Cloning And Expression Of Differentially Expressed Genes That Related To Anaerobic Respiration During Persimmon Fruit Deastringent

Persimmon?Diospyros kaki?can be generally divided into nonastringent and astringent persimmon.The astringent persimmon can only be eaten after deastringency.Artificial high CO₂ treatment is the most commonly used post-harvest deastringency treatment.Anaerobic respiration of the fruit leads to an increase in acetaldehyde content,acetaldehyde and soluble tannin are condensed to form an insoluble polymer,thereby removing the astringency of persimmon fruit.In this study,the previously collected'Jingmianshi'were used as materials,which were treated with artificial high CO₂.In order to obtain new target genes related to the deastringency of persimmon fruit,transcriptomic analysis were applied to identify three gene families related to the acetaldehyde metabolism?pyruvate kinase,PK;pyruvate decarboxylase,PDC;alcohol dehydrogenase,ADH?.The main results are as follows:1.Genes related to acetaldehyde metabolism during the astringency of persimmon fruit were cloned.The results of KEGG analysis based on transcriptome data show that the differentially expressed genes related to anaerobic respiration during the deastringent process of persimmon fruit are enriched in the glycolysis pathway,including 15 structural genes,belonging to the ADH,PDC and PK families,respectively.Using RACE?Rapid amplification of cDNA ends?technology,12 genes were obtained?4 members in each of the three families?,and their nucleotide sequence,amino acid structure and evolutionary relationship were analyzed.DkADH1?JX117841.1?,DkPDC1/2/3?JX117844.1-6.1?and DkPK1/2/6/8?KU130358.1-9.1/KU130363.1/KY292503.1?are reported genes,and the remaining genes are named DkADH4/5,?EVM0023012/EVM0010955.1?,DkADH6 and DkPDC6?EVM0028451?.The resulting ADH and PDC members are highly conserved.Four of the ADH members belong to the mid-chain ADH subfamily,which contains the ADH catalytic domain,Zn²⁺binding site,and the gene domain of the NAD binding protein.Only the types of NAD binding proteins differ;The four PDC members have the characteristics of TPP-dependent oxidase domain.However,the four PK members were clustered into two subfamilies due to sequence differences.The results of cluster analysis showed that DkPK1,DkPK2 and DkPK8 belonged to the PK_c subfamily,while DkPK6 belonged to the PK_p subfamily.2.The expression patterns of differentially expressed genes related to anaerobic respiration during the astringency of persimmon fruit were analyzed.Firstly,real-time quantitative PCR was used to compare and analyze the transcription patterns of 12differentially expressed genes in'Jingmianshi'treated with control and artificial high CO₂ treatment.It was found that DkADH1,DkPDC1/2 and DkPK1/2 not only had higher transcription abundance in their respective families,but also had higher fold induction induced by high-concentration CO₂ treatment.When treated for 1 d,they were enhanced by 14.1,22.6,29.9,3.2 and 3.7 times,respectively.This result is consistent with the reported expression patterns of DkADH1 and DkPDC1/2,and also verifies the possibility of DkPK1/2 as a new target gene related to the astringency of persimmon fruits after harvest.3.Transcription factors regulating DkPK1 were screened.With reference to the'Youshi'genome?http://www.kakiwi.zju.edu.cn/cgi-bin/persimmon/index.cgi?,the sequence of-1225bp^-1bp upstream of the start codon of the DkPK1 gene was cloned and analyzed.After the analysis of its cis-acting elements,it proved that the number of elements recognized by the transcription factor ERF was the largest.The experiment also tried to clone the upstream sequence of the DkPK2 start codon,but no ideal results were obtained.In addition,After the transcriptome analysis of DkPK1 expression correlation,it found that the regression coefficient was greater than 0.9968 and the differential expression was up-regulated by more than 8 times,te number of ERF was the highest.Furthermore,the dual luciferase system was used to analyze the regulatory effects of ERF transcription factors on the DkPK1 promoter.The results showed that DkERF18 and DkERF24 could induce DkPK1 promoter activity,and there was a synergistic effect.At the same time,the BIFC showed that DkERF18 and DkERF24had protein interaction,so it was speculated that the two transcription factors may synergistically regulate DkPK1 by forming a protein complex.In summary,this paper discovered a new target gene?DkPK1/2?involved in de-astringency of persimmon fruit in response to hypoxia treatment,and preliminary analyzed the regulatory effect of DkERF on the DkPK1 promoter.These results expand the target gene range of the persimmon fruit post-harvest de-astringent process.