Response Of MiR528a To Abiotic Stress And Low Nitrate And Regulatory Mechanism In Maize

Abiotic stresses, such as low nitrogen, are limiting factors in crop yield. Basicresearch of genetic and physiological in abiotic stresses will provide the theoreticaland technical basis for breeding resistance varieties of crop. Maize is an importantcereal, fodder and industrial crop, which is the first crop in China. But nitrogenrecycling efficiency is only25%and nitrogen use efficiency is low, thereby improvingnitrogen use efficiency of maize and cultivating a new variety of low nitrogenresistance are the great demand of sustainable developing agriculture in China. So ithas important significance to research on basic research of genetic and physiologicalof low nitrogen tolerant in maize. MicroRNA (MiRNA) is an important regulatoryfactor of the plant, which involved in plant development and adjusted the stressadaption through post-transcriptional level. The research shows that Zm-miR528amay be involved the low nitrogen adaptation in maize roots and leaves, but the lack ofclear experimental evidence. This study was to search for and identify the target genesof miR528a with RLM-5’RACE method, to analyze the response to abiotic stressesand the regulation patterns between miR528a and its targets, and to over-expressmiR528a and its targets in model organism system——Arabidopsis thaliana. Thesestudies would provide important theory and experiment foundation of miR528a andits target genes for molecular breeding application. The main research results are asfollows:1. Four miR528a target genes, which are GRMZM2G062069_T02,GRMZM2G169033_T01, GRMZM2G004106_T01and ZmLac5were amplified byRLM-5’RACE. The results show that the splicing sites of four target genes all locatedin the coding region, therefore the regulations between miR528a and the four targetgenes occur in post-transcriptional level. Except the functional site of2069is outsidebase1, the other three targets’ functional site are located in base10or11, incomplementary to mature miR528a. It was confirmed that miR528a degraded itstarget genes mRNA by cutting it in the specific site, so as to give play to its functionin regulation process.2. Overexpression vector of pCUB-Ubi::ZmLac5was constructed using In-FusionTMAdvantage PCR Cloning Kit. The plasmid was transformed into Agrobacterium tumefacien strain LBA4404by electrotransformation and then transformed intoArabidopsis thaliana (Columbia ecotype) wild type plants.62T1and60T2transformants were screened from80independent positive T0transformation eventsthrough PCR screening approach. The transformation rate was1.28%. The adequatehomozygote T3tranformants will be available soon to analyze the gene function in amodel system of Arabidopsis.3. The MIR528a gene was cloned from inbred lines B73, and then constructed theoverexpression vector pCPB-35S::MIR528a. The overexpression vector wastransformed into Arabidopsis thaliana (Columbia ecotype) wild type plants, mediatedby Agrobacterium tumefacien strain LBA4404. T1transformants were collected fromindependent positive T0transformation events through PCR screening approach. Theadequate homozygote T3tranformants will be available soon to analyze the genefunction in a model system of Arabidopsis.4. The expression pattern of miR528a and its target genes under eight kinds of abioticstresses was verified through Real-time PCR.The results indicated that miR528aresponded to low nitrogen. MiR528a was down-regulated and its target genes9033and ZmLac5were all up-regulated under low nitrate stress in maize leaves. MiR528awas up-regulated and its target genes2069and4106were all down-regulated underlow ammonium stress in maize. The results also indicated that miR528a and its fourtarget genes responded to many kinds of abiotic stresses such as drought,submergence and salt stresses.This research shows that miR528a and its target genes response to many kinds ofabiotic stresses such as low nitrogen, and they are involved in the adaptabilityregulation. The study of the low nitrogen tolerance, nitrogen molecule mechanism ofcorn provides an important experimental basis for the molecular breeding of corn.