Molecular Mechanism Of LncRNA1717-miR408-TaPLC33 Module Regulating Winter Wheat In Response To Low Temperature

Low temperature stress is one of the common abiotic stresses that affect crop growth and development.Overwintering crops are scarce in the extremely cold areas of northern China,but Dongnong Dongmai 1（Dn1）is the first winter wheat variety in Heilongjiang Province that can be safely overwintered with a return rate of>85%.Therefore,it is particularly important to study its cold resistance mechanism.It is known that miRNAs are involved in plants response to multiple stress,and miR408 can mediate plant responses to environment.Lnc RNAs are a class of non-coding RNAs that are indispensable for normal plant growth and development and can participate in stress response as competing endogenous RNAs（ceRNAs）,which competitively bind miRNAs and co-regulate the expression of target genes.Our preliminary RNA database analysis revealed that tae-miR408 and lncRNA1717 were significantly differentially expressed in Dn1 at different temperatures.How they coordinate to participate in the wheat cold response needs to be investigated in depth.In this study,we used Dn1 as the material and verified the regulatory relationship among tae-miR408,lncRNA1717 and TaPLC33 by bioinformatics analysis,qRT-PCR,dual luciferase assay,5’RACE and RAP.Their cold resistance was verified by obtaining the overexpression of lncRNA1717,tae-miR408 and TaPLC33,as well as the silent expression of mlncRNA1717 and miR408 in Arabidopsis plants,thus revealing the molecular mechanism of the lncRNA1717-tae-miR408-TaPLC33 module in response to low temperature stress,and providing new ideas for molecular breeding of wheat for cold resistance.The main findings were as follows:（1）Bioinformatics analysis revealed that winter wheat pre-tae-miR408a and pre-tae-miR408c had typical stem-loop structures and were highly conserved in the mature miRNA region.tae-miR408 target gene was predicted to be TaPLC33,a member of the Plantacyanin family,which was 289 amino acids in length and contained a plastocyanin structural domain;lncRNA1717was 825 bp in length and was predicted to interact with tae-miR408.（2）The results of 5’RACE verified that tae-miR408 targeted the cleavage site of TaPLC33;the partial TaPLC33 sequence containing the cleavage site,the m TaPLC33 sequence and tae-miR408were cloned into dual luciferase system expression vector containing the reporter gene LUC,respectively.These recombinant plasmids were transiently expressed in tobacco.The results showed that tae-miR408 could target cleavage of TaPLC33.（3）The results of RAP verified that lncRNA1717 interacted with tae-miR408;lncRNA1717,mlncRNA1717 and tae-miR408 were cloned into the dual luciferase system expression vector containing the reporter gene LUC,respectively.These recombinant plasmids were transiently expressed in tobacco.The results showed that lncRNA1717 interacted with tae-miR408 interacted with each other.（4）Partial TaPLC33 sequences containing cleavage sites,lncRNA1717,mlncRNA1717 and tae-miR408 were constructed into expression vectors containing the GUS reporter gene for tobacco cotransformation transient expression.The results showed that that lncRNA1717 can act as a tae-miR408 sponge by binding to tae-miR408 through the e TM binding site,inhibiting the activity of tae-miR408 and thus blocking the negative regulatory effect of tae-miR408 on TaPLC33.（5）qRT-PCR analysis was performed on Dn1 tiller nodes treated with natural cooling in the field（5°C,0°C,-10°C and-25°C）and low temperatures indoors（5°C,0°C,-10°C and-25°C）.The results showed that the expression of Dn1 tae-miR408 decreased significantly with lower temperature in winter in the field,and the expression of its target gene TaPLC33 rose and then fell,reaching a peak at-10°C and the opposite trend to that of tae-miR408;the expression of lncRNA1717 decreased significantly.The expression of Dn1 in indoor culture decreased significantly with the decrease in temperature of tae-miR408,and the expression of its target gene TaPLC33 rose and then fell,reaching a peak at-10°C and the opposite trend of tae-miR408;the expression of lncRNA1717 rose and then fell,reaching a peak at-10°C.（6）The vectors of overexpressing pre-tae-miR408a,pre-tae-miR408c,lncRNA1717 and T a PLC33 as well as the vector of overexpressing mlncRNA1717,which mutating the recipro cal region of lncRNA1717 and tae-miR408,and the miR408 silencing vector were construc ted and transformed into Arabidopsis thaliana,respectively.T₃generation positive plants w ere obtained;Agrobacterium mediated methods and STTM-tae-miR408-silenced T₀generatio n transgenic Dn1 plants were obtained by resistance screening.（7）The aforementioned transgenic Arabidopsis plants were subjected to cold stress treatment,we observed the phenotype and found that OEmiR408 has strongest cold resistance,OElncRNA1717 had weaker cold resistance than the wild type（WT）,and STTM-miR408 had the weakest cold resistance;physiological index assays showed that OEmiR408 had lower conductivity,MDA,H₂O₂and O₂.^-content than WT,and its DAB and NBT chemical staining was lighter than that of WT.In contrast,the conductivity,MDA,H₂O₂and O₂.^-content of STTM-miR408 were higher than those of WT,and its DAB and NBT chemistry staining was lighter than that of the wild type.These results showed that STTM-miR408 had lower survival rate,higher cell membrane damage and membrane lipid peroxidation.Analysis of antioxidant enzyme activities and related gene expressions showed that the enzyme activities of SOD,POD and CAT and their gene expressions were significantly higher in OEmiR408 than that in WT under low temperature stress;while the antioxidant enzyme activities and their gene expressions were significantly lower in STTM-miR408 than that in WT.The overexpression of STTM-miR408enhanced the cold resistance of Arabidopsis plants.In summary,this experiment provides preliminary evidence that tae-miR408 in Dn1 is involved in freezing stress and can be regulated by lncRNA1717 to further regulate the expression of the target gene TaPLC33,thereby affecting the expression of other oxidative stress genes.This experiment provides new insights into the molecular mechanism of the lncRNA1717-tae-miR408-TaPLC33 module regulating the response to freezing stress in wheat.