【作者】 李艳；

【导师】 许为钢； 崔党群；

【作者基本信息】 河南农业大学 ， 作物遗传育种， 2009， 博士

【摘要】 小麦为我国的重要粮食作物,其生产能力及供需状况始终关系到我国国民经济发展和粮食安全等重大战略问题。随着我国社会经济的发展和人口的增加,人们对于粮食的需求日益增长。光合作用是一切绿色植物物质生产的基础,因此,提高作物产量的关键是提高光能利用效率,从而提高单位面积的产量。C4植物如玉米、高粱、甘蔗等具有CO2浓缩机制,光补偿点高,CO2补偿点低,光呼吸弱,特别在高温、干旱等逆境条件下,比C3植物小麦、水稻等有较高的光合效率。将C4途径关键酶基因引入C3植物以提高其光合效率和籽粒产量,一直是国内外生物学研究领域的前沿问题。磷酸烯醇式丙酮酸羧化酶(PEPC)是C4光合途径中的关键酶,其主要分布在C4植物的叶肉细胞的细胞质内,形成CO2的浓缩机制为维管束鞘细胞的C3途径提供CO2,起“CO2泵”的作用。磷酸丙酮酸二激酶(PPDK)催化的反应是C4代谢途径中的限速步骤,是C4光合途径过程中另一个备受关注的酶,此酶位于叶肉细胞的叶绿体中,催化CO2最初受体PEP的再生。许多研究已表明,将C4型pepc基因转入C3作物可以提高C3作物的光合效率和产量潜力。为获得具有C4光合特征的高光效转基因小麦新品系,我们从光合效率和PEPC酶活性均较高的玉米自交系Z561中克隆出C4型pepc基因和ppdk基因的cDNA,通过基因枪介导法将玉米C4型pepc基因导入到小麦中,为提高小麦品种的光合效率和转基因高光效育种提供必要的理论和技术材料支撑。主要结果如下:1.利用同源克隆技术,从玉米中克隆了C4型磷酸烯醇式丙酮酸羧化酶(PEPC)基因的cDNA。PEPC的核苷酸序列分析表明,该片段全长3053bp,包含一个2910bp长的开放阅读框,编码970个氨基酸。与已登陆的玉米pepc基因(X15238)相比,序列同源性可达99%。核苷酸序列中存在16处碱基差异,导致蛋白质水平上有5处非连续性排列的氨基酸差异,并未影响到该酶的酶活性。编码区核苷酸序列与甘蔗C4型、高粱C4型、谷子C4型同源率分别达到91%、90.2%、83.2%,与水稻C3型、高粱C3型、谷子C3型、稗草C3型同源率分别为74.1%、75.1%、76.8%、75.7%。氨基酸序列进化树分析表明,所克隆的玉米C4型PEPC与甘蔗和高粱的C4型遗传距离最近,而与谷子C4型次之。编码蛋白质的功能位点分析表明,玉米PEPC蛋白质氨基酸序列有7个保守区。2.利用同源克隆技术,从玉米中克隆了C4型磷酸丙酮酸二激酶(PPDK)基因的cDNA。PPDK序列测定结果表明,PCR合成片段长度为3014bp,包括一个2757bp长的开放阅读框。与已登陆的玉米ppdk基因(J03901)相比,序列同源性可达92.94%,3.利用从玉米中克隆的C4型磷酸烯醇式丙酮酸羧化酶(PEPC)基因(GenBank接受号为FJ415327)构建高效双元表达载体。将玉米C4型PEPC cDNA序列插入双元表达载体pCAMBIA3301中,构建了p3301-pepc融合表达载体。4.利用基因枪介导法将p3301-pepc高效表达载体导入6个小麦品种(系)中,共获得409株抗性再生植株,其中有357株PCR扩增结果呈阳性。Southern杂交结果表明玉米C4型pepc基因已整合到小麦基因组中。利用实时荧光定量PCR方法分析转基因小麦植株的拷贝数,在15株转基因植株中7株拷贝数为1,3株拷贝数为2,2株拷贝数为3,拷贝数为5、8和17的分别为1株。RT-PCR分析结果表明外源基因在小麦基因组中得到正确转录,且不同转基因植株pepc基因的表达量存在较大差异。5.对T1代转基因小麦植株进行PCR检测和遗传分析,结果表明大部分转基因植株的分离比例符合孟德尔显性单基因遗传分离规律,外源基因在T0代转基因植株中整合情况良好,而且能够稳定的遗传给后代;对T1代部分转基因植株进行PEPC酶活性测定,结果显示部分转基因植株的PEPC酶活性得到了提高,最高的酶活性是对照植株的2.17倍,SDS-PAGE凝胶电泳分析结果表明不同转基因植株间pepc基因表达水平存在差异,酶活性较高的转基因植株,其pepc基因表达水平亦高;对田间种植的T1代转基因小麦植株和对照植株进行叶片净光合速率测定,结果显示,与对照植株相比,除个别T1代转基因小麦植株净光合速率与对照差异不明显,大部分转基因小麦植株的光合速率得到明显提高,最高值达到31.95μmolCO2m-2s-1,比对照提高了26%,明显高于本生态区小麦物种已有报道的最高表达值,为高光效转基因小麦新品种的选育、小麦高光效转基因育种理论与方法的研究工作提供了重要材料。更多还原

【Abstract】 Wheat is one of most important crops in the world, which productivity ability and supply-demand situation always impact national economic development and the food safety. With the development of the society and the increase of population, demand for food keeps increasing in the past, today and future. Photosynthesis is the basis of all green plants, and enhancement of light use efficiency is essential for higher yield in major crops. Comparative to C3 crops, C4 crops, such as Zea mays、Sorghum vulgare and Saccharum officinarum have CO2 concentration mechanism with higher light compensation point, lower CO2 compensation point, and weaker photorespiration, especially under high light intensity, high temperature and drought. Introduction of C4 key enzyme genes into C3 plants to improve their photosynthetic characteristics and grain yield has been an active research area in plant biology at home and abroad.Phosphoenolpyruvate carboxylase (PEPC) is one of the key enzymes in C4 photosynthetic pathway, it main distribute to cytosol of mesophyll cell in C4 plants. The C4 pathway forms CO2 concentration mechanism, providing CO2 for C3 pathway in bundle sheath cells, severs as a“CO2 pump”. Pyruvate, orthophosphate dikinase (PPDK) is potentially rate limiting in C4 pathway, also plays important roles in C4 pathway. PPDK is located in the chloroplast of mesophyll cell, catalyzing the reproduction of PEP that is CO2 first receptor. To date, many studies indicated that the photosynthesis efficiency and the grain yield of C3 crops can be improved by introducing C4-specific pepc gene into C3 plants.In order to obtain high photosynthetic efficiency transgenic wheat germplasms with C4 photosynthetic characteristics, a full-length cDNA for PEPC and a full-length cDNA for PPDK were isolated from maize inbred line Z561, which was high photosynthetic efficiency and PEPC activity. Furthermore, plant high efficient expression vector p3301-pepc containing maize pepc gene and select marker bar gene was introduced into wheat mediated by particle bombardment transformation. Hopefully, we may thereby be able to provide the theoretical and technical support for the improvement of photosynthetic efficiency of wheat varieties and accomplish high photosynthetic efficiency transgenic breeding.The results were as follows:1. A full-length cDNA for PEPC was isolated from Zea mays by homology-based cloning technology. The nucleotide sequence was characterized that the sequence length was 3053 bp, containing one ORF with 2910 bp which coded a peotide 970 amino acids. After alignment on NCBI by Blast, the identity of the cloned fragment with pepc genes from X15238 was 99%. There were 16 differences in the nucleotide sequence, resulting in 5 differences in the amino acid sequence, and which had no effect on the PEPCase activity. After alignment on DNAMAN program, the identities of the cloned fragment with PEPC genes from Saccharum officinarum C4-form, Sorghum vulgare C4-form and Setaria italica C4-form were about 91%、90.2% and 83.2%, respectively; and that from Oryza sativa C3-form, Sorghum vulgare C3-form, Setaria italica C3-form and Echinochloa crus-galli C3-form were about 74.1%、75.1%、76.8% and 75.7%. Alignment and phylogenetic analysis of the amino acid sequence deduced from the fragment and the PEPCase sequence of other plants retrieved from GenBank were carried out by DNAMAN program showing the sequence was closer to Saccharum officinarum C4-form and Sorghum vulgare C4-form than to Setaria italica C4-form. The function sites of the PEPC protein were analysised using Interproscan program on EMBL , and the resultes showed that the amino acid sequence of Zea mays C4-form PEPC protein had seven conserved domains.2. A full-length cDNA for PPDK was isolated from Zea mays by homology-based cloning technology technology. The nucleotide sequence was characterized that the sequence length was 3014bp, containing one ORF with 2757 bp. After alignment on NCBI by Blast, the identity of the cloned fragment with ppdk genes from J03901 was 92.94%.3. The full-length cDNA sequence coding for maize PEPCase gene (GenBank accession number: FJ415327) was cloned into the high-efficiency binary expression vector pCAMBIA3301, named as p3301-pepc.4. The binary vector p3301-pepc was introduced into six wheat varieties using particle bombardment transformation method. A total of 409 PPT resistant plants were obtained, among them the expected 668bp fragment was present in 357 of the 409 PPT resistant plants. The integration of maize pepc gene in transgenic wheat plants was confirmed by Southern blotting analysis. The copy number of different transgenic lines was analyzed by real-time fluorescence quantitative PCR. Among the fifteen positive transgenic lines, seven had one copy number, three had two copies, two had three copies, one had five copies, one had eight copies and one had seventeen copies. RT-PCR analysis results showed that the foreign gene could expressed normally in transgenic wheat plants, and expression strength in transgenic plants was distinct remarkably. Physiological studies showed that the transformants displayed higher photosynthesis than non-transformed controls.5. Genetic analysis of T1 transgenic lines showed that the foreign pepc gene could stability transmit to the progeny, with the segregation ratios of most transgenic lines following Mendelian manner. Expression level of the maize C4-spcific pepc gene in T1 transgenic lines was determined firstly by assaying the activity of PEPC, and the results displayed that partly transgenic lines had higher PEPC activities than untransformed control, with the highest enhancement of 2.17 fold. SDS-PAGE electrophoresis analysis of leaf protein in transgenic and untransformed plants revealed that expression level of pepc gene was different in transgenic lines, in accord with the results of PEPCase activity; Photosynthetic physiological analysis Under field condition showed that net photosynthesis rates of most T1 transgenic lines were significant higher than non-transformed control, the highest photosynthesis was (31.95μmolCO2m-2s-1) distinctly higher than the max value reported under normal condition in Huaihuaihai planting area, and increased by 26% than non-transformed control.The results described above provid important research materials, theoretical and methodology for high photosynthetic efficiency transgenic wheat breeding.更多还原