板栗核黄素合成通路关键基因鉴定及功能验证

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

摘要/Abstract

摘要：

目的二氧四氢蝶啶合成酶（lumazine synthase, LS）和核黄素合成酶（riboflavin synthase, RS）是板栗核黄素合成的关键基因，探究其对板栗核黄素合成的影响，为板栗核黄素合成提供分子理论基础。方法以模式植物拟南芥核黄素合成相关基因的蛋白序列为诱饵，在板栗基因组数据库中通过BLAST方法鉴定核黄素合成相关酶基因，利用转录组数据分析相关基因在板栗果实发育3个时期的表达模式。利用超高效液相色谱仪测定8个板栗品种中核黄素含量，结合基因表达量进行组间差异分析和相关性分析，利用生物信息学方法分析关键基因的结构、蛋白理化性质、系统进化和启动子顺式作用元件。通过板栗愈伤组织瞬时转化体系初步验证关键基因功能。结果板栗中有2个GTP环水解酶Ⅱ（GCHⅡ），2个嘧啶脱氨酶（PYRD），1个嘧啶还原酶（PYRR），1个嘧啶磷酸酶（PYRP），2个3，4-二羟基-2-丁酮-4-磷酸合酶（DHBPs），2个二氧四氢蝶啶合成酶（LS）和1个核黄素合成酶（RS），二氧四氢蝶啶合成酶和核黄素合成酶在板栗果仁中表达量最高。板栗中核黄素含量范围在0.054‒0.104 mg/100 g，CmLS1和CmRS的基因表达量与核黄素含量分别呈显著正相关和极显著正相关。CmLS1全长693 bp，编码231个氨基酸，CmRS全长843 bp，编码281个氨基酸。CmLS1与欧榛（Corylus avellana）同源性最高，CmRS与栓皮栎（Quercus suber）、白栎（Quercus lobata）和夏栎（Corylus avellana）的同源性最高，CmLS1和CmRS启动子上主要包含激素响应顺式作用元件、光响应顺式作用元件和低温响应元件还有厌氧诱导响应元件。沉默CmLS1和CmRS后板栗愈伤组织中核黄素含量分别下降33.1%和49.1%。结论 CmLS1和CmRS是板栗核黄素合成关键基因，正调控板栗核黄素合成。

关键词: 板栗, 核黄素, 二氧四氢蝶啶合成酶, 核黄素合成酶, 愈伤组织, 瞬时转化

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

于文杰, 范斯然, 高文丽, 邢宇, 秦岭. 板栗核黄素合成通路关键基因鉴定及功能验证[J]. 生物技术通报, 2025, 41(9): 232-241.

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.

图/表 10

表1 HPLC参数

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

表2 CmLS1和CmRS目的基因特异性片段克隆引物

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

表3 CmLS1和CmRS荧光定量PCR引物

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

图1 板栗核黄素合成酶基因鉴定和果实不同发育时期基因表达量分析花后68、81和95 d的板栗果实基因表达量

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

图2 板栗不同品种核黄素含量以及与CmLS1和CmRS基因表达量的相关性分析A：板栗核黄素含量和组间差异分析；B：CmLS1在板栗不同品种中表达差异和组间差异分析；C：CmRS在板栗不同品种中表达差异和组间差异分析；D：CmLS1和CmRS和核黄素含量相关性分析；**** P<0.001；** P<0.01；* P<0.05； n=3；下同

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

图3 板栗CmLS1 （A）和CmRS （B）基因保守结构域分析

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

表4 板栗CmLS1和CmRS蛋白理化性质

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

图4 二氧四氢蝶啶合成酶LS和核黄素合成酶RS蛋白序列比对和进化树分析A：板栗二氧四氢蝶啶合成酶蛋白序列比对； B：板栗核黄素合成酶蛋白序列比对；C：板栗二氧四氢蝶啶合成酶进化树分析；D：板栗核黄素合成酶进化树分析

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

表5 板栗二氧四氢蝶啶合成酶CmLS1和核黄素合成酶CmRS启动子顺式作用元件分析

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

图5 CmLS1和CmRS瞬时沉默和核黄素含量测定A：愈伤组织荧光观察；B：CmLS1相对表达量；C：CmRS相对表达量；D：阳性愈伤组织核黄素含量检测；标尺=1 000 μm

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

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