Mining Cold-tolerance Related Genes And Functional Verification In Melon

Melon,an important horticultural crop with plenty of nutrients in fruit,is widely cultured all over the world.Melon originated from tropical and subtropical regions and is sensitive to low temperature.In China,melon is usually cultivated in early spring to maximize benefit by going market early.However,the frequent occurrence of “late spring cold” in north China is one of the critical factors restricting the development of melon industry.It is extremely likely to cause melon to suffer from cold stress at seedling stage or early planting stage,which seriously affects the growth and development of melon and even causes delays in the fruit market.Therefore,it is necessary to mine cold-tolerance related genes and study the regulatory network of cold response in melon.Those findings will facilitate a solid foundation and provide excellent genes for cultivating cold tolerance and high-quality melon.In this study,forward genetics and multi-omics methods were used to explore the key genes that regulate seedling cold tolerance or trade-off between seedling cold tolerance and fruit quality in melon.Besides,functions verification of the key candidate genes were conducted and molecular mechanisms in cold tolerance response were analyzed in melon.The main findings are as follows:1.The F₂ and F_2:3 populations constructed from cold-resistant line H581 and cold-sensitive line H906 were used to create a genetic linkage map.Then,a genetic map spanning 1,462.30 c M with an average marker density of 1.39 c M and 12 linkage groups were constructed by dd RAD-Seq and genotyping of 140 F₂ populations.Two major QTLs（q CL3.1 and q CL11.1）for cold tolerance in melon seedlings were identified by combining the chilling injury index（CII）of four environments in two years as trait indexes.Based on genotypic variations in the regions of q CL3.1 and q CL11.1,combined with the transcriptome data and previous researches,21 and 12 preliminary candidates were obtained from q CL3.1 and q CL11.1,respectively.Further,two key cold tolerance regulatory genes Cmb ZIP53（MELO3C010967.2）and Cm RR14（MELO3C022469.2）were identified by cold-induced expression analysis and transient transformation.Both of Cmb ZIP53 and Cm RR14 are negative regulators of seedling cold tolerance in melon.2.In this study,a total of 18 Cm RR genes were identified from melon genome and classified into three types（type A,type B,and clock PRR）according to phylogenetic analysis.Expression analysis showed that most of Cm RR genes exhibited tissue-specific expression patterns and some of Cm RRs were cold-induced.In addition,a variety of stresses and hormone-related cis-acting elements were found in the promoters of Cm RR genes,implying that these genes could respond to multiple stresses.Further,Cm RR6 and Cm PRR3 were verified as negative regulators of seedling cold tolerance in melon by virus-induced gene silencing（VIGS）.Moreover,the interaction between Cm RR6 and Cm ARF5 was verified by luciferase complementary assay.3.Cmb ZIP53,a candidate gene from q CL3.1,had two non-synonymous mutations in the CDS between H581 and H906.We analyzed the functions of the two transcripts（Cmb ZIP53-H581 and Cmb ZIP53-H906）in Arabidopsis and melon,respectively.Overexpression of Cmb ZIP53-H581/H906 decreased cold tolerance in both of Arabidopsis and melon.These results indicated that Cmb ZIP53 was a negative regulator of seedling cold tolerance in melon,and the non-synonymous mutations of H581 and H906 did not affect gene function.Subcellular localization showed that both Cmb ZIP53-H581 and Cmb ZIP53-H906 were located in the nucleus.Meanwhile,the expression patterns of Cmb ZIP53-H581 and Cmb ZIP53-H906 were consistent,but the expression levels were significantly different.Y1 H results indicated that both of them could bind Cm CBF1/2/3 promoters and G-box element to inhibit the reporter expression in yeast.Similarly,LUC experiment further demonstrated that they could directly target Cm CBF1 promoter and negatively regulate the transcription of the reporter gene in tobacco.Y2 H and split luciferase complementation experiment demonstrated that Cmb ZIP53/Cmb ZIP53 and Cmb ZIP53/Cmb ZIP9 could form homodimer or heterodimer,regulating melon response to cold stress.In general,promoter activity affects gene expression.SNPs and Indels were found in Cmb ZIP53 promoters of H581 and H906,among which SNP at-1,592 bp and-1,527 bp in H906 resulted in two new AE-Box and MYC cis-acting elements,respectively,while SNP at-67 bp in H581 caused a new TATA-Box.However,the promoter activity of Cmb ZIP53 gene in H581 was higher than that in H906,indicating that that the low expression of Cmb ZIP53 in H581 was not due to its promoter activity.So,we speculated that the difference of Cmb ZIP53 expression between H581 and H906 might be caused by mi RNA,which might be the main reason for different cold tolerance of H581 and H906.4.Metabolic profiles of mature fruit flesh of eight materials with different cold tolerance were performed by GC-MS.A total of 31 primary metabolites were detected by 48 standards,including twelve amino acids,ten organic acids and nine soluble sugars.Overall,most primary metabolites in cold-tolerance melons were generally lower than those in cold-sensitive melons.Typically,the content of metabolites in cold-resistant line H581 and moderately cold-resistant line HH09 were significantly different.Weighted gene co-expression network analysis（WGCNA）was performed basing on metabolites and transcriptome data in H581 and HH09.Five candidate genes MELO3C016749.2（Cm WEB1）,MELO3C012147.2（Cm EAF7）,MELO3C026791.2（Cm WD40）,MELO3C010343.2（Cm SPX1）,and MELO3C024337.2（Cm TMEM78B）were obtained.Then,the function of the key candidate gene Cm EAF7 was analyzed by transient expression.Under cold stress,silence of Cm EAF7 significantly decreased the cold tolerance at seedling stage.And transient overexpression in fruits increased sucrose and fructose content in flesh,while silencing Cm EAF7 decreased sucrose and fructose content,but did not affect glucose content.In summary,four negative regulators of seedling cold tolerance（Cmb ZIP53,Cm RR14,Cm RR6,and Cm PRR3）and one “ideal” trade-off gene（Cm EAF7）that improve seedling cold tolerance and fruit quality in melon were identified in this study,and gene function was verified through heterologous overexpression in Arabidopsis or transient transformation in melon.At the same time,we explored the molecular mechanism of Cmb ZIP53 gene regulating cold tolerance in melon.It was found that Cmb ZIP53 formed homodimer or heterodimer with Cmb ZIP53 or Cmb ZIP9,and directly targeted Cm CBF1 promoter to inhibit gene transcription to negatively regulate seedling cold tolerance in melon.Our findings will provide new gene resources for cold-tolerance molecular breeding or cooperative improvement breeding of melon,and lay a theoretical foundation for the research of cold tolerance regulation network in melon.