不结球白菜的抗旱与抗寒特性、作用机制及抗寒关键基因CBF1的克隆与功能研究
发布时间：2019-01-25 21:52
【摘要】：干旱和低温胁迫是导致植物生长发育受阻和作物减产的重要环境因素。研究植物对干旱和低温的响应具有重要的理论意义和实践价值。我们对小白菜的三个品系苏州青(BcL.1)、黄心乌菜(BcL.2)和黑心乌菜(BcL.3)对干旱和低温胁迫的耐受性进行了研究,主要研究结果如下：1.黑心乌菜比苏州青和黄心乌菜更加耐寒,表现出较好的低温耐受性。低温胁迫后黑心乌菜的存活率明显高于苏州青和黄心乌菜(2-3倍),黑心乌菜的相对电导率明显低于苏州青和黄心乌菜,低温胁迫下黑心乌菜相对于前两者受到的伤害水平较低,植株组织的冻害较小。2.黑心乌菜相对于苏州青和黄心乌菜对干旱胁迫更加耐受,黑心的存活率明显高于苏州青和黄心乌菜(2-3倍),单株离体实验表明,黑心乌菜叶片失水率明显小于苏州青和黄心乌菜。3.为了研究黑心乌菜对低温、干旱胁迫耐受的机制,对三种植株体内可溶性糖含量进行检测,其胁迫处理后可溶性糖含量均有上升但三者之间无显著差异,表明黑心乌菜株系对低温、干早的耐受性可能与可溶性糖积累关系不大。4.干旱处理后,植株体内脯氨酸含量均有上升,黑心乌菜中脯氨酸含量显著高于苏州青和黄心乌菜。但在低温胁迫下三种植株的脯氨酸含量无明差异。表明黑心乌菜对干旱胁迫的耐受性可能与脯氨酸积累有关。5.为了进一步验证黑心乌菜抗旱和抗寒的机制,我们检测了胁迫下脯氨酸代谢途径上相关基因的表达,干旱处理后,黑心乌菜中脯氨酸合成关键基因P5CS表达水平明显上升,脯氨酸降解途径中PDH基因的表达与苏州青和黄心乌菜无显著差异,说明黑心乌菜中P5CS基因高度表达增加脯氨酸含量导致其对干旱胁迫的耐受。进一步检测P5CS酶活,也支持上述结论。而低温处理后,三种植株中P5CS和PDH在转录水平和P5CS酶活均无显著差异。6.为了进一步研究黑心乌菜相对于苏州青和黄心乌菜对低温胁迫更加耐受的原因,我们检测了在低温胁迫下CBF/DREB1依赖的信号途径中相关基因的表达水平。结果表明在低温胁迫下,黑心乌菜中CBF/DREB1途径中关键基因CBF1在其上游调控因子LOS4和CBF自身调控因子CBF2调控下高度表达,激活其下游COR15A基因表达,使黑心乌菜具有比苏州青及黄心乌菜更强的抗寒性。7.以黑心乌菜的cDNA为模板扩增BrCBF1基因全长,将该基因连接到pART27载体上。获得的重组载体转化至农杆菌菌株GV3101,通过浸花转化法将BrCBF1重组载体转化到拟南芥野生型植株中,利用转基因筛选与遗传鉴定。
[Abstract]:Drought and low temperature stress are the important environmental factors leading to plant growth and development hindrance and crop yield reduction. It is of great theoretical and practical value to study the response of plants to drought and low temperature. The tolerance of three lines of pakchoi, BcL.1, BcL.2 and BcL.3 to drought and low temperature stress were studied. The main results were as follows: 1. Acanthopsis nigra showed better cold tolerance and lower temperature tolerance than those from Suzhou and Huangxin. After low temperature stress, the survival rate of Acanthopsis nigra was significantly higher than that of Soochow and Huangxin (2-3 times), and the relative electrical conductivity was significantly lower than that of Soochow and Huangxin. Under low temperature stress, the injury level of Acanthopsis nigra was lower than that of the former two, and the freezing injury of plant tissue was less than that of the former. 2. 2. Acanthopsis nigra was more tolerant to drought stress than that of Soochow and Huangxin, and the survival rate of black heart was significantly higher than that of Soochow and Huangxin (2-3 times). The experiment in vitro showed that the survival rate of black heart was 2 ~ 3 times higher than that of Soochow and Huangxin. The rate of water loss in leaves of Acanthopsis nigra was significantly lower than that of Qingqing and Huangxin in Suzhou. In order to study the mechanism of tolerance to low temperature and drought stress, the content of soluble sugar in three plants was detected. The content of soluble sugar increased after stress treatment, but there was no significant difference among them. The results showed that the tolerance to low temperature and early drying of the black heart black cabbage strain may not be related to the accumulation of soluble sugar. 4. After drought treatment, the proline content in the plants increased, and the proline content in Acanthopsis nigra was significantly higher than that in Soochow and Huangxin. However, there was no obvious difference in proline content among the three plants under low temperature stress. The results suggested that the tolerance of Acanthopsis nigra to drought stress might be related to the accumulation of proline. 5. In order to further verify the mechanism of drought resistance and cold resistance, we detected the expression of proline metabolic pathway related genes under stress. After drought treatment, the P5CS expression level of proline biosynthesis key gene was significantly increased. There was no significant difference between the expression of PDH gene in proline degradation pathway and that in Soochow Qingxinwu and Huangxinqingcai, which indicated that the high expression of P5CS gene in Acanthopsis nigra led to its tolerance to drought stress due to the increase of proline content. Further detection of P5CS activity also supports the above conclusion. However, there was no significant difference in transcription level and P5CS enzyme activity between P5CS and PDH in the three plants after low temperature treatment. In order to further study the reason of the higher tolerance to low temperature stress, we detected the expression level of related genes in CBF/DREB1 dependent signaling pathway under low temperature stress. The results showed that CBF1, the key gene of CBF/DREB1 pathway, was highly expressed under the control of upstream regulatory factor LOS4 and CBF self-regulatory factor CBF2 under low temperature stress, and the downstream COR15A gene expression was activated. To make the black heart black cabbage has stronger cold resistance than Suzhou green and yellow black. 7. The full length of the BrCBF1 gene was amplified by using the cDNA of Acanthopsis nigra as a template, and the gene was ligated to the pART27 vector. The recombinant vector was transformed into Agrobacterium tumefaciens strain GV3101, to transform BrCBF1 recombinant vector into Arabidopsis wild-type plants by means of flower soaking. Transgenic screening and genetic identification were used.
【学位授予单位】：合肥工业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S634.3
本文编号：2415226
[Abstract]:Drought and low temperature stress are the important environmental factors leading to plant growth and development hindrance and crop yield reduction. It is of great theoretical and practical value to study the response of plants to drought and low temperature. The tolerance of three lines of pakchoi, BcL.1, BcL.2 and BcL.3 to drought and low temperature stress were studied. The main results were as follows: 1. Acanthopsis nigra showed better cold tolerance and lower temperature tolerance than those from Suzhou and Huangxin. After low temperature stress, the survival rate of Acanthopsis nigra was significantly higher than that of Soochow and Huangxin (2-3 times), and the relative electrical conductivity was significantly lower than that of Soochow and Huangxin. Under low temperature stress, the injury level of Acanthopsis nigra was lower than that of the former two, and the freezing injury of plant tissue was less than that of the former. 2. 2. Acanthopsis nigra was more tolerant to drought stress than that of Soochow and Huangxin, and the survival rate of black heart was significantly higher than that of Soochow and Huangxin (2-3 times). The experiment in vitro showed that the survival rate of black heart was 2 ~ 3 times higher than that of Soochow and Huangxin. The rate of water loss in leaves of Acanthopsis nigra was significantly lower than that of Qingqing and Huangxin in Suzhou. In order to study the mechanism of tolerance to low temperature and drought stress, the content of soluble sugar in three plants was detected. The content of soluble sugar increased after stress treatment, but there was no significant difference among them. The results showed that the tolerance to low temperature and early drying of the black heart black cabbage strain may not be related to the accumulation of soluble sugar. 4. After drought treatment, the proline content in the plants increased, and the proline content in Acanthopsis nigra was significantly higher than that in Soochow and Huangxin. However, there was no obvious difference in proline content among the three plants under low temperature stress. The results suggested that the tolerance of Acanthopsis nigra to drought stress might be related to the accumulation of proline. 5. In order to further verify the mechanism of drought resistance and cold resistance, we detected the expression of proline metabolic pathway related genes under stress. After drought treatment, the P5CS expression level of proline biosynthesis key gene was significantly increased. There was no significant difference between the expression of PDH gene in proline degradation pathway and that in Soochow Qingxinwu and Huangxinqingcai, which indicated that the high expression of P5CS gene in Acanthopsis nigra led to its tolerance to drought stress due to the increase of proline content. Further detection of P5CS activity also supports the above conclusion. However, there was no significant difference in transcription level and P5CS enzyme activity between P5CS and PDH in the three plants after low temperature treatment. In order to further study the reason of the higher tolerance to low temperature stress, we detected the expression level of related genes in CBF/DREB1 dependent signaling pathway under low temperature stress. The results showed that CBF1, the key gene of CBF/DREB1 pathway, was highly expressed under the control of upstream regulatory factor LOS4 and CBF self-regulatory factor CBF2 under low temperature stress, and the downstream COR15A gene expression was activated. To make the black heart black cabbage has stronger cold resistance than Suzhou green and yellow black. 7. The full length of the BrCBF1 gene was amplified by using the cDNA of Acanthopsis nigra as a template, and the gene was ligated to the pART27 vector. The recombinant vector was transformed into Agrobacterium tumefaciens strain GV3101, to transform BrCBF1 recombinant vector into Arabidopsis wild-type plants by means of flower soaking. Transgenic screening and genetic identification were used.
【学位授予单位】：合肥工业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S634.3
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1 王韧;不结球白菜的抗旱与抗寒特性、作用机制及抗寒关键基因CBF1的克隆与功能研究[D];合肥工业大学;2016年
，本文编号：2415226
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