| 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. |
