抗盐碱基因OsCYP2转化水稻的研究
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摘要
水稻是我国和世界上最重要的粮食作物之一。在我国,水稻作为最重要的粮食作物,种植面积占全国粮食作物种植面积的30%左右,稻米产量占粮食总产量的50%左右,水稻生产在国民经济发展中发挥着巨大作用,直接关系着国民生计。
目前我国人口不断增加,而可耕地面积不断减少,这促使人们对盐碱地的开发与利用产生了极大关注。在盐碱地的开发与利用方面虽然有多种方法,但培育适宜在盐碱地上生长的植物新品种,无疑是利用盐碱地最经济有效的措施之一。随着分子生物学和转基因技术的大发展,克隆功能基因,鉴定基因功能,并将功能基因转化到目标植物中,以获得具有优良目标性状的转基因植株,使植物育种工作取得突破性进展。目前,以上方面的研究已有很多报道,为选育耐盐植物品种开辟了新途径。
本研究克隆得到水稻盐诱导基因OsCYP2,通过农杆菌介导法将其转入水稻,使其在水稻中超表达,通过分子鉴定、生理生化指标鉴定及田间农艺性状鉴定,获得了高世代耐盐水稻新种质,为转基因耐盐水稻的种质创新和新品种培育奠定了基础。取得如下结果：
(1)从北方优质粳稻品种中克隆了全长519bp的盐诱导基因OsCYP2,经分析表明该基因编码173个氨基酸,具有cyclophilinee-ABH-like结构域,属于cyclophilin基因家族,与已知的稻属OsCYP2基因相似性达到97.5%。
(2)将新获得的9sCYP2基因在模式植物烟草中进行功能验证,结果表明新得到的OsCYP2基因可以提高转基因烟草对钠盐的耐受性。
(3)对农大7、吉农大838、吉农大10-41等10份粳稻材料进行筛选,确定吉农大838在农艺性状和再生方面均好于其他品种,可以作为转基因的受体材料,并建立吉农大838转化再生体系。
(4)构建植物表达载体pCAMBIA3301-OsCYPO2,通过农杆菌介导法转化水稻,获得再生转基因水稻植株。
(5)对转OsCYP2基因的转基因水稻进行southern杂交、RT-PCR,等分子鉴定,结果表明目的基因整合在受体基因组中,并得到表达。
(6)以表达载体pCAMBIA3301-OsCYPO2中bar基因为锚定基因,初步建立了通过单引物PCR获得转基因水稻外源基因插入位置侧翼序列的方法,并获得了转基因水稻外源基因插入位置的侧翼序列。
(7)对转基因水稻材料进行脯氨酸含量、SOD酶活性等生理生化指标检测,结果表明OsCYP2基因的超表达能够提高水稻对盐碱的耐受性。
(8)对转基因水稻进行耐盐试验,结果表明转基因水稻的耐盐性较非转基因对照明显提高。
(9)对转基因水稻的主要田间农艺性性状进行观察,结果表明转基因水稻的生育期较非转基因受体延长。
(10)对T2代、T3代转基因水稻遗传稳定性分析,结果表明目标性状在转基因水稻中可以稳定遗传给后代,其分离比符合孟德尔遗传规律。
Not only in China, but also over the globe, rice (Oryza Sativa L) is one of the most important crops. As most influent crops, directly rice own many things to do with national economy development and the people's livelihood. In China, area of rice cultivation is round thirty percent of all crops, and yield of rice is around fifty percent of all others. The production system of rice plays a major role among whole national economy development.
One side the population increases, another side cultivated land decreases, there is much practical value to make use of saline and alkaline land at high efficiency. To develop and cultivate high salt-tolerance plants which contain much economy and ecological wealth is one of ways for deep development of the saline and alkaline land. With progressing of modern molecular biology and gene transformation technique, a breakthrough has been made for breeding of plant, until now, there are numbers of gene which are involved with salt-tolerance already had been isolated and transferred into lots of target plants, and meantime to established a new channel to selected and developed strong salt-tolerance plant variety.
In this research, salt-induced gene OsCYP2has been cloned from Japonica varieties, and transformed into rice by Agrobacterium-mediated way. The target gene expressed inside the rice, and identified with molecular methods, physiological examinations and agricultural traits investigations. Obtained multiple-generations of new rice germplasm, created a efficient system to develop new rice germplasms and new rice varieties. The outputs are as follow:
(1) Isolated519base pair of salt-induced gene OsCYP2from Northern good-quality Japonica cultivar, the analytical result demonstrates the gene code172amino acids of peptide, contain cyclophilinee-ABH-like domain, belong to cyclophilin gene family,97.5%similar to whole known gene OsCYP2.
(2) For detecting the function, transformed the target gene into model plant of tobacco, the consequence show the ability of salt tolerance was high improved.
(3) Cultivated and selected Jinong7, Jinongda838, Jinongda1-41and others varieties of rice, finally chose Jinongda838which own more excellent agricultural traits than others material as acceptor, and then established regenerated system for Jinongda838.
(4) Constructed the plant expressed vector pCAMBIA3301-OsCYPO2, transformed the target gene into rice with Agrobacterium-mediated way to over-expressed in the rice, and achieved transgenic plants.
(5) Force on bar gene inside recombinant plant expressed vector, using single-prime PCR way, created a primary system to locate the foreign gene by finding out the flank sequence of inserted fragment.
(6) Force on bar gene inside recombinant plant expressed vector, using single-prime PCR way, created a primary system to locate the foreign gene by finding out the flank sequence of inserted fragment.
(7) Investigated a number of physiology index such as content of praline, assay of Superoxide Disemutase and others, the data showed actually the gene help rice organism to improve the resistance of salt-tolerance.
(8) Characterized the resistance of salt-tolerance on transgenic rice and found the ability was precisely elevated at high level.
(9) Examination on major agricultural traits of the transgenic rice, contrasted to control, nothing but reproductive period prolonged.
(10) Analysis of inheritance stable ability of the rice on generations of T2and T3, figure out that target gene in progeny stably transmitted into next generation, and distribution data corresponded to Mendel's law of inheritance.
目前我国人口不断增加,而可耕地面积不断减少,这促使人们对盐碱地的开发与利用产生了极大关注。在盐碱地的开发与利用方面虽然有多种方法,但培育适宜在盐碱地上生长的植物新品种,无疑是利用盐碱地最经济有效的措施之一。随着分子生物学和转基因技术的大发展,克隆功能基因,鉴定基因功能,并将功能基因转化到目标植物中,以获得具有优良目标性状的转基因植株,使植物育种工作取得突破性进展。目前,以上方面的研究已有很多报道,为选育耐盐植物品种开辟了新途径。
本研究克隆得到水稻盐诱导基因OsCYP2,通过农杆菌介导法将其转入水稻,使其在水稻中超表达,通过分子鉴定、生理生化指标鉴定及田间农艺性状鉴定,获得了高世代耐盐水稻新种质,为转基因耐盐水稻的种质创新和新品种培育奠定了基础。取得如下结果：
(1)从北方优质粳稻品种中克隆了全长519bp的盐诱导基因OsCYP2,经分析表明该基因编码173个氨基酸,具有cyclophilinee-ABH-like结构域,属于cyclophilin基因家族,与已知的稻属OsCYP2基因相似性达到97.5%。
(2)将新获得的9sCYP2基因在模式植物烟草中进行功能验证,结果表明新得到的OsCYP2基因可以提高转基因烟草对钠盐的耐受性。
(3)对农大7、吉农大838、吉农大10-41等10份粳稻材料进行筛选,确定吉农大838在农艺性状和再生方面均好于其他品种,可以作为转基因的受体材料,并建立吉农大838转化再生体系。
(4)构建植物表达载体pCAMBIA3301-OsCYPO2,通过农杆菌介导法转化水稻,获得再生转基因水稻植株。
(5)对转OsCYP2基因的转基因水稻进行southern杂交、RT-PCR,等分子鉴定,结果表明目的基因整合在受体基因组中,并得到表达。
(6)以表达载体pCAMBIA3301-OsCYPO2中bar基因为锚定基因,初步建立了通过单引物PCR获得转基因水稻外源基因插入位置侧翼序列的方法,并获得了转基因水稻外源基因插入位置的侧翼序列。
(7)对转基因水稻材料进行脯氨酸含量、SOD酶活性等生理生化指标检测,结果表明OsCYP2基因的超表达能够提高水稻对盐碱的耐受性。
(8)对转基因水稻进行耐盐试验,结果表明转基因水稻的耐盐性较非转基因对照明显提高。
(9)对转基因水稻的主要田间农艺性性状进行观察,结果表明转基因水稻的生育期较非转基因受体延长。
(10)对T2代、T3代转基因水稻遗传稳定性分析,结果表明目标性状在转基因水稻中可以稳定遗传给后代,其分离比符合孟德尔遗传规律。
Not only in China, but also over the globe, rice (Oryza Sativa L) is one of the most important crops. As most influent crops, directly rice own many things to do with national economy development and the people's livelihood. In China, area of rice cultivation is round thirty percent of all crops, and yield of rice is around fifty percent of all others. The production system of rice plays a major role among whole national economy development.
One side the population increases, another side cultivated land decreases, there is much practical value to make use of saline and alkaline land at high efficiency. To develop and cultivate high salt-tolerance plants which contain much economy and ecological wealth is one of ways for deep development of the saline and alkaline land. With progressing of modern molecular biology and gene transformation technique, a breakthrough has been made for breeding of plant, until now, there are numbers of gene which are involved with salt-tolerance already had been isolated and transferred into lots of target plants, and meantime to established a new channel to selected and developed strong salt-tolerance plant variety.
In this research, salt-induced gene OsCYP2has been cloned from Japonica varieties, and transformed into rice by Agrobacterium-mediated way. The target gene expressed inside the rice, and identified with molecular methods, physiological examinations and agricultural traits investigations. Obtained multiple-generations of new rice germplasm, created a efficient system to develop new rice germplasms and new rice varieties. The outputs are as follow:
(1) Isolated519base pair of salt-induced gene OsCYP2from Northern good-quality Japonica cultivar, the analytical result demonstrates the gene code172amino acids of peptide, contain cyclophilinee-ABH-like domain, belong to cyclophilin gene family,97.5%similar to whole known gene OsCYP2.
(2) For detecting the function, transformed the target gene into model plant of tobacco, the consequence show the ability of salt tolerance was high improved.
(3) Cultivated and selected Jinong7, Jinongda838, Jinongda1-41and others varieties of rice, finally chose Jinongda838which own more excellent agricultural traits than others material as acceptor, and then established regenerated system for Jinongda838.
(4) Constructed the plant expressed vector pCAMBIA3301-OsCYPO2, transformed the target gene into rice with Agrobacterium-mediated way to over-expressed in the rice, and achieved transgenic plants.
(5) Force on bar gene inside recombinant plant expressed vector, using single-prime PCR way, created a primary system to locate the foreign gene by finding out the flank sequence of inserted fragment.
(6) Force on bar gene inside recombinant plant expressed vector, using single-prime PCR way, created a primary system to locate the foreign gene by finding out the flank sequence of inserted fragment.
(7) Investigated a number of physiology index such as content of praline, assay of Superoxide Disemutase and others, the data showed actually the gene help rice organism to improve the resistance of salt-tolerance.
(8) Characterized the resistance of salt-tolerance on transgenic rice and found the ability was precisely elevated at high level.
(9) Examination on major agricultural traits of the transgenic rice, contrasted to control, nothing but reproductive period prolonged.
(10) Analysis of inheritance stable ability of the rice on generations of T2and T3, figure out that target gene in progeny stably transmitted into next generation, and distribution data corresponded to Mendel's law of inheritance.
引文
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