Analysis Of Gene Expression Of Brassica Napus Under Low Phosphorus Condition

Phosphorus (P) is one of essential macronutrients required by plants.The concentration of Pi in thesoil solution is often low (2 to 10μmol/L) that limit the yield of crops. Rapeseed is one of the most important oilcrops in the world.But the rapeseed is sensitive to P. The output and quality of rapeseed reduce because of p deficiency .Under these situations, it is urgent to breed low-P-tolerance crop by bio-techniques.In this thesis, our goal is to study the low phosphorus-tolerance mechanism of and investigate the genetic basis of its low p-tolerance. The P nutrition characteristics of the phosphorus-tolerance zhongyou No.821 and phosphorus-sensitive No. 1182 were investigated under sand culture conditions. Analysis of cDNA fragments responded to low- p-stress was showed by using mRNA differential display technique and their expression characters were analyzed. The results were shown as follows:1. Effects of low-phosphorus-stress on membrane lipid peroxidation and protection enzyme and the chlorophyll content of different genotype rapeseed.During the period of low-p-stress, the activities of SOD maintained relatively stable in low-p-tolerant cultivars. whereas those increased obviously at early stage and subsequently decreased rapidly in the low-p-sensitive cultivars, suggesting that the absolute activities of protective enzymes had no relation with the low-p-stress, while the changing trend was reverse. The increased MDA content in low-phosphorus tolerant cultivars was obviously less than that in low p-sensitive cultivars. The chlorophyll content is stable during low phosphorus stress2. Establishment and optimization of sliver staining differential display of rapeseedThe results showed: Many clear bands were observedwhen the amount of RNA used was about 2ug and the amount of reverse transcription products used was 1.5μl. The annealing temperature on DDRT-PCR amplification was 40°C, which produced many cDNA bands.3. Analysis of gene expression of rapeseed under low phosphorus conditionIn this research work, the different expression in low-phosphorus stressed and unstressed between low-p-tolerant rapeseed zhongyou No. 821 and low-p-sensitive No.1182 were analyzed by the silver staining mRNA differential display method. Total 61 low-phosphorus-induced cDNA fragments expressing differentially were isolated. 16 cDNA fragments were sequenced and analyzed. They were induced or suppressed. The deduced amino acid of bnr2 exhibited 85% identity to xyloglucan endotransglycosylase. The deduced amino acid of bull showed homologous to putative photosystem II type I chlorophyll a/b binding protein.The deduced amino acid of bnr3 showed homology to DNA binding protein. In addition, a lot of genes, in relation to protein synthesis and metabolism (such as structural constituent of ribosome mRNA, RNA binding protein, chloroplast ribosomal large subunit protein L21) and ESTs in relation to abiotic stresses (such as bnll0) were expressed under low-p stress. And some genes, which were involved in low-p-stress response (bnr-4,6,15 and bnl-5,9), but their function were unknown, need to be verified next. 4. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses of differential display cNDA fragmentsThe analysis of the deduced amino acid sequence of bull indicated homologous to putative photosystem II type I chlorophyll a/b binding protein, the expression level in zhongyou No.821 leaves increased after 6 hour treatment with low phosphorus by Northern blotting and RT-PCR. The deduced amino acid of bnl4 exhibited 85% identity to Arabidopsis thaliana RNA binding/structural constituent of ribosome. After 6 hour under low-p the level of bnl4 mRNA in leaves increased significantly. Based on the analysis of the deduced amino acid sequence, it is inferred that bnl8 encodes thylakoid membrane phosphoprotein of 14 kda. The expression of bnl8 in the leaves of zhongyou No.821 was induced by low-p stress by semi-quantitative RT-PCR, and the expression was highest after 24 hour treatment with low-p.The deduced amino acid of bnr2 exhibited 88% identity to Arabidopsis thaliana xyloglucan endotransglycosylase (XET). XET is the key gene related to the root hair forming. The expression of bnr2 in the roots of No.1182 was suppressed under low-p stress by semi-quantitative RT-PCR. The analysis of the deduced 44 amino acid sequence of bnr8 indicated homologous to FAD-binding domain-containing protein. This protein in the mitochondria endomembrane takes part in electric transport.