福建农业学报2025，Vol.40Issue(3)：225-233,9.DOI:10.19303/j.issn.1008-0384.2025.03.002
硼毒害柑橘酵母单杂交文库构建及MIR397上游调控因子筛选
Yeast One-hybrid Library and Upstream Regulators of MIR397 in Boron-treated Citrus Leaves
摘要
Abstract
[Objective]Upstream regulatory factors of boron toxicity-related gene MIR397 in citrus were identified to understand the underlying mechanism for possible genetic improvement on the plants.[Method]A homogenized yeast one-hybrid(Y1H)three-frame cDNA library on Xuegan(Citrus sinensis)and sour pumelo(C.grandis)was constructed by SMART RACE technology.Upstream regulators of MIR397 were screened based on the cis-elements,including ABRE,GCN4,Box Ⅱ-like,and ERE,as baits.[Results]The capacity of the 3 frame cDNA libraries were 1.5×106,1.5×106,and 1.6×106 CFU,respectively.The amplification sizes of the inserted fragments in the cDNA library ranged from 500 to 4 000 bp with a recombination rate of 100％and zero redundancy.With a titer of 4×109 CFU·mL-1,the requirements for constructing Y1H cDNA library was met.Twenty-three expression sequence tags(ESTs)were obtained from the Y1H screening.Bioinformatics analysis revealed that 60.87％of the ESTs were related to plant growth and development,stress response,signal transduction,and transcriptional regulation,17.39％to protein translation,and the remainders annotated as proteases.HAP3,BBX,and EIN3 might participate in the citrus response to boron toxicity by interacting with ERE cis elements.Point-to-point Y1H verification showed that HAP3,BBX,and EIN3 interacted with ERE elements.[Conclusion]Three transcription factors that interacted with the ERE element of the MIR397 promoter were identified through Y1H library screening.The information obtained paved the way for further studies on the molecular networks involved in the citrus responses to boron toxicity.关键词
柑橘/硼毒害/cDNA三框文库/启动子/调控因子Key words
Citrus/boron toxicity/three-frame cDNA library/promoter/regulator分类
园艺学与植物营养学引用本文复制引用
黄镜浩,林雄杰,陈木兰,饶文华,和静怡,高芳銮,范国成..硼毒害柑橘酵母单杂交文库构建及MIR397上游调控因子筛选[J].福建农业学报,2025,40(3):225-233,9.基金项目
国家自然科学基金面上项目(32472676) （32472676）
福建省自然科学基金项目(2023J01371) （2023J01371）
福建省农业高质量发展超越"5511"协同创新工程项目(XTCXGC2021006) （XTCXGC2021006）
福建省农业科学院科技创新团队建设项目(CXTD2021003-3) （CXTD2021003-3）
福建科技计划引导性项目(2022N0021) （2022N0021）
