| Walnut(Juglans regia L.)and jujube(Ziziphus jujuba Mill.)are the two largest tree species in the characteristic forest and fruit industry of Xinjiang.As medicine homologous fruits,walnut kernels and jujube fruits have important edible values as well as nutritional and healthy functions,which are considered as important sources of bioactive compounds.In recent years,the composition,content and health benefits of functional components from walnut oil and jujube polyphenols have been extensively studied.However,there are few reports on the molecular mechanisms involved in oil accumulation of different walnut varieties,and the biosynthesis of jujube proanthocyanidins.Therefore,based on the determination of walnut oil content and its fatty acid composition,as well as the physical and chemical components of jujube,the different walnut and jujube varieties at different developmental stages were used as experimental materials to reveal the potential molecular mechanisms of walnut oil and jujube proanthocyanidin biosynthesis by transcriptome analysis in this study.The key genes in the biosynthetic pathway were further identified.The target genes were further validated by real-time quantitative PCR(qRT-PCR).The main conclusions were as follows:1.Comparative transcriptome analysis of oil accumulation in defferent walnut cultivars at different developmental stages in XinjiangThe oil content and fatty acid composition of 5 walnut cultivars(i.e.,“Zhipi”,“Xinwu 417”,“Xinwen 81”,“Xinwen 179”and“Xincuifeng”)over kernel development were tested.The results showed that the oil content of the five walnut cultivars all showed a“fast followed by slow”change pattern.The oil content of mature walnuts was close to 70%,and the proportion of saturated fatty acids(FAs)reached more than 90%.A total of 6 fatty acids including palmitic acid,stearic acid,oleic acid(OA),linoleic acid(LA),?-linolenic acid(ALA)and eicosenoic acid were detected in walnut oil.Among them,the content of LA and OA were the highest,which showed a simultaneous trend of declining and increasing,respectively.It was speculated that there was a mutual transformation process between LA and OA during walnut development.In addition,according to the change pattern of oil content and fatty acid composition,the three developmental stages,namely,84 days(T1),98 days(T2)and119 days(T3)after flowering were selected for subsequent transcriptome analysis.Transcriptome sequencing was performed on 5 walnut varieties at T1 stage,and 3varieties(i.e.,"Zhipi"(ZP),"Xinwu 417"(W417)and"Xinwen 81"(W81))with relatively large differences were chosen for subsequent transcriptomics analysis through correlation analysis between samples based on gene expression levels.Transcriptome sequencing was performed on 27 samples from 3 developmental stages of 3 walnut varieties.The results showed that 10 key enzymes(ACCase,LACS6,LACS8,SAD,FAD2,FAD3,LPAAT1,DGAT2,PDAT2 and PLC)encoded by 19 genes were highly related to lipid biosynthesis.Among them,SAD,FAD2 and FAD3 were highly transcribed in walnut kernels,which may be responsible for high content of unsaturated FAs.Five transcriptional regulators(TFs)including WRI1,ABI3,FUS3,PKL and VAL1 related to lipid biosynthesis were identified.Further correlation analysis between these genes based on the expression levels in 27 samples were conducetd.The results showed that the main genes regulated by these TFs included ACCase,KAS?,LCS,FAD3 and LPAAT.Nine key genes(ACCase?LACS6?LACS8?SAD?FAD2?FAD3?DGAT2?PDAT2 and PLC)were validated by qRT-PCR in ZP(5 genes were validated in W417and W81),showing a strong correlation between the relative expression by qRT-PCR and the transcriptome sequencing data(R2=0.90889,p=1.62×10-32).2.Physicochemical composition and antioxidant capacity of eight jujube cultivars at five developmental stagesThe 8 physical and chemical indexes including moisture,titratable acid(TA),ascorbic acid,sugars,total phenol content(TPC),total flavonoid content(TFC)and antioxidant activity of 8 jujube cultivars(i.e.,Dabailing,Junzao,Junyou,Spinosa,Huizao,Zanhuang,Fushuai and Fucuimi)at five typical developmental periods(S1-S5)were determined.During the ripening of jujube fruit,moisture,ascorbic acid,TPC,TFC,major phenolic compounds(catechin,epicatechin,proanthocyanidin and rutin)and antioxidant activity(DPPH and ABTS)of eight cultivars decreased,while sugars,especially sucrose content increased.TA,fructose and glucose content experienced straight increases from S1 to S3,and then decreased until to final stage(S5).At the early stages,there are obvious differences in ascorbic acid sugars,phenolic compounds and antioxidant among all jujube cultivars,but the gap among jujube cultivars could be gradually reduced with fruit ripening.This suggested that development stage rather than cultivar is the major factor affecting phytochemicals and antioxidant activity accumulation in jujube fruit.In conclusion,un-coloring jujube fruit is rich in ascorbic acid,phenolic compounds and antioxidant activity,which may be used as potential functional food,while full red jujube fruit is rich in sugars,and contains less phenolic compounds,correspondingly resulting in the sweet and pleasant flavor of jujube fruit.Therefore,the suitable harvested time of jujube fruit should be made according to the intended commercial purpose.3.Transcriptome analysis of proanthocyanidin metabolism during jujube fruit developmentTranscriptome sequencing of 8 jujube cultivars at S1 period was conducted.Three varities(i.e.,Dabailing(DB),Junzao(JJ)and Fushuai(FS))with relatively big difference evaluated by principal component analysis were selected for subsequent transcriptome analysis.The catechin,epicatechin and proanthocyanidin profiling of these three varieties during the maturity period were analyzed.The results showed that the catechin and proanthocyanidin content of the three varieties showed a downward trend in general,but the epicatechin content increased first,and subsequently decreased.According to the change pattern of these three indicators,three developmental stages of S1(T1),S3(T2)and S4(T3)were selected for subsequent transcriptome analysis.Transcriptome sequencing was performed on 27 samples from 3 developmental stages of 3 jujube varieties.The results showed that 17 differentially expressed genes encoding PAL,CHS,CHI,CHS,F3'H,LAR,ANR,C4H,4CL,FLS,DFR and UFGT were the key genes involved in the flavonoid biosynthetic pathway.The high expression of LAR in jujube may contribute much to the increase in epicatechin content.Weighted co-expression gene network analysis(WGCNA)was performed on 27jujube samples.The results showed that the MEblue module containing 1620 genes was significantly correlated with the content of proanthocyanidins(r=0.90,p=1×10-10)and the content of catechins(r=0.87,p=3×10-9).In this module,58 key genes including 49 transcription factors and 9 structural genes involved in the metabolic pathway of proanthocyanidins were identified.Further correlation analysis was conducted to explore the interactions between these geness.The results showed that 16 genes encoding 9 transcription factors(MYB,bHLH,ERF,bZIP,NAC,SBP,MIKC,HB and WRKY)have extremely high connectivity,which are considered as hub genes in the regulatory metabolism netwotk of proanthocyanidins.Ten key genes(C4H?4CL?F3H?F3'H?LAR?GST?ERF?bHLH?MYB and WRKY in DB were validated by qRT-PCR(5 genes were validated in FS and JJ).The regression analysis results showed there was good linear relationship(R2=0.93286,p=6.31×10-36)between qRT-PCR data and transcriptome sequencing data.In this study,based on the transcriptome analysis of walnut and jujube from Xinjiang,we identified the key genes in oil accumulation and biosynthesis unsaturated FAs,and the molecular mechanism of proanthocyanidin accumulation and regulation in jujube was revealed.These findings will promote the research of Xinjiang characteristic fruit and the development of the local fruit industry,and provide valuable theoretical knowledge for the genetic research and molecular breeding of walnuts and jujubes. |
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