Identification And Function Analysis Of Cold Responsive-related MiRNA And Their Target In Wheat

Wheat（Triticum aestivum L.）is a kind of widely cultivated food crop.In recent years,due to the frequent occurrence of extreme weather,the risk of wheat suffering from low temperature injury continued to increase,which seriously threatened the growth and yield of wheat.Therefore,improving the cold resistance of wheat has became one of the key problems in breeding.MicroRNA（miRNA）,as a class of endogenous non-coding small single-stranded RNA molecules in eukaryotes,degrades target genes or inhibits their translation through complementary pairing and binding with specific target genes,participates in gene expression regulation at the post-transcriptional level,and plays an important role in plant growth and stress response.In this study,the low temperature tolerant wheat cultivar "HeNong 8184"（HN8184）and low temperature sensitive wheat cultivar "Chinese Spring"（CS）were used as materials.miRNA sequencing and degradation sequencing techniques were used to identify the miRNAs and their target genes related to low temperature stress response in wheat stem base.The function of tae-miR160 and its target gene TaARF11-7A,which were significantly differentially expressed at low temperature was verified.The main research results are as follows:1.miRNAs differentially expressed under low temperature stress in varieties with different cold tolerance were identified.A total of 1298 miRNAs,including 502 known miRNA and 796 new miRNA（novel-miRNA）,were identified by analysis of miRNA expression profiles in wheat stem base under low temperature（4℃）and normal temperature（22℃）,respectively.In"HN8184",71 miRNAs were identified as significantly different in expression（23 up-regulated and 48 down-regulated）,and in CS only 26 significantly different expressed miRNA（15 up-regulated and 11 down-regulated）were found.two miRNAs（tae-MIR444b-p3₁ss18CA and tae-MIR164-p3）were identified in both cultivars and were significantly up-regulated miRNAs.69 miRNAs,including PC-3p-38746₁45,tae-miR160 and tae-miR164,were identified as key candidates for low temperature response which only existed in HN8184.2.The target of differentially expressed miRNA was determined by degradation group sequencing.Four degradation group libraries were constructed by degradation group sequencing after normal and cold treatment of "HN8184" and "CS",respectively.In the wheat reference genome（IWGSC v1.1）,932 and 512 target genes of differentially expressed miRNA were compared between "HN8184" and "CS" after cold treatment,respectively.GO functional annotation and analysis showed that target genes were mainly concentrated in GO categories such as transcriptional regulation,DNA-template,REDOX process,protein phosphorylation,integral component of membrane,nucleus,membrane system,protein binding,ATP binding and DNA binding.3.tae-miR160 was found to be involved in the regulation of cold resistance of plants.The expression of tae-miR160 in "HN8184" was significantly up-regulated at low temperature,which was the key miRNA for low temperature response.Under cold stress,plants overexpressing tae-miR160 had higher freezing survival rate and lower O2-and H2O2 contents,compared with wild plants.The expression levels of cold-related genes were significantly increased,and plants overexpressing tae-miR 160 enhanced low temperature tolerance.Compared with control group,tae-miR160 silenced wheat plants showed decreased survival rate,increased relative conductivity and decreased ROS scavenging related enzyme activity under cold stress,and tae-miR160 silenced wheat plants were more sensitive to low temperature.4.The targeting relationship between tae-miR160 and the target gene TaARF11-7A was verified.By using psRNATarget online and degradation group sequencing we predicted that tae-miR160 targeted cleaved base 1716 of TaARF11-7A（TraesCS7A02G4469001）transcript.qRT-PCR results showed that tae-miR160 expression was up-regulated at low temperature,while TaARF11-7A expression was significantly down-regulated,showing opposite expression patterns.Results from the dual luciferase reporting system showed that tae-miR 160 can cleave transcripts of TaARF11-7A.5.The role of TaARF11-7A in plant cold resistance was identified.Under low temperature stress,compared with control group,the survival rate of frozen wheat plants with silenced TaARF11-7A increased,the relative electrical conductivity increased less,ROS scavenging related enzyme activity enhanced,and the low temperature tolerance of silenced wheat plants significantly improved.In conclusion,this study identified and screened miRNAs and their target genes at the base of wheat stem on a genome-wide scale,differentially expressed miRNAs related to wheat hypothermia response,and the function of tae-miR160/TaARF11-7A,as a key hypothermia response module,in plant hypothermia stress response.The results of this study are helpful to further explore the regulatory function of miRNA on genes related to wheat low temperature response,and provide gene resources for genetic improvement of cold-resistant wheat varieties.