| Camellia oleifera,lablled with 'eastern olive oil',has been cultivated widely in southern china.Selecting superior clones and the best hybrid combination is the universal and effective breeding method in practice.However,superior clones have a general low selfing fecundation index together with poor genetic stability in selfing filial generation,leading to a serious flower and fruit drop,thus decreasing the rate of seed setting.To investigate camellia oleifera selfing-compatibility,'Min43'?'Min48' and Camellia meiocarpa were provided to be pollination and powder species respectively.3 pollen grain morphology were observed with scanning electron microscopy.Setting 3 pollination combination,that is selfing,intraspecific cross and outraspecific cross,pistils in 7 different pollination(1?4?7?24?48?72?96 h)period were obtained after control pollination.Fluorescence microscopy was applied to study pollen growth in 7 different pollination period among selfing,intraspecific cross and outraspecific cross,aming to find out the growth difference period;then combing with the proteomic technology to anlysis differetial proteins participating in the process,working out whom and what fuction they are.The purpose of this investigation is to build a theoretical model for the process of pollination in camellia,such as cell signal recognition,interaction et,hoping to make for paractical pollination configuration and genetic improvement in camellia.Otherwise,a suitable 2DE system for camellia pistil was optimized in some aspects,including sample protein extraction,IEF program,SDS-PAGE pouring and so on.The major results were as follows:1.3 pollen grain morphology was observed by scanning electron microscopy shows that all of them have features in following:actinomorphic and long spherical shape,oval equatorial view and 3 equally distributed germination furrows in polor view.It seems that they were in a certain similar but for the surface ornamentation.Camellia oleifera 'Min43'?'Min48' were belong to striatal brain ornamentation,Camellia meiocarpa performed a cell texture.2.Fluorescence microscopy observation indicated that pollen grain might hydrate normally in stigma and germinated polle tube no matter which pollination method.What is more,there was no any callose block happening in whole fertiliazation process.While pollen tubes growth speed were different in different period?It took totally 96 hours for selfing pollen tube to reach the pitil base,approximately later than cross-pollination 1d.The growth tendency exhibited significant difference.intraspecific cross showed a 'up-down-up' tendency but selfing and outraspecific cross'S' tendency.In addition,a lag phase was occurred in the period of 4?7 h after pollination.3.Conclusions made from the 2DE system for camellia oleifera pistil were said that method of combing TCA/Acetone with SDS/phoel was better suitble for camellia oleiferal pistil protein extraction.And protein powder dissolved in lysate containing 7mol·L-1 urea,2mol·L-1 thiourea,80mmol·L-1 DTT,4%(W/V)CHAPS and 2%(V/V),IPG buffer(pH 3?10L)performed a better solubility.A Beckman J-30I centrifuge was used for the centrifugation of protein sample made an contribution to eliminating impurities and more proteins dissociation.Beside,sample in-gel rehydration as well as IEF program with low voltage for getting rid of salt ion and 62000vhr total focusing time was benefit for protein horizontal segregation.In vertical segregation,10%concentration of SDS-PAGE gel displayed a better result.4.2DE coupling with MS,applied to study camellia oleifera selfing compatibility,showed that 44 protein spots from 55 detectable protein were successfully identified by MS.These proteins were categorized into the following six groups:energy metabolism,protein transpose and synthesis,signal transduction,cell growth/division and unclasses.Furthermore,one differential protein identifided as 26S proteasome was highlly expressed in selfing pollen tube,inferring as a factor preventing pollen tube growth. |
PDF Full Text Request