Expressive And Functional Analysis Of MicroRNA399 In Tomato Under Phosphorus Deficiency

microRNA399 is an important phosphorus homeostasis molecule in plants,which induces the degradation of gene transcripts through post-transcriptional regulation.miR399 has long been reported to respond to phosphorus starvation in plants.The regulation of miR399-PHO2 pathway in Arabidopsis thaliana on the adaptive response to phosphorus deficiency stress has been described successively.However,there are still few reports in tomatoes,and more studies need to be carried out urgently.Accordingly,this study combined the high-throughput sequencing results of tomato obtained in the laboratory with genetic engineering technology and bioinformatics analysis to study the expression patterns and physiological functions of Sly-miR399,which is functionally conserved in tomatoes.The biological functions of Sly-miR399 in tomatoes were revealed,which provided theoretical basis for the breeding of Phosphorus-Efficient crops and environmental protection.To understand the spatiotemporal expression pattern oftomato Sly-miR399 under phosphorus deficiency,wild type tomatoes were hydroponically cultured using hoagland nutrient solution as growth substrate and treated with complete phosphate-deficiency.The time of phosphorus-deficiency treatment included 6 hours,12 hours,1 day,3 days and 7days.At the same time,phosphorus supply was restored after 7 days of phosphorus deficiency,including 6 hours,12 hours and 1 day of phosphorus supply.High-throughput sequencing was performed on the materials of the treatment group and the control group,and the sequencing results were verified by qRT-PCR.The quantitative results were in good agreement with the sequencing results.The results showed that,like the previous reports in many plant species,the Sly-miR399 family of tomatoes had multiple members,and the precursors of each member could produce a mature Sly-miR399 at 3'and 5' ends,and named Sly-miR399-5P ? Sly-miR399-3P,respectively.According to whether the mature Sly-miR399 is the same or not,seven members of the Sly-miR399 family were divided into three groups,whichnamed Sly-miR399 A,Sly-miR399 B and Sly-miR399 C,respectively.From the temporal expression pattern of Sly-miR399,it is inferred that there are functional redundancies between Sly-miR399-5P and Sly-miR399-3P members.Based on the newly sequenced findings,this study constructed each expression vector related to Sly-miR399 for further study of the biological functions of tomato Sly-miR399.These expression vectors include Sly-miR399 promoter fused with GUS/GFP gene expression vector,Sly-miR399 gene knockout or STTM silencing expression vector,Sly-miR399 artificial overexpression vector.Up to now,the obtained transgenic plants include promiR399A:GUS fusion expression plants,pYLCas9:Sly-miR399 geneknockout plants,35S:amiR399A/B/C-5 overexpression plants and 35S: STTM-Sly-miR399B/C-3P gene silencing plants.In order to analyze the function of Sly-miR399 under phosphorus deficiency in tomato,different physiological experiments were carried out simultaneously on35S:amiR399A overexpressed strains and wild-type tomatoes preserved in our laboratory.The results showed that over-expression of Sly-miR399 A could shorten the germination time of tomato seeds compared with wild type.and the increase of root to shoot ratio and decrease of protein content of Sly-miR399 A overexpression reduced in phosphate-deficient environment,which implied that the resistance of Sly-miR399 A strains to phosphorus deficiency stress was enhanced.This may be because overexpression of Sly-miR399 A in tomatoes can significantly affect the physiological indicators related to phosphorus uptake,suggesting that Sly-miR399 A is an important regulator of phosphorus uptake in plants.