Study On Effects Of Low Temperature On Starch Accumulation In Wheat Caryopsis At Early Filling Stage Based On High-Throughput Sequencing

Wheat is an important food crop widely cultivated around the world.With the continuous global warming and abnormal ocean circulation,extreme weather events occur frequently,with spring low temperatures having a significant impact on wheat.Low temperature and cold damage have led to a significant decrease in wheat production in major wheat growing areas around the world.Starch is the main storage substance in wheat grains,accounting for approximately 70-80%of the weight of the grains.The accumulation and physicochemical properties of starch are related to the quality formation of wheat.At present,high-throughput sequencing technology is widely used to analyze the response of plants to abiotic stress.However,the regulation characteristics of starch accumulation genes during the development of wheat caryopsis under low temperature after anthesis are still unclear.Based on three types of high-throughput sequencing technologies（RNA sequencing,miRNA sequencing,and degradation group sequencing）,this study studied the miRNAs related to starch accumulation in wheat caryopsis under low temperature after anthesis.This study analyzed the pathways in which miRNAs and their target genes participate in response to starch accumulation under low temperature stress,revealing the regulatory mechanism of wheat caryopsis response to low temperature stress at the post transcriptional level.The research results of this study provide new theoretical support for the breeding of wheat varieties with low temperature tolerance,the exploration of stress resistance genes,and efficient cultivation.The main research results of this paper are as follows:（1）Effect of low temperature at early filling stage on starch accumulation in wheat caryopsisUnder low temperature conditions after flowering,the content of YM15 amylose significantly decreased compared to the control group,and the content of YN19 amylose also showed a downward trend.The content of amylopectin in YM15 significantly increased,while there was a downward trend in YN19.The total starch content of YM15 shows an increasing trend,while YN19 shows a decreasing trend.The swelling power of starch in both varieties of wheat significantly increased.The large starch particles in the YM15 lowtemperature treatment group significantly increased,while the small starch particles in the YN19 low-temperature treatment group significantly increased.However,the endosperm plumpness of both varieties of wheat decreased.In addition,low-temperature treatment increased the content of short chain branched starch in both varieties of wheat,while the content of medium and long chains decreased.The above results indicate that low temperature can affect the process of starch accumulation in wheat caryopsis.（2）Analysis of DEGs in wheat caryopsis under low temperature at early filling stageIn this study,high-throughput sequencing was used to sequence the transcriptome of wheat grains under low temperature stress and the control.6440 differentially expressed genes（DEGs）were identified in YM15,of which 2361 genes were up-regulated and 4079 genes were down regulated;A total of 7842 DEGs were identified in YN19,with 3820 genes upregulated and 4022 genes downregulated.97 and 117 candidate genes related to starch accumulation were screened in YM15 and YN19,respectively.Among them,42 candidate genes are simultaneously involved in the starch accumulation related processes of two wheat varieties,YM15 and YN19.Screen these candidate genes and identify GLGB,GLGB3,SSY3A,SSY22,and SSG1B gene encodes SBEⅠ,SBEⅠ,SSSⅢ,SSSⅡ,and GBSSⅠ,respectively.Through GO and KEGG enrichment analysis of candidate genes,it was found that YM15 and YN19 were both enriched at 1,4-α-Glucose branching enzyme activity,starch and sucrose metabolism and glycolysis/glucose metabolism pathways.The functions of candidate genes in YM15 are concentrated in glucose binding,glucose kinase activity,carbohydrate phosphatase activity,and β-glucosidase activity.The candidate genes in YN19 are functionally enriched in biological processes such as starch biosynthesis、amylase activity,carbohydrate phosphatase activity,and glucan catabolism.（3）Differential expression analysis of miRNAs in wheat caryopsis under low temperature at early filling stageIn this study,we used high-throughput miRNAs sequencing technology to analyze four sRNA libraries of two wheat varieties,YM15 and YN19,in the low temperature treatment group and the normal growth control group.A total of 321.56 M Raw Reads were obtained,and the fragment length showed a normal distribution in the range of 18-32 nt,of which 24 nt was the highest sRNA abundance.A total of 217 miRNAs were identified,including 117 new miRNAs and 100 known miRNAs belonging to 33 miRNA families.This study identified 14 and 27 differentially expressed miRNAs in YM15 and YN19,respectively.Among them,YM15 had a total of 10 up-regulated and 4 down-regulated miRNAs,while YN19 had a total of 19 up-regulated and 8 down-regulated miRNAs.Six miRNAs（1D₁28₂54662315₂54662335,5A₇1₁16585637₁16585657,5D546₁10367926₁10367946,tae miR531,tae miR9657b-5p,tae miR9659-3p）and two miRNAs（5D546₁10367926 110367946,tae miR531）related to starch accumulation were screened out among the differentially expressed miRNAs YM15 and YN19,respectively.（4）Degradation group sequencing validation of miRNA target genes in wheat caryopsis under low temperature at early filling stageThis study used YM15 and YN19 as materials to construct a degradation group library and conduct deep sequencing.Combining with the miRNAs sequencing obtained in the early stage of wheat response to low temperature stress,bioinformatics analysis was conducted on the degradation group data.Genes related to starch accumulation were screened,and 42 and 29 candidate genes were screened in YM15 and YN19,respectively.There are 15 upregulated genes and 27 down-regulated genes in YM15;There are 15 up-regulated genes and 14 down-regulated genes in YN19.Among them,7 pairs of miRNA-target gene are involved in starch accumulation.Through the analysis of GO functional significance enrichment of candidate genes,it was found that GO entries enriched in both varieties of wheat include starch binding and polysaccharide metabolism processes.In addition,the enriched entries of these target genes in YM15 mainly include glycogen（starch）synthase activity and the synthesis and metabolism of trehalose and glucan,etc.The enriched entries of these target genes in YN19 mainly include the activities of glucosyltransferases and hydrolases,the synthesis and metabolic pathways of glucose,polysaccharides.Through KEGG pathway enrichment analysis,it was found that the target genes in YM15 and YN19 responding to low temperature stress miRNAs were significantly enriched in pentose and glucuronic acid conversion and starch,sucrose,galactose,fructose and mannose metabolism,glycolysis/glucose metabolism and pentose phosphate pathway.In summary,this study identified 7 pairs of miRNA-target gene pairs that may be involved in starch accumulation in wheat under low temperature stress by analyzing the differential expression of caryopsis genes and miRNAs in two wheat varieties under spring low temperature stress.Among them,in YM15,6 pairs of miRNA-target gene pairs were identified to be closely related to starch accumulation,including tae-miR531-SSY23,1D₁28₂54662315₂54662335-HXK3,tae-miR531-INV2,tae-miR9657b-5p-GLGS2,5A₇1₁16585637₁16585657-SUS 5D₅46₁10367926₁10367946-SUS2,and taemiR9659-3p-SSY3A.In YN19,two pairs of miRNA target gene pairs（tae-miR531-INV2,5D₅46₁10367926₁10367946-SUS2）were identified as closely related to starch accumulation.The above research results not only deepen the molecular mechanism of starch accumulation in wheat grains in response to temperature,but also provide theoretical support for molecular breeding of cold resistant wheat varieties.