| Phosphorus (P) is one of the essential macronutrients for plant growth and development, which directly participates in an array of processes. So it plays an important role in plant growth, metabolism and yield increase. Although P is quite abundant in the soil, yet it is largely unavailable for intake because it rapidly forms insoluble complexes with cations, especially with ions of aluminum and iron under acid conditions. Phosphorus deficiency in soil is a global problem. In the long adaption to the phosphorus-deficient condition in soils, the plants have evolved series of adaptive mechanisms, involving morphological, developing, biochemical and genetic changes that are governed by gene. Studies on Maize tolerance to low phosphorus have been gradually deepened from low-P tolerant genotype selection, morphological changes, physiological and biochemical changes to genetic traits analysis of tolerating low phosphorus, QTL orientation. However, few researches have been reported, concerning gene regulation and molecular mechanisms of Maize tolerance to low phosphorus.An elite maize inbred line with high tolerance to low phosphate,178, was studied in the research. After germination, the seedlings with four leaves were cultivated in normal (control) and low phosphorus nutrient solution (check). The time when the seedlings were transplanted into low P solution were regarded as zero. At five time points of 6h,12h,24h, 48h and 72 h, the materials of seedling roots were harvested from check and control. These materials were prepared for total RNA extraction. Then purified mRNA from total RNA was used for further root library construction of differentially expressed genes in Maize tolerance to low P by Suppression Subtractive Hybridization (SSH). After PCR testing, sequencing and blasting by bioinformatics, unique EST sequences were divided into categories by their speculated functions according to its homology gene function. To investigate their agreement degree, single-copy EST sequences and low phosphate-related QTL locus were co-localized by bioinformatics methods. Genes with important function were studied for expression models during low phosphate response in Maize by semi-quantitative RT-PCR. The research results were the follows:1. Under low P, the forward and reverse libraries of different expression genes from inbred line 178 were constructed with 3648 clones and 2498 clones, respectively, by SSH and library construction technology. The length of cDNA fragments in the library ranged from 100 to 500 bp.2. From the forward and reverse library,103 and 15 clones, respectively, were randomly selected for sequencing. By blast analysis based on B73 maize whole genome, we obtained 65 unique EST sequences, including 44 function-known and 21 unknown sequences. Based on precious studies, the 44 sequences were divided into 9 categories according to their homology gene functions, namely energy metabolism category (including 2,3%), amino acid and protein metabolism category (including 18,28%), P recycle category (including 4,6%), signal transduction category (including 3,5%) cell division and composition category (including 2,3%), stress related category (including 5, 8%), transcriptional regulation category (including 7,11%) and channel protein category (including 2,3%). SSH results suggested, in Maize roots under low P, the transfer of low P signal stress involved multiple signal transduction pathways, energy metabolism and protein synthesis increased, transcriptional regulation and decomposition of organic phosphorus rose, phosphorus consumption decreased, auxin synthesis increase accelerated cell division, the protective mechanism mounted in response to phosphorus threat on cells.3. By bioinformatics alignment,24 single-copy EST sequences were found from 65 EST sequences, after the single-copy EST sequences and phosphorus stress-related QTL locus from other researches were located in the map of maize chromosomes, the co-localization analysis showed 45.8%overlap between them. It showed that SSH was a certain reliable and reasonable method for differential expression gene library construction under phosphorus stress because the responsible genes to low phosphate with significant genetic effects were filtered. The co-location on chromosome between EST and QTL could be more obviouse with the increase of EST and QTL number combined into integrative linkage map.4. The five genes with important functions from different types were studied for their expressions in different time periods by semi-quantitative RT-PCR. The results showed that the expressions of five genes were in the overall upward trend, but in each time period there were some differences in expression. According to the up-expression time and the increase range of each gene expression, it could preliminarily speculate the expression order of 5 genes in maize roots, namely purple acid phosphatase precursor gene (PAP),γ-glutamylcysteine synthetase gene (GCS), putative target of mybl gene (TOM), protein disulfide isomerase gene (PDI), and auxin-induced protein gene (AIP). The order showed that physiological and biochemical changes occurred prior to morphological changes such as root system architecture changes in maize roots under low P stress. Phosphorus demand pressure was first triggered by phosphorus deficiency, and then induced cell imbalance between internal and external environment, required large-scale synthesis of related proteins and regulators which activated related downstream genes in tolerance to low phosphorus, and finally a large number of auxin induced protein synthesizing, which led to changes in root architecture. The results were consistent with field observations and previous researches.5. The findings by SSH in this study are consistent with previous results by gene chips, proteome analysis, cDNA-AFLP et al., and also with the researches in wheat by Gu (2009). and on rice by Li (2009). Thus, SSH technology was highly reliable and reasonable for differentially-expressed gene library construction under low phosphorus stress and the research of tolerance mechanism to low phosphorus.6. Though the overall, low phosphorus stress response in maize included genes of many other aspects involving root morphology, energy metabolism, metabolism, signal transduction, transcription regulation, and transportation and so on. The interacting and closely linking genes constituted the gene regulatory networks, responding to low phosphorus in Maize, which is consistent with previous findings.7. Low P stress response in Maize roots was a process of a gene expression in order. At first, phosphorus cycle genes started and were followed by stress related genes, amino acids and protein metabolism gene, auxin synthesis genes and target sequences of transcriptional regulatory genes. The later-promoted genes were root cell division and composition category. In this study, gene expression sequence was similar to the results of field observations and previous studies, namely the physiological and biochemical changes occur earlier than the root morphological changes such as root system architecture in maize roots under low P stress.8. In this study the maize core adaptive capacity of tolerating low phosphorus was speculated in areas as follows:the phosphorus cycle, cell protection and root architecture, which agrees with the tolerance mechanism to low phosphate from previous researches in wheat and rice. The regulatory network, which was composed of three core adaptive capacity of tolerating low phosphorus, and signal transduction systems, transcription systems, amino acid and protein synthesis and energy metabolism together, comprise the maize adaptive mechanism to low phosphate. |
PDF Full Text Request