The Molecular Mechanism And Action Mode Of Wheat Physiological Male Sterility Induced By Chemical Hybridizing Agent SQ-1

Wheat, as one of the most main grain crops in the world, takes an important role in the production of food crops in our country. The applications of wheat heterosis can increase wheat yield and improve the wheat quality. Chemical hybrid agent SQ-1 prevents wheat from normal pollen development without affecting female fertility, and the wheat seed setting rate is also good, most importantly, allows many thousands of hybridized combinations to be combined freely. However, the mechanism by which SQ-1 induces male sterility in wheat remained unclear, especially the action mode of chemical hybrid agent SQ-1(ion transport or information transfer), the key gene and metabolic pathways related to wheat male sterility were unknown. They greatly restricted the development and application of new type wheat chemical hybrid agent. A method for the determination of chemical hybridizing agent SQ-1 has been established, and we analyzed the dynamic resolution of chemical hybridization agent SQ-1 in wheat plant, transfer direction and the action mode. The analysis of complete transcriptome and small RNA between wheat physiological male sterile lines and fertile lines was carried out using high-throughput sequencing technology, it can identify differentially expressed unigenes and s RNA between fertile lines and SQ-1–induced male sterile lines, the key gene and metabolic pathways related to wheat male sterility. It provided potential targets and theoretical basis for developping new type wheat chemical hybriding agent, promoted the popularization and application of chemical hybrid agent in our country. The main results and conslusions are as follows:(1) The process for extracting SQ-1 in wheat seed was improved, using solid phase extraction instead of simple liquid-liquid extraction. This method was simple and the cost is low. A precess for detecting SQ-1 in wheat leaves and anthers was established using Qu ECh ERS method. The proportional band of the technology was 1.0~60.0 mg/L(correlation index 0.9999), and the mean recoveries rate of SQ-1 in wheat leaves was 91.27~96.48%, at three spiked levels(10 mg/kg, 25 mg/kg and 50 mg/kg), with RSD of 1.60~1.99%. The average recoveries of SQ-1 in wheat anthers was 90.77~97.21%, with RSD of 1.27~2.45%. The technology had some merits of simple operation, low cost, greatly improved the detection efficiency of chemical hybridization agent SQ-1.(2) Chemical hybridization agent SQ-1 was evenly sprayed on the leaves of the wheat, it was transported to anthers from leaves, and it was rapidly accumulated in wheat anther. The concentrations of chemical hybridization agent SQ-1 in wheat anthers was high in the important period of microspore growth(from meiosis period to mononuclear period), which caused a series of physiological and biochemical changes in reproductive organs anthers and seriously hindered the normal growth and development of microspores, eventually leaded to microspores abortive. Chemical hybridization agent SQ-1 amostly can not transport from main stem to tillers, from tillers to main stem and from tillers stem to tillers. Chemical hybridization agent SQ-1 amostly can transport from roots to wheat leaves, but it amostly can not transport from wheat leaves to wheat roots. The time effect and dose effect of chemical hybridization agent SQ-1 was investigate, it can not rain 6 hours(at least 4 hours) after chemical hybridization agent SQ-1 was evenly sprayed on the leaves of the wheat. If the dose of chemical hybridization agent SQ-1 was too high, it can cause wheat earing difficultly, and even caused plant death. If the dose of chemical hybridization agent SQ-1 was too low, the abortive degree of wheat hybrids will drop, it can seriously influence the purity of wheat hybrids. So we needed to choose the right dose to spray chemical hybridization agent SQ-1.(3) Small RNA that were differentially expressed between wheat physiological male sterile lines and fertile lines were analyzed and the functional annotation of differentially expressed si RNA was also made. A total of 36 known si RNA were differentially expressed between wheat physiological male sterile lines and fertile lines, tae-mi R156 and tae-mi R159 a were slightly down-regulated, tae-mi R167 a was slightly up-regulated, and tae-mi R160 was markedly up-regulated. In addition, a total of 86 significant differentially expressed mi RNAs were obtained between wheat physiological male sterile lines and fertile lines, 55 mi RNAs were up-regulated, 31 mi RNAs were down-regulated. The prediction and functional annotation of the significant differentially expressed mi RNAs target genes were made. A total of 95 differentially expressed mi RNAs target genes were annotated, 24 target genes were annotated by COG database, 62 target genes were annotated by GO database, 11 target genes were annotated by KEGG database, these differentially expressed mi RNAs target genes were related to “Glycolysis/Gluconeogenesis”, “Fructose and mannose metabolism”, “Amino sugar and nucleotide sugar metabolism”, “Protein export”, they were not only involved in energy and materials metabolism, but also were closely related to plant stress resistance, and also participated in the synthesis of some important material, such as the biosynthesis of carotenoids.(4) Unigene that were differentially expressed between wheat physiological male sterile lines and fertile lines were analyzed and the functional annotation of differentially expressed genes was also made. A total of 1088 unigenes were differentially expressed between wheat physiological male sterile lines and fertile lines. Wheat fertile lines, acting as a control, 643 unigenes were up-regulated and 445 unigenes was down-regulated in SQ-1–induced male sterile lines. A total of 967 differentially expressed unigenes were annotated, 333 target genes were annotated by COG database, accounted for 30.61 percent; 637 target genes were annotated by GO database, accounted for 58.55 percent; 156 target genes were annotated by KEGG database, accounted for 14.34 percent; 756 target genes were annotated by Swiss-Prot database, accounted for 69.49 percent; 967 target genes were annotated by NR database, accounted for 88.88 percent. A total of 126 differentially expressed unigenes were mapped onto 60 pathways in the KEGG database. In all, 54 up-regulated unigenes were assigned to 42 pathways and 72 down-regulated unigenes were assigned to 40 pathways. After a lot of chemical hybridization agent SQ-1 was gathered in the anther, it not only impeded ribosome biosynthesis and DNA metabolism, but also impeded photosynthesis and aerobic respiration, it affected plant active oxygen metabolism at the same time. On the other hand, enhanced anaerobic respiration and abnormal RNA degradation, and the unigenes related to plant stress resistance became active, such as “glutathione metabolism”, “ubiquitin mediated proteolysis”. These abnormal metabolic processes collectively caused pollen sterility.