TalncR3017 As CeRNA Mediates MiR408 Regulating Winter Wheat Response To Low Temperature

Low temperature is one of the main abiotic stresses that affect crop growth and limit yield.In extreme cold regions,it is difficult for crops to stabilize through the winter.Dongnongdongmai 1（Dn1）is the first winter wheat that can be safely overwinter ed in extreme cold regions of Heilongjiang Province.It is important to study its cold resistance mechanism.It is known that miRNA is involved in many stress response of plants,and miR408 can mediate plant response to environment.LncRNA is a type of non-coding RNA that is indispensable for the normal growth and development of plants.It can be used as competitive endogenous RNAs（cceRNA）.It can compete with miRNA to regulate the expression of target genes and participate in stress responses.Our database analysis found that the differential expression of TamiR408 and TalncR3017 in Dn1 was significant at different temperatures.How to coordinate the participation of wheat in cold resistance response needs to be further studied.In this study,Dn1 was used as the experimental material.Firstly,we predicted that TalncR3017 could be used as cceRNA to competitively bind to TamiR408with m RNA and regulate its expression.Then,Dn1 leaves and tillering nodes were obtained in the temperature that naturally decreased to 5℃,-10℃,and-25℃for 10 consecutive days.The gene expression levels of TamiR408,TalncR3017 and TaCLP were measured,and the tae-pre-miR408and TalncR3017 genes were isolated from Dn1.They were overexpressed in Arabidopsis,and the TalncR3017 mutation and miR408 silencing vector were transferred to Arabidopsis used as a complementary experiment.To explore the molecular mechanism of TalncR3017-TamiR408-TaCLP module in response to cold stress.The experimental results are as follows:（1）Bioinformatics analysis shows that winter w heat tae-pre-miR408 has a typical stem-loop structure,and the mature is well conserved.The TamiR408 target gene is predicted to be TaCLP,TaCLP is 126 amino acids in length,containing the plantacyanin domain,and it is a member of the phytocyanin family,and TalncR3017 is a lnc RNA with a length of 397 bp.It is predicted to be TamiR408’s cceRNA and can adsorb TamiR408.（2）We analysis the expression patterns of TamiR408,TaCLP,and TalncR3017 in winter wheat leaves and tillering nodes at 5℃,-10℃,and-25℃:In leaves,firstly,the expression pattern of TamiR408 decreased significantly and then did not change significantly.The expression pattern of TaCLP gradually increased with decreasing temperature,and the target gene TaCLP and TamiR408had opposite expression pattern.The expression pattern of TalncR3017 showed a significant decrease and then did not change significantly.In the tillering nodes,TamiR408,TaCLP,and TalncR3017 all showed an expression pattern of descending and then rising.Exogenous ABA can significantly affect the expression levels of TamiR408,TalncR3017 and TaCLP under Dn1 cold stress.Compared with the control,exogenous ABA inhibited the expression of TalncR3017 and TamiR408 in leaves at 5℃,while TaCLP was induced,and all three genes expression peaked at-10℃,and then suddenly dropped;Compared with the control,exogenous ABA made the expression of the three genes at 5℃consistent with that in the leaf,but the peak expression of the three genes was at-25℃.（3）Construct overexpression vectors of tae-pre-miR408 and TalncR3017 genes,and construct overexpression of mlnc R3017 that mutate the interaction region of TalncR3017 and TamiR408,and construct miR408 silencing vectors.They were transferred into Arabidopsis to obtain T₂-positive plants,and they were treated at 4℃8 h,-10℃6 h,and normal temperature for 3 days.Phenotypic observations showed that Arabidopsis over-expressing TamiR408 is the most resistant to cold,Arabidopsis over-expressing TalncR3017 is less resistant to cold than wild type,and Arabidopsis silencing miR408 is the least resistant to cold.The expression patterns of miR408,miR408 target genes and oxidative stress related genes were detected.The results showed that compared to wild type,miR408 was up-regulated about 100-fold after overexpression,and the miR408 silencing plant decreased to about 0.2-fold of wild-type.Mi R408 down-regulates its target genes to increase the expression level of oxidative stress genes in Arabidopsis.Overexpression of TalncR3017 in Arabidopsis showed that it can increase the expression of miR408 target genes by adsorbing miR408.After it was mutated to the miRNA binding site,the expression level of the miR408 target gene did not change significantly,proving that TalncR3017 acts as cceRNA to regulate the expression of miR408.The above experiments preliminarily demonstrated that TamiR408 in Dn1 participates in cold stress,and can interact with TalncR3017 to further regulate the expression of the target gene TaCLP,thereby affecting the expression of other oxidative stress genes.This experiment provides insights into how the TalncR3017-TamiR408-TaCLP module regulates wheat response to cold stress.