The Molecular Basis Of Important Quality Formation In Walnut (Juglans Regia L.) Nut

Walnut(Juglans regia)is an important woody oil tree species in China.The kernel contains 50-69%of oil,in which linoleic acid and linolenic acid account for more than 71%,and the seed coat is rich in secondary metabolites such as polyphenols.Walnut kernels are known as the super health food in the 21st century.At present,the genetic regulation mechanism of kernel nutrition has not been clarified,and it is necessary to improve the quality of walnut at the molecular level.This research focuses on the important components in the development of walnut nuts.Through the analysis of transcriptome and metabolome data during the development of embryo and seed coat,it explored the key genes involved in the accumulation of important components,and explored the formation and regulation mechanisms of important quality components such as fatty acids and polyphenols.In order to establish a gene regulatory network related to nut quality,screen key regulatory factors,and provide support for efficient walnut breeding and cultivation.The main research results are as follows:(1)Based on the observation of walnut fruit development,2 endosperm,8 embryos,5 seed coat and 5 shell samples were sequenced.A total of 60 transcriptome libraries were constructed,and the sequencing data of each library was no less than 6 GB,and a total of 458 GB transcriptome data were obtained.The analysis of gene expression profile showed that the transcriptome data of nut tissue had both tissue specificity and development specificity,and the early and late development samples were far apart from each other.The shell and seed coat samples were similar,but different from endosperm and embryo.We described how to collect samples,extract m RNAs,build c DNA libraries,and control the quality of transcriptome data so that it can be used for subsequent analysis.(2)The oil content of the mature kernel is 69%,of which 71%is polyunsaturated fatty acid,64%is linoleic acid,7%is linolenic acid.The embryo transcriptome data showed that 39 384unigene were obtained from 24 c DNA libraries.Cluster analysis of PCA and differential genes showed that the total samples could be divided into three groups:49 DAP,63-119 DAP and133-147 DAP.A total of 108 unigenes associated with lipid synthesis were identified,of which60 were associated with fatty acid synthesis,33 were associated with triglyceride assembly,7were associated with oil body synthesis,and 8 were associated with transcription factors.The genes encoding WRI1,ACCASE,ACP,KASII,SAD,FAD2,FAD3 and PDAT were upregulated in 63-119 DAP,which was also the period of rapid lipid accumulation and the oil content increased from 15%to 69%.WRI1 gene was co-expressed with ACP,ENO,VAMP727and IDD14 by weighted gene co-expression network analysis.RNA-seq was highly correlated with q PCR data(R~2=0.80,P<0.01),indicating that the expression level of RNA-seq was reliable.In addition,SAD,FAD2 and FAD3 were highly expressed in walnut kernel,and the FPKM value could reach 834,2205 and 9038,respectively.The high expression of FAD2 and FAD3 may be the reason why walnut is rich in polyunsaturated fatty acids.The subcellular localization of Jr FAD3 protein was confirmed to be located in the endoplasmic reticulum rather than in the plastid,suggesting that the synthesis of linolenic acid is mainly in the endoplasmic reticulum.The Jr FAD3 gene was successfully cloned and expressed in tobacco.(3)The content of polyphenols in the seed coat increased from 5.68 mg/g(35 DAP)to9.78 mg/g(147 DAP)with development,which was much higher than that in the embryo(2.22mg/g).A total of 760 metabolites were detected in the metabolome,and the polyphenols in the seed coat were higher than those in the embryo.The contents of flavonoids and proanthocyanidins in the seed coat were the highest at mature stage.A total of 15proanthocyanidins,10 kaempferol glycosides and 21 quercetin glycosides were identified,and they accumulated continuously with the development of seed coat.The analysis of phenylpropane metabolic pathway showed that with the development of seed coat,phenylalanine was gradually transformed into secondary metabolites such as proanthocyanidins.Through transcriptome analysis of seed coat,a total of 49 unigene related to the synthesis of polyphenols were identified,among which PAL,C4H,4CL,CHS,CHI,F3H,LDOX,and ANR showed similar expression patterns,and the highest expression levels were at mature stage.The results of transcriptome and metabolome analysis were consistent.The mature stage was the key period for the synthesis of polyphenols in the seed coat.Moreover,the transcription factor MYB111 plays an important role in the synthesis of polyphenols,and its expression pattern is positively correlated with the accumulation pattern of quercetin,kaempferol and proanthocyanidins.MYB111 is co-expressed with NAP,NAC,ATR1 and other genes involved in cell aging and abiotic stress response.Based on all the experimental results and analysis,a high-quality walnut tissue development transcriptome database was established in this study,which will provide a basis for future research on the molecular mechanism.The high expression of FAD2 and FAD3genes is an important reason for the high content of polyunsaturated fatty acids in walnut kernels.Combined transcriptome and metabolome analysis revealed that the phenolic species and quantity in the seed coat were higher than those in embryo,and the polyphenol biosynthesis related genes were highly expressed in the late development of the seed coat.These results reflect the molecular mechanism of walnut oil and polyphenols formation.These results provide important genetic resources for molecular breeding of walnut,and also provide new perspectives and insights for the study of nut development.