Screening And Analyzing The Key Genes For Arginine Accumulation In Rosa Roxburghii Tratt.According To Transcriptome

Arginine is a semi essential amino acid for human body,which has medicinal and health value.Rosa roxburghii Tratt is a native fruit tree of Rose of Rosaceae from China.R.roxburghii fruit is rich in arginine,which is a good natural raw material for arginine.But now,the key genes that regulate arginine accumulation during fruit development of R.roxburghii are not clear.Therefore,in this study,we cleared the change of arginine in the fruit development period and the key genes that regulate arginine accumulation.Second,we analysed the effect of shading and exogenous growth regulator on arginine and its key genes.Third,Throught transient overexpressed and silenced key genes,we explored the role of key genes on arginine accumulation.It has important value for further understanding the formation mechanism of special quality of R.roxburghii fruit.The specific research results are as follows:1)Screening key genes for arginine accumulation in R.roxburghii fruit based on transcriptomeBased on the genome of ‘Rr-5’ and transcriptome of ‘Rr-5’ on 30 DAA,60DAA and 90 DAA,21 expressed genes in arginine synthesis pathway and 4 expressed genes in decomposition pathway were found.Measured the relative expression levels of these 25 genes on 8 different developmental stages fruits through q RT-PCR,and analyzed their correlation with arginine content.Confirmed that the Rr NAGS1 gene of synthesis pathway and Rr ADC1 of decomposition pathway were important structural genes.By analysis the upstream promoter elements of Rr NAGS1 and Rr ADC1 we found that Rr NAGS1 and Rr ADC1 have many binding sites of MYB transcription factor.Then,We performed a correlation analysis on the expression levels of Rr NAGS1,Rr ADC1 and Rr MYBs in the transcriptome data of three fruit development stages,suggested that 13 Rr MYBs may be potential regulatory transcription factors affecting arginine accumulation.The expression level of 13 Rr MYBs in eight development stages was tesed by q RT-PCR,and it was clear that Rr LHY and RrDMTF1 is important transcription factors.Rr LHY is positively correlated with arginine accumulation,while RrDMTF1 is negatively correlated with arginine accumulation.2)Response of key genes for arginine accumulation under shading and growth regulator treatment15 days after flowering,the fruit of ‘Rr-5’ was treated with shading(20%,50%,90%),ABA(0.5 mmol/L,1mmol/L)and Me JA(2.5 mg/L,25 mg/L).We found that shading 90%significantly increased the content of hydrolyzed arginine and free arginine in mature fruit of R.roxburghii,reached 207.6mg/100 g and 45.3mg/100 g,respectively,It was 1.3 and 7.0 times higher than the control.Under all treatments,the content of hydrolyzed arginine and free arginine in mature fruit of R.roxburghii always showed a consistent change with the expression level of Rr NAGS1.The expression level of Rr ADC1 was up-regulated in all treatments compare to the control,and the expression was consistent with Rr LHY at mature fruit,while RrDMTF1 showed no regular change pattern.3)Functional verification of key genes for arginineHigh arginine R.roxburghii ‘Rr-1’,low arginine R.roxburghii ‘Rr-5’ and strawberry were selected as study materials.The Rr NAGS1 gene and Rr ADC1 gene were transiently overexpressed and VIGS in fruit by Agrobacterium tumefaciens.Over-expressed Rr NAGS1 fruit and silenced Rr ADC1 fruit significantly increased Rr NAGS1 expression level and decreased Rr ADC1 and Rr LHY expression level,and significantly increased the hydrolyzed arginine and free arginine content.Over-expressed Rr ADC1 and silenced Rr NAGS1 fruit significantly decreased Rr NAGS1 expression and hydrolyzed arginine and free arginine content.RrDMTF1 was no regular change.