【作者】 叶凌凤；

【导师】 康定明；

【作者基本信息】 中国农业大学 ， 作物遗传育种， 2013， 博士

【摘要】 合理株型是作物高产品种的形态特征和生育基础,其中矮生性状是理想株型的一个重要方面。培育矮化玉米杂交种,可以降低株高,改善株型结构,增加种植密度,提高群体光合效能,进而达到高产的目的。在玉米上共定位到了100多个和株高相关的QTL,但很少被克隆并应用于遗传改良。本研究组前期在玉米的l号染色体上1.07bin位置定位一个控制株高QTL-qph1, qph1目标区段位于玉米Br2(Brachytic2/ZmPGP1)基因的第5外显子上,SNP5259造成由精氨酸至亮氨酸的替换可能为其功能位点。该QTL产生较为温和的矮化表型,并且在育种上表现出极大的潜力。本研究在前期研究的基础上,进一步对定位到QPH1基因进行鉴定和功能分析,阐明其影响株高的分子机制,同时将SNP5259恢复突变,转化同源的拟南芥的突变体以及相应玉米材料,进一步验证其调控株高的功能位点。为理解玉米株高发育的分子机理与培育适宜株型奠定相关理论基础。研究结果如下：1.定位的QPH1基因为玉米Br2基因的新等位基因,编码的蛋白质含有1379个氨基酸,含有两个跨膜结构域和两个ATP结合区。克隆出该基因上游1kb的启动子区域,序列分析表明该区域含有光响应元件、激素响应元件等相关顺式作用元件。2.通过PromoterQPH1::GUS以及Real-time PCR分析表明QPH1基因主要在玉米的根、茎、叶器官中表达,并且两个玉米材料在不同部位的基因表达量并没有显著差异；亚细胞定位表明QPH1编码的蛋白定位在细胞膜上。采用[3H]IAA同位素示踪法测定生长素运输量表明qph1突变体材料生长素运输量降低程度小于br2的无效突变体。3.采用定点突变PCR将SNP5259恢复突变后的基因mqph1构建过量表达载体转化拟南芥atpgp1突变体和玉米qph1突变体RIL88材料,结果表明过量表达mqph1可以部分恢复拟南芥突变体在下胚轴长度、株高以及生长素运输量方面的缺陷,其恢复程度和QPH1基因相当,但好于qph1基因。转基因玉米也表现出了茎部粗壮,株高明显增高的表型。证实了SNP5259确实为qph1影响株高的功能位点。4.本研究在鉴定拟南芥三个atpgp1突变体中发现一个不同于研究报道的突变体,该突变体表现出较短的下胚轴长度和明显的矮化表型,生长素在根尖的分布和含量以及对NPA敏感性都不同于野生型,生长素运输量也低于野生型。T-DNA插在AtPGP1基因第三个外显子上,RT-PCR分析表明该突变体为一个完全突变体,之前报道研究所用的两个突变体中AtPGP1基因可以编码一个含有623个氨基酸的截短蛋白,可能并不是无效突变体。更多还原

【Abstract】 Rational plant type is the morphological characteristics and reproductive basis of high-yield variety. Dwarfism is an important aspect of ideal plant type. Cultivating dwarf maize hybrids, can improve the structure of plants, increase planting density and improve group photosynthetic efficiency, thus to achieve the goal of high yield. Although more than100of maize plant height QTLs have been identified, few were cloned and applied to the genetic improvement. One maize plant height QTL-qphl has been identified by our study group, which was located at1.07bin chromosomes1. The confidence interval of qphl locates on the5th exon of maize Br2gene (Brachytic2/ZmPGPl); SNP5259cause an amino acid substitution from Arginine to Leucine and is proposed to be the functional site of qphl. The QTL caused moderate dwarf phenotype and exhibited great potential in breeding. This study is based on the previous studies, the QPH1gene identification and function analysis for further study and elucidated the molecular mechanism of plant height control. To further identify the functional site of SNP5259, we did back mutation of SNP5259and transformed to the Arabidopsis thaliana pgpl mutant and maize qph1mutant. These studies laid the theoretical basis of molecular mechanism of plant height growth and cultivation of suitable plant type. Results for these studies include:1. QPH1gene is a new allele of maize Br2gene, which encode a protein containing1379amino acids. The protein has two transmembrane domains and two nucleotide-binding domains. We cloned the1Kb promoter region of QPH1gene; sequence analysis showed that this region contained light response element and hormone response element.2. PromoterQPH1::GVS and Real-time PCR analysis indicated that QPH1gene express in root, stem and leaf, and there is no expression significant difference at different part between two maize lines. The protein encoded by the QPH1gene located in the cell membrane. The decreasing degree of auxin transport in qphl mutant is smaller than the br2null muatant.3. Site-directed mutagenasis by PCR, mqph1(mutagenized qphl) was transformed to the Arabidopsis thaliana pgpl mutant and maize qphl mutant.Over expression of mqph1can restore the plant height,hypocotyl length and auxin transport defect, and no difference between QPH1and mqphl transgenic plants. The transgenic maize exhibit stronger stem and higher plant height. The results confirm that SNP5259is the functional site of QTL-qphl.4. We found a different atpgpl muant from the mutants reported before, the mutant show shorter hypocotyls length and dwarfism phenotype. The distribution and content of auxin in root tip, and the NPA sensibility are different from the wild type. The relative auxin transport is lower than wild type. The mutant contained a T-DNA insertion in the third exon and was a null mutant, the muants used in the studies before may encode a truncated protein contained623amino acids.更多还原