Study On Expression Analysis Of Tapdk From Energy Metabolism Pathway And Its Interactive Protein Of Physiological Male-Sterility In Wheat(Triticum Aestivum L.)

Currently, the physiological male sterility (PHYMS) induced by chemical hybridizing agent (CHA) is an important source of male sterile line in wheat (Triticum aestivum L.). SQ-1, a new chemical hybridizing agent, can induce95%-100%spikelet sterility in wheat. In practice, a series of new hybrid wheat cultivars with high yield, elite quality, and multiple resistance were developed by making use of SQ-1. However, very little is known about the molecular mechanism of PHYMS. The challenge remains to understand how intermediary metabolism can be regulated during pollen abortion. To elucidate energy metabolism mechanism of the abnormal pollen development in PHYMS, the cytological observation of tapetum development, cell division, starch accumulation in PHYMS were performed, and the content of pyruvic acid was detected. Furthermore, the molecular characteristics of TaPDK, as the research starting point, were analyzed via common clone, hiTAIL-PCR, and prokaryotic expression. Then the expression pattern of TaPDK and its substrate protein gene TaPDC-E1α were detected via semiquantitative RT-PCR analyses. In addition, the proteins interacting with TaPDK were screened via yeast two-hybrid technique. Subsequently, a few candidate proteins in nucleotide expression levels were detected by real-time quantitative PCR, and further discussed the relationship between these genes expression and pollen development.The main results and conclusions were as follows:1. Cytological observations showed that tapetum was premature degeneration at early microspore stage in PHYMS, and the majority of microspores failed to divide and remained bicellular status. Additionally, there was no starch accumulation in the mature pollen of PHYMS. Subsequently, the results from the pyruvic acid detection showed that the content of pyruvic acid was dramatically down-regulated in the PHYMS lines than that of the fertile lines. Collectively, the abnormal microspore development was associated with energy shortage.2. The TaPDK encoded364amino acid residues, and protein molecular weight was41KD. The protein molecular weight of TaPDK from prokaryotic expression was consistent with software prediction. Conservative structure analysis found that TaPDK contains BCDHK-Adom3superfamily and HATPase-c superfamily. Subsequently, the upstream regulation sequences of TaPDK contained multiple cis-elements. Therefore, the structure diversity of TaPDK molecular sequence suggested that the TaPDK may be involved in the metabolic regulation of multiple pathway.3. According to semiquantitative RT-PCR analyses, the expression levels of TaPDC-E1α was lowest in PHYMS, demonstrating that the energy metabolism pathway was more susceptible in the PHYMS. TaPDK was obviously down-regulated expression in PHYMS, and up-regulated expression in genetic male sterile lines, which indicated that the upstream signal mechanism of mediating TaPDK was inconsistent between the PHYMS and the genetic male-sterile lines.4. We obtained twenty-four colonies coding for eight different proteins via yeast two-hybrid screen, namely non specific lipid transfer protein precursor, polyubiquitin, glyceraldehyde-3-phosphate dehydrogenase, proliferating cell nuclear antigen, CBS domain containing protein, actin, guanine nucleotide-binding protein beta subunit, chalcone synthase, and unknown function protein. These proteins involved in protein degradation, energy supply, cell division, and pollen formation, which further suggested that TaPDK plays multiple roles in pollen development, besides participating in regulating pyruvate dehydrogenase complex activity.5. The quantitative RT-PCR analysis indicated that the expression level of TaPUbi was only obviously up-regulated in PHYMS lines, but no significant difference in TaPUbi transcripts was observed in the corresponding genetic male sterility and fertility. It suggested that the SQ-1spraying induced TaPUbi expression, and ubiquitin proteasome pathway may participate in regulating the energy status of TCA pathway in PHYMS. The TaPCNA expression in sterile and fertile lines gradually exhibited down-regulated pattern with the pollen developmemt, but its expression was still higher in PHYMS lines and genetic male sterile lines than in fertile lines. Whereas the expression of TaCBS maintained stable, no significant differences were detected in leaf and anther at the uninucleate and binucleate stage between PHYMS and fertile lines. Intriguingly, TaCBS expression was much more increased at the tricellular stage in PHYMS lines than in fertile lines. It was speculated that the energy supply was serious shortage in later pollen development of PHYMS lines, and TaCBS, as cell energy sensor, adjusted energy levels to deal with energy crisis during pollen abortion in PHYMS lines. The expression of TanLTP was higher in PHYMS lines than in fertile lines. This indicated that the highest expression of TanLTP might protect pollen against potential disruption after SQ-1treatment.