Study On The Role Of Tomato SlGolS2 In Fruit Development

Galactinol synthase(Gol S)plays a key rate-limiting role in the biosynthesis of raffinose family oligosaccharides(RFOs).RFOs are important soluble carbohydrates in plants,including trisaccharide raffinose,tetrasaccharide stachyose and pentasaccharide verbascose.and have functions such as reserve carbohydrates,stabilizing membrane structure,resisting stress,and participating in osmoregulation.Gol S participates in regulating plant growth and development and resisting stress by affecting the content of RFOs.There are 4 Gol S genes in tomato,and the functions of these 4 genes have not been reported yet.In this study,wild-type tomato Ailsa Craig was used as the experimental material to study the function of the SlGolS2 gene with the following results:1.Preliminarily clarified the expression pattern and potential function of SlGolS2 gene in tomato.The expression of SlGolS2 gene in tomato tissues was determined by q RT-PCR and revealed that it was expressed in tomato roots,stems,leaves,flower buds,flowers,various floral organs,and fruits at all stages,particularly,abundantly expressed in the ovary at the flower bud stage,indicating that SlGolS2 genes may be closely involved in tomato fruit growth and development.Therefore,we constructed a CRISPR-Cas9 knockout vector of SlGolS2 gene to further explore the biological function of SlGolS2 gene,which was transferred into Ailsa Craig tomato by Agrobacterium mediated method,and homozygous stably inherited slgols2 T2 and T3 knockout mutant lines were screened for subsequent experiments2.Clarified the influence of SlGolS2 gene on tomato fruit development and maturity.The green mature fruit of the slgols2 knock-out mutant lines are uniform light green,and the dark green shoulders disappeared.slgols2 mutant tomato fruits turned color earlier compared to wild-type tomato.It shows that the SlGolS2 gene can affect the color and lustre of tomato fruit during the mature green period and fruit ripening.3.Clarified the influence of SlGolS2 gene on tomato fruit parenchymal structure and metabolites.The chlorophyll content in the pericarp during the mature green period was reduced,the plastids appeared light green,the chloroplast volume is reduced,the thylakoid lamella structure is reduced,and the contents of ALA,PBG,Urogen III、Proto IX、Mg-Proto IX and Pchlide a are decreased.It shows that the SlGolS2 gene affects the content change of chlorophyll by affecting the accumulation of precursors of chlorophyll biosynthesis,chloroplast structure,and finally leading the fruit to appear uniform Ly light green.Tomato color index confirmed that the color change of the slgols2 mutant was earlier than that of the wild-type tomato fruit.The total carotenoids and lycopene accumulated in a large amount,the fruit hardness decreased earlier,and the ethylene release surge occurred earlier.It shows that SlGolS2 gene affects tomato fruit ripening by affecting carotenoid accumulation,ethylene release and firmness.4.The genes related to chlorophyll synthesis and chloroplast development in tomato fruits that are affected by the SlGolS2 gene are preliminarily identified.Using q RT-PCR technology,the chlorophyll biosynthesis-related genes affected by the SlGolS2 gene were selected.The expression of HEMA1,GSA,HEMB,HEMC,HEME1,HEME2,HEMF,HEMG1,FC1,CHLH,CHLI,CHLD,CRD1,DVR,PORA,PORB,PORC,CAO1,CAO2,CHLG in the slgols2 mutant tomato pericarp was significantly down-regulated.Through RNA-seq,the related genes of chlorophyll biosynthesis and chloroplast development affected by SlGolS2 were further screened,indicating that the SlGolS2 influences the expression of the above genes and ultimately leads to the formation of light green fruits in the mature green period.5.Preliminary identification of genes related to cucurbitacin and ethylene accumulation in tomato fruits affected by the SlGolS2 gene.Using q RT-PCR technology,genes related to carotenoid biosynthesis and ethylene biosynthesis affected by the SlGolS2 gene were obtained.In the slgols2 mutant tomato fruits,the expression of PSY1,NOR,RIN and AP2 A genes increased significantly,indicating that the SlGolS2 gene affects the accumulation of carotenoids by affecting the expression of these genes.The significant up-regulation of ACS2,ACS4,E4,E8 and ACO3 gene expression indicates that the SlGolS2 gene affects the release of ethylene by affecting the expression of these genes.