breaker.The expression in the leaves was stable but less and the expression level is lower in rest of the other organs.3.The expression pattern of Sl CPRF2 gene in fruit ripening mutants rin,nor,Nr is significantly different from that of wild type.The expression level of Sl CPRF2 gene in the fruit mutant rin during the IMG and B+4 phases was significantly higher than that of AC⁺⁺.In nor,the expression of Sl CPRF2 gene was significantly higher than that of AC⁺⁺during the IMG period only.In the Nr mutant,the gene expression of Sl CPRF2 was significantly higher than that of AC⁺⁺in the B period,and significantly decreased in the B+4 period.The results showed that the expression pattern of Sl CPRF2 was significantly different from that in the wild type,and the Sl CPRF2 gene may be involved in the regulation of fruit ripening.4.By applying different exogenous hormones（Eth,ABA,GA,SA,JA）to wild-type tomato AC⁺⁺,collecting leaf samples and extracting RNA,using q RT-PCR to analyze the factors that induce Sl CPRF2 gene expression:（1）Under ABA treatment,the expression of Sl CPRF2 gene showed a trend of first increase and then decrease,and was significantly higher than the control at 1h,6h,and 12h;（2）When Eth treatment for 6h,the expression of Sl CPRF2 gene increased significantly.There was no significant difference in the rest of the time;（3）The expression of Sl CPRF2 gene was significantly lower than that of control at 1h after GA treatment;（4）The expression of Sl CPRF2 gene was significantly lower than that of control when treated with JA exogenous hormone for 12h;（5）Under SA exogenous hormone treatment,Sl CPRF2 gene expression has no significant difference compared with the control.5.The overexpression vector,p VCT2024-Sl CPRF2-X1 and p VCT2024-Sl CPRF2-X2,and the Sl CPRF2 CRISPR knockout vector were constructed respectively.And were transformed wild tomato AC⁺⁺through the Agrobacteria.Obtained 20 independent Sl CPRF2-X1 lines,15 independent Sl CPRF2-X2 lines,and 30 independent CRISPR knockout lines.Select three Sl CPRF2-X1 and Sl CPRF2-X2 overexpression lines,respectively,and 4 transgenic tomato lines knocked out Sl CPRF2 genes at different sites.We found that the transgenic tomato lines knocked out by CRISPR were not significantly different from the wild-type tomato AC⁺⁺,while the overexpression lines,both of the two transcripts of the Sl CPRF2 gene,accelerated the maturation of tomato fruits.The fruits of the Sl CPRF2-X1 overexpression lines showed dark spots around 30 days after pollination.No bacteria or fungi were isolated after culturing the dark spots.The fruit epidermal cells were observed by paraffin sectioning and found that the epidermal cells of the black-spotted fruit were significantly larger than the wild-type tomato fruit cells,and the arrangement was more compact,and the top epidermal cells were dented.6.In order to further explore the reasons for the black spots on the surface of the Sl CPRF2-X1-OE fruit,the fruit samples of the Sl CPRF2-X1 overexpression line during pollination MG and B+4 were analyzed by transcriptome sequencing,and the AC fruit samples of the same period were used as controls.There were 955 genes expressed differentially in the fruits of MG,of which 608 genes were up-regulated and 347 genes were down-regulated.There were 1175 genes expressed differentially in the fruits of B+4stage,of which 548 genes were up-regulated and 627 genes were down-regulated.These differential genes are mainly related genes such as ethylene,MYB,cytochrome p450,etc.It is speculated that Sl CPRF2-X1 may affect the formation of dark spots on the fruit epidermis by regulating these genes.Using q RT-PCR to detect the expression of ethylene pathway-related genes and key genes of the carotenoid pathway.The results showed that:ethylene biosynthesis-related genes（ACO1,ACO3,ACS2）and ethylene response genes（E4,E8,ERF1）in transgenic tomato fruits.The overall expression level was up-regulated at 30 days and down-regulated after 37 days;the expression of carotenoid pathway genes PSY1,PDS（D）and LYCB genes in transgenic tomato fruits were also significantly different compared with wild-type fruits.These results indicate that the Sl CPRF2 gene may affect the ripening of tomato fruits by affecting the expression of genes related to the ethylene pathway and the carotenoid pathway.7.Yeast two-hybrid screening from the tomato c DNA library,42 of the 145 possible interaction proteins with Sl CPRF2 were selected for point-to-point verification.Among them,23 proteins interacted with Sl CPRF2.By analyzing the functions of the interacting proteins,the results showed that these interacting proteins are photosynthesis pathway proteins,stress pathway related proteins,ubiquitin synthesis related proteins,cytochrome synthesis proteins,sucrose synthesis proteins,fruit maturation proteins,and growth and development related proteins.It indicates that Sl CPRF2 may interact with these proteins to regulate the formation of tomato fruit ripening.