Expression Of Duplicated ClR2R3-MYB Genes In Watermelon Response To Low Temperature Stress

Watermelon(Citrullus lanatus)is a widely cultivated and edible horticultural crop all around the world,with high economic value.Low temperature is a common environmental stress factor,which seriously restricts agricultural production.Watermelon is a thermophilic crop.In the early spring and off-season protected cultivation process,watermelon is often subjected to low temperature stress,which affects yield and economic benefits.Studying the function and regulation network of watermelon low temperature response gene can not only analyze the mechanism of watermelon low temperature tolerance,but also provide theoretical basis for genetic improvement of watermelon varieties,which has important theoretical and practical significance.Studies on model plants showed that R2R3-MYB transcription factors play an important role in plant response and tolerant to low temperature.Besides,gene duplication plays an important role in family gene expansion and functional diversity.However,whether R2R3-MYB duplication gene can regulate low temperature tolerance in watermelon and its regulatory network are still unclear.In order to answer the above questions,the watermelon 97103 seedlings were treated under low temperature stress.The phenotypic and physiological responses of watermelon seedlings were observed.RNA-seq data of watermelon seedlings under low temperature stress were analyzed at different time points.Identification of R2R3-MYB transcription factor family and its duplicated genes.Differential expressed duplicated genes co-expression network were constructed under low temperature stress.Predicting downstream interaction cis-elements of duplicated genes,and construct downstream interaction regulatory network.The main results are as follows:1.The phenotypic of watermelon seedlings at 0 h and 6 h,12 h,24 h,36 h and 48 h under low temperature stress,and the determination of malondialdehyde(MDA)and peroxidase showed that with the delay of low temperature treatment,watermelon seedlings were still seriously damaged by low temperature for a long time,although the plants had some ability to recover and adapt.2.The analysis of low temperature transcriptome data of watermelon showed that there were 584 differential expressed genes of 0 h with 6 h,12 h,24 h,36 h and 48 h.The number of differential expressed genes of 0 h and 36 h was the largest.The number of differential expressed genes of 0 h and 48 h was the smallest.There are 10 differential expressed patterns of watermelon genome under the time series of low temperature stress,of which four are more abundant.GO enrichment of differential expressed genes are in biological regulation,metabolic process,cellular process and response to stimulation.KEGG pathway showed that differential expressed genes under low temperature stress are mainly enriched in metabolic and secondary metabolic biosynthesis pathways,it was also found that these genes participated in hormone signal transduction and circadian rhythm process.3.A total of 89 members of the R2R3-MYB transcription factor family in watermelon were identified.48 pairs of R2R3-MYB family duplicated genes were detected,of which 27 pairs(56.25%)were segmental duplicated gene pairs,19 pairs(39.58%)were transposed duplicated gene pairs,1 pair of proximal duplicated gene pairs and 1 pair of tandem duplicated gene pairs.The Ka/Ks values of all the duplicated gene pairs were less than 1,indicating that ClR2R3-MYB duplicated genes were purified selection during evolution process.4.According to RNA-seq and qRT-PCR,only one pair of duplicated genes Cla005982-Cla005982(ClR2R3-MYB50 and ClR2R3-MYB58)were found to be highly differential expressed genes under low temperature stress treatment.Co-expression analysis showed that the target genes ClR2R3-MYB50 and ClR2R3-MYB58 appeared in the blue module,indicating that their expression patterns were similar,and they both have high expression at 48 h after low temperature stress.The co-expression value of ClR2R3-MYB58 was higher than ClR2R3-MYB50.GO enrichment analysis,and KEGG pathway showed that same module and two genes with high correlation have the functions of regulating the activity of enzymes,which are enriched in metabolic pathways and biosynthesis of secondary metabolites5.Screening the promoter region of genes with high correlation with ClR2R3-MYB50 and ClR2R3-MYB58 in co-expression network contains MBS elements.Constructing the regulatory network of ClR2R3-MYB50 and ClR2R3-MYB58 at low temperature.Predicting the downstream gene which binding of ClR2R3-MYB50 and ClR2R3-MYB58,revealed that Cla004399 and other downstream genes may be involved in the regulation of membrane lipid unsaturation and play roles in CBF pathways.It is suggested that ClR2R3-MYB50 and ClR2R3-MYB58 duplicated genes evolve to retain their functions under low temperature stress,and play similar regulatory roles.