【作者】 胡轶；

【导师】 李晚忱；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 干旱是玉米生产的主要非生物限制因素。耐旱性是众多形态结构与生理生化性质综合作用的结果,其分子机制非常复杂。研究玉米耐旱性的分子遗传特性,克隆与耐性相关的基因,研究其在干旱胁迫条件下的表达特性,可为功能基因选择标记的开发提供依据,以期通过分子标记辅助选择改良提高耐旱性。本研究以玉米耐旱自交系“81565”和不耐旱自交系“200B”为材料,用16%PEG模拟干旱处理,正常浇水为对照,分别于胁迫17,24和48h,取芯叶下第一片叶提取总蛋白。用等电聚焦和SDS-聚丙烯酰胺凝胶双向电泳分离总蛋白样品,GS-170光密度成相系统扫描2-DE凝胶,然后用PDQuest-6.0图象分析软件比对分析耐旱自交系和不耐旱自交系在干旱胁迫与对照条件下叶片蛋白质的表达差异。从12块样品胶生成的参照图谱中鉴定出的500多个蛋白点中,有58个在干旱胁迫条件下差异表达。其中,蛋白点2506,3507和4506在干旱胁迫下被诱导表达,最高表达量出现在胁迫24h。将这3个蛋白点酶切后,用基质辅助激光解吸离子化-飞行时间质谱(MALDI-TOF-MS)根据酶切片段荷质比的不同来测定其分子量。然后在Mascot和Ms-Fit数据库中比对,根据MOWSE分值来鉴定蛋白质。点2506、3507和4506与拟南芥肉桂醇脱氢酶(CAD)、拟南芥细胞色素96A8(CYP96A8)和Elaeagnus umbellata的S-腺苷酸-L-甲硫氨酸合成酶(SAMS)的序列比对结果,MOWSE分值分别为66、68和68。因为上述三个功能蛋白都参与木质素生物合成,所以在分析木质素合成途径生化过程的基础上,我们用实时定量PCR技术分析了耐旱性不同的玉米自交系,在干旱胁迫条件下,木质素合成关键酶—肉桂醇脱氢酶(CAD)和咖啡酸-O-甲基转移酶(COMT)的mRNA表达差异。在耐旱自交系“81565”、“200B”和“87-1”中,CAD和COMT的表达量在胁迫初期都有大幅度下降,但随着胁迫时间延长表达量又会明显回升;而在不耐旱自交系“丹340”中,CAD和COMT的表达量在胁迫初期大幅度下降后就再也没有明显的回升。为在表型上进一步分析木质素合成与耐旱性的关系,我们选用“81565”、“87-1”、“200B”、“18-599”、“18-599M”和“ES40”6个耐旱性不同的玉米自交系,在不同干旱胁迫强度及正常浇水条件下,测定叶片本质素含量,同时调查雌雄花期间隔和单株籽粒产量,并转换为耐旱系数进行相关分析。结果表明,耐旱性不同的自交系的叶片木质素含量对干旱胁迫的反应大不相同。在干旱胁迫条件下,耐旱性较强的自交系木质素含量显著升高,平均耐旱系数较大,耐旱性中等的自交系木质素含量比正常灌水对照略有升高,耐旱性较弱的自交系木质素含量与对照相当或下降。在中等干旱和严重干旱条件下,叶片木质素含量平均耐旱系数与单株籽粒产量和雌雄花期间隔耐旱系数的相关系数,分别为r=0.848和r=0.889,均达显著水平。综上所述,叶片的木质化是玉米对干旱胁迫的一种主动防御反应,木质素合成相关基因的差异表达与不同自交系的耐旱性有关,是造成其耐旱性差异的原因之一。在进一步研究这些基因与耐旱性分子遗传机制关系的基础上,可根据其在不同自交系中的单核苷酸多态性及表达调控方面的差异,开发功能基因选择标记,应用于玉米耐旱育种的分子标记辅助选择。更多还原

【Abstract】 Drought is the most widespread abiotic constraints in maize production. The molecular mechanism is believed to be complex because drought tolerance is the result of cooperative interactions among multiple morphological, physiological and biochemical characters. Information of molecular genetic properties, cloning and differential expression of drought-tolerant candidate genes is useful to selection marker development of functional genes and their application to marker assisted selection to improve drought tolerance in maize.Drought-tolerant inbred line ’81565’ and sensitive line ’200B’ were treated by using 16% PEG to simulate drought stress at 7 leaf stage, with well-watering control. The top first fully open leaf was sampled at 17, 24 and 48 h after drought treatment for total protein extraction. The total protein samples were separated by two-dimensional electrophoresis of isoelectric focusing and SDS-polyacrylamide gel, scaned by GS-170 scan system and analysed by PDQuest-6.0 image analying software for their differential expression in drought tolerant and sensitive inbred lines under drought stress and well-watering control. More than 500 protein spots were identified from the consensus master map constructed from 12 scaned images. Fifty-eight of them were found in response to drought stress. The expression of spot 2506, 3507 and 4506 was induced under drought stress. The highest expression amount was found at 24 h of drought stress. These three protein spots were digested and used for measurement of mass to charge ratio and molecular weight by MALDI-TOF-MS. Alignment was conducted against Mascot and Ms-Fit databases and MOWSE scores were used for protein identification. The result showed that spots 2506, 3507 and 4506 had MOWSE scores of 66, 68 and 68 with cinnamyl alcohol dehydrogenase (CAD), cytochrome protein 96A8 (CYP96A8) and S-adenosyl-L-methionine synthase (SAMS), respectively.Because these three functional proteins are involved in lignin biosynthesis, we used real time PCR technique to analyse the mRNA differential expression of two key enzymes in lignin biosynthesis pathway, cinnamyl alcohol dehydrogenase (CAD) and caffeic acid 3-O-methyltransferase (COMT), in different inbred lines under drought stress. In drought-tolerant lines ’81565’, ’200B’ and ’87-1’, the expression of CAD and COMT showed an obvious picking up after the significant decrease at the early stage of drought stress. However, in sensitive line ’Dan340’, this kind of picking up was not found after the decrease at the early stage.To analyse the phenotypic relationships between lignin biosynthesis and drought tolerance, leaf lignin content, anthesis-silking interval and per plant grain yield were investigated with inbred lines ’81565’, ’87-1’, ’200B’, ’18-599’, ’18-599M’ and ’ES40’ of different drought tolerance under strong and moderate drought stress as well as well-watered control, and converted into drought tolerant coefficients for correlation analysis. Significant difference was found in the response of leaf lignin content of inbred lines with different drought tolerance to drought stress. Under drought stress, the leaf lignin content of highly tolerant inbred lines increased significantly with a high average drought coefficient, the leaf lignin content of intermediate tolerant inbred lines increased slightly, while the leaf lignin content of drought sensitive inbred lines matched with the well-watered control or decreased slightly. Under the strong and moderate drought stress, significant correlation coefficients (r = 0.848 and r = 0.889) were estimated between the average drought tolerant coefficient of leaf lignin content and the drought tolerant coefficients of per plant grain yield and anthesis-silking interval.As a conclusion, lignification of leaf can be speculated to be a kind of active response of maize to drought stress. The differential expression of the candidate genes to lignin biosynthesis is relative to drought tolerance of different inbred lines. Based on detail research on the relation of these genes to molecular genetic mechanism of drought tolerance, selection markers of functional genes can be developed according to the difference of single nucleotide polymorphism and expression regulation among different inbred lines, and applied to molecular assisted selection of drought tolerance breeding in maize.更多还原