Construction And Analysis Of SNAC4/9 Gene Knockout Tomato Mutant By CRISPR/Cas9 System

The NAC transcription factors are the plant-specific transcription factors,which can regulate the different stages of plant growth and development.The previous research of the laboratory showed that SNAC4/9 is closely related to fruit ripening.However,the specific mechanism of SNAC4/9 regulating tomato fruit ripening is still unclear.In this study,GO enrichment analysis was performed on the SNAC4 target genes obtained by Ch IP-seq in the previous research of our laboratory,and the SNAC4 target genes related to fruit ripening were explored deeply.Based on the CRISPR/Cas9 technology,the SNAC4/9 single gene and double gene knockout vectors were constructed,and the vectors were transformed into Micro-Tom tomato to obtain SNAC4/9 single gene and double gene knockout mutants,which provided techniques and effective materials for exploring the differential mechanism of SNAC4 and SNAC9 transcription factors regulating tomato fruit ripening and senescence.The main findings are as follows:1.GO enrichment analysis showed that the target genes of SNAC4 were mainly enriched in phytohormone signal transduction pathways such as auxin,ethylene,abscisic acid and jasmonic acid.2.Through the screening of ripening-related pathways,in the mature green tomato fruits,48,23,29,53 and 11 SNAC4 target genes related to auxin,ethylene,ABA,cell wall metabolism,and fruit coloring were screened respectively;in the red ripening tomato fruits,18,15,14,33 and 4 SNAC4 target genes related to auxin,ethylene,ABA,cell wall metabolism,and fruit coloring were screened respectively.Thirteen target genes were selected for Ch IP-q PCR verification experiments.The results were basically consistent with the Ch IP-seq results,proving the reliability of the Ch IP-seq data.3.Five(4T1-4T5)and six(9T1-9T6)targets in the CDS region of SNAC4/9 gene were designed respectively,and it was successful in construction of four SNAC4 single gene single target vectors(named No.1/3/4/5),five SNAC9 single gene single target vectors(named No.6～10),one SNAC4 single gene double target vector(named No.11),two SNAC9 single gene double target vectors(Named No.13/14),two SNAC4/9double gene double target vectors(named No.15/16)and one SNAC4/9 double gene four target vector(named No.17).4.Three SNAC4/9 single gene editing vectors(No.5,No.11 and No.14)and two double gene editing vectors(No.15 and No.16)were selected to carry out tomato genetic transformation,and the target sites and potential off-targets sites were sequenced.Finally,24 SNAC4 gene mutants,9 SNAC9 gene mutants,and 1 SNAC4/9double gene mutant were obtained.Among them,5-8/5-9/5-11/14-12/14-14/14-17/14-61/16-20 mutants had a homozygous mutation.5.Preliminary phenotypic observation of 11-31/16-70/14-57/14-61/16-20 mutant fruits at different ripening stages revealed that the knockout of SNAC4/9 gene resulted in tomato fruits not being fully ripe.