南极株塔玛亚历山大藻产毒营养生理及毒素生理功能研究
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摘要
南极株塔玛亚历山大藻(Alexandrium tamarense Polar)是一株分离自南极海区的有毒亚历山大藻,曾在南极海区形成大规模赤潮,对海区的生态系统和海洋生物资源构成了威胁。本论文以A.tamarense Polar为主要研究对象,运用藻类生理学、分析化学和蛋白质组学等方法和技术,比较研究了A.tamarense Polar和无毒塔玛亚历山大藻(A.tamarense CCMP2023)营养,特别是氮营养利用特性上的差异、不同营养盐及培养条件下A.tamarense Polar的产毒生理、以及高氨氮胁迫下A.tamarense Polar细胞蛋白质组的差异表达,探讨了亚历山大藻中麻痹性贝毒的生理功能及合成机制。取得的主要结果如下:
1、比较研究了有毒亚历山大藻A.tamarense Polar和无毒亚历山大藻A.tamarenseCCMP2023氮营养盐吸收同化的差异,重点研究了营养吸收同化的两种关键酶—硝酸还原酶和谷氨酰胺合成酶的活性。结果表明,相对于无毒株塔玛亚历山大藻A.tamarense CCMP2023,有毒塔玛亚历山大藻A.tamarense Polar在硝酸盐和氨氮的吸收、细胞生长和同化酶活性等方面均存在一定的差异,有毒亚历山大藻对氮营养盐的吸收、转换速率更高,导致A.tamarense Polar细胞内氨盐的大量累积,而毒素的合成可能是亚历山大藻细胞降低氨毒性和储存氮的一种途径;
2、研究了不同浓度氨氮和硝氮营养盐条件下的A.tamarense Polar产毒生理,表明在一定浓度范围内,硝氮对其细胞增长和毒素产生起促进作用,但氨氮的作用及其机制则比较复杂,中、低浓度(100μmol/L以下)组,其促进作用比较明显,各指标均普遍高于相应的硝氮组,但高浓度氨氮(150μmol/L以上)对细胞有明显毒性作用,其作用强度与氨氮浓度正相关,高氨培养条件下A.tamarense Polar的单细胞毒素含量和毒素产量都明显增高,证明细胞内PSP毒素的合成是细胞降低氨氮毒性的一个重要途径,是细胞生命活动中的一个重要生理功能;
3、不同氮源和培养条件,如磷限制、低温(13℃)和谷氨酰胺合成酶抑制剂作用下A.tamarense Polar毒素含量和组成变化结果表明:在A.tamarense Polar中,细胞可能首先合成C_(1,2)毒素,在N-硫酸酯酶的作用下转化为GTX_(2,3),这一途径受到营养盐、温度等多种环境因子特别是温度的调控。在低温条件下A.tamarensePolar细胞中GTX毒素消失可能与N-硫酸酯酶的合成受到抑制或使酶失活有关;
4、比较研究氨氮培养条件下有毒亚历山大藻A.tamarense Polar不同生长阶段细胞蛋白质组的表达差异,在高氨氮培养后期的细胞内发现一组与细胞重要生命活动相关的蛋白质,如与细胞代谢相关尿卟啉原脱羧酶(ATP1)、三羧酸循环中的琥珀酸脱氢酶(ATP3)和脂肪酸代谢中的乙酰辅酶A羧化酶(ATP7);与细胞分裂相关SMC蛋白(ATP6)以及与细胞运动相关的Rod蛋白FlgB(点2)等消失,同时发现了两个与牛磺酸代谢途径相关的新蛋白,ABC转运器(ATP4和5)。这些蛋白的消失和出现是细胞对氨毒性的一种生理响应,可作为一类潜在的生物标志物,用于指示氨的毒性。
Alexandrium tamarense Polar is a paralytic shellfish toxins (PSTs)-producing dinoflagellate which ever formed large scale toxic blooms in the Sourth Polar region, and caused adverse impacts on marine ecosystem and marine organisms. This study investigated nutrient physiology of toxin production and physiological functions of toxin in A. tamarense Polar using a combination approach of algal physiology, analytical chemistry and proteomics, including comparative studies on nutrient utilization characteristics between toxic and none-toxic A. tamarense strains, toxin production physiology of A. tamarense Polar under various nutrients and culture conditions, and proteomics of A. tamarense Polar under high ammonium concentration. The main results were as follows:
1. Compared nutrient-utilization characteristics between toxic (A. tamarense Polar) and none-toxic (A. tamarense CCMP2023) Alexandrium strains with emphasis on two nitrogen assimilation enzyme, nitrate reductase (NR) and glutamine synthestase (GS) activities. The results showed that the toxic strains, A. tamarense Polar exhibited both higher NR and GS activities which made it more efficiently in utilizing different nitrogen sources, which might also lead to the accumulation of ammonium in cells of A. tamarense Polar. The biosynthesis of PSP toxins may be a celluar detoxification pathway to alleviate the ammonium toxicity to cells and a nitrogen storage ;
2. Investigated the toxin production of A. tamarense Polar under various concentrations of nitrate and ammonium, the results showed that within a certain concentration range of nitrate enhanced cell growth and toxin production of A. tamarense Polar. However, the effects of ammonium on A. tamarense Polar were much more complex: low concentrations (under 100μmol/L) of ammonium had positive effects while high concentrations (above 150μmol/L) of ammonium exhibited an inhibitory effect on the growth of A. tamarense Polar and the inhibiting effect was increased with the concentrations of ammonium within a certain range. High concentrations of ammonium induced more PSP toxin productivity, indicating biosynthesis of PSP toxins might be a detoxification pathway of ammonium to cells and is an important physiological function of A. tamarense Polar;
3. Variations of toxin content and composition of A. tamarense Polar under different nitrogen sources and various growh conditions, such as phosphate-limitation, low temperature (13℃) and GS-inhibitor (MSX) indicated that Cl/2 might be the first synthesized PSP toxin in cells of A. tamarense Polar, which was converted into other PSP derivatives, such as GTXs with the action of N- sulphatase. This biosynthesis pathway of PSP toxins was affected by nutrients, temperatre and other environmental factors. Low temperature might inhibite the biosynthesis of N-sulphortase or activity of N-sulphortase which resulted in undectation of GTXs;
4. Compared differential expressions of proteomes of A. tamarense Polar under low (50μmol/L) and high (150μmol/L) concentrations of ammonium, and identified and characterized a group importanr proteins related to metabolism, cell division and cell motivation which were not observed in the later growth phase of A. tamarense Polar under high ammonium concentration while two proteins related taurine metabolism were found.. These proteins reflected the physiological reponse of A. tamarense Polar cells to the toxicity of ammomum and could be used as potential biomarkers as cells response to ammonium toxicity.
1、比较研究了有毒亚历山大藻A.tamarense Polar和无毒亚历山大藻A.tamarenseCCMP2023氮营养盐吸收同化的差异,重点研究了营养吸收同化的两种关键酶—硝酸还原酶和谷氨酰胺合成酶的活性。结果表明,相对于无毒株塔玛亚历山大藻A.tamarense CCMP2023,有毒塔玛亚历山大藻A.tamarense Polar在硝酸盐和氨氮的吸收、细胞生长和同化酶活性等方面均存在一定的差异,有毒亚历山大藻对氮营养盐的吸收、转换速率更高,导致A.tamarense Polar细胞内氨盐的大量累积,而毒素的合成可能是亚历山大藻细胞降低氨毒性和储存氮的一种途径;
2、研究了不同浓度氨氮和硝氮营养盐条件下的A.tamarense Polar产毒生理,表明在一定浓度范围内,硝氮对其细胞增长和毒素产生起促进作用,但氨氮的作用及其机制则比较复杂,中、低浓度(100μmol/L以下)组,其促进作用比较明显,各指标均普遍高于相应的硝氮组,但高浓度氨氮(150μmol/L以上)对细胞有明显毒性作用,其作用强度与氨氮浓度正相关,高氨培养条件下A.tamarense Polar的单细胞毒素含量和毒素产量都明显增高,证明细胞内PSP毒素的合成是细胞降低氨氮毒性的一个重要途径,是细胞生命活动中的一个重要生理功能;
3、不同氮源和培养条件,如磷限制、低温(13℃)和谷氨酰胺合成酶抑制剂作用下A.tamarense Polar毒素含量和组成变化结果表明:在A.tamarense Polar中,细胞可能首先合成C_(1,2)毒素,在N-硫酸酯酶的作用下转化为GTX_(2,3),这一途径受到营养盐、温度等多种环境因子特别是温度的调控。在低温条件下A.tamarensePolar细胞中GTX毒素消失可能与N-硫酸酯酶的合成受到抑制或使酶失活有关;
4、比较研究氨氮培养条件下有毒亚历山大藻A.tamarense Polar不同生长阶段细胞蛋白质组的表达差异,在高氨氮培养后期的细胞内发现一组与细胞重要生命活动相关的蛋白质,如与细胞代谢相关尿卟啉原脱羧酶(ATP1)、三羧酸循环中的琥珀酸脱氢酶(ATP3)和脂肪酸代谢中的乙酰辅酶A羧化酶(ATP7);与细胞分裂相关SMC蛋白(ATP6)以及与细胞运动相关的Rod蛋白FlgB(点2)等消失,同时发现了两个与牛磺酸代谢途径相关的新蛋白,ABC转运器(ATP4和5)。这些蛋白的消失和出现是细胞对氨毒性的一种生理响应,可作为一类潜在的生物标志物,用于指示氨的毒性。
Alexandrium tamarense Polar is a paralytic shellfish toxins (PSTs)-producing dinoflagellate which ever formed large scale toxic blooms in the Sourth Polar region, and caused adverse impacts on marine ecosystem and marine organisms. This study investigated nutrient physiology of toxin production and physiological functions of toxin in A. tamarense Polar using a combination approach of algal physiology, analytical chemistry and proteomics, including comparative studies on nutrient utilization characteristics between toxic and none-toxic A. tamarense strains, toxin production physiology of A. tamarense Polar under various nutrients and culture conditions, and proteomics of A. tamarense Polar under high ammonium concentration. The main results were as follows:
1. Compared nutrient-utilization characteristics between toxic (A. tamarense Polar) and none-toxic (A. tamarense CCMP2023) Alexandrium strains with emphasis on two nitrogen assimilation enzyme, nitrate reductase (NR) and glutamine synthestase (GS) activities. The results showed that the toxic strains, A. tamarense Polar exhibited both higher NR and GS activities which made it more efficiently in utilizing different nitrogen sources, which might also lead to the accumulation of ammonium in cells of A. tamarense Polar. The biosynthesis of PSP toxins may be a celluar detoxification pathway to alleviate the ammonium toxicity to cells and a nitrogen storage ;
2. Investigated the toxin production of A. tamarense Polar under various concentrations of nitrate and ammonium, the results showed that within a certain concentration range of nitrate enhanced cell growth and toxin production of A. tamarense Polar. However, the effects of ammonium on A. tamarense Polar were much more complex: low concentrations (under 100μmol/L) of ammonium had positive effects while high concentrations (above 150μmol/L) of ammonium exhibited an inhibitory effect on the growth of A. tamarense Polar and the inhibiting effect was increased with the concentrations of ammonium within a certain range. High concentrations of ammonium induced more PSP toxin productivity, indicating biosynthesis of PSP toxins might be a detoxification pathway of ammonium to cells and is an important physiological function of A. tamarense Polar;
3. Variations of toxin content and composition of A. tamarense Polar under different nitrogen sources and various growh conditions, such as phosphate-limitation, low temperature (13℃) and GS-inhibitor (MSX) indicated that Cl/2 might be the first synthesized PSP toxin in cells of A. tamarense Polar, which was converted into other PSP derivatives, such as GTXs with the action of N- sulphatase. This biosynthesis pathway of PSP toxins was affected by nutrients, temperatre and other environmental factors. Low temperature might inhibite the biosynthesis of N-sulphortase or activity of N-sulphortase which resulted in undectation of GTXs;
4. Compared differential expressions of proteomes of A. tamarense Polar under low (50μmol/L) and high (150μmol/L) concentrations of ammonium, and identified and characterized a group importanr proteins related to metabolism, cell division and cell motivation which were not observed in the later growth phase of A. tamarense Polar under high ammonium concentration while two proteins related taurine metabolism were found.. These proteins reflected the physiological reponse of A. tamarense Polar cells to the toxicity of ammomum and could be used as potential biomarkers as cells response to ammonium toxicity.
引文
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