Identification and Functional Verification of Key Genes in Riboflavin Synthesis Pathway in Chinese Chestnut

doi:10.13560/j.cnki.biotech.bull.1985.2025-0064

Abstract

Abstract:

Objective Lumazine synthase (LS) and riboflavin synthase (RS) are key enzymes in riboflavin biosynthesis in Chinese chestnut. This work aims to explore the effects of LS and RS on the synthesis of Chinese chestnut riboflavin, and to provide a molecular basis for the synthesis of Chinese chestnut riboflavin. Method Having the protein sequences of riboflavin synthesis-related genes in the model plant Arabidopsis thaliana as bait, BLAST method was used to identify the riboflavin synthesis-related enzyme genes in the Chinese chestnut genome database. Transcriptome data was used to analyze the expression patterns of related genes in three stages of Chinese chestnut fruit development. Ultra-performance liquid chromatography (UHPLC) was applied to determine the riboflavin contents in eight Chinese chestnut varieties. Combined with inter-group difference and correlation analysis based on gene expression levels. Bioinformatics methods were adapted to analyze the structure, protein physicochemical properties, systematic evolution and promoter cis-acting elements of key genes. A chestnut callus transient transformation system was for verifying the functions of key genes. Result The Chinese chestnut contained GTP cyclohydrolase Ⅱ (GCHⅡ) , along with two pyrimidine deaminases (PYRD), one pyrimidine reductase (PYRR), one pyrimidine phosphatase (PYRP), two 3,4-dihydroxy-2-butanone 4-phosphate synthases (DHBPS), two lumazine synthases (LS), and one riboflavin synthase (RS) . The expressions of LS and RS were the highest in the kernel of Chinese chestnut. The riboflavin content in Chinese chestnut was between 0.104 and 0.054 mg/100 g. The expression of CmLS1 was significantly and positively correlated with the riboflavin content, the expression of CmRS was extremely significantly and positively correlated with the riboflavin content. CmLS1 was 693 bp in length and encoded 231 amino acids. CmRS was 843 bp in length and encoded 281 amino acids. CmLS1 had the highest homology with Corylus avellana. CmRS had the highest homology with Quercus suber,Quercus lobata and Corylus avellana. The promoters of CmLS1 and CmRS mainly contained hormone-responsive, light-responsive, low-temperature responsive and anaerobic induction cis-acting elements. After silencing CmLS1 and CmRS, riboflavin content in chestnut callus decreased by 33.1% and 49.1% respectively. Conclusion The riboflavin synthetases CmLS1 and CmRS of Chinese chestnut positively regulate riboflavin synthesis in the kernel and play an important role in riboflavin synthesis.

Key words: Chinese chestnut, riboflavin, lumazine synthase, riboflavin synthetase, callus, transient transformation

YU Wen-jie, FAN Si-ran, GAO Wen-li, XING Yu, QIN Ling. Identification and Functional Verification of Key Genes in Riboflavin Synthesis Pathway in Chinese Chestnut[J]. Biotechnology Bulletin, 2025, 41(9): 232-241.

Figures/Tables 10

Table 1 HPLC parameters

参数名称 Parameter name	参数 Parameter
色谱柱 Chromatographic column	C₁₈柱150 mm × 4.6 mm × 5 μm
流动相 Mobile phase	乙酸钠溶液（0.05 mol/L）-甲醇（80∶20）
流速 Velocity of flow （mL/min）	1
柱温 Column temperature （℃）	30
检测波长 Test wavelength	激发波长462 nm，发射波长522 nm
进样体积 Injection volume （μL）	20

Table 2 Primers for cloning specific fragments of CmLS1 and CmRS target genes

引物名称 Primer name	引物序列 Primer sequence （5'‒3'）	退火温度 Annealing temperature （℃）
CmRS RNAi-F	GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCTCTCACATCATTCTCCAA	56.5
CmRS RNAi-R	GGGGACCACTTTGTACAAGAAAGCTGGGTACGCGGCTATTCTCATATCG	57
CmLS1 RNAi-F	GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCCAGAACTCAGGGTCTTC	60.3
CmLS1 RNAi-R	GGGGACCACTTTGTACAAGAAAGCTGGGTACCAGCCGAATTAGCAACAG	60.4

Table 3 Primers for fluorescence quantitative PCR of CmLS1 and CmRS

引物名称 Primer name	引物序列 Primer sequence （5'‒3'）	退火温度 Annealing temperature （℃）
Actin-F	TTGACTATGAGCAGGAACTT	58.9
Actin-R	TTGTAGGTGGTCTCGTGAAT	60.8
CmRS QP-F	CGGCATAGTTGAAGAAGT	56.8
CmRS QP-R	GCGGCTATTCTCATATCG	57.0
CmLS1 QP-F	GCCATCCTACACCTTAACAG	58.4
CmLS1 QP-R	CTTCCTCTCAACAGCATAGC	59.0

Fig. 1 Identification of riboflavin synthetase gene and analysis of gene expression at different developmental stages of Chinese chestnutGene expression in Chinese chestnut (Castanea mollissima) at 68, 81, and 95 d after anthesis

Fig. 2 Correlation analysis of riboflavin contents and expressions of CmLS1 and CmRS genes in different Chinese chestnut cultivarsA: Analysis of riboflavin content in Chinese chestnut and differences between groups. B: Differential expressions of CmLS1 in Chinese chestnut cultivars and analysis of differences between groups. C: Differential expression of CmRS in Chinese chestnut cultivars and analysis of differences between groups. D: Correlation analysis of CmLS1, CmRS and riboflavin content. **** P<0.001; ** P<0.01; * P<0.05; n=3. The same below

Fig. 3 Analysis of conserved domainsof gene CmLS1 （A） and CmRS （B）in Chinese chestnut

Table 4 Physicochemical properties of CmLS1 and CmRS proteins in Chinese chestnut

蛋白

Protein

蛋白长度

Length of protein （aa）

分子量

Molecular weight （Da）

等电点

Isoelectric point

不稳定系数

Coef ficient of instability

最大亲水性

Derophilic index

亚细胞定位

Subcel lular localization

Fig. 4 Protein sequence alignment and phylogenetic tree analysis of lumazine synthase LS and riboflavin synthetase RSA: Protein sequence alignment of lumazine synthase in Chinese chestnut. B: Protein sequence alignment of riboflavin synthetase in Chinese chestnut. C: Phylogenetic tree analysis of lumazine synthas in Chinese chestnut. D: Phylogenetic tree analysis of riboflavin synthetase in Chinese chestnut

Table 5 Analyses of cis-acting regulatory elements in the promotersoflumazine synthase CmLS1 andriboflavin synthetase CmRS

元件名称 Name of element	元件序列 Sequence of element	元件类型 Type of element	CmLS1启动子元件数量 Number of CmLS1 promoter element	CmRS启动子元件数量 Number of CmRS promoter element
G-box	CACGTT	光响应元件	2	1
AuxRE	TGTCTCAATAAG	生长素响应元件	0	2
TGACG-motif	TGACG	茉莉酸甲酯响应元件	2	1
ABRE	ACGTG	脱落酸响应元件	1	1
TCA-element	CCATCTTTTT	水杨酸响应元件	0	1
ARE	AAACCA	厌氧诱导响应元件	3	2
LER	CCGAAA	低温响应元件	2	0

Fig. 5 CmLS1 and CmRS transient silencing and determination of riboflavin contentA: Fluorescence observation of callus. B: Relative expression of CmLS1. C: Relative expression of CmRS. D: Riboflavin content detection of positive callus. Bar =1 000 μm

References 34

[1]	Averianova LA, Balabanova LA, Son OM, et al. Production of vitamin B2 (riboflavin) by microorganisms: an overview [J]. Front Bioeng Biotechnol, 2020, 8: 570828.
[2]	李俊霖, 边祥雨, 姚站馨, 等. 膳食核黄素参考摄入量国内外研究进展 [J]. 营养学报, 2022, 44(6): 530-533.
	Li JL, Bian XY, Yao ZX, et al. Revision of dietary riboflavin reference intake: an update [J]. Acta Nutr Sin, 2022, 44(6): 530-533.
[3]	万旻. 核黄素营养状况对骨骼健康影响的研究 [D]. 北京: 军事科学院, 2023.
	Wan M. Study of the effect of riboflavin nutritional status on bone health [D]. Beijing: Academy of Military Science, 2023.
[4]	胡海涛, 郭龙彪. 植物核黄素的生物合成及其功能研究进展 [J]. 植物学报, 2023, 58(4): 638-655.
	Hu HT, Guo LB. Progress in the research on riboflavin biosynthesis and function in plants [J]. Chin Bull Bot, 2023, 58(4): 638-655.
[5]	Ozbekova Z, Kulmyrzaev A. Study of moisture content and water activity of rice using fluorescence spectroscopy and multivariate analysis [J]. Spectrochim Acta Part A Mol Biomol Spectrosc, 2019, 223: 117357.
[6]	Villareal CP, Juliano BO. Variability in contents of thiamine and riboflavin in brown rice, crude oil in brown rice and bran-Polish, and silicon in hull of IR rices [J]. Plant Foods Hum Nutr, 1989, 39(3): 287-297.
[7]	武妍妍, 史文石, 石新如, 等. 板栗坚果营养物质和抗氧化成分综合评价 [J]. 林业科学研究, 2022, 35(6): 12-22.
	Wu YY, Shi WS, Shi XR, et al. Comprehensive evaluation of nutrients and antioxidant components in nuts of chestnut [J]. For Res, 2022, 35(6): 12-22.
[8]	徐志祥, 高绘菊. 板栗营养价值及其养生保健功能 [J]. 食品研究与开发, 2004, 25(5): 118-119.
	Xu ZX, Gao HJ. Nutritional value of chestnut and its health care function [J]. Food Res Dev, 2004, 25(5): 118-119.
[9]	张瑞菊, 孙强, 张洪坤. 板栗的营养、生产现状及前景展望 [J]. 山东商业职业技术学院学报, 2014, 14(4): 106-107.
	Zhang RJ, Sun Q, Zhang HK. Nutrition, production status and prospect of Chinese chestnut [J]. J Shandong Inst Commer Technol, 2014, 14(4): 106-107.
[10]	乔艳杰, 刘巍, 吴瑞刚, 等. 北京市板栗产业发展问题及对策研究 [J]. 中国果树, 2024(6): 125-129.
	Qiao YJ, Liu W, Wu RG, et al. Research on the development problems and countermeasures of Chinese chestnut industry in Beijing [J]. China Fruits, 2024(6): 125-129.
[11]	韩元顺, 许林云, 周杰. 中国板栗产业与市场发展现状及趋势 [J]. 中国果树, 2021(4): 83-88.
	Han YS, Xu LY, Zhou J. Status and trend of the development of chestnut industry in China [J]. China Fruits, 2021(4): 83-88.
[12]	蔡荣, 虢佳花, 祁春节. 板栗产业发展现状、存在问题与对策分析 [J]. 中国果菜, 2007, 27(1): 52-53.
	Cai R, Guo JH, Qi CJ. Present situation, existing problems and countermeasures of chestnut industry development [J]. China Fruit Veg, 2007, 27(1): 52-53.
[13]	Tolar JG, Li SL, Ajo-Franklin CM. The differing roles of flavins and quinones in extracellular electron transfer in lactiplantibacillus plantarum [J]. Appl Environ Microbiol, 2023, 89(1): e0131322.
[14]	Bacher A, Eberhardt S, Eisenreich W, et al. Biosynthesis of riboflavin [M]//Cofactor Biosynthesis. Amsterdam: Elsevier, 2001: 1-49.
[15]	郭长江, 顾景范. 核黄素 [J]. 营养学报, 2013, 35(2): 119-121.
	Guo CJ, Gu JF. Riboflavin [J]. Acta Nutr Sin, 2013, 35(2): 119-121.
[16]	王林静, 黄亿明. 核黄素与健康 [J]. 广东药学院学报, 2000, 16(3): 223-225, 228.
	Wang LJ, Huang YM. Riboflavin and health [J]. Acad J Guangdong Coll Pharm, 2000, 16(3): 223-225, 228.
[17]	Xu ZB, Lin ZQ, Wang ZW, et al. Improvement of the riboflavin production by engineering the precursor biosynthesis pathways in Escherichia coli [J]. Chin J Chem Eng, 2015, 23(11): 1834-1839.
[18]	Harale B, Kidwai S, Ojha D, et al. Synthesis and evaluation of antimycobacterial activity of riboflavin derivatives [J]. Bioorg Med Chem Lett, 2021, 48: 128236.
[19]	Tani T, Sobajima H, Okada K, et al. Identification of the OsOPR7 gene encoding 12-oxophytodienoate reductase involved in the biosynthesis of jasmonic acid in rice [J]. Planta, 2008, 227(3): 517-526.
[20]	Sa N, Rawat R, Thornburg C, et al. Identification and characterization of the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana [J]. Plant J, 2016, 88(5): 705-716.
[21]	Xiao S, Dai LY, Liu FQ, et al. COS1: an Arabidopsis coronatine insensitive1 suppressor essential for regulation of jasmonate-mediated plant defense and senescence [J]. Plant Cell, 2004, 16(5): 1132-1142.
[22]	Hu HT, Ren DY, Hu J, et al. WHITE AND LESION-MIMIC LEAF1, encoding a lumazine synthase, affects reactive oxygen species balance and chloroplast development in rice [J]. Plant J, 2021, 108(6): 1690-1703.
[23]	任秀艳, 乔洁, 张江丽. 核黄素合酶的研究进展 [J]. 西北植物学报, 2011, 31(9): 1917-1926.
	Ren XY, Qiao J, Zhang JL. Research progress of riboflavin synthase [J]. Acta Bot Boreali Occidentalia Sin, 2011, 31(9): 1917-1926.
[24]	黄伟志, 黄桂东, 钟先锋. 高效液相色谱法测定功能性饮料中维生素B₁、维生素B₂含量 [J]. 食品研究与开发, 2019, 40(24): 191-197.
	Huang WZ, Huang GD, Zhong XF. Determination of vitamins B₁ and vitamins B₂ in energy drinks by high performance liquid chromatography [J]. Food Res Dev, 2019, 40(24): 191-197.
[25]	朱丽, 钱前. 虾青素功能米: 生物强化新思路, 优质米培育新资源 [J]. 植物学报, 2019, 54(1): 4-8.
	Zhu L, Qian Q. Astaxanthin functional rice: new idea of biofortification, new perspectives for high-quality rice breeding [J]. Chin Bull Bot, 2019, 54(1): 4-8.
[26]	Suwannasom N, Kao I, Pruß A, et al. Riboflavin: the health benefits of a forgotten natural vitamin [J]. Int J Mol Sci, 2020, 21(3): 950.
[27]	Tuan PA, Kim JK, Lee S, et al. Riboflavin accumulation and characterization of cDNAs encoding lumazine synthase and riboflavin synthase in bitter melon (Momordica charantia) [J]. J Agric Food Chem, 2012, 60(48): 11980-11986.
[28]	Zhao YY, Wang DF, Wu TQ, et al. Transgenic expression of a rice riboflavin synthase gene in tobacco enhances plant growth and resistance to Tobacco mosaic virus [J]. Can J Plant Pathol, 2014, 36(1): 100-109.
[29]	Namba J, Harada M, Shibata R, et al. AtDREB2G is involved in the regulation of riboflavin biosynthesis in response to low-temperature stress and abscisic acid treatment in Arabidopsis thaliana [J]. Plant Sci, 2024, 347: 112196.
[30]	Wang TZ, Wang J, Zhang D, et al. Protein kinase MtCIPK12 modulates iron reduction in Medicago truncatula by regulating riboflavin biosynthesis [J]. Plant Cell Environ, 2023, 46(3): 991-1003.
[31]	Xu XP, Zhang CY, Xu XQ, et al. Riboflavin mediates m⁶A modification targeted by miR408, promoting early somatic embryogenesis in Longan [J]. Plant Physiol, 2023, 192(3): 1799-1820.
[32]	Bosch G, Fuentes M, Erro J, et al. Hydrolysis of riboflavins in root exudates under iron deficiency and alkaline stress [J]. Plant Physiol Biochem, 2024, 210: 108573.
[33]	Deng BL, Dong HS. Ectopic expression of riboflavin-binding protein gene TsRfBP paradoxically enhances both plant growth and drought tolerance in transgenic Arabidopsis thaliana [J]. J Plant Growth Regul, 2013, 32(1): 170-181.
[34]	Wu YZ, Cheng SR, Ding XF, et al. Exogenous riboflavin application at different growth stages regulates photosynthetic accumulation and grain yield in fragrant rice [J]. Agriculture, 2024, 14(11): 1979.