摘要
油菜是中国最重要的油料经济作物之一,土壤有效磷缺乏严重影响油菜的产量和品质。人们通过大量施用磷肥来提高油菜的产量,然而我国的磷矿储存量有限,而且磷肥的大量施用会污染环境,因此对植物暨油菜响应低磷胁迫的分析有助于解析植物对磷营养的吸收和利用,为作物的抗逆机理及遗传育种研究打下理论基础。
本研究首先建立油菜幼苗营养液水培的方法,在此基础上以甘蓝型油菜(中油821)为材料,结合前期研究基础,对低磷胁迫下油菜幼苗形态、相关生理特征进行分析；建立了基于管状胶的油菜蛋白双向电泳技术体系,得到了低磷胁迫下幼苗叶片、根系蛋白差异表达图谱并进行分析,为本实验室后续开展的油菜幼苗响应低磷胁迫的分子机理研究奠定基础。主要研究结果如下：
1、优化了油菜幼苗低磷胁迫的水溶液培养方法。结合后续实验要求和低磷营养液处理剂量、培养时间、培养步骤等因素,采取预培养环节并降低营养液浓度至四分之一,确定了适合于低磷胁迫的油菜幼苗水溶液培养方法。
2、考察了低磷胁迫下油菜幼苗的形态学指标。低磷胁迫下,油菜幼苗外部形态与对照比有明显变化。地上部分真叶叶片较小、颜色暗绿;根系变细,低磷胁迫下幼苗根系长度显著高于对照(处理后14天达到对照的122%)。
3、低磷胁迫下油菜幼苗根系相关生理学分析表明：低磷胁迫促进了油菜幼苗根系活力,低磷胁迫处理期间,油菜幼苗根系活力均有不同程度的提高,21天时为对照的127%；低磷胁迫诱导根系分泌磷酸酶,酸性磷酸酶活显著高于对照,在低磷肋、迫21天后为对照的187%；低磷胁迫在一定范围内提高保护酶活性,低磷胁迫下过氧化物酶和过氧化氢酶活性均有不同程度的升高,14天后达到最大值,分别为对照的205%和190%。
4、建立和优化了基于管状胶的油菜幼苗双向电泳体系(two-dimensional gel electrophoresis,2D-PAGE)。采用三氯醋酸-丙酮法提取总蛋白；选取7M尿素裂解液裂解(含7M尿素,4%Chaps,2M硫脲)蛋白干粉；确定叶片和根系蛋白的最佳上样量分别为400μg和260μg。
5、利用上述适合幼苗生长的水培方法和优化的双向电泳技术体系,对低磷胁迫处理的油菜幼苗根系、叶片蛋白双向电泳分析,获得了蛋白丰度、解析度较高的差异蛋白表达图谱。图像解析表明：差异蛋白稳定表达,图谱重复性较好,叶片和根系的相关系数分别为0.86和0.72；低磷胁迫下叶片中稳定上调表达的差异点为11个,下调表达的为11个；低磷胁迫下根系中稳定上调表达的蛋白点为25个,下调表达的为37个。
6、分析了低磷胁迫对油菜幼苗生长和生理影响的相关机理；探讨了有关自制管状胶双向凝胶电泳及蛋白图谱分析中存在的问题,为后续的实验奠定了良好的基础。
Brassica Napus L. is one of the most the important oil-bearing plants in China. The production and quality of Brassica Napus L. will decrease sharply when there is not enough phosphorous in soil. However, with too much phosphate fertilizer being used to improve the yield of crops, the environment will be polluted. The study on brassica seedings under phosphorus deficiency will help to understand the molecular mechanism of Brassica napus L. endured phosphorus deficiency stress, which consequently setted a foundation for a breeding research on Brassica Napus L.
For this study, an hydroponics method for culturing Brassica Napus L.(Zhongyou821) was first established. The young seedling characters of morphology under the stress phosphorus deficiency were surveyed. Also the root activity, the secreted acid phosphatase(Apase), the catalase(CAT) enzyme and the peroxide(POD) in seeding roots were evaluated. An system of two-dimensional gel electrophoresis (2D-PAGE) with tube gels suitable for brassica seedings was established. The leaf and root proteins under low-phosphorus stress were seperated using2D-PAGE, and the differential protein patterns were acquired and analysised. The main results were summarizeal as follows:
1. The different treatment dosage of phosphorus deficiency stress, the time cultured and the procedure cultured were evaluated. The concentration was optimized to1/4dosage of nutrient solution, and the pre-culture for3days before the treatment of phosphorus deficiency stress was proved to be available. And the modified hydroponics method of culturing brassica young seedlings adapted to the physiology and differential proteome analysis was established finally.
The morphological characteristics of brassica seedings under low phosphorus deficiency stress were investigated. The exterior aspects of the young seedlings showed an obvious difference after stress low phosphorus deficiency. The leaves of seedling subjected to phosphorus deficiency were small and dark green compared with control, while the root was longer and slender. The length of root under phosphorus deficiency stress was measured up to an increasement by22%compared to control.
2.The effects of low-phosphorus treatment on the physiology parameters of brassica seedings were conducted. The root activity presented an rapid increasement and then a slight decreasement at the treatment course of phosphorus deficiency. The root activity showed a constant enhancement against untreatment, with a1.27times activity as that of control after21-days'phosphorus deficiency. The acid phosphatase (Apase) of seeding root was secreted in low-phosphorus environment. And the activity of acid phosphatase showed a highest increasement at the21th day during treat course, which is1.87times as of control. The activity of protective enzymes, peroxide (POD) enzyme and the catalase(CAT), were promoted under phosphorus deficiency stress. The activity of POD was achived the highest folds of2.05to control at14th day, while the CAT1.90to control at14th day.
3. The two-dimensional electrophoresis method based tube gels for the separation of brassica young seedlings was established and optimized. Two protein extraction protocol from leaf and root were evaluated, and the Optimization is TCA-acetone precipitation method. Four protein lysis buffers for isoelectric focusing electrophoresis (IEF) suitable for tubel gels were compared together. The optimized lysis buffer is7M urea lysis buffer (including7M urea,4%Chaps,2M thiourea). After the comparison of protein quantity subjected to tube gel, the loading quantity of400and260p.g were present for2D-PAGE separation of leaf protein and root proteins, respectively.
4. The whole proteins extreated from hydroponics young seedlings under phosphorus deficiency stress were seperated by2D-PAGE. After dying by Coomassie Brilliant Blue, the proteomics image patterns were resolved by PDQuest8.0.1software. The correlation ratio of leaf protein differential patterns between21-day-treatment by phosphorus deficiency and its control is0.86.22proteins showed diffenently expressed under salt stress, with11up and11down. The correlation ratio of root protein differential patterns between16-day-treatment by phosphorus deficiency and its control is0.72.62proteins showed diffenently expressed under salt stress, with25up and37down.
5. In the paper, the effect of low-phosphorus treatment on the physiology parameters of brassica seedings were also discused. The feasible suggestions on procedure of2D-PAGE and image analysis were presented.
引文
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