强光下一氧化氮对三角褐指藻EPA产量的影响

摘要

Eicosapentaenoic acid(EPA,5,8,11,14,17-cis-eicosapentaenoic acid)is an n-3 C20-polyunsaturated fatty acid that has a variety of functions in the field of medicine.In this research,the effect of exogenous nitric oxide(NO)on EPA yield in Phaeodactylum tricornutum under high light irradiance was studied.The relationship between light intensity and photosynthetic rate of Phaeodactylum tricornutum was determined with oxygen electrode method,and the results indicate that the light saturation point of Phaeodactylum tricornutum is 126.3 μmol/(m2·s).The Phaeodactylum tricornutum was cultivated in F/2 medium.15 days later,the EPA content was determined by gas chromatography.It was found that EPA yield in Phaeodactylum tricornutum under high light irradiance was increased by exogenous NO,which depended on the effect of NO promoting Phaeodactylum tricornutum growth,but NO had no significant effect on EPA content per g dry algae powder.The growth rate of Phaeodactylum tricornutum was detected by cell count technique,and the results showed that there was a light inhibition in the growth of Phaeodactylum tricornutum under 200 μmol/(m2·s)light irradiance,and SNP could relieve the inhibition.Glycolic acid oxidase(GO)inhibitor oxalic acid decreased Phaeodactylum tricornutum growth rate which was promoted by exogenous NO which increased GO activity.It indicated that exogenous NO accelerated Phaeo-dactylum tricornutum growth rate by increasing GO activity.After purification,the GO was identified with AMCA switch method,and it was found that both GOX and GLY(two isozyme of GO)were S-nitrosylated by NO.The SNO site positions in GLY and GOX were analyzed by the software of iSNO-AAPair,GPS-SNO and iSNO-PseAAC.The 238th cysteine in GLY,the 237th cysteine and the 332th cysteine in GOX were probably S-nitrosylated by NO.In addition,GOX and GLY gene expression levels detected by Real-Time PCR were increased by exogenous NO.The data demonstrate that NO can increase GO activity both by stimulating gene expression and by post-transcriptional S-nitrosylation.In summary,NO,by enhancing GO activity,can quicken the growth of Phaeodactylum tricornutum under high light condition and then improve the overall EPA yield.%二十碳五烯酸(EPA)是一种长链多不饱和脂肪酸,具有重要的医药价值.本文研究了光照强度和亚硝基铁氰化钠(SNP)分解产生的NO对三角褐指藻EPA产量的影响及机理.采用氧电极法探究光照强度与三角褐指藻光合速率的关系,结果显示三角褐指藻的光饱和点为126.3μmol/(m2·s).采用气相色谱法检测EPA的含量,结果显示外源NO可通过促进强光下三角褐指藻的快速生长来提高强光下三角褐指藻EPA的产量,而对单位生物量中EPA含量并没有影响;用血球计数板法检测三角褐指藻的生长速率,结果显示在光强200μmol/(m2·s)时三角褐指藻出现了光抑制,而SNP能解除这种抑制.探究NO促进三角褐指藻快速生长的机理,结果显示NO提高了乙醇酸氧化酶(GO)的活性.用实时荧光定量PCR(Real-time PCR)检测外源NO对GO基因表达量的影响,结果显示GO的两个同工酶"GOX和GLY"的基因表达量均增强了.采用AMCA开关法对纯化的GO进行S–亚硝基化鉴定,结果显示GOX和GLY均发生了S–亚硝基化修饰.分别采用ISNO-AAPair、GPS-SNO和ISNO-PseAAC方法预测S–亚硝基化位点,结果显示GLY的238位、GOX的237位和332位很可能是NO的修饰位点.因此,NO提高GO活性的途径有两条:提高GO基因的表达量;对GO酶蛋白进行S–亚硝基化修饰.综上所述,NO通过提高GO活性促进强光下三角褐指藻的快速生长从而提高三角褐指藻EPA的产量.