生物技术通报2025，Vol.41Issue(9)：232-241,10.DOI:10.13560/j.cnki.biotech.bull.1985.2025-0064
板栗核黄素合成通路关键基因鉴定及功能验证
Identification and Functional Verification of Key Genes in Riboflavin Synthesis Pathway in Chinese Chestnut
摘要
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.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Key words
Chinese chestnut/riboflavin/lumazine synthase/riboflavin synthetase/callus/transient transformation引用本文复制引用
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):232-241,10.基金项目
国家自然科学基金项目(32471917) （32471917）
