Studies On In Vitro Conservation And Relative Molecular Mechanism In Chinese Olive (Canarium Album)

Chinese olive （Canarium album） is one of the famous specific woody fruit trees in southChina. Different Canarium album cultivars were used as the materials in the experiment. First, theanther callus induction and immature embryo culture, mature embryo culture and the furtherresearch of in vitro conservation of Canarium album plantlets were conducted; then, the cloning ofPPO and Cab genes from the leaves of the cultivar "Huiyuan" in vitro plantlets was carried out;next, the PPO and Cab gene transcription expressions were analysed in the process of in vitroconservation of Canarium album plantlets by real time fluorescence quantitative PCR; finally, thePPO acitivities of the cultivar "Huiyuan" plantlets in the process of in vitro conservation anddifferent cultivars were measured. The main results were as follows:1In vitro culture of Canarium album①Induction of anther callus in Canarium albumAnthers of Canarium album "Huiyuan" were used as materials. Different disinfectionmethods were conducted to research the effects of the buds and the effects of different media onanther callus induction rate were conducted. The results indicated that the best disinfection way fotanther was to use0.1%HgCl2disinfection for8min. The MS medium supplemented with30g·L^-1sucrose,2.0mg·L^-12,4-D,0.5mg·L^-1KT,5.0mg·L^-1AgNO3was suitable for callus inductionof anthers in Canarium album.②Immature embryo culture and obtaining of plantlets in Canarium albumThe immature embryo germination and plantlet proliferation of three cultivars wereconducted respectively in Canarium album.The results showed that the cultivars and discrepanciesin size resulted in the differences of immature embryonic germination rates; however, differentlight intensities produced little influence on immature embryonic germination rates. Differentsampling time had little influence on immature embryonic germination rates. The1/2MS mediumsupplemented with30g·L^-1sucrose had the highest germination rate, or the MS mediumsupplemented with30g·L^-1sucrose,1.0mg·L^-16-BA,0.5mg·L^-1KT,0.5mg·L^-1IBA, was suitablefor plantlet proliferation of three different Canarium album cultivars and lateral bud germinationrate was the highest. ③Mature embryo culture and obtaining of plantlets in Canarium albumCanarium album "Huiyuan" was used as materials to evaluate the bud induction effects ofdifferent illumination time, concentration of sucrose and orthogonal design （four factors and threelevels） different factors （6-BA, IBA, KT, sucrose）. The results showed that, the effective lateralbud number was the highest under illumination time for12h/d; the medium wtih sucroseconcentration30g·L^-1or40g·L^-1, the number of effective lateral bud from plantlets was also high.In multiple factor test, the MS medium supplemented with30g·L^-1sucrose,0.5mg·L^-16-BA,0.5mg·L^-1KT,0.5mg·L^-1IBA was suitable for inducing lateral buds of Canarium album cultivar"Huiyuan", and effective lateral maximum number was the highest.2In vitro conservation of Canarium album plantletsThe experiment was first to study the in vitro germplasm preservation methods in Canariumalbum, and the effects of different media on in vitro conservation were compared. The resultsindicated that the MS medium supplemented with30g·L^-1sucrose was suitable for in vitroconservation of Canarium album plantlets, and the seedling survival rate reached88.35%withoutsubculture for12months.56accessions of Canarium album germplasm resources werepreserved in vitro using the culture medium, and the growth situation of Canarium albumresources that had been kept for a year and a half was observed and researched.3PPO gene cloning and expression analysis in the process of in vitro conservation ofCanarium album plantlets by real time fluorescence quantitative PCRThe leaves of Canarium album in vitro plantlets were firstly used as the materials for RNAextraction for further cloning PPO gene. PPO1gene fragments of376bp were obtained via3’RACE and homologous cloning, the sequence of PPO1gene was highly homologous with thatother plants reported in GenBank, and reached95%. It was registered in GenBank, and accessionnumbers was JF811036. The full-length cDNA sequence of PPO2gene was obtained1934bp. Itwas registered in GenBank, and accession numbers was JQ319005. The PPO2gene contained13bp5’UTR,100bp3’UTR, and3’-end involved24bp poly（A） tails. The sequence of PPO2genewas highly homologous with that other plants reported in GenBank. The cDNA contained the1818bp open reading frame, encoded606amino acids, with ATG as start codon and TAA as stopcodon.The predict of protein’s physical and chemical properties showed that, the molecular formulawas C3079H4741N835O897S20; the molecular weight of protein PPO2was68448.7Da; the isoelectricpoint was8.47; So, PPO2was a kind of the alkaline protein; The protein is a hydrophilic protein, which didn’t contain signal peptide and probably not the secreted proteins. Comparison ofhomologous protein family, the discovery of the PPO2gene open reading frame of606aminoacid residues encoded proteins belonging to the Tyrosinase super family member. PPO2mightform from the outside to the inside of the transmembrane helices structure with the N-terminalinside of the membrane. The maximum credibility that its sub-cellular localization was inmitochondrial matrix space was0.681.and the phosphorylation sites were30; PPO2protein in theamino acid sequence of492-519region might form coiled-coil.Secondary structure predictionshowed that the PPO2protein was with15.02%α-helices,14.85%extended strand and70.13%irregular curl. The main structural elements of Canarium album in vitro plantlets PPO2proteinwere irregular curl, which were spreaded into the whole protein.Three-dimensional structureprediction showed that Canarium album in vitro plantlets PPO2protein was surrounded withα-helice and irregular curl. These studies laid the important foundation for the study of PPO2molecular mechanism Canarium album in vitro plantlets conservation. The genomic PPO2genewas also cloned from Canarium album genome, and the result showed that it was no intron.The PPO2gene expression was analysed in mRNA transcription during the process of invitro conservation of Canarium album plantlets. The results showed that, preservation for1month,the plantlets with more young leaves, and the PPO2gene expression was at the higher level; for2³months, as the leaves matured, the PPO2gene expression level dropped; for4months, as thelateral buds germinated, PPO2gene expression rapidly rose; the expression declined sharply for5months; for6months, lateral bud grew well and the young leaves increased, the level of PPO2gene expression rose sharply again; for7months it was down slightly; for8months, the plantletsaged, the PPO2gene expression increased. PPO2gene was closely related to the process of invitro conservation of Canarium album plantlets. The PPO2gene likely played an important role inthe process of plantlet growth and development in Canarium album.4Cab gene cloning and expression analysis in the process of in vitro conservation ofCanarium album plantlets by real time fluorescence quantitative PCRThe leaves of Canarium album in vitro plantlets were firstly used as the materials for RNAextraction for further cloning Cab gene. Cab gene fragments of676bp were obtained via3’RACEand homologous cloning, contained135bp3’UTR, and3’-end involved11bppoly （A） tails. Thesequence of Cab gene was highly homologous with that other plants reported in GenBank.andreached90%, then a pair of primers were designed to clone the ORF of Cab gene gained a792bpopen reading frame, encoded264amino acids.The full-length cDNA sequence of Cab gene wasobtained946bp. It was registered in GenBank, and accession numbers was JN661689.The predict of protein’s physical and chemical properties showed that, the molecular formula was C1282H1949N329O364S11; molecular weight of protein Cab was28147.2Da; the theoretical PIwas5.29; So,Cab was a kind of the acidic protein; The protein is a hydrophilic protein, whichdidn’t contain signal peptide and probably not the secreted proteins.Comparison of homologousprotein family, the discovery of the Cab gene open reading frame of264amino acid residuesencoded proteins belonging to the Chloro a_b-bind super family member. Cab might form fromthe inside to the outside of the transmembrane helices structure with the N-terminal inside of themembrane. The the maximum credibility that its sub-cellular localization was in microbody（peroxisome） was0.640and the phosphorylation sites were10; Cab protein might not formcoiled-coil. Secondary structure prediction showed that theCab protein was with29.17%α-helices,10.23%extended strand and60.61%irregular curl.Three-dimensional structure prediction showedthat Canarium album in vitro plantlets Cab protein was surrounded with α-helices and irregularcurl. These studies laid the important foundation for the study of Cab molecular mechanismCanarium album in vitro plantlets conservation. The genomic Cab gene was also cloned fromCanarium album genome, and the result showed that it was no intron.Cab gene expression was analysed in mRNA transcription during the process of in vitroconservation of Canarium album plantlets. The results showed that, The results showed that,preservation for1month, the plantlets with more young leaves, and the Cab gene expression wasat the higher level; for2³months, as the leaves matured, the Cab gene expression level dropped;for4months, as the lateral buds germinated, Cab gene expression slightly rose; the expressiondeclined sharply for5months; for6months, lateral bud grew well and the young leaves increased,the level of Cab gene expression rose slightly; for7months it was down slightly; for8months,the plantlets aged, the Cab gene expression increased sharply.5The activity changes of PPO during the process of in vitro conservation of Canarium albumplantletsThe activity changes of PPO during the process of in vitro conservation of Canarium albumplantlets were measured to analyse the PPO gene expression at the protein level analysis. Theresults showed that the PPO activities of Canarium album plantlets changed slightly afterconservation for1⁵months; after conservation for6⁸months, the PPO activities increasedsharply. The PPO activities of different cultivars were compared after in vitro conservation for8months and the resulted showed that the PPO activities diferred among different cultivars, and thePPO activities of Changying C （184）, Canarium album284, Mei Pu （M141） and HuangaChangying （M167） were significantly higher than those of the other8cultivars.In summary, in this experiment, the in vitro culture and conservation were comprehensivelyand systematically studied in Canarium album; at the same time, the relationship between the PPO, Cab genes and the in vitro conservation was also analysed. Firstly, the leaves of Canarium albumin vitro plantlets were used as the materials for RNA extraction and further cloning of PPO andCab genes were conducted, which filled the blank about gene cloning of PPO and Cab inCanarium album; secondly, the PPO and Cab gene expressions were analysed in the process of invitro conservation of Canarium album plantlets by real time fluorescence quantitative PCR;finally, the enzyme activity changes of PPO were used as the index of the Canarium album PPOprotein expression, which laid a foundation for discussing the molecular mechanism of in vitroconservation in Canarium album.