| Self-incompatibility (SI) not only is a hotspot in the research of plant molecular biology and genetics breeding, but also is important in research of cell signal transduction and heterosis. Because the SI molecular mechanism has not been understood clearlily, most researchers have try to clone the SI gene using map-base cloning method. In this study, a thermo-sensitive self-incompatibility line HE97, it exhibited complete self-incompatibility when the daily minimum temperature was below 20°C in the floret differentiation of staminate inflorescence, whereas plants exhibited self-compatibility when the daily maximum temperature was higher 24°C.For dissect the genetic basis of the thermo-sensitive self-incompatible line, the inbred line HE97 was sowed in the farm of Henan Agricultural University from April 19, 2008 to June 10, 2008 by stages. Silk and pollen of the plants for the inbred line HE97was carried out for each stage, and then self-pollinated by hand with its ample pollen. The pollen and silk of the inbred line HE97, which represent the self-incompatibility and self-compatibility completely, were used to identify the different expression proteins by means of two-dimensional electrophoresis and mass spectrometry method. The major results in this study were as follows:1. The tassel shedd and silk emergement of the inbred line HE97 exhibited normally in all sowing stages, . However the fecundity for SI plants has significant difference by different stages, which coincided with those obtained by predecessors.2. The effects of different protein extraction methods, including TCA/acetone extraction, phenol extraction and TCA/acetone/phenol extraction, the loading quantity of sample and IEF conditions as well as the gel concentrations were optimized for the analysis of 2-DE system on the silk and pollen in maize. The results showed that samples prepared by TCA/acetone extraction had observed more protein spots than phenol extraction method and TCA/acetone/phenol extraction, and clearer background and protein spots were obtained in the 2-DE map.3. Two-dimensional electrophoresis was performed to generate the images of proteins of pollen and silk at self-incompatibility and incompatibility stage, respectively. Most protein spots lay in PH4-7 position of the gel. There were about 340 detectable spots for pollens and 670 detectable spots for silks on each 2D-gel. Studying the protein expression pattern, the results showed that there were 9 protein spots with differential expression. Among them, the protein P2 just appeared in the pollen of the self-incompatible stage, the protein P1 had low expression in the pollen of the self-compatible stage. There were 3 protein spots (S1, S2, S7) appeared in the silk of the self-incompatible stage. Two proein spots (S4, S5) just exhibited at the self-compatible stage. The protein S6 ans S3 had high expression in the silk at the self-incompatible and self-compatible stage respectively.4. By means of LC/ESI-MS/MS analysis and protein database searching, the nine different protein spots were identified from the 2D protein maps of the HE97 pollens and silks during the self-incompatible and self-compatible stages, and seven different protein amino acid sequences were obstained, out of them, two candidate gene related to signal transduction were identified.5. After function analysis, two self-compatible related genes, P1and S7, were cloned according to B73 genomic database sequence. The gene P1 have very high similarity with a cDNA named LlANK of Lily, and it was found that the over expression of LlANK gene would lead to abnormal growth of the pollen tube. On the other hand, transient silencing of LlANK impaired pollen germination and tube growth. For another gene S7, its protein has been tested playing an important role in transfering cell growth and differentiation signals.
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