水稻OsNCED4基因的克隆及功能初探  

Cloning and Function Analysis of OsNCED4 Gene in Rice

作　　者：周丽 李梦婷 朱骞[1,2] Sadia Nadir 李伟[1] 郭效琼 冯天 陈丽娟[1,2] 李东宣[1,2] Zhou Li;Li Mengting;Zhu Qian;Sadia Nadir;Li Wei;Guo Xiaoqiong;Feng Tian;Chen Lijuan;Lee Dongsun(Rice Research Institute,Yunnan Agricultural University,Kunming,650201;State Key Laboratory for Conservation and Utilization of Bio-Re sources in Yunnan,Yunnan Agricultural University,Kunming,650201;Department of Chemistry,University of Science and Technology Bannu,Bannu,28100,Pakistan;Centre for Mountain Ecosystem Studies,Kunming Institute of Botany,Chinese Academy of Sciences,Kunming,650201)

机构地区：[1]云南农业大学,稻作研究所,昆明650201 [2]云南农业大学,省部共建云南生物资源保护与利用国家重点实验室,昆明650201 [3]本努科技大学化学系,巴基斯坦本努28100 [4]中国科学院昆明植物研究所山地生态系统研究中心,昆明650201

出　　处：《分子植物育种》2020年第7期2087-2096,共10页Molecular Plant Breeding

基　　金：国家重点研发计划项目(2017YFD0100205);国家自然科学基金NSFC-云南合基金(U1136604);国家自然科学基金项目(31560115,31860108);云南省重点基金项目(2015FA023);云南省重大专项(2018ZG005)共同资助。

摘　　要：脱落酸(abscisic acid,ABA)是一种抑制植物生长的植物激素,因能促使叶子脱落而得名,在植物逆胁迫中发挥关键作用。由NCED基因编码的9-顺-环氧类胡萝卜素双加氧酶(NCED),是ABA在高等植物中的关键酶。本研究已成功从水稻Ilmibyeo(O.sativa L.ssp.japonica)基因组中克隆得到Os NCED4,生物信息学分析表明,Os NCED4基因位于水稻第7号染色体上,ORF全长1749 bp,无内含子结构,编码582个氨基酸,具有NCED家族的RPE65保守结构;以该基因和其启动子序列构建组织表达载体pOsNCED4::GUS、pCaMV35S::OsNCED4:EGFP和RNAi干扰载体p Ca MV35S::OsNCED4:RNAi,用于基因表达和功能分析,并利用农杆菌介导的方法,成功获得了相应遗传载体的转基因植株;GUS染色结果表明,该基因在叶片、叶耳、小穗等组织部位均有表达;RT-PCR结果显示,Os NCED4在水稻不同生育期的根、茎、叶、叶鞘和穗子中均有不同程度的表达,但表达量具有明显的差异;亚细胞定位结果显示,OsNCED4基因编码的蛋白定位于细胞核;与野生型植株相比,RNAi植株的花粉育性无明显差异,而结实率显著下降。该研究结果为进一步研究水稻OsNCED4基因表达的调控,以及为水稻生长发育上的功能研究提供了科学依据。Abscisic acid(ABA)is a plant hormone that inhibits plant growth and is named for its ability to cause leaves to fall off.And plays an important role in plant’s responses to various environmental stresses.9-cis-epoxycarotenoid dioxygenase(NCED)encoded by the NCED gene,it is the key regulatory enzyme involved in the biosynthetic pathway of ABA in higher plants.In this study,a gene OsNCED4 with unknown function was cloned from the genome of Ilmibyeo(O.sativa L.ssp.japonica).The bioinformatics analysis showed that the rice Os NCED4 gene was located on rice chromosome 7.The full length of the gene ORF is 1749 bp,with no introns.The gene and encodes for a 582 amino acid containing protein with a conserved domain of RPE65 in NCED family.The gene and its promoter part were fused to binary vectors and expression vectors pOsNCED4::GUS,pCaMV35 S::OsNCED4:EGFP and pCaMV35 S::OsNCED4:RNAi.These vectors were used to generate transgenic plants.GUS staining results showed that OsNCED4 was differently expressed in leaf blade,leaf sheath,and panicle in different tissues.RT-PCR results showed that OsNCED4 was differently expressed in root,stem,leaf and leaf sheath in different growth stages.Subcellular localization indicated that the protein encoded by OsNCED4 gene is located in the nucleus.When compared with the wild type,there was no significant difference in pollen fertility,but the seed setting rate of the RNAi transgenic lines was significantly decreased.This study laid a foundation for further studying the role of OsNCED4 gene in the regulation of gene expression and rice plant growth and development.

关 键 词：水稻(Oryza SATIVA L.) OsNCED4 基因克隆 生物信息学分析 基因表达 

分 类 号：S511[农业科学—作物学]