| Maize is one of the important crops in the industrial and agricultural production. Lacking of available phosphorus in soil will limit its yield and quality. People can increase its production by using phosphate fertilizer to increase the effective phosphorus content in soil; however, it causes a series of harm to our environment and the waste of phosphate rock resources. At present, the scientific method is that viewing from the point of controlling expression and regulation of genes to control the mechanism of adaptation to low phosphorus stress of maize, we are able to discover the key genes of tolerance to low phosphorus, improve the use efficiency of soil phosphorus on crop and in the meantime achieve stable output with low investment. In this study, we used two maize genotypes as materials, one is tolerant to low phosphorus (DSY-2) and the other is sensitive to phosphorus (DSY-79). We studied the effects of low phosphorus stress on the leaf chlorophyll content in the two kinds of maize seedling, the activity of root POD and the soluble protein content in roots, and at the same time, by using gene chip technology, we analyzed the expression of different genes in the roots.Combining with enzyme activity, real-time fluorescence quantitative PCR (qRT-PCR) and other methods, we also analyzed and confirmed some of the candidate genes; the main results we obtained are as follows:1) Effects of low phosphorus stress on the chlorophyll content in maize seedling leavesComparing with the control group, the leaf chlorophyll a, b and the total chlorophyll content in a new leaf of two maize cultivars declined to different degrees under low phosphorus stress for 8 days, and the chlorophyll content in the new leaf of phosphorus sensitive genotype DSY-79 fell more; Comparing the chlorophyll content in the positive fifth leaf of two maize seedlings, the chlorophyll a, b and the total chlorophyll contents in DSY-2 genotype that is tolerant to low phosphorus were lower than sensitive counterparts. This indicates that under phosphorus-deficient condition, maize seedlings of the DSY-79 genotype, which is sensitive to phosphorus, cannot acquire enough phosphorus, and acquire a large number of phosphorus in old leaves to ensure the normal growth of new leaves. This also indicates that 8 days treatment under low phosphorus stress is able to result in obvious changes in aboveground characters of the materials. Therefore it is appropriate to choose those maize seedling roots which are treated under the low phosphorus for 8 days to do differential gene expression analysis.2) Effects of low phosphorus stress on the POD and soluble proteins in maize seedling rootsThe POD activity in maize seedling root under low phosphorus stress for 8 days was measured in this experiment. Activities in the DSY-2 genotype that is tolerant to low phosphorus and the DSY-79 genotype that is sensitive to phosphorus both increased, and DSY-2genotype had more obvious increase. It suggests that the DSY-2 genotype is expressing POD to overcome the low phosphorus stress. This is consistent with the results form gene chip and qPCR that gene expression of peroxidase in the DSY-2 genotype increased obviously and an increase in the peroxidase secretion. This shows that in the low phosphorus conditions, maize seedlings generate a large number of POD to maintain the normal physiological and biochemical reactions in the body as well as its ecological balance. Under the abduction of low phosphorus, soluble protein content of the DSY-2 genotype increased while the DSY-79 genotype did not show a significantly change, which indicates that gene expression has changed in the DSY-2 genotype.3) Gene chip and fluorescence quantitative PCR analysis on the expression level of key genes that are tolerant to low phosphorusUnder low phosphorus stress for 8 days, there were totally 102 effective expression differences genes in phosphorus sensitive DSY-79 root to low phosphorus stress compared with a control (signal ratio=signal value of transcription in treatment group/transcription signals in the control group, the signal rates that are less than or equal to 0.5 or more than 2 can be identified as a differentially expressed gene), including 36 up-regulated genes and DSY-2 down-regulated genes; while there were 336 genes in phosphorus tolerant the DSY-2 genotype, including 251 up-regulated genes and 85 down-regulated genes. Then, according to the level of gene transcription and the possible functional differences, totally 27 possible key genes with low phosphorus tolerance were screened respectively into:phosphorus uptake, mobilization and energy metabolism; substance metabolism related genes; cell growth and structure of cell wall related genes; transcription regulation and factor related genes; material transport and transfer related genes; stress response and defense related genes, etc. Detecting the relative expression amount of these genes by fluorescence quantitative PCR under low phosphorus stress for 8 days indicates that, for the low-phosphorus-tolerance DSY-2 genotype,17 genes of selecting 20 up-regulated genes showed significant upregulation, genes relates to inorganic phosphate transporter 3 and β expansin (expB7) induced under low phosphorus stress particular significantly, and genes relates to peroxidase (peroxidase 72), acid phosphatase (acid phosphatase), plant stress tolerance (endochitinase PR4), jasmonic acid synthesis (12-oxo-phytodienoic acid reductase) showed significant adjustment. But only 6 genes in phosphorus sensitive varieties showed significant upregulation:inorganic phosphate transporter DSY-79 roots of maize seedlings (inorganic phosphate transporter 3) and β expansin (expB7) related genes induced very significantly under low phosphorus stress.After treating under low phosphorus, expression of genes that relates to dormancy (ARP) and ubiquitin like modifiers (ubiqultin-like proteins) in the two maize varieties significantly decreased. Low phosphorus tolerance of the DSY-2 genotype had a higher decrease, and part of genes of hypothetical proteins with the verification of qPCR also showed a significant upward or down. It needs scholars to do further study to confirm whether they play an important role in the tolerance to low phosphorus in maize.In this study, we verified genes that relate to the uptake of phosphorus, mobilization and energy metabolism; metabolism; growth of cell and genes related to the structure of cell wall; genes related to transcription regulation and factor; genes related to material transport and gene transfer; genes related to stress response and defense play an important role in the tolerance of maize to low phosphorus stress.4) Isolation and purification of secreted proteins in maize rootsFor a long time, progress in the study of secreted protein in roots is slow because of its low secretion and difficulty to collect due to mixture in the root exudates. At present, it is difficult to grow sterile culture seedling in the premise of ensuring crop grow under sufficient light. In this study we minimized the effect of microorganism on experiment based on the normal growth of plants, enriched the sample by using Millipore filtering cup and ultrafiltration cup, and then obtained secreted protein that the root secreted into the culture fluid with modified TCA acetone precipitation. The content of root secreted proteins was obtained over 300μg/mL.These work lay the basis for follow-up two-dimensional electrophoresis and identification of the secretory proteins of interest. |
PDF Full Text Request