Signal Pathway Analysis Of MiR164 Regulating Maize Kernel Development

As one of the four main crops that people eat,Maize(Zea may L.)plays a very important role in food security,industrial and economic development.It is the most important multi-purpose crop in the world.Maize seed is an important energy source for human diet and animal feed.At the same time,the starch in seeds can also be used as an important industrial raw material.With the increase of population and energy shortage in the world,it is very important to raise the maize yield.As a trans-acting factor,microRNAs can bind to the target gene mRNA molecule and degrade the latter through complementary base sequence pairing,so as to achieve the negative regulation of target gene after transcriptional level.It plays a vital role in the process of plant growth and development.Therefore,exploring microRNAs and their target genes related to maize grain development and the exploration of their molecular mechanisms can help people to understand the regulatory network of maize seed development,improve the maize seed traits from the level of molecular biology and increase the yield of maize.The research team analyzed the small RNA-seq in grain filling from start to finish(Zheng et al.2019),It was found that the differential expression of miR164 during maize grain development,miR164 may regulate grain development.Previous studies have shown that miR164 plays an important regulatory role in plant growth and development(Kim et al.2009).However,miR164 has little knowledge on the regulation of maize grain development,and its regulation mechanism is not clear.In this study,RNA-seq,RT-PCR,gene gun-mediated transient expression,yeast one-hybrid,transgenic and other techniques were used to analyze miR164 expression patterns,select and identify downstream target genes of miR164,The regulatory network of miR164 in maize grain development was primary confirmed;Through the phenotypic analysis of MIR164 overexpression lines,the function of mi164 in maize grain development was clarified.The regulatory network of miR164 in maize kernel development was analyzed to provide theoretical basis for further research on the effect of miR164 on maize kernel development and the regulatory mechanism.The discovery of the regulation mechanism of miR164 in maize grain development provides a theoretical basis for maize molecular breeding,heterosis breeding and high yield breeding in the future.The main results are as follows:1.To explore the important role of miR164 in maize seed development,we first analyzed the expression pattern of MIR164.Sequencing of small molecular RNA libraries in maize grains showed that MIR164 was differential expression from start to finish grain filling.The expression pattern showed that the expression of MIR164 e was down-regulated during maize grain development.2.In order to identify the target genes of miR164 in grain,we determined that ZmNAC32 and ZmNAC40 are the target genes of miR164 by degradation sequencing,transient expression,qPCR and other experiments.3.In order to explore the downstream genes of ZmNAC32 and ZmNAC40,we verified the indirect regulation of ZmEXPB14 and ZmEXPB15 by RNA-Seq and transient expression of gene gun and yeast one-hybrid,The family of ZmEXPB14 and ZmEXPB15 is related to cell enlargement and growth.4.In order to clarify the function of miR164 in maize grain development.We created MIR164 overexpression strains and selected three MIR164 overexpression strains for phenotypic identification.5.Through phenotypic identification of wild type and MIR164 overexpressing strains,we found that the 100-seed weight,seed length,seed width and endosperm of MIR164 overexpressing strains were significantly smaller than wild type.These results suggest that miR164 may be involved in the regulation of maize seed development by regulating target genes ZmNAC32 and ZmNAC40.