| The various organs of walnut（Juglans L.）are rich in phenolic substances with strong biological activity,which can remove excess free radicals in the human body and prevent the oxidation of biological macromolecules such as DNA,protein and lipids.As an important signal molecule and allelochemicals in the process,it can resist the adverse effects of ultraviolet rays,low temperature,drought and insect foraging,or interact with other plants,and has an important regulatory effect on the physiological metabolism of plants.Studies of phenolic metabolism have focused mainly on the physiological level,while few studies the molecular level have been conducted.In this study,the fruit of walnut（J.sigillata Dode cv?Qianhe-7‘）was used as a test material to study the changes of phenolic substances in the development process of green husks and kernels,and the fruit was sequenced by SMRT.At the same time,30 D,90 D,140 D green husks and 90 D,120 D,140 D kernels expression profiles were constructed.and analysis of genes involved in the regulation of phenol metabolism during the development of walnut fruit The relationship between differentially expressed genes in phenolic metabolism and changes of phenolic substances in different stages of green husks and kernels.It provides a theoretical basis for the discovery,regulation mechanism and breeding of new walnut varieties of functional genes related to phenol metabolism in walnut and other plants.The main results are as follows:1.The full-length transcriptome sequencing of the fruit of‘Qianhe 7‘was carried out,and finally 75232 do away redundant full-length transcripts were obtained with an average length of 2890.52 bp;the length of N50 was4178 bp.The reference gene sequences and a great quantity of genes which is peculiar to walnut fruit growth and development and transcripts with ORF have been detected.Functional annotation of Isoform obtained by full-length transcriptome sequencing,a total of 74,053 transcripts were annotated,accounting for 98.43%of all Isoforms;a total of 75,101 transcripts were annotated into the GO database,and 19948 transcripts were annotated to 133 KEGG pathway,54 genes are involved in the regulation of phenolic metabolism in walnut fruit.2.Trend analysis and functional annotation of differentially expressed genes showed that the number and expression trends of phenol metabolism-related differential genes in the development of green husks and kernels during the development of‘Qianhe 7‘were different.There are 19 differential genes involved in the regulation of quercetin metabolism（BGLU24,SHT,PER43,FLS,PAL,CYP84A1,4CL1,BACOVA02659,PER64,F3′5′H、COMT1,CAD9,PER17,F3H,BAN,PER73,CCOAOMT,DFR and ANS）,there are 7 differential genes involved in the regulation of phenolic metabolism（PAL,PER17,CAD9,COMT1,BGLU40,GSVIVT00023967001 and TAT）.The q RT-PCR validation of 23 differentially expressed genes related to phenol metabolism showed that the23 genes were consistent with the trend analysis results,indicating that the transcriptome sequencing results were reliable.WGCNA analysis established that PAL,4CL and FLS are the hub genes for the regulation of fruit phenol metabolism.3.The content of total phenolics and total flavonoids in the green husks of walnut was generally decreased,and the variation in kernels was opposite,and The content of catechins in green husks and kernels was the highest,and the content of catechin and epicatechin was increasing,the content of other monomeric phenols was higher at30 D.In addition to coumaric acid and ferulic acid,the content of other monomeric phenols was higher at 140 d;the content of rutin and chlorogenic acid was higher in the seed kernel,but the walnut was only found in the green skin;during the development of the green skin,differential expression The activities of PAL,4CL,FLS and F3H regulated by gene were positively and negatively correlated with the content of total phenols,total flavonoids and most of the monomeric phenols,while the F3′5′H activity with walnut quinone and other flavonoids except arbutus pigment free phenol content of positive and negative correlation is higher.4.Differentially expressed genes have a regulatory effect on the content of phenolic substances.The expression of BGLU24,SHT,PER43,FLS,PAL,CYP84A1,4CL1,BACOVA02659 and PER64 in the green skin is beneficial to the accumulation of most phenolic substances,when the accumulation of catechins,epicatechin and myricetin is reversed;the expression of F3′5′H,COMT1,CAD9,PER17,BAN,PER73 and CCOAOMT is negative in phenolic substances except chlorogenic acid,syringaldehyde and myricetin correlation;the expression of PER73and CCOAOMT promoted the synthesis of catechin and epicatechin,but had a high negative correlation with rutin content.The expression of CAD9 and COMT1 in the kernels reduced the accumulation of phenolic substances;the expression of BGLU40,GSVIVT00023967001 and TAT was beneficial to the accumulation of other monomeric phenols,total phenols and total flavonoids except vanillic acid,ferulic acid and quercetin.Although the expression of PER17 attenuated the accumulation of total phenols,total flavonoids,gallic acid,epicatechin,rutin,myricetin and quercetin,it was beneficial to the synthesis of other monomeric phenols.5.The functions and pathways of phenol metabolism related genes in walnut fruit indicate that the phenolic metabolism of fruit begins in the shikima te pathway and proceeds to the synthesis of flavonoids and lignin substances via the phenylpropane pathway.In addition,Phenolic acid and hydrolyzed tannins can be directly synthesized by shikimic acid pathway the results confirmed that the t yrosine metabolism pathway in walnut fruit can synthesize phenolic acids and regulate the metabolism of flavonoids and lignin. |
