Expression Analysis Of PPO,GLK1 And NADH In Lotus And Identification Of Its Interaction Domain

Lotus root(Nelumbo nucifera Gaertn.)is a perennial herbaceous aquatic plant with swollen rhizome.It is a kind of medicinal food homology food that is popular among people.Because of its rich medicinal and nutritional value,people’s demand for processed products and fresh food is increasing.However,in the process of processing,storage and transportation of lotus root,water loss and wilting,browning and other problems are common,which lead to the decline of commodity,short shelf life of fresh food,and seriously affect the export earnings of lotus root fresh food and processed products.It has been discovered by predecessors that polyphenol oxidase(PPO)is one of the key enzymes that cause enzymatic browning of lotus root.This study used bioinformatics methods to identify and analyze all members of the PPO gene family.The kits and q RT-PCR methods were used to detect the activity of PPO and response of its family genes in different tissues,different growth times,and different inducing factors.Based on the previous work in the laboratory,the expression and interaction sites of PPO1 and its interacting genes GLK1 and NADH were analyzed by q RT-PCR and yeast two hybrid method.The main results are as follows:1. According to the domain structure of the lotus PPO family sequence,an alignment search was performed in the lotus genome database,and then four gene family members were verified through the Pfam website:PPO1,PPO4,AS-1,and AS-2.Using bioinformatics methods to predict and analyze its physicochemical properties,transmembrane regions,signal peptides,gene structure,conserved motifs,secondary and tertiary structures.It was found that PPO1 and PPO4 were more similar in gene,protein sequence and structure.Similarly,AS-1 and AS-2 are also very similar in gene,protein sequence and structure.2. Taking different test materials,using the kit and q RT-PCR method to detect the activity of PPO and the response of its family genes in different tissues,different inducing factors,and different growth times.The results showed that PPO family genes had tissue expression specificity in lotus root,PPO1 and PPO4 were highly expressed in bud,AS-1was highly expressed in root,and AS-2 was highly expressed in leaf.The expression of PPO family genes was closely related to the activity of PPO.The expression levels of PPO1,PPO4,AS-1 genes and the activity of PPO increased in different degrees under different induction treatments,but AS-2 did not change significantly.The expression of PPO family genes was obviously time-specific.The expression of PPO1,PPO4 and AS-1 began to increase rapidly at the 30th day of lotus growth,but AS-2 had no significant change.3. The leaves and stems of lotus treated with different inducements were used as experimental materials to determine the content of chlorophyll and superoxide anion free radicals.And q RT-PCR was used to detect the expression of PPO1 and its interaction genes GLK1 and NADH in different tissues and induction of lotus.The results showed that yeast extract,Ag~+,ascorbic acid and L-cysteine could induce the accumulation of free radicals in different degrees and stimulate the abiotic stress response in different degrees of lotus,and all of them increased the expression of PPO1 and GLK1,while NADH had no significant change.The expression of GLK1 under yeast extract and ascorbic acid treatments increased significantly,but the content of chlorophyll decreased.GLK1 had tissue-specific expression,and the expression of GLK1 was high in the stem of lotus in hydroponic culture,but there was no significant difference in the expression of NADH in each tissue.4. On the basis of the previous work in the laboratory,according to the domain and specific binding site of PPO1 and GLK1,the amino acid sequence was truncated to construct the truncated bait carrier and prey carrier respectively,and then they were transferred into Y2H gold yeast for verification.The results showed that the Cu A and Cu B catalytic active centers of PPO1 and the second half of GLK1 containing the GCT-box domain were the interaction sites of the two.