CN110184278A - 一种柽柳盐胁迫响应基因TcNAC2及其miRNA抗性靶标rTcNAC2和应用 - Google Patents
一种柽柳盐胁迫响应基因TcNAC2及其miRNA抗性靶标rTcNAC2和应用 Download PDFInfo
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Abstract
本发明公开了一种柽柳盐胁迫响应基因TcNAC2及其miRNA抗性靶标rTcNAC2和应用。所述的柽柳盐胁迫响应基因TcNAC2的核苷酸序列如SEQ ID No.1所示,其表达蛋白的氨基酸序列如SEQ ID No.2所示。本发明通过大引物突变技术将TcNAC2基因的miR164响应元件同义突变,获得了miR164抗性靶标rTcNAC2,其核苷酸序列如SEQ ID No.3所示。在盐胁迫下,TcNAC2受miR164的转录后调控,而rTcNAC2则不受其调控。柽柳盐胁迫响应基因TcNAC2及其miR164抗性靶标rTcNAC2在植物耐盐性或林木抗性育种领域的研究和应用中有重要价值。
Description
技术领域
本发明属于植物基因工程技术领域,具体涉及一种柽柳盐胁迫响应基因TcNAC2及其miRNA抗性靶标rTcNAC2的应用。
背景技术
我国存在大面积无法有效利用的盐碱地,通过抗逆育种方法培育耐盐种质,可以增加可耕种地面积、提高粮食产量作。尽管在在拟南芥、水稻等模式物种中已经阐明了部分植物耐盐机理,挖掘出相关耐盐基因,但在抗性育种应用中效果不显著,转基因植物耐盐性提高的效果并不理想,不能满足在实际生产中的需求(Zhu,2002)。相比草本植物和禾本科作物,耐盐树种可以在盐渍化土壤、滨海滩涂上正常生长,具有独特的高耐盐特性,其耐盐机制是抗逆育种的理论基础,相关的耐盐基因资源对高耐盐性的作物品种培育的指导意义巨大。柽柳属(Tamarix)植物约90种,主要分布区集中在亚洲和北非,我国西北及华北地区共有18个种,台湾地区有1变种无叶柽柳。中国柽柳(Tamarix chinensis Lour.)是我国柽柳属植物中分布区最广的,以灌木形式丛生于内陆荒漠区和滨海滩涂,辽、冀、豫、鲁、苏北、皖北、新疆等地都有野生群体分布(张鹏云,1990)。中国种子、叶子的解剖形态和茎叶表面的盐腺结构是适应于盐渍、干旱环境的典型特征,能够在2%的盐碱地上正常生长,是耐盐性最强的树种之一。中国柽柳独特的耐高盐特性,目前仅在生理层面解析了相关机制,如泌盐、离子区域化等机制,但是其分子层面了解较少,目前在柽柳属的其他物种中已经克隆得到了几十个抗逆相关的转录因子,但中国柽柳尚未有耐盐基因的分离鉴定报道,急需开展相关研究。挖掘中国盐胁迫响应相关的转录因子基因并研究其表达调控模式,不仅可以加深理解柽柳耐盐分子机理,同时可以为盐渍地脱盐、抗逆植物育种提供分子工具,
NAC转录因子家族广泛分布于高等植物中,成员数量巨大,拟南芥中有138个成员。NAC转录因子的N端较为保守,含有保守的NAC结构域,C端高度可变,发挥转录激活作用和寡聚化作用,决定了NAC以同源或异源二聚体形式发挥功能。NAC家族功能广泛,参与植物的非生物胁迫响应、器官形成、次生组织发育、果实成熟、叶片衰老等生物过程,由于数量巨大,很多成员功能尚不明确(Olsen et al.,2005)。非生物胁迫相关的NAC中,很多NAC报道为耐盐性正调控因子,柽柳属植物中,两个刚毛柽柳(Tamarix hispida)NAC基因进行了功能研究,其中刚毛柽柳ThNAC23异源表达提高了拟南芥盐胁迫和渗透胁迫耐受能力(Wang,Li,Luand Wang,2017),刚毛柽柳ThNAC7的过表达类似ThNAC23提高了植株的耐盐性(张明意,2015)。部分研究报道了NAC为耐盐性负调控因子,如水稻中一个miR164的靶基因NAC负调控水稻抗逆性,胡杨中三个NAC胁迫下表达模式有不一致甚至相反的情况,其中一个NAC在根中负调控耐盐性。近年来研究表明,含有miR164响应元件的NAC基因,愈发显示出和非生物胁迫密切相关的关系,如拟南芥AtNAC4(ANAC080)最初报道为影响侧根发生和叶片衰老进程,近年研究发现和N、P等营养元素胁迫密切相关且含有保守的miR164响应元件(Lee etal.,2016)。
上述结果表明,NAC在植物盐胁迫响应方面有重要作用,并且受到miR164的转录后调控的影响。目前,尚未有中国柽柳的NAC转录因子相关报道,克隆和开发利用中国柽柳NAC转录因子,不仅有助于阐明其高度耐盐的分子机制,而且可以为筛选重要耐盐基因和抗逆遗传育种提供理论基础和分子工具,对盐碱地利用和农业综合生产能力提高具有重要价值。
发明内容
发明目的:针对现有技术中存在的不足,本发明的目的是提供一种柽柳盐胁迫响应基因TcNAC2,满足植物耐盐性或林木抗性育种的应用需求。本发明的另一目的是提供一种上述柽柳盐胁迫响应基因TcNAC2改造后的miRNA抗性靶标rTcNAC2。本发明还有一目的是提供一种上述柽柳盐胁迫响应基因TcNAC2或miRNA抗性靶标rTcNAC2的应用。
技术方案:为了解决上述问题,本发明所采用的技术方案如下:
一种柽柳盐胁迫响应基因TcNAC2,其核苷酸序列如SEQ ID No.1所示。
所述柽柳盐胁迫响应基因TcNAC2的表达蛋白,其氨基酸序列如SEQ ID No.2所示。
一种miR164抗性靶标rTcNAC2,由所述柽柳盐胁迫响应基因TcNAC2通过miR164响应元件同义突变获得,其核苷酸序列如SEQ ID No.3所示。
含有所述柽柳盐胁迫响应基因TcNAC2的载体。
含有所述miR164抗性靶标rTcNAC2的载体。
优选地,所述柽柳盐胁迫响应基因TcNAC2载体为双荧光素酶报告载体35sGLO:TcNAC2,在TcNAC2的miR164响应元件的5′端上游组装有组成型强表达启动子P35S和萤火虫荧光素酶报告基因,3′端下游组装有海肾荧光素酶报告基因。
优选地,所述miR164抗性靶标rTcNAC2的载体为双荧光素酶报告载体35sGLO:rTcNAC2,在rTcNAC2的miR164响应元件同义突变的5′端上游组装有组成型强表达启动子P35S和萤火虫荧光素酶报告基因,3′端下游组装有海肾荧光素酶报告基因。
优选地,所述miR164抗性靶标rTcNAC2的载体为rTcNAC2的过表达载体PBI121GW:rTcNAC2,组装35S启动子;组装NPTII基因表达盒,组装LB和RB序列,LB和RB序列之间为rTcNAC2基因表达框架和基因NPTII。
所述的柽柳盐胁迫响应基因TcNAC2或所述柽柳盐胁迫响应基因TcNAC2的表达蛋白或所述柽柳盐胁迫响应基因TcNAC2的载体在植物耐盐性或林木抗性育种中的应用。
所述的miR164抗性靶标rTcNAC2或所述的miR164抗性靶标rTcNAC2的载体在植物耐盐性或林木抗性育种中的应用。
有益效果:与现有技术相比,本发明以中国柽柳(Tamarix chinenses)为材料,通过RACE技术克隆了TcNAC2基因;通过双荧光素酶报告***鉴定了TcNAC2的miR164响应元件;通过实时荧光定量PCR技术检测了TcNAC2基因在盐胁迫下的时空表达模式;通过大引物突变技术将miR164响应元件同义突变,获得了miR164抗性靶标rTcNAC2。在盐胁迫下,TcNAC2的miR164响应元件受到miR164调控,TcNAC2特异性地在柽柳根中迅速下调,突变该元件后的rTcNAC2不受miR164的转录后调控,柽柳盐胁迫响应基因TcNAC2及其miR164抗性靶标rTcNAC2在植物耐盐性或林木抗性育种领域中有重要应用价值。
附图说明
图1是TcNAC2基因盐胁迫下的表达模式图;
图2是双荧光素酶报告基因载体35sGLO结构图;
图3是TcNAC2基因的miR164响应元件荧光检测图;
图4是过表达载体PBI121GW结构图。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。以下实施例中未作具体说明的分子生物学实验方法,均可参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的方法或本领域的常规方法进行,或者按照试剂盒和产品说明书进行。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1:通过RACE技术克隆TcNAC2基因
基于柽柳RNA-seq数据的TcNAC2转录本,设计5′和3′的RACE引物,通过巢式PCR扩增两段特异性产物,通过T-载体克隆测序,测序结果通过重叠区拼接,得到cNDA全长。具体如下:
I.引物设计
3′RACE正向引物:
Outer Primer:5′-GAGGTGATGAAGAACCAATAGAG-3′;
Inner Primer:5′-TAGACATGGTTGGTACAGACTC-3′;
3′RACE反向引物:
Outer Primer:
5′-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3′;
Inner Primer:5′-CTAATACGACTCACTATAGGGC-3′;
5′RACE正向引物:
Outer Primer:
5′-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3′;
Inner Primer:5′-CTAATACGACTCACTATAGGGC-3′;
5′RACE反向引物:
Outer Primer:5′-TATGGCCTTTTGGAGCCCGCCCCTT-3′;
Inner Primer:5′-TTACGCTCACCACTTGCTC-3′。
II.3′RACE反应过程:
(1)反转录,将以下成分加入到一个置于冰上的RNase-free的小离心管中:1μgTotal RNA(植物材料为柽柳叶片),4μL dNTP Mix,2μL 3′RACE Adapter,2μL 10X RTBuffer,1μL RNase Inhibitor,1μL M-MLV Reverse Transcriptase,Nuclease-freeWater补至20μL。
(2)轻轻混匀,短暂离心,42℃温育1小时,进入PCR步骤;
(3)3′RACE巢式PCR;
3′RACE Outer PCR反应体系(50μL)组成:5.0μL 10×LAPCR Buffer(Mg2+ Free),5.0μL MgCl2(25mM),8.0μL dNTP Mixture(each 2.5mM),2.0μL 3′RACE Outer PrimerF(10μM),2.0μL 3′Outer PrimerR(10μM),1μL RT reaction product,0.5μL TakaRa LATaq(5U/μL),26.5μL Nuclease-free Water。
3′RACE Inner PCR反应体系(50μL)组成:5.0μL 10×LA PCR Buffer(Mg2+ Free),5.0μL MgCl2(25mM),8.0μL dNTP Mixture(each 2.5mM),2.0μL 3′RACE Inner PrimerF(10μM),2.0μL 3′RACE Inner PrimerR(10μM),1μL Outer 3′RACE PCR product,0.5μLTakaRa LA Taq(5U/μL),26.5μL Nuclease-free Water。
反应程序:94℃ 3分钟;94℃ 30秒,60℃ 30秒,72℃ 1分钟,35 cycles;72℃ 7分钟。
(4)纯化片段的连接反应
采用TaKaRa公司的pMD19-T simple Vetor克隆目的DNA分子,反应体系(5μL):2.2μL纯化回收的PCR产物,0.3μL pMD-19 Simple Vector,2.5μL Solution I。反应条件:16℃30分钟;4℃过夜。
(5)大肠杆菌转化:将新鲜制备或-70℃冻存的大肠杆菌TOP10感受态细胞在冰上融化;取5μL纯化片段与克隆载体的连接产物,加入到100μL感受态细胞中,并轻轻混匀,冰浴30分钟;42℃水浴中热击90秒,迅速置于冰上3-5分钟;加入800μLLB液体培养基,37℃100转/分钟摇菌1小时;4000转/分钟离心3分钟,吸掉上层800μL培养基,混匀剩余菌液;将菌液涂抹于含有Amp的LB筛选培养板上,37℃倒置培养过夜。
(6)阳性克隆筛选及测序分析
从筛选培养板上挑选单菌落接种于LB液体培养基中,37℃ 250转/分钟摇菌过夜;直接以培养过夜的菌液为模板进行重组转化子的PCR检测。
反应体系(20μL):2.0μL 10×PCR Buffer(Mg2+ free),1.5μL MgCl2(25mM),1.3μLdNTP Mixture(each 2.5mM),1.0μL 3′RACE gene specific inner primer(10μM),1.0μL3′RACE Inner Primer(10μM),0.1μL菌液,1.0μL rTaq,12.1μL Milli-Q Water。反应程序:94℃ 3分钟;94℃ 30秒,60℃ 30秒,72℃ 1分钟,28 cycles;72℃ 7分钟。
阳性克隆通过Sanger测序法测序得到碱基序列。
III.5′RACE反应过程
(1)RNA处理:CIP反应,将如下成分加入到RNase-free的小离心管中:10μg TotalRNA,2μL 10X CIP buffer,2μL Calf Intestine Alkaline Phosphatase(CIP),Nuclease-free Water至20μL。
(2)轻轻混匀,短暂离心;37℃温育1小时;
(3)加入以下试剂到CIP反应离心管:15μL Ammonium Acetate Solution,115μLNuclease-free Water,150μL acid phenol:chloroform。
(4)充分涡旋,室温高速离心(≥10000g)5分钟;
(5)转移上层水相到一个新的离心管中,加入150μL氯仿,充分涡旋,室温高速离心(≥10000g)5分钟;
(6)转移上层水相到一个新的离心管中,加入150μL异丙醇,充分涡旋,冰浴10分钟;
(7)最大转速离心20分钟,用0.5ml预冷的70%乙醇冲洗沉淀,最大转速离心5分钟,小心弃乙醇,气干沉淀;
(8)以11μL Nuclease-free Water重悬沉淀,即得CIP’RNA,冰上放置进一步用于TAP反应,或者-20℃保存;
(9)TAP反应,把以下成分加入到一个RNase-free的小离心管中:5μLCIP’d RNA,1μL 10X TAP buffer,2μL Tobacco Acid Pyrophosphatase(TAP),2μL Nuclease-freeWater;
(10)轻轻混匀,短暂离心,37℃温育1小时,即得CIP/TAP-treated RNA;进入接头连接步骤,或-20℃保存反应物;
(11)5′RACE接头连接,把以下成分加入到一个RNase-free的小离心管中:2μLCIP/TAP-treated RNA,1μL 5′RACE Adapter,1μL 10×RNA Ligase Buffer,2μL T4 RNALigase(2.5U/μL),4μL Nuclease-free Water。
(12)轻轻混匀,短暂离心,37℃温育1小时,即得Ligated RNA;进入反转录步骤,或-20℃保存反应物。
(13)将以下成分加入到一个置于冰上的RNase-free的小离心管中:2μL LigatedRNA,4μL dNTP Mix,2μL Random Decamers,2μL 10X RT Buffer,1μL RNase Inhibitor,1μL M-MLV Reverse Transcriptase,Nuclease-free Water补至20μL。
(14)轻轻混匀,短暂离心;42℃温育1小时,即得RT reaction;进入PCR步骤,或-20℃保存反应物。
(15)5′RACE巢式PCR:反应体系、反应条件与3′RACE的巢式PCR一致。
(16)PCR产物克隆测序,操作与3′RACE克隆一致。
IV.ORF扩增
对3′RACE和5′RACE序列进行拼接,并利用NCBI-ORF finder工具预测其阅读框。根据基因全长序列设计引物(扩增子包含起始密码子及终止密码子),进行TcNAC2基因的全长克隆。其中,TcNAC2 ORF正向引物:5′-ATGACCATGATTACGCCAAG-3′,TcNAC2 ORF反向引物:5′-TCAGTTACAATAACTCCAAAAC-3′,高保真PCR反应体系如下:10×LA PCR Buffer 5.0μL;2.5mM dNTP Mixture 8.0μL;25mM Mg2+ 5.0μL;LA Taq DNA Polymerase(5U/μL)0.5μL;正向引物(10μM)2μL;反向引物(10μM)2μL;模板(柽柳cDNA)1μL;加无菌ddH2O补足50μL。反应程序:预变性94℃ 3分钟-(94℃ 40秒-55℃ 30秒-72℃ 30秒)×35个循环-72℃ 10分钟。
TcNAC2全长cDNA序列为1579bp,其序列如SEQ ID No.1所示,包含一个1362bp的完整阅读框(CDS),对应的TcNAC2蛋白的氨基酸序列为453aa,其序列如SEQ ID No.2所示。
实施例2:通过荧光定量PCR分析TcNAC2基因表达模式
通过荧光定量PCR技术验证TcNAC2基因的盐胁迫响应,基于TcNAC2基因的CDS区域设计荧光定量PCR引物,基于柽柳的TIF基因设计内参引物,序列如下:
TcNAC2正向引物:5′-GATCCAAGCTCGCGGAAGCC-3′;
TcNAC2反向引物:5′-CACGAATGTCGGTGGCCGAT-3′;
TIF正向引物:5′-ACCACAGGAGTGTCCACCACA-3′;
TIF反向引物:5′-TGATGCTTTGCGTGCCAGTG-3′。
利用饱和染料evagreen(Biotium公司)和荧光定量PCR仪Viia7(ABI公司),实时检测PCR过程的荧光强度,具体PCR体系参照evagreen说明书,通过比较TcNAC2基因和内参的达到荧光阈值的循环数来计算确定TcNAC2基因的相对表达量。其中,PCR模板为不同组织的mRNA反转录得到的cDNA,取样来自柽柳扦插苗,盐处理0.5小时、1小时的根、茎、叶,以无胁迫处理的根为对照,计算相对定量,结果如图1所示,TcNAC2基因在柽柳根中显著下调(盐胁迫0.5小时下调约0.5,1小时后表达量大大下调,其丰度接近于零难以检测),在叶和茎中表达量较低且为组成型表达(表达量在0.05-0.2之间微弱变化),说明TcNAC2特异性地在柽柳根中迅速响应盐胁迫。另测得柽柳miR164被盐胁迫显著诱导,TcNAC2和miR164表现为盐胁迫下表达负相关关系,表明TcNAC2基因可能受到柽柳miR164的调控。
实施例3
采用大引物PCR法进行多点突变,获得TcNAC2的miR164响应元件同义突变后的ORF,具体步骤如下:
根据密码子简并性,设计同义突变引物,引入尽可能多的突变位点,但不要有超过3碱基的poly结构,同时侧翼保留至少10nt的互补碱基,TcNAC2突变引物为5′-ACAGAATCATCTCATGTAAGCTGTTTCAGTAATCCCATGATC-3′,在一轮PCR反应中,通过TcNAC2突变引物和实施例1的TcNAC2 ORF反向引物,扩增得到的一轮PCR产物片段。在二轮PCR反应中,利用一轮反应产物作为大引物,实施例1的TcNAC2 ORF正向引物,进行PCR获得引入多位点突变的抗性靶标rTcNAC2片段,第一轮和第二轮PCR均采用高保真PCR反应体系如下:10×LA PCRBuffer5.0μL;2.5mM dNTP Mixture 8.0μL;25mM Mg2+ 5.0μL;LA Taq DNA Polymerase(5U/μL)0.5μL;正向引物(10μM)2μL;反向引物(10μM)2μL;模板(柽柳cDNA)1μL;加无菌ddH2O补足50μL。反应程序:预变性94℃ 3分钟-(94℃ 40秒-55℃ 30秒-72℃ 30秒)×35个循环-72℃ 10分钟。将第二轮PCR产物连接T载体,转化大肠杆菌后进行克隆测序,回收包含阳性片段的T载体。
测序得到TcNAC2的miR164抗性靶标rTcNAC2,其核苷酸序列如SEQ ID No.3所示。
实施例4:TcNAC2基因的miRNA响应元件鉴定
实施例2的结果表明TcNAC2基因可能受到柽柳miR164的调控,结合序列互补性,预测到TcNAC2基因含有一个miR164响应元件(5′-TCACGTGTCCTGCTTCTCCA-3′),实施例3采用大引物PCR法进行多点突变,获得TcNAC2的miR164响应元件同义突变后的ORF,即rTcNAC2。本实施例通过构建双荧光素酶报告基因载体(图2),测定荧光强度变化,通过定量miRNA164和响应元件间的互作强度,验证TcNAC2响应元件。
具体如下:
I.35sGLO:TcNAC2重组载体构建
1)寡核苷酸设计
TcNAC2响应元件侧翼添加酶切位点,按照以下要求设计两对寡核苷酸:
TcNAC2正义链:酶切位点A+Notl酶切位点+TcNAC2响应元件+酶切位点B残基;TcNAC2反义链:酶切位点B+反义TcNAC2响应元件+Notl酶切位点+酶切位点A残基;
rTcNAC2正义链:酶切位点A+Notl酶切位点+rTcNAC2突变序列+酶切位点B残基;rTcNAC2反义链:酶切位点B+反义rTcNAC2突变序列+Notl酶切位点+酶切位点A残基。
2)双酶切组合选择
35sGLO的多克隆位点存在多种酶切组合选择,原则上避开同尾酶和同裂酶,如SalI和XhoI,XbaI和NheI,且两个酶切位点距离不要太近,侧翼至少6nt的保护碱基。NheI(酶切位点A)和SalI(酶切位点B)符合以上规则,可以进行高效双酶切。利用NheI(酶切位点A)和SalI(酶切位点B)为酶切位点,设计寡核苷酸如下,利用PAGE合成寡核苷酸
TcNAC2+:5′-CTAGATAGCGGCCGCTATTCACGTGTCCTGCTTCTCCAG-3′;
TcNAC2-:5′-TCGACTGGAGAAGCAGGACACGTGAATAGCGGCCGCTAT-3′;
rTcNAC2+:5′-CTAGATAGCGGCCGCTATCATGTAAGCTGTTTCAGTAG-3′;
rTcNAC2-:5′-TCGACTACTGAAACAGCTTACATGATAGCGGCCGCTAT-3′。
3)35sGLO载体线性化
通过NEBcloner(https://nebcloner.neb.com)获取理论最佳酶切体系(50μL):1μL 35sGLO(1μg/μL),5μL 10X CutSmart Buffer,0.5μL NheI(20U/μL),0.5μL SalI(20U/μL),43μL Nuclease-free Water。
配制反应体系(50μL),吹打混匀,37℃孵育5-15min,电泳检测酶切产物,回收线性化质粒。
4)获***片段
在2个0.2ml离心管中,按照以下体系(50μL),分别进行两对寡核苷酸杂交:
2μL TcNAC2+/rTcNAC2+(1μg/μL),2μL TcNAC2-/rTcNAC2-(1μg/μL),46μL OligoAnnealing Bufier。
90℃反应3min,37℃退火15min后得到的TcNAC2和rTcNAC2杂交产物,即为***片段。
5)载体重组
配制T4(NEB)连接体系(10μL):1μL TcNAC2或rTcNAC2杂交产物(4ng/μL),0.5μLT4连接酶(400U/μL),1μL T4 10X bufier,XμL线性化35sGLO(50ng),7.5-X μL ddH2O。
25℃孵育10min(或16℃过夜连接)
转化后提取质粒,NotI酶切得到140bp左右的片段为阳性质粒。
6)阳性质粒测序验证正确后,得到35sGLO:TcNAC2和35sGLO:rTcNAC2重组载体。
II.miR164瞬时表达载体构建
通过gateway体系,将柽柳miR164(5′-UGACAGAAGAGAGUGAGCAC-3′)***带有35S启动子的P2GW7载体,构建miRNA瞬时表达载体。
首先利用BP反应,将miR164片段***PCR8/GW/TOPO载体,测序验证***片段,得到miR164入门载体。然后利用LR反应,通过LR ClonaseTM II Enzyme Mix试剂盒,进行miR164入门载体和适用于植物原生质体的P2GW7超表达载体的重组反应,测序验证***片段,抽提阳性质粒,得到P2GW7:miR164a重组载体。
III.转染原生质体并测定荧光强度
将对照组:9μL的35sGLO:TcNAC2载体;
TcNAC2组:6μL的mi164:P2FGW7载体(1μg/μL)和3μL的35sGLO:TcNAC2载体(1μg/μL);
rTcNAC2组:6μL的mi164:P2FGW7载体(1μg/μL)和3μL的35sGLO:rTcNAC2载体(1μg/μL);
分别共转染到100μL杨树原生质体(6×105/ml)中,重复4次,室温黑暗培养16~24h充分进行转染后,进行荧光检测。
1)配置荧光素酶反应底物(Promega E1910试剂盒),冰上待用;
LAR配制:10mL Luciferase Assay Buffer II溶解Luciferase AssaySubstrate,混匀,现用或-70冻存;
Stop&Glo配制:Stop&Glo Buffer和Substrate按照50∶1配制,混匀,现配现用,或者-20冻存;
2)4000rpm离心收集转染后的原生质体,加入100μL 1X Passive Lysis Buffer100ml裂解沉淀;
3)裂解后的转染液转移至96孔酶标板孔(100μL),对照组,TcNAC2组和rTcNAC2组各重复4孔;
4)接通Glomax-96化学发光仪电源,打开GloMax软件,按照软件说明书,建立GloMax自动化测定程序,测定荧光强度;
将对照组的荧光强度设为100%,计算得到TcNAC2组和rTcNAC2组相对荧光强度,绘制为柱状图,如图3,可以看出TcNAC2组荧光显著受到抑制,说明TcNAC2被柽柳miR164抑制,而rTcNAC2组荧光恢复为34%,说明突变后的rTcNAC2受到miR164抑制一定程度被解除,证明了TcNAC2的miR164响应元件受到miR164抑制。
实施例5:rTcNAC2基因植物表达载体构建
利用通路克隆技术构建rTcNAC2基因的过量表达载体。使用特异PCR引物(实施例1的TcNAC2 ORF引物),以实施例3中包含rTcNAC2片段的T载体为模板,进行PCR扩增,将rTcNAC2基因ORF构建到入门载体。入门载体为pCR8/GW/TOPOTM vector(Invitrogen)。反应体系为:Fresh PCR product(purified)10-20ng;Salt solution 1μL;pCR8/GW/TOPOTMvector 1μL;加无菌ddH2O补足6μL。反应程序为:室温静置30分钟。
从筛选培养板上挑取阳性克隆进行测序验证,阳性入门载体与植物表达载体PBI121GW进行LR反应。载体质粒如图4所示。反应体系为:入门载体100ng;PBI121GW vector(100ng/μL)1.5μL;LR Clonase II enzyme mix 2μL;加TE(pH 8.0)补足10μL。反应条件:25℃ 1小时。经LR反应后,rTcNAC2基因导入植物表达载体PBI121GW中。PBI121GW组装有通路克隆attR1和attR2元件,快速组装rTcNAC2基因表达框并确保准确翻译;组装有LB和RB序列,促使组装于其间的rTcNAC2基因和筛选标记基因NPTII整合至侵染的拟南芥染色体中。另外,在rTcNAC2基因的5′端组装组成型强表达启动子P35S,它能使TcNAC2高效表达。通过PCR检测及测序验证,确认过量表达载体构建成功,命名为PBI121GW:rTcNAC2,该基因位于启动子P35S之后,在启动子P35S的驱动下,rTcNAC2可在植物体内高效表达。
由于rTcNAC2不含有miR164响应元件,通过所述载体,该基因在植物体内的表达水平不受miR164转录后调控,过表达后的盐胁迫响应效用更为显著,是林木转基因耐盐研究的重要分子工具。
以上说明对本发明而言只是说明性的,而非限制性的,本领域普通技术人员理解,在不脱离所附权利要求所限定的精神和范围的情况下,可做出许多修改、变化或等效,但都将落入本发明的保护范围。
序列表
<110> 南京林业大学
<120> 一种柽柳盐胁迫响应基因TcNAC2及其miRNA抗性靶标rTcNAC2和应用
<130> 100
<160> 22
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1579
<212> DNA
<213> Tamarix chinenses
<400> 1
tcacacagga aacagctatg accatgatta cgccaagctc agaattaacc ctcactaaag 60
ggactagtcc tgcaggttta aacgaattgg cccttctaat acgactcact atagggcaag 120
cagtggtatc aacgcagagt acatggggat tttactcttt ttttttctct gacaaaagaa 180
gaagaagaag aagaaggatt agtattctta ttattagaag gaaagaagaa atggtaaaca 240
ttaatcctgg atccgttgtt ggaggtgatg aagaaccaat agagttacct cctggattcc 300
gattccatcc gacggacgaa gagctgatca ctcactatct ttcaccaaaa gtagctgata 360
acagcttctc tgctatagct gttggtgaag ttgatttgaa caactgtgaa ccctgggact 420
tgcctaagca ttcaaagatg ggagaaaagc agtggtattt cttctgtgtt agaggcagga 480
aatacccaac tggttcaagg ataaatagag ctactgatgc tggttattgg aaggcaaccg 540
gtatggacaa ggaaatttac agaggaaaac agctggctgg tatgaagaaa actttggttt 600
tctacaaggg gcgggctcca aaaggccata aatctaattg ggtcatacat gaatatagat 660
tggaggaaaa attctccttc caaaacctct ctgattcatc taagaaggat gagtgggtct 720
tatgccgggc cttcgagaag agtgcaggag agaagaagat accttaccca ggaccgattc 780
catccaattc tatccatttt caaaatccat ttgcaagctt acctcccttg atagaatcct 840
cgtcacgcat cactgaccat ctcaaaccaa caagtataac agaatcatct cacgtgtcct 900
gcttctccaa tcccatgatc aaaacccctc aacaaaccat catcaatgac aattcctttg 960
attcttaccg taagcataac atccacagcc ctttcatgcc cggctactat gctgaattac 1020
aatcatcgac ggaggactca tactgtggat ctcaaattag ttcaagcttc gcctatggca 1080
ctttgccgta tccaggtgga gaatacatgt cggatcagtc aatcttgagg tctatcctta 1140
agaacaatgg aacagaggga gcaatgaaaa cagagagtaa tcattgtaat atggttactg 1200
ctattgaatc tcgggatact ggtttgtcta gtgatatgta tgctgaaata tcatctgtgg 1260
tcgataaata taagacgagg ggtactagag atgcatttga tgatcatcag ggtcaccagg 1320
gctgttctat ttctggcgga cctcaagata ttgattggtt ttggagttat tgtaactgaa 1380
tcagtcagaa gaagatgccg atggcccatg gtgcttgctg ttatggcgat gagataatga 1440
ctcgctaggg tcattagatt caacagaacc ggaaaacagg aaagaaagga agaaaacact 1500
atccttcact gtatgcaaaa tatatatgaa aactcaggtc atgtggatac agaaaaaaaa 1560
aaaaaaaaaa aaaaaaaaa 1579
<210> 2
<211> 453
<212> PRT
<213> Tamarix chinenses
<400> 2
Met Thr Met Ile Thr Pro Ser Ser Glu Leu Thr Leu Thr Lys Gly Thr
1 5 10 15
Ser Pro Ala Gly Leu Asn Glu Leu Ala Leu Leu Ile Arg Leu Thr Ile
20 25 30
Gly Gln Ala Val Val Ser Thr Gln Ser Thr Trp Gly Phe Tyr Ser Phe
35 40 45
Phe Phe Ser Asp Lys Arg Arg Arg Arg Arg Arg Arg Ile Ser Ile Leu
50 55 60
Ile Ile Arg Arg Lys Glu Glu Met Val Asn Ile Asn Pro Gly Ser Val
65 70 75 80
Val Gly Gly Asp Glu Glu Pro Ile Glu Leu Pro Pro Gly Phe Arg Phe
85 90 95
His Pro Thr Asp Glu Glu Leu Ile Thr His Tyr Leu Ser Pro Lys Val
100 105 110
Ala Asp Asn Ser Phe Ser Ala Ile Ala Val Gly Glu Val Asp Leu Asn
115 120 125
Asn Cys Glu Pro Trp Asp Leu Pro Lys His Ser Lys Met Gly Glu Lys
130 135 140
Gln Trp Tyr Phe Phe Cys Val Arg Gly Arg Lys Tyr Pro Thr Gly Ser
145 150 155 160
Arg Ile Asn Arg Ala Thr Asp Ala Gly Tyr Trp Lys Ala Thr Gly Met
165 170 175
Asp Lys Glu Ile Tyr Arg Gly Lys Gln Leu Ala Gly Met Lys Lys Thr
180 185 190
Leu Val Phe Tyr Lys Gly Arg Ala Pro Lys Gly His Lys Ser Asn Trp
195 200 205
Val Ile His Glu Tyr Arg Leu Glu Glu Lys Phe Ser Phe Gln Asn Leu
210 215 220
Ser Asp Ser Ser Lys Lys Asp Glu Trp Val Leu Cys Arg Ala Phe Glu
225 230 235 240
Lys Ser Ala Gly Glu Lys Lys Ile Pro Tyr Pro Gly Pro Ile Pro Ser
245 250 255
Asn Ser Ile His Phe Gln Asn Pro Phe Ala Ser Leu Pro Pro Leu Ile
260 265 270
Glu Ser Ser Ser Arg Ile Thr Asp His Leu Lys Pro Thr Ser Ile Thr
275 280 285
Glu Ser Ser His Val Ser Cys Phe Ser Asn Pro Met Ile Lys Thr Pro
290 295 300
Gln Gln Thr Ile Ile Asn Asp Asn Ser Phe Asp Ser Tyr Arg Lys His
305 310 315 320
Asn Ile His Ser Pro Phe Met Pro Gly Tyr Tyr Ala Glu Leu Gln Ser
325 330 335
Ser Thr Glu Asp Ser Tyr Cys Gly Ser Gln Ile Ser Ser Ser Phe Ala
340 345 350
Tyr Gly Thr Leu Pro Tyr Pro Gly Gly Glu Tyr Met Ser Asp Gln Ser
355 360 365
Ile Leu Arg Ser Ile Leu Lys Asn Asn Gly Thr Glu Gly Ala Met Lys
370 375 380
Thr Glu Ser Asn His Cys Asn Met Val Thr Ala Ile Glu Ser Arg Asp
385 390 395 400
Thr Gly Leu Ser Ser Asp Met Tyr Ala Glu Ile Ser Ser Val Val Asp
405 410 415
Lys Tyr Lys Thr Arg Gly Thr Arg Asp Ala Phe Asp Asp His Gln Gly
420 425 430
His Gln Gly Cys Ser Ile Ser Gly Gly Pro Gln Asp Ile Asp Trp Phe
435 440 445
Trp Ser Tyr Cys Asn
450
<210> 3
<211> 1362
<212> DNA
<213> rTcNAC2(Artificial)
<400> 3
atgaccatga ttacgccaag ctcagaatta accctcacta aagggactag tcctgcaggt 60
ttaaacgaat tggcccttct aatacgactc actatagggc aagcagtggt atcaacgcag 120
agtacatggg gattttactc tttttttttc tctgacaaaa gaagaagaag aagaagaagg 180
attagtattc ttattattag aaggaaagaa gaaatggtaa acattaatcc tggatccgtt 240
gttggaggtg atgaagaacc aatagagtta cctcctggat tccgattcca tccgacggac 300
gaagagctga tcactcacta tctttcacca aaagtagctg ataacagctt ctctgctata 360
gctgttggtg aagttgattt gaacaactgt gaaccctggg acttgcctaa gcattcaaag 420
atgggagaaa agcagtggta tttcttctgt gttagaggca ggaaataccc aactggttca 480
aggataaata gagctactga tgctggttat tggaaggcaa ccggtatgga caaggaaatt 540
tacagaggaa aacagctggc tggtatgaag aaaactttgg ttttctacaa ggggcgggct 600
ccaaaaggcc ataaatctaa ttgggtcata catgaatata gattggagga aaaattctcc 660
ttccaaaacc tctctgattc atctaagaag gatgagtggg tcttatgccg ggccttcgag 720
aagagtgcag gagagaagaa gataccttac ccaggaccga ttccatccaa ttctatccat 780
tttcaaaatc catttgcaag cttacctccc ttgatagaat cctcgtcacg catcactgac 840
catctcaaac caacaagtat aacagaatca tctcatgtaa gctgtttcag taatcccatg 900
atcaaaaccc ctcaacaaac catcatcaat gacaattcct ttgattctta ccgtaagcat 960
aacatccaca gccctttcat gcccggctac tatgctgaat tacaatcatc gacggaggac 1020
tcatactgtg gatctcaaat tagttcaagc ttcgcctatg gcactttgcc gtatccaggt 1080
ggagaataca tgtcggatca gtcaatcttg aggtctatcc ttaagaacaa tggaacagag 1140
ggagcaatga aaacagagag taatcattgt aatatggtta ctgctattga atctcgggat 1200
actggtttgt ctagtgatat gtatgctgaa atatcatctg tggtcgataa atataagacg 1260
aggggtacta gagatgcatt tgatgatcat cagggtcacc agggctgttc tatttctggc 1320
ggacctcaag atattgattg gttttggagt tattgtaact ga 1362
<210> 4
<211> 23
<212> DNA
<213> 3' RACE 正向引物 Outer Primer(Artificial)
<400> 4
gaggtgatga agaaccaata gag 23
<210> 5
<211> 22
<212> DNA
<213> 3' RACE 正向引物 Inner Primer(Artificial)
<400> 5
tagacatggt tggtacagac tc 22
<210> 6
<211> 45
<212> DNA
<213> 3' RACE 反向引物 Outer Primer(Artificial)
<400> 6
ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt 45
<210> 7
<211> 22
<212> DNA
<213> 3' RACE 反向引物 Inner Primer(Artificial)
<400> 7
ctaatacgac tcactatagg gc 22
<210> 8
<211> 45
<212> DNA
<213> 5' RACE正向引物 Outer Primer(Artificial)
<400> 8
ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt 45
<210> 9
<211> 22
<212> DNA
<213> 5' RACE正向引物 Inner Primer(Artificial)
<400> 9
ctaatacgac tcactatagg gc 22
<210> 10
<211> 25
<212> DNA
<213> 5' RACE反向引物 Outer Primer(Artificial)
<400> 10
tatggccttt tggagcccgc ccctt 25
<210> 11
<211> 19
<212> DNA
<213> 5' RACE反向引物 Inner Primer(Artificial)
<400> 11
ttacgctcac cacttgctc 19
<210> 12
<211> 20
<212> DNA
<213> TcNAC2 ORF正向引物(Artificial)
<400> 12
atgaccatga ttacgccaag 20
<210> 13
<211> 22
<212> DNA
<213> TcNAC2 ORF反向引物(Artificial)
<400> 13
tcagttacaa taactccaaa ac 22
<210> 14
<211> 20
<212> DNA
<213> TcNAC2正向引物(Artificial)
<400> 14
gatccaagct cgcggaagcc 20
<210> 15
<211> 20
<212> DNA
<213> TcNAC2反向引物(Artificial)
<400> 15
cacgaatgtc ggtggccgat 20
<210> 16
<211> 21
<212> DNA
<213> TIF正向引物(Artificial)
<400> 16
accacaggag tgtccaccac a 21
<210> 17
<211> 20
<212> DNA
<213> TIF反向引物(Artificial)
<400> 17
tgatgctttg cgtgccagtg 20
<210> 18
<211> 42
<212> DNA
<213> TcNAC2突变引物(Artificial)
<400> 18
acagaatcat ctcatgtaag ctgtttcagt aatcccatga tc 42
<210> 19
<211> 39
<212> DNA
<213> TcNAC2+引物序列(Artificial)
<400> 19
ctagatagcg gccgctattc acgtgtcctg cttctccag 39
<210> 20
<211> 39
<212> DNA
<213> TcNAC2-引物序列(Artificial)
<400> 20
tcgactggag aagcaggaca cgtgaatagc ggccgctat 39
<210> 21
<211> 38
<212> DNA
<213> rTcNAC2+引物序列(Artificial)
<400> 21
ctagatagcg gccgctatca tgtaagctgt ttcagtag 38
<210> 22
<211> 38
<212> DNA
<213> rTcNAC2-引物序列(Artificial)
<400> 22
tcgactactg aaacagctta catgatagcg gccgctat 38
Claims (10)
1.一种柽柳盐胁迫响应基因TcNAC2,其核苷酸序列如SEQ ID No.1所示。
2.权利要求1所述柽柳盐胁迫响应基因TcNAC2的表达蛋白,其氨基酸序列如SEQ IDNo.2所示。
3.一种miR164抗性靶标rTcNAC2,由权利要求1所述柽柳盐胁迫响应基因TcNAC2通过miR164响应元件同义突变获得,其核苷酸序列如SEQ ID No.3所示。
4.含有权利要求1所述柽柳盐胁迫响应基因TcNAC2的载体。
5.根据权利要求4所述载体,其特征在于:所述载体为双荧光素酶报告载体35sGLO:TcNAC2,在TcNAC2的miR164响应元件的5′端上游组装有组成型强表达启动子P35S和萤火虫荧光素酶报告基因,3′端下游组装有海肾荧光素酶报告基因。
6.含有权利要求3所述miR164抗性靶标rTcNAC2的载体。
7.根据权利要求6所述载体,其特征在于:所述载体为双荧光素酶报告载体35sGLO:rTcNAC2,在rTcNAC2的miR164响应元件同义突变的5′端上游组装有组成型强表达启动子P35S和萤火虫荧光素酶报告基因,3′端下游组装有海肾荧光素酶报告基因。
8.根据权利要求6所述载体,其特征在于:所述载体为rTcNAC2的过表达载体PBI121GW:rTcNAC2,组装35S启动子;组装NPTII基因表达盒,组装LB和RB序列,LB和RB序列之间为rTcNAC2基因表达框架和基因NPTII。
9.权利要求1所述的柽柳盐胁迫响应基因TcNAC2或权利要求2所述柽柳盐胁迫响应基因TcNAC2的表达蛋白或权利要求4所述柽柳盐胁迫响应基因TcNAC2的载体在植物耐盐性或林木抗性育种中的应用。
10.权利要求3所述的miR164抗性靶标rTcNAC2或权利要求6所述的miR164抗性靶标rTcNAC2的载体在植物耐盐性或林木抗性育种中的应用。
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CN111500579A (zh) * | 2020-04-23 | 2020-08-07 | 九圣禾种业股份有限公司 | 棉花miR164a和NAC100L及其在调控植物黄萎病抗性中的应用 |
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CN111500579A (zh) * | 2020-04-23 | 2020-08-07 | 九圣禾种业股份有限公司 | 棉花miR164a和NAC100L及其在调控植物黄萎病抗性中的应用 |
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