转ICE1基因提高水稻抗寒性的生理机制研究
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摘要
本研究以实验室获得的阳性转ICE1基因水稻再生植株的T2代植株为试验材料,利用分子生物学方法检测了外源基因ICE1在转基因水稻后代的遗传情况;利用离体叶片检测水稻植株潮霉素抗性,同时用PCR方法检测转基因水稻的HPT基因,比较两种检测结果的一致性,探讨了离体叶片法的可靠性和可行性。本试验研究了低温环境下外源基因ICE1对T2代植株抗氧化系统酶(SOD、CAT、POD、APX和GR)活性变化的影响,并利用蛋白双向电泳技术研究低温处理后非转基因和转基因水稻植株蛋白质组的变化情况,在蛋白质组水平上分析ICE1基因对转基因水稻植株的影响。本试验对转ICE1基因水稻T2代植株抗寒性的研究结论如下:
1.本试验检测了转基因水稻T2代的15个株系,共439个单株。比对了三个生育时期中,HPT基因的PCR检测和离体叶片潮霉素检测结果的一致性。在水稻不同生育期两种检测方法的一致性有所不同,苗期和孕穗期符合率为分别为96.19%和94.94%,但乳熟期的符合率仅为75.11%。试验结果说明,利用离体叶片检测筛选标记基因在苗期和孕穗期具有可行性和可靠性。
2.常温下,四周龄的非转基因和转基因水稻在表型上没有明显差异,低温(0℃)处理2d,非转基因水稻表现冷害症状,转基因水稻表型没有变化;低温处理10d,非转基因水稻叶片几乎全部发黄枯萎,转基因水稻叶片卷缩,但仍保持绿色。说明转ICE1基因水稻抗寒性高于非转基因水稻。
低温处理前,非转基因和转基因水稻的抗氧化酶活性水平相似。0℃胁迫后,水稻抗氧化酶活性升高。非转基因水稻的SOD、CAT、APX和GR酶活性在低温处理0.5d达到最大值,随后开始下降,POD在2d达到最大值,随后下降。转基因水稻的SOD、CAT、APX和GR分别在低温处理2d、4d、4d和6d达到最大值。在酶活性水平上,除POD外转基因水稻植株要高于非转基因植株,在低温处理后期表现更为突出。说明转ICE1基因水稻抗氧胁迫能力高于非转基因水稻。
3.以非转基因和转ICE1基因水稻为材料,提取0℃处理0d及2d的水稻总蛋白,利用蛋白双向电泳技术分析低温处理前后两种水稻蛋白质组的变化。本试验利用质谱鉴定了35个差异表达的蛋白。这些蛋白参与植株体内的多个生理过程,包括信号转导、蛋白翻译后修饰及加工、光合作用、氧化还原平衡等各种生理代谢。它们共同作用,使水稻在低温条件下建立了新的代谢平衡。转基因水稻的Rubisco大亚基的降解速率低于非转基因,烯醇化酶、新的低温响应蛋白(hypothetical protein OsJ_28200)等的含量高于非转基因水稻,这些试验结果暗示低温胁迫下转ICE1基因水稻的生理生化代谢水平优于非转基因植株。
In this research, the positive T2 generation of transfering ICE1 rice and the rice (Oryza sativa L. cv Kenjiandao No.10) were used as the materials. We used molecular biology methods to test the hereditable character of exogenous gene ICE1 in transgenic rice. By the methods of leaf in vitro and PCR the T2 transfer ICE1 rice which has hygromycin tolerance was sifted, and we discussed the feasibility and reliability of the method in vitro leaf due to the statistics of the test. In addition, this test researched the influence of low temperature conditions on the activity of antioxidant system (SOD, CAT, POD, APX, GR) of T2 generation transgenic ICE1 rice through the physiological and biochemical experiments. Then, we used the method of protein two-dimensional electrophoresis to study rice proteomics of T2 transgenic rice and non-transgenic rice which had been treated with low temperature, and analysised the effect of ICE1 upon the transgenic rice at the proteomics level. The experiment results of the study on the cold tolerance of T2 transgenic rice show as follows:
1. 15 lines of T2 transgenic rice, a total of 439 plants were put to the test in the first experiment section. The results of PCR detection of HPT gene and leaves HPT tolerance test during three different stages of rice were compared. The consistency of two detection methods was different at different growth stages of rice. Seedling and booting stage were consistent with the rate of 94.94% and 96.19%, but the consistent was 75.11% in Milky stage. The test results showed that using the method of leaf in vitro detection in the seedling stage and booting stage possessed the feasibility and reliability.
2. In the normal condition, there is no significant phenotype difference in the four-week age non-transgenic and transgenic rice. The performance of non-transgenic rice treated 2d with cold showed the chilling injury symptomslow; the phenotype of transgenic rice did not chang, simultaneously. Low-temperature treated 10d, non-genetically modified rice leaves wilted and almost became entirely yellow; in the meantime, the leaves of transgenic rice crimpled, but still green. The experiment result demonstrated that the cold tolerance of the transgenic ICE1 rice was higher than non-transgenic rice.
The level of activity of antioxidant enzymes of non-transgenic and transgenic rice was similar before low-temperature treated. The activity of antioxidant enzymes of rice plants raised with low temperature stress. SOD, CAT, APX and GR activity of non-transgenic rice went up to the maximum in 0.5d and then began to decline during the low temperature condition; the activity of POD reached the maximum in 2d and then declined. The activity of SOD, CAT, APX and GR of the transgenic rice reached the maximumin in the low-temperature treated 2d, 4d, 4d and 6d, respectively. The level of antioxidant enzymes activity of the transgenic rice plants was higher than non-transgenic plants, except POD, especially in the latter of the low temperature treated. The results above all implied that the antioxidant ability of the transgenic ICE1 rice was stronger than non-transgenic rice.
3. Non-transgenic and transgenic rice ICE1 were used as the materials. We analyzed the variation of the rice proteomics after low temperature(0℃) treated 0d and 2d through the technology of two-dimensional gel electrophoresis. In this study, using mass spectrometry identified 35 proteins which differentially expressed. These proteins involved in the plant physiological processes in vivo, including signal transduction, protein post-translational modification and processing, photosynthesis, redox metabolism in various physiological balance. Their common role in the low-temperature(0℃) condition was to establish a new metabolic balance. The degradation rate of Rubisco large subunit of Transgenic rice was lower than non-genetically modified; the content of enolase, the new low-temperature response protein (hypothetical protein OsJ_28200) was higher than non-genetically modified rice. The results of these experiments suggested that physiological and biochemical metabolism of transgenic plants was higher than non-transgenic plants under low temperature stress.
1.本试验检测了转基因水稻T2代的15个株系,共439个单株。比对了三个生育时期中,HPT基因的PCR检测和离体叶片潮霉素检测结果的一致性。在水稻不同生育期两种检测方法的一致性有所不同,苗期和孕穗期符合率为分别为96.19%和94.94%,但乳熟期的符合率仅为75.11%。试验结果说明,利用离体叶片检测筛选标记基因在苗期和孕穗期具有可行性和可靠性。
2.常温下,四周龄的非转基因和转基因水稻在表型上没有明显差异,低温(0℃)处理2d,非转基因水稻表现冷害症状,转基因水稻表型没有变化;低温处理10d,非转基因水稻叶片几乎全部发黄枯萎,转基因水稻叶片卷缩,但仍保持绿色。说明转ICE1基因水稻抗寒性高于非转基因水稻。
低温处理前,非转基因和转基因水稻的抗氧化酶活性水平相似。0℃胁迫后,水稻抗氧化酶活性升高。非转基因水稻的SOD、CAT、APX和GR酶活性在低温处理0.5d达到最大值,随后开始下降,POD在2d达到最大值,随后下降。转基因水稻的SOD、CAT、APX和GR分别在低温处理2d、4d、4d和6d达到最大值。在酶活性水平上,除POD外转基因水稻植株要高于非转基因植株,在低温处理后期表现更为突出。说明转ICE1基因水稻抗氧胁迫能力高于非转基因水稻。
3.以非转基因和转ICE1基因水稻为材料,提取0℃处理0d及2d的水稻总蛋白,利用蛋白双向电泳技术分析低温处理前后两种水稻蛋白质组的变化。本试验利用质谱鉴定了35个差异表达的蛋白。这些蛋白参与植株体内的多个生理过程,包括信号转导、蛋白翻译后修饰及加工、光合作用、氧化还原平衡等各种生理代谢。它们共同作用,使水稻在低温条件下建立了新的代谢平衡。转基因水稻的Rubisco大亚基的降解速率低于非转基因,烯醇化酶、新的低温响应蛋白(hypothetical protein OsJ_28200)等的含量高于非转基因水稻,这些试验结果暗示低温胁迫下转ICE1基因水稻的生理生化代谢水平优于非转基因植株。
In this research, the positive T2 generation of transfering ICE1 rice and the rice (Oryza sativa L. cv Kenjiandao No.10) were used as the materials. We used molecular biology methods to test the hereditable character of exogenous gene ICE1 in transgenic rice. By the methods of leaf in vitro and PCR the T2 transfer ICE1 rice which has hygromycin tolerance was sifted, and we discussed the feasibility and reliability of the method in vitro leaf due to the statistics of the test. In addition, this test researched the influence of low temperature conditions on the activity of antioxidant system (SOD, CAT, POD, APX, GR) of T2 generation transgenic ICE1 rice through the physiological and biochemical experiments. Then, we used the method of protein two-dimensional electrophoresis to study rice proteomics of T2 transgenic rice and non-transgenic rice which had been treated with low temperature, and analysised the effect of ICE1 upon the transgenic rice at the proteomics level. The experiment results of the study on the cold tolerance of T2 transgenic rice show as follows:
1. 15 lines of T2 transgenic rice, a total of 439 plants were put to the test in the first experiment section. The results of PCR detection of HPT gene and leaves HPT tolerance test during three different stages of rice were compared. The consistency of two detection methods was different at different growth stages of rice. Seedling and booting stage were consistent with the rate of 94.94% and 96.19%, but the consistent was 75.11% in Milky stage. The test results showed that using the method of leaf in vitro detection in the seedling stage and booting stage possessed the feasibility and reliability.
2. In the normal condition, there is no significant phenotype difference in the four-week age non-transgenic and transgenic rice. The performance of non-transgenic rice treated 2d with cold showed the chilling injury symptomslow; the phenotype of transgenic rice did not chang, simultaneously. Low-temperature treated 10d, non-genetically modified rice leaves wilted and almost became entirely yellow; in the meantime, the leaves of transgenic rice crimpled, but still green. The experiment result demonstrated that the cold tolerance of the transgenic ICE1 rice was higher than non-transgenic rice.
The level of activity of antioxidant enzymes of non-transgenic and transgenic rice was similar before low-temperature treated. The activity of antioxidant enzymes of rice plants raised with low temperature stress. SOD, CAT, APX and GR activity of non-transgenic rice went up to the maximum in 0.5d and then began to decline during the low temperature condition; the activity of POD reached the maximum in 2d and then declined. The activity of SOD, CAT, APX and GR of the transgenic rice reached the maximumin in the low-temperature treated 2d, 4d, 4d and 6d, respectively. The level of antioxidant enzymes activity of the transgenic rice plants was higher than non-transgenic plants, except POD, especially in the latter of the low temperature treated. The results above all implied that the antioxidant ability of the transgenic ICE1 rice was stronger than non-transgenic rice.
3. Non-transgenic and transgenic rice ICE1 were used as the materials. We analyzed the variation of the rice proteomics after low temperature(0℃) treated 0d and 2d through the technology of two-dimensional gel electrophoresis. In this study, using mass spectrometry identified 35 proteins which differentially expressed. These proteins involved in the plant physiological processes in vivo, including signal transduction, protein post-translational modification and processing, photosynthesis, redox metabolism in various physiological balance. Their common role in the low-temperature(0℃) condition was to establish a new metabolic balance. The degradation rate of Rubisco large subunit of Transgenic rice was lower than non-genetically modified; the content of enolase, the new low-temperature response protein (hypothetical protein OsJ_28200) was higher than non-genetically modified rice. The results of these experiments suggested that physiological and biochemical metabolism of transgenic plants was higher than non-transgenic plants under low temperature stress.
引文
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