本文選題：硫 + 茉莉酸��； 參考：《浙江師范大學(xué)》2016年碩士論文

【摘要】：地球上60%以上的人以水稻(Oryza sativa L)為主要的糧食作物,但是鹽脅迫卻是導(dǎo)致水稻產(chǎn)量大幅降低的一種全球性威脅。水稻在其苗期階段對鹽較為敏感,高濃度鹽(300mM-500mMNaCl)會擾動維持蛋白結(jié)構(gòu)間疏水性靜電平衡力,從而抑制大多數(shù)酶的活性,因此在高濃度鹽的情況下,植物體內(nèi)的很多代謝反應(yīng)和膜功能都會受到強烈的影響。為了改善鹽脅迫條件下的水稻產(chǎn)率,人們需要去探討其耐受機理。在過去幾年中,與鹽脅迫、干旱和堿性等相關(guān)的基因已被分離和表征,最近研究表明,硫營養(yǎng)在植物抗擊外界環(huán)境壓力方面起到了重要的作用。然而鹽、干旱和堿性的耐受性是通過控制多基因表達來調(diào)控,總的來說,這些基因的產(chǎn)物,可以減輕環(huán)境壓力造成的損害,或直接增強植物耐受性。SAC蛋白是一種雙功能性的酶復(fù)合體,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,在硫酸鹽同化過程中起到關(guān)鍵的作用。SAC是僅有硫酸鹽通道效應(yīng)的蛋白,硫酸鹽在硫酸化酶的作用下形成APS,進而在APSK的作用下形成PAPS, PAPS能夠提高植物體內(nèi)半胱氨酸的積累,而半胱氨酸,谷氨酸與甘氨酸會組合形成谷胱甘肽。GSH是一種能夠消除體內(nèi)氧化環(huán)境地化合物,GSH在植株代謝與脅迫耐受ROS方面起到關(guān)鍵的用途。在鹽脅迫下,植物生長可以調(diào)節(jié)影響GSH和抗氧化酶的合成機制。同時很多研究表明,植物在遭受非生物脅迫時,茉莉酸(JA)可以直接通過信號網(wǎng)來調(diào)節(jié)植物細胞死亡過程中產(chǎn)生的活性氧,外源JA能促使植物的自身防御,這方面讓人們更加的去關(guān)注以及研究。另外的一些證據(jù)表明,S元素的氧化還原代謝和茉莉酸的響應(yīng)存在著一定的聯(lián)系,例如,用外源茉莉酸處理植株能夠上調(diào)一些參與半胱氨酸,蛋氨酸,和谷胱甘肽(GSH)合成以及S還原過程中相關(guān)基因的表達,最近的研究更進一步的闡述了S的氧化代謝是怎么影響茉莉酸反應(yīng)的。在本實驗的研究中,我們通過利用GUS以及Western Blotting等技術(shù)手段對轉(zhuǎn)基因植株分別鑒定,得到兩種高表達的轉(zhuǎn)基因植株SHC-7以及SHC-10,我們分析了兩者之間的表達水平,選定SHC-10以及WT進行進一步的深入研究,在生理水平方面,經(jīng)不同的鹽濃度處理時結(jié)果顯示,植株SHC-10相比WT,SHC-10中丙二醛的含量下降,谷胱甘肽,過氧化物酶和葉綠素都有明顯的提高。導(dǎo)致這種結(jié)果的原因,我們認為有可能SAC蛋白的活性影響水稻內(nèi)某種激素水平的變化。為了驗證上述假設(shè),我們尋找出反映水稻體內(nèi)茉莉酸水平含量變化的響應(yīng)基因OsJAZ8,并通過RT-PCR以及SqRT-PCR手段測出WT,SHC-10相對轉(zhuǎn)錄水平結(jié)果表明,0mM鹽濃度處理時,WT內(nèi)源茉莉酸的相對表達量是SHC-10的0.54倍,然而,在60mM時,兩種水稻之間的差異性非常明顯,SHC-10是WT的19倍,同時在100mM時,SHC-10是WT的1倍,OsJAZ9在三個鹽濃度的條件之下都表明SHC-10的相對表達量要比WT高,其在0mM兩者之間的差異性最大,大概是WT的140倍。并且,與對照相比,我們發(fā)現(xiàn)水稻體內(nèi)的SAC蛋白雖已被修飾,但其活性為APSK的Vmax為344.5(±28.611)μM/min,Km為0.23(±0.103)μM,而ATPS活性Vmax為236.5(±3.19)μM/min,Km為0.04(±0.005)mM綜上所述,結(jié)果SAC蛋白活力的測定表明,活力的增加提高了水稻對外界抗壓的作用,SAC基因的表達促進植物體內(nèi)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體內(nèi)的氧化環(huán)境,有助于還原環(huán)境的形成。同時茉莉酸在植物非生物脅迫條件下起到重要的作用,GSH含量的升高也影響著水稻體內(nèi)茉莉酸響應(yīng)基因OsJAZ8表達,而OsJAZ9表達量的分析得出SHC-10耐鹽性優(yōu)于WT。這些結(jié)果表示水稻體內(nèi),硫代謝相關(guān)基因和茉莉酸以及GSH在抗氧化代謝中有一定的聯(lián)系,并且為我們提供了新的見解。
[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.
【學(xué)位授予單位】：浙江師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S511

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1 季科研;轉(zhuǎn)SAC基因水稻耐鹽性的分析[D];浙江師范大學(xué);2016年

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