Studies On Ultrastructure Features, Endogenous Phytohormones Contents And Relative Genes Isolation Of The Male Sterile Sesame (Sesamum Indicum L.)

Sesame (Sesamum indicum L.—Pedaliaceae) is one of the most important oil-seed crops in the world. Many researches confirmed the remarkable heterosis in sesame in various aspects including production, qualities and stress-resistance. Heterosis application is the most important way for improving the unit productivity and the qualities of sesame. And utilizing male sterility is the optimal way for extensive production of hybrid sesame due to its high planting density and indefinite inflorescence characteristic. Since the first available GMS (genetic male sterile) line of sesame was discovered by Osman and Yermanos, researchers in Henan academy of agriculture sciences have done a lot of work in its improvement and application for breeding. They created the GMS two-line method for breeding in the Late of 1980s. But the existing problem that 50% male fertile plantlets need to be extracted before artificial pollination greatly limited the development of sesame hybridization breeding. In order to explore new ways for maintaining male sterility and improve the hybrid production efficiency, we carried out the further investigation research on GMS abortion mechanism in sesame using GMS line ms86-l. Our present study included three aspects: observation of the abortion process in ultrastructure level using TEM (transmission electron microscopy) techniques; Comparation of the endogenous phytohormones contents in fertile and sterile anthers; Isolation of relative male sterile genes. The results are as follows:1. Abortion process observation on GMS sesameA comparative study was conducted on the fertile and sterile microsporogenesis of ms86-l using TEM techniques. According to the morphologic characteristics of the microspores, our results showed that the wholly developmental process of sesame pollen could be tentatively divided into seven stages, including microspore mother cell formation stage, microspore mother cell meiosis stage, tetrads stage, early microspore stage, middle microspore stage, late microspore stage, and pollen maturation stage, respectively.The abnormal phenomena observed in the sterile anthers indicated that male sterility might arise in microspore mother cell formation stage. With the growth and development of the sterile anthers, the following abnormal features were observed evidently. The cell wall morphology of the sterile microsporocytes was irregular during their formation stage; In the meiosis stage, the cell walls were getting distorted obviously, and the microsporocytes failed to form the early exine component designated probaculums on the outside of the plasma membrane; Subsequently, abnormal villiform deposits were observed on the outside of the callose wall during the tetrads stage; As soon as the tetrad cells disintegrated, the released sterile microspores represented aberrantly dumbbell-shaped, and their exines were undergrown without any baculum. Accompanying with the delay of the degeneration process, inclusions of the tapetum cells became more abundant and few abnormal ubisch bodies were secreted; Later on, the sterile microspore was gradually crimpled as its cytoplasm agglomerating to an electron dense clot, and ultimately degraded to the complete abortion.2. Endogenous phytohormones contents comparation in GMS line of sesameContents of 4 endogenous phytohormones, IAA , GA₃, ZR and ABA , were measured by ELISA (enzyme-linked immunosorbent assay) in anthers of genetic male sterile and fertile sesame during archesporialcell differentiation stage, microspore mother cell formation stage, microspore mother cell meiosis to tetrads stage, early microspore stage, middle microspore stage, late microspore stage, to study the relationship between male sterility and phytohormone changes. The results of the sterile anthers showed that the IAA content was deficit after microspore mother cell formation stage; The GA₃ content kept low during meiosis, and its sharp increase was delayed from meiosis stage to early microspore stage compared with male fertile anthers; the ZR content was serious deficit during meiosis to tetrads stage and middle microspore stage; the ABA content was lower from meiosis stage to middle microspore stage, and higher in late microspore stage. The differences of the 4 phytohormones contents between the fertile and sterile anthers being more significant during the abortion stages indicated their roles in male sterility occurrence.3. Relative male sterile genes isolationWe obtained 40 cDNA fragments differentially expressed in fertile and sterile anthes of GMS sesame ms86-1 using mRNA differential display technology. By BLASTX analysis and semi-quantitative RT-PCR verification, we finally selected out four fragments which maybe relative to male sterility for further study. Semi-quantitative results showed all the four genes are strongly expressed in fertile anthers, and nearly not expressed in sterile anthers. BLASTX analysis showed one fragment had 74% identity with xyloglucan endotransglucosylase (XET), one had 79% identity with small heat shock protein (HSP), and the other two had high homologies with GRAS (GRAS was named after the three founding members: GIBBERELLIC ACID INSENSITIVE, GAI; REPRESSOR OF GA1, RGA; SCARECROW, SCR) family transcription factors. Two differential expressed fragments being GRAS family members implied the family might play a role in male sterility occurrence. They were 400bp and 510bp in length respectively via differential display PCR and 5'RACE techniques, and further investigations are needed.The full length cDNA encoding a polypeptide of 292 amina acids, designated SiXET, was isolated from the fertile anthers via homologous cloning and 5'RACE technologies. SignalP analysis indicated that SiXET had a signal peptide containing 30 amina acids in the N terminal. Real-time quantitative PCR results showed that SiXET was predominantly expressed in floral organ, its expression level in fertile anthers was very high while in sterile anthers was the lowest. According to our results, the SiXET, as a key factor in cell wall degradation and synthesis, maybe important in causing male sterility. Hence, we constructed the sense expression vector containing 35S promoter using pBI121 plasmid and RNAi vector for SiXET. The work of transferring these recombined vectors into Sesamum Indicum L. by Agrobacterium tumefaciens will be done in the near future.The full length cDNA encoding a polypeptide of 239 amina acids, designated SiHSP, was isolated from the fertile anthers via 5'RACE technology. Real-time quantitative PCR results showed that SiHSP was predominantly expressed in floral organ, its expression level in fertile anthers was the highest while in sterile anthers was very low. We constructed the prokaryotic expression vector pET30a (+)-SiHSP, and SDS-PAGE analysis showed that the target protein of 32.5KD in size was expressed. Many researches indicated that the HSPs were closely related to male sterility, therefore, the work of protein purification and functional verification awaits further study.