Study On Cryopreservation Of Siraitia Grosvenorii Shoot Tips By Encapsulation Dehydration And Its Proteome

Encapsulation dehydration is based on artificial seed technology which uses calcium alginate to wrap the sample to protected sample from chemical agents and dehydration.This method is good at its convenient experiment process,low using cost,and plant have high survival pencentage.Siraitia grosvenorii is the typical medicinal and food plant in our country,there is rare reports of its germplasm cryopreservation now.This paper studies on cryopreservation of four varieties of Siraitia grosvenorii shoot tips(longji,dongguahan,qingguan,bolin3hao.)by encapsulation dehydration using one factor experiment and orthogonal experiment.And extracted proteins of Siraitia grosvenorii shoot-tips during encapsulation dehydration cryopreservation and play protein two-dimensional electrophoresis,then the proteomes were analyzed,some different proteins were identified by mass spectrometry in order to explore the effects of encapsulation dehydration on their gene expression regulation which could supply the molecular and theoretical basis for S.grosvenorii germplasm cryopreservation.The main results are as follows:single experiment:1.Effects of different preculture times on survival rates were compared.Results showed that the suitable preculture time was 2 days,survival rate of Dongguahan shoot tips was 55%.2.Using different sucrose concentrations of preculture medium on cryopreservation,the suitable sucrose concentration was 0.6mol/L,the best survival rates of Dongguahan shoot tips was 58%.3.Results of 10%glycerol adding to different concentration of sucrose in preculture mediums on cryopreservation showed that the medium supplemented with 0.6mol/L sucrose adding 10%glycerol was the best,its survival rate of Dongguahan shoot tips was 78%.4.Following the elongation of dehydration time,survival rates were less than 10%when dehydration time was shorter than 4 hours,then survival rates increased,arrived peak value at dehydration 6 hours(61%),the moisture content of bead was 23%,after that,survival rate decreased.orthogonal experiments:5.Using 3 factors(dehydration time,sucrose concentration and preculture time)and 3 levels orthogonal experiments,a suitable combination of Dongguahan shoot tips cryopreservation was screened which was that shoot-tips precultured for 1d on MS medium supplemented with 0.6mol/L sucrose,and dehydrated 6h in room temperature.On which,the survival rate was 60%.Using the method on Qingguan,Bolin 3 hao,Longji cryopreservation,their survival rates were 42%,45%,59%,respectively,which indicated that the relationship of Qingguan and Bolin 3 hao was closer than Longji.6.Proteins of Dongguahan shoot-tips after and before LN frozen and subculture(CK)were extracted and run two-dimensional electrophoresis,analyzed their 2-DE spectrum.Results showed that,there were obviously difference among three groups treatment,the protein numbers of them were 1400?1500,1900?2000,2200?2400,respectively.The minimum protein match percent was 60%,the maximum value was 84%.Comparing of each experimental group,the minimum value was 76%,the maximum value was 84%,which indicated that distinguish and repeat ability of 2-DE were higher.7.The different analysis of three experimental groups 2-DE tables showed that,there were different proteins emerging during crypreservation.Differences were greater between after LN frozen and CK groups which had total 283 different protein points,181 and 72different protein points were between before LN frozen and CK,after and before LN frozen groups,respectively.After statistical analysis,a total of 22 protein points with good repeatability and high diversity were found.8.Based on different analysis of proteome,choosing 21 proteins(A23,A08,C10,B01,C19,D60,D111,D119,D134,D124,D136,D163,E84,C21,C49,C58,D61,D128,E57,B18,D44):were identified by mass spectrometry,the results were as follows A23 was malate dehydrogenase;A08 was glutamine synthetase;C10 was heat shock protein 70;B01 was Patellin1;C19 was Chaperonin CPN60-2;D60 was 26S proteasome non-ATPase regulatory subunit(Fragment);D111 was Ribulose bisphosphate carboxylase/oxygenase activase 1;D119 was Glyceraldehyde-3-phosphate dehydrogenase;D124 was Ankyrin repeat domain containg protein 2;D134 was S-adenosyl-L-methionine:delta 24-sterol-C-methyltransferase(Fragment);D136 was Ran-binding protein 1;D165 was Chlorophyll a-b binding protein,chloroplastic;E84 was HSP17.4;C21,C49,C58,D61,D128,E57,B18 and B44,were uncharacterized protein.