Mining and Analysis of Proteins Associated with Neoxanthin Biosynthesis in Chlamydomonas reinhardtii

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

Abstract

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

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.

Figures/Tables 9

References 34

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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

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

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

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

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