【作者】 王立静；

【导师】 刘保申； 金危危；

【作者基本信息】 山东农业大学 ， 作物遗传育种， 2012， 博士

【摘要】 玉米是世界重要粮食作物，其产量的高低在国民经济的发展中起着重要的作用。玉米产量和品质受多种环境胁迫和生物胁迫的影响。玉米的倒伏是严重影响玉米生产的限制因素，分离矮化基因并研究其功能具有重要的理论实践意义。除此之外，玉米的生长发育还受微生物感染、合成或降解途径的基因突变，甚至细胞程序性死亡等很多因素影响，分离坏死基因并研究其功能也具有重要的理论意义。本文对矮秆突变体和坏死突变体的表型、候选基因和功能做了分析，结果如下:1.矮秆突变体属GA缺陷型矮化突变体：矮秆突变体株高较矮只有高秆株高的49.57%，节间均匀缩短，结实正常，种子变小，生育期与高秆相比略有推迟。扫描电镜观察表明矮化是由节间纵向细胞的伸长受阻引起的。通过不同浓度外源激素GA、BR和IAA处理，结果发现外源GA能促进突变体第二叶鞘的伸长，有效回复矮化的表型；外源BR在黑暗或是光照下均不能回复矮化的表型；外源生长素IAA也不能促使突变体第二叶鞘的伸长。据此，我们认为矮化突变体是一个GA缺陷型矮化突变体。2.矮化突变候选基因Dt的克隆分析与确定：前期的定位结果将Dt基因定位在分子标记187790-4与c187790-15之间物理距离68Kb的区间，对区间的基因分析找到3个开放阅读框（ORF）：GRMZM2G040359（植物脂肪酸水解酶）、GRMZM2G040673（植物特异TIGR01568蛋白家族）、GRMZM2G016477（油菜素类固醇不敏感受体激酶BAK1前体）。对三个基因cDNA和DNA测序分析，发现突变体中基因GRMZM2G040673的第663个碱基发生突变，导致蛋白质第221个氨基酸由赖氨酸突变为甲硫氨酸。8个玉米自交系的测序结果进一步验证了该基因的特异点突变，初步确定GRMZM2G040673为矮秆候选基因Dt。Dt基因组序列全长1720bp，编码383个氨基酸。Dt蛋白是一个含高度保守OVATE结构域的转录因子蛋白家族。3.矮秆基因Dt的时空表达分析：半定量RT-PCR和Real-time PCR结果发现Dt基因在根、茎、叶、雄穗、雌穗中都有表达，在茎和雌、雄穗中的表达量较高，矮秆突变体中Dt基因的表达与自交系P11中没有明显差异；RNA原位杂交结果表明Dt基因的表达信号遍布整个分生组织区，顶端分生区中心信号最强，表达水平最高，与突变体中Dt基因表达差异不明显。4.矮秆基因Dt的功能验证：OVATE基因是植物特有的一类转录抑制因子。Dt-GFP融合蛋白转染洋葱表皮细胞的亚细胞定位结果显示Dt蛋白定位于细胞核内，是转录因子蛋白。通过构建RNAi、过表达载体和功能互补载体转化玉米验证Dt基因功能，结果显示Dt基因干扰后，功能改变，出现矮化表型；Dt基因过表达和互补载体转化玉米，得到T0代转基因植株，表型有待于鉴定。通过对突变体中GA合成代谢途径中一些关键酶基因的表达分析，发现突变体中与GA合成相关的基因ZmKS的表达量降低，推测ZmKS是Dt基因的靶基因。5.黄化坏死突变基因nec-t的鉴定：黄化坏死叶突变体nec-t两叶期表现黄化，叶绿素含量降低，有坏死斑点，并在正常光照下死亡。透射电镜结果表明：突变体nec-t中类囊体分散，基粒片层减少，叶绿体数目也减少。前期将nec-t基因定位131.7Kb的区间内，有4个开放阅读框（ORF）：LOC100274241、LOC100280469、AC215678.3和LOC100192977。测序发现突变体中基因LOC100192977的5′非翻译区有7bp的插入突变。初步确定LOC100192977为黄化坏死基因的候选基因nec-t。该基因在玉米叶片中表达量最高，在根和茎中也有表达，但表达量相比叶片中的要弱很多，几乎检测不到；突变体中该基因的表达相比B73有所降低。nec-t基因编码的甲硫氨酸氨肽酶，负责切除新生肽链N端的起始甲硫氨酸，对叶绿体的定位和功能调节极其重要。更多还原

【Abstract】 Maize (Zea mays L.) is an important crop in world. Its yield plays a role in nationaleconomy. The yield and quality are influenced by environmental stress and biological stress.Maize lodging happened frequently, which influenced the yields seriously. The creation andscreening of new maize dwarf genes and functional analysis have important theoretical andpractical significance. Beyond that its growth and development can be reduced by mutationsof biosynthetic and degradative pathways which can cause necrosis or even programmed celldeath. Hence, Cloning necrotic genes and functional analysis have important theoreticalsignificance. In present study we described the phenotypes of dwarf mutant and necroticmutant, candidate genes and functional analysis. The results were as follows:1. The dwarf mutant was a GA-deficient mutant. The plant height of dwarf plants wasonly49.57%of high individuals. The dwarf mutant plant had shortened internodes, normalcorn, small seed and delayed growth period. Cytological observation revealed that theinternodes were shortened because the stretches of cells in dwarf individuals were inhibited.The exogenous bioactive GAs can successfully made the second sheath elongate and reversedthe dwarf phenotype. But the exogenous BR and IAA did not make the second sheathelongated. So, the dwarf mutant was a GA-deficient mutant.2. Determining and cloning analysis of the candidate gene Dt. We mapped the Dt genebetween markers187790-4and c187790-15. The physical distance was about68Kb andcontained three candidate genes：GRMZM2G040359(GDSL-motif lipase/hydrolase familyprotein), GRMZM2G040673(Plant-specific domain TIGR01568family protein) andGRMZM2G016477(BRASSINOSTEROID INSENSITIVE1-associated receptor kinase1precursor). We sequenced the cDNA and DNA of the three genes and found a singlenucleotide mutant in the663bp of GRMZM2G040673. This led to the lysine change intomethionine. It was a solid foundation for cloning the dwarf gene and studying its functions. We also sequenced eight lines and confirmed the specific mutant. The full length of Dt gene is1720base pair, and encoded383amino acid. The Dt protein belong to OVATE FamilyProtein.3. Temporal and spatial expression patterns of dwarf gene Dt. Dt was specificallyexpressed in stems, tassels and ears by semi-quantitative RT-PCR and quantitative PCR. Theexpression in mutant had no difference with P11. The in situ hybridization of Dt indicated thetranscripts were detected in all meristematic tissue. But expression of Dt in apical meristemwas highest. There was no significant difference between dwarf mutant and P11.4. Functional verification of dwarf gene Dt. OVATE gene was a transcription repressor inplant. Dt-GFP sub-cellular localization analysis indicated that Dt gene was localized in thenucleus. In order to analysis the function of Dt, we constructed Dt-RNAi interfere vector,overexpression vector, and complementary vector. There were a few of dwarf plants intransgenic offspring after Dt was interfered. We also acquired the T0transgenic seedlings ofoverexpression vector and complementary vector. The expression level of ZmKS which wasthe key enzyme of the GA biosynthesis pathway was down regulated. We inferred ZmKS wasthe target of Dt gene.5. Identification of yellow and necrotic mutant gene nec-t. The nec-t mutant plant hadyellow leaves with necrotic spot and reduced level of chlorophyll content at the two-leaf stage,and later on etiolated seedlings died under normal growth conditions. In the mutant leaves,transmission electron microscopy revealed scattered thylakoids, and reduced number of thegrana lamellae and chloroplasts per cell. The nec-t was mapped to a131.7kb region and fourpredicted genes were identified as candidates in the target region. Cloning and sequencing ofthe target region showed that the same7bp insertion mutation occurred in the5′-untranslatedregion (UTR) of the locus LOC100192977. RT-PCR analysis indicated that the nec-ttranscriptional level was mainly expressed in leaves and less in roots and stems both in B73and nec-t mutant. And transcriptional level was significantly reduced in the leaves of mutant.Methionine aminopeptidase that removes the initiator methionine and exposes the penultimateN-terminal amino acid residue plays important role in determining the chloroplast proteinstability. Further studies on functional confirmation of nec-t and the mechanism of lesionformation are currently underway.更多还原