Functional Analysis Of Citrus CrHSFC1 Gene Involved In The Resistance To Penicillium Italicum

As the world’s most productive fruit,citrus is rich in organic acids and vitamins,and has high nutritional and economic value.However,during the storage and transportation of citrus after harvest,it is susceptible to penicillosis,resulting in rot,inedibility and processing of the fruit.As a fungus,Penicillium is currently mainly controlled by chemical agents,but its toxicity is greater,so it is of great significance to find non-toxic,harmless and efficient biotechnological means to control Penicillium to improve the economic value of citrus fruits.Most of the functional studies of HSF family transcription factors focus on stress stress,and the HSF family genes related to disease resistance are rarely reported,and a large number of studies show that plant hormones as a natural substance can regulate the mechanism of plant disease resistance.Therefore,from the perspective of endogenous hormones,the mining of citrus HSF transcription factors that improve penicillium resistance is not only of great significance for the innovation of biological control methods of penicillium,but also enriches the functional research of HSF family.In this study,citrus was used as plant material,and the mining and functional study of HSF family transcription factors involved in anti-penicillium disease were carried out through bioinformatics analysis,transgenic technology and RT-q PCR screening.The main research results are as follows:(1)Mining of HSF transcription factors associated with anti-penicillosis.In this study,CrHSFC1,the HSF family gene with the greatest response to penicillosis,was mined by q RT-PCR,and subsequent multiplex sequence alignment results showed that citrus CrHSFC1 may be involved in hormonal signaling pathways like HSFC1 in plants such as wheat.Further transcriptional regulation analysis and endogenous hormone analysis showed that the transcription factor CrHSFC1 may bind to thesalicylate synthase Cr ICS2 promoter to regulate the SA signaling pathway,which in turn affects the disease resistance of fruits.(2)Correlation between expression of transcription factor CrHSFC1 and penicillium resistance.The function of CrHSFC1 was verified from both sides by gene overexpression and gene silencing technology,and the results showed that the expression of CrHSFC1 in silent fruits was significantly lower than that of control fruits,and the incidence,disease spot diameter and decay degree were slightly higher than those in control fruits.The expression of CrHSFC1 in overexpressed fruits was significantly higher than that of control fruits,and the incidence,disease spot diameter and decay degree were lower than those of control fruits.The results showed that the overexpression of CrHSFC1 could weaken the activity of cell wall degrading enzymes PG and PME,reduce the content of watersoluble pectin(WSP)in the peel,and delay the softening of the peel.At the same time,the overexpression of CrHSFC1 can also enhance the activity of antioxidant enzymes such as SOD,POD and APX,and combined with the decrease of malondialdehyde content and conductivity of the peel,it indicates that CrHSFC1 can reduce the damage of organelles and cell membranes caused by membranous peroxidation after infection with penicillosis.In addition,the results showed that CrHSFC1 could bind to the promoter of Cr ICS2,a key synthetase gene of SA,causing an increase in endogenous SA content in fruits.(3)Study on the inhibition of Salicylic Acid on Penicillium.In order to analyze whether CrHSFC1 improves the disease resistance of citrus fruits to penicillosis through the SA pathway,in this study,it was first found that 2m Mol/L SA can destroy the cell membrane and cause leakage of intracellular substances by culturing Penicillium ex vivo,thereby inhibiting the growth of Penicillium bacteria.In order to further prove that SA can inhibit the occurrence ofpenicillosis in citrus fruits,in this study,different concentrations of SA were used to treat the fruits,and it was found that SA treatment could increase the total phenolic and total flavonoid content of the peel,increase the activity of antioxidant enzymes such as POD and SOD,and reduce the damage of membrane peroxidation.In addition,exogenous SA treatment can also induce endogenous SA synthesis in citrus fruits and activate the expression of NPR,PR and other defense genes mediated by salicylic acid pathway,thereby improving the resistance of citrus fruits to penicillosis.The results showed that CrHSFC1 could bind to the promoter of Cr ICS2,a key synthetase gene of SA,and the above results showed that CrHSFC1 could participate in the salicylic acid signaling pathway in a transcriptionally regulated manner,thereby improving fruit resistance to penicillosis.In sum Mary,the study found that the transcription factor CrHSFC1 was positively correlated with penicillium resistance,and the mechanism of action was that overexpression of CrHSFC1 could reduce peel oxidative damage,improve the expression of related resistance genes and enzyme activity by increasing endogenous salicylic acid content,thereby enhancing the defense ability of fruits against penicillosis.