雨生红球藻（Haematococcus pluvialis）虾青素酯和脂肪酸的鉴定及差异表达基因的分析
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摘要
以雨生红球藻(Haematococcus pluvialis)为研究对象，利用高效液相色谱—大气压化学电离正离子质谱鉴定了虾青素酯的各个成份，并建立了测定虾青素含量的HPLC方法；用HPLC及GC-MS的方法研究了雨生红球藻四种类型细胞的虾青素酯和脂肪酸的种类及含量；利用分光光度法和HPLC研究了雨生红球藻虾青素的稳定性；比较研究了酶解和皂化过程中游离虾青素数量变化规律及其得率；并利用抑制性差减杂交技术构建了雨生红球藻中与孢子发育和虾青素形成相关的差减cDNA文库。主要结果如下。
1．利用LC-(APCI)MS鉴定出了雨生红球藻提取物中的4个游离类胡萝卜素，15个虾青素单酯，12个虾青素双酯及3个虾红素单酯。另外，经高效液相色谱—DAD检测到一组化合物的最大吸收在400 nm，正离子模式二次质谱出现有较强的m／z 871和593的分子离子碎片离子特征峰。这组化合物可能是虾红素的亚麻酸或ω-6-γ-亚麻酸酯的异构体，这是以前未见报道的。
2．用HPLC外标法对雨生红球藻中虾青素的含量进行测定。虾青素在0.1-2.0μg线性范围内线性关系良好，其回归方程为：y=1027.3x+245.55，相关系数R~2=0.9997，平均加样回收率为100.277，RSD(％)为2.14。本方法定量准确，重复性好，鉴定出的种类较多，可以作为测定虾青素酯的一种方法。
3．红色游动细胞和红色孢子中的虾青素酯的种类是一致，而且四种类型细胞中的脂肪酸的种类是一致的。另外，红色游动细胞和红色孢子的虾青素酯中主要的几种脂肪酸的含量变化与细胞中总的脂肪酸的含量变化趋势一致。油酸(C18：1)和亚油酸(C18：2)是红色孢子和红色游动细胞中的主要脂肪酸，而且二者在红色孢子和红色游动细胞中的含量远大于绿色孢子和绿色游动细胞中的，说明虾青素酯的形成需要油酸和亚油酸大量地合成。绿色孢子的HPLC图谱与绿色游动细胞的相似，都没有检测到虾青素酯和游离的虾青素。而且，绿色孢子中的脂肪酸组成与绿色游动细胞基本相同，显然，脂肪酸的积累与虾青素的积累密切相关，而与孢子的形成关系不大。红球藻红色孢子中含有30％以上的脂肪酸，并且不饱和的脂肪酸占总脂肪酸的81％，因此，雨生红球藻不仅可以作为珍贵虾青素的较好来源，也可以作为具有较高品质的脂肪酸的来源。
4．氧、温度对虾青素稳定性的作用显著。抗氧化剂VE或VC对虾青素的稳定性起了负作用，加了抗氧化剂的藻粉中虾青素的含量都低于无抗氧化剂的。保存1.5年时，破壁藻粉抽真空后在20℃下保存和藻泥-20℃保存，虾青素的含量和虾青素酯中的各个成份间的相对含量变化不大。保存2年时，虾青素的含量都下降了约10％，藻粉中的虾青素各酯的相对含量变化较大，藻泥中各酯的相对含量仍较稳定。
5．最佳的酶解条件是在反应体系中加入1.02 units的胆固醇酯酶后在37℃下反应75 min，酶解得率是72.04％；最佳皂化条件是加入1.07 mol L~(-1)NaOH在22℃下反应40 min，皂化得率为51.48％。无论是通过酶解还是碱水解，对虾青素酯都具有一定的破坏作用。先水解，特别是先皂化水解虾青素酯，然后用HPLC测定雨生红球藻虾青素含量的方法误差太大，是不可行的。
6．雨生红球藻细胞在胁迫条件下能够积累大量的虾青素，细胞由绿色游动细胞变为红色不动孢子。提取绿色游动细胞和红色孢子中的mRNA，利用抑制性差减杂交技术，成功构建了雨生红球藻中与孢子发育和虾青素形成相关基因的差减cDNA文库。以管家基因α-tubulin作为差减指标，检测到差减cDNA文库的差减效率可高达2~(10)-2~(15)倍，表明在红色孢子中某些差异表达基因包括与孢子发育和虾青素形成相关的基因的富集效率也接近2~(10)-2~(15)倍。雨生红球藻中与孢子发育和虾青素形成相关基因的差减cDNA文库的建立对快速分离、克隆孢子发育及虾青素形成相关基因和认识雨生红球藻中积累虾青素的分子机理有重要意义。
The astaxanthin ester composition of Haematococcus pluvialis were characterized by LC-(APCI)MS, and a HPLC method was developed to determine the astaxanthin content. The astaxanthin ester composition and the fatty acid composition in four different cell types of H. pluvailis were characterized by HPLC and GC-MS. The stability of astaxanthin and astaxanthin esters was studied. The content of free astaxanthin obtained by saponification and enzymatic hydrolysis were examined. A subtractive cDNA library of genes related to cyst formation and astaxanthin synthesis was contracted by SSH.
1. Four free carotenoids, fifteen astaxanthin monoesters, twelve astaxanthin diesters and three astacin monoesters in H. pluvialis were identified by LC-(APCI)MS. In addition, a set of compounds with maximum absorbance at 400 nm, detected by HPLC-DAD, had strong characteristic fragment ions at m/z 871 and m/z 593 in the positive ion mode MS_2. They were presumed to be linolenic acid or an isomer ofω-6-γ-linolenic acid esters of astacin. The presence of these compounds has not been reported ever in H. pluvialis cells.
2. The astaxanthin ester contents in H. pluvialis were quantified by the external standard calibration curve method through HPLC. The linear calibration curves were y = 1027.3x+245.55 (R~2=0.9997) when the astaxanthin content ranging from 0.1 to 2.0μg. The average recovery was 100.277 and the RSD (%) was 2.14. This method, with better sensitivity and better reproducibility, could identify more astaxanthin esters than those ever reported
3. The astaxanthin ester types were identical in red mobile cells and red cysts, and the fatty acid types were identical in four types of H. pluvialis cells. The major fatty acid contents in astaxanthin esters had the same tendency with the contents of the total fatty acids in red motile cells and red cysts. The oleic acid (C18:1) and linoleic acid (C18:2) were dominant in both red mobile cells and red cysts, and red mobile cells and red cysts contained much more oleic acid and linoleic acid than green motile cells and green cysts. It indicated that the active synthesis of oleic acid and linoleic acid was necessary to astaxanthin ester synthesis. The HPLC chromatogram of green cysts was similar to that of green motile cells, and neither free astaxanthin nor astaxanthin esters were detected in these two cells. Furthermore, the fatty acid composition was the same in green cysts and green motile cells. It suggested that astaxanthin ester accumulation coordinated with the synthesis of fatty acids from another point of view. The synthesis and accumulation of fatty acids was independent of the formation of cysts, but was highly correlated with the synthesis and accumulation of astaxanthin. The red cysts contained more than 30% of fatty acids, in which the unsaturated fatty acids accounted for about 81%. Therefore, H. pluvialis was not only a good resource of precious astaxanthin, but also would become a good resource of valuable fatty acids in future.
4. Both oxygen and temperature had strong effects astaxanthin stability. Both vitamin C and vitamin E, the common antioxidant, decreased the astaxanthin stability. There were no significant changes in the astaxanthin content or the relative content of each astaxanthin ester after the dry algal meal with broken cell wall stored under vacuum at -20℃for 1.5 years or the algal meal stored at -20℃for 1.5 years. After stored for 2 years, the astaxanthin content of each treatment decreased about 10%, and the relative content of each astaxanthin ester was stabile in algal meal, but decreased significantly in dry algal meal.
5. 72.04% free astaxanthin was obtained after the extraction mixture was treated at 37℃for 75 min with the presence of 1.02 units cholesterol. And it was the optimal treatment for hydrolysis by enzyme. However, only 51.48% free astaxanthin was obtained after the reaction mixture was treated at 22℃for 40 min with the presence of 1.07 M NaOH. And it was the optimal treatment for hydrolysis by alkali. Therefore, it was not accurate to determine astaxanthin content by HPLC after hydrolysis especially after saponification, for hydrolysis by enzyme or by alkali would destroy astaxanthin in some degree.
6. H. pluvialis formed red cysts with astaxanthin accumulation in cells under stresses. In order to analyze the change of gene expressions and isolate these related genes inducing astaxanthin synthesis, a subtractive cDNA library of cyst formation and astaxanthin synthesis was constructed by using the Suppression Subtractive Hybridization (SSH) techniques. In this paper, the quality of cellular RNAs and adaptor ligation efficiency were analyzed, and the housekeeping gene,α-tubulin, was used to estimate the efficiency of subtractive cDNA. In this subtractive cDNA library,α-tubulin was subtracted very efficiently at appropriate 2~(10)-2~(15) fold, indicating that some differentially expressed genes including the genes related to the cyst formation and astaxanthin synthesis were also enriched at the same folds. The construction of the subtractive cDNA library would be essential for rapid isolation and clone of the genes related to cyst formation and astaxanthin synthesis, and would be helpful to find out the molecular mechanism of the astaxanthin accumulation in H. pluvialis.
1．利用LC-(APCI)MS鉴定出了雨生红球藻提取物中的4个游离类胡萝卜素，15个虾青素单酯，12个虾青素双酯及3个虾红素单酯。另外，经高效液相色谱—DAD检测到一组化合物的最大吸收在400 nm，正离子模式二次质谱出现有较强的m／z 871和593的分子离子碎片离子特征峰。这组化合物可能是虾红素的亚麻酸或ω-6-γ-亚麻酸酯的异构体，这是以前未见报道的。
2．用HPLC外标法对雨生红球藻中虾青素的含量进行测定。虾青素在0.1-2.0μg线性范围内线性关系良好，其回归方程为：y=1027.3x+245.55，相关系数R~2=0.9997，平均加样回收率为100.277，RSD(％)为2.14。本方法定量准确，重复性好，鉴定出的种类较多，可以作为测定虾青素酯的一种方法。
3．红色游动细胞和红色孢子中的虾青素酯的种类是一致，而且四种类型细胞中的脂肪酸的种类是一致的。另外，红色游动细胞和红色孢子的虾青素酯中主要的几种脂肪酸的含量变化与细胞中总的脂肪酸的含量变化趋势一致。油酸(C18：1)和亚油酸(C18：2)是红色孢子和红色游动细胞中的主要脂肪酸，而且二者在红色孢子和红色游动细胞中的含量远大于绿色孢子和绿色游动细胞中的，说明虾青素酯的形成需要油酸和亚油酸大量地合成。绿色孢子的HPLC图谱与绿色游动细胞的相似，都没有检测到虾青素酯和游离的虾青素。而且，绿色孢子中的脂肪酸组成与绿色游动细胞基本相同，显然，脂肪酸的积累与虾青素的积累密切相关，而与孢子的形成关系不大。红球藻红色孢子中含有30％以上的脂肪酸，并且不饱和的脂肪酸占总脂肪酸的81％，因此，雨生红球藻不仅可以作为珍贵虾青素的较好来源，也可以作为具有较高品质的脂肪酸的来源。
4．氧、温度对虾青素稳定性的作用显著。抗氧化剂VE或VC对虾青素的稳定性起了负作用，加了抗氧化剂的藻粉中虾青素的含量都低于无抗氧化剂的。保存1.5年时，破壁藻粉抽真空后在20℃下保存和藻泥-20℃保存，虾青素的含量和虾青素酯中的各个成份间的相对含量变化不大。保存2年时，虾青素的含量都下降了约10％，藻粉中的虾青素各酯的相对含量变化较大，藻泥中各酯的相对含量仍较稳定。
5．最佳的酶解条件是在反应体系中加入1.02 units的胆固醇酯酶后在37℃下反应75 min，酶解得率是72.04％；最佳皂化条件是加入1.07 mol L~(-1)NaOH在22℃下反应40 min，皂化得率为51.48％。无论是通过酶解还是碱水解，对虾青素酯都具有一定的破坏作用。先水解，特别是先皂化水解虾青素酯，然后用HPLC测定雨生红球藻虾青素含量的方法误差太大，是不可行的。
6．雨生红球藻细胞在胁迫条件下能够积累大量的虾青素，细胞由绿色游动细胞变为红色不动孢子。提取绿色游动细胞和红色孢子中的mRNA，利用抑制性差减杂交技术，成功构建了雨生红球藻中与孢子发育和虾青素形成相关基因的差减cDNA文库。以管家基因α-tubulin作为差减指标，检测到差减cDNA文库的差减效率可高达2~(10)-2~(15)倍，表明在红色孢子中某些差异表达基因包括与孢子发育和虾青素形成相关的基因的富集效率也接近2~(10)-2~(15)倍。雨生红球藻中与孢子发育和虾青素形成相关基因的差减cDNA文库的建立对快速分离、克隆孢子发育及虾青素形成相关基因和认识雨生红球藻中积累虾青素的分子机理有重要意义。
The astaxanthin ester composition of Haematococcus pluvialis were characterized by LC-(APCI)MS, and a HPLC method was developed to determine the astaxanthin content. The astaxanthin ester composition and the fatty acid composition in four different cell types of H. pluvailis were characterized by HPLC and GC-MS. The stability of astaxanthin and astaxanthin esters was studied. The content of free astaxanthin obtained by saponification and enzymatic hydrolysis were examined. A subtractive cDNA library of genes related to cyst formation and astaxanthin synthesis was contracted by SSH.
1. Four free carotenoids, fifteen astaxanthin monoesters, twelve astaxanthin diesters and three astacin monoesters in H. pluvialis were identified by LC-(APCI)MS. In addition, a set of compounds with maximum absorbance at 400 nm, detected by HPLC-DAD, had strong characteristic fragment ions at m/z 871 and m/z 593 in the positive ion mode MS_2. They were presumed to be linolenic acid or an isomer ofω-6-γ-linolenic acid esters of astacin. The presence of these compounds has not been reported ever in H. pluvialis cells.
2. The astaxanthin ester contents in H. pluvialis were quantified by the external standard calibration curve method through HPLC. The linear calibration curves were y = 1027.3x+245.55 (R~2=0.9997) when the astaxanthin content ranging from 0.1 to 2.0μg. The average recovery was 100.277 and the RSD (%) was 2.14. This method, with better sensitivity and better reproducibility, could identify more astaxanthin esters than those ever reported
3. The astaxanthin ester types were identical in red mobile cells and red cysts, and the fatty acid types were identical in four types of H. pluvialis cells. The major fatty acid contents in astaxanthin esters had the same tendency with the contents of the total fatty acids in red motile cells and red cysts. The oleic acid (C18:1) and linoleic acid (C18:2) were dominant in both red mobile cells and red cysts, and red mobile cells and red cysts contained much more oleic acid and linoleic acid than green motile cells and green cysts. It indicated that the active synthesis of oleic acid and linoleic acid was necessary to astaxanthin ester synthesis. The HPLC chromatogram of green cysts was similar to that of green motile cells, and neither free astaxanthin nor astaxanthin esters were detected in these two cells. Furthermore, the fatty acid composition was the same in green cysts and green motile cells. It suggested that astaxanthin ester accumulation coordinated with the synthesis of fatty acids from another point of view. The synthesis and accumulation of fatty acids was independent of the formation of cysts, but was highly correlated with the synthesis and accumulation of astaxanthin. The red cysts contained more than 30% of fatty acids, in which the unsaturated fatty acids accounted for about 81%. Therefore, H. pluvialis was not only a good resource of precious astaxanthin, but also would become a good resource of valuable fatty acids in future.
4. Both oxygen and temperature had strong effects astaxanthin stability. Both vitamin C and vitamin E, the common antioxidant, decreased the astaxanthin stability. There were no significant changes in the astaxanthin content or the relative content of each astaxanthin ester after the dry algal meal with broken cell wall stored under vacuum at -20℃for 1.5 years or the algal meal stored at -20℃for 1.5 years. After stored for 2 years, the astaxanthin content of each treatment decreased about 10%, and the relative content of each astaxanthin ester was stabile in algal meal, but decreased significantly in dry algal meal.
5. 72.04% free astaxanthin was obtained after the extraction mixture was treated at 37℃for 75 min with the presence of 1.02 units cholesterol. And it was the optimal treatment for hydrolysis by enzyme. However, only 51.48% free astaxanthin was obtained after the reaction mixture was treated at 22℃for 40 min with the presence of 1.07 M NaOH. And it was the optimal treatment for hydrolysis by alkali. Therefore, it was not accurate to determine astaxanthin content by HPLC after hydrolysis especially after saponification, for hydrolysis by enzyme or by alkali would destroy astaxanthin in some degree.
6. H. pluvialis formed red cysts with astaxanthin accumulation in cells under stresses. In order to analyze the change of gene expressions and isolate these related genes inducing astaxanthin synthesis, a subtractive cDNA library of cyst formation and astaxanthin synthesis was constructed by using the Suppression Subtractive Hybridization (SSH) techniques. In this paper, the quality of cellular RNAs and adaptor ligation efficiency were analyzed, and the housekeeping gene,α-tubulin, was used to estimate the efficiency of subtractive cDNA. In this subtractive cDNA library,α-tubulin was subtracted very efficiently at appropriate 2~(10)-2~(15) fold, indicating that some differentially expressed genes including the genes related to the cyst formation and astaxanthin synthesis were also enriched at the same folds. The construction of the subtractive cDNA library would be essential for rapid isolation and clone of the genes related to cyst formation and astaxanthin synthesis, and would be helpful to find out the molecular mechanism of the astaxanthin accumulation in H. pluvialis.
引文
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