Rao Gundra Sivakrishna, Deveshwar Priyanka, Sharma Malini, Kapoor Sanjay, Rao Khareedu Venkateswara
Centre for Plant Molecular Biology, Osmania University, Hyderabad, T.S., 500007, India.
Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, 110021, India.
Plant Mol Biol. 2018 Jan;96(1-2):35-51. doi: 10.1007/s11103-017-0678-5. Epub 2017 Oct 31.
We have developed a unique male-sterility and fertility-restoration system in rice by combining Brassica napus cysteine-protease gene (BnCysP1) with anther-specific P12 promoter of rice for facilitating production of hybrid varieties. In diverse crop plants, male-sterility has been exploited as a useful approach for production of hybrid varieties to harness the benefits of hybrid vigour. The promoter region of Os12bglu38 gene of rice has been isolated from the developing panicles and was designated as P12. The promoter was fused with gusA reporter gene and was expressed in Arabidopsis and rice systems. Transgenic plants exhibited GUS activity in tapetal cells and pollen of the developing anthers indicating anther/pollen-specific expression of the promoter. For engineering nuclear male sterility, the coding region of Brassica napus cysteine protease1 (BnCysP1) was isolated from developing seeds and fused to P12 promoter. Transgenic rice plants obtained with P12-BnCysP1 failed to produce functional pollen grains. The F seeds obtained from BnCysP1 male-sterile plants and untransformed controls showed 1:1 (tolerant:sensitive) ratio when germinated on the MS medium supplemented with phosphinothricin (5 mg/l), confirming that the male sterility has been successfully engineered in rice. For male fertility restoration, transgenic rice plants carrying BnCysP1Si silencing system were developed. The pollination of BnCysP1 male-sterile (female-fertile) plants with BnCysP1Si pollen resulted in normal grain filling. The F seeds of BnCysP1 × BnCysP1Si when germinated on the MS basal medium containing PPT (5 mg/l) and hygromycin (70 mg/l) exhibited 1:1 (tolerant:sensitive) ratio and the tolerant plants invariably showed normal grain filling. The overall results clearly suggest that the customized male-sterility & fertility-restoration system can be exploited for quality hybrid seed production in various crops.
我们通过将甘蓝型油菜半胱氨酸蛋白酶基因（BnCysP1）与水稻花药特异性P12启动子相结合，开发了一种独特的水稻雄性不育和育性恢复系统，以促进杂交品种的生产。在多种作物中，雄性不育已被用作生产杂交品种的一种有用方法，以利用杂种优势。水稻Os12bglu38基因的启动子区域已从发育中的稻穗中分离出来，并命名为P12。该启动子与gusA报告基因融合，并在拟南芥和水稻系统中表达。转基因植物在发育中的花药的绒毡层细胞和花粉中表现出GUS活性，表明该启动子具有花药/花粉特异性表达。为了构建核雄性不育，从发育中的种子中分离出甘蓝型油菜半胱氨酸蛋白酶1（BnCysP1）的编码区，并与P12启动子融合。用P12-BnCysP1获得的转基因水稻植株未能产生功能性花粉粒。从BnCysP1雄性不育植株和未转化对照获得的F种子在添加了草丁膦（5毫克/升）的MS培养基上萌发时，显示出1:1（耐受:敏感）的比例，证实水稻中的雄性不育已成功构建。为了恢复雄性育性，开发了携带BnCysP1Si沉默系统的转基因水稻植株。用BnCysP1Si花粉对BnCysP1雄性不育（雌性可育）植株进行授粉，导致正常的籽粒充实。BnCysP1×BnCysP1Si的F种子在含有PPT（5毫克/升）和潮霉素（70毫克/升）的MS基础培养基上萌发时，表现出1:1（耐受:敏感）的比例，耐受植株总是表现出正常的籽粒充实。总体结果清楚地表明，定制的雄性不育和育性恢复系统可用于各种作物的优质杂交种子生产。
