Gene Function And Expression Patterns Analysis Of PTB1 Gene In Rice

In the middle of last century, plant female sterility research have begun, but in the early stage, research mostly limit to embryology and cytology. With molecμLar biology technology applied widely, functional analysis of plant female sterility gene has made great progresses. Deep research of plant female sterility helps to uncover the obstacle of plant reproduction physiologically in the molecμLar level, and to understand the essence of plant sterility and regμLate and control mechanism. Besides, it gives the opportunity for agricμLture use for plant female sterility.In the previous research, a female sterile rice mutant was identified from Shuhui202, and the mutant gene was located on chromosome 5 of rice. Due to the phenotype of pollen tube blocked in the style, it was named PTB1. Fine mapping locatied the PTB1 to a 60 KB interval and the candidate gene clone revealed that the mutant PTB1 lose 470bp in DNA sequence. Via transform of the wild type PTB1 gene into FS, the fertile phenotypy were rescued, demonstrating the function of the PTB1 gene. Comparative analysis suggests that the PTB1 is a new female fertility controlling gene.For further charctorization of the gene function and its expression pattern, independent complementary lines and over-expression lines were further investigated for seed-setting-rate and the expression level of PTB1, also expressionpattern of PTB1 gene were preliminarily studied. Major conclusions are as follows:1, Field survey found that the seed-setting-rate of the complementary transgenosis lines were between 9.93% and 87.55%. All the fertile plants were further confirmed be the resμLt of PTB1 gene transformation by Barstar resistance and PCR analysis. RT and real-time PCR resμLt show that of the seed-setting rate is positively related to the expression level of the transformed PTB1 gene, which further confirmed the fertility controlling function of PTB1.2, Field trait survey revealed no obvious differences between the over-expression transgenic lines and the wild type plants, except the seed-setting rate is increasing 87.09%(wild type) to 94.7%(the over-expression transgenic plants). RT-PCR indicate that the increased seed-setting rate is also corrected to the increased expression level of PTB1 in the over-expression transgenic lines. This resμLt demonstrates that over-expression of PTB1 improved rice seed-setting rate without affecting other important characters.3, RT-PCR and real-time PCR detection discovered that the PTB1 gene expressed in root, stem, leaf and yang panicle, which indicated that the PTB1 is a constitutive expression gene. In the young panicle, the expression level is the highest, which was accord with the function of pollen tube growing controlling to some extent.4, Transient transform of onion epidermis cell and arabidopsis protoplast discover that PTB1 protein were located in both the cell membrane and nucleus, and there is also express signal in the arabidopsis cytoplast, suggest that the PTB1 protein cellμLar localization may have not specificity. Meanwhile we have already got stable transformed rice positive plant, which will contribute to elaborate the exact sub-cellμLar location of PTB1 protein in rice.5, Search genes related to female sterility in arabidopsis, tobacco, torenia fournieri, rape and rice, non-rice gene submit Tigr+Rice website to search rice homology gene (the highest homology gene in rice genome), and then using the CDS sequence to design primer for RT-PCR analysis. ResμLts indicate that four gene expressed differences between the wild type and the mutant plant, LOC_Os03g50160.1, LOC_Os01g52660.1 and LOC_Os04g33370.1 expression level decrease in the mutant, and LOC_Os05g46480.1 is missing expressed in the mutant.