Excavation And Functional Verification Of Maize MiRNAs As Well As Target Genes Involved In Drought Stress

MicroRNAs(miRNAs)are a class of small,non-coding regulatory RNAs that are involved in growth and development,differentiation and proliferation,cell apoptosis and many other biological processes by guiding target mRNA cleavage or translational inhibition in plants and animals.At present,the study of plant miRNAs is concentrate on model plants such as Arabidopsis and rice,there still have a lot of species specific miRNAs were not found in other plants.Maize is the most widely planted crops and also one of the important food crops.Drought is one of the main limiting factors of maize yield so that breeding for drought resistance has been the important breeding target of breeding researchers.To explore drought resistance related functional genes by identifying drought-tolerant miRNAs not only helps us to understand maize drought resistance mechanism,but also provides an effective way to cultivate maize new drought-resistant varieties.This research identified new miRNAs differentially expressed between two maize inbred lines through high throughput sequencing,then analyzed these new miRNAs and predicted their target genes.And on this basis we over expressed the target gene by transgenic method to proceed functional verification and analyze the regulatory mechanism of ZmmiR139 and ZmDST44.The following are the main results:1.A total of 192 maize mi RNAs,including 124 known miRNAs and 68 new miRNAs,were identified through high-throughput sequencing technology.18 of 68 new miRNAs which low abundance and more than 24 nt in length were selected for validation by stem-loop real-time PCR and the results showed that 14 miRNAs were detected the expression quantities in two maize inbred lines.2.A total of 29 miRNAs differentially expressed between two maize inbred lines were screened from 192 identified maize miRNAs,with 18 down-regulated and 11up-regulated.Among these 29 miRNAs,5 miRNAs(PC-3p-190,PC-3p-104764,PC-3p-129630,PC-3p-552502 and PC-5p-139812)were novel miRNAs.After predicting their target genes and analyzing the GO annotation of target genes,we found 7 of 9 target genes could be classified into 21 biological processes including carbon fixation,photosynthesis and proteolysis,while the remaining two were of unknown function.In addition,4 of 5 novel miRNAs had common target gene(GRMZM2G448344)and PC-3p-104764 and PC-3p-129630 also shared common target genes(GRMZM2G360821 and GRMZM2G308907).Interestingly,these target genes are all involved in photosynthesis.3.According to the publicly available maize transcriptome data,we analyzed the expression patterns of the target genes of the five novel miRNAs in 18 tissues from five organs using RNA sequencing(RNA-Seq).The results showed that 9 target genes had distinct expression profiles and were appeared to be expressed only in the leaves.Moreover,GRMZM2G360821 and GRMZM2G308907 exhibited the highest expression levels,suggesting important roles in the photosynthesis of plants.These results indicated that these genes may participate in photosynthesis,which was consistent with the GO analysis.4.The expression quantities of ZmmiR139 and ZmDST44 detected through the Stem-loop RT-PCR method present a negative correlation,which preliminarily verified that ZmDST44 was the target gene of ZmmiR139.Then using 5 'RACE method to validate the shear action of ZmmiR139 against to ZmDST44,there were 41 correct sequencing results among 50 sequencing samples and their shear sites were the same position.Therefore,the results of Stem-loop RT-PCR and 5 'RACE validated the interaction between ZmmiR139 and ZmDST44.5.The phenotype of transgenic Arabidopsis overexpressed ZmDST44 and wild-type Arabidopsis under normal conditions had no significant difference,while transgenic Arabidopsis overexpressed ZmDST44 compared with wild type had obvious drought resistant phenotype under drought stress condition.After measuring the germination rates of wild type plants and different transgenic plants under the treatment of PEG 6000 and the MDA content of wild type plants and different transgenic plants under drought treatment,we found that the germination rate of transgenic plants under the treatment of PEG 6000 was significantly higher than wild type plants,and the MDA content of different transgenic plants under drought treatment were significantly lower than wild type plants.The results indicated that over expressed ZmDST44 could significantly improve drought tolerance of transgenic Arabidopsis.6.The phenotype of transgenic rice overexpressed ZmDST44 and wild-type rice under normal conditions had no significant difference,while transgenic rice overexpressed ZmDST44 compared with wild type had obvious drought resistant phenotype under drought stress condition.In addition,the relative water content of transgenic rice overexpressed ZmDST44 was significantly higher than wild type,which suggested that overexpressed ZmDST44 could improve the water retention of plants under drought stress conditions.The results of water recovery test showed thatthe survival rate of transgenic rice was also significantly higher than wild type.Combined with the above results,over expressed ZmDST44 could significantly improve drought tolerance of transgenic rice.To sum up,this research explored drought resistance related functional genes by identifying drought-tolerant miRNAs and validated the interaction between ZmmiR139 and ZmDST44 through molecular biology experiment method.Finally,we suggested that over expressed ZmDST44 could significantly improve drought tolerance of transgenic Arabidopsis and rice using transgenic method.Our study not only provides good gene resources for improving stress resistance of crop by genetic engineering,but also lays the foundation for analyzing molecular mechanism of maize drought resistance.