Screening Cold-resistant LncRNA Of Winter Wheat And Through Interaction With Tae-miR398 In Response To Cold Stress

Wheat is one of the most important human food crops,and low temperature stress has a huge impact on crops.Dongnongdongmai 1（Dn1）is the first cultivar in the northern high-cold area that can safely overwinter,and it has important long non-coding RNA（lncRNA）resources.MicroRNA（miRNA）is closely related to the cold resistance of winter wheat.Cold-associated miRNAs can cause gene silencing by binding to the target mRNA,thereby initiating the cold-resistance response mechanism of winter wheat.Plant miRNAs play an important role in various stress responses.lncRNAs can be used as competing endogenous RNAs（ceRNAs）to competitively bind miRNAs to control the expression of target genes.There are close interactions between miRNA and lncRNA.However,there is no report on the regulation of winter wheat cold-resistance mechanism by lncRNA-mediated ceRNA.It is necessary to commence relevant research work.In this study,Dn1 was divided into experimental materials.Under natural cooling conditions in Datian,the tillers were harvested at three different temperatures of 5°C,-10°C,and-25°C,respectively.RNA-seq sequencing was performed using HiSeq.The lengths of the fragments were statistically analyzed and screened by ORF,Rfam,and CPC databases,and the repeats were removed.All lncRNAs at three temperature points were found and analyzed by family.Then,the lncRNA differential expression profiles were constructed.On the basis of the Wenn analysis,120lncRNAs with extremely different expressions were screened.The interactions with miRNAs were predicted,and the miRNAs that could interact with each other were used to predict the target mRNA.To construct a regulatory interaction network of cold-resistant lncRNA-miRNA-target mRNA,Cytoscape software was used to integrate the RNA.Selecting miR398 with cold-resistant expression in the previous study further established a lncRNA-miR398-target mRNA regulatory network based on the regulatory interaction network of lncRNA-miRNA-target mRNA,and verified the interaction between lncRNA-miR398-target mRNA.And related lncRNA expression analysis.The overexpression vector was constructed by cloning lncRNA,and the expression level of related RNA was detected in low-temperature treatment in model plants.The lncRNA regulation of the cold resistance of miR398 was initially verified.The main findings are as follows:1.lncRNA sequencing data processing and statistical analysis of Dn1High-throughput sequencing using HiSeq resulted in RNA fragments with lengths between200-4656 nt,most of which（77.7%）were less than 1,000 nt,and finally obtained clean at 5°C,-15°C,-25°C.The total number of reads was 65,483,181,67,567,197 and 61,436,657 respectively.These lncRNAs are more evenly distributed on chromosomes in Dn1,with no obvious position preference and a more complete coverage depth.The reads aligned to the chromosome are annotated to exonic,intronic,and intergenic.We found that the ratio of reads in exonics was81.05%-86.83%higher than that in exonics,while the ratio of reads to intronic（introns）was the lowest,ranging from 2.33%to 3.27%.Using Cufflinks for assembly statistics of the number of new transcripts for each sample and calculation of new transcript expressions,it was found that New transcript at-10°C.yields the highest number,averaging 13875,with the lowest number of New transcripts at-25°C.It is 12328.The average number of detected genes among the 3temperature samples is 5°C>-10°C>-25°C,that is,the transcript abundance is highest at 5°C.2.Identification and differential expression analysis of lncRNA in Dn1 under low temperature stressUsing bioinformatics,9971 highly-recognized Dn1 Candidate IncRNAs were screened and identified.Some lncRNAs in the tiller showed different responses to low temperature stress（control:5°C library;experiments:-10°C and-25°C library）.At-10°C,1260 lncRNAs showed significant differential expression,of which 591 were up-regulated and 669 were down-regulated,while l382 lncRNAs were significantly different at-25°C,of which 1539 were up-regulated,3843down-regulated expression.In addition,there was a significant difference in the expression levels of 5072 lncRNAs in the-25°C pool compared to-10°C and-25°C,with 1378 up-regulated and3694 down-regulated.A total of 471 lncRNAs with simultaneous changes in expression levels at three temperatures were used to verify the expression of some of the selected IncRNAs by RT-PCR method.The results were basically the same.3.Screening the construction and functional prediction of cold-resistant lncRNA-miRNA interaction networkWe selected 120 lncRNAs（60 up and 60 down）with extremely significant differences in expression at-10°C and-25°C,and screened them by miRanda,PITA,and RNAhybrid,which are the lncRNA and miRNA interaction screening software.The obtained lncRNA and miRNA prediction results serve as the basis for the verification of this experiment.A total of 80 miRNAs were predicted,and target miRNAs were predicted for the relevant miRNAs.The correlations between miRNAs-target mRNAs and miRNA-lncRNAs were integrated and analyzed using Cytoscape bioinformatics software.The regulatory relationship.To investigate the potential function of lncRNAs and tae-miRNAs,GO genes targeted to target miRNAs interacting with cold-resistant lncRNAs were subjected to GO analysis to analyze biological functions that may be exerted by Dn1 at low temperature.The results showed that lncRNA may be involved in biological processes such as signal transduction,energy synthesis,molecular metabolism,detoxification,transcription,and redox.Our study shows that lncRNAs can regulate plant response and adapt to cold stress by regulating protein coding genes.4.Construction and expression analysis of the interaction network of cold-resistant lncRNA-miR398-target mRNAIn the above results,the cold-resistant lncRNA interacting with tae-miR398 was selected to form an lncRNA-miR398-target mRNA interaction network.Then RACE and RAP techniques were used to verify the interactions between miR398 and its target RNA（TaCSD1）and its interactionwiththreelncRNAgenes:TRAES7AS9A759DD28.1（lncR9A）,TRAES3BF117100150CFD_t1（lncR117）and TRAES3BF061600100CFD_t1（lncR616）.（1）The 5’RACE results demonstrate that TaCSD1 is the target gene of tae-miR398 and is cleaved by tae-miR398.The cleavage site is located in the coding region and is located 110bp downstream of the interaction site of tae-miR398 and the target gene.RAP verification of the interactions between ceRNAs showed that lncRNAs（lncR9A,lncR117,and lncR616）can be used as ceRNAs to regulate the synergistic effects of Tae-miR398 and TaCSD1 under low temperature stress.lncR9A,lncR117,and lncR616 are involved in the regulation of TaCDS1 and tae-miR398 at the RNA level.（2）Three lncRNAs,lncR9A,lncR117,and lncR616 genes,which interact with tae-miR398,were successfully cloned and constructed.Three expression vectors were constructed to form three recombinantplasmids（pCAMBIA3300sU-tae-lncR9A,pCAMBIA3300sU-tae-lncR117,pCAMBIA3300sU-tae-lncR616）,transformed into Arabidopsis thaliana,screened positive plants,and harvested T₁,T₂ generation seeds.The T₂ plants were treated with low temperature（0 h blank bacteria inoculation as a control）,4°C 3 h→-10°C 5 h→4°C 3 h→normal culture for 5 days,and the leaves were taken and detected by real-time PCR.Compared to transgenic Arabidopsis thaliana and lncR616 transgenic Arabidopsis thaliana plants and their low temperature stress,RNA expression was different:miR398 showed a decreasing trend overall,target genes CSD1 and miR398 showed the opposite trend.（3）The recombinant plasmid of lncR9A was then used to transform Poaceae,a graminaceous plant species,and T₂ seeds were obtained by resistance screening.The T₂ plants were treated with low temperature,4°C 3 h→-10°C 5 h→4°C 3 h→normal culture for 5 days and the leaves were taken for RT-PCR detection.The results showed that after cold treatment the expression of CSD1,while the expression of miR398 showed a decreasing trend.Both miR398 and target mRNA（CSD1）were able to respond to low temperature stress,and the expression levels under low temperature treatment were different and coordinated.Through the above experiments,a complex biological regulation network between miR398and lncRNA and target gene CSD1 is preliminarily formed.miR398 can regulate its target gene CSD1 to initiate the cold resistance response,and is also competitively combined by lncRNA to regulate the expression of the target gene CSD1 and thereby affect Dn1 The cold resistance.This study provides new ideas for the regulation mechanism of miR398 in Dn1.At the same time,miRNA-lncRNA can influence the morphological characteristics and growth process of plants,and it can also provide theoretical guidance for the cultivation of crops.