转OsbHLH1或AhCMO基因水稻的性状鉴定与分析
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摘要
盐胁迫、低温胁迫是水稻生产中常见的非生物逆境。为了选育耐低温、耐盐的水稻新品种,提高水稻抗逆性,本研究对粳稻恢复系淮C17为受体的转OsbHLH1+Bar基因和转AhCMO+Bar基因的T1代及其后代进行了研究,获得如下结果：
(1)使用相同方法分别对转OsbHLH1+Bar基因和转AhCMO+Bar基因的植株进行草铵膦筛选。再生植株T1代的叶片用1g/L的草铵膦涂布,结果表明再生植株具有抗草铵膦的能力,说明Bar基因在水稻中已经表达。转基因T2代和T3代植株在三叶一心时期喷施0.75g/L的草铵膦,结果表明Bar基因在T2代和T3代均正常表达。转OsbHLH1+Bar基因T3代的少数株系中出现全活的,说明已经筛选出了Bar基因纯合株系。
(2)分别对转OsbHLH1+Bar基因和转AhCMO+Bar基因植株进行PCR和Southern检测,结果表明外源基因已经整合到水稻基因组中。分别对转OsbHLH1+Bar基因和转AhCMO+Bar基因株系T3代进行RT-PCR和实时荧光定量PCR检测,结果表明外源基因已经在水稻中表达,同一外源基因在不同株系之间表达量不同,并且转OsbHLH1+Bar基因植株的表达量远高于非转基因对照。
(3)分别对T2代转OsbHLH1+Bar基因和转AhCMO+Bar基因水稻部分株系的农艺性状进行考察。转OsbHLH1+Bar基因株系考察结果如下：7号株系的株高显著比对照矮；转基因株系的穗长与对照无显著差异；6、7、9、11号株系的千粒重显著比对照重；6、7号株系的结实率显著比对照高；6、9号株系的理论产量显著比对照高。转AhCMO+Bar基因株系考察结果如下：转基因株系的株高显著比对照矮；转基因株系的穗长与对照无显著差异；转基因株系的千粒重显著比对照重；2、6、7号株系的结实率显著比对照低；2、5、6、7号株系的理论产量显著比对照低,但10号株系的理论产量显著比对照高。综上所述,外源基因的导入对水稻的农艺性状产生了重要影响。
(4)以2°C/6 d为低温处理条件,对转OsbHLH1+Bar基因水稻T3代进行芽期耐低温的鉴定,幼芽恢复生长6d后,转基因水稻的死苗率明显小于非转基因对照。结果证明OsbHLH1基因在水稻中过表达提高了水稻芽期的耐低温能力。对转OsbHLH1+Bar基因水稻T3代苗期进行低温胁迫试验,结果表明OsbHLH1基因在水稻中过表达显著提高了水稻苗期的耐低温能力。
(5)以1.4%、1.6%、1.8% NaCl对转AhCMO+Bar基因水稻进行盐胁迫发芽试验,统计成苗率。结果表明转基因水稻的成苗率明显高于非转基因对照,说明转AhCMO基因能提高水稻芽期耐盐能力。
Salt stress and low temperature are common abiotic stresses in rice production. In order to breed new rice varieties with salt and low temperature tolerance and improve theeir stress tolerance in rice, the characters of transgenic sinica (japonica) restorer line Huai C17 with Bar-OsbHLH1 or Bar-AhCMO gene and their progenies were studied. The main results were as follows:
(1) The regenerated plants of transgenic T1 generation were smeared with 1 g/L of glufosinate on the leaves and the results showed that the regenerated plants were resistant to glufosinate, which indicated that the Bar gene have expressed in rice. T2 and T3 generation of transgenic plants at three complete leaves plus one young leaf stage were sprayed 0.75 g/L of glufosinate and the results showed that the Bar gene expressed normally also. The lines homozygous in Bar gene were selected out because all individuals of these lines with Bar-OsbHLH1gene in T3 generation were alive after sprayed 0.75 g/L of glufosinate.
(2) Transgenic plants were detected by PCR and Southern blotting and the results showed that the foreign genes had integrated into the rice genome. T3 generation of transgenic lines were detected by RT-PCR and real-time fluorescence quantitative PCR, the results showed that the foreign genes (OsbHLHl and AhCMO) had expressed in rice. But there were different expressions in the different lines, and the expressions of OsbHLHl gene in transgenic plants were significantly higher than these of non-transgenic control.
(3) The agronomic traits of the transgenic lines in T2 generation were investigated, the results showed that there were significant differences between transgenic lines and control in plant height, panicle length,1000-grain weight, seed setting and theoretical yield. The results of OsbHLH1-transgenic lines were as followes:the plant height of the 7th transgenic line was shorter than that of non-transgenic control; there was no significant difference in panicle length between transgenic lines and control; the 1000-grain weight of the 6th,7th,9th and 11th transgenic line were higher than these of control; the seed setting of the 6th and 7th line was higher than that of non-transgenic control; the theoretical yield of the 6th,9th and 11th transgenic line were higher than these of control. The results of AhCMO-transgenic lines were as followes:the plant height of transgenic lines were shorter than those of non-transgenic control; there was no significant difference in panicle length between transgenic lines and control; the 1000-grain weight of the transgenic lines were higher than these of control; the seed setting of the 2nd,6th and 7th line were lower than those of non-transgenic control; the theoretical yield of the 2nd,5th,6th and 7th transgenic line were lower than those of control, but the theoretical yield of the 10th transgenic line was higher than that of control. Overall, the agronomic traits of transgenic lines were greatly influenced by foreign genes or transformation.
(4) After stressed in low temperature (2℃for 6 d) and recovered growth for 6 days at germination stage, the mortality rates of T3 generation of OsbHLH1-transgenic lines were significantly lower than those of non-transgenic control. After stressed in low temperature (8-10℃for 7 d) and recovered growth for 3 days at seedling stage, the result showed that over-expression of OsbHLH1 gene could be able to enhance the cold tolerance of rice.
(5) After stressed in 1.4%,1.6%,1.8% NaCl at germination stage for 20 days, the seedling rate of AhCMO-transgenic lines were significantly higher than those of non-transgenic control which indicated that AhCMO gene could improve salt tolerance in rice at seedling stage.
(1)使用相同方法分别对转OsbHLH1+Bar基因和转AhCMO+Bar基因的植株进行草铵膦筛选。再生植株T1代的叶片用1g/L的草铵膦涂布,结果表明再生植株具有抗草铵膦的能力,说明Bar基因在水稻中已经表达。转基因T2代和T3代植株在三叶一心时期喷施0.75g/L的草铵膦,结果表明Bar基因在T2代和T3代均正常表达。转OsbHLH1+Bar基因T3代的少数株系中出现全活的,说明已经筛选出了Bar基因纯合株系。
(2)分别对转OsbHLH1+Bar基因和转AhCMO+Bar基因植株进行PCR和Southern检测,结果表明外源基因已经整合到水稻基因组中。分别对转OsbHLH1+Bar基因和转AhCMO+Bar基因株系T3代进行RT-PCR和实时荧光定量PCR检测,结果表明外源基因已经在水稻中表达,同一外源基因在不同株系之间表达量不同,并且转OsbHLH1+Bar基因植株的表达量远高于非转基因对照。
(3)分别对T2代转OsbHLH1+Bar基因和转AhCMO+Bar基因水稻部分株系的农艺性状进行考察。转OsbHLH1+Bar基因株系考察结果如下：7号株系的株高显著比对照矮；转基因株系的穗长与对照无显著差异；6、7、9、11号株系的千粒重显著比对照重；6、7号株系的结实率显著比对照高；6、9号株系的理论产量显著比对照高。转AhCMO+Bar基因株系考察结果如下：转基因株系的株高显著比对照矮；转基因株系的穗长与对照无显著差异；转基因株系的千粒重显著比对照重；2、6、7号株系的结实率显著比对照低；2、5、6、7号株系的理论产量显著比对照低,但10号株系的理论产量显著比对照高。综上所述,外源基因的导入对水稻的农艺性状产生了重要影响。
(4)以2°C/6 d为低温处理条件,对转OsbHLH1+Bar基因水稻T3代进行芽期耐低温的鉴定,幼芽恢复生长6d后,转基因水稻的死苗率明显小于非转基因对照。结果证明OsbHLH1基因在水稻中过表达提高了水稻芽期的耐低温能力。对转OsbHLH1+Bar基因水稻T3代苗期进行低温胁迫试验,结果表明OsbHLH1基因在水稻中过表达显著提高了水稻苗期的耐低温能力。
(5)以1.4%、1.6%、1.8% NaCl对转AhCMO+Bar基因水稻进行盐胁迫发芽试验,统计成苗率。结果表明转基因水稻的成苗率明显高于非转基因对照,说明转AhCMO基因能提高水稻芽期耐盐能力。
Salt stress and low temperature are common abiotic stresses in rice production. In order to breed new rice varieties with salt and low temperature tolerance and improve theeir stress tolerance in rice, the characters of transgenic sinica (japonica) restorer line Huai C17 with Bar-OsbHLH1 or Bar-AhCMO gene and their progenies were studied. The main results were as follows:
(1) The regenerated plants of transgenic T1 generation were smeared with 1 g/L of glufosinate on the leaves and the results showed that the regenerated plants were resistant to glufosinate, which indicated that the Bar gene have expressed in rice. T2 and T3 generation of transgenic plants at three complete leaves plus one young leaf stage were sprayed 0.75 g/L of glufosinate and the results showed that the Bar gene expressed normally also. The lines homozygous in Bar gene were selected out because all individuals of these lines with Bar-OsbHLH1gene in T3 generation were alive after sprayed 0.75 g/L of glufosinate.
(2) Transgenic plants were detected by PCR and Southern blotting and the results showed that the foreign genes had integrated into the rice genome. T3 generation of transgenic lines were detected by RT-PCR and real-time fluorescence quantitative PCR, the results showed that the foreign genes (OsbHLHl and AhCMO) had expressed in rice. But there were different expressions in the different lines, and the expressions of OsbHLHl gene in transgenic plants were significantly higher than these of non-transgenic control.
(3) The agronomic traits of the transgenic lines in T2 generation were investigated, the results showed that there were significant differences between transgenic lines and control in plant height, panicle length,1000-grain weight, seed setting and theoretical yield. The results of OsbHLH1-transgenic lines were as followes:the plant height of the 7th transgenic line was shorter than that of non-transgenic control; there was no significant difference in panicle length between transgenic lines and control; the 1000-grain weight of the 6th,7th,9th and 11th transgenic line were higher than these of control; the seed setting of the 6th and 7th line was higher than that of non-transgenic control; the theoretical yield of the 6th,9th and 11th transgenic line were higher than these of control. The results of AhCMO-transgenic lines were as followes:the plant height of transgenic lines were shorter than those of non-transgenic control; there was no significant difference in panicle length between transgenic lines and control; the 1000-grain weight of the transgenic lines were higher than these of control; the seed setting of the 2nd,6th and 7th line were lower than those of non-transgenic control; the theoretical yield of the 2nd,5th,6th and 7th transgenic line were lower than those of control, but the theoretical yield of the 10th transgenic line was higher than that of control. Overall, the agronomic traits of transgenic lines were greatly influenced by foreign genes or transformation.
(4) After stressed in low temperature (2℃for 6 d) and recovered growth for 6 days at germination stage, the mortality rates of T3 generation of OsbHLH1-transgenic lines were significantly lower than those of non-transgenic control. After stressed in low temperature (8-10℃for 7 d) and recovered growth for 3 days at seedling stage, the result showed that over-expression of OsbHLH1 gene could be able to enhance the cold tolerance of rice.
(5) After stressed in 1.4%,1.6%,1.8% NaCl at germination stage for 20 days, the seedling rate of AhCMO-transgenic lines were significantly higher than those of non-transgenic control which indicated that AhCMO gene could improve salt tolerance in rice at seedling stage.
引文
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