Differential Expression Analysis Of Microgametocyte Responding To Temperature On Meiosis Stage In Oryza Sativa L

The utilization of the two-line system hybrid rice that based on photoperiod-and thereto-sensitive genic male sterile (PTGMS), is an important aspect of heterosis in rice. So the molecular mechanism of PTGMS is significant for enriching the theory of rice pollen development and directing hybrid rice breeding. Forward TGMS were sterile under the condition of high temperature and fertile under low temperature, while the reverse TGMS lines act in the opposite way. So far, studies on molecular level were focusing on gene and protein expression of TGMS, but stuty on using microarray and proteomic technology to analyse the pollen development of normal cultivar responding to temperature was few.Here, the normal fertility rice 93-11 was studied.Via different temperature treating on its meiosis stage, rice spikelet proteins were obtained to carry out the proteomics research, then some differential expression proteins were selected to verify by Real-Time PCR. Meanwhile, gene chip of male gametophyte in meiosis stage was carried out to screen differential expression genes. Results were showed as follows:(1)The proteins were separated by two-dimensional electrophoresis with immobilized pH (3-10 non-linear, NL)gradients as the first dimension and SDS-PAGE as the second. The silver-stained protein spots,which was analyzed using PDQuest software, showed that 884 and 867 protein spots identified in respect to different conditions, and most proteins with pH 4.0～8.5 and molecular weight ranged from 15 to 80 kD.61 proteins were found to be differentially expressed in H and D, With direct MALD I-TOF mass spectrometry analysis and protein database searching,22 protein spots out of 40 were identified. While the up-regulation proteins in H are structural maintenance of chromosomes,rev, heat shock protein 90, triosephosphate isomerase, tubulin,the down-regulation proteins are L-ascorbate peroxidase,UDP-glucuronic acid decarboxylase and some unkown proteins.(2) Through real-time SYBR Green quanti-tative PCR(RQ-PCR) to quantify D3、D7、H11 and H19 in transcription level, the result showed:the amplification curve had flat baseline, distinct exponential area; There was a linear relationship between threshold cycle value at which sample crosses threshold and the logarithmic value of template concentration; The expression of H11and H19 was enhanced by high temperature treating in comparison to supply of low temperature treating in pollen,while the expression of D3 was enhanced in pollen of low temperature treating, these results are in good correlation with those obtained by two-dimensional electrophoresis.However, D7 was not significantly enhanced in comparison with results of other genes mentioned above.(3) We sampled the microgametophyte which was treated under the different temperature on the meiosis stage, obtaining 45 differentially expressed genes by using cDNA microarray hybridizing, in which 30 was up-regulated in low temperature treating,while 15 was down-regulated. Further analysis of the 45 related genes showed that they involved in several metabolism pathways,such as glycometabolism, protein metabolism, regulation of gene expression, signal transduction, stress response and so on.