莱茵衣藻新黄素合成相关蛋白质的挖掘与分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0809

摘要/Abstract

摘要：

目的新黄素是一种广泛存在于绿色植物中的脂溶性类胡萝卜素，不仅是光合作用中不可或缺的关键色素，还具有抗氧化等多种重要的生理功能。深入分析影响新黄素合成的培养条件及相关蛋白质，对其产量提升具有重要意义。方法以模式微藻莱茵衣藻为对象，考察不同培养条件（缺氮、缺硫、光照强度）对新黄素合成的影响；基于生物信息学分析、RNA干扰技术、转录水平和HPLC检测相结合的方法，挖掘影响莱茵衣藻中新黄素合成的相关蛋白质。结果光照强度从3 000 lx提高到4 000 lx时，莱茵衣藻的新黄素含量提高16.1%，而缺氮和缺硫条件下的新黄素含量分别比正常条件下降37.2%和42.6%。3个含有DUF4281功能域的假定蛋白质中，nxs2和nxs3转录水平的下降使得新黄素含量分别提高17.4%和33.7%，而nxs1的干扰无明显影响。进一步分析发现，4 000 lx光照和缺硫条件下，nxs3的转录水平分别降低30.4%和提高34.0%，该变化分别与新黄素含量在4 000 lx条件下的上升和缺硫条件下的下降趋势对应。结论 4 000 lx光照条件比3 000 lx更有利于新黄素合成，缺氮和缺硫条件则产生抑制作用。nxs2和nxs3负向调控莱茵衣藻的新黄素合成，而nxs1无显著影响。

关键词: 新黄素, 莱茵衣藻, 培养条件, 相关蛋白质, RNAi, 转录水平

Abstract:

Objective Neoxanthin is a lipid-soluble carotenoid widely found in green plants. It serves not only as an essential pigment in photosynthesis but also presents various important physiological functions, such as antioxidant activity. Thus, analyzing the influences of culture conditions and associated proteins on neoxanthin synthesis is critical for enhancing its production. Method Using the model microalga Chlamydomonas reinhardtii as object, the effects of different culture conditions, including nitrogen deficiency, sulfur deficiency, and light intensity, on neoxanthin synthesis were examined. Through the integrated analysis of bioinformatics, RNA interference (RNAi), transcriptional level, and HPLC quantification, proteins associated with neoxanthin synthesis in C. reinhardtii were mined. Result Compared to normal conditions, the increase in light intensity from 3 000 lx to 4 000 lx promoted neoxanthin content in C. reinhardtii by 16.1%, while nitrogen deficiency and sulfur deficiency reduced the amount by 37.2% and 42.6%, respectively. Among three putative DUF4281 domain-containing proteins, the knockdown of nxs2 and nxs3 enhanced neoxanthin content by 17.4% and 33.7%, respectively, whereas the interference with nxs1 showed no significant effect. Further analysis revealed that under 4 000 lx light intensity and sulfur-deficient conditions, the transcript level of nxs3 decreased by 30.4% and increased by 34.0%, respectively. These changes correlated well with the increased neoxanthin content under 4 000 lx and the decreased content under sulfur deficiency. Conclusion 4 000 lx light conditions are more conducive to neoxanthin synthesis than 3000 lx, while nitrogen deficiency and sulfur deficiency yield inhibition effects. In addition, nxs2 and nxs3 negatively regulate neoxanthin synthesis in C. reinhardtii, whereas nxs1 has no significant effect.

Key words: neoxanthin, Chlamydomonas reinhardtii, culture conditions, associated proteins, RNA interference (RNAi), transcriptional level

梁颖怡, 赵安宿, 王瑞曦, 沈天虹, 马欣荣, 王敏, 骆健美. 莱茵衣藻新黄素合成相关蛋白质的挖掘与分析[J]. 生物技术通报, 2025, 41(11): 190-200.

LIANG Ying-yi, ZHAO An-su, WANG Rui-xi, SHEN Tian-hong, MA Xin-rong, WANG Min, LUO Jian-mei. Mining and Analysis of Proteins Associated with Neoxanthin Biosynthesis in Chlamydomonas reinhardtii[J]. Biotechnology Bulletin, 2025, 41(11): 190-200.

图/表 9

表1 本实验所用引物

Table 1 Primers used in this experiment

名称 Name	引物序列 Primer sequence （5′-3′）	用途 Application
aR-nxs1-F	*ctagt*CCGGCTCCATTATCATGCTAAtctcgctgatcggcaccatgggggtggtggtgatcagcgctaTTAGGATGATAATGGAGCCGGg	干扰藻株构建引物
aR-nxs1-R	*ctagc*CCGGCTCCATTATCATCCTAAtagcgctgatcaccaccacccccatggtgccgatcagcgagaTTAGCATGATAATGGAGCCGGa	干扰藻株构建引物
aR-nxs2-F	*ctagt*ACGGACGCTATGATTTTTGTAtctcgctgatcggcaccatgggggtggtggtgatcagcgctaTACAGGAATCATAGCGTCCGTg	干扰藻株构建引物
aR-nxs2-R	*ctagc*ACGGACGCTATGATTCCTGTAtagcgctgatcaccaccacccccatggtgccgatcagcgagaTACAAAAATCATAGCGTCCGTa	干扰藻株构建引物
aR-nxs3-F	*ctagt*ATGGCAATAAACTCAGTAACAtctcgctgatcggcaccatgggggtggtggtgatcagcgctaTGTTTCTGAGTTTATTGCCATg	干扰藻株构建引物
aR-nxs3-R	*ctagc*ATGGCAATAAACTCAGAAACAtagcgctgatcaccaccacccccatggtgccgatcagcgagaTGTTACTGAGTTTATTGCCATa	干扰藻株构建引物
AmiRNAprecF	GGTGTTGGGTCGGTGTTTTTG	菌落PCR引物
SpacerR	TAGCGCTGATCACCACCACCC	菌落PCR引物
Luciferase-F	CCATATGGTCAACGGCGTGAAGGTG	干扰藻株初筛引物
Luciferase-R	GCCGCCGGCGCCCTTGATCTTGTCC	干扰藻株初筛引物
nxs1-qF	TCATTCTCACCGTGGTAGCTGG	RT-qPCR引物
nxs1-qR	AAGAACTGCGGGTTGAAGC	RT-qPCR引物
nxs2-qF	GCTCACCCTGGATGTAGTCAT	RT-qPCR引物
nxs2-qR	CGGTGGGTCCGAAGAAGAAG	RT-qPCR引物
nxs3-qF	CGGACTTCAGCGAGATGTTC	RT-qPCR引物
nxs3-qR	CAACCATCATGCACAGGAACAG	RT-qPCR引物
α-tubulin-qF	CTCGCTTCGCTTTGACGGTG	内参
α-tubulin-qR	CGTGGTACGCCTTCTCGGC	内参

表2 新黄素的液相梯度洗脱条件

Table 2 Liquid phase gradient elution conditions for neoxanthin

时间Time （min）	流速 Flow rate （mL/min）	A（%）	B（%）	C（%）
0	1.0	100	0	0
4	1.0	0	100	0
18	1.0	0	20	80
21	1.0	0	100	0
29	1.0	100	0	0

图1 培养条件对莱茵衣藻生长和色素合成的影响A：生长曲线；B：对数中期的培养液状态；C：新黄素含量；D：叶绿素含量；E：类胡萝卜素含量；F：油脂含量；柱状图的数值为平均值±标准差（n=3）；*，**，***分别表示P<0.05，P<0.01，P<0.001，下同

Fig. 1 Effects of culture conditions on the growth and pigment biosynthesis in C. reinhardtiiA: Growth curves. B: Culture status at mid-logarithmic phase. C: Neoxanthin content. D: Chlorophyll content. E: Carotenoid content. F: Lipid content. The values in the bar charts are of the mean±standard error (n=3); *, **, *** indicate P<0.05, P<0.01, P<0.001, respectively, the same below

表3 莱茵衣藻中3个新黄素合成相关候选基因的染色体位置

Table 3 Chromosomal locations of three neoxanthin synthase candidate genes in C. reinhardtii

基因

Gene

染色体

Chromosome

基因长度

Gene length （bp）

染色体初始位置

Start locus on chromosome

染色体结束位置

End locus on chromosome

nxs1

nxs2

nxs3

表4 莱茵衣藻中3个候选蛋白质与拟南芥新黄素合成酶的氨基酸序列及功能域分析

Table 4 Analysis of amino acid sequences and functional domains between three candidate proteins in C. reinhardtii and A. thalian neoxanthin synthase

基因 Gene	氨基酸长度 Amino acid length （aa）	与文献鉴定的新黄素合成酶氨基酸序列一致性 Amino acid sequence identity to the identified neoxanthin synthase in literature （%）		DUF4281功能域位置 DUF4281 domain position	与文献鉴定的新黄素合成酶的DUF4281的氨基酸序列一致性DUF4281 amino acid sequence identity to identified neoxanthin synthase in literature （%）
基因 Gene	氨基酸长度 Amino acid length （aa）	拟南芥 A. thalian	羽衣甘蓝 Chinese kale	DUF4281功能域位置 DUF4281 domain position	拟南芥 A. thalian	羽衣甘蓝 Chinese kale
nxs1	258	49.1	48.9	91-222	55.3	54.9
nxs2	226	46.8	46.6	75-214	46.2	46.2
nxs3	105	21.5	21.8	3-72	31.1	31.1

图2 新黄素合成相关蛋白质生物信息学分析A：莱茵衣藻中3个候选蛋白质和文献已鉴定的新黄素合成酶的氨基酸序列比对，图中AtNXS，拟南芥新黄素合成酶；BoaNXS，羽衣甘蓝新黄素合成酶；CrNXS1-3，莱茵衣藻中3个新黄素合成相关候选蛋白质；红色框表示为DUF4281功能域；B：氨基酸组成；C：跨膜区预测分析；D：三级结构预测，绿色表示α-helix，粉色表示β-sheet，红色表示DUF4281功能域

Fig. 2 Bioinformatics analysis of proteins associated with neoxanthin synthesisA: Amino acid sequence alignment of three candidate proteins in C. reinhardtii with the identified neoxanthin synthase in literature;AtNXS (neoxanthin synthase in A. thaliana), BoaNXS (neoxanthin synthase in Brassica oleracea var. acephala), CrNXS1-3 (neoxanthin synthase in C. reinhardtii). The conserved DUF4281 domain is boxed in red. B: Amino acid composition. C: Prediction analysis of transmembrane domain. D: The predicted tertiary structure is depicted as α-helices (green), β-sheets (pink), and the DUF4281 domain (red)

表5 莱茵衣藻中3个新黄素合成相关候选蛋白质的理化性质和亚细胞定位预测

Table 5 Physicochemical properties and subcellular localization prediction of three candidate neoxanthin synthase proteins in C. reinhardtii

基因

Gene

分子式

Molecular formula

等电点

Isoelectric point （pI）

不稳定指数

Instability index

疏水性Hydropathicity

（GRAVY）

脂肪族指数

Aliphatic index

亚细胞定位

Subcellular localization

nxs1

C₁₂₂₈H₁₉₆₈N₃₁₆O₃₂₈S₁₃

nxs2

C₁₀₂₄H₁₆₅₉N₂₈₉O₂₇₂S₁₀

nxs3

C₅₂₀H₈₁₆N₁₃₀O₁₃₇S₉

图3 RNAi藻株的转录水平和色素含量A-C：分别为nxs1、nxs2、nxs3基因RNAi藻株的转录水平；D：新黄素含量；E：叶绿素含量；F：类胡萝卜素含量

Fig. 3 Transcriptional level and pigment content of the RNAi strainsA: nxs1. B: nxs2. C: nxs3. D: neoxanthin content. E: Chlorophyll content. F: Carotenoid content

图4 不同培养条件下nxs3基因相对转录水平（Control为1）

Fig. 4 Relative transcriptional levels of the nxs3 gene under different culture conditions (normalized to Control as 1)

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