Functional Studies Of A Fertility Related Gene BnATA20 In Rapseed (Brassica Napus L.)

Genic Male Sterility (GMS) system has the wide prospect in utilizing heterosis in rapeseed breeding, due to the stable and complete male sterility, no negative cytoplasmic effects and potential threat of using only one cytoplasmic source. However, the mechanism research of male sterility has been lagged behind. The anther development is a complicated process, including stamen primordium initial, archesporial cell formation and division, somatic cells (epidermis, endothecium, middle layer and tapetum) successive formation, division and apoptosis. The fertility would be affected whenever defects occur in the development. So far, abundant genes controlling male sterility have been screened in Arabidopsis, Rice, Tomato, and Tobacco, which are specially expressed in anther, and the research of these genes provide a platform to study the mechanism of fertility in B. napus.In this study, BnATA20 gene was obtained from suppression substractive hybridization (SSH) between Rs1046A and Rs1046B. The BnATA20 antisense suppression vector was transformed to Brassica napus Huashuang 5 according to the Agrobacterium-mediated method.The functions and characteristic of this gene were studied in detail. Main conclusions are listed as follows:1. To optimizing Agrobacterium-mediated genetic transformation system, the effects of different combination of BAP/Zentin concentration on buds induction from calli directly were studied. The results indicated that BAP (4 mg/L)/Zentin (2 mg/L) and BAP (3 mg/L)/Zentin (2 mg/L) were the most evidently to induce callus and shoot regeneration, respectively. The frequency of transformation of transgenic plants is about 17.5%.2. 63 individuals were obtained by the way of transforming the BnATA20 antisense suppression vector to B. napus, eleven were positive by PCR analysis, three of which showed male sterile phenotype eventually, the filaments were greatly reduced in length, and the stamens were shriveled, produced no pollen grains with respect to wild type, and thus were male sterility completely. When these were used to cross with wide type of Huashuang 5, likewise, the F1 plants from the crosses were characterized with male sterility and their sterility were stable.3. The sterile plants could be detected with cytological observations taking advantage of light microscopy, scanning and transmission electron microscopy techniques. We could find aberrations in the tapetum at the late tetrads stage when BnATA20 was knocked-out, further leading to microspore cell death. RT-PCR analysis showed that the BnATA20 gene solely expressed in the tapetum of monocyte stage, with tapetal cells that have excess and/or enlarged vacuoles and lack the densely stained cytoplasm typical of normal tapetal cells, controlling the formation and proliferation of tapetum. Real-time PCR demonstrated that most of genes including MS1, NEF, BnA6 and BnA9 were down-regulated in the BnATA20 knock-out individuals, specially expressed in the tapetum. Our results support the hypothesis that BnATA20 is a crucial component of a genetic network that controls anther development and function.