小黑麦转录因子TwNAC01互作蛋白筛选及其抗旱性验证OA
Screening of Interacting Proteins of Transcription Factor TwNAC01 in Triticale and Its Function under Drought Stress
NAC转录因子在植物响应干旱胁迫时发挥关键调控作用,本课题组前期克隆到小黑麦转录因子TwNAC01全长cDNA.为了鉴定TwNAC01的互作蛋白,并解析其在植物应答干旱胁迫中的分子机制,本试验采用40℃、4℃、20%PEG6000、200 mmol L-1 NaCl以及叶面喷施100 μmol L-1 茉莉酸甲酯(MeJA)与100 μmol L-1脱落酸(ABA)处理的两叶一心期小黑麦幼苗,提取总RNA,构建基因组均一化cDNA文库.以TwNAC01为诱饵通过酵母双杂筛选互作蛋白,并通过双分子荧光互补(BiFC)、酵母逆境表型验证对筛选到的部分与抗旱相关蛋白的抗旱性进行验证.结果表明,构建了库容为1.436×108 CFU·mL-1、总克隆数为2.872×108的小黑麦均一化cDNA文库.共获得了33个与TwNAC01蛋白潜在互作的蛋白,涉及光合作用、氧化还原反应、防御反应以及泛素化修饰等多种生物学进程,其中经BiFC验证,确定TwNAC01与E2泛素结合酶(UbcE2)及丝氨酸羧肽酶类蛋白SCPL1存在互作.含有TwNAC01、UbcE2和SCPL1的酵母较对照能适应更高浓度的渗透势胁迫,同时TwNAC01、UbcE2和SCPL1在根系中优势表达,且表达量均随着干旱胁迫的增强而升高.TwNAC01蛋白在小黑麦响应干旱胁迫中发挥胁迫信号传导、蛋白代谢调控的作用,TwNAC01及其互作蛋白基因SCPL1、UbcE2均具有增强植物抗旱性的功能.本研究结果为揭示小黑麦抗旱的分子机制奠定了理论基础.
The NAC transcription factor plays a crucial regulatory role in plant drought stress responses.Our research group previously cloned the full-length cDNA of the triticale transcription factor TwNAC01.This study is aimed to identify interacting proteins with TwNAC01 and investigate its molecular mechanisms under drought stress.At cotyledon stage,triticale seedlings were subjected to various treatments including high temperature stress(40℃),low temperature stress(4℃),Osmotic stress(20%PEG6000),Salinity stress(200 mmol·L-1 NaCl),and hormonal foliar spray treatment[100 μmmol·L-1 methyl jasmonate(MeJA)and 100 μmol·L-1 abscisic acid(ABA)].Total RNA of these seedlings was extracted,reverse-transcribed into double-stranded cDNA,and a normalized cDNA library was constructed.Yeast two-hybrid(Y2H)screening was performed to identify interacting proteins with TwNAC01 as bait.The drought resistance conferred by selected stress-related proteins was substantiated through bimolecular fluorescence complementation(BiFC)and yeast stress phenotype assays.A normalized triticale cDNA library was constructed,yielding a titer of 1.436×108 CFU·mL-1 and 2.872×108 total recombinant clones.A total of 33 potential TwNAC01-interacting proteins were identified,which are involved in various biological processes including photosynthesis,redox reactions,defense responses,and ubiquitination modification.BiFC analysis corroborated the interaction between TwNAC01 and two key proteins:ubiquitin-conjugating enzyme E2(UbcE2)and serine carboxypeptidase 1-like protein(SCPL1).Yeast strains expressing TwNAC01,UbcE2,or SCPL1 exhibit enhanced tolerance to high osmotic stress compared to controls.Additionally,TwNAC01,UbcE2,and SCPL1 were predominantly expressed in the roots,with high expression levels under drought stress.Both TwNAC01 and its interacting partners,SCPL1 and UbcE2,contribute to enhanced plant drought resistance,thereby providing a theoretical foundation for elucidating the molecular mechanisms of drought resistance in triticale.
甘长波;卫小勇;宗金和;孔广超
石河子大学农学院/绿洲生态农业兵团重点实验室,新疆 石河子 832000石河子大学农学院/绿洲生态农业兵团重点实验室,新疆 石河子 832000石河子大学农学院/绿洲生态农业兵团重点实验室,新疆 石河子 832000石河子大学农学院/绿洲生态农业兵团重点实验室,新疆 石河子 832000
小黑麦干旱胁迫转录因子酵母双杂TwNAC01
triticaledrought stresstranscription factoryeast two-hybridTwNAC01
《核农学报》 2026 (5)
884-896,13
国家自然科学基金地区科学基金项目(32160478),兵团重点领域科技攻关项目(2024AB006),石河子大学自然科学研究与技术创新项目(校20200209)
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