【作者】 刘君；

【导师】 韩烈保；

【作者基本信息】 北京林业大学 ， 草业科学， 2008， 博士

【摘要】 乙烯（ethylene,ETH）在植物生长发育过程中具有重要的生理作用。本研究的目的是在分析乙烯（ETH）对麦冬（Ophiopogon japonicus）光合作用影响的基础上,利用SSH等分子生物学实验技术,从麦冬中克隆与植物衰老相关的基因,为培育绿期长的草坪草新品种奠定基础。通过测定麦冬的光响应曲线和CO₂响应曲线得知麦冬的光饱和点在为500μmol·m^-2·s^-1,光补偿点为10μmol·m^-2·s^-1,CO₂的饱和点为1200μmol·m^-2·s^-1,CO₂补偿点为20μmol·m^-2·s^-1。乙烯可明显抑制麦冬的净光合速率,使其随处理时间的延长呈下降趋势,乙烯利各浓度间、处理时间之间差异极显著,即随着处理浓度的增加和处理时间的延长,麦冬净光合速率迅速地降低。在乙烯利不同浓度和时间的胁迫下,Fo、Fv/Fm、Fv’/Fm’、Φ_PSⅡ、qP、ETR等荧光参数逐渐下降,NPQ逐渐升高,说明乙烯利在植物体内不断分解释放出乙烯,对光系统Ⅱ结构及光系统Ⅱ的光合电子传递链有较大的影响。为进一步从分子水平探明乙烯对麦冬光合作用的影响,通过SSH方法,以0.5g/L乙烯利处理6h的麦冬一年生叶片的cDNA为Tester,对照为Driver,构建了一个含有576个克隆的差减cDNA文库,差异片段大小在100～1500bp之间。通过斑点杂交,从576个克隆中筛选出259个阳性克隆,经测序分析,最终得到237条EST序列,经BLASTx分析,发现有16条EST序列在GenBank中没有找同源序列,可能代表新基因;74条EST序列在数据库中检索到同源性序列,但其功能尚不清楚;147条序列在GenBank中能找到与其它物种已知基因部分区域的同源序列,同源性为25～100%。在建立全长cDNA文库的基础上,根据获得的EST序列设计引物,从全长cDNA文库中筛选出叶绿素结合蛋白（CAB）基因和一个编码未知蛋白（UNP）基因,经测序分析:CAB基因的全长序列为1055bp,其编码区为18～1812之间,编码区为795bp,5’UTR为1～17bp,包含17个bp;3’UTR为813～11055bp,包含243bp。同源性比较结果为:麦冬CAB基因与二球悬铃木(（Platanus xacerifolia）、水稻（Oryza sativa）、烟草（Nicotiana tabacum）等植物有86%～73%的同源性。系统发育分析:其与龙舌兰（Agave tequilana）CAB基因亲缘关系较近,与烟草CAB基因最远。其编码的蛋白质由264个氨基酸组成,分子量为28kDa,理论等电点pI为5.29,Asp+Glu为26个,Arg+Lys为21个,分子组成为C₁₂₇₂H₁₉₄₃N₃₂₇O₃₆₇S₁₀。CAB基因编码蛋白的疏水性的最大值为2.222、最小值为-1.844。三维结构具有3个α螺旋结构。UNP基因全长序列为916bp,其编码区为55-762之间,编码区为708bp,5’UTR为1～54bp,包含54bp;3’UTR为763-～916bp,包含154bp。BLAST分析:与非冗余核苷酸数据库（nr）、STS、GSS、HTGS比对,未发现相似性高的同源序列;与EST数据库比对,发现与鹅掌楸（Liriodendron tulipifera）花芽cDNA文库中的四条EST序列同源性为73%;随后又将这一序列与非冗余蛋白数据库（nr）比对分析,其与葡萄（Vitis vinifera）、黑杨（Populus trichocarpa）等植物有37～54%同源性。系统发育分析:麦冬UNP编码蛋白与葡萄、黑杨、拟南芥（Arabidopsis thaliana）等UNP编码蛋白亲缘关系较近,和其他植物之间的亲缘关系比较远。其编码的蛋白质由235个氨基酸组成,分子量为27kDa,理论等电点pI为6.47,Asp+Glu为37个,Arg+Lys为36个,原子组成为C₁₁₈₈H₁₈₇₁N₃₃₃O₃₆₁S₁₃。UNP基因编码蛋白的疏水性最大值为1.511、最小值为-3.267。以pCAMBIA1303为基础,引入BamHⅠ和KpnⅠ的酶切位点,构建了pCAMBIA1303-CAB（+）、pCAMBIA1303-CAB（-）、pCAMBIA1303-UNP（+）、pCAMNIA1303-UNP（-）等四种植物表达载体,并导入到土壤农杆菌LBA4404中。以pET-30a（+）为基础,构建了原核表达载体pET-30a-CAB和pET-30-UNP,转化到大肠杆菌BL21中,经不同浓度IPTG分别诱导0、1、2、3、4和5h后,SDS-PAGE电泳分析结果为:在IPTG的诱导下,外源基因CAB与UNP都能在大肠杆菌BL21中表达,CAB基因表达的蛋白的电泳条带在28kDa左右,UNP基因表达的蛋白的电泳条带在27kDa左右,理论推测结果相一致。在一定的IPTG浓度和诱导时间的范围内,随着浓度和时间的延长,CAB与UNP基因表达的蛋白量增大。更多还原

【Abstract】 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_PSⅡ,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-systemⅡand 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 BamHⅠand KpnⅠsite,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.更多还原