Mutant Phenotype And Protein Localization Analysis Of PTB1Gene In Rice

Rice is one of the most important crops, and it feed on nearly half the population of the world. Fertility is an important agronomic trait, and is also the breakthrough point of research about mutant. The preliminary study has found a new female sterile rice mutant FS-202R in our laboratory, and the gene has been located into60kb interval on the short arm of fifth chromosome by map-based cloning. Sequence analysis indicated that one of candidate genes losed470bp in DNA sequence, and the missing caused a premature termination codon in the mutant gene, then, it made mutant protein truncated. By functional complementation and excessive expression, we cloned the gene of controlling fertility, and found that the has a positive effect on the seed setting rate.In the meantime, we obtained some positive results for a preliminary study about the mutant phenotype. The results showed that mutant pollen-tube growth in style was blocked, which caused incomplete fertilization, and it was named Pollen Tube Blocked1(PTB1). This study intends to do a comprehensive survey about cytological, anatomy and the growth characteristics of pollen tube, which can lay the foundation for determining the biological mechanism of rice fertility. The concrete results are as follows:1.Fluorescence scanning confocal microscopic observation results showed that, the mutant’s ovary embryo sac was normal and identical with normal Shuhui202rice Polygonum type. In the micropylar end, an egg cell and two help cell made up of the egg apparatus. The top of the egg apparatus have two polar nuclei, and the chalazal end had antipodal cells group, which didn’t belong to the embryo sac structural abortion.2. Fluorescence microscopic used to observe the process of rice pollination, the results showed that the mutant pollen tube was indifferent from wild-type in0-10minutes; but during10-20min, the pollen tube extended into the style, and the growth rate of mutant was slower than wild-type; After20min, the difference is extremely obvious, the mutant growed extremely slow, or even stopped. Whereas, wild-type pollen tube reached the intersection of ovary and chapiter in20min, reached into micropyle in30min, respectively. Thereafter, the pollen tube was getting into the ovary, which suggestted that the mutant sterile is basically due to the growth arrest of pollen tube in the style 3. Fluorescence scanning confocal microscopic was used to observe the structure oi mutant style. It discovered that the female sterile mutant had normal structure partition in style tissue, the stylar canal morphology was also normal and identical with Shuhui202. which suggested that sterility phenotypes should be independent on the structure of style.4. Alcian blue and neutral red double staining results indicated that, both style ECM (extracellular matrix) and wild type material had not deletion and dystopia, which showed that mutant sterility had nothing to do with ECM.5. The complementarily transgenic plants, previously created in the mutant background, whose wild-type PTB1genes were in different expression level, were used tc observe their pollen tube growth dynamic. The results showed that, the fertility of these transgenic plants were getting to recover in different degrees. The seed setting rate was significantly related to the depth and number of pollen tube.6. Natural variation materials were used to identify their fertility. The results showed that, the seed setting rate was unrelated to pollen fertility, but correlated with the rate ol pollen tube getting into style. It indicated that, low fertility of natural variation materials was mainly due to that the growth of the tube was hampered in the style, and unable to complete fertilization.7.Scanning the published genes that related with self-incompatibility, and retrieved them in the rice genome. The results showed that our genes(S1、S2、S4、S5) were getton. which were homologous with gametophytic SLG genes.RT-PCR and realtime qPCR were used to analysis these genes, the result showed thai these four genes in FS-202R had downward trend in different degrees, and the S5did not express in mutant.8. Transient and stable expressive vector were constructed, and then transformed them into onion epidermal cells and rice protoplasts. Fluorescence scanning confocal microscopic was used to observe the subcellular localization of PTB1protein. The result; showed that the PTB1protein was expressed in cell membrane and nucleus in onion epidermal cells, bui the expression in nucleus was more than cell membrane; PTB1was expressed in cytoplasm in the rice protoplast. These results suggestted that this expression of protein had no strong preferences about cellular localization.