| The compact(dwarf)plant architecture is an important trait in cucumber(Cucumis sativus L.)breeding.The compact gene(cp)from cucumber WI7201 participates in regulating the cucumber plant architecture,causing reduced height and compact phenotype.Analysis and verification of the cp gene is important to understand the regulation mechanism of plant height and germplasm innovation.Fine genetic mapping had delimited the cp gene locus to a9 kb genomic DNA region.We had already cloned the candidate gene and found it to be Cs ERECTA gene,one member of the LRR receptor-like kinase gene family.Based on the previous study,we selected the vining CCMC and the compact germplasm WI7201 as the research objects,prepared the polyclonal antibody by prokaryotic expression of CsERECTA protein,used western blot to examine the differences between the two species on transcription level,and genetically transformated CsERECTA gene in cucumber,mainly got the following results:1.Prokaryotic expression and polyclonal antibody preparation of CsERECTA gene.The prokaryotic expression vector of pET21a-CsERECTA was successfully constructed and then it was transformed and shifted into E.coli BL21(DE3),realizing efficiently expression and the purpose protein was obtained.The results showed that the CsERECTA protein expressed in two forms of soluble and inclusion,and low temperature can contribute to abundantly express in soluble form.In 23℃,most of the CsERECTA recombinant protein is soluble.The induced temperature,duration and the concentration of IPTG for the Cs ERECTA recombinant protein were optimized and found that the optimum induced temperature was 23℃,the optimum concentration of IPTG was 0.5mmol/L and the optimum induced duration was 6h.The recombinant protein of MBP-CsERECTA was purified through Ni-chelating affinity chromatography.After rTEV enzyme digestion,CsERECTA protein was prepared and then it was used to make polyclonal antibody by a biotechnology company.Western blot detection showed that both CsERECTA protein and proteins of cucumber could have idiosyncratic reaction with prepared serum,which demonstrated that the polyclonal antibody was successfully prepared.Therefore,the polyclonal antibody can be used for detecting protein expression of CsERECTA gene on transcription level in the next step.2.Analysis of CsERECTA gene expression on transcription level.In the whole growth cycle of cucumber,CsERECTA protein expression level of vining germplasm CCMC and dwarf germplasm WI7201 dispalyed a rule: seedling stage >cotyledon stage > blossom stage.This showed that the expression level of ERECTA gene was moderate in the early development stage,higher in vegetative growth transiting to reproductive growth phase,and low in blossom period.However,especially in cotyledon stage and seeding stage,the CsERECTA protein content of CCMC was significantly higher than that of WI7201 in the stem apex,and the CsERECTA protein content was slightly higher than that of WI7201 in the stem.There was no obvious difference on the protein expression level in other organs except for stem apex and stem.Thus,the dwarf trait of WI7201 is likely to be due to the low expression of CsERECTA gene on transcription level in the stem apex.3.Preliminary genetic transformation analysis of Cs ERECTA gene in cucumber.The plant overexpression vector of pCAMBIA3301-CsgERECTA-flag was successfully constructed by our research group.Using the optimized agrobacterium-mediated genetic transformation program of cucumber,through preculture,coincubate culture,regeneration culture and rooting culture,we intended to insert CsERECTA gene into the cucumber genome.Finally,we used the method of PCR test to inditify in the transgenic plants.We had obtained nineteen transformed plants which were detected positive for PCR test.But none of T0 transgenic plants displayed phenotypic changes compared to untransformed cucumber.In order to further identify,western blot of flag label test should be taken to confirm T0 and T1 transgenic plants. |
PDF Full Text Request