Luo Hong, Lee Jang-Yong, Hu Qian, Nelson-Vasilchik Kimberly, Eitas Timothy K, Lickwar Colin, Kausch Albert P, Chandlee Joel M, Hodges Thomas K
Department of Genetics, Biochemistry and Life Science Studies, Clemson University, 100 Jordan Hall, Clemson, SC 29634, USA.
Plant Mol Biol. 2006 Oct;62(3):397-408. doi: 10.1007/s11103-006-9031-0. Epub 2006 Aug 1.
A tapetum-specific gene, RTS, has been isolated by differential screening of a cDNA library from rice panicles. RTS is a unique gene in the rice genome. RNA blot analysis and in situ hybridization indicates that this gene is predominantly expressed in the anther's tapetum during meiosis and disappears before anthesis. RTS has no introns and encodes a putative polypeptide of 94 amino acids with a hydrophobic N-terminal region. The nucleotide and deduced amino acid sequence of the gene do not show significant homology to any known sequences. However, a sequence in the promoter region, GAATTTGTTA, differs only by one or two nucleotides from one of the conserved motifs in the promoter region of two pollen-specific genes of tomato. Several other sequence motifs found in other anther-specific promoters were also identified in the promoter of the RTS gene. Transgenic and antisense RNA approaches revealed that RTS gene is required for male fertility in rice. The promoter region of RTS, when fused to the Bacillus amyloliquefaciens ribonuclease gene, barnase, or the antisense of the RTS gene, is able to drive tissue-specific expression of both genes in rice, creeping bentgrass (Agrostis stolonifera L.) and Arabidopsis, conferring male sterility to the transgenic plants. Light and near-infrared confocal microscopy of cross-sections through developing flowers of male-sterile transgenics shows that tissue-specific expression of barnase or the antisense RTS genes interrupts tapetal development, resulting in deformed non-viable pollen. These results demonstrate a critical role of the RTS gene in pollen development in rice and the versatile application of the RTS gene promoter in directing anther-specific gene expression in both monocotyledonous and dicotyledonous plants, pointing to a potential for exploiting this gene and its promoter for engineering male sterility for hybrid production of various plant species.
通过对水稻穗部cDNA文库进行差异筛选，分离出了一个绒毡层特异基因RTS。RTS是水稻基因组中的一个独特基因。RNA印迹分析和原位杂交表明，该基因在减数分裂期间主要在花药绒毡层中表达，并在开花前消失。RTS没有内含子，编码一个由94个氨基酸组成的推定多肽，其N端区域具有疏水性。该基因的核苷酸和推导的氨基酸序列与任何已知序列均无显著同源性。然而，启动子区域的一个序列GAATTTGTTA与番茄两个花粉特异基因启动子区域的一个保守基序仅相差一两个核苷酸。在RTS基因的启动子中还鉴定出了其他一些在其他花药特异启动子中发现的序列基序。转基因和反义RNA方法表明，RTS基因是水稻雄性育性所必需的。RTS的启动子区域与解淀粉芽孢杆菌核糖核酸酶基因barnase或RTS基因的反义序列融合后，能够驱动这两个基因在水稻、匍匐翦股颖（Agrostis stolonifera L.）和拟南芥中进行组织特异性表达，使转基因植物产生雄性不育。对雄性不育转基因植物发育中的花朵进行横截面的光和近红外共聚焦显微镜观察表明，barnase或反义RTS基因的组织特异性表达会中断绒毡层发育，导致花粉变形且无活力。这些结果证明了RTS基因在水稻花粉发育中的关键作用，以及RTS基因启动子在指导单子叶和双子叶植物花药特异性基因表达方面的广泛应用，表明利用该基因及其启动子来构建雄性不育用于各种植物物种的杂交生产具有潜力。
