The Functional Study Of Long Non-coding RNA-CRR1 In Cassava Responds To Low Temperature

Cold is an abiotic stress that seriously affects plants’ growth and development.It is a major limiting factor for global crop yields.In order to overcome the losses of crop yield caused by stress,it is necessary to develop stress-tolerant crop cultivars.Plants respond to cold stress and adapt to the situation through complex physiological and biochemical processes,including altering gene expression levels,signal transduction pathways and cellular metabolic rates to obtain resistance.At present,researchers have conducted a large number of studies to explore the coping mechanisms and regulatory networks of plants’ response towards cold.The researches of plants’ IncRNAs have shown that IncRNAs have become an important class of regulators and they play a key role in almost all plants’ development processes.They are able to be involved in transcriptional and post-transcriptional regulation of gene expression through a number of complicated mechanisms.In this study,we use the method which called strand-specific RNA sequence(ssRNA-seq)to study the genome-wide transcriptome reconstitution of cassava that attacked by low-temperature stress in a high-throughput manner.Particular attention is paid to the expression pattern of stress response exhibited by a selected lncRNA-CRR1 which related to chilling injury.Meanwhile,we perform the functional study of lncRNA-CRR1.The specific research results are as follows:1.Through the improved sequencing method of RNA-seq,we identify a total of 318 lncRNAs that respond to cold stress and drought from the RNA-seq database.Further analysis reveals that there are 198 cold-specific differentially expressed lncRNAs(DE-lncRNAs).Finally,combined with bioinformatics analysis,the low temperature-regulated IncRNA-CRR1 is screened.The lncRNA-CRR1 is 976 bp in length and contains three exons.The chromosomal location is in Chromosome 01:26346926-26350713,negative strand expressed.2.Homology analysis of lncRNA-CRR1 is performed by BLAST sequence alignment provided on the NCBI website.Finally,we find that the similarity of CRR1 in this study to histidine acyltransferase in cassava is above 90%.In addition,the homology of CRR1 and an unknown mRNA in cassava is the closest.At the same time,we analyze the secondary structure characteristics of the CRR1’s base pairs.The result shows that the secondary structure of CRR1 is a multi-loop hairpin structure and the number of stem loops is large which can indicate that CRR1 is easy to bind to RNA molecules,proteins,etc.The structure is not stable and not highly conserved.We can compare the number of stem loops and conservation of lncRNA-CRR1 in cassava with those of low temperature resistant IncRNA in plants such as Arabidopsis thaliana to explore its function.3.The result of real-time quantitative PCR(qRT-PCR)shows that after different low temperature treatment time of cassava tissue culture seedlings,it is found that the relative expression of CRR1 increases first and then decreases,especially after low temperature treatment for 24 hours,the expression is the most obvious,the relative expression amount is about 20 times that of the control group.This indicates that lncRNA-CRR1 in cassava responds to low temperature stress and the relative expression reaches the peak after 24h of low temperature treatment.4.Southern hybridization analysis shows that lncRNA-CRR1 has been successfully transferred into the genome of cassava transgenic lines:OE-1 and OE-5.And it integrates into the chromosome of the cassava receptor cells with a single copy Real-time quantitative PCR shows that the relative expression of lncRNA-CRR1 in most transgenic lines are much higher than that of wild type which can indicate that lncRNA-CRR1 is overexpressed.5.After chilling 4℃ treatment of cassava wild-type and transgenic lines for 24h,the phenotypic observation shows that cassava transgenic lines OE-1 and OE-5 have no significant changes in phenotype before and after low temperature treatment,while after cold treatment,the wild type’s leaves appears wilting and shrinking.It indicates that the cold-tolerant ability of transgenic cassava is stronger than that of wild-type.Under low temperature induction,lncRNA-CRR1 would respond to stress.6.Determination of four physiological indexes before and after low temperature treatment of cassava transgenic lines OE-1,OE-5 and wild type WT,including soluble sugar content,MDA content,proline content and hydrogen peroxide content.The result indicates that the contents of soluble sugar,proline and hydrogen peroxide increase obviously and the content of MDA decreases in transgenic lines compared with WT.It shows that the transgenic lines are more resistant to cold.In addition,between transgenic lines,it is found that the contents of soluble sugar,proline and hydrogen peroxide in OE-5 increase significantly compared with OE-1 before and after low temperature treatment,while the content of MDA is lower.It shows that the cold resistance of OE-5 is higher than that of OE-1.7.Identify the network regulation of IncRNA by co-expression gene analysis to further understand its function.The result shows that a total of 12,651 protein-coding genes are co-expressed with cold-responsive IncRNA and the 16 co-expressed genes related to the lncRNA-CRR1 are screened.The co-expression coefficients are all above 0.95 which can indicate that the reliability is high and show a positive correlation mode among them.It is found that both the WRKY DNA binding domain and the basic leucine zipper(bZIP)transcription factor are induced by low temperature,so we predict that lncRNA-CRR1 is also regulated by chilling injury and significantly differentially expressed at low temperature.It could be used for further functional verification..8.The KEGG pathway enrichment analysis is performed on the differentially expressed target genes of lncRNA-CRR1 in the transgenic lines OE-1 and OE-5.The result shows that OE-1 is found to respond to cold stress in the significantly differentially expressed lncRNA 20 ways.Among them,the most abundant pathways include the biosynthesis and metabolic pathways of transcriptionally regulated secondary metabolites;in OE-5,twenty pathways in response to cold stress are found in the significantly differentially expressed lncRNA.Among them,the most abundant pathways include the biosynthesis and metabolic pathways of secondary metabolites regulated by transcription;sugar metabolism,amino sugar and nucleotide sugar metabolism,and biosynthesis and metabolic pathways of secondary metabolites.These results suggest that the main role of lncRNA-CRR1 may be related to the transcriptional regulation of gene expression,especially genes which get involved in hormone signal transduction and secondary metabolic pathways.