Analysis Of CsAN1 Role In Citrate Accumulation And The Effect Of Different Factors On CsAN1 Expression

The low expression of the citrus plasma membrane proton pump gene CsPH8 is an important reason for the low acid character of Honganliu(HL,Citrus sinensis cv.Honganliu).In this experiment,the correlation between CsAN1 and CsPH8 genes and organic acid content was studied by using Anliu and Honganliu as research materials.It is speculated that there may be a correlation between CsAN1,CsPH8 and organic acid content.Eight pairs of citrus varieties were used as experimental materials to compare the relationship between their titratable acid content and CsAN1 gene expression level,thus further confirming the conjecture.Subsequently,we studied the effects of different environmental factors on the expression of CsAN1,CsPH8 gene and titratable acid in vivo and in vitro.The relationship between CsAN1 and CsPH8 gene expression levels and organic acid content was preliminarily determined.The experimental results are as follows:1.The relationship between CsAN1 and organic acids during citrus fruit developmentIt was found that the content of titratable acid and citric acid rise first and then fall in Anliu and Honganliu,and the titratable acid content and citric acid content in Anliu were significantly higher than Honganliu;The expression levels of CsAN1 and CsPH8 genes also rise first and then fall in the development of the fruit of Anliu and Honganliu,and the gene expression level in Anliu was significantly higher than that of Honganliu.The titration acid and citric acid content showed a very similar trend,but the soluble solids,glucose,fructose,sucrose and malic acid content did not show this similar trend.2.Analysis of titratable acid content and CsAN1 gene expression in multi-variety citrus fruitsThe titratable acid content and CsAN1 gene expression levels were determined in 8 pairs of citrus varieties.It was found that fruits of all varieties showed titratable acid content is proportional to the CsAN1 gene expression level except for Fengjie navel orange 72-1 and Fengjie navel orange 72-1 late maturity.It is further confirmed that CsAN1 has an important role in citric acid accumulation.3.Effects of environmental factors on the expression level of CsAN1 gene in vivoThe results showed that the application of higher nitrogen and calcium fertilizers to Satsuma orange could increase the expression of CsAN1 gene and titratable acid content,but the application of phosphorus and potassium could not significantly increase the expression of CsAN1 gene and titratable acid content;Injecting ABA(abscisic acid)and drought stress on the fruit of Satsuma orange can significantly increase the expression level of CsAN1 and CsPH8 genes and the titratable acid content,while spraying ABA on the surface of Satsuma orange,the expression levels of CsAN1 and CsPH8 genes and the titratable acid content were significantly decreased,but the difference was not obvious.4.Effects of environmental factors on the expression of CsAN1 gene in vitroThe effects of temperature,pH,mineral elements and NaCl stress on the expression level of CsAN1 gene were studied in the tissue culture of Satsuma orange and ponkan juice cells.It was found that the expression level of CsAN1 gene gradually decreased in the range of 4°C-15°C-25°C-35°C in the temperature treatments;In pH treatments,the expression level of CsAN1 gene in ponkan fruits showed a decreasing trend at pH 4.8 and pH 6.8 treatments.The expression level of CsAN1 gene in Satsuma orange fruit decreased first and then increased at pH 4.8,pH 5.8 and pH 6.8 treatments,showing a gradual decreased trend under the condition of pH 7.8;The expression level of CsAN1 gene in Satsuma orange was higher than that in 1/2Mg treatment,2Ca was higher than 1/2Ca treatment,2N was higher than 1/2N treatment,and 2P was lower than 1/2P treatment,2K is lower than 1/2K treatment;In NaCl stress treatment,the expression level of CsAN1 gene in citrus fruits showed an increasing trend in 5g and 15 g treatments,and decreased in 10 g and 20 g treatments,and the difference was significant.Based on the above results,it is suspected that there is a correlation between CsAN1 and CsPH8 gene and organic acid content,and external environmental factors such as temperature,pH,mineral elements,drought and NaCl stress have significant effects on the expression level of CsAN1.