Screening And Analysis Of Cold Stress Response And Its Related Factors In Tomato Fruit Under Low Temperature Stress

Low-temperature storage is an important technology to maintain the postharvest quality of fruit and vegetables.However,some tropical and subtropical fruits and vegetables are susceptible to chilling injury(CI)during cold storage,which leads to signifcant degradation of quality.The cold response of fruits and vegetables a complicated process.In addition to the well-known C-repeat binding factor(CBF)pathway,there are still a large number of anti-cold factors whose functions are still unknown.In this study,the model plant tomato fruit was used as the material to analyze the effect of cold stress response induced by low temperature pretreatment on the cold resistance of tomato fruits.In the present work,we screened the key anti-cold factors in the process of cold stress response,and predictd the nature,structure and function of the encoded protein.Finally,the target genes silenced tomato fruits were constructed by the virus-induced gene silencing technology(VIGS)to preliminarily verify the roles of these genes in fruit cold resistance.In addition,the long noncoding RNA104647961(lnc RNA7961)knockout expression vector was constructed using CRISPR/Cas9 gene editing technology to lay the foundation for further study of its mechanism in the process of tomato fruit cold resistance.The main results were as follows:(1)The optimal treatment time of inducing tomato fruit cold stress response with 2~oC was 12 h.Pretreatment at 2~oC for 12 h reduced the CI index and CI incidence,enhanced the degree of fruit color change,inhibited the increase of fruit electrical conductivity and MDA content,induced the activities of antioxidant enzymes(SOD,POD,CAT,APX)and the expression of cold resistance genes(Sl ICE1,Sl ICEa,Sl CBF1,Sl COR),and enhanced the cold tolerance of fruits.(2)Using transcriptome sequencing technology analysis,we found that tomato fruits pretreated with 2~oC had 6,185 up-regulated genes and 6,039 down-regulated genes,which were significantly annotated to metabolic pathways such as ribosomal pathways,transport,and protein processing.In addition,seven cold resistance related factors,including Sl WRKY45,Sl JRE2,Sl GST,Sl STPK,Sl ODD,lnc RNA4504 and lnc RNA7961,were screened.(3)Bioinformatics analysis predicted that Sl WRKY45 was a transcription factor responsive to jasmonic acid,which could regulate ethylene synthesis and heat shock protein expression;Sl JRE2,as a transcription factor of the jasmonic acid signal pathway,could participate in the resistance response of plants and regulate the ethylene signal transduction pathway;Sl GST could participate in the two processes of glutathione metabolism and activation of the growth hormone signaling pathway;Sl STPK,as a plant protein kinase,not only catalyzed the protein phosphorylation process,but also combined with ATP to participate in the defense reaction process;Sl ODD,as a kind of oxygenase,could combine with metal ions to catalyze the redox process.(4)The expressions of Sl WRKY45,Sl JRE2,Sl STPK,Sl ODD,lnc RNA4504 and lnc RNA7961 were successfully silenced in tomato fruits by VIGS technology,and the silence of these genes led to severe symptoms of CI in tomato fruits.In addition,the binary expression vector of p YLCRISPR/Cas9-lnc RNA7961 was successfully constructed to provide a basis for further construction of lnc RNA7961 knockout tomato materials.