Study On The Role Of Litchi Pericarp Epigallocatechin Synthase(ANR) In The Accumulation Of Peel Browning Substrate

Litchi(Litchi chinensis Sonn.)is one of the most competitive fruits of China in the international fruit market.The skin of litchi fruit is easy to brown,resulting in a shorter shelf life,which is the main factor that reduces the commercial value of litchi fruit.Although a lot of researches have been done on the browning mechanism of litchi pericarp at home and abroad,no satisfactory explanation has been provided.In this study,an anthocyanidin reductase(ANR),a key gene in the epicatechin synthesis pathway,was cloned from litchi pericarp using molecular biology methods,the function of litchi epicatechin synthesis gene(LcANR1)in the accumulation of pericarp browning substrates was carried out by gene complementation of Arabidopsis ANR deletion mutant(ban);The activity of prokaryotic expression of LcANR1 fusion protein was analyzed;The expression of ANR gene in litchi pericarp was analyzed,and the content of procyanidin monomers and oligomers was determined.The results of the study supply information for understanding the role of ANR in the accumulation of peel browning substrate.The main research results obtained are as follows:1.Gene cloning and bioinformatics analysis of litchi LcANRs.In this study,five LcANR genes were first screened from the litchi genome database,ANRs were cloned from the peel using RT-PCR for sequencing and bioinformatics analysis.Phylogenetic analysis showed that the genetic relationship between ANR1 and ANR2 genes were of high identity.The three-dimensional structures of ANR1,ANR2,and ANR3 were similar,with NADH-binding domains and three unique catalytic sites.Sequencing results revealed that,except for the ANR1 and ANR2 transcripts other 3 ANR transcripts had early termination of protein translation.It is speculated that there may be only two functional ANR genes(ANR1 and ANR2)in the litchi peel,and the two members are extremely similar.Sequence analysis showed that the open reading frame of the ANR1 gene was 1011 bp long,encoding 336 amino acids,and the predicted molecular weight of the protein was about36.4 k D.2.Arabidopsis genetic transformation system to verify the LcANR1 function of litchi.LcANR1 gene was used to complement Arabidopsis ban mutant plants by genetic means,and then transgenic Arabidopsis seeds were stained by DMACA staining method.The results showed that the 35 SLcANR1/ban seeds recovered the same phenotype as wild-type Arabidopsis seeds,while the mutants supplemented with the GFP control gene had the same phenotype as the mutant ban,indicating that the LcANR1 gene complemented the function of ban and restored the epicatechin biosynthesis of Arabidopsis mutant ban.3.Enzyme activity analysis of prokaryotic expression of litchi LcANR1.The LcANR1-6XHis was expressed in purified prokaryotic cells.The fusion protein was detected by SDS-PAGE electrophoresis and mass spectrometry,indicating that the target protein was successfully expressed and purified.Using Cyanidin(CYA)as a substrate and NADPH as a coenzyme,the activity of the LcANR1 fusion protein was detected in vitro.Spectrophotometric detection revealed that the CYA content of the LcANR1 active enzyme was significantly reduced compared to the reaction of the LcANR1 inactivating enzyme.High performance liquid chromatography(HPLC)was used to detect the reaction products.LcANR1 enzyme produced(Epicatechin,EC)in the reaction,suggesting that the enzyme encoded by LcANR1 can catalyze the production of EC from CYA in the presence of coenzyme NADPH.4.Localization of subcellular litchi LcANR1.ORF of LcANR1 gene fused with GFP was inserted into the transient expression vector(p YL322-N-GFP)and transformed into the protoplasts of Arabidopsis seedlings using the method of subcellular localization of transient expression plants.The non-colocalization of green florescence and chlorophyll florescence was observed under a confocal microscope,indicating that LcANR1 may be localized in the cytoplasm.5.Proanthocyanidin detection in Huaizhi pericarpExtract the extractable polyphenols(EPP)from the peels of Huaizhi(L.Chinensis.cv.Huaizhi)after flowering and postharvest browning at six stages.The epicatechin in the skin was detected by HPLC during fruit development.Epicatechin(EC)and cyanidin-3-O-rutinoside(Cy3R)and other proanthocyanidin monomers,oligomers and anthocyanins content changes,found that the content of these procyanidins with in the process of fruit development,the content decreased,and the content was the highest in the30 days of young fruit development.During the postharvest browning process of Huaizhi,the content of the peel of these proanthocyanidins also decreased with the deepening of the browning of the peel,which was significantly negatively correlated with the browning of the peel.6.Expression analysis of LcANR1 gene in different development stages of Huaizhi peel.The expression of LcANR1 gene in different developmental stages of Huaizhi was detected by real-time quantitative PCR technology.The expression of LcANR1 was highest in litchi 30 days after flowering,and it gradually decreased with the developmental period.The results showed that the expression level of LcANR1 gene was consistent with the accumulation pattern of EC in litchi peel.It showed a downward trend with the development and maturity of the fruit.The expression level and EC content of LcANR1 gene reached the highest levels after 30 days of litchi flowering.At maturity(full red stage),its expression level and content are very low,indicating that the expression of LcANR1 gene is related to epicatechin accumulation.