Lipids And Phenolics Identification During Fruit Quality Formation Of Carya Illinoensis And Cloning And Expression Of Related Genes

Pecan[Carya illinoensis(Wangech.)K.Koch]nuts are rich in lipid and have high antioxidant capacity.In recent years,pecan had been identified as a new source of woody oil plant and had raised a lot of attention.Cultivation area was increased rapidly.The analysis of the dynamic changes of nutrients in the process of fruit quality formation and the clarification of the accumulation of key metabolites are the theoretical foundation of mechanism research and quality breeding.In this study,pecan(cultivar Pawnee)was used as main test material,and dynamic sampling were carried out during the formation period of fruit quality,which is from the middle to the end of fruit development.Quality related key phenotypic characters were measured.Physical,chemical,gas chromatography-mass spectrometry(GC-MS),gas chromatography(GC),liquid chromatography-mass spectro-metry were used to monitor metabolists content.Transcriptomics were also carried on to identify candidate genes.Key genes were cloned and the expression patterns were measured.The major results are as follow:1.Pawnee was used as experiment subject.Morphology changes and nutrients contents were measured.The results showed that during 105?155 d,fruit external and internal morphology are changing rapidly.During 145?155 d after full blossom,fruit weight,nut weight,fruit length,nut length,fruit width,nut shape index,kernel weight,kernel percentage and shell thickness all reached the stable value.The fat contents in the kernel increased at the early stage and reached the stable value.Soluble sugar,protein,fiber,water cotents were gradually decreased.The content of Ca,Mg,Mn,Fe,Cu,Zn and Se decreased rapidly during 95?125 d.Nutrients in pecan changed most obvious during 95?145 d.2.GC method was used to indentify metabolites in lipid extracted from kernels of pecan Pawnee.A total of 14 metabolites were identified,which were 4 fatty acids,including palmitic acid,stearic acid,oleic acid and linoleic acid;4 glyceryl esters,including 2-monopalmitoylglycerol,1-monopalmitoylglycerol,2-monostearoylglycerol and 1-mono stearoylglycerolsterol;2 sterols,including ?-sitosterol and 3.?-gorgost-5-en-3-ol;?-L-galactopyranose,sucrose,squalene and y-tocopherol.Further determined of FA were carried out by GC.A total of 25 FAs were quantified.The most abundant 5 FAs were oleic acid,linoleic acid,palm acid,stearic acid and linonleic acid.The highest oleic acid content appeared at 125 d of 77.80%.There are more types of FAs at the middle stage of fruit development,and decreased along with fruit mature.High performance liquid chromatography(HPLC)was used to determine the tocopherol contents in the oil extracted from 5 varieties of Pawnee,Stuart,Wichita,Jinhua and Shaoxing.The results showed that the content of 3 kinds of tocopherol in each variety was higher in the middle period of fruit development and decreased significantly in the later period.The content of ?-tocopherol is the highest,and the content of ?-tocopherol is the lowest.3.The UPLC-Q/TOF-MS method was used to analyse the phenolic metabolites of Pawnee pecan kernels.A total of 40 phenolic metabolites were identified,in which 1 was identified in the genus Carya for the first time,6 were identified from pecan for the first time,and 1 was identified for the first time from the kernel of pecan.These 8 newly identified phenolic compounds included 4 phenolic acids and 4 ellagic tannins.The dynamic changes of total phenolic content,condensed tannin,total flavonoid content,5 main phenolic acids and antioxidant properties of 5 cultivars of Pawnee,Stuart,Wichita,Jinhua and Shaoxing of ten different developing times were analyzed.The results showed that the content of total phenolic content,condensed tannin,total flavonoid content,5 main phenolic acids and antioxidant activities were all similar to the development of nuts,which were the highest in water or milk stage,the lowest in milk or hard core stage,and a slight increase or gentle in the mature stage of kernel.Total phenolic content,condensed tannin,total flavonoid content and phenolic acid were significantly positive correlated with antioxidant activity,especially catechin,epicatechin and total phenolic content.4.Four key points of kernel development-115 d,135 d,145 d and 165 d of Pawnee were selected for transcriptome analysis,and the results were verified by qRT-PCR.A total of 73,262 unigenes were identified from four libraries.507 unigenes were found involved in lipid metabolism pathway.93 potential candidate genes encoding 29 enzymes were identified in the further screening of de novo synthesis of fatty acid to TAG related unginenes.Through the co-analysis with the results of fatty acids,the main enzyme genes in the formation of high oleic acid proportion in the kernel of pecan were SAD and FAD2,and ACCase and GPAT played a major role in the lipid metabolism of pecan too.A total of 164 unigene related to phenolic metabolism were obtained.25 unigenes encoding 9 enzymes were identified by further screening of de novo synthesis of phenolics to proanthocyanidins related unginenes.Among them,DFR,F3H,CHS,ANS and ANR have higher expression levels,which may play important roles in phenolic metabolism of pecan kernel.5.Using the mixed Pawnee pecan kernel samples of 115 d and 135 d,the gene sequences of CiSAD,CiFAD2 and CiGPAT of pecan lipid metabolism pathway and the gene sequences of CiDFR,CiLAR and CiANR of pecan phenolic metabolism pathway were obtained by RT-PCR amplification,cloning and sequencing.The bioinformatics analysis was carried out too.The CiSAD gene is 1240 bp long,including a 1194 bp complete open reading frame(ORF),encoding 397 amino acids.The CiFAD2 gene is 1329 bp long,containing a complete ORF of 1155 bp,encoding 384 amino acids.The length of the CiGPAT gene is 1671 bp,including a complete ORF of 1626 bp,encoding 541 amino acids.The CiDFR gene is 1148 bp long,containing a complete ORF of 1020 bp,encoding 339 amino acids.The CiLAR gene is 1390 bp long,including a complete ORF of 1050 bp,encoding 349 amino acids.The length of the CiANR gene is 1104 bp,including a complete ORF of 1014 bp,encoding 337 amino acids.All of the cloned proteins have corresponding conserved domains,indicating that the proteins encoded by these genes should have corresponding functions in organism.The qRT-PCR result showed that the expression of CiDFR and CiANR gene in 95?105 d samples were higher and then decreased rapidly to the lower value.The expression of CiLAR gene in 95 d sample was higher,then decreased rapidly,and the expression in 155 d sample increased to the highest point again.There was a strong correlation between the expression of CiDFR and CiANR gene and the content of phenolicss,while CiLAR gene was relatively weak.