Functional Identification And Regulation Analysis Of Low Temperature Responsive ClMYB46 In Watermelon

Watermelon(Citrullus lanatus) is an important cucurbitaceae crop with high economic value.Watermelon is a typical thermophilic crop,in the process of cultivation it often comes across low temperature stress,so its production and economic value are affected severely.Low temperature has become the main limiting factor of abiotic stress in the production of watermelon,which seriously restrict the annual supply and planting time of watermelon.MYB transcription factors play an important role in plant response to external stress environment,which is involved in regulating downstream expression of multiple genes.In this study,we analyzed the transcriptome data about the watermelon 97103 seeding leaves with low temperature treatment of 10?,found a MYB transcription factor Cla005622,further analysis showed that the expression of Cla005622 was significantly up-regulated and belong to MYB-related transcription factor,Its roles in resistant to low temperature stress is not clear.In order to answer the above-mentioned questions,this study will identify the MYB-related family members,analyze the structural characteristics of Cla005622,qRT-PCR identified the response of Cla005622 in watermelon at low temperature,subcellular localization and transcriptional activity identified the characteristics of Cla005622.Identification of resistance in transplants under low temperature treatment and found the interaction proteins of Cla005622 by using yeast two-hybrid system.The results showed that Cla005622 was not only participating in the resistance under low temperature stress,but also analysing the low temperature tolerance mechanism in watermelon.What's more,these results can improve the molecular breeding of watermelon,finally will increase watermelon production and quality of genetic resources.The main results of this paper are as follows:1.Structure analysis of Cla005622 and identification of MYB-related family members in watermelon.Using NCBI to predict the conserved domains of Cla005622 protein,which predict it is a MYB-related gene.In order to further explore its characteristics in the MYB-related family,50 MYB-related family genes were identified by HMM in the whole genome of watermelon.The coding sequence length was 240bp-4986 bp,the isoelectric point was 4.8-9.97,the molecular weight was 5890.67-181178.4.It is given the name of ClMYB1-ClMYB50 according to the distribution of genes on chromosomes.Cla005622 corresponds to ClMYB46,the coding sequence is 1040 bp in length,encodes 334 amino acids with an isoelectric point of 6.70 and a molecular weight of 35890.61,located on chromosome 10.2.The response of ClMYB46 at low temperature in watermelon.Trancriptomic data and qRT-PCR revealed that the expression of ClMYB46 can be induced by up-regulating under low temperature.Using watermelon 97103 as the experimental material,gene expression was first increased then decreased,after that increased again.Among them,after 6h point of expression reached the highest.Thus,ClMYB46 was significantly up-regulated by low temperature stress induction.3.The characteristic analysis of ClMYB46.We constructed plant expression vector of GV3101 [pH7LIC5.0-N-eGFP-ClMYB46] and transformed it into Nicotiana benthamiana for transient expression,at last we observed leaf epidermal cells with confocal microscopy that the green fluorescence was distributed in the nucleus and overlapped the fluorescence of the nucleus dye DAPI.Transcriptional activity was identified by the constructed expression vector of Y2 HGold [pGBKT7-ClMYB46],the reporter genes of HIS3,ADE2 be activily expressed.Thus,ClMYB46 is expressed in the nucleus and has transcriptional activity.4.Identification the resistance of ClMYB46 under low temperature.Tobacco genetic transformation using the vector LBA4404 [pHellsgate8-ClMYB46].The expression of ClMYB46 was detected by qRT-PCR and the subsequent experiments were carried out to screen OE1 and OE2 strains.Phenotypic observation and chilling injury index showed that the wilting degree of wild type plants was higher than that of transgenic lines at different time points.ClMYB46 could increase the resistance of tobacco in low temperature stress.Using the kit to determine the conductivity and malondialdehyde content at different time points,it was found that the damage of wild type cell membrane was more serious,and overexpression of ClMYB46 could improve the resistance of tobacco to low temperature stress.The results showed that ABA signal transduction pathway related gene(rab18,ABI1,ABI2)with cold stress related genes(DREB transcription factor DREB2 A,Proline synthesis promoting gene P5CS)were detected by qRT-PCR at different time points by using the wild type tobacco and transgenic lines as materials.ClMYB46 upregulated caused upregulation of rab18,ABI1/ABI2 down-regulated expression,DREB2 A and P5 CS up-regulated expression.5.Identified the interaction proteins of ClMYB46 and point to point verification.Construction of non-toxic bait vector Y2 HGold [pGBKT7-ClMYB46],use AbA inhibition self-activated before Mating screening and sequencing.There are 10 functional intercellular proteins were screened,mainly including: maintaining the balance of intracellular environment,salt stress response,maintaining ion channel balance,responding to ABA signal transduction pathway,and participating in jasmonate stress response.Among them,ClMYB46 interacting protein Cla014285(At CPK28)was involved in protein phosphorylation pathway,ABA signal transduction pathway and Ca2 + signal transduction pathway,which induced the response of plant to stress.