| Rice is an important food crop and an important model plant to study the growth and development of monocotyledonous crops.Nitrogen is necessary for plant growth,and its proper application is vital to improve crop yield.Increasing the nitrogen utilization efficiency of crops has important theoretical and production significance for improving varieties,increasing yields and reducing environmental pollution.Over the past few decades,numerous genes associated with nitrogen use efficiency has been identified in plants,including the small RNA family genes.Small RNA(s RNA)is a type of endogenous non-coding RNA with a length of about20～24bp,which can regulate the virtually every aspect of animal and plant growth and development.It can be divided into three categories: micro RNA(mi RNA),small interfering RNA(si RNA)and other small RNAs.In the present study,small RNA sequencing was conducted on wild-type(WT),Os NAR2.1 silencing material(Os NAR2.1-RNAi)and Os NAR2.1 overexpression material(OE-Os NAR2.1)in Nipponbare backgound to identify small RNAs related to nitrogen regulation and its possible target genes.We then analyzed the expression patterns of the identified candidate s RNAs and studied its physiological function in relation to nitrogen regulation through transgenic approach.The main findings were summarized as follows:1.A small RNA chr4-27017,which may be involved in the N response in rice,has been identified through in silico analysis on the small RNA sequencing data.chr4-27017 is predicted to be a mi RNA by different bioinformatic approaches,including stem-loop method and the prediction was further supported by the lower abundance of chr4-27017 in DCL1-like mutant.DCL1 is the core component for small RNA biogenesis and the knockout of DCL1-like will significantly affect the formation of small RNA.In order to study the biological function of chr4-27017,the mutant lines of chr4-27017 were also generated using “Target mimicry Method”.Under field condition,the mutant lines of chr4-27017 exhibited a late flowering phenotype and rwlatively increased yield per plant compared with the wild-type.Moreover,five possible target genes of chr4-27017 were also identified using mi RNA target prediction tool and RNA-seq analysis.These putative target genes included LOC＿Os08g09080,LOC＿Os05g14220,LOC＿Os02g41954,LOC＿Os01g34920 and LOC＿Os09g39430.Expression profile analysis on these target genes showed that the expression level of LOC＿Os08g09080 、 LOC＿Os01g34920 and LOC＿Os05g14220 increased significantly in chr4-27017 mutant compared to the wild type.Hence,these genes could be the potential target genes for chr4-27017.These phenotype suggested that chr4-20107 may play an important role in nitrogen uptake and utilization as well as the growth of rice.2.Two small RNAs s NRT2.3-1 and s NRT2.3-2 were identified to be located near Os NRT2.3 gene.The lower expression level of s NRT2.3-1 and s NRT2.3-2 in DCL3 mutant suggested that these small RNAs were si RNA.We then generated the target mimic lines for s NRT2.3-1 and s NRT2.3-2 using “Target mimicry method”.Under high nitrogen supply condition,we found that the heading time of the field-grown sm NRT2.3-1-m ATG mutants(the target mimic line for s NRT2.3-1)was two weeks later than that of the wild-type.Furthermore,the plant height and grain yield of sm NRT2.3-1-m ATG were 6 cm and 35%higher than that of wild-type at mature stage,respectively.Furthermore,the plant height and grain yield of sm NRT2.3-1-m ATG at the mature stage were 6 cm and 35% higher than those of wild-type,respectively.These observed phenotypes were highly similar with the phenotype of Os NRT2.3b overexpression lines(delayed heading time and increased grain yield).Quantitative RT-PCR analysis showed that the expression level of Os NRT2.3b increased significantly in sm NRT2.3-1-m ATG mutant,as compared with the wild-type.These findings indicated that s NRT2.3-1 may involve in the regulation of Os NRT2.3b gene expression level,leading to the observed phenotype. |
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