Functional Analysis Of Tomato Cell Wall Invertase Inhibitor

Tomato(Solanum lycopersicum L.)belongs to the herbaceous plant of the Solanaceae tomato genus and is one of the important vegetable crops in China.With the continuous development of agricultural technology and the requirements of genetic breeding,the demand for tomato breeding and improvement is increasing day by day.(1)This study cloned the sucrose invertase inhibitory protein genes of three different tomato varieties,wild tomato 124039,cherry tomato 118407,and cultivated tomato 15-196,and obtained three vacuolar sucrose invertase inhibitory proteins(Soly VIF)and three cell wall sucrose invertase inhibitory proteins(Soly CIF),respectively.The basic physical and chemical properties,protein secondary structure,hydrophobicity,signal peptide,phosphorylation site,conservative domain,homology and phylogeny of tomato invertase inhibitor protein were systematically analyzed by bioinformatics methods;The Soly CIF gene fragment is 1411 bp,with 175 amino acids,a molecular weight of 19.92 k Da,and an isoelectric point of 5.53,indicating acidity.Both belong to the PMEI superfamily and are water-soluble proteins.(2)Using CRISPR/Cas9 technology,genome editing of tomato Soly CIF gene targeting site was carried out.This study selected the Bsa I cleavage site adjacent to the PAM sequence on the exon of the Soly CIF gene as the guiding sequence target,and successfully constructed the Cas9-Soly CIF plant expression vector.The experiment used Agrobacterium tumefaciens mediated leaf disc transformation method to introduce the tomato Soly CIF gene into chestnut tomato.Mutants were detected in the target mutation region of the transgenic tomato plants.According to the sequencing results,tomato Cas9-Soly CIF transgenic plants were successfully obtained,resulting in two transgenic tomato plants with Cas9-Soly CIF gene.The transgenic plant grew and received T1 generation seeds.Measure the glucose,fructose,and sucrose content of tomato Soly CIF transgenic plants during their ripening period.By analyzing the difference of soluble sugar content between the transgenic tomato and the control group,it showed that genome editing Soly CIF could increase the enzyme activity of INV and the soluble sugar content in tomato fruit.(3)Through transcriptome RNA Seq technology,high-throughput sequencing was carried out on tomato fruit(INVINH1)and control group Alisa(WT)after genome editing.After sequencing,a total of 42.75 Gb data was obtained,and high-quality data was obtained for quality control and analysis.The differential gene expression levels of INVINH1 and WT are within the range of 0-1FPKM,while the expression patterns of differentially expressed genes are opposite.A total of 3977 differentially expressed genes were screened,with 1501 upregulated differentially expressed genes and 2476 downregulated differentially expressed genes.GO annotation obtained 34151 differential genes,11628 up-regulated differential genes and 22523 down-regulated differential genes.Pathways with high degree of enrichment of differential genes include: response to hypoxia,cell response to oxygen level,response to extracellular stimulation calmodulin binding,secondary activity transmembrane transport protein activity,DNA packaging complex,nucleosome and protein complex.Soly CIF is involved in cell growth,glucose metabolism and synthesis,and affects the growth and development of different organ organisms in tomatoes.KEGG annotation obtained 2776 differentially expressed genes and participated in136 pathways.The pathways with significant differential enrichment included plant hormone signal transduction(a total of 156 differentially expressed genes,up regulated67 genes,and down regulated 89 genes),plant pathogen interaction(a total of 156 differentially expressed genes,up regulated 67 genes,and down regulated 89 genes),carotenoid biosynthesis(a total of 20 differentially expressed genes,up regulated 7 genes,and down regulated 13 genes),Amino sugar and nucleotide sugar metabolism(44differential genes,6 up-regulated and 38 down regulated).This indicates that Soly CIF is involved in metabolic pathways and stress defense in tomatoes,and its expression and activity are influenced by different plant hormone factors and environmental factors.This indicates that Soly CIF is involved in the metabolic pathway and synthesis of secondary metabolites in tomatoes,and its expression and activity are influenced by different plant hormone factors and environmental factors.KOG annotation obtained 26 gene functional descriptions,among which the functions with a large number of differential genes were: energy generation and conversion,amino acid transport and metabolism,carbohydrate transport and metabolism,lipid transport and metabolism,transcription,post translation modification,protein turnover,indicating that Soly CIF is involved in tomato photosynthesis and metabolic transport,inhibits INV activity after transcription and translation,and is influenced by endogenous signals.This study analyzed the physiological and biochemical properties of Soly VIF 和Soly CIF,completed the genome editing of tomato Soly CIF,measured the relationship between the soluble sugar content of transgenic tomato and the activity of Soly CIF,and preliminarily completed the functional analysis of tomato cell wall sucrose invertase inhibitor protein.