天女木兰MsPHYA基因的表达及休眠解除功能研究  

The Study on Expression of MsPHYA Gene and Its Function of Dormancy Relieving in Magnolia sieboldii K.Koch

作　　者：张晓林[1,2,3] 张若晰[1] 曾莞棋 孙宏涛 郝昕 雷鸣雷 陆秀君 ZHANG Xiao-lin;ZHANG Ruo-xi;ZENG Wan-qi;SUN Hong-tao;HAO Xin;LEI Ming-lei;LUXiu-jun(College of Forestry,Ministry of Education,ShenyangAgricultural University Shenyang 110161,China;Key Laboratory of Facility Horticulture,Ministry of Education,ShenyangAgricultural University Shenyang 110161,China;Research Institute of Forestry,Chinese Academy of Forestry,Beijing 100091,China;Chaoyang Forest Pest Control And Quarantine Station,Chaoyang Liaoning 122000,China)

机构地区：[1]沈阳农业大学林学院,沈阳110161 [2]沈阳农业大学设施园艺教育部重点实验室,沈阳110161 [3]中国林业科学研究院林业研究所,北京100091 [4]朝阳市森林病虫害防治检疫站,辽宁朝阳122000

出　　处：《沈阳农业大学学报》2022年第6期650-657,共8页Journal of Shenyang Agricultural University

基　　金：国家自然科学基金青年基金项目(31600505);国家自然科学基金面上项目(31971647,32271858)。

摘　　要：光敏色素通过感知光信号来参与调控植物种子萌发,其中PHYA是吸收远红光信号的主要光受体。为了探究天女木兰(Magnolia sieboldii K.Koch)种子休眠解除的分子调控机制,对天女木兰光敏色素相关基因PHYA基因(MsPHYA)展开研究。从天女木兰种子中克隆MsPHYA基因(MZ221928)cDNA全长序列,采用qRT-PCR的方法测定MsPHYA基因和MsPIF3基因在不同光质处理下在天女木兰种子中的表达量,MsPHYA基因在天女木兰不同组织中表达量,测定不同光质诱导的种子萌发率,构建pGS-21T-MsPHYA原核表达载体,纯化并复性重组蛋白。研究结果表明:MsPHYA蛋白序列包含PAS结构域、GAF结构域、Phytochrome Region和His kinase结构域。MsPHYA基因在子叶和长枝中高表达,在远红光处理下MsPHYA和MsPIF3基因表达量先升高后下降。不同光质诱导种子萌发率从高到低顺序为红光>黑暗>远红光。天女木兰MsPHYA基因受远红光刺激而上调表达,但远红光处理并没有促进天女木兰种子萌发,推测MsPHYA基因并不直接调控天女木兰种子休眠,其休眠解除同时受MsPIF3调控。成功构建pGS-21T-MsPHYA载体并获得纯化蛋白,制备的MsPHYA抗体具有较好的特异性,可用于蛋白丰度分析。研究结果为揭示光解除种子休眠机制提供理论依据。Phytochrome regulates seed germination by sensing light signals,and PHYA is the main light receptor for absorption of farred light signals.In order to investigate the molecular mechanism of seed dormancy release in Magnolia Sieboldii K.Koch,we investigated the PHYA gene(MsPHYA)associated with photosensitivity.In this study,the full-length cDNA sequence of MsPHYA(MZ221928)was cloned from the seeds of M.Sieboldii K.Koch.The expression levels of MsPHYA and MsPIF3 in the seeds of M.Sieboldii K.Koch were determined by qRT-PCR under different light quality treatment.The expression levels of MsPHYA in different tissues of M.Sieboldii K.Koch were determined by qRT-PCR.The seed germination rate induced by different light quality was determined,the prokaryotic expression vector pGS-21T-MsPHYA was constructed,and the recombinant protein was purified and refold.The results showed that MsPHYA protein sequence contained PAS Domain,GAF domain,Phytochrome Region and His kinase domain.MsPHYA was highly expressed in cotyledons and long branches,and the expression levels of MsPHYA and MsPIF3 were firstly increased and then decreased under far-red light treatment.The order of germination rate induced by different light quality was red light>dark>far-red light.The expression of MsPHYA was up-regulated by the stimulation of far red light,but the far-red treatment did not promote seed germination.It suggested that MsPHYA did not directly regulate seed dormancy,and its dormancy release was also regulated by MsPIF3.pGS-21T-MsPHYA vector was successfully constructed and purified protein was obtained.The MsPHYA antibody had good specificity and could be used for protein abundance analysis.This study provides a theoretical basis for revealing the mechanism of seed dormancy release by light.

关 键 词：天女木兰(Magnolia sieboldii K.Koch) 光敏色素蛋白PHYA 基因克隆 原核表达 抗体制备 

分 类 号：S792.99[农业科学—林木遗传育种]