Study On Expression Analysis Of TaPCNA From Regulation Of Microspore Cell Cycle And Its Interactive Protein Of Physiological Male-sterility In Wheat

In hybrid crop breeding, hybrid vigor or heterosis is a common phenomenon, which crossing different inbred lines produces F1 hybrids that usually have higher yields than the parents. Many studies and practice showed that the wheat hybrids have obvious advantages. As the male and female parents, it can be chosen from wheat conventional varieties freely. CHA system is an important method to produce hybrids wheat seed. CHA system freedom of choice means parents can use the advantages of conventional varieties directly. Wheat hybrids become the main advantage of using the channels. SQ-1 a new proprietary wheat gametocide solely developed by Chinese scientists. A proper dose can achieve 95%-100% of the male sterility at appropriate spraying times, and the saturation pollination rate can reach 85% or higher. 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 physiological male sterility. In order to reveal how male sterility was induced in wheat by chemical hybridizing agent and to provide solid theoretical support on the heterosis in wheat. In this paper, the cytological observation of cell division, starch accumulation in physiological male sterility(PHYMS) were performed, and then study of reactive oxygen species(ROS), antioxidant enzymes and DNA damage. Furthermore, the molecular characteristics of TaPCNA, as the research starting point, the sequence of cDNA of TaPCNA gene was cloned from wheat using RT-PCR approach with degenerate primers based on the conserved motifs found in a number of plant PCNA genes,and analyzed via real-time quantitative PCR, prokaryotic expression, and hiTAIL-PCR. In addition, the proteins interacting with TaPCNA were screened via yeast two-hybrid technique. Subsequently, the 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.The scanning electron microscopy(SEM) indicated that the PHYMS line pollen grain was seriously empty and shrunken. At the same time, the majority of microspores failed to divide and remained bicellular status.2.The study of ROS metabolism of physiological male sterile was showed that anthers of male sterility wheat had higher contents of O2?-, H2O2 and MDA than those of normal wheat. However, here were lower activities of superoxide dismutase(SOD), peroxidase(POD) and catalase(CAT) in scavenging ROS in the anthers of the male sterility wheat than in normal wheat. In vivo, if ROS are not promptly cleared, the organism will suffer oxidative stress, resulting in protein and nucleic acid damage, lipid peroxidation and even necrocytosis. The result of DNA hyperchromic effects was showed that the DNA hyperchromic effects of male sterility wheat was increased. It was suggested that the DNA of male sterility wheat was broken. Under the optimum conditions, six suitable primers were sereened, which could obtain clear, high polymorphism and reproducible RAPD products and studying the genomic DNA damages of anther of PHYMS. The result showed that different bands were amplified by seven primers between male sterility and normal wheat. The binding sites of the random primer in genomic DNA were start to be destroyed and the RAPD amplified bands of male sterility wheat were increased or dilute and even disappeared. This shows that DNA of the physiological male sterile line was damaged.3.In this reseach, the cDNA sequences of PCNA genes of wheat were cloned by RT-PCR and designated as TaPCNA(GenBank NO. KM087781). Sequence analysis showed that it included an open reading frame of 792 bp encoding 263 amino acids with the predicted molecular mass of 29.16 kD and the theoretical point of 4.53. To study cell cycle, the expression of cell cycle regulatory genes was evaluated. This well-characterized cell gene Histone H4 is widely used to predict the cell cycle progression, a gene whose transcription was regarded as a marker of the S phase in cell division. The result of real-time quantitative PCR was indicated that the expression of Histone H4, exhibited lower transcript level in male sterility wheat at binucleate and trinucleate anther. However, the TaPCNA transcript level was higher in male sterility wheat than in normal wheat in all three stages. By the technology of DNA recombination, the wheat TaPCNA clone was subcloned to Sac I/Hind III sites of the expression plasmid pET-32 a vector. The recombined plasmid, named pET32a-PCNA, was transformed into E.coli BL21(DE3) competent cells and induced with IPTG. SDS-PAGE illuminated that about 50 kD protein was expressed in the induced recombinant E.coli BL21(DE3), with nothing in the control group. The expression product was purified by nickel affinity column, and used as antigen to immune rabbits. Western blot analysis showed that SQ-1 can increase the expression of TaPCNA in male sterility wheat. Based on the mRNA sequence of TaPCNA gene reported, a 894 bp DNA sequence was isolated from wheat by high-efficiency thermal asymmetric interlaced PCR(hiTAIL-PCR). Sequence analysis revealed that the promoter contained several cis-acting elements such as light, heat, abscisic acid, pollen development, E2F-binding site, and so on. The CaMV 35 S promoter of pBI121 was replaced by the upstream sequence of TaPCNA to build a fusion plasmid in purpose to verify the promoter activity of the sequence. The results of Agrobacterium-mediated transient expression showed that the promoter exhibited obvious activity to drive GUS gene expression under SQ-1.4.Yeast expression vector(Bait) of TaPCNA was successful constructed, which no toxicity and autoactivation in Yeast cells.We obtained six different proteins via yeast two-hybrid screen, and these candidate proteins are mainly involved in cell cycle regulation and DNA damage repair. The result of real-time quantitative PCR was indicated that the expression of TaCycD2, exhibited lower transcript level in male sterility wheat. However, the TaICK1, TaKu70, TaXRCC1 and TaFen-1 transcript level were higher in male sterility wheat than in normal wheat in all three stages. BiFC assay was applied to confirm the interactions in vivo between TaPCNA and TaICK1 in onion epidermis cells.The results showed that the nYFP(which linked to TaPCNA) and cYFP(which linked to TaICK1) could complement to emit fluorescence in the nucleus,indicating the proteins they fused to Can interact with each other in the plant cells.