Functional Characterization Of ALDH2 Genes Involved In Natural Deastringency In Chinese PCNA Persimmon

Chinese pollination-constant and non-astringent persimmon(C-PCNA)has important application value in genetic improvement of PCNA for its traits of natural deastringency controlled by single dominant gene,but the key genes and its regulatory networks is not fully clear.The process of C-PCNA naturally deastringency may associated with the coagulation of soluble tannins which mediated by acetaldehyde,but the function of ALDH2 genes related to metabolism of acetaldehyde is not clear.In this work,three types of persimmon cultivars: ‘Eshi 1’ and ‘Luotian Tianshi’(C-PCNA type),‘Youhou’(J-PCNA type)and ‘Mopanshi’(non-PCNA type)were sampled.Firstly,two members of ALDH2 family genes,DkALDH2 a and DkALDH2 b,were isolated from ‘Eshi 1’ persimmon fruit.Gene expression patterns and transient expression in ‘Eshi 1’ leaves were used to analyze the function of ALDH2 genes.Secondly,yeast one-hybrid(Y1H)screen was performed in search of the transcription factors binding the DkALDH2 b for figuring out the regulatory mechanism of astringency removal in PCNA cultivars.Lastly,transcriptome sequencing analysis was executed to look for key genes in natural deastringency process after transient over-expression ALDH2,LAC1 and ANR in ‘Eshi 1’ leaves.The study will be helpful for providing more scientific evidence for regulation of deastringency mechanism in C-PCNA and creation of PCNA new germplasm.The main results are as following: 1.Full-length cDNAs of DkALDH2 a and DkALDH2 b genes isolated from C-PCNA persimmon fruit were obtained by RACE and RNA-Seq data.They were 1,984 bp and 1,933 bp in length,and encoded 542 and 540 amino acids,respectively.The contents of both soluble and insoluble tannins were markedly increased with the transient over-expression of the two ALDH2 genes.Soluble tannin content showed no remarkable changes with the silencing of DkALDH2 a and a marked decline with the silencing of DkALDH2 b.The two ALDH2 genes can led to reduce consumption of soluble tannin and inhibit the astringency removal process.2.DkALDH2 a and DkALDH2 b gene expression was negatively correlated with natural deastringency in C-PCNA.The former mainly acted on seeds,its expression level was gradually decline in fruit development,resulting a large amount of acetaldehyde generated from seed to pulp,and then promoted the coagulation effect leading to deastingency.The later was expressed abundantly in pulp,which might due to the accumulation of acetaldehyde,but the consumption was not sufficient to restrain deastringency of C-PCNA.3.Based on cDNA full length,ALDH2 a and ALDH2 b promoter sequence,515 bp and 1,209 bp,respectively,were obtained through genome walking.ALDH2 b promoter was used to construct pALDH2b-AbAi prey vector,which was transformed into Y1 HGold strain to construct Y1HGold[pALDH2b-AbAi] strain.16 monoclonal were isolated from yeast library based on the Yeast one hybrid screening,1 upstream regulate factor BBR/BPC1 were finally identified.4.The specific expression genes of ALDH2,LAC1 and ANR in the leaves of C-PCNA were obtained by the combination of RNA-seq and transient transformation system.A total of 119.63 Gb Clean Data were obtained.Through Trinity software de novo assembly,192,597 transcripts and 100,371 Unigene were obtained.56,009 Unigene(about 55.8%)got functional annotation information.Transcription factor of ERF family genes has the highest number,and it was up-expressed in three treatments.It may play an important role in natural deastringency process of C-PCNA.In conclusion,we identified the relationship between the two members of ALDH2 gene family,DkALDH2 a and DkALDH2 b,and the natural deastringency of C-PCNA.Then we found the two types of transcription factors,BBR/BPC1 and ERF,may regulate the process of its natural deastringency.Our research provide a reference for the analysis of natural deastringency mechanism in C-PCNA and its genetic improvement.