Characterization And Expression Analysis Of MiR167e In Wheat

The regulation of wheat grain development directly affects 1000 grains weight and yield of wheat,and its important to study the molecular regulation mechanism of wheat grain development for high-yield wheat breeding.In the previous study,differential expression of miRNAs was systematically analyzed and it was found that miR167 was up-regulated during grain development.Here,a new member of the wheat miR167 family,tae-miR167 e,was identified and its sequence characteristics,target genes and their spatio-temporal expression patterns were studied,and the regulatory function of this miRNA in the process of grain development was discussed.The main results are as follows:1.The partial homologous gene fragments of TaMIR167 e were cloned by RT-P CR using the wheat cultivars of Chinese spring,Yumai 18 and Aikang58 and their s equence characteristics were analyzed.MIR167 e gene was located on the short arm o f chromosome 5,and its three homologous gene from genome A,B and D were des ignated as TaMIR167e-5AS,TaMIR167e-5BS and TaMIR167e-5DS,respectively.Seque nce analysis showed that the region of miRNA precursor was highly conserved amon g tested varieties and only SNPs were detected among the three homologous.while t he sequence corresponding to the mature miR167 e was identical.RNA folding analys is show that three partial homologous genes could form a specific hairpin structure.Target genes of candidate miR167 e were screened and analyzed by using bioinformat ics analysis.The resulted showed that a total of 80 target genes were predicted.NC BI was used to analyze the sequences of these target genes,and 32 target gene sequ ence were annotated completely in the database of Chinese spring,and the sequence of the remaining target genes is unknown.One target gene,Ta PHT4;3,was analyzed of protein characteristics and cluster analysis of gene families were carried out.2.The expression analysis of tae-miR167 e in grain development showed that the expression level of miRNA in all the 6 varieties showed a trend of up-regulation with the grain-filling process.One of the target genes,Ta PHT4;3,was contrary to the expression pattern of miR167 e,and showed a down-regulated expression trend during the grain development of 6 varieties.The analysis of expression characteristics in different tissues showed that tae-miR167 e was highly expressed in leaves and roots,while it was lower in the inferior panicle.Overall,the expression level of target gene Ta PHT4;3 was lower in different tissues.Under the drought stress,tae-miR167 e was up-regulated and the induction responding to drought stress was faster in root tissues of Aikang58 than that of Chinese spring,and the expression level in leaves was higher than that in Chinese spring,as well.Under the salt stress,the tae-miR167 e was down-regulated in root tissues,while up-regulated in leaf tissues.Our results indicated that tae-miR167 e play important roles during the process of developmental regulation and osmotic stress response in wheat.3.In order to investigate the biological function of tae-miR167 e,a STTM-based miR167 e inhibitor expression vector was constructed,and the effect of miR167 e inhibition on the grain development was analyzed by barley striped mosaic virus(BSMV)mediated STTM technique.The results showed that this technique could effectively inhibit the expression of endogenous miR167 e in leaves.Five days after flowering,spike inoculations significantly inhibited the expression of miR167 e,and resulted in signifcant changes in the development characteristics of wheat grains,and the 1000 grain weight increased by 3%.The above results indicated that miR167 e plays an important regulatory role in the process of grain development and stress response,and the relevant regulatory mechanisms still need to be further studied.