The Mechanism Of Reverse Thermo-sensitive Male Sterility In Oryza Sativa L.

Rice is the main staple crop for more than one third of human population on the earth.The research and utilization of hybrid rice provided a way to solve the starvation problem of human beings. Rice is a typical self-pollinated plant; the application of male sterility is convenient and essential for rice hybrid. The photoperiod/thermo-sensitive genic male sterile lines (P (T) GMS) show great advantages for hybrid rice production. P (T) GMS rice can propagate itself under low temperature conditions and be used to produce hybrid seeds through interplanting with normal fertile lines under high temperature conditions. P(T)GMS rices therefore provide an opportunity to replace the convenient "three-line" (male sterile, maintainer, and restorer) system by a "two-line" system that promises to reduce the costs, labor, time in the production of hybrid rice seed. P(T)GMS rice has a broad spectrum of restoration; almost all normal rice strains restore the fertility of the F1 hybrid. Another advantage is that the performance of P (T) GMS hybrid does not suffer from the adverse effects of male-sterile cytoplasm such as has commonly been the case with the three-line hybrids. Use of P(T)GMS rice to found two-line hybrids system has been therefor becoming a major goal in many rice breeding projects in China. However, the two-line hybrids system still face some difficulties in stabilization of sterility, which is easily effected by the factors of light and temperature in environmental condition. The mechanism of P (T) GMS is still less known. In order to accelerate the practical use of two-line hybrids system and make it possible to control the rice fertilization by artificial tool instead of light and temperature, it is urgently to reveal the molecular mechanism of male sterilization. P (T) GMS is usually under the control of one or two genes, which make it convenient to investigate the mechanism of male sterilization. There is still less reports about the mechanism of rice reverse TGMS, so far, and no associated gene of RTGMS has been cloned or reported. Data shown in this work may provide with important value for theories and practical application. In this work, a reverse TGMS line, Tb7S, which was the offspring derived from the hybrid between the wild rice and cultivar, was studied on its fertility change, cytological observation on its male sterility, inheritance analysis to its male sterility. At the meantime, an associated geneof the reverse TGMS, F32, was identified through RNAi.The result were showed as follow:At first, The correlation between the Tb7S pollen fertility and the air temperature value under Haikou natural environmental conditions was analyzed. The results showed that the temperature-sensitive time of fertility conversion was twelve to fifteen days before heading, when male gametophyte in meiophase of pollen mother cell. The critical temperature of its fertility conversion was 25-26℃(average value of daily temperature), daily maximum ground temperature 28℃, and daily minimum ground temperature 24℃. The result also indicated that the fertility appeared a positive correlation with temperature value, especially with the overlapping averages value of continuous 3 days maximum ground temperature, in Tb7S thermo-sensitive phase of the fertility conversion. The pollen developments of TB7S and a normal fertility cultivar Zhonghua 11 were compared by cytological microscopy. The results revealed that pollen mother cell of TB7S did not equally divided in the dyad phase of meiosis, which, might result in pollen sterility. Furthermore, abnormal tapetal cells and disordered microspores in TB7S may also response for the sterility of Tb7S. The genetic researches suggested that the sterility of TB7S was regulated by recessive genic gene with the segregation ratio of fertility 9:7, indicated that the phenotype was under control of two independent genes.F32 was cloned through constructing a SSH library by screening the differential expression between high and low temperature treatment in our laboratory previous work. The F32 gene is similar to SIT4 phosphatase-associated family protein. The RNAi vector targeting F32 was successfully constructed. The expression of RNAi cassette was under the control of a strong monocotyledon promoter. TO generation of F32 RNAi transformation exhibited the stained abortive pollen and subsequent low seeding rate when they were planted at lower temperature (18-24℃) and the fertility restored normally at high temperature (>28℃). Their sterility was significant correlated to the temperature value at 15-17 days before heading. Cytological observation indicated that tapetal cells degraded earlier than wild type, which resulted in stained abortive pollen. According to analysis of relative data, F32 gene was proposed to be a OsSitPPAL1, a homologous gene of the Saccharomyces cerevisiae phosphatase SIT4 protein. RT-PCR and Northern detection of small RNA proved that the transformed plant existed OsSUPPAL1 silence. The F32 RNAi transgenic T1 generation met Mendel's genetic laws.