Construction Of Tomato Multiple Transcription Factor CRISPR/Cas9 Mutant Library And Analysis Of Nf-y Gamma Like Mutants

Tomato(Solamum lycopersicum),an important economic and widely cultivated vegetable crop,has become an ideal model plant owing to its important economic value,smaller genome size,easier to modify the transformation and regenerate.et.al.CRISPR/Cas gene editing technology has developed rapidly in recent years,and has been continuously improved.The CRISPR/Cas9 genome engineering tool system has been developed,which is simple and efficient,and can create small insertions and deletions(insert deletions)of specific target genes.CRISPR/Cas9 mutant libraries have been developed for screening of genome-wide mutations in cultured eukaryotic cells,rice,and screening of tomato immune-related genes,and related gene editing systems have gradually matured.In this study,CRISPR/Cas9 technology was used to construct a tomato multiple transcription factor deletion mutant,and a NF-Y gamma like deletion mutant was obtained from it,and the function of the NF-Y gamma like gene was initially explored.The main results of the paper are as follows:CRISPR/Cas9 technology was used to construct a tomato transcription factor plasmid library,Agrobacterium transformation method was used to obtain deletion mutants.A total of 926 tissue culture plants and 452 positive plants were obtained.T0 generation DNA was extracted using CTAB,and amplified by PCR.There are 65 types of transcription factors involved in the results.Then primers are designed for the full length of corresponding transcription factors,and using primers PCR sequencing,effectively there are 20 types of mutations and 16 types of transcription factors involved.The CRISPR/Cas9 gene transformation systems of two tomato varieties,M82 and AC,were optimized.Use water containing different concentrations of hygromycin to germinate and irrigate germinated seedlings,determine the seed germination and growth status,and determine the lethal concentration of hygromycin.Under different OD600 values and different durations of infection,determine the explant healing.The formation rate of wound tissue was screened for the appropriate infection combination.Under the selected infection combination,different concentrations of acetylsyringone were added to the suspension to determine the callus formation and positive rate,and the appropriate AS concentration was selected for subsequent experiments.The lethal concentration of hygromycin is 3mg/L under M82 background,the most suitable infection combination is OD600=0.8,the infection time is 4 minutes,and the suspension efficiency of AS concentration 100 μmol/L is the best.Under AC background,hygromycin The lethal concentration is 2mg/L,the most suitable infection combination is OD600=0.6,the infection time is 6min,and the suspension efficiency with the AS concentration of 50 or 100μmol/L is the best.The callus formation rate of explants was measured under different OD600 values and different infection durations.Under the M82 background,when OD600=0.8 and the infection duration is 4min,the callus formation rate is significantly different from other infection combinations;under AC background,when OD600=0.6 and the infection duration is 6min,the callus formation rate and there were significant differences in other infection combinations.Under the appropriate infection combination screened,different concentrations of acetylsyringone were added to the suspension to determine the callus formation and positive rate.Under the M82 background,a suspension with an AS concentration of 100 μmol/L was used to callus the tissue formation rate and positive rate were significantly higher than those of other treatments.Under AC background,using a suspension with AS concentration of 50 or 100μmol/L,the callus formation rate and positive rate were significantly higher than those of other treatments.T1 generation of NF-Y gamma like deletion mutant was randomly selected and sequenced as 13 homozygous mutants.Compared with the wild type,the lack of NF-Y gamma like leads to early seed germination,plant dwarfing,accelerates seedling growth,promotes primary root elongation,and affectes the growth of lateral roots.Under 150mM NaCl salt stress,the phenotypic changes of NF-Y gamma like deletion plants and wild type were observed.Compared with the wild type,the salt tolerance of mutants was reduced;RT-PCR was used to analyze tomato NF-Y gamma like gene expression changes under low salt stress,in the initial stage of stress,the expression increased rapidly,and then decreased,and after recovery treatment,the expression increased.