Influence Of Ethylene To Photosynthesis Of Drawf Lilyturf (Ophiopogon Japonicus) And Clone Of Correlated Genes

Ethylene(ETH)presents an important physiology effect during plants growth and development. Based on its influence to photosynthesis of Drawf lilyturf(Ophiopogon japonicus)(DL),genes related on plants consenescence were cloned by SSH technology.The photoresponse curve of DL and CO₂ were measured.In conclusion,the light saturation point and compensation point of DL were about 500μmol·m^-2·s^-1and 10μmol·m^-2·s^-1while they were 1200μl·L^-1and 20μl·L^-1for CO₂,respectively.The photosynthetic speed of DL was depressed dramatically by ethephon(CEPHA)which declined it with the elongate of treating time.There was a notable interaction between the treating time and concentration of CEPHA which showed huge significant difference.The photosynthetic speed of DL was prompt declined with the increasing disposal density and the prolonged treating time.Fo,Fv/Fm,Fv'/Fm',Φ_PSâ…¡,qP and ETR fell off while NPQ arose gradually under different CEPHA consistency and time explained that CEPHA decomposed continuously and released ETH in plants which exhibited a rather huge contribution to photo-systemâ…¡and its photosynthetic electron transfer chain.A cDNA subtractive library containing 576 clones was constructed using the cDNA of annual DL leaves treated with 0.5g/L CEPHA as tester and the control as driver by SSH method in order to prove up the effect of ETH to photosynthesis of DL.The contrast sector was between 500 to 1500bp.259 light demanding clones of total were selected by spot blotting.Eventually,we obtained 237 pieces of EST sequence through sequence measurement analysis.According to the analysis of BLASTx:16 pieces of EST sequence could not find homologous sequence in the GenBank and they might be new genes;74 pieces of sequence could index homologous sequence but their function were not clear yet; 147 pieces of sequence had found the homologous sequence with known gene section of other species and the homology degree was 25～100%.CBA and UNP gene was screened form full-length cDNA library according to the EST sequence design primer and based on the construction of full-length cDNA library.The full-length sequence of CAB gene was 1055bp and its code area was 795bp between 18 and 812.5'UTR was 1-17bp containing 17bp while 3'UTR was 813-1055bp containing 243bp.The CAB gene showed 86～73%homology with sycamore(Platanus xacerifolia),rice(Oryza sativa)and tobacco(Nicotiana tabacum)and presented a close affinity relationship with tequila(Agave tequilana),a furthest with tobacco.Its coded protein was consisted of 264 cistine and its molecular weight was 28kDa.pI,Asp+Glu and Arg+Lys of CBA gene were 5.29,26 and 21,respectively.The molecular composition of this gene was C₁₂₇₂H₁₉₄₃N₃₂₇O₃₆₇S₁₀. The max and min valuation of hydrophobicity of coded protein by CAB was 2.222 and -1.844, respectively.The three dimensional structure exhibited 3αspiral conformations.The full-length sequence of UNP gene was 916bp and the code area was 708bp between 55 and 762.5'UTR was 1-54bp containing 54bp while 3'UTR was 763-916bp containing 154bp.Compared with non-redundant nucleotide database(nr),STS,GSS and HTGS,no homologous sequence could be found.UNP sequence exhibited 73%homology with 4 pieces of EST sequences of flower bud cDNA of litiodendron(Liriodendron tulipifera)by compared with EST database.Check this sequence with nr,it represented 54～37%homology with grape(Vitis vinifera)and black poplar(Populus trichocarpa).The coded protein of UNP from DL had a further affinity relationship with grape,black poplar and mouseearcress(Arabidopsis thaliana)and their divergence time on the phylogenesis tree were very close.The coded protein of UPN gene was consisted by 235 cistine and its molecular weight was 27kDa. The pI,Asp+Glu and Arg+Lys were 6.47,37 and 36 and its molecular composition was C₁₁₈₈H₁₈₇₁N₃₃₃O₃₆₁S₁₃.The max and min valuation of hydrophobicity of coded protein by UNP was 1.511 and -3.267,respectively.Based on pCAMBIA1303 and introduce BamHâ… and Kpnâ… site,four plants expression vector pCAMBIA1303-CAB(+),pCAMBIA1303-CAB(-),pCAMBIA1303-UNP(+)and pCAMNIA1303-UNP(-)were constructed and leading-in to Agrobacterium.Based on pET-30a(+),procaryon expression vector pET-30a-CAB and pET-30-UNP were constructed and introduced into colibacillus BL21.After induction under 0,1,2,3,4 and 5h by different concentration of IPTG,SDS-PAGE catphoresis concluded that both CAB and UNP could express in colibacillus BL21.The catphoresis band of the protein expressed by CAB gene showed about 28kDa while UNP was 27kDa.The amount of the protein of these two genes increased with the elevation of the concentration and time under the instructed time and IPTG density.