皱波角叉菜CaM、HSP70基因的克

发布时间：2018-03-25 04:15

  本文选题：皱波角叉菜　切入点：CaM　出处：《辽宁师范大学》2016年硕士论文

【摘要】：植物生长环境的变化会影响植物的生长与代谢。藻类应答环境胁迫的信号通路及相应蛋白质功能的研究已成为热点。作为机体受到一系列如高温、干旱和严寒等非生物胁迫后大量表达的应激蛋白,HSP70(Heat Shock protein 70)广泛参与生物体内变性蛋白质的复性和降解,对机体的代谢调控和响应环境胁迫具有重要的作用。随着温室效应的加剧,灾害性高温气候成为影响藻类生长的一种主要的非生物胁迫。面对高温胁迫,植物体会快速启动复杂的调控机制来应对热刺激,钙-钙调素热激信号转导通路作为响应热胁迫主要的调控机制,可以调节HSP70的表达。本实验以经济海藻皱波角叉菜的叶状体为实验材料,运用RT-PCR技术克隆皱波角叉菜的CcCaM、CcHSP70基因的ORF序列。利用生物软件对序列进行分析,结果显示CcCaM基因的ORF全长为453bp,共编码150个氨基酸;CcHSP70基因的ORF全长为1971bp,共编码656个氨基酸,并预测出在CcHSP70的氨基酸序列中包含一段由21个氨基酸残基组成的CaM结合区域,这表明在皱波角叉菜中可能存在着通过CaM与HSP70相结合进而来调节热激信号转导的下游事件,这为进一步研究皱波角叉菜的热激信号转导通路提供依据。为了探讨HSP70在皱波角叉菜抵抗高温与冷胁迫中的作用及响应特点,本研究采用Real-Time PCR技术检测皱波角叉菜叶状体在28℃高温胁迫与4℃冷胁迫下CcHSP70基因相对表达量的变化。并将CcHSP70基因转入大肠杆菌内进行原核表达与分离纯化,随后检测了CcHSP70蛋白质的ATPase活性,测定45℃高温与10℃冷胁迫下转pET28a-CcHSP70大肠杆菌的生长情况。结果显示:CcHSP70基因的表达受到高温胁迫与冷胁迫的显著诱导,在持续28℃高温胁迫下,CcHSP70的表达量在4 h时达到峰值,之后一直保持在较高的水平与对照差异显著。在持续冷胁迫下,在8 h时CcHSP70表达量与对照组相比达到显著差异。CcHSP70具有ATPase活性,其可通过ATP的水解循环来控制多肽的结合和释放。在45℃高温与10℃冷胁迫下CcHSP70的过表达提高了大肠杆菌的抗高温能力与耐冷性。结果表明CcHSP70受热激与冷胁迫的诱导表达,在高温胁迫与冷胁迫中维持了细胞蛋白的稳定,对细胞具有重要的保护作用。
[Abstract]:The changes of plant growth environment will affect the growth and metabolism of plants. The research on the signal pathway of algae response to environmental stress and the corresponding protein function has become a hotspot. As a series of organisms subject to a series of high temperatures, The stress protein (HSP70 Heat Shock protein 70), which is widely expressed after abiotic stress such as drought and cold, is widely involved in the renaturation and degradation of denatured proteins in organisms. With the aggravation of Greenhouse Effect, catastrophic high temperature climate has become a major abiotic stress affecting algae growth. Plants experience the rapid initiation of complex regulatory mechanisms to respond to heat stimulation, calcium calmodulin heat shock signal transduction pathway as the main regulatory mechanism in response to heat stress, The RT-PCR technique was used to clone the ORF sequence of the CcCaMU CcHSP70 gene from the striatum of economic algal carrageenia crassifolia, and the sequence was analyzed by biological software. The results showed that the total length of ORF of CcCaM gene was 453bpand the total length of ORF encoding 150 amino acids CcHSP70 gene was 1971bp. the total length of ORF encoding 656 amino acids was predicted to include a CaM binding region composed of 21 amino acid residues in the amino acid sequence of CcHSP70. This suggests that there may be downstream events that regulate heat shock signal transduction through the combination of CaM and HSP70 in Carrageenia crassifolia. This provides a basis for further study on the heat shock signal transduction pathway of carrageenia crassifolia. In order to investigate the role and response of HSP70 in the resistance to high temperature and cold stress of Carrageenia crassifolia, In this study, Real-Time PCR technique was used to detect the relative expression of CcHSP70 gene in the striatum of Carrageenia crassifolia under high temperature stress at 28 鈩，

本文编号：1661496