摘要
磷是植物所必须的大量元素之一,土壤中有效磷的浓度偏低(2-10μmol/L),已成为作物生长的重要限制因子。油菜是全球重要的油料作物之一。而油菜是对磷敏感的作物,缺磷导致油菜产量和品质下降,因此培育耐低磷作物新材料已迫在眉睫。
为进一步揭示油菜耐低磷机理以及其耐低磷性的遗传基础,本工作以大田筛选的耐低磷油菜和磷敏感油菜品种为材料,研究磷素营养特征以及它们在低磷胁迫前后基因表达情况。主要工作内容与结果如下:
1.不同基因型油菜在低磷胁迫下膜脂过氧化、保护酶活性和叶绿素含量的变化
研究表明耐低磷品种的SOD活性在实验期间相对稳定,而低磷敏感品种在低磷胁迫初期有明显的增加,尔后又快速下降,这表明保护酶的绝对活力与低磷胁迫无关,而变化趋势与低磷胁迫有关。低磷引起耐低磷品种MDA的增量明显小于低磷敏感品种,而叶绿素含量在整个胁迫期间并没有显著变化。
2.甘蓝型油菜银染mRNA差别显示条件的建立及优化
本实验对油菜差别显示体系进行了筛选,最终获得了优化的差别显示体系。即RNA模板在2μg,反转录产物用量为1.5μl时,PCR退火温度在40℃扩增条带清晰。
3.甘蓝型油菜低磷胁迫应答cDNA差异表达分析
本研究用银染mRNA差异显示技术分析了耐低磷油菜品种中油821和磷敏感品种1182在低磷胁迫前后基因的差异表达情况。共获得差异表达cDNA片段61个。从中挑选差异显著的16个片段进行克隆分析,它们在低磷胁迫下表达增强或受到抑制。bnr2与拟南芥编码木葡聚糖转葡糖苷酶基因相似达到85%。bnl1与推测的光合系统Ⅱ类型Ⅰ叶绿体a/b结合蛋白基因同源性很高,bnr3编码的产物为DNA结合蛋白。低磷胁迫还调动了蛋白质合成与代谢(核糖体蛋白基因、RNA结合蛋白基因、叶绿体核糖体大亚基蛋白基因)及其与胁迫相关的EST如bnr10等相关基因的差异表达。还有一些未知功能的基因(bnr4,bnr6,bnr9,bnr15,bnl5,bnl9)还需进一步研究。
4.差异显示片段的半定量RT-PCR分析
bnl1推导的氨基酸序列是叶绿体a/b结合蛋白靠近3’端一保守区,Northern杂交及半定量RT-PCR分析表明该基因低磷胁迫6h后表达增强。bnl4cDNA片段推测的氨基酸序列与拟南芥核糖体结构蛋白的相似性为98%。RT-PCR分析表明该基因在胁迫后6h表达增强。bnl8的核苷酸序列与拟南芥的类囊体膜蛋白磷酸酯酶的基因同源性较高,且在胁迫后24h表达量最大。
bnr2推测的氨基酸序列与拟南芥木葡聚糖转葡糖苷酶(xyloglucan endotransglycosylase,XET)相似性为88%,XET在植物根毛形成过程中起到非常重要的作用,半定量RT-PCR分析表明该基因在磷敏感品种1182根中受低磷胁迫的抑制。bnr8cDNA片段推测的44个氨基酸序列与拟南芥的FAD-binding domain-containing蛋白有较高的同源性,该蛋白位于线粒体内膜上,参与电子传递。
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 stress
2. Establishment and optimization of sliver staining differential display of rapeseed
The 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 condition
In 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 fragments
The 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.
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