Research On MiRNA390 Mediated Response To Low Temperature Stress In Solanum Tuberosum L

winter potato's production in southern China is playing an increasingly important role in promoting China's grain revenue and safeguarding national food security.The problem of unstable yield and delayed harvest due to cold seasoning and off-season cultivation is one of the major problems facing potato production in winter.MicroRNAs(miRNAs),a group of highly conservednon-coding RNA in eukaryotes,regulate target genes via multiple approaches,such as inhibition of translation,mRNA splicing,mRNA degradation.Therefore,they play important roles in plant organ morphogenesis,growth,signal transduction and abiotic stress response.The use of molecular biology to explore the expression characteristics of cold-resistance-related genes and the pattern of regulation by MicroRNAs(miRNAs)is a great significance for the future to use the genetic engineering methods for the breeding of cold-tolerant potato varieties.MicroRNAs(miRNAs),a group of highly conservednon-coding RNA in eukaryotes,regulate target genes via multiple approaches,such as inhibition of translation,mRNA splicing,mRNA degradation.Therefore,they play important roles in plant organ morphogenesis,growth,signal transduction and abiotic stress response.In this study,potato GS393(Solanum commersonii-LZ3.4)was used as the research material to analyze the interaction of potato miR390(stumiR390)and its target genes under low temperature stress;clone related target genes and perform functional analysis.The main findings are as follows: 1.Analysis of promoter elements showed that stumiR390 has low-temperature regulatory elements that can be induced by low temperature.Bioinformatics analysis predicts that its target gene has two leucine-rich repeat-like receptor-like protein kinases(PGS-10618,stuBG70158)and one endo-1,3;1,4-?-D-glucan Carbohydrase(stuBG09701),4 receptor-like serine/threonine protein kinases(PGS-45969,PGS-20698,PGS-66895,PGS-64235)and 1 PGR5-like protein 1B(PGS-58521).Real-time PCR and semi-quantitative PCR showed that the expression of stumiR390,stuBG09701 and stuBG70158 was strongly induced at low temperature,and the expression of stumiR390 was positively correlated with stuBG09701,and negatively correlated with the expression of stuBG70158,PGS-10618,PGS-45969 and PGS-20698.RLM-RACE confirmed that the cleavage site of miR390 regulating stuBG70158 transcription was ATTCCT CCTGAGTT;however,the cleavage site of stumiR390 regulated transcription of target gene stuBG09701 was inconsistent with the prediction result,and no corresponding fragment was found in the sequence of stuBG09701.This may be related to that stuBG09701 is within the membrane.Proteins are positively regulated by stumiR390,and the specific mechanism of action needs further study.2.A specific band of approximately 720 bp in length(stuBG09701,MG763883)and a specific length of approximately 2685 bp(stuBG70158,MG763884)were obtained by cloning.A specific band with a length of approximately 4925 bp(stuBG09701)and a specific band with a length of 3549 bp(stuBG70158)was obtained by PCR using potato GS393 DNA as a template.Bioinformatics analysis showed that stuBG09701 is an endo-1,3-1,4-?-D glucanohydrolase that belongs to intracellular hydrophilic proteins and contains one ELM2,A1 pp,KNOX2,and Abhydrolase structure,respectively.Domain;stuBG70158 is a leucine-rich repeat receptor-like protein kinase that is a typical transmembrane hydrophobic protein with multiple extracellular LRR domains,a single transmembrane domain,and an intracellular kinase structure.area.The analysis of promoter regulatory elements revealed that stuBG09701 and stuBG70158 contain various promoter regulatory elements such as hypothermia,hormones,and growth and development.stuBG09701 and stuBG70158 may be induced by low temperature and other abiotic stresses.3.Quantitative PCR and semi-quantitative PCR analysis of the expression of GS393 in roots,stems,leaves,stolons and tubers of potato showed that stumiR390 and stuBG09701 and stuBG70158 were expressed in all tissues and organs of potato,but the expression abundance was significantly different.The expression of stumiR390 and stuBG09701 was highest in leaves,followed by roots,and lowest in stolons.On the contrary,stuBG70158 had the lowest expression in leaves and the highest expression in stolons,followed by tubers.At the same time,the expressions of stuBG09701 and stuBG70158 under 6-BA,ABA,GA3,IAA,Al,Cd,Cu,Hg,Nacl and PEG were also analyzed.The results showed that the expression of stuBG09701 under ABA,Nacl,PEG,Hg,and Cu stress increased significantly within 48 h;the expression of Cd,6-BA,IAA,and GA3 increased significantly within 36 h;the response under Al stress was not significant.The expression of stuBG70158 increased significantly under PEG stress within 24 h;Hg,ABA,Al,and IAA increased the expression level significantly within 36 h;Cu and Cd decreased the expression level significantly within 48 h;6-BA and GA3 stress induced stuBG70158 Gradually rise.