Effects Of SlARG2 Silence On Postharvest Storage Quality And Arginine Metabolism Of Tomato Fruit

Arginine,a basic amino acid with a high N/C ratio,serves as an important nitrogen reserve in many plants.Polyamines,proline and nitric oxide(NO),the metabolite of arginine played an important role in fruit growth and development,stress metabolism and mature aging.Arginase is one of the key enzymes involved in the metabolism of arginine,and the change of its activity has a potential effect on the metabolic direction of arginine,and as a result the quality and postharvest aging process of fruit may also be influenced by arginine metabolsim,but the related research rarely reported.Virus-induced gene silencing(VIGS)as a fast,effective,high-throughout reverse genetics technology has played an important role in the function study of gene.In the present study,we silenced the SlARG2 gene which encoded the arginase to research the role of arginase in regulating the storage quality and and the metabolism of arginine of postharvest tomato.The main results obtained were summarized in the following portions.(1)In this study,the VIGS system was constructed with Tobacco rattle virus(TRV)in the tomato plant and fruit by using the phytoene desaturase gene(SlPDS)as the marker gene.SlPDS gene silence resulted in photo bleaching phenomenon of the leaves,flowers and fruits of tomato plants.Thet lycopene content in silenced tomato fruit was 77.8% lower than that in TRV empty vector treated fruit.Moreover,real-time fluorescence quantitative PCR and semi-quantitative PCR analysis showed that the relative expression of SlPDS in the silenced tomato was only 20% of the TRV treatment.The results all above indicated that we have successfully constructed the VIGS system in tomato plants and fruits.(2)In the follow-up experiment,we silenced SlARG2 gene with the same method.The real-time fluorescence quantitative PCR and semi-quantitative PCR analysis showed that the relative expression of SlARG2 in the silenced tomato was significantly inhibited,the relative expression of which was only 38% of TRV empty vector treated fruit.The result indicated that the target gene SlARG2 was highly silenced,laying the foundation for the follow-up experiment.(3)In this experiment,the SlPDS gene was used as the indicator gene to construct the tandem vector with the target gene SlARG2,and the results showed that the leaves,flowers and fruits of tomato plants treated with tandem vector showed the same photo bleaching phenomenon as SlPDS silenced.Real-time quantitative PCR and semi-quantitative PCR analysis showed that the expression of SlPDS and SlARG2 was significantly inhibited,indicating that we successfully constructed a tandem vector containing both the marker geneand the target gene.This result provided the technical support for studying the function of target gene without obvious phenotype after silence.(4)Silencing of SlARG2 gene reduced the firmness of the fruit,promoted the increase of cell conductivity and malondialdehyde content,and reduced the activity of antioxidant enzymes such as superoxide disproportionate,catalase and ascorbate peroxidase activity,as well as total phenol and vitamin C and other antioxidant substances,so that the antioxidant capacity of the fruit decreased;accelerated fruit aging process.While the tobacco embryo virus empty vector treatment did not have a significant impact on fruit storage quality.(5)The gene expression and enzymes activity analysis indicated that silencing of SlARG2 gene inhibit the expression of SlARG2,SlADC and SlODC;determination of polyamine and arginine metabolism related amino acids high performance liquid chromatography,and the result induced that the silencing of SlARG2 gene resulted in the decrease of arginine,ornithine decarboxylase and ornithine aminotransferase activity,arginine metabolism of the direct product of ornithine,proline and polyamine content decreased;induced arginine decarboxylase activity increased;however,the NO content and the nitric oxide synthase activity showed that SlARG2 silencing did not significantly affect nitric oxide synthase pathway.Moreover,Tobacco brittle virus empty vector treatment did not significantly affect the arginine metabolism of tomato fruit.