Spationtemporal Specific Expression Of Tomato Sly-miR164 Regulates Fruit Development And Maturation

MicroRNAs(miRNAs)are a class of single-stranded,endogenous,non-coding small RNAs of approximately 20-24 nt in length.By pairing with specific bases of the target gene mRNA,the degradation of its mRNA can be induced or its translation can be inhibited,so as to regulate the gene after transcription.miRNAs play an important regulatory role in plant growth and development.At the same time.Tomato is an important economic crop and model plant for studying fruit development and maturity.As an important gene expression regulatory molecule,miRNAs have regulatory effects on tomato compound leaves,inflorescence morphology,flower organ development and other important characters,but they are still poorly understood in the regulation of fruit development and maturatio.In this paper,tomato was used as the research material to analyze the expression and biological functions of Sly-miR164 family members through morphological,genetic transformation and molecular biology methods,in order to reveal the regulation mechanism of Sly-miR164 expression on tomato fruit development and maturation.These results provid a theoretical basis for the production practice in order to elucidate the mechanism by which Sly-miR164 mediates the regulation of target genes in post-transcriptional regulation of tomato fruit development and maturation.The main results of this paper are as follows:1?By fluorescent quantitative PCR,Northern blot hybridization testing,we found that both Sly-miR164 and target gengs had spatio-temporal specific expression patterns.Mature Sly-miR164 was less abundant in leaves and more abundant in floral organs and fruits.The expression was mainly concentrated in the petals and anthers in the floral organs and in the fruit at the mature stage.In the expression mode of target genes,GOB,NAC1,NAM2 and NAM3 were all low in leaves,and were highly expressed in ovary,sepals and anther of flower organs respectively,while NAM3 was highly expressed in petals,ovary and anther.In different stages of fruit development,both GOB and NAC1 are low expressed in each stage of fruit development,but the expression patterns of NAC1 in fruit ripening stage are similar to Sly-miR164 expression patterns.NAM2 and NAM3 are expressed from the day of flowering to 8d of flowering.The GUS staining of transgenic lines with fused expression of proMIR164a/b:GUS revealed that the spatio-temporal specific transcription level of proMIR164b:GUS in various tissues and organs was higher than that of proMIR164a:GUS,suggesting that the expression abundance of mature Sly-mir164 was mainly generated by the precursor MIR164b2 ? Build the component type and the fruit of the specific promoter expression(amiRNAs-Sly-miR164),silence suppression(STTM-Sly-miR164)expression vector and Crispr/Cas9 knockout expression vector,and successful transformation of agrobacterium..The tomato cotyledons are mediated by Agrobacterium,genetically transformed,and corresponding transgenic materials are obtained.Through agrobacterium mediates tomato cotyledon,carries on the genetic transformation,and obtains the corresponding transgenic material.3?It was found that the time of fruit development and ripening was affected in the over-expression and silencing inhibited transgenic lines,and the time from flowering to color breaking and color breaking to maturity was significantly delayed compared with the wild type.Studies have shown that overexpression of Sly-miR164 delays fruit development and ripening in tomatoes.4?Identification of function by Crispr / Cas9 knockout transgenic lines found,Crispr / Cas9 presence knockout phenotypes pri-Sly-miR164 a,and pri-Sly-miR164 b plants differences.In the pri-Sly-miR164 a transgenic plants,only the number of side branches and chambers of the plants increased compared with the wild type;in the pri-Sly-miR164 b transgenic plants,the lateral branches of the plants increased,and the number of serrated deepening of the leaves decreased.The deformed parts of the organs were developed.Compared with the wild type,the number of petals increased,the bracts were severely missing,the number of bracts decreased,the anthers were filiform,the number of ovary increased,and the mature fruit did not have an internal chamber.Therefore,Sly-miR164,as a negative regulatory factor,is mainly produced from the precursor pri-Sly-miR164 b during fruit maturation and development,and plays an important regulatory role in tomato growth and development.