Identification And Cold Tolerance Analysis Of Sweet Orange NADPH Oxidase Gene Family

Plant NADPH oxidase is a homologous of macrophage NADPH oxidase gp91phox,which usually exists in the form of gene family.Studies have shown that NADPH oxidase plays an important role in biological stress and abiotic stress.At present,the role of NADPH oxidase in citrus stress response is unclear.Accordingly,the present study screened by bioinformatics the NADPH oxidase gene family orange;analyzing the gene expression profile of the family members in different tissues,different stress and stress hormone treatment using qRT-PCR method;using virus-induced the preliminary analysis of gene silencing method cold function NADPH oxidase gene;and finally analyzed physiological and biochemical changes in orange under low temperature stress.The main findings are as follows:1.There are 14 NADPH oxidase family members were found in the sweet orange genome by bioinformatics analysis.These NADPH oxidase genes are distributed on five chromosomes;they are mainly concentrated on chromosomes 5,7,and 8.The 14 members of the amino acid sequence have low homology,and the exon-intron structure has a large difference,and the number of exons is between 5 and 14.Promoter analysis revealed that the promoter region of the gene contains a large number of cis-acting elements Such as light response element,defense stress response element,etc.This suggests that the NADPH oxidase gene is involved in a variety of functions such as growth and development,adaptation to abiotic and biotic stresses.qRT-PCR analysis of the expression levels of NADPH oxidase gene under different stress treatments in different tissues of sweet orange showed that the NADPH oxidase gene expression was tissue-specific and could be induced by various hormones and abiotic stress,which was consistent with the analysis results of the cis-acting elements of the promoter.2.A NADPH oxidase gene(Cs5g11890),which was significantly up-regulated by low temperature treatment,was selected.Several Cs5g11890 interference plants were obtained by virus-induced gene silencing.The low temperature resistance analysis indicated that Cs5g11890 expression was interfered with,and the plant was cold-resistant.The cold resistance of the plant decreased significantly.3.The changes of ROS content,reactive oxygen species(Ros)and antioxidant enzyme activity of sweet orange leaves treated with NADPH oxidase inhibitor(DPI)for 48 h were analyzed,and the changes of MDA content,reactive oxygen species content and antioxidant enzyme activity of sweet orange leaves treated with DPI and distilled water for 12 h,6 h,12 h and 24 h,respectively,were analyzed at 4 0C and low temperature for 0 h,6 h,12 h and 24 h,respectively.The results showed that most of the NADPH oxidase gene expression was significantly up-regulated after low temperature stress.It is speculated that DPI can inhibit NADPH oxidase for a certain period of time before it can inhibit the growth of plants after DPI inhibition of NADPH oxidase activity.Prolonged,the damage rate of cell membrane is higher;the ROS content in plants first increases,then decreases and then rises,which is consistent with the trend of antioxidant enzyme activity.The aim of this study was to explore the members of the sweet orange NADPH oxidase gene family and their expression characteristics,and to analyze its role in stress,in order to provide a theoretical basis for citrus genetic breeding and stress resistance improvement.