转SAC基因水稻耐盐性的分析

发布时间：2018-05-26 23:43

本文选题：硫 + 茉莉酸　；参考：《浙江师范大学》2016年硕士论文

【摘要】：地球上60%以上的人以水稻(Oryza sativa L)为主要的粮食作物,但是盐胁迫却是导致水稻产量大幅降低的一种全球性威胁。水稻在其苗期阶段对盐较为敏感,高浓度盐(300mM-500mMNaCl)会扰动维持蛋白结构间疏水性静电平衡力,从而抑制大多数酶的活性,因此在高浓度盐的情况下,植物体内的很多代谢反应和膜功能都会受到强烈的影响。为了改善盐胁迫条件下的水稻产率,人们需要去探讨其耐受机理。在过去几年中,与盐胁迫、干旱和碱性等相关的基因已被分离和表征,最近研究表明,硫营养在植物抗击外界环境压力方面起到了重要的作用。然而盐、干旱和碱性的耐受性是通过控制多基因表达来调控,总的来说,这些基因的产物,可以减轻环境压力造成的损害,或直接增强植物耐受性。SAC蛋白是一种双功能性的酶复合体,具有ATP硫酸化酶(ATP sulfurylase)和腺苷-5'-磷酰硫酸激酶(Adenosine-5'-phosphosulfate kinase)活性,在硫酸盐同化过程中起到关键的作用。SAC是仅有硫酸盐通道效应的蛋白,硫酸盐在硫酸化酶的作用下形成APS,进而在APSK的作用下形成PAPS, PAPS能够提高植物体内半胱氨酸的积累,而半胱氨酸,谷氨酸与甘氨酸会组合形成谷胱甘肽。GSH是一种能够消除体内氧化环境地化合物,GSH在植株代谢与胁迫耐受ROS方面起到关键的用途。在盐胁迫下,植物生长可以调节影响GSH和抗氧化酶的合成机制。同时很多研究表明,植物在遭受非生物胁迫时,茉莉酸(JA)可以直接通过信号网来调节植物细胞死亡过程中产生的活性氧,外源JA能促使植物的自身防御,这方面让人们更加的去关注以及研究。另外的一些证据表明,S元素的氧化还原代谢和茉莉酸的响应存在着一定的联系,例如,用外源茉莉酸处理植株能够上调一些参与半胱氨酸,蛋氨酸,和谷胱甘肽(GSH)合成以及S还原过程中相关基因的表达,最近的研究更进一步的阐述了S的氧化代谢是怎么影响茉莉酸反应的。在本实验的研究中,我们通过利用GUS以及Western Blotting等技术手段对转基因植株分别鉴定,得到两种高表达的转基因植株SHC-7以及SHC-10,我们分析了两者之间的表达水平,选定SHC-10以及WT进行进一步的深入研究,在生理水平方面,经不同的盐浓度处理时结果显示,植株SHC-10相比WT,SHC-10中丙二醛的含量下降,谷胱甘肽,过氧化物酶和叶绿素都有明显的提高。导致这种结果的原因,我们认为有可能SAC蛋白的活性影响水稻内某种激素水平的变化。为了验证上述假设,我们寻找出反映水稻体内茉莉酸水平含量变化的响应基因OsJAZ8,并通过RT-PCR以及SqRT-PCR手段测出WT,SHC-10相对转录水平结果表明,0mM盐浓度处理时,WT内源茉莉酸的相对表达量是SHC-10的0.54倍,然而,在60mM时,两种水稻之间的差异性非常明显,SHC-10是WT的19倍,同时在100mM时,SHC-10是WT的1倍,OsJAZ9在三个盐浓度的条件之下都表明SHC-10的相对表达量要比WT高,其在0mM两者之间的差异性最大,大概是WT的140倍。并且,与对照相比,我们发现水稻体内的SAC蛋白虽已被修饰,但其活性为APSK的Vmax为344.5(±28.611)μM/min,Km为0.23(±0.103)μM,而ATPS活性Vmax为236.5(±3.19)μM/min,Km为0.04(±0.005)mM综上所述,结果SAC蛋白活力的测定表明,活力的增加提高了水稻对外界抗压的作用,SAC基因的表达促进植物体内半胱氨酸含量的提高,而其又是GSH原料之一,所以谷胱甘肽因此得到升高并消除体内的氧化环境,有助于还原环境的形成。同时茉莉酸在植物非生物胁迫条件下起到重要的作用,GSH含量的升高也影响着水稻体内茉莉酸响应基因OsJAZ8表达,而OsJAZ9表达量的分析得出SHC-10耐盐性优于WT。这些结果表示水稻体内,硫代谢相关基因和茉莉酸以及GSH在抗氧化代谢中有一定的联系,并且为我们提供了新的见解。
[Abstract]:More than 60% of the people on the earth have rice (Oryza sativa L) as the main grain crops, but salt stress is a global threat to a large reduction in rice yield. Rice is sensitive to salt during its seedling stage, and high concentration salt (300mM-500mMNaCl) may disturb the hydrophobic electrostatic balance between the protein structures, thus inhibiting the majority of them. In the case of high concentration of salt, many metabolic reactions and membrane functions in plants are strongly affected. In order to improve the yield of rice under salt stress, people need to explore its tolerance mechanism. In the past few years, genes related to salt stress, drought and alkalinity have been separated and characterized. It shows that sulfur nutrition plays an important role in plant resistance to environmental pressure. However, the tolerance of salt, drought and alkaline is regulated by controlling polygene expression. In general, the products of these genes can reduce the damage caused by environmental pressure, or direct connection of plant tolerance.SAC protein is a bifunctional. The enzyme complex, with the activity of ATP sulphate enzyme (ATP sulfurylase) and adenosine -5'- phosphoryl kinase (Adenosine-5'-phosphosulfate kinase), plays a key role in the process of sulfate assimilation,.SAC is the only sulfate channel effect protein, sulfate forms APS under the action of sulfonylase, and then forms PAPS under the action of APSK. PAPS can improve the accumulation of cysteine in plants, while cysteine, glutamic acid and glycine combine to form glutathione.GSH, a compound that can eliminate the oxidative environment in the body. GSH plays a key role in plant metabolism and stress tolerance to ROS. Under salt stress, plant growth can regulate the effects of GSH and antioxidant enzymes. At the same time, many studies have shown that when plants suffer from abiotic stress, JA can directly regulate the reactive oxygen species produced during plant cell death through the signal network, and the exogenous JA can promote the plant's self defense. In this respect, people are more concerned and studied. Some other evidence suggests that the oxygen of the S element. There is a certain relationship between the reduction metabolism and the response of jasmonic acid. For example, exogenous jasmonic acid treatment plants can increase the expression of some genes involved in cysteine, methionine, glutathione (GSH) synthesis and S reduction. Recent studies have further elaborated how S's oxidation metabolism affects jasmonic acid. In this study, we identified two highly expressed transgenic plants, SHC-7 and SHC-10 by using GUS and Western Blotting. We analyzed the level of expression between them, selected SHC-10 and WT for further in-depth study, in physiological level, The results showed that the content of malondialdehyde in SHC-10 decreased, glutathione, peroxidase and chlorophyll increased significantly in the plant SHC-10 compared with WT, and the cause of this result was that it was possible that the activity of SAC protein could affect the changes in a certain hormone level in rice. In order to verify the hypothesis, we The response gene OsJAZ8, which reflects the changes in the level of jasmonic acid in rice, was found and WT was measured by RT-PCR and SqRT-PCR. The relative transcriptional level of SHC-10 showed that the relative expression of endogenous jasmonic acid in WT was 0.54 times that of SHC-10 when 0mM salt concentration was treated. However, the difference between the two kinds of rice was very obvious at 60mM, SHC-, SHC-. 10 is 19 times of WT, and at the same time, at 100mM, SHC-10 is 1 times as high as WT, and OsJAZ9 is higher than WT in the three salt concentration conditions. The difference between 0mM and 0mM is maximum, probably 140 times of WT. And, compared with the control, we found that the SAC protein in rice has been modified, but its activity is APSK Vmax. For 344.5 (+ 28.611) mu M/min and Km 0.23 (+ 0.103) mu M, ATPS activity Vmax was 236.5 (+ 3.19) mu M/min and Km was 0.04 (+ 0.005) mM. The result of the determination of SAC protein activity showed that the increase of vitality increased the effect of rice on the pressure of the outside world. The expression of SAC gene promoted the increase of cysteine content in the plant, and it was also a GSH raw material. Therefore, glutathione increases and eliminates the oxidation environment in the body and helps to reduce the formation of the environment. At the same time, jasmonic acid plays an important role in plant abiotic stress. The increase of GSH content also affects the OsJAZ8 expression of jasmonic acid response gene in rice, and the analysis of the expression of OsJAZ9 results in the salt tolerance of SHC-10 The results above WT. indicate that the gene for thioselxie related genes, jasmonic acid and GSH are associated with antioxidant metabolism in rice, and provide us with new insights.
【学位授予单位】：浙江师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S511

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