新麦草异戊烯基转移酶PjIPT基因的功能验证  

Functional Verification of Isopentenyl Transferases PjIPT Gene in Psathyrostachys juncea

作　　者：任晓敏 云岚 艾芊 赵乔 REN Xiao-min;YUN Lan;AI Qian;ZHAO Qiao(College of Grassland,Resources and Environment,Inner Mongolia Agricultural University,Hohhot 010018;Key Laboratory of Grassland Resources of Education Ministry,Hohhot 010011;Shenzhen Institute of Advanced Technology,Chinese Academy of Sciences,Shenzhen 518055)

机构地区：[1]内蒙古农业大学草原与资源环境学院,呼和浩特010018 [2]草地资源教育部重点实验室,呼和浩特010011 [3]中国科学院深圳先进技术研究院,深圳518055

出　　处：《生物技术通报》2024年第7期207-215,共9页Biotechnology Bulletin

基　　金：国家自然科学基金项目(32371762);内蒙古自治区自然科学基金重点项目(2023ZD07);内蒙古自治区揭榜挂帅项目(2022JBGS00400303)。

摘　　要：【目的】异戊烯基转移酶(isopentenyl transferases,IPT)参与反式玉米素(trans-zeatin,tZ)的生物合成。tZ是调节植物生长发育和抵抗逆境胁迫的一种细胞分裂素类型,在促进芽生长和调控植物侧枝发育方面具有重要作用。探索IPT的功能,为后续新麦草产量性状改良提供了理论依据。【方法】通过对新麦草IPT进行系统发育和多序列比对,探究其编码氨基酸序列的特征和同源性。通过花粉管通道法获得过表达PjIPT拟南芥(Arabidopsis thaliana)植株,并运用逆转录PCR(RT-PCR)和蛋白质印迹(Western blot,WB)对转基因植株进行验证,利用实时荧光定量逆转录PCR(RT-qPCR)对其组织表达模式进行分析,使用液质联用系统测定反式玉米素。【结果】IPT在新麦草(Psathyrostachys juncea)和二粒小麦(Triticum dicoccoides)等麦类作物中具有高度同源性,且AtIPT9是PjIPT的同源蛋白。通过花粉管通道法获得过表达PjIPT拟南芥(Arabidopsis thaliana)植株,证明PjIPT上调植株分枝数。RT-PCR和蛋白质印迹分析显示,PjIPT在转录水平和蛋白水平均能够正常表达。利用不同部位组织RT-qPCR分析PjIPT的空间特异性表达,结果显示,PjIPT在拟南芥的分蘖节和叶中特异性表达。此外,反式玉米素的定量分析表明PjIPT通过参与tZ的生物合成去调控拟南芥植株分枝数。【结论】PjIPT是上调植株分枝数的关键基因。【Objective】The isopentenyl transferase(IPT)enzyme plays a crucial role in the biosynthesis of trans-zeatin(tZ),a cytokinin that regulates plant growth,development,and resistance to stress.It significantly contributes to bud growth promotion and regulation of lateral branch development in plants.Exploring the function of IPT provides a theoretical basis for the subsequent improvement of new wheat grass yield traits.【Method】Through phylogeny and multiple sequence alignment,the features and homology of the coding amino acid sequence were explored.PjIPT-overexpressed Arabidopsis thaliana plants were obtained by pollen tube passage,and the transgenic plants were verified by reverse PCR(RT-PCR)and protein blot(Western blot,WB),their tissue expression patterns were analyzed by real-time quantitative reverse PCR(RT-qPCR),and trans-zeatin was determined by liquid mass combination system.【Result】The IPT proteins in Psathyrostachys juncea with Triticum dicoccoides and other wheat crops had high homology,and AtIPT9 is a homologous protein of PjIPT.PjIPT-overexpressed in A.thaliana plants was achieved through the pollen tube pathway method,demonstrating an upregulation in the number of branches.RT-PCR and Western blot analysis confirmed the normal expressions of PjIPT at both transcriptional and protein levels.Spatial-specific expression analysis using RT-qPCR indicated that PjIPT demonstrated specific expression patterns in the tillering nodes and leaves of Arabidopsis.Furthermore,quantitative analysis of tZ revealed that PjIPT regulated the number of branches in Arabidopsis by participating in the biosynthesis of tZ.【Conclusion】PjIPT is a key gene involved in upregulating plant branching.

关 键 词：新麦草 异戊烯基转移酶 反式玉米素 分蘖 功能验证 

分 类 号：S543.9[农业科学—作物学]