Differential Proteins Analysis On The Early Compatible Pollination In SI Brassica Oleracea L.

Self-incompatibility (SI) is an important mechanism to prevent plants from inbreeding, keep diversity of genetic; it is widespread in the flowering plants. Self-incompatibility signal pathway has become the important model system of plant intercellular signaling, which is attached much importance in the international. The most popular and clear machanism is the spores SI of Brassica plant. SI Brassica Oleracea L. Style can discriminate self-and non-self pollen, and then make a swift response on protein level. And this made the self-and non-self pollen present a different fate. self-pollen (the same S haplotype) Can’t hydration, germination, or it’s pollen tube could not penetrate into the papilla cell, but non-self pollen (different S haplotype) can hydrate and germination successfully and complete the normal fertilization process. Self-incompatibility of Brassica Oleracea L. is controlled by a single S locus. After pollination, SCR combined SRK with the same haplotype, and then, the complex activates function of ARC1 and degrades some factors which are relative to compatible pollination, such as Exo70A1. And with some unknown signal transduction process, which at last result in pollination fails. This process also involves a lot of other factors, such as MLPK, rdr6, THL1, KAPP, SNX1, Calmodulin, PUB8, sp locus, etc. Compatible pollination mechanism is largely unknown relative to SI mechanism. Some reports shows that Long chain lipid, SLG, SLR1, pollen oleosin are involved in pollen adhesion, hydration, and germination; Ca2+ plays an important role in stigma response regulation; Exo70A1 is a necessary component when pollen tube penetrate the stigma. Overexpression of Exo70A1 leads SI Brassica oleracea tends to self-compatible, it suggests that there is an intersection at ARC1 in SI pollination and compatible pollination, and both proceeding share a subsequent path.Proteomics research technologies have advantages in expression patterns analysis, especially in a particular organization at a particular period. After comparison of the different period expression patterns, differentially expressed proteins can be screened quickly and intuitively. So it was widely used in the separation of key factors which is involved in plant disease resistance, resistance and plants sterility lives. In order to provide some new content for the compatible mechanism of SI Brassica Oleracea, using Brassica Oleracea L. as material, here explored and set up a proteomics method that was used for the first time to study differentially expressed protein of the stigma at the early stage of compatible pollination. The main work and the results are as follows:1. Compatibility and affinity of Brassica oleracea such as A4, F1 was identified through identification of SRK haplotype, morphologic observation and affinity index method. A4 was identified as S28 haplotype, F1 was identified as S7 haplotype, both haplotypes are belong to Class I which are strong SI haplotype, and their relationships were far apart, they can outbreeding successfully.2. TCA/acetone method was used in protein extraction. We optimized the parameters of 2-DE including SDS-PAGE gel concentration, isoelectric focusing conditions, staining method and protein extraction methods. Combined someone other’s experience, the optimized system include the following steps:separating the proteins with 17cm pH 3-10 NL IPG strips, loading proteins samples of 150μg followed by isoelectric focusing program 2, and SDS-PAGE gel concentration selected 12%. The 2-D images with good repeatability and high resolution were obtained.3. Compared 3-5min with 1 h group,116 differential expressed proteins were found, but there’s no significant difference between 1 h and 2 h group. The possible reason is that the early events of pollen-stigma interaction was dramatically increase within 1 h, and tend to be gentle afte 1 h.71 protein spots were picked and analyzed through MALDI-TOF-MS mass spectrometry, and 37 of them were identified homologous to known proteins in various databases related to MALDI-TOF-MS and MASCOT analysis. Compared with 3-5min group, 1 h group including 21 specifically expressed ones,12 up-regulated and 4 down-regulated ones.4. According to bioinformatical analysis, two of 37 proteins were identified as some kinds of proteins involving in pollen tube development which were reported. The predicted functions of the rest 35 proteins were related to regulation of translation, Carbohydrate and energy metabolism, transmembrane transport, response of defence and stress. 5. By combining protein function which is successful identified and the bioprocess of pollen-pistil interaction in the early stage. Potential role of these proteins were infered the in this physiological progress. Ca2+ plays an important role in pollen germination and extension, regulation, direction of pollen tube. Ca2+ binding protein may indirectly function in pollen-stigma interaction process by participating in the calcium transportation. Plants defensin like proteins are not only function in defense response, but also involved in the pollen tube growth and guidance. It is suggest that penetration of pollen tube probably accompanied by the improvement of style resistance.