AtICE1等多价外源基因表达对番茄耐寒性影响

发布时间：2018-07-28 17:25

【摘要】：番茄(Solanum lycopersicum L.)不仅是一种重要的蔬菜,而且也是一种重要的模式植物。在我国种植番茄已经成为部分农民的主要经济来源,在经济和农村社会发展中发挥着越来越重要的作用。番茄是冷敏感植株,周期性的寒害给我国番茄产业造成巨大的经济损失,培育耐寒品种是解决番茄寒害的重要途径。传统的育种方法存在着生产周期长、工作量大、资源匮乏等缺点,因此难以通过传统的育种方式培育耐寒品种,而转基因技术为培育耐寒品种提供了很好的途径。转基因技术培育的耐寒材料有着试验周期短和低成本等优点,但植物的耐寒性是由多个微效基因控制的复杂数量性状,转入1-2个耐寒性基因并不能显著提高植物的耐寒性,具有一定的局限性。因此,本研究将多价基因导入番茄,以分析转基因番茄植株的耐寒性。主要的研究结果如下:1.整合有AtCBF1等四价转基因番茄和野生型番茄各12株,置于4℃低温处理7天后,随之2℃低温处理14天,观察冷害症状。4℃低温处理7天,野生型番茄萎焉,番茄顶端甚至出现严重脱水症状,四价转基因番茄叶片稍微卷曲,表现出比野生型植株有更强的耐寒性,在此期间,二者均没有冷害致死;2℃处理14天时,野生型番茄已经冷害致死,四价转基因番茄植株叶片出现严重脱水症状并且顶端倒伏但没冷害致死,表现出比野生型植株有更强的耐寒性。最后,将这些番茄植株在25℃光照培养箱进行恢复处理,在恢复7天后,野生型番茄依然死亡,而四价转基因番茄萎焉枝叶恢复正常,在恢复14天时,四价转基因番茄的长出新叶。这些结果显示四价外源基因的表达增强了番茄的耐寒性。2.利用qRT-PCR对四价外源基因和番茄内源基因的表达量进行测定,结果验证了四价外源基因在转基因番茄中均获得表达,而且在6h表达量达到最高,其中AtCBF1-2提高了约12倍;AtCBF3提高了约30倍;AtICE1提高了约2倍,番茄内源基因SlCi7、Sl Ci7、SlDEHL和SlCAT1相对表达量也在6h达到最高,提高了约10倍。3.在4℃低温下处理5天,对四价转基因番茄和野生型番茄进行耐寒性相关的生理生化指标测定。结果显示,四价转基因番茄的相对离子泄漏、丙二醛含量均要低于野生型番茄;而四价转基因番茄的脯氨酸含量和抗氧化酶(CAT、POD和SOD)活性均高于野生型番茄。4.经PCR扩增检测,在获得的AtHOS10等十一价转基因番茄(T2代)中检测到3个纯合株系。5.整合有AtHOS10等十一价转基因番茄和野生型番茄各12株置于4℃低温处理7天后,随之2℃低温处理14天,观察冷害症状。4℃低温处理7天,野生型番茄严重倒伏并出现脱水症状,而十一价转基因番茄叶片稍微卷曲,表现出比野生型植株有更强的耐寒性,在此期间,二者均没有冷害致死。随之,将这些植株进行2℃低温处理,2℃处理14天时,十一价转基因番茄植株叶片出现严重脱水症状并且顶端倒伏但没冷害致死,表现出比野生型植株有更强的耐寒性。最后,将这些番茄植株在25℃光照培养箱进行恢复处理,在恢复7天后,野生型番茄依然死亡,而十一价转基因番茄萎焉枝叶恢复正常,在恢复14天时,十一价转基因番茄长出新的枝条并正常生长这些结果显示十一价外源基因的表达增强了番茄的耐寒性。6.利用qRT-PCR对十一价外源基因和番茄内源基因的表达量进行测定,结果验证了十一价外源基因在转基因番茄中均获得表达,而且在6h表达量达到最高值,其中AtCBF1-3和At COR15A提高了约20倍;At ICE1提高了约10倍,AtCOR47、AtLTI45和AtRD29A提高了约5倍;AtCBR15B、AtHOS10和AtZAT12提高了约2倍,番茄内源基因SlCi7、SlCi7和SlDEHL相对表达量也在6h达到最高值,分别提高了约5、3和10倍。7.在4℃低温下处理5天,对十一价转基因番茄和野生型番茄进行耐寒性相关的生理生化指标测定。结果显示,十一价转基因番茄的相对离子泄漏、丙二醛含量均要低于野生型番茄;而十一价转基因番茄的脯氨酸含量和抗氧化酶(CAT、POD和SOD)活性均高于野生型番茄。该研究为今后深入研究AtCBF1、AtCBF2、AtCBF3、AtICE1、AtCOR15A、AtCBR15B、AtCOR47、At HOS10、AtZAT12、AtLTI45和AtRD29A基因的调控机制及培育优异的番茄耐寒育种材料奠定了基础。
[Abstract]:Tomato (Solanum lycopersicum L.) is not only an important vegetable but also an important model plant. Planting tomatoes in China has become the main economic source of some farmers and plays a more and more important role in the economic and rural social development. The tomato is a cold sensitive plant, and the cyclical cold damage to the tomato production in China. It is an important way to solve the cold damage of tomato. The traditional breeding methods have the disadvantages of long production cycle, heavy workload and lack of resources. Therefore, it is difficult to cultivate cold resistant varieties by traditional breeding methods, and transgenic technology provides a good way to cultivate cold resistant varieties. The cold resistant materials developed by technology have the advantages of short period of test and low cost, but the cold resistance of plants is a complex quantitative trait controlled by multiple microsatellite genes. The transfer into 1-2 cold resistant genes can not significantly improve the cold resistance of plants. Therefore, this study introduces the polyvalent genes into tomato to analyze transgenic plants. The main research results were as follows: 1. integrated with AtCBF1 and 12 strains of transgenic tomato and wild tomato, 7 days at 4 degrees centigrade, 7 days at low temperature and 14 days at low temperature at 2, and 7 days for cold treatment at low temperature. The tomato leaves were slightly curly, showing a stronger cold resistance than the wild type plants. During this period, both of the two had no cold damage to death. At 2 C 14 days, the wild type tomato had been killed by cold damage. The leaves of the tetravalent transgenic tomato plants had severe dehydration symptoms and the top lodged but did not kill the cold damage, showing a stronger than wild type plants. Finally, the tomato plants were recovered at 25 degrees centigrade light culture box. After 7 days, the wild tomato was still dead, and the tetravalent transgenic tomato leaves returned to normal, and the tetravalent transgenic tomato grew out of the new leaf at 14 days. These results showed that the expression of tetravalent foreign genes enhanced the tolerance of tomato. Cold.2. used qRT-PCR to determine the expression of tetravalent foreign gene and tomato endogenous gene. The results showed that the expression of tetravalent exogenous gene in transgenic tomato was all expressed, and the expression of 6h reached the highest, of which AtCBF1-2 increased about 12 times, AtCBF3 increased about 30 times, AtICE1 increased about 2 times, tomato endogenous gene SlCi7, Sl C The relative expression of i7, SlDEHL and SlCAT1 reached the highest in 6h, and increased about 10 times of.3. at 4 C for 5 days. The physiological and biochemical indexes related to cold resistance of tetravalent transgenic tomato and wild tomato were measured. The results showed that the relative ion leakage and malondialdehyde content of the tetravalent transgenic tomato were lower than that of the wild type tomato; and four. The proline content and antioxidant enzyme (CAT, POD and SOD) activity of the transgenic tomato were higher than that of the wild type tomato.4. by PCR amplification. In the obtained AtHOS10 and other eleven valent transgenic tomatoes (T2 generation), 3 homozygous strains were integrated with AtHOS10 and eleven wild tomato and 12 wild tomato plants at 4 temperature for 7 days. After 14 days of low temperature treatment at 2 degrees centigrade, the symptoms of cold injury.4 C were observed for 7 days. The wild type tomato was lodged seriously and dehydrated, and the leaves of the eleven transgenic tomato were slightly curly, showing a stronger cold resistance than the wild type. During this period, the two had no cold damage to death. Then, the plants were at 2 degrees centigrade at low temperature. At 2 C for 14 days, the leaves of eleven price transgenic tomato plants had severe dehydration symptoms and the apex was lodged but did not kill the cold damage. It showed a stronger cold resistance than the wild type plants. Finally, the tomato plants were recovered at 25 centigrade light culture box, and the wild tomato was still dead after the restoration of 7 days, and the eleven price turned. The gene tomato leaves returned to normal, and at the time of recovery 14 days, the eleven valence transgenic tomatoes grew new branches and normal growth. The results showed that the expression of eleven valence foreign genes enhanced the cold tolerance of tomato.6., and the expression of eleven valence foreign gene and tomato endogenous gene was measured by qRT-PCR. The result proved eleven price. Exogenous genes were expressed in transgenic tomatoes, and the expression of 6h reached the highest value, in which AtCBF1-3 and At COR15A increased about 20 times; At ICE1 increased about 10 times, AtCOR47, AtLTI45 and AtRD29A increased by about 5 times; AtCBR15B, AtHOS10 and AtZAT12 increased about 2 times, and the relative expression of the tomato endogenous gene was also 6 H reached the highest value, raised about 5,3 and 10 times.7. at 4 centigrade for 5 days. The physiological and biochemical indexes related to cold resistance of eleven transgenic tomato and wild tomato were measured. The results showed that the relative ion leakage and malondialdehyde content of eleven valent transgenic tomatoes were lower than those of wild type tomatoes; and the eleven price transgenic tomatoes were lower than those of the wild type tomato. The activity of proline and antioxidant enzyme (CAT, POD and SOD) was higher than that of wild tomato. This study laid the foundation for further study of AtCBF1, AtCBF2, AtCBF3, AtICE1, AtCOR15A, AtCBR15B, AtCOR47, At HOS10, regulating mechanism and breeding of excellent cold resistant breeding materials for tomato.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S641.2

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