Expression Vector Construction And Preliminary Function Analysis Of A Female Sterility Gene, PTB1, In Rice

The main food for people in the world mostly generated from the seed of crops. Thus the crop fertility is directly corrected to the output. Although female sterility is detrimental to the plant itself, it offers a good chance for the study of mechanism for plant fertility. Since the last century, a large number of research concerning the plant female sterility were reported. However, little is known about it's mechanism. In recent years, scientists have identified a number of reproductive-related mutant genes. Some relevant mechanisms of reproductive development also becomes clear, through most of the molecular mechanism underline reproductive development remains unknown.In the previous study, a female sterility material, generated from a spontaneous mutation, was identified from a rice restore line,202R. By employing a map-based clone strategy, the mutant gene was located to a 60kb interval on the short arm of chromosome 5, and the candidate gene were further comfirmed by sequencing. In the present study, several expression vetctor PHB-PT-OVE, PHB-Ppt-PT, PHB-PI1 pHB-PI1, pHB-PI2, P1300-Ppt-GUS, PA7-35s-PT-YFP for functional analysis of the mutant gene were construct. Through these vectors, several transgenic rice, Arabidopsis and onion plants or cells were generated for functional analysis.The main research results are as follows:1. The intermediate vector PBS-PT, PBS-Ppt, PBS-PI1, PBS-PI2 were used as target gene donor, and plant expression vector PHB for the receptor to construct over-expression vectors PHB-PT-OVE, complementation expression vectors PHB-Ppt-PT, interfere expression vector pHB-PI1 and pHB-PI2. The intermediate vector PBS-Ppt was used as donor and expression vector P1300-GUS for the receptor to construct tissue-expression vectors P1300-Ppt-GUS. The intermediate vector PBS-PT was used as donor and expression vector PA7-YFP for the receptor to construct cellular localization-expression vectors.All the the recombinant plasmids were identified by enzymes digestion and direct sequencing. 2. The complement expression vector PHB-Ppt-PT was transferred into callus of fs-202R through Agrobactetium-mediated transformation. After tissue culture, HPT resistance screening and callus differentiation, the regeneration plants were gained.And finally,14 positive plants are obtained by PCR Screening and Basta resistance analysis.3. Five To Ppt-PT transgenic rice plants were selected and plant singlely to generate the T1 family lines. Fertility investigation of the 5 lines indicated that and the mutant phenotype was rescued by the transgene in all line. The target gene was confirmed tightly linked to the fertile phenotype by Screening and Basta resistance analysis of each T1 transgenic rice plants in every line. The results showed that it is The transgene that restore the mutant fertility.4. The tissue-specific expression vector PHB-Ppt-GUS was transferred into Arabidopsis through Agrobactetium-mediated transformation. And the transgenic plants were obtained. Gus staining of transgenic plant seedlings and mature plants tissues indicated that the mutant gene was expressed in vascular tissue of the Arabidopsis seedlings and in vegetative organs, also expressed in the pistil and stamen.5. The sub-cellular localization expression vector PA7-35s-PT-YFP was transferred into onion epidermal cells through Particle bombardment. The onion cells by transient expression observed through Laser scanning confocal microscope sugested that the YFP gene mainly located in the onion cell membrane, cytoplasm and nucleus.6. The over-expression vector PHB-PT-OVE was transferred into callus of fs-202R and kasalath through Agrobactetium-mediated transformation. After tissue culture, HPT resistance screening and callus differentiation, the regeneration plants were gained. Screening these regeneration plants by PCR, 3 positive seedlings of fs-202R and 10 positive plants of kasalath are obtained. The RNAi expression vector PHB-PI1, PHB-PI2 was transferred into callus of Nipponbare through Agrobactetium-mediated transformation.60 PHB-PI1 transgenic HPT resistant regeneration plants were gained.62 PHB-PI2 transgenic HPT resistant regeneration plants were gained.