本文選題：硫化氫 + Cd脅迫��； 參考：《山西大學》2016年博士論文

【摘要】：近年來,H_2S作為生理信號分子得到了研究者越來越多的關注,已有的報道表明在植物中它具有促進根生長和形態(tài)建成、增強光合作用、促進種子萌發(fā)、刺激開花、延遲衰老等多方面生理功能。H_2S在植物抵抗各種環(huán)境脅迫過程中,也扮演了積極的角色。其中,在植物適應重金屬脅迫的過程中發(fā)揮如下方面的作用：H_2S可以促進還原型谷胱甘肽、抗壞血酸的合成,并提高一系列抗氧化酶的活性來維持細胞內氧化還原狀態(tài)的平衡,降低脅迫造成的氧化損傷；研究表明H_2S提高植物中Na+-ATPase、 K+-ATPase和H+-ATPase的活性來抑制對重金屬離子的吸收,同時增加植物對硫、磷、鈣、鎂和鐵的吸收,H_2S還可以抑制受H202調控的Ca通道來減少Cd進入質膜,并且激活液泡膜上的轉運通道,將胞質中Cd轉運至液泡,從而降低胞質內的重金屬離子的濃度；外源H_2S通過提高植物的葉綠素含量,光合作用強度,以及促進細胞內葉綠體內囊的發(fā)育,增加線粒體數(shù)目來緩解Cd對植物造成的毒害：H_2S還會和其他信號分子、多種激素相互作用來緩解重金屬對植物造成的傷害。本研究以重要的十字花科蕓薹屬作物大白菜(Brassica rapa L. ssp. Pekinensis)為材料,運用藥理學、細胞生物學、生理生化等實驗技術,從表型、細胞、分子多個水平對H_2S在提高白菜根部耐受重金屬Cd脅迫過程中的生理作用進行系統(tǒng)研究。1.H_2S對Cd脅迫下白菜根部細胞死亡的影響。Cd脅迫下,隨著處理濃度的增加,白菜幼苗根的生長受到抑制,同時根部細胞的死亡率也迅速提高,且根長和根部細胞死亡率呈負相關。Cd脅迫下白菜幼苗根部內源H_2S產(chǎn)率和H_2S生成關鍵酶的編碼基因表達量都顯著上調。外源H_2S處理(NaHS為供體),可以緩解Cd對白菜幼苗根的生長抑制,并且顯著減少根部細胞的死亡和DNA的降解。進一步研究表明,H_2S信號可以減少白菜幼苗根中Cd脅迫誘導的活性氧(ROS)積累、抑制ROS誘導的根部膜脂過氧化損傷并維護質膜結構的完整,從而抑制根部細胞死亡。2.H_2S對Cd脅迫下白菜根部抗氧化系統(tǒng)的影響。對白菜根部抗氧化系統(tǒng)指標檢測表明,H_2S可以降低Cd脅迫下根部H202的含量,減少細胞內脂質過氧化產(chǎn)物丙二醛(MDA)和蛋白質羰基化產(chǎn)物(PCO)的積累。進一步對ROS產(chǎn)生和清除系統(tǒng)進行研究后發(fā)現(xiàn)：H_2S抑制了質膜上02·-的產(chǎn)生酶NADPH氧化酶(NOX)編碼基因BraRohD、BraRohF的表達,同時對NOX的活性也有明顯抑制；Cd脅迫中ROS清除系統(tǒng)中超氧化物歧化酶(SOD)和過氧化氫酶(CAT)也受到H_2S的調節(jié),轉錄水平和酶活水平都有不同程度的增加,而過氧化物酶(POD)和抗壞血酸過氧化物酶(APX)則沒有明顯變化；H_2S預處理可以促進Cd脅迫下抗氧化劑谷胱甘肽(GSH)的合成,減少了氧化型谷胱甘肽(GSSG)的產(chǎn)生,這有效地提高了GSH/GSSG比值,相應地檢測到GSH-AsA循環(huán)系統(tǒng)中抗壞血酸(AsA)含量的顯著增加。這些結果說明H_2S信號可從酶和非酶系統(tǒng)多方面調節(jié)植物體內ROS的平衡,降低植物在Cd脅迫下的氧化損傷程度。3.H_2S對Cd脅迫白菜幼苗根尖細胞遺傳損傷的影響。Cd脅迫可使白菜幼苗根的生長受到嚴重抑制,根尖細胞的有絲分裂也受到了抑制。生理濃度H_2S處理可以促進Cd脅迫中根尖細胞有絲分裂的發(fā)生,而且可以顯著降低有絲分裂過程中染色體的斷裂、滯后、染色體橋等異�，F(xiàn)象,細胞的微核發(fā)生率也顯著降低。H_2S對Cd脅迫造成的DNA損傷也有明顯的抑制作用,DNA-蛋白質交聯(lián)程度降低,基因組DNA的斷裂減少,與DNA損傷修復相關的多聚腺苷二磷酸-核糖聚合酶(PARP)家族中的PARP1和PARP2編碼基因顯著上調。這些結果表明H_2S在抑制Cd脅迫對白菜根尖細胞造成的遺傳損傷過程中發(fā)揮了積極作用。4.H_2S對Cd脅迫下白菜幼苗根系滲透脅迫的影響在Cd脅迫過程中,隨處理時問的延長,白菜根系的膜脂過氧化產(chǎn)物MDA含量增加,根細胞的相對電導率增大,細胞膜透性增大；H_2S預處理可使Cd脅迫引起的白菜根相對電導率顯著下降,提高根部的相對含水量；進一步研究表明,H_2S增加Cd脅迫下幼苗根部可溶性糖和脯氨酸的含量來抵抗?jié)B透脅迫,但可溶性蛋白和甜菜堿含量無明顯變化。綜上所述：Cd脅迫下白菜幼苗根部的內源H_2S合成增加；H_2S預處理可以減少Cd脅迫下根部細胞ROS誘導的細胞死亡；根部細胞ROS的積累減少源自H_2S從多方面調節(jié)ROS的產(chǎn)生和清除；H_2S可以降低Cd脅迫對根部細胞造成的遺傳損傷,促進根尖細胞有絲分裂；H_2S通過調節(jié)Cd脅迫過程中白菜根部多種滲透調節(jié)物質的變化來抵抗Cd脅迫。因此,H_2S提高白菜幼苗根部耐受Cd脅迫的能力是通過多層次的防御和調節(jié)機制實現(xiàn)的。
[Abstract]:In recent years, more and more attention has been paid to H_2S as a physiological signal molecule. Reports have shown that in plants it has many physiological functions, such as promoting root growth and morphogenesis, enhancing photosynthesis, promoting seed germination, stimulating flowering, delayed senescence, and so on,.H_2S also plays a role in the resistance of plants to various environmental stresses. Positive roles. Among them, plants play the following role in the process of plant adaptation to heavy metal stress: H_2S can promote the synthesis of reduced glutathione, ascorbic acid, and increase the activity of a series of antioxidant enzymes to maintain the balance of redox state in cells and reduce oxidative damage caused by stress; studies show that H_2S increases The activities of Na+-ATPase, K+-ATPase and H+-ATPase in plants inhibit the absorption of heavy metal ions and increase the absorption of sulfur, phosphorus, calcium, magnesium and iron in plants. H_2S also inhibits the Ca channel regulated by H202 to reduce Cd into the plasma membrane and activates the transport channel on the vacuolar membrane and transtransport Cd in the cytoplasm to the vacuole, thus reducing the cytoplasm in the cytoplasm. The concentration of heavy metal ions; exogenous H_2S can alleviate the toxic effects of Cd on plants by increasing the chlorophyll content of plants, the intensity of photosynthesis, and promoting the development of the internal capsule of the cells in the cells, and increasing the number of mitochondria to alleviate the damage to plants. H_2S will also interact with other signaling molecules and various hormones to alleviate the damage caused by heavy metals to plants. In this study, Brassica rapa L. ssp. Pekinensis, an important cruciferous Brassica plant, was used as the material, using pharmacological, cell biology, physiological and biochemical techniques to systematically study the physiological role of H_2S in improving the physiological role of Cd stress tolerance in the root of Chinese cabbage from the phenotypic, cell and molecular levels of H_2S in the process of increasing the tolerance to heavy gold in the root of Chinese cabbage. The effects of Cd stress on the cell death of cabbage root cells under.Cd stress, with the increase of treatment concentration, the root growth of cabbage seedlings was inhibited and the mortality of root cells increased rapidly. The root length and root cell mortality were negatively correlated with the endogenous H_2S yield in root of cabbage seedlings and the encoding genes of key enzymes of H_2S generation under.Cd stress. Exogenous H_2S treatment (NaHS as donor) could alleviate the growth inhibition of Cd on the root of cabbage seedlings, and significantly reduce the death of root cells and the degradation of DNA. Further research shows that H_2S signal can reduce the accumulation of active oxygen (ROS) induced by Cd stress in the root of cabbage seedlings and inhibit the membrane lipid peroxidation induced by ROS. Damage and maintenance of the integrity of the plasma membrane structure, thus inhibiting the effect of cell death.2.H_2S on the antioxidant system in root of Chinese Cabbage under Cd stress. The detection of antioxidant system in the root of Chinese cabbage shows that H_2S can reduce the content of H202 at the root of Cd stress, reduce the malondialdehyde (MDA) and protein carbonylation products of the lipid peroxylation products in the cells. After further study on the ROS production and scavenging system, it was found that H_2S inhibited the 02. - producing enzyme NADPH oxidase (NOX) encoding gene BraRohD, the expression of BraRohF, and the activity of NOX in the membrane of the plasma membrane, and also inhibited the activity of NOX, and the superoxide dismutase (SOD) and catalase (CAT) in the ROS scavenging system in Cd stress were also affected. The regulation of _2S, transcriptional level and enzyme activity level increased in varying degrees, while peroxidase (POD) and ascorbic acid peroxidase (APX) did not change significantly; H_2S pretreatment could promote the synthesis of antioxidant glutathione (GSH) under Cd stress and reduced the production of oxidized glutathione (GSSG), which effectively improved GSH/GSS. The G ratio, correspondingly detected the significant increase in the content of ascorbic acid (AsA) in the GSH-AsA cycle system. These results show that H_2S signals can regulate the balance of ROS in plants in various aspects of enzymes and non enzyme systems, and reduce the oxidative damage degree of plants under Cd stress.3.H_2S on the effect of.Cd stress on the genetic damage of the root tip cells of the cabbage seedlings under the stress of Cd. The root growth of cabbage seedlings was severely inhibited and the mitosis of root tip cells was inhibited. Physiological concentration H_2S treatment could promote the mitosis of root tip cells in Cd stress, and could significantly reduce chromosome breakage, lag, chromosome bridge and other abnormal phenomena in the process of mitosis, and the incidence of micronucleus in cells. It also significantly reduced the inhibitory effect of.H_2S on the DNA damage caused by Cd stress, the decrease of DNA- protein crosslinking degree and the decrease of genomic DNA. The PARP1 and PARP2 encoding genes in the polyadenosine two phosphoric ribose polymerase (PARP) family associated with DNA damage repair were significantly up-regulated. These results indicate that H_2S is inhibiting Cd stress. In the process of genetic damage caused by root tip cells of Chinese cabbage, the effect of.4.H_2S on the root osmotic stress of Cabbage Seedlings under the stress of Cd stress under the stress of Cd stress in the process of Cd stress, with the prolongation of treatment time, the content of MDA content of membrane lipid peroxidation product in root of Chinese cabbage increased, the relative conductivity of root cells increased, cell membrane permeability increased; H_2S pretreatment The relative conductivity of cabbage root caused by Cd stress decreased significantly and increased the relative water content of root. Further studies showed that H_2S increased the content of soluble sugar and proline in root of seedling under Cd stress to resist osmotic stress, but there was no obvious change in soluble protein and betaine content. Under Cd stress, the root of Cabbage Seedlings under Cd stress The endogenous H_2S synthesis increased; H_2S pretreatment could reduce the cell death induced by ROS in root cells under Cd stress; the accumulation of ROS in root cells decreased from H_2S to regulate the production and removal of ROS in many ways; H_2S could reduce the genetic damage caused by Cd stress to root cells and promote mitosis in the root tip cells; H_2S by regulating Cd. In the course of stress, the changes of various osmotic regulators in the root of Chinese cabbage resist Cd stress. Therefore, the ability of H_2S to improve the tolerance to Cd stress in the roots of Chinese cabbage is achieved through a multi-level defense and regulation mechanism.
【學位授予單位】：山西大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S634.1

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