Measurement Of Hyperoside Content,Cloning And Expression Of The Synthesis Genes In Canarium Album

Chinese Olive(Canarium album(Lour.)Raeusch)is a health fruit which can used both edible and medical.It has various effects,however the bitter mouthfeel is an important limiting factor of its quality.Previous studies speculated that hyperoside may be the main cause of its bitter mouthfeel.So in this experiment,two olive strain('Qinglanyihao' and'Meipuerhao')and two sweet olive strain('Changying' and 'Tanxiang')was used as the materials to study the content of hyperoside and the expression patterns of it's related genes in different period of fruit.It can lay a foundation to cultivate less bitter olive in the future.Main results of the study are as follows:1.In this experiment,eight mature stage of 'Qinglanyihao'?'Meipuerhao'?'Changying' and 'Tanxiang' were used as the materials to study the contain of hyperoside in which the HPLC technics was used.Results shows that:(1)The tendency of the hyperoside contain can be separated into olive group and sweet olive group and they have a similar trend in each group;(2)The contain of hyperoside in 'Changying'and 'Tanxiang' are higher than other two species;(3)The tendency of the the hyperoside contain are similar in early and late mature stage(50-70 d and 150-190 d after flowering)in four species.2.The 'Qinglanyihao' olive was used as the materials to clone four common reference genes:ACTB7,TUB5,GAPDH3 and G6PDH2.In order to study the expression level of them,four different olive varieties,eight mature stage of fruit were used.The three computational procedure:geNorm,Bestkeeper and NormFinder were applied to analyze the stability of expression level of the four reference genes.The results showed that ACTB7 was the single gene with the highest expression in the comprehensive comparison of the variety and period,while the combination was ACTB7/TUB5.3.The homology cloning was used to obtain the cDNA of F3'H,F3H and FLS which take part in the metabolic pathway of hyperoside.The full-length of three genes are 1 649 bp?1 280 bp and 1191 bp.The open reading frame of them are 1 554 bp?1 095 bp and 1011 bp.Among them F3'H encodes 517 amino acids in which the P450 domain and cysteine heme binding region were contained;F3H encoded 364 amino acids,in the encoded amino acid sequence 20G-FeII conserved domain was contained and it belongs to the PcbC family.FLS encodes 336 amino acids and contains 20G-FeII-Oxy conserved domains in the encoded amino acid sequence,belonging to the PcbC family.Comparing the cDNA with its gDNA,we found that there are two introns in all three genome DNA.4.In this experiment,the flesh of eight mature stage of'Qinglanyihao'?'Meipuerhao'?'Changying' and 'Tanxiang' was used as the materials to study the expression level of F3'H,F3H and FLS in which Real-time PCR was used.Results shows that:(1)The expression level of F3 'H and F3H are similar in same species,but different with FLS;(2)The expression of F3 'H and F3H are declining with the fruit matures in 'Qinglanyihao'?'Meipuerhao' and 'Tanxiang' strains,however they are alternating up and down in 'Changying';(3)The expression of FLS are declining with the fruit matures in 'Tanxiang' and 'Changying' strains,but they have upward trendency in late mature stage in 'Qinglanyihao'?'Meipuerhao' strains.5.By comparing the content of hyperoside with the expression levels of three biosynthesis-related genes,we speculate that FLS had a greater effect on hyperoside synthesis than F3'H and F3H.