| Watermelon(Citrullus lanatus)is a temperature-friendly and heat-resistant crop,which is also a typical cold-sensitive plant.The most suitable temperature for its growth is 25-30℃,and the growth and development of plants becomes slow or even stops below 10℃.Low temperature can affect the normal growth of watermelon,which reduces the quality and yield of watermelon.In recent years,many miRNAs families have been identified in plants,the miR160 family is highly conserved,and many studies have shown that it targets auxin response factor(ARF).In addition,miR160a and its ARF target genes have also been reported to be involved in the regulation of seed development,signal transduction,root tip development and abiotic stress processes such as drought,salt,chilling and heat stress.Based on transcriptome data,We screened miR160a and its candidate target genes involved in low temperature response,but their regulation mechanism with chilling stress in watermelon remains unclear.In this study,the Cl-miR160a and ARF genes were analyzed by bioinformatics,and virus-induced gene silencing(VIGS)was used to study them.Cl-miR160a silencing plants(TRSV-STTM160a)and ARF target genes silencing plants(pV190-ClARFs)were constructed to observe their phenotypes in chilling treatment,determine the related physiological indexes,and analyze the expression characteristics of genes related to cold tolerance pathway,preliminarily explored the regulation mechanism of Cl-miR160a on chilling tolerance,which provided reference for further research on the molecular mechanism and regulatory network related to low temperature in watermelon.The main research results of this paper are as follows:1、Using bioinformatics analyzed the sequence of Cl-miR160a,identify the members of ARF genes family in watermelon and predict the ARF target genes of Cl-miR160a,and the targeting relationship was verified by transient infection of tobacco.By analyzing the promoter sequence of Cl-miR160a and its ARF target gene,it was found that there were many stress related cis-acting elements.2、The germplasm "LB2" suitable for VIGS was selected from the watermelon materials in our laboratory,and the vacuum negative pressure method was optimized to inoculate and silence the germinated seeds.The results showed that the infection success rate of "LB2" inbred line could reach 90%.Vacuum negative pressure method can greatly advance the time of virus-induced gene silencing,so that it can play a role when two leaves are in one mind,which is conducive to functional verification at seedling stage.3、The silencing vector of Cl-miR160a was successfully constructed based on STTM technology and TRSV vector and the silencing watermelon seedlings of Cl-miR160a were obtained.Silencing plants of ARF target genes were constructed using CGMMV vector,and the silencing success rate of TRSV reached 88.9%,the silencing success rate of CGMMV could reach 100%.4、Under chilling stress,Cl-miR160a silencing material TRSV-STTM160a seedlings were more sensitive to low temperature.Fluorescence quantitative analysis showed that Cl-miR160a silencing would lead to changes in the expression of genes related to CBF-dependent low temperature signal transduction pathway and antioxidant genes such as SOD,POD,GST and CAT.At the same time,the expression pattern analysis of related genes preliminarily suggested that Cl-miR160a may affect the low temperature tolerance of watermelon mainly through negative regulation of ClARF08 and CIARF10 under low temperature stress.5、Through chilling treatment of ClARFs silent plants,it was found that ClARFs silent plants had different tolerance degrees under low temperature stress,and pV190-ClARF08 and pV190-ClARF10 watermelon seedlings had relatively better tolerance to low temperature.It is further indicated that Cl-miR160a may be involved in the low temperature resistance regulation mainly through the regulation of ClARF08 and ClARF10. |
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