| Given low solubility and immobility in many soils of the world, phosphorus(P) may be the most widely studied macronutrient for plants. Rapeseed is an important oil crop in China, in which more than 80% of the plant area is occupied by Brassica napus. Brassica napus is sensitive to P deficiency and has high P requirement for growth and development. However, the available P of the soil in cultivated zone is generally low(< 10 mg/kg). Thus, the study on the mechanisms of Brassica napus in response to P deficiency has important significance for the genetic improvement of P nutrition in rapeseed production. Brassica napus P-efficient genotype “Eyou Changjia” and P-inefficient genotype “B104-2” were screened in our previously studies. In the present study, the differential abundance proteins in roots and leaves of the two genotypes, in response to long-term low P stress and short-term P-free starvation, respectively, were investigated by 2-DE. As well as the plasma membrane proteins in roots were investigated by label-free. Finally, the comparative genomics was performed to map the identified proteins to the linkage maps. The relationship between the differential proteins and QTLs for P efficiency was established, which provided us for novel information in understanding the adaptability of Brassica napus to P deficiency. The main results were obtained as follows: 1. Proteome analysis of Brassica napus under long-term low P stress Total proteins in roots and leaves were extracted from two genotype Brassica napus, P-efficient genotype “Eyou Changjia” and P-inefficient genotype “B104-2”, under long-term low P stress, respectively. And proteome analysis was performed by 2-DE to investigate proteome alterations in response to the long-term low P stress. Totally, 84 proteins whose abundance were significantly changed in response to long-term low P stress, which included 19 proteins in analysis set A(root proteins of “B104-2”), 24 proteins in analysis set B(root proteins of “Eyou Changjia”), 20 proteins in analysis set C(leaf proteins of “B104-2”) and 21 proteins in analysis set D(leaf proteins of “Eyou Changjia”), respectively. 2. Proteome analysis of Brassica napus under short-term P-free starvation Total proteins in roots and leaves were extracted from two genotype Brassica napus,P-efficient genotype “Eyou Changjia” and P-inefficient genotype “B104-2”, under short-term P-free starvation, respectively. And proteome analysis was performed by 2-DE to investigate proteome alterations in response to the short-term P-free starvation. Totally, 75 proteins whose abundance were significantly changed in response to short-term P-free starvation, which included 16 proteins in analysis set E(root proteins of “B104-2”), 24 proteins in analysis set F(root proteins of “Eyou Changjia”), 17 proteins in analysis set G(leaf proteins of “B104-2”) and 18 proteins in analysis set H(leaf proteins of “Eyou Changjia”), respectively. 3 Functional analysis of the identified differential abundance proteins The identified proteins were categorized into several groups including defense and stress response, carbohydrate and energy metabolism, signaling and regulation, amino acid and fatty acid metabolism, protein process, biogenesis and cellular component, and function unknown. There were 43 proteins involved in defense and stress response, and 76.7% of which were up-accumulated proteins under P deficiency. Compared to P-inefficient genotype “B104-2”, P-efficient genotype “Eyou Changjia” suffered less oxidative damage and lipid peroxidation, and inhibited O2·- generation more efficiently under P deficiency. sqd1 protein can facilitate the biosynthesis of sulfolipid and instead of phospholipid for phosphatidylglycerol under P limitation. And the abundance of sqd1 protein was up-accumulated in “Eyou Changjia” in response to P-deficiency. There were 37 proteins involved in carbohydrate and energy metabolism, and the proteins involved in the cytosolic bypass metabolism of production of UDP-glucose was activated in the P-efficient genotype “Eyou Changjia” but not in P-inefficient genotype “B104-2”. These results indicated that the P-efficient genotype Brassica napus through enhancing the antioxidative ability and membrane lipid remould, as well as the carbohydrate metabolism changes to adaptation to P deficiency. 4. Construction of plasma membrane purification for plasma membrane proteomic study A series of aqueous two-phase system containing 5.8-6.4%(w/v) dextran-PEG polymer were conducted for plasma membrane(PM) enrichment from Brassica napus roots. And a phase partition system consisted of 6.2%/6.2% dextran T-500/PEF 3350would give the higher puritied PM(ca. 90%) and higher protein yield(ca. 6.5 μg/g FW). The PM protein was extracted after the root PM was purified. And the proteome analysis was performed by label-free to investigate the changes of the roots PM proteins in two Brassica napus, “Eyou Changjia” and “B104-2” suffering from P-free starvation, respectively. Totally, 71 roots PM proteins were significantly changed their abundance, and among these identified proteins, 31 proteins were from “Eyou Changjia” and 40 proteins were from “B104-2”. The identified plasma membrane proteins were categorized into several groups including cytoskeleton, cell defense, transportation, signal transduction, protein modification, protein synthesis/degradation, metabolism and energy, and unclassified, based on their function and biological process. 5. Construction of the relationship between QTLs for P efficiency and differential abundance proteins by comparative genomics All the identified proteins were mapped to the linkage map of Brassica napus “B104-2” × “Eyou Changjia” RIL population through comparative genomics. Finally, 94 proteins that identified by 2-DE and 35 PM proteins identified by label-free were successfully mapped to the lingkage map. And 72 proteins identified by 2-DE and 30 PM proteins identified by label-free were mapped into the confidence interval of QTLs that associated with P efficiency coefficient, P content and dry weight et al. |
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