【作者】 杨海荣；

【导师】 胡秀丽；

【作者基本信息】 河南农业大学 ， 植物学， 2011， 硕士

【摘要】 为确定脱落酸（ABA）和H₂O₂在干旱高温复合胁迫诱导的玉米（Zea mays L.）叶片和根系小热休克蛋白（sHSPs）和抗氧化防护中的作用,本研究以玉米郑单958及ABA缺失突变体vp5为实验材料,首先通过引物设计和RT-PCR分析筛选出干旱高温复合胁迫诱导产生的sHSPs基因,其次采用ABA抑制剂钨酸钠（T）和H₂O₂清除剂KI（I）,通过RT-PCR分析ABA和H₂O₂对玉米叶片和根中sHSPs和抗氧化防护酶基因表达的影响,最后通过Western blot分析验证了sHSP26的表达。具体研究结果如下:1.从NCBI数据库（http://www.ncbi.nlm.nih.gov/）GenBank中查找出玉米sHSPs已发表的全部基因序列,利用软件Dnaman对不同家族的sHSPs基因序列分别进行比对分析,并利用软件Oligo6.0分别在其保守区设计引物,最后通过RT-PCR筛选出诱导型sHSPs基因,本实验筛选出的诱导型sHSPs基因分别有:sHSP16.9、sHSP17.2、sHSP17.4、sHSP17.5、sHSP22和sHSP26。另外还分别对四种抗氧化防护酶的基因设计了引物:过氧化氢酶1（catalase 1,CAT1）、抗坏血酸过氧化物酶（cytolic ascorbate peroxidase,cAPX）、谷胱甘肽还原酶1（glutathione reductase 1,GR1）和超氧化物歧化酶4（superoxide dismutase 4,SOD4）。2.ABA抑制剂钨酸钠和H₂O₂清除剂KI预处理结果表明:（1）六种sHSPs基因在玉米叶片中的表达分别都受到T不同程度的抑制,而根中只有sHSP16.9、sHSP17.2、sHSP17.5和sHSP22表达稍微受到抑制,CAT1、cAPX、GR1和SOD4四种抗氧化防护酶基因表达在玉米叶片和根中均受到T明显抑制。（2）与未用100μM ABA预处理的vp5相比,100μM ABA预处理明显增强了四种抗氧化防护酶基因在突变体vp5叶片和根中的表达,但是仅稍微提高了六种sHSPs基因在叶片中的表达和sHSP17.2在根中的表达。（3）KI预处理明显抑制了四种酶基因在玉米叶片和根中的表达,但是稍微抑制了六种sHSPs基因在玉米叶片中的表达和sHSP16.9、sHSP17.2、sHSP17.5、sHSP22和sHSP26五种基因在根中的表达。3. Western blot实验检测分析sHSP26的表达,结果显示,在玉米叶片中T和I均明显抑制了sHSP26的表达,而在根中,T对sHSP26的表达几乎没有影响而I仅稍微抑制了sHSP26的表达。这些研究结果为进一步研究植物抗逆机制及基因克隆奠定基础,并为揭示植物在多胁迫条件下耐逆机理提供重要的科学证据。更多还原

【Abstract】 In order to determine the effects of H₂O₂ and ABA on small heat shock protein （sHSPs） and antioxidant defense enzymes induced by the combination of drought and heat stress in maize （Zea mays L.） leaves and roots, Zheng dan 958 and ABA deletion mutant vp5 maize plants as experimental materials were used in this study. Fristly, through primer designing and RT-PCR analyzing, selected out sHSPs that induced by the combination of drought and heat stress. Secondly, ABA inhibitor tungstate （T） and H₂O₂ scavenger KI（I） were used to analyse the influences of H₂O₂ and ABA on sHSPs and antioxidant defense enzymes genes expression induced by the combination of drought and heat stress in maize leaves and roots by RT-PCR. Finally, the expression of sHSP26 was analysed by western blot. The detailed research results are as follows.1、Searching out all of maize sHSPs gene sequences published in NCBI database GenBank. Fristly, sHSPs gene sequences with different families were compared and analysised by Dnaman software; Then, primers in its conservative districts were designed by Oligo6.0 software. In the end, sHSPs genes induced by combined drought and heat stress were selected out by RT-PCR, which incluced sHSP16.9、sHSP17.2、sHSP17.4、sHSP17.5、sHSP22 and sHSP26. In addition, the four antioxidant defense enzymes such as catalase 1（ CAT1）、cytolic ascorbate peroxidase （ cAPX）、glutathione reductase 1 （ GR1） and superoxide dismutase 4 （SOD4） primers were also designed.2、The results of pretreatment with ABA inhibitor T and H₂O₂ scavenger KI showed that: （1） The six sHSPs genes expression in maize leaves were all inhibited by T more or less, but only sHSP16.9、sHSP17.2、sHSP17.5 and sHSP22 were slightly inhibited in maize roots. The expression of four antioxidant defense enzymes gene SOD4, GR1, CAT1 and cAPX were all significantly inhibited by T both in maize leaves and roots. （2） Compared with vp5 maize plant that without 100μM ABA pretreatment, the pretreatment significantly increased the four enzymes genes expression in leaves and roots, but only slightly increased the six sHSPs genes expression in maize leaves and sHSP17.2 expression in roots. （3） The pretreatment with KI significantly inhibited the genes expression of four antioxidant defense enzymes both in maize leaves and roots and slightly inhibited the genes expression of six sHSPs in maize leaves, but only slightly inhibited the genes expression of sHSP16.9、sHSP17.2、sHSP17.5、sHSP22 and sHSP26 in maize roots.3、The results of sHSP26 expression analysed by western blot showed that, sHSP26 was significantly inhabited by T and KI in maize leaves, but T was almostly no influence to sHSP26 expression and KI only slightly inhabited sHSP26 expression in maize roots.These results supplied the basis for further researching the mechanisms of plant response to stress and gene cloning, and also offered important scientific evidences to reveal the mechanism of plants tolerant to combination stress.更多还原