应用差异蛋白质组学方法分析作物化感作用的分子机理

Analyzing the molecular mechanism of crop allelopathy by using differential proteomics

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。

In this paper, differential proteomic method was employed to study the molecular mechanism of crop allelopathy. The traditional QTL method was also compared. Experiments were conducted to determine the molecular mechanism of rice allelopathy under the biotic stresses induced by barnyardgrass （Echinochloa crus-galli L. ）. One of the two cloning tillers, obtained from single rice plant at 5 leaf-stage, was cultivated in pots with the addition of root exudates of either rice or barnyardgrass, respectively. At 7d, their leaf proteins were extracted for two-dimensional electrophoresis （2-DE）. More than 800 proteins were resolved in each 2-DE gel and four leaf proteins displayed differentially. The four proteins showed high degree of reproducibility in the allelopathic rice response to the stresses of barnyardgrass. Individual protein spots were excised respectively from the 2-DE gel and measured by MALDI-TOF MS. Their Peptide Mass Finger Prints （PMF） were obtained. Further SWlSS-PROT database search identified four matched proteins： peroxidase 2 precursor （POD）, phenylalanine ammonia-lyase （PAL）, 3-hydroxy-3-methylglutaryl-coenzyme A reductase 3 （HGMR） and thioredoxin M-type （Trx-m）. The genes ecoding these four differential proteins were located on the chromosome 4, 7, 8,and 12 of rice. The QTLs of rice allelopathy varied with the donor plants, receiver plants and bioassay methods used. QTL analysis based on its approximate map locations and its small effects on phenotype proved to be inefficient. Advances in proteomics provide opportunities for accurate identification of positional, functional and expressional genes. By comparison of 2-DE protein patterns obtained from the treated and untreated plants, a set of stress-responsive proteins encoded by expressional candidate genes could be identified. Sequencing of these stressed-responsive proteins will then reveal the gene functions associated with the stress tolerant trait. The encoding genes will thus be regarded as both expressional and functional candidate genes.