CFPS-M及CFPS-2M基因在拟南芥中的过表达及抗逆性研究

发布时间：2018-05-30 05:44

  本文选题：植物抗逆性 + 基因工程技术　； 参考：《西南科技大学》2016年硕士论文

【摘要】：耐辐射奇球菌(Deinococcus radiodurans)是迄今为止发现的辐射耐受性最强的生物之一,CFPS是位于耐辐射奇球菌R1属的一段基因。通过NCBI数据比对发现CFPS与LEA76基因家族同源,属于LEA第三族的基因。第三族LEA蛋白基因包含多个拷贝的11个氨基酸组成的基元序列(TAQAAKEKAGE)。本研究将耐辐射奇球菌CFPS全长基因分为5个结构域,其中含有11个氨基酸重复区段的第104位至160位氨基酸结构域,命名为CFPS-M;含有双倍CFPS-M基因的串联序列,命名为CFPS-2M。我们构建了植物表达载体PCAMBIA2300-M和PCAMBIA2300-2M,分别将两个基因转入拟南芥中,获得T3代转基因阳性苗,并对其进行了多种非生物胁迫下的抗逆性研究分析。主要结果如下:(1)成功构建植物表达载体PCAMBIA2300-M和PCAMBIA2300-2M,并用冻融法将它们分别导入农杆菌GV3103中,经Kan抗性筛选和菌落PCR鉴定成功导入农杆菌中。(2)选择拟南芥作为受体材料,用农杆菌介导法将CFPS-M和CFPS-2M基因转入拟南芥中,经Kan抗性筛选和对基因组DNA进行PCR鉴定,以及实时荧光定量PCR鉴定,筛选到18株转CFPS-M基因阳性苗和21株转CFPS-2M基因阳性苗,并不断繁育筛选到T3代纯合阳性苗。(3)在多种非生物胁迫下,转基因拟南芥种子的相对发芽率和相对根长都大于非转基因植株(P0.05)。并且包含双拷贝的CFPS-M基因的CFPS-2M转基因拟南芥抗逆能力更强,在干旱、盐渍、高温、低温等非生物胁迫下均较为突出。(4)在多种非生物胁迫下,转基因拟南芥成苗的生理生化指标分析表明转基因植株的丙二醛含量比野生型对照显著降低(P0.05),可溶性糖、可溶性蛋白及脯氨酸等渗透调节物质的含量显著高于非转基因植株(P0.05),且抗氧化酶系统(SOD、POD、CAT)的活性也显著高于野生型对照(P0.05)。说明CFPS-M和CFPS-2M基因通过降低植物体内积累的有害物质MDA的含量及提高植物体内渗透调节保护物质含量、抗氧化酶系统的活力来提高植物抵御非生物胁迫的能力。(5)转CFPS-2M基因拟南芥植株的抗逆性比转CFPS-M基因植株的抗逆性更强,说明增加了外源抗逆基因的拷贝数增强了该抗逆基因表达的效果,使植株有更强的抗逆性。
[Abstract]:Deinococcus radiodurans is one of the most radiation tolerant organisms found so far. CFPS is a gene located in the genus R1 of A. radiodurans. By comparing NCBI data, we found that CFPS is homologous to LEA76 gene family and belongs to the third family of LEA gene. The third group of LEA protein genes contain multiple copies of 11 amino acid motifs (TAQAAKEKAGEG). In this study, the full-length CFPS gene was divided into five domains, including the 104th to 160th amino acid domain with 11 amino acid repeats, and the tandem sequence containing double CFPS-M gene, named CFPS-2M. We constructed plant expression vectors PCAMBIA2300-M and PCAMBIA 2300-2Mrespectively and transferred two genes into Arabidopsis thaliana to obtain T3 generation transgenic positive seedlings. The main results were as follows: (1) the plant expression vectors PCAMBIA2300-M and PCAMBIA 2300-2M were successfully constructed, and they were introduced into Agrobacterium GV3103 by freeze-thaw method. Arabidopsis thaliana was selected as the recipient material by Kan resistance screening and colony PCR identification. CFPS-M and CFPS-2M genes were transferred into Arabidopsis thaliana by Agrobacterium tumefaciens mediated. After Kan resistance screening, PCR identification of genomic DNA and real-time fluorescent quantitative PCR identification, 18 CFPS-M gene positive seedlings and 21 CFPS-2M gene positive seedlings were screened. The relative germination rate and root length of transgenic Arabidopsis thaliana seeds were higher than that of non-transgenic plants P0.05under various abiotic stresses. The transgenic Arabidopsis thaliana containing double copy of CFPS-M gene is more resistant to stress, especially under abiotic stress, such as drought, salinity, high temperature, low temperature and so on. The analysis of physiological and biochemical indexes of transgenic Arabidopsis thaliana seedlings showed that the malondialdehyde content of transgenic plants was significantly lower than that of wild type control. The contents of soluble protein, proline and other osmotic regulators were significantly higher than that of non-transgenic plants (P0.05N), and the activity of antioxidant enzyme system (SODPOD) CATwas significantly higher than that of wild type control (P0.05). It is suggested that CFPS-M and CFPS-2M genes decrease the content of MDA accumulated in plants and increase the content of osmotic regulatory protective substances in plants. The activity of antioxidant enzyme system to improve the ability of plants to resist abiotic stress.) the resistance of transgenic Arabidopsis thaliana plants with CFPS-2M gene is stronger than that of transgenic plants with CFPS-M gene. The results showed that increasing the copy number of exogenous stress resistance gene enhanced the expression of the stress resistance gene and made the plants more resistant to stress.
【学位授予单位】：西南科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2
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本文编号：1954147