【作者】 杨丽；

【导师】 彭镇华；

【作者基本信息】 中国林业科学研究院 ， 林木遗传育种， 2014， 博士

【摘要】 光合作用是生物界获取能量、食物以及氧气的根本途径，而叶绿素是植物光合作用吸收光能的主要载体，其生物合成需要15步反应，涉及15种酶，其中镁原卟啉单酯环化酶（MPEC）在叶绿素生物合成的碳环形成过程中发挥着重要的作用，如果将编码MPEC的基因敲除，植物会出现致死现象。毛竹（Phyllostachys edulis）基因组草图的发布为基因功能的研究，分子标记的开发与应用奠定了基础。本研究以重要经济竹种毛竹为材料，从中分离MPEC同源基因，研究其时空表达模式，通过转基因技术验证其功能。然后根据毛竹基因组数据，搜索与毛竹光合作用途径相关基因中搜索SSR位点，开发SSR分子标记，对刚竹属植物的遗传多样性分析。主要结果如下：1基因获得采用同源比对方法从毛竹全长cDNA文库中得到1个MPEC基因同源序列（Genbank：FP092998），命名为PeMPEC。该基因全长1474bp，5′和3′非编码区分别长23bp和206bp，编码区为1248bp，编码415个aa，推断其分子量为49.3kD。PeMPEC编码区对应的基因组序列为1864bp，其中含有5个外显子和4个内含子。蛋白结构分析表明，PeMPEC基因编码的蛋白具有两个拷贝的EXnDEXRH和一个亮氨酸拉链结构，该蛋白序列与拟南芥（Arabidopsis thaliana）中MPEC的同源性为73.6%，与水稻（Oryza sative）等禾本科植物的MPEC同源性均在85.0%以上，进化上具有较高的保守性。2基因时空表达特异性半定量PCR检测表明， PeMPEC基因主要在毛竹光合器官叶片中表达，而根中检测不到表达。qRT-PCR结果表明，黑暗条件可诱导PeMPEC基因的表达，随着光照强度的增强（100-1500μmol m-2s-1）基因的表达受到抑制；高温（42℃）抑制基因的表达，而短时间内（0.5h）基因表达受低温（4℃）的诱导，随着低温处理时间增加，基因表达受到抑制；PeMPEC基因在毛竹黄化苗中仅检测到微弱表达，随着光照（200μmol m-2s-1）时间延长，基因表达量逐渐上升，8h的表达量上升为对照的80%。3基因在拟南芥中异位表达构建PeMPEC正、反义植物表达载体，分别转化拟南芥。表型分析显示，正义转基因植株与野生型拟南芥表型没有明显的区别；反义转基因植株则生长缓慢，叶片出现叶绿素缺乏症状，呈浅黄色。半定量RT-PCR结果表明，PeMPEC基因在5个转基因株系中均有表达，其中在正义转基因株系S-5中最高，反义转基因株系A-4中最低。色素含量分析表明，正义转基因株系叶绿素含量比野生型增加了20%以上，而反义转基因株系叶绿素含量降低，其中A-2株系的叶绿素含量仅为野生型的58%。叶绿素荧光参数测定结果显示，与野生型相比反义转基因株系A-1和A-2的Fv/Fm、Y(Ⅱ)和ΔI/I值均显著下降（p<0.05），而转正义基因株系虽有所增加，但差异不显著。由此表明，转基因植株叶绿素的含量受到了的影响，PeMPEC基因对叶绿素的生物合成具有调控作用。4基因功能互补实验在拟南芥T-DNA插入Crd1突变体（SALK-024716C）中过量表达PeMPEC基因，获得了恢复到野生型表型的转基因植株。半定量结果表明，PeMPEC基因在转基因植株中均有表达，其中转在基因株系M-4中表达量最高，M-1中最低。叶绿素含量测定结果表明，转基因植株的总叶绿素含量比突变体的高22-50%，比野生型的高7-20%左右。叶绿素荧光参数测定结果显示，转基因植株的Fv/Fm、Y(Ⅱ)和ΔI/I值比突变体显著增加（p<0.05），而与野生型植株相近。证明PeMPEC基因的过量表达可以提高叶绿素的含量，对维持PSⅠ和PSⅡ的稳定具有重要的作用。5Western Blotting检测构建PeMPEC基因的原核表达载体，转化大肠杆菌表达菌株（Rosetta-gamin B (DE3)）感受态细胞，并进行诱导表达。通过条件优化，在28℃条件下用0.4mmol·L-1IPTG诱导4h，获得了表达丰度较高的可溶蛋白。蛋白纯化结果表明，在大肠杆菌中分离出一个60kDa的重组蛋白，并利用该蛋白制备多克隆抗体。分别提取毛竹根、茎、叶鞘和叶片组织的总蛋白，SDS-PAGE电泳后，以PeMPEC的多克隆抗体为探针进行杂交。Western Blotting结果表明，PeMPEC主要于叶片组织中积累。6毛竹基因组SSR特点对毛竹全基因组数据进行SSR搜索，共筛选出302615个SSR位点，平均1Mb序列中出现147.5个SSR，其中单核苷酸和二核苷酸重复类型为主，占总SSR的87.33%。在单核苷酸重复单元中，以A/T的单核苷酸重复占主导地位，占单核苷酸重复总数的72.00%，；二核苷酸重复基序中AG/CT重复类型占得比例最高，达到二核苷酸重复的41.16%，其次为AT/AT、CG/CG重复类型；SSR出现的数目随SSR的长度增加逐渐减少，当SSR长度为10-20bp时，所检测到的SSR的数量最多，达到79.76%；两个SSR位点之间碱基数为4-100的SSR位点数量最多，占复合型SSR标记的39.54%。7SSR在刚竹属中的多态性分析选择73对引物对刚竹属的种或变异类型进行了遗传多样性的扩增，其中22对引物具有多态性，占总引物的29.79%。在78个样品中共检测到64个等位位点，平均每个引物有2.78个等位位点。对22个位点的遗传参数进行了估算，期望杂合度分布范围为0.09-0.93，平均值为0.44，表现出中度的遗传多样性，观测杂合度分布范围为0.02-0.92，平均值为0.68。Shannon多样性指数的分布范围为0.25-1.30，平均为0.74。与其他物种相比，所检测刚竹属样品的遗传多样性水平较低，这可能与其无性繁殖方式有关。8SSR在刚竹属分类中的辅助应用根据遗传距离进行聚类分析，结果表明78个样品共聚为3个大类，第Ⅰ类和第Ⅱ类的竹种主要属于刚竹属刚竹组，第Ⅲ类竹种主要属于刚竹属水竹组。STRUCTURE结果显示78个样品共分为4个类群，结果与根据遗传距离的聚类结果部分相似，但也有部分的出入。STRUCTURE的结果没有将刚竹组和水竹组分离，但是可用于种下等级的分类，而根据遗传距离的聚类结果比较容易的区分刚竹组和水竹组，但是有些种下等级之间的遗传距离较远，因此，结合这两种方法的分类结果，与传统分类学类似，吻合程度较高。PeMPEC基因是毛竹叶绿素生物合成过程中的关键酶基因，本研究为通过基因工程调控叶绿素的生物合成提供了新的功能基因资源，为培育出具有高效光合效率的植物新品种提供了理论依据。毛竹光合途径相关基因中SSR位点的开发及应用，不仅为刚竹属的分类提供了佐证，而且对于在分子标记水平挖掘光合作用相关位点提供遗传资源，揭示竹子光合作用的遗传多态性，筛选特有遗传因子具有重要意义。更多还原

【Abstract】 Photosynthesis is the basic way to obtain the energy, food and oxygen in the biologicalworld. Chlorophyll is the main carrier for absorption of light energy in plant photosynthesis.Chlorophyll biosynthesis takes15-step reaction, involving15kinds of enzymes, among whichMg-protoporphyrin Ⅸ monomethyl ester cyclase (MPEC) plays an important role in theformation of isocyclic ring. Plants will appear the phenomenon of death if the gene encodingMPEC was knocked out. The published draft genome of moso bamboo laid a good foundationfor the study of gene function, development and application of molecular marker.Phyllostachys edulis, an important economical bamboo, was selected for the isolation ofMPEC homologous gene. The spatio-temporal expression patterns of MPEC in Ph. edulis wereanalyzed, and the gene function was verified through transgenic technology. SSR molecularmarkers in the genes of related with photosynthesis pathway were developed according to thegenome of Ph. edulis and used to analyze the genetic diversity of bamboos in Phyllostachysgenus. The results were as follows:First, Isolation of PeMPEC gene A homologous gene of MPEC was abtained from thefull length cDNA library of Ph. edulis by alignment method and designed as PeMPEC(Genbank: FP092998). The cDNA of PeMPEC was1474bp including5′untranslated region(UTR)23bp,3′UTR206bp and an open reading frame (ORF) of1248bp which encoded aprotein with415aa. The corresponding genomic sequence of ORF was1854bp including5exons and4introns. The analysis of protein structure showed that PeMPEC had one conservedleucine zipper domain and two copies of the EXnDEXRH motif. The result of sequencealignment demonstrated that PeMPEC shared73.6%homology with that of Arabidopsisthaliana, and more than85.0%homology with those of gramineous plants such as Oryza sative,which indicated that MPEC had a high conservative in evolution.Second, Spatio-temporal expression pattern of PeMPEC Semi-quantitative PCRanalysis showed that PeMPEC was transcribed at high level in leaf which is main photosynthesis organ and could not be detected at all in root. The result of real-timequantitative PCR suggested that the expression of PeMPEC was induced by darkness andsupressed with increase of light from100μmol m-2s-1to1500μmol m-2s-1. Hightemperature (42℃) also could suppress the gene expression. However, it was induced by lowtemperature (4℃) in a short time(0.5h) and then was suppressed by the prolonged treatment.Thhough expression of PeMPEC was very weak in etiolated seedlings, it was increasedgradually with the treatment of light (200μmol m-2s-1) and reached to80%of the controlafter8h treatment.Third, Ectopic expression of PeMPEC in A. thaliana The sense and antisenseexpression vectors of PeMPEC were constructed and transferred into A. thaliana. The analysisof phenotype showed that there was no obvious difference between sense transgenics and wildtype. However chlorophyll deficient leaf with light yellow was found in the antisensetransgenic lines which grew slowly. The semi-quantitative PCR analysis confirmed thatPeMPEC had been expressed in transgenic plants, among which the most abundant was insense line S-5and the least was in antisense line A-4. The analysis of pigments showed thatchlorophyll content in sense plants was20%higher than that of of wild type, while that ofantisense plants all was reduced, especially for that of line A-2was only58%as that of wildtype. The data of chlorophyll fluorescence parameters showed that Fv/Fm, Y(Ⅱ) and ΔI/Ivalue of the antisense line A-1and A-2decreased significantly (p<0.05) compared with thoseof wild type, however there were no significantly different between parameter values of senselines and those of wild type. These indicated that the chlorophyll content of transgenic plantshas been influenced by PeMPEC which involved in the regulation of chlorophyll biosynthesis.Forth, Functional complementation analysis of PeMPEC PeMPEC was transferredinto Crd1(SALK-024716C) which is a T-DNA mutant of A. thaliana, the phenotype oftransgenic plants was similar to that of wild type. The semi-quantitative PCR of transgenicplants confirmed that PeMPEC had been integrated into genome and its expression was mostabundant in line M-4and least in line M-1. The analysis of pigments showed that chlorophyll content in sense plants was22-50%higher than that of mutant and7-20%higher than that ofwild type. The measurement of chlorophyll fluorescence parameters showed that there were noobvious difference between transgenic plants and wild type for the value of Fv/Fm, Y(Ⅱ) andΔI/I, however significant difference (p<0.05) were presented between transgenic plants andmutant. These results indicated that the overexpression of PeMPEC could increased the contentof chlorophyll, which played an important role in keeping stability of PS Ⅰ and PSⅡ.Fifth, Western Blotting The prokaryotic expression vector of PeMPEC was constructedand transferred into Escherich coil (Rosetta-gamin B (DE3)). The high expression of solute proteinwas obtained with0.4mmol·L-1IPTG induced4h at28℃. The purification of protein showedthat a60kDa recombinant protein was isolated from E. coli and used to prepare polyclonalantibody. The total protein of root, stem, sheath and leaf were extracted respectively andseparated with SDS-PAGE. The polyclonal antibody of PeMPEC was used as probe forwestern blotting. The result suggested that PeMPEC was mainly detected in leaf tissue.Sixth, Characteristic of SSR in Ph. edulis genome Through genome-wide search, a totalof302615SSR loci were identified from the genome sequence of Ph. edulis, in which1Mbsequence appeared an average147.5SSRs. Mononucleotide and dinucleotide repeats SSR weredominant, accounting for87.33%. The most abundant motif in mononucleotide repeats is A/Taccounted for72.00%, and the most one in dinucleotide is AG/CT accounted for41.16%,followed by AT/AT, CG/CG motif repeats. The number of SSR was decreased gradually withthe increased length of SSR. The number of SSR with length of10-20bp was the most one,accounting for79.76%. When the bases were4-100bp between two SSR loci the number ofSSR was the most abundant, accounting for39.54%of that of compound SSR loci. Thenumber of compound SSR with4-100bp between two SSR loci was the most abundant,accounting for39.54%.Seventh, Polymorphism analysis of SSR in Phyllostachys A total of73primer pairswere designed and used to precede genetic diversity analysis of78samples from Phyllostachys,in which22pairs of primers were polymorphic, accounting for29.79%. A total64alleles were detected in78samples, there is an average of2.78alleles per primers. Genetic diversityparameters of22loci were calculated, the excepted heterozygosity was ranged from0.09-0.93with average value of0.44, the observed heterozygosity ranged from0.02~0.92with averagevalue of0.68, and the Shannon index ranged from0.25to1.3with average value of0.74. Allthe genetic parameters indicated that the genetic diversity level of Phyllostachys was lowerthan that of other species, it may be related with its asexual reproduction.Eighth, Auxiliary application of SSR in Phyllostachys classification The result ofcluster analysis showed78samples were clustered into3classes according to the geneticdistance. The samples clustered into the first and second classes belonged to Sect.Phyllostachys group and those of the third classs belonged to Sect. Heterocladae group.However, the result of STRUCTURE demonstrated that78samples were divided into4classes,which was consistent some but inconsistent portion with cluster analysis. Though the result ofSTRUCTURE method was not distinguished Sect Phyllostachys and Sect Heterocladae, itcould be used to classify the species and its variants. It was easy to distinguish SectPhyllostachys and Sect Heterocladae using cluster analysis, but difficult for the variants for thereason of genetic distance. Therefore, it was similar to the traditional taxonomy with highdegree of agreement by combining the results of two methods.PeMPEC was an essential gene in chlorophyll biosynthesis. This study provided a newgenetic resource for the regulate of chlorophyll biosynthesis through genetic engineering, and atheoretical basis for the breeding of new varieties with high photosynthetic efficiency. SSR lociin the genes related to photosynthesis pathway were not only provided the evidence fortaxonomy of Phyllostachys but also genetic resource for development of the loci related withphotosynthesis at molecular marker level, which had important value for revealing geneticpolymorphism of bamboo photosynthesis and screening the unique genetic factors.更多还原