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试验旨在分析运用分子标记技术(QTL)和差异蛋白组学技术研究作物化感作用分子机理的差异性。首先运用差异蛋白组学技术探讨在生物胁迫(稗草)下水稻化感作用潜力变化的内在分子机理。分别用稗草和水稻的根系分泌物培养切自一株5叶龄化感水稻P I312777植株并经恢复的2个分蘖。7d后,提取处理和对照相同叶位叶片的全蛋白质并进行双向电泳,每张电泳胶片上获得800多个电泳胶点,其中差异表达的蛋白质点有4个。采用M ALD I-TOF-M S对各差异蛋白质点进行肽质量指纹图谱分析,经过SW ISS-PROT数据库查询,结果表明化感水稻P I312777在稗草胁迫下的特异蛋白分别与苯丙氨酸氨解酶(PAL)、硫还原型蛋白(T rx-m)、3-羟基-3-甲基戊二酰辅酶A还原酶(HM GR)和过氧化物酶(POD)相匹配。根据编码以上4个差异蛋白质的DNA序列,发现编码以上4个差异蛋白的基因分别位于水稻染色体4、7、8和12上的特定克隆位点,这就是与化感作用相关基因。前人也运用QTL方法开展作物化感作用的分子机理研究,但由于所采用的供体材料、受体植物及对表型性状的评价方法等的不同,定位结果存在较大的差异。综合比较两种方法后认为,运用差异蛋白组学技术分析水稻化感作用的分子机理,比QTL技术更加直接和深入。因为比较胁迫处理和对照植物组织的2-DE图谱将能鉴定出由表达候选基因编码的胁迫蛋白质,氨基酸残基序列的测定将揭示那些功能与胁迫性状密切相关的蛋白质,这种编码的基因就是兼具功能与表达的候选基因。
The purpose of the experiment was to analyze the molecular mechanism of crop allelopathy using molecular marker techniques (QTLs) and differential proteomics techniques. First of all, differential proteomic techniques were used to explore the underlying molecular mechanism underlying the potential of rice allelopathy under biotic stress (barnyardgrass). Respectively, two tillers which were cut from a 5-leaf-sensitive rice P I312777 plant were cultured with root exudates of barnyardgrass and rice. After 7 days, the total protein of the leaves of the same leaf position was extracted and compared by two-dimensional electrophoresis, and more than 800 electrophoresis gel spots were obtained on each electrophoresis film, of which four protein spots were differentially expressed. Peptide mass fingerprinting analysis was carried out using M ALD I-TOF-M S, and the results of SW ISS-PROT database showed that the specific proteins of allelopathic rice P I312777 under barnyardgrass stress were compared with phenylalanine (PAL), sulfur-reduced protein (T rx-m), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HM GR) and peroxidase (POD). According to the DNA sequences encoding the above four different proteins, it was found that the genes encoding the above four different proteins were located at specific cloning sites on rice chromosomes 4, 7, 8 and 12, respectively, which is the allele-related gene. Previous studies also used QTL methods to study the molecular mechanism of crop allelopathy. However, due to the different donor materials, receptor plants and methods used to evaluate phenotypic traits, the localization results are quite different. After a comprehensive comparison of the two methods, it is considered that the molecular mechanism of rice allelopathy using differential proteomics technology is more direct and in-depth than the QTL technology. Because comparing the 2-DE maps of stress treatment and control plant tissues will identify stress proteins encoded by candidate genes for expression, the determination of the amino acid residue sequence will reveal those proteins that are functionally associated with stress traits, which are Candidate genes with both function and expression.