AtICE1等多價(jià)外源基因表達(dá)對番茄耐寒性影響
發(fā)布時(shí)間：2018-07-28 17:25
【摘要】：番茄(Solanum lycopersicum L.)不僅是一種重要的蔬菜,而且也是一種重要的模式植物。在我國種植番茄已經(jīng)成為部分農(nóng)民的主要經(jīng)濟(jì)來源,在經(jīng)濟(jì)和農(nóng)村社會(huì)發(fā)展中發(fā)揮著越來越重要的作用。番茄是冷敏感植株,周期性的寒害給我國番茄產(chǎn)業(yè)造成巨大的經(jīng)濟(jì)損失,培育耐寒品種是解決番茄寒害的重要途徑。傳統(tǒng)的育種方法存在著生產(chǎn)周期長、工作量大、資源匱乏等缺點(diǎn),因此難以通過傳統(tǒng)的育種方式培育耐寒品種,而轉(zhuǎn)基因技術(shù)為培育耐寒品種提供了很好的途徑。轉(zhuǎn)基因技術(shù)培育的耐寒材料有著試驗(yàn)周期短和低成本等優(yōu)點(diǎn),但植物的耐寒性是由多個(gè)微效基因控制的復(fù)雜數(shù)量性狀,轉(zhuǎn)入1-2個(gè)耐寒性基因并不能顯著提高植物的耐寒性,具有一定的局限性。因此,本研究將多價(jià)基因?qū)敕?以分析轉(zhuǎn)基因番茄植株的耐寒性。主要的研究結(jié)果如下:1.整合有AtCBF1等四價(jià)轉(zhuǎn)基因番茄和野生型番茄各12株,置于4℃低溫處理7天后,隨之2℃低溫處理14天,觀察冷害癥狀。4℃低溫處理7天,野生型番茄萎焉,番茄頂端甚至出現(xiàn)嚴(yán)重脫水癥狀,四價(jià)轉(zhuǎn)基因番茄葉片稍微卷曲,表現(xiàn)出比野生型植株有更強(qiáng)的耐寒性,在此期間,二者均沒有冷害致死;2℃處理14天時(shí),野生型番茄已經(jīng)冷害致死,四價(jià)轉(zhuǎn)基因番茄植株葉片出現(xiàn)嚴(yán)重脫水癥狀并且頂端倒伏但沒冷害致死,表現(xiàn)出比野生型植株有更強(qiáng)的耐寒性。最后,將這些番茄植株在25℃光照培養(yǎng)箱進(jìn)行恢復(fù)處理,在恢復(fù)7天后,野生型番茄依然死亡,而四價(jià)轉(zhuǎn)基因番茄萎焉枝葉恢復(fù)正常,在恢復(fù)14天時(shí),四價(jià)轉(zhuǎn)基因番茄的長出新葉。這些結(jié)果顯示四價(jià)外源基因的表達(dá)增強(qiáng)了番茄的耐寒性。2.利用qRT-PCR對四價(jià)外源基因和番茄內(nèi)源基因的表達(dá)量進(jìn)行測定,結(jié)果驗(yàn)證了四價(jià)外源基因在轉(zhuǎn)基因番茄中均獲得表達(dá),而且在6h表達(dá)量達(dá)到最高,其中AtCBF1-2提高了約12倍;AtCBF3提高了約30倍;AtICE1提高了約2倍,番茄內(nèi)源基因SlCi7、Sl Ci7、SlDEHL和SlCAT1相對表達(dá)量也在6h達(dá)到最高,提高了約10倍。3.在4℃低溫下處理5天,對四價(jià)轉(zhuǎn)基因番茄和野生型番茄進(jìn)行耐寒性相關(guān)的生理生化指標(biāo)測定。結(jié)果顯示,四價(jià)轉(zhuǎn)基因番茄的相對離子泄漏、丙二醛含量均要低于野生型番茄;而四價(jià)轉(zhuǎn)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.經(jīng)PCR擴(kuò)增檢測,在獲得的AtHOS10等十一價(jià)轉(zhuǎn)基因番茄(T2代)中檢測到3個(gè)純合株系。5.整合有AtHOS10等十一價(jià)轉(zhuǎn)基因番茄和野生型番茄各12株置于4℃低溫處理7天后,隨之2℃低溫處理14天,觀察冷害癥狀。4℃低溫處理7天,野生型番茄嚴(yán)重倒伏并出現(xiàn)脫水癥狀,而十一價(jià)轉(zhuǎn)基因番茄葉片稍微卷曲,表現(xiàn)出比野生型植株有更強(qiáng)的耐寒性,在此期間,二者均沒有冷害致死。隨之,將這些植株進(jìn)行2℃低溫處理,2℃處理14天時(shí),十一價(jià)轉(zhuǎn)基因番茄植株葉片出現(xiàn)嚴(yán)重脫水癥狀并且頂端倒伏但沒冷害致死,表現(xiàn)出比野生型植株有更強(qiáng)的耐寒性。最后,將這些番茄植株在25℃光照培養(yǎng)箱進(jìn)行恢復(fù)處理,在恢復(fù)7天后,野生型番茄依然死亡,而十一價(jià)轉(zhuǎn)基因番茄萎焉枝葉恢復(fù)正常,在恢復(fù)14天時(shí),十一價(jià)轉(zhuǎn)基因番茄長出新的枝條并正常生長這些結(jié)果顯示十一價(jià)外源基因的表達(dá)增強(qiáng)了番茄的耐寒性。6.利用qRT-PCR對十一價(jià)外源基因和番茄內(nèi)源基因的表達(dá)量進(jìn)行測定,結(jié)果驗(yàn)證了十一價(jià)外源基因在轉(zhuǎn)基因番茄中均獲得表達(dá),而且在6h表達(dá)量達(dá)到最高值,其中AtCBF1-3和At COR15A提高了約20倍;At ICE1提高了約10倍,AtCOR47、AtLTI45和AtRD29A提高了約5倍;AtCBR15B、AtHOS10和AtZAT12提高了約2倍,番茄內(nèi)源基因SlCi7、SlCi7和SlDEHL相對表達(dá)量也在6h達(dá)到最高值,分別提高了約5、3和10倍。7.在4℃低溫下處理5天,對十一價(jià)轉(zhuǎn)基因番茄和野生型番茄進(jìn)行耐寒性相關(guān)的生理生化指標(biāo)測定。結(jié)果顯示,十一價(jià)轉(zhuǎn)基因番茄的相對離子泄漏、丙二醛含量均要低于野生型番茄;而十一價(jià)轉(zhuǎn)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。該研究為今后深入研究AtCBF1、AtCBF2、AtCBF3、AtICE1、AtCOR15A、AtCBR15B、AtCOR47、At HOS10、AtZAT12、AtLTI45和AtRD29A基因的調(diào)控機(jī)制及培育優(yōu)異的番茄耐寒育種材料奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S641.2
本文編號：2151046
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S641.2
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