CN112048505B - 水稻OsLSD3基因的5’UTR内含子在转基因水稻中增强目的基因表达的用途 - Google Patents

水稻OsLSD3基因的5’UTR内含子在转基因水稻中增强目的基因表达的用途 Download PDF

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CN112048505B
CN112048505B CN202010810698.9A CN202010810698A CN112048505B CN 112048505 B CN112048505 B CN 112048505B CN 202010810698 A CN202010810698 A CN 202010810698A CN 112048505 B CN112048505 B CN 112048505B
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潘刚
吴鑫
贺焕焕
陈艳艳
程方民
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Abstract

本发明公开了水稻OsLSD3基因的5'UTR内含子在转基因水稻中增强目的基因表达的新用途。本发明首次发现核苷酸序列如SEQ ID NO.1所示的水稻OsLSD3基因的5’UTR内含子具有IME效应，可在转基因植物中显著增强外源目的基因GUS的表达，为开发目的基因高表达的转基因产品奠定了基础。

Description

水稻OsLSD3基因的5’UTR内含子在转基因水稻中增强目的基 因表达的用途
技术领域
本发明涉及植物内含子和水稻基因工程技术领域，主要涉及水稻OsLSD3基因的5’UTR内含子在转基因水稻中增强目的基因表达的用途。
背景技术
内含子(Intron)又称间隔顺序，指一个基因或mRNA分子中无编码作用的片段，是真核生物细胞DNA中的间插序列(intervening sequence)。这些序列可以转录，但在基因转录后，这些间插序列转录的部分经剪接加工而被去除，最终不存在于成熟RNA分子中。内含子不仅存在于前体mRNA的编码区(the coding sequences)，还存在于5′UTR(5′-untranslated region)以及3′UTR(3′-untranslated region)。内含子是真核生物区别原核生物的重要特征之一，其不仅介导了基因的选择性剪接，致使一个基因可以产生多种不同的蛋白质；还通过无义介导的mRNA降解(nonsense-mediated mRNA decay,NMD)监控真核细胞的重要RNA，识别并降解开放阅读框中含有提前终止密码子(premature terminationcodon,PTC)的mRNA，以避免因截短的蛋白产物积累对细胞造成毒害；此外，部分内含子还可以调控真核细胞中的基因表达，这种内含子增强基因表达的效应被称之为内含子增强效应，即IME效应(intron-mediated enhancement,IME)，该效应广泛存在于植物、动物、昆虫、线虫、真菌等真核生物中(Shaul O.How introns enhance geneexpression.International Journal of Biochemistry and Cell Biology,2017,91:145-155；Laxa M.Intron-mediated enhancement:A tool for heterologous geneexpression in plants.Frontiers in Plant Science,2017,7:1977)。
在植物中，1987年Callis等(Callis et al.,Introns increase gene incultured maize cells.Genes Dev.1987,1:1183-1200)首次报道了玉米Adhl基因的第一个内含子以及玉米Bz1基因的唯一内含子均能增强外源CAT基因的表达。此后，具有IME效应的内含子相继从单双子叶植物中分离克隆(Shaul O.How introns enhance geneexpression.International Journal of Biochemistry and Cell Biology,2017,91:145-155；Laxa M.Intron-mediated enhancement:A tool for heterologous geneexpression in plants.Frontiers in Plant Science,2017,7:1977)，如水稻(McElroyD.,et al.Isolation of an efficient actin promoter for use in ricetransformation.Plant Cell,1990,2:163-171)、油菜(Xiao G.,et al.Characterizationof the promoter and 5′-UTR intron of oleic acid desaturase(FAD2)gene inBrassica napus.Gene 2014,545:45-55)、胡麻(Kim M.J.,et al.Seed-specificexpression of sesame microsomal oleic acid desaturase is controlled bycombinatorial properties between negative cis-regulatory elements in theSeFAD2 promoter and enhancers in the 5′-UTR intron.Mol.Genet.Genomics 2006,276:351-368)、茜草(Tanaka A.,et al.Enhancement of foreign gene expression by adicot intron in rice but not in tobacco is correlated with an increased levelof mRNA and an efficient splicing of the intron.Nucleic Acids Res.1990,18:6767-6770)、拟南芥(Curie C.,et al.Cis and trans-acting elements involved inthe activation of Arabidopsis thaliana A1 gene encoding the translationelongation factor EF-1 alpha.Nucleic Acids Res.1991,19:1305-1310)、碧冬茄和玉米(Vain P.,et al.Intron-mediated enhancement of gene expression in maize(Zeamays L.)and bluegrass(Poa pratensis L.).Plant Cell Rep.1996,15:489–494)。其中玉米Ubi1内含子被广泛用于培育高表达转基因玉米和水稻(Pan YY,et al.Utilizingmodified ubi1 introns to enhance exogenous gene expression in maize(Zea maysL.)and rice(Oryza sativa L.).Journal of Integrative Agriculture,2016,15:1716-1726)。
进一步研究发现，内含子的IME效应具有明显的位置效应，插入位置越靠近翻译起始密码子，其增强效应更强(Rose AB.The effect of intron location on intron-mediated enhancement of gene expression in Arabidopsis.Plant J.2004,40:744-751；Parra G,et al.Comparative and functional analysis of intron-mediatedenhancement signals reveals conserved features among plants.Nucleic AcidsResearch,2011,39:5328–5337)，尤其是5′UTR内含子更能提高外源基因的表达(SamadderP,et al.Transcriptional and post-transcriptional enhancement of geneexpression by the 5′UTR intron of rice rubi3 gene in transgenic ricecells.Molecular Genetics and Genomics,2008,279:429–439；Akua T.,et al.TheArabidopsis thaliana MHX gene includes an intronic element that booststranslation when localized in a 5'UTR intron.J Exp Bot.2013,64:4255-4270)。而且，研究认为单子叶的内含子IME效应强于双子叶植物，其增强效应多为2-10倍，但部分内含子的增强效应可以增加100倍(Mass C,et al.The combination of a novelstimulatory element in the first exon of the maize Shrunken-1gene with thefollowing intron 1enhances reporter gene expression up to 1000-fold.PlantMolecular Biology,1991,16:199–207；Parra G,et al.Comparative and functionalanalysis of intron-mediated enhancement signals reveals conserved featuresamong plants.Nucleic Acids Research,2011,39:5328–5337)。因此，具有IME效应的内含子在转基因植物培育中具有很大的应用价值。
发明内容
本发明的目的在于提供水稻OsLSD3基因的5’UTR内含子在转基因水稻中增强目的基因表达的新用途，为开发目的基因高表达的转基因产品奠定基础。
具体技术方案如下：
本发明提供了水稻OsLSD3基因的5’UTR内含子在转基因水稻中增强目的基因表达的用途，所述5’UTR内含子的核苷酸序列如SEQ ID NO.1所示。
具体的，所述目的基因为GUS基因。
本发明首先根据OsLSD3基因的5’UTR内含子序列信息设计特异引物，从钱江6号B的叶片基因组DNA中扩增获得核苷酸序列如SEQ ID NO.1所示的5’UTR内含子；然后，构建表达载体并进行水稻遗传转化，获得转基因水稻；通过GUS活性的组织化学染色证实含有5’UTR内含子的转基因植株的GUS活性显著提高，说明核苷酸序列如SEQ ID NO.1所示的5’UTR内含子可极显著增强外源目的基因的表达。
本发明首次发现核苷酸序列如SEQ ID NO.1所示的水稻OsLSD3基因的5’UTR内含子具有IME效应，可在转基因植物中显著增强外源目的基因的表达，为开发目的基因高表达的转基因产品奠定了基础。
附图说明
图1为实施例2中质粒pCP1、pCP2和pCP3的结构示意图。
图2为实施例2中转基因水稻的PCR鉴定；
其中，M：DNA标准物；P：质粒；N1-N2：非转基因水稻；1-8：抗潮霉素转基因水稻。
图3为实施例2中的不同质粒的转基因水稻不同组织器官的GUS染色。
图4为实施例2中的不同质粒的转基因水稻幼苗的GUS活性测定。
具体实施方式
下面结合具体实施例对本发明作进一步描述，以下列举的仅是本发明的具体实施例，但本发明的保护范围不仅限于此。
实施例中所涉及的分子生物学和生物化学方法均为已知的技术，主要参考由Ausubel编写的Current Protocols in Molecular Biology,以及Green MR和Sambrook J编写的Molecular Cloning:ALaboratory Mannual,4th ED.。实施例中所用的实验材料如无特殊说明均为市售产品。
实施例1水稻OsLSD3基因5’UTR内含子的克隆
根据OsLSD3基因的登录号LOC_Os12g41700，在线查找(rapdb.dna.affrc.go.jp/viewer/gbrowse/build5/)该基因5’UTR内含子的基因组序列并设计特异引物LSD3(表1)，以钱江6号B的叶片基因组DNA为模板，利用高保真酶Phanta Max(Vazyme)PCR扩增并测序获得DNA片段，其核苷酸序列如SEQ ID NO.1所示，序列全长347bp。
实施例2水稻OsLSD3基因5’UTR内含子增强GUS基因表达的研究
步骤1：遗传转化载体构建
根据OsLSD3基因的翻译起始位点上游的基因组序列信息，设计3对特异引物，分别为PCP1、PCP2和PCP3(表1)，以钱江6号B的叶片基因组DNA为模板，利用高保真酶Phanta Max(Vazyme)PCR扩增并测序获得相应DNA片段。
PCR反应成分：
Figure BDA0002630881210000041
反应参数：98℃，3分；98℃，10秒，56℃，15秒，72℃，2分，40个循环；72℃延伸5分钟。
PCR产物经琼脂糖胶确定片段大小后，利用DNA纯化试剂盒纯化分别获得DNA片段CP1、CP2和CP3。用HindIII和NcoI双酶切农杆菌双元T-DNA载体pCAMBIA1305，纯化获得线性化的pCAMBIA1305，再利用Clontech的
Figure BDA0002630881210000043
HD Cloning Kit将DNA片段CP1、CP2和CP3克隆到线性化后的pCAMBIA1305载体中，而后酶切鉴定获得阳性克隆并测序验证(PE公司，377测序仪，杭州擎科梓熙生物技术有限公司)，测序正确的质粒分别命名为pCP1、pCP2和pCP3(图1)，其中，表达载体pCP2和pCP3不含有5’UTR内含子。
表1 5’UTR内含子克隆、载体构建和转基因植物鉴定的特异引物序列
Figure BDA0002630881210000042
步骤2：表达载体的水稻遗传转化
根据Pan等方法(Pan G.,et al.,Map-based cloning of a novel ricecytochrome P450 genes CYP81A6 that confers resistance to two differentclasses of herbicides.Plant Molecular Biology,2006,61:933-943)进行水稻遗传转化。
具体操作简述如下：首先用粳稻空育131的成熟胚诱导培养获得初生愈伤；其次利用电激法将pCP1、pCP2和pCP3导入农杆菌EHA105，获得含有相应质粒的EHA105于LB培养基中培养，获得对数生长期的农杆菌并用于水稻转化；而后将待转化的水稻愈伤组织在无菌滤纸上稍微吸后侵入OD600＝0.1的上述农杆菌菌液中(含200μM乙酰丁香酮)，室温下放置40分钟后弃菌液，于无菌滤纸上吸去多余菌液，将已侵染的愈伤组织转移到共培养基上培养50-55小时后于抑菌培养基上培养5-7天；而后再将表面没有农杆菌的愈伤组织转入筛选培养基上培养4-6周(每两周继代一次)；将抗性愈伤组织转移到预分化培养基上(先暗培养5-7天，而后16小时光照分化发芽)4-6周，待抗性幼苗长成后转移到生根培养基上生根，用潮霉素磷酸转移酶基因的特异引物HPH(表1)鉴定(图2)获得阳性转基因植株，最后将阳性植株种植于田间，直至收获种子。
步骤3：转基因水稻的GUS组织化学染色及其活性分析
GUS活性的组织化学染色方法参考Jefferson方法(Jefferson RA.Assayingchime rice genes in plants:The GUS genes fusion system plant.Mol BiolRep.1987,5:387-405)。
具体操作简述如下：将待检测的水稻愈伤、根、叶和叶鞘等组织器官置于GUS染液中(50mM PBS buffer(pH＝7.0),0.1M K3[Fe(CN)6]，0.1M K4[Fe(CN)6],0.5M EDTA,1mMX-Gluc)，室温下抽真空30分钟后，37℃过夜,叶片和叶鞘经70％乙醇脱色处理后拍照。
结果显示，缺失5’UTR内含子的载体pCP2和pCP3的转基因后代中仅在愈伤、叶片和叶鞘等组织器官中检测到较浅的GUS染色，而转含有5’UTR内含子的载体pCP1的转基因后代的不同组织器官中均能检测到很深的GUS染色(图3)。
进一步分析转基因幼苗的GUS活性，结果证实转pCP1的转基因一叶一心幼苗的GUS活性分别比转pCP2和pCP3的转基因后代高近30和80倍(图4)。这些结果均说明5’UTR内含子具有IME效应并能显著增强外源目的基因的表达。
序列表
<110> 浙江大学
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catgtgtatc actggcaaac 20

Claims (2)

1.水稻OsLSD3基因的5’UTR内含子在转基因水稻中增强目的基因表达的用途，其特征在于，所述5’UTR内含子的核苷酸序列如SEQ ID NO.1所示。
2.如权利要求1所述的水稻OsLSD3基因的5’UTR内含子在转基因水稻中增强目的基因表达的用途，其特征在于，所述目的基因为GUS基因。
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