正交设计的诱导子组合对三角褐指藻岩藻黄素含量和光合生理及基因表达的影响

Effects of Orthogonal Design Optimized Elicitor Combinations on Fucoxanthin Content and Photosynthetic Physiology and Gene Expression of Phaeodactylum Tricornutum

目的 研究光合诱导因子(PIF)、茉莉酸甲酯(MeJA)、硫酸铈铵(ACS)和乙酰水杨酸(ASA)不同浓度组合对三角褐指藻岩藻黄素含量的影响,筛选促进岩藻黄素积累的最佳诱导子组合,并研究最佳诱导子组合处理下岩藻黄素含量积累的光合生理及基因表达变化。方法 采用4因素4水平正交试验法筛选出对三角褐指藻岩藻黄素含量促进效果最佳的诱导子组合,调制叶绿素荧光(PAM)检测最佳诱导子组合处理下三角褐指藻的叶绿素荧光参数,反转录-实时荧光定量聚合酶链反应(RT-qPCR)检测zds、lcyb、rbcL及psbA基因的表达水平。结果 正交试验结果表明,MeJA是影响三角褐指藻藻细胞岩藻黄素积累最显著的因素,其次是ASA。促进三角褐指藻岩藻黄素积累的最佳诱导子组合为1μg·L^(-1)PIF+0.2 mg·L^(-1)ACS+40 mg·L^(-1)ASA。该诱导子组合处理下三角褐指藻岩藻黄素含量达到21.77 mg·g^(-1),较对照组提高了104%。光合生理研究结果表明,诱导子组合显著提高了三角褐指藻藻细胞的最大相对电子传递速率(rETRmax)值(P<0.05)。RT-qPCR分析结果表明,诱导子组合促进三角褐指藻岩藻黄素合成相关基因zds、lcyb及光合作用PSⅡ碳同化相关基因rbcL、psbA基因的表达,其中,rbcL基因的表达量最高,是对照处理组的2.0倍(P<0.01)。结论 三角褐指藻岩藻黄素含量与zds基因呈显著正相关,与rETRmax、chla含量、lcyb、rbcL、psbA基因表达量呈极显著正相关,三角褐指藻可通过促进光合作用基因表达和PSⅡ相对电子传递速率来提高光合作用效率、促进岩藻黄素合成相关基因表达来促进岩藻黄素积累。

OBJECTIVE To study the effects of different concentrations of photosynthetic induction factor(PIF),methyl jasmonate(MeJA),ceric ammonium sulfate(ACS)and acetylsalicylic acid(ASA)on the accumulation of fucoxanthin in Phaeodactylum tricornutum,screen the best elicitor combination for fucoxanthin accumulation,and study the photosynthetic physiology and gene expression changes of fucoxanthin accumulation in Phaeodactylum tricornutum under the treatment of the best inducer combination.METHODS A four-factor,four-level orthogonal test was used to screen the best inducer combination to promote the accumulation of fucoxanthin in Phaeodactylum tricornutum,and the chlorophyll fluorescence parameters of Phaeodactylum tricornutum under the treatment of the best elicitor combination were detected by pulse-amplitude-modulation(PAM).The expressions of zds,lcyb,rbcL and psbA were detected by RT-qPCR.RESULTS The results of the orthogonal test showed that MeJA was the most significant factor affecting the accumulation of fucoxanthin in Phaeodactylum tricornutum cells,followed by ASA,and the best elicitor combination to promote the accumulation of fucoxanthin in Phaeodactylum tricornutum was 1μg·L^(-1) PIF+0.2 mg·L^(-1) ACS+40 mg·L^(-1) ASA.The photosynthetic physiological study showed that the inductor treatment was able to increase the fucoxanthin content by 104%compared with the control group,which was 21.77 mg·g^(-1).The results of photosynthetic physiological study showed that the inducer combination significantly increased the rETRmax value(P<0.05)of Phaeodactylum tricornutum.The expression of rbcL gene was the highest,which was 2.0 times higher than that of the control treatment(P<0.01).CONCLUSION The expressions of rETRmax,chla content,lcyb,rbcL and psbA genes are highly significantly correlated with zds gene,and the photosynthetic efficiency can be improved by promoting the expression of photosynthetic genes and the relative electron transfer rate of PSⅡ,and the expression of genes related to the synthesis of lutein can promote the accumulation of fucoxanthin in Phaeodactylum tricornutum.