Biochemical Function Of Female Fertility Controlling Protein PTB1and It’s Related Protein Identification In Rice

In the middle of last century, humans began to work on phenomenon of plant female sterility. However, the majority of previous studies were limited to the level of embryology and cytology. With the developments and widely applications of molecular biology technology, significant progress has been made on function analysis of plant female infertility genes, especially on the structural molecular mechanism of infertility.In our earlier study, work has been done on a female sterile mutant originated from Shuhui202and results indicated that this infertility was controlled by a pair of recessive gene located on rice chromosome5. The female sterility was caused by the blocked pollen tube which failed to complete normal fertilization process. Accordingly, it was named as Pollen tube blocked1(PTB1) gene. Based on the intermediate expression vector pBS-Ppt, we constructed the complementary vector for PTB1gene. The fertile of fs-202was recovered by successful transformation of PTB1gene.In this study, structure analysis of PTB1protein was performed based on TMHMM website and related software. The results showed that PTB1protein contained four transmembrane structures. The mutant had only two transmembrane structures for a fragment (470bp in length) was missing which led to generate terminator codon TAA in advance. The database analysis indicated that PTB1protein contained RING-FINGER structure domain and belonged to the type of C3H2C3. It was reported that most of the proteins with RING-FINGER structure domain had E3ligase enzyme activity which was involved in ubiquitin-proteasome pathway (an important system of protein degradation). Therefore, we inferred that the protein encoded by PTB1may have E3ligase enzyme activity.In order to verify the speculation above and further understand the biochemical function of PTB1gene, related expression vectors of PTB1was constructed. Then the expression product was purified and the ubiquitin-proteasome experiments were performed in vitro. Meanwhile, proteomic analysis was done to detect translation differences between wild type and PTB1mutant. The main research results were as follows: 1. Prokaryotic expression vectors of PTB1were constructed using intermediate vector PBS-PT as donor, and expression vector pET-28a (+), pET-32a (+), pET-43.1a (+), pET-GST, pGEX-6P-1as receptors respectively. These recombinant vectors were expressed in E.coli respectively. Although expression conditions, such as IPTG concentration, temperature and induction time, were optimized and the expression host bacteria were replaced, no target protein was detected. Additionally, pEU-EO1-MCS employed as the receptor was used to construct cell-free expression vector pEU-202, which was expressed by the wheat germ cell-free expression system in vitro, but no target protein was detected as well. Lastly, specific primers were designed based on the pET28a-202to remove transmembrane structure by PCR amplification but to keep its function domain RING-FINGER structure. Then prokaryotic expression vector pET-GST was used as the receptor to construct GST-RING which was successfully expressed. Therefore, we speculate that the difficulty of expressing full length of PTB1is likely to associate with the transmembrane structure.2. GST-RING fusion protein was purified by glutathione agarose gel (glutathione sepharose4B resins) and affinity chromatography column. Then the small molecules and other impurities were removed and the target protein was concentrated with the help of ultrafiltration technology. Finally, recombination protein with high concentration and purity was obtained.3. In vitro ubiquitination, the necessary components for ubiquitination process were manually provided, including El, E2, Ub, ATP and etc., and it was detected by WESTERN BLOT. The result showed that GST-RING fusion protein had E3ligase enzyme activity and participated in the ubiquitin-proteasome process, which confirmed that ubiquitination is involved in control of female sterility in rice for the first time.4. Proteome analysis was conducted between the wild type and mutant. By total spike proteins of wild type202and mutant FS extract in pollination period, two-dimensional gel electrophoresis and mass spectrometry analysis,80proteins with significant difference in expression were identified. Among them, there were62proteins with difference in up-regulated or down-regulated expression (more than2times),18proteins with difference in expression or no expression. Of their specific proteins, the wild type had5types, while the mutant had13types.5. According to the function of PTB1gene, our work focused on the up-regulated and specific proteins for the function prediction in mutant. Of the mutant up-regulated proteins, gi|149392357may be related to RNA degradation, gi|218196880and gi|125524449may be related to restoring force, gi|8468043may be related to multiple developmental process of male sterility, gi|39546274participated in SUMO decoration, and gi|297719697involved in epigenetic hereditary, such as gene expression regulation, which may be related to sterility. Of the mutant specific proteins, gi|38344480was related to restoring force, gi|52076555was related to growth of pollen tube, and gi|125525605was related to cell elongation, etc. In addition, it was reported that RNA enzyme of gi|90399221was able to interacte with proteins with RING domain. We intended to investigate whether there would be direct substrates of PTB1among these candidate proteins by protein interaction methods.