Identification And Function Anaylsis Of Specific MicroRNAs During Young Panicle Development Of Rice

Plant microRNAs are a class of 21nt-length RNA molecules,which are single-stranded and regulate the expression of genes at transcriptional and translational levels.It was proved that microRNAs play important roles in regulating the growth and development of plants,response to adverse stresses.It was shown that the development of rice flower is closely related to the regulation of miRNAs.However little is known about the specific regulatory mechanisms.Therefore,the study on miRNA and its target gene function of rice flower is one of the key points in the development of rice flower.In this study,young panicles of three different developmental stages were selected as materials from Nipponbare.The length of young panicles were < 2mm,3-5mm and 6-10 mm,respectively.Based on high-throughput sequencing and bioinformatics analyze,the nine specifically-expressed osa-miRNAs and their corresponding target genes were identified.Then the expression patterns and functions were studied,the main results are as follows:High-throughput sequencing data analysis.A total of 9 sequencing libraries were constructed during the three stages of young panicles from Nipponbare rice and high-throughput sequencing of miRNAs was performed.Small RNA sequences of 21 nt,22 nt,23 nt,and 24 nt were obtained and 24 nt was the most abundant,followed by 21 nt.About 762 osa-miRNAs were predicted for all samples,including 650 known osa-miRNAs and 112 novel ones.The miRNA expression differences during the three panicle stages were screened.There are differential expressions 25 osa-miRNAs in three stages,of which 22 are known osa-miRNAs,except osa-miR319a-3p.2-3p,osa-miR319 b,osa-miR5509,osa-miR5516 a and osa-miR5516 b,the rest are osa-miR2118 family members.A total of 359 target genes were predicted,of which osa-miRNAs were known to correspond to 328 target genes and 31 new target genes were predicted.The differentially analyzed target genes were annotated and analyzed.The relationship between osa-miRNA and its target mRNA expression pattern.Sequencing based on the high-throughput sequencing and the degradative group,9 osa-miRNAs with target mRNA specific expression were screened out.The qRT-PCR validation was performed for their expression levels results showed that in addition to osa-miR5499 with target gene LOC_Os07g07610,the remaining 8 miRNAs were negatively correlated with their target expression.The construction of osa-miRNA overexpression vector.The overexpression vector pOX was used to construct 8 pre-miRNA overexpression vectors for: Ubi::pre-mir5506,Ubi::pre-miR5499,Ubi::pre-miR5530,Ubi::pre-miR2275 d,Ubi::pre-miR5522,Ubi::pre-miR5518,Ubi::pre-miR5496,and Ubi::pre-miR5514 were constructed.The construction of osa-miRNA silencing expression vector.Using the short tandem target simulant(STTM)technology,the selected 9 osa-miRNA STTM sequences were designed and linked to the overexpression vector pOX.And four silent expression vectors Ubi::STTM-miR5518,Ubi::STTM-miR5530,Ubi::STTM-miR5485 and Ubi::STTM-miR2275 d were constructed.The construction of target genes overexpression vector.The CDS sequences of 8 selected target genes were amplified from Nipponbare rice DNA,and two target gene overexpression vectors Ubi::LOC_Os07g07610 and Ubi::LOC_Os03g31290 were successfully constructed through the linkage with the overexpression vector pOX.The obtaining and molecular identification of transgenic seedlings.Using the method of agrobacterium-mediated,the successfully constructed overexpression vector was transferred into Nipponbare rice callus,and after co-cultivation,selection,and differentiation culture,The transformed seedlings of Ubi::pre-miR5514,Ubi::pre-miR5499,Ubi::pre-miR5522,Ubi::pre-miR5506 and Ubi::pre-miR5518 were obtained and were transferred by PCR reaction.In this study,small RNA libraries at different developmental stages of young panicles of rice were constructed using high-throughput sequencing technology and bioinformatics analysis was carried out.The screening 9 phase-specific expression of osa-miRNA and target mRNA vectors were constructed to provide a basis for the functional analysis of miRNAs and their target genes.