Molecular Mechanisms Of Interaction Between Winter Wheat MiR5049-3p And MiR1120a And Their Target Genes 6PGL And FBA In Response To Low Temperature Stress

MicroRNAs are a class of endogenous and non-coding small RNAs which of length is about 22 nt and can regulate gene expression in a variety of eukaryotes.In plants,it is involved in many biological processes,including plant growth,development,and biological and abiotic stress(including disease,cold,high salt,drought,etc.)responses.Wheat is one of the major food crops,can be produced all over the world.Cold stress has a negative impact on the growth of wheat and other economic crops.has The strong cold-tolerant “Dongongdongmai 1”(Dn1)which can overcome the cold weather conditions in Heilongjiang Province autonomously cultivated by Northeast Agricultural University,has an important coldresistance-related miRNA resource.The study of the mechanism of response to glucose metabolism under the cold stress from the miRNA direction has important theoretical and practical significance for enriching the cold resistance theory of wheat and cultivating cold-resistant varieties.This study used Dn1 planted under natural cooling conditions in the field as a basic material to investigate the effects of low temperature on the expression of tae-miR5049-3p and tae-miR1120 a,and their target genes 6PGL and FBA.Pre-tae-miR5049-3p was transformed into wild type Arabidopsis thaliana(Columbia type)using inflorescence inoculation.The expression patterns of tae-miR5049-3p and its target genes in Arabidopsis thaliana were verified,and the physiological indicators of Arabidopsis thaliana were examined.Obtaining the following main results:(1)The tae-miR5049-3p and tae-miRNA1120 a expression patterns of Dn1 in the four temperature regions(5°C,0°C,-10°C,-25°C)were:Tillering: As temperature decreases,the expression of miR5049-3p increases at first and then decreases,while the expression of miR1120 a shows a pattern of “rise-fall-rise”.Both have highest relative expression at 0°C.Leaves: miR5049-3p showed a trend of “first increase and then decrease” when the temperature decreased,and the highest expression was at-10°C,and the lowest expression at 5°C;miR1120a gradually increased as the temperature decreased(-25°C Reached thehighest).(2)Bioinformatics analysis was used to study the structure of proteins encoded by 6PGL and FBA genes.It was found that 1)proteins encoded by 6PGL and FBA have their typical domains respectively;6PGL contains SugarP_isomerase in its a mino acid sequence.The superfamily domain,which is a typical phospho-isomerase family domain,and the phosphoglucoisomerase family contains 6PGL.The FBA protein has a typical ICL_KPHMT superfamily domain which is a fructose diphosphate aldolase domain structure.2)6PGL and FBA proteins may separately be synthesized in the chloroplast and cytoplasm and they cannot be transported,so both are non-secretory proteins.3)6PGL protein is mainly composed of a variety of spatial structures;and the 3D structure of FBA protein is mainly based on helices.(3)Arabidopsis thaliana was successfully transfected using inflorescence inoculation and oxpre-miR5049-3p T3 transgene positive plants were obtained.After being treated with low temperature stress at 4°C for 24 hours,the expression of miR5049-3p and its target gene 6PGL was detected and showed negative regulation.;meanwhile,the physiological indexes(chlorophyll content,injury rate,MDA content)of the transgenic plants were detected.The results showed that the cold resistance of ox-pre-miR5049-3p transgenic plants was higher than that of wild type plants.This study confirmed that tae-miR5049-3p can response to cold stress by negatively regulating the expression of 6PGL and mediating the glycometabolism pathway by RT-PCR and Overexpressed plant verification.This study initially revealed the mechanism of miRNA regulation of Dn1 glycometabolism in response to cold stress.It has an important theoretical significance and application prospects for molecular breeding of new winter wheat cultivars.