Functional Study Of ClTRX H2 And ClTRX H7 Genes In Response To Chilling Stress In Watermelon

Watermelon(Citrullus lanatus)originated in the tropics and is an important horticultural crop of the cucurbitaceae.Watermelon is often subjected to low temperature damage during winter and early spring cultivation,which not only causes the yield reduction of watermelon,but also affects the flavor quality of watermelon.Low temperature stress affects the balance of redox state in plant cells,and Thioredoxins(TRXs)are important components of the cell redox system.TRXs are widely involved in plant responses to environmental stresses,but there are few reports on the response mechanisms of TRXs under low temperature stress.In order to answer the above questions,this paper uses watermelon 97103 and Nicotiana benthamiana as experimental materials,and uses bioinformatics,molecular biology and plant physiology and biochemistry to study the gene excavation and low temperature response function of watermelon ClTRX family.The main research results are as follows:1.Identification and characteristics analysis of watermelon TRX family members.Through the Hidden Mar-kov Model(HMM)algorithm,19 TRX genes were retrieved from the Cucurbitaceae genome database,and all of them contained the conserved functional domain of WC(G/P)PC.Characterization of ClTRXs family members,the coding sequence of ClTRXs gene is 315 bp-594 bp,encoding protein isoelectric point 4.22-9.66,molecular weight 12 kDa-21 kDa.Among them,h-type ClTRXs gene has 12 members,and the expression site is predicted to be located in a variety of organelles mainly composed of plasma membrane and cytoplasm.ClTRX f,ClTRX y,ClTRX z have only one member and ClTRX m has three members,mainly located in the chloroplast.Only one member of ClTRX o is mainly located in the mitochondria.The members of the ClTRXs family are widely distributed on the chromosome.2.Responding to low temperature watermelon ClTRX family gene mining.Using watermelon 97103 as experimental material,qRT-PCR was used to detect the expression of ClTRX family gene in watermelon leaves after low temperature treatment at 5 °C/5 °C.It was found that the expression level of most h-type ClTRXs gene was significantly higher than that of normal temperature control after low temperature treatment,indicating that htype ClTRXs gene may be involved in the response of watermelon to low temperature stress.In the N.benthamiana database we identified 10 members of tobacco h-type NbTRXs.Then using Tobacco rattle virus(TRV)-mediated transient-gene silencing(VIGS)technology,the h-type NbTRXs gene was silenced by selecting different silencing fragments.The low temperature sensitivity of the gene silencing material of h-type NbTRXs was evaluated.It was found that TRV: NbTRX h2/2 had the highest chilling injury index,and the maximum photosynthetic efficiency of PS II decreased at low temperature,and the relative electrolyte leakage rate and malondialdehyde(membrane lipid peroxidation product)content were also significantly higher than the control,the comprehensive index TRV: NbTRX h2/2 showed the most significant low temperature sensitive phenotype compared to other silent lines and controls.3.Functional analysis of ClTRX h2 and ClTRX h7 genes.By constructing a phylogenetic tree of h-type TRXs in tobacco and watermelon,it was found that NbTRX h2/2 and ClTRX h2 and ClTRX h7 had the highest homology.The results of protein high-order structure prediction and promoter element analysis showed that the protein secondary structure of ClTRX h2 and ClTRX h7 was mainly α-helix and random coil,and the tertiary structure was helix-turn-helix.The promoter regions of ClTRX h2 and ClTRX h7 have many elements involved in abiotic stress and hormonal regulation.Both ClTRX h2 and ClTRX h7 are hydrophilic proteins with many phosphorylation sites,none of which have transmembrane domains and belong to intracellular proteins.Subcellular localization was used to construct a transient expression vector,and the fluorescence signals of ClTRX h2 and ClTRX h7 were observed in the plasma membrane and cytoplasm.The model plant N.benthamiana was used as experimental material to construct the overexpression vector of ClTRX h2 and ClTRX h7 gene,and then the tobacco was genetically transformed.After positive identification,the OE strain was obtained.Pretreatment with 10μM SA for 24 h before low temperature treatment can increase the expression of ClTRX h2 gene in watermelon leaves and the expression of ClTRX h7 gene in watermelon leaves,stems and roots.SA pretreatment can increase the expression of ClTRX h2 gene in watermelon stems and roots at low temperature,and increase the expression of ClTRX h7 gene in watermelon leaves and stems.The non-toxic bait vector NMY51 [pBT3-STE-ClTRX h2] was recombinantly constructed,and the concentration of the antibiotic(3-AT)which inhibited the self-activation of the bait vector was 1 mM.The results of protein point-to-point validation interaction showed that ClTRX h2 interacted with five proteins in the ClMAPKK(Mitogen-activated protein kinase kinase)subfamily.The above results were aimed at exploring the regulation of ClTRXs gene in the low temperature response of watermelon.This study not only provides a new way to analyze the low temperature response mechanism of watermelon,but also provides important theoretical basis and candidate gene information for improving the low temperature resistance of watermelon and improving the yield and quality of watermelon from the perspective of molecular breeding and hormone regulation.