Identification And Study On The Role Of Polyphenol Oxidase(PPO) Gene In The Browning Of Luffa Cylindrical

Loofah(Luffa cylindrical)is popular with people for its tender and juicy fruit and heat-clearing effect.However,the browning reaction occurs during cooking,which greatly reduces sensory quality,food value,and product value,and also hinders product circulation,shortening the shelf life,even seriously affecting people's visual experience and appetite,and making them unacceptable to consumers,which has become one of the major obstacles to the development of fruit and vegetable storage and transportation processing industry.Polyphenol oxidase(PPO)is a key enzyme that causes browning of loofah.It can catalyze the oxidation of phenolic substrates to quinones with the participation of O2,and cause the browning reaction.To understand the mechanism of the browning reaction and control it from the perspective of molecular biology has become a hot spot for postharvest research of fruits and vegetables.This study based on transcriptome squencing use common loofah as material,after obtaining the loofah PPO gene family members by RT-PCR,to test the tissue organs,species,storage period and the changes of PPO gene when it is placed in frech-cut status by QPCR,and then analyze the relationship between PPO gene expression,enzyme activity and browning.To do a preliminary study on the function of PPO gene and its role in browning,and structure a recombinant vector for prokaryotic expression at the same time,which provides material basis and theoretical support for further isolation,purification and bioassay of PPO protein inloofah and also offers the possibility for further exploring its functions.The main results are as follows:(1)To test the browning degree of two Rough loofah and six kinds of common loofah by using Eliminating light wave method,and screen out test materials with higher browning.The results indicates that the common loofah is easier to browning,and has the biggest R3 browning degree than the luffa gourd.(2)To use common loofah R3 as test material and test its enzyme activity and total phenolic changes when placed under storage time 4?and room temperature 25 ? in fresh-cut status.The results shows that With the increase of storage time at 4? and the time of fresh-cutting at 25?,the browning of loofah increased,the total phenolic content of loofah increased first,and reached a certain value and then tends to stability;The overall change trend of PPO enzyme activity in loofah increases first and then decreased.(3)To use a common loofah fruit pulp as material,and obtain the cDNA sequences of three PPO gene families by transcriptome sequencing and RT-PCR,both have a complete reading frame(ORF).The LcPPO1 gene is 2 026 bp in length and contains a 1 794 bp ORF,encodes 598 amino acids.The LcPP02 gene has a full length of 2 071 bp and anORF of 1 722 bp,encoding 574 amino acids.The LcPP03 gene has a full length of 2 189 bp and an ORF of 1 779 bp,encoding 593 amino acids.The homology of the three PPO gene-encoded protein sequences is 47.5%.The nucleotide sequence of full-length cDNA sequence is 56.4%.The homology of LcPPO1 and LcPP03 is higher,reaching 71.7%.The similar parts are relatively densely distributed at the ORF.(4)To do a bioinformatics anaylsis of LcPPO encoded proteins by online analysis software.It shows that the three proteins contain no signal peptide sequence and belong to hydrophilic and non transmembrane proteins.LcPPO subcellular locate in chloroplasts;LcPPO has typical characteristics of PPO protein.Homology analysis shows that different sources of PPO have high homology in amino acid sequence.To anaylse the PPO protein affinity and find that LcPPO1,LcPPO2 and LcPPO3 are all clustered with cucumbers and melon etc..They have a close protein affinity.It can be seen that the PPO phylogenetic relationships of different plants have obvious species characteristics;The spatial structure of the three proteins is similar.(5)Using loofah 18s rRNA as a reference gene,to anaylize the expression of PPO mRNA related to browning in loofah by QPCR.The results shows that the three genes of loofah PPO gene family have tissue specificity.It is expressed in roots,stems,leaves,flowers and fruits.LcPPO1,LcPPO2,and LcPPO3 express the highest levels in fruits,leaves,and flowers respectively.With the increase of storage time at 4?and fresh-keeping at room temperature of 25?,the expression of LcPPO1 and LcPPO2 increase at first and then decreased.The expression of LcPP03 gene after fresh-cut is lower than that after harvest,and the expression level is slightly up-and-down.(6)To combine quantitative expression of LcPPO with enzyme activity and total phenol content analysis.The results shows that in the process low temperature of 4?,the expression of the three genes is positively correlated with the PPO enzyme activity.The correlation coefficient is LcPPO2>LcPPO1>LcPPO3.But it is negatively correlated with total phenol content.During the storage period of 25 ? after postharvest,During the storage period of 25 ? after postharvest,the browning of loofah occurs obviously.The total phenol content of loofah increases gradually with the storage time.There is a significant positive correlation between LcPPO1 expression and PPO activity.LcPPO2 expression is positively correlated with PPO enzyme activity.LcPPO2 gene expression is most strongly correlated with enzyme activity.The correlation between LcPPO3 expression and PPO enzyme activity is not obvious.It can be speculated that LcPPO1 and LcPPO2 may play an important role in the browning process of common loofah and promote the occurrence of browning in loofah.(7)To use E.coli prokaryotic expression system,construct browning-related LcPPO2 into pET-21d(+)vector plasmid.and successfully obtain highly efficient expression plasmid pET-21d-LcPPO2.To transfer pET-21d-LcPPO2 into BL21(DE3)competent cells.In the condition of 25?,the target protein begins to express at 65kDa when the final concentration of IPTG(isopropyl thiogalactoside)inducer is 0.2 mmol/L.And the protein expression increases with the concentration of inducer.When the final concentration of IPTG reaches 0.8 mmol/L,the protein expression is the highest.