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磷化氢对海洋微藻的影响及作用机制研究
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摘要
磷化氢是广泛存在于海洋环境中的磷的还原性气态化合物,主要以基质结合态存在于海洋沉积物中,可动态释放到上层海水和空气中,成为一种可能的磷的补充形式。磷化氢易被氧化为磷的其他形态,可对海洋浮游植物产生影响,其参与磷的生物地球化学循环过程,对海洋中磷的循环补充具有重要意义。本论文通过室内实验,考察了磷化氢对伪矮海链藻(Thalassiosira pseudonana)细胞比增长率、高亲和力磷酸盐转运蛋白基因表达量、碱性磷酸酶活性及抗氧化酶活性的影响,从微藻细胞对磷的吸收利用的角度及氧化压力、自由基的角度,结合以往研究基础,较为系统的分析了磷化氢对海洋微藻的作用机制,以期解释磷化氢对海洋微藻的作用,为完善磷的海洋生物地球化学循环过程中磷化氢的作用和地位提供科学依据。主要结果如下:
     磷化氢对微藻生长具有一定的影响,但并不能作为唯一磷源供细胞增长。磷缺乏及磷酸盐充足条件下,磷化氢对伪矮海链藻藻细胞密度及细胞比增长率的影响研究发现,低浓度的磷化氢对微藻生长有一定的刺激作用,高浓度磷化氢具有一定的抑制作用。
     磷化氢具有直接作用于海洋微藻的途径,影响海洋微藻吸收利用磷的能力是磷化氢是磷化氢对海洋微藻的作用机制之一。一方面,磷缺乏条件下,伪矮海链藻碱性磷酸酶(AKP)活性随磷化氢浓度的增加先升高后下降,表明磷化氢促进了伪矮海链藻细胞的缺磷信号的表达与传导,同时其活性的升高也说明磷化氢除转化为磷酸盐被微藻利用外,可能还具有直接作用于海洋微藻的途径。另一方面,在磷缺乏及4μmol/L磷酸盐条件下,磷化氢对伪矮海链藻高亲和力磷酸盐转运蛋白基因(TpPHO)基因表达具有一定的促进作用,也表明磷化氢促进了伪矮海链藻细胞的缺磷信号的表达与传导。对比研究磷化氢和相同磷浓度的磷酸盐对TpPHO基因表达量的影响发现,磷化氢处理组的TpPHO基因表达量显著大于磷酸盐处理组的TpPHO基因表达量,进一步证明磷化氢与磷酸盐对TpPHO基因的表达具有不同的作用途径,磷化氢可直接作用于藻细胞而并非完全转化为磷酸盐被吸收。因此,一定浓度的磷化氢对AKP活性和PHO基因表达具有促进作用,进而可促进微藻对磷的吸收利用,有益于细胞增长。
     磷化氢对海洋微藻的作用机制之一是通过改变细胞内的活性氧水平而作用于微藻细胞的抗氧化酶系统。在本论文磷化氢的实验浓度范围内,伪矮海链藻超氧化物歧化酶(SOD)活性随磷化氢浓度的增加先升高后降低。推测磷化氢诱导细胞产生了较多的超氧阴离子自由基(O2),进一步诱导了SOD活性升高,使得细胞清除O2
     的能力增强。磷化氢诱导产生的O2
     被SOD歧化为过氧化氢(H2O2后,进一步诱导了伪矮海链藻的过氧化氢酶(CAT)活性,伪矮海链藻对磷化氢胁迫产生的H2O2主要依靠强化CAT的活性来清除。实验前期,磷化氢所致的氧化压力激活了伪矮海链藻抗氧化防御系统,保护细胞免受损伤,表现为磷化氢处理组伪矮海链藻细胞丙二醛(MDA)含量较对照组低;而实验后期MDA含量表现为磷化氢处理组高于对照组,说明磷化氢的作用具有一定的时间效应。伪矮海链藻细胞MDA含量与细胞比增长率呈一定的负相关关系,证明了抗氧化酶活性对磷化氢的响应是微藻细胞对磷化氢响应的一种途径。根据本文及以往研究结果,较为系统的探讨了磷化氢对海洋微藻的作用机制分析推断磷化氢对海洋微藻的作用兼具直接作用和转化为磷酸盐后被吸收利用
     的两种方式。磷化氢对微藻细胞生长、抗氧化酶活性及磷代谢的影响表明了其对微藻的双重作用:一方面,低浓度磷化氢诱导抗氧化酶活性的升高,对TpPHO基因的表达及AKP活性均具有促进作用,从而对细胞增长具有一定的刺激作用;另一方面,磷化氢具有毒性,高浓度的磷化氢会对微藻的酶活性和基因表达等产生抑制作用,损伤细胞膜系统,抑制细胞增长。
Phosphine, as a reduced form of phosphorus, is a ubiquitous constituent in themarine environment. It mainly exists as matrix-bound phosphine (MBP) which canhydrolyze to form free phosphine gas. The free phosphine gas can release intoseawater and air to be the probable supplementary form of phosphorus. Phosphine canbe easily oxidized to other forms of P and it could affect marine microalgae andparticipate in the oceanic biogeochemical cycle of phosphorus. It is of greatsignificance for phosphorus cycle. In this article, the effect of phosphine on specificgrowth rates, transcriptional level of high affinity phosphate transporter gene (PHO),alkaline phosphatase activity and antioxidant enzyme activities were studied inThalassiosira pseudonana. Based on these parameters and previous studies, themechanism of phosphine on marine algae was analyzed from the point of phosphateuptake and utilization, and the point of oxidative stress and free radical. We aimed toprovide a basis for future research on the biological and ecological significance ofphosphine in the marine environment. The main results were as follows:
     The effects of phosphine on the growth of marine microalgae wereexperimentally studied. The results showed that the adaptive amount of phosphinemight stimulate the growth of T. pseudonana, and high levels of phosphine mightinhibit the growth of algae. Generally, phosphine has influence on the growth ofmarine algae, however, phosphine cannot be the sole phosphorus to marine algae.
     According to variation in the transcriptional level of phosphate transporter gene(TpPHO) and activity of alkaline phosphatase (AKP) in relation to differentconcentrations of phosphine, the effects of phosphine on phosphorus uptake andutilization in T. pseudonana were studied. TpPHO expression and AKP activity werepromoted by phosphine. AKP activity was higher in the phosphine treatment groupsthan that of the control. It increased with increasing phosphine concentration in the range of0to0.056μmol/L, but was inhibited by higher levels of phosphine. What’smore, TpPHO expression was markedly promoted by phosphine in both thephosphate-deficient and phosphate-4μmol/L culture. However, high phosphineconcentrations can inhibit TpPHO transcription in the declining growth phase.Theseresults revealed that phosphine could promote the signal transduction of phosphorusdeficiency. Consequently, it can benefit T. pseudonana to absorb and utilizephosphorus. Comparison of the phosphine treatment with the phosphate treatment ofthe same concentration revealed that the TpPHO transcriptional level of the formerwas much higher than that of the latter. Therefore, phosphine appeared to influencethe algae in a manner other than by being oxidized to phosphate. The increasing AKPactivity with increasing phosphine also supported this point. The effect of phosphineon the transcriptional level of TpPHO and AKP activity would be one influencing wayof phosphine to algae.
     The activity of superoxide dismutase (SOD) increased with increasing phosphineconcentration when the concentration was below0.11μmol/L. Phosphine induce theproduction of superoxide radical (O2
     ), and stimulate SOD a higher activity todisproportionate O2
     to hydrogen peroxide (H2O2). And then, the activity of catalase(CAT) was further induced by H2O2. The content of malonyldialdehyde (MDA)decreased with the increasing concentration of phosphine during the early cultureperiod. During the decline phase, high phosphine concentrations had inhibitory effectson SOD and CAT, and the content of MDA increased with increasing phosphineconcentration. The dual effects of phosphine on antioxidant system of T. pseudonanaand the negative correlation between MDA and specific growth rate indicated thatphosphine may affect algae through impacting the ROS level and antioxidant defensesystem.
     In conclusion, phosphine can both influence algae directly and be oxidized to bephosphate to influence algae. The variations of specific growth rate, antioxidantenzyme activities, AKP activity, and the transcriptional level of TpPHO indicated thedual effect of phosphine on T. pseudonana. Phosphine can stimulate cells to reproduce via promote SOD, CAT, AKP, and TpPHO gene expression at a low concentration,while has inhibitory effect on cell vitality at a high concentration because of itstoxicity.
引文
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