CN113584047B - 大麦HvNAT2基因及其用途 - Google Patents
大麦HvNAT2基因及其用途 Download PDFInfo
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- CN113584047B CN113584047B CN202110817737.2A CN202110817737A CN113584047B CN 113584047 B CN113584047 B CN 113584047B CN 202110817737 A CN202110817737 A CN 202110817737A CN 113584047 B CN113584047 B CN 113584047B
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Abstract
本发明公开了大麦HvNAT2基因及其在调控大麦对镉胁迫耐受性方面的应用,属于基因工程技术领域。大麦HvNAT2基因的CDS区核苷酸序列如SEQ ID NO.1所示。本发明通过对大麦HvNAT2基因的克隆和分析,并结合同源遗传转化过表达和RNAi技术在大麦品种黄金希望上对该基因进行功能验证发现,HvNAT2过表达植株的耐镉性显著增强,而HvNAT2‑RNAi沉默植株的耐镉性显著降低。本发明为大麦耐镉胁迫育种与生产提供了理论依据和相关基因。
Description
技术领域
本发明涉及基因工程技术领域,具体涉及大麦HvNAT2基因及其在调控大麦对镉胁迫耐受性方面的应用。
背景技术
土壤镉污染不仅影响作物的产量和品质,并可通过食物链危害人体健康,严重影响粮食安全。而工业化的发展导致土壤镉污染日渐严重,镉米、镉麦的存在已是不争事实,镉中毒事件亦有发生(Hu等,2016;Wang等,2019;沈凤斌,2017)。显然,土壤镉污染已对作物生产和农产品安全及人类健康构成了严重威胁,镉污染治理已经成为各界普遍关注的热点问题。提高作物对镉耐性和减少食用器官镉积累是持续高效利用土地资源和解决“饭碗里重金属”之殇的一项重要科学任务。
对于中、轻度大面积污染的农田,培育耐镉且食用器官低积累作物品种,是有效利用自然资源和保证农产品安全生产最经济和重要的途径之一。发掘耐镉与低镉积累种质与相关基因,明确其调控机制是培育耐镉且低镉积累品种的基础和关键。植物的耐镉性或对镉的适应能力是一个复杂的性状,涉及很多代谢途径和表型特征;同时,同一作物不同的基因型间可能拥有不同的镉响应模式,因此解析植物的耐镉机制仍然是一个具有挑战性的任务。
大麦是全球各地普遍栽培的第四大禾谷类作物,也是人类直接或间接摄取镉的主要来源之一(Hayes等,2020;Lei等,2020),其产品安全与人类健康密切相关。同时,大麦是二倍体自花授粉作物,其染色体数目少,可作为其它麦类作物的模式植物,十分适合生理和遗传机理研究(Forster等,2000)。因此挖掘镉耐性相关基因,探究大麦镉耐性机制,培育耐镉-低积累品种极为重要。
NATs(Nucleobase-Ascorbate Transporters)转运蛋白广泛存在于生物体内,主要转运碱基以及抗坏血酸。在植物中有关NAT的具体生物学功能鲜有报道;NAT蛋白与植物抗逆途径的研究刚起步,孙婷婷(2018)研究发现,苹果中MdNAT1和MdNAT7参与了干旱和盐胁迫响应机制。在大麦中还未有关于NAT基因的研究,因此鉴定大麦NAT家族,探究其调控镉胁迫响应和功能,对提高大麦抵御非生物胁迫能力和大麦的遗传改良与育种具有重要意义。
发明内容
本发明的目的在于提供一种从大麦中克隆得到的具有耐镉胁迫特性的基因,为大麦耐镉胁迫育种与生产提供了理论依据和相关基因。
为实现上述目的,本发明从大麦(Hordeum vulgare L.)Zhenong8和W6nk2中鉴定克隆到具有耐镉胁迫特性的基因HvNAT2,ZN8和W6nk2分别为本课题组前期筛选得到的耐镉和镉敏感基因型。
HvNAT2基因克隆与分析:以镉耐性差异大麦基因型Zhenong8(ZN8;耐镉基因型)和W6nk2(镉敏感基因型)为材料,5μM CdCl2镉胁迫试验,比较分析了大麦根系miRNA及其靶基因响应镉胁迫的基因型差异,鉴定到与ZN8耐镉性相关的候选miRNA(miR156g-3p_3)及其靶基因HORVU3Hr1G073180.2。高通量测序和qRT-PCR验证结果表明,镉胁迫下,保守miRNAmiR156g-3p_3在ZN8中上调表达,但在W6nk2中表达不变(Cd vs CK);而其靶基因HORVU3Hr1G073180.2在ZN8中下调表达,在W6nk2中表达不变。miR156g-3p_3和HORVU3Hr1G073180.2表达变化与miRNA调控靶基因的典型机制吻合。将靶基因HORVU3Hr1G073180.2的核酸序列翻译成蛋白序列,其在NCBI中注释为Nucleobase-ascorbate transporter(NAT),是以碱基类物质为底物的转运蛋白。基于隐马尔可夫模型和BLAST分析,在大麦基因组中共发现了9个NAT家族成员,分别位于大麦第2、3、4、5和6号染色体上,因此将位于3号染色体上的该基因命名为HvNAT2。HvNAT2的CDS全长为1575bp,核苷酸序列如SEQ ID NO.1所示。
本发明还提供了所述的大麦HvNAT2基因编码的蛋白质,该蛋白质的氨基酸序列如SEQ ID NO.2所示。
HvNAT2蛋白由524个氨基酸残基组成,该蛋白分子量为57.15kDa,等电点pI=8.13。含有保守的(Q/E/P)NXGXXXXT(R/K/G)和QH结构域。通过SMART和Protter网站的蛋白结构域预测显示,HvNAT蛋白含有1个Pfam结构域Xan_ur_permease,属于APC(Amino-polyamine-organocation)超家族,转运核酸类物质,预测有11个跨膜结构域(Transmembrane domains,TMDs)。蛋白三级结构预测表明具有嘌呤/H+同向转运结构。
利用oneKP植物转录组数据库分析表明,HvNAT2与小麦TaNAT2和山羊草AetNAT2的关系最近,其次是二穗短柄草的BdNAT2。HvNAT2和TaNAT2、OsNAT2、ZmNAT2蛋白序列比对结果,HvNAT2蛋白序列与TaNAT2蛋白序列有99.05%的相似度,与OsNAT2蛋白序列有95.61%的相似度,与ZmNAT2有91.48%的相似度。
HvNAT2基因的表达模式分析:通过荧光定量PCR技术分析,HvNAT2基因在叶片中的表达量高于茎和根系,而且在灌浆期的种子中表达量最高。进一步分析HvNAT2基因对镉胁迫时间的响应变化,耐镉品种ZN 8叶片中HvNAT2基因的相对表达量在10μM Cd胁迫1h后即达到最大值。
采用地高辛(DIG)为标记的原位PCR技术分析,HvNAT2主要在维管束中表达。HvNAT2的亚细胞定位分析显示,HvNAT2定位于细胞质膜上。
进一步地,利用农杆菌介导的大麦幼胚遗传转化技术,获得了HvNAT2基因的过表达和RNAi沉默植株。通过水培镉胁迫试验,对遗传转化植株进行了表型鉴定。结果显示:
在无镉正常生长条件下,HvNAT2过表达植株、HvNAT2-RNAi沉默植株的长势与野生型(GP)没有明显差异。在镉胁迫下,过表达植株的长势显著优于GP。生物量的测量结果表明,两者在正常生长条件下地上部和地下部干重无明显差异,但是镉胁迫下过表达植株的地上部和地下部下降幅度显著低于GP;过表达植株的分蘖数大于GP,且GP枯死的老叶显著多于过表达植株。而相反的,在镉胁迫下,HvNAT2-RNAi植株长势显著低于野生型(GP),生物量的测量结果表明,沉默植株的下降幅度显著高于GP。这些结果表明HvNAT2基因过表达后植株的耐镉性显著增强,而RNAi沉默后的使大麦对镉的耐受性显著降低。
本发明提供了所述的大麦HvNAT2基因在调控大麦对镉胁迫耐受性方面的应用,大麦HvNAT2基因可以增强大麦对镉胁迫耐受性。大麦HvNAT2基因过表达使植株耐镉性增强;大麦HvNAT2基因沉默使植株对镉的耐受性降低。
具体的,所述应用包括:将所述大麦HvNAT2基因插入过表达载体中构建重组质粒,然后利用农杆菌介导技术将目的片段转入受体大麦中,筛选获得功能性获得的转基因植株。
优选的,所述过表达载体为pBract214。
重组质粒的构建可采用常规方法,如采用Gateway系统,先将HvNAT2基因和基因片段通过BP反应连入入门载体pDONR(Zeo)中,再经过LR反应连入原始目标载体中。
优选的,所述农杆菌介导技术采用的宿主菌为农杆菌AGL1。遗传转化材料采用大麦幼胚。所述受体大麦的品种为黄金希望。
本发明具备的有益效果:
本发明通过对大麦HvNAT2基因的克隆和分析,并结合同源遗传转化过表达和RNAi技术在大麦品种黄金希望(Golden Promise,GP;H.vulgare L.)上对该基因进行功能验证发现,HvNAT2过表达植株的耐镉性显著增强,而HvNAT2-RNAi沉默植株的耐镉性显著降低。本发明为大麦耐镉胁迫育种与生产提供了理论依据和相关基因。
附图说明
图1为两个大麦基因型(ZN8和W6nk2)的HvNAT2基因的核苷酸序列比对。
图2为两个大麦基因型(ZN8和W6nk2)的HvNAT2蛋白的氨基酸序列对比。
图3为两个大麦基因型(ZN8和W6nk2)的HvNAT2启动子顺式作用元件分析。
图4为HvNAT2蛋白功能域预测图,其中(A)为SMART预测的蛋白功能域,不重要或者重合的结构域并未显示;(B)为Protter预测的蛋白结构域;(C)为HvNAT2的蛋白三级结构预测。
图5为HvNAT2与玉米、水稻、二穗短柄草、拟南芥等植物和藻类NAT2蛋白家族成员的进化树分析。
图6为HvNAT2与TaNAT2、ZmNAT2、OsNAT2的氨基酸序列对比图。
图7为HvNAT2基因的表达模式:(A)HvNAT2基因的组织定位;(B)HvNAT2基因表达对镉胁迫响应;(C)大麦叶片和根系中HvNAT2基因的细胞定位(基于原位PCR技术);(D)HvNAT2蛋白的亚细胞定位(农杆菌介导的烟草表皮细胞异源表达)。
图8为大麦HvNAT2遗传转化载体构建图。(A)pBract214-HvNAT2载体示意图;(B)pANDA-HvNAT2载体示意图。pUbq-ubiquitin promoter,T-terminator。
图9为DNA水平上验证遗传转化阳性植株;M-marker,P-positive。
图10为利用同源过表达技术验证HvNAT2基因在大麦耐镉性中的作用,其中(A)为自然生长条件下和镉处理(10μM Cd)30d后,野生型GP、过表达OX植株的生长表型;(B)为镉处理(10μM Cd)30d后,野生型GP、过表达OX植株的株高、根长、地上部干重、根系干重的差异;(C、D)为镉处理(10μM Cd)30d处理后,野生型GP、过表达植株SPAD和分蘖数的差异;(E)为遗传转化阳性验证(荧光定量)。
图11为利用RNAi技术验证HvNAT2基因在大麦耐镉性中的作用,其中,(A)为自然生长条件下和镉处理(10μM Cd)15d后,野生型GP和RNAi沉默植株的生长表型;(B)为镉处理(10μM Cd)15d后,野生型GP和RNAi沉默植株株高、根长、地上部干重、根系干重的差异;(C)为镉处理(10μM Cd)15d后,野生型GP和RNAi沉默植株SPAD的差异;(D)遗传转化阳性验证(荧光定量)。
具体实施方式
下面通过具体实施例,对本发明的技术方案作进一步的具体说明。应当理解,本发明的实施并不局限于下面的实施例,对本发明所做的任何形式上的变通和/或改变都将落入本发明保护范围。
在本发明中,若非特指,所有的份、百分比均为重量单位,所采用的设备和原料等均可从市场购得或是本领域常用的。下述实施例中的方法,如无特别说明,均为本领域的常规方法。
本发明以课题组前期筛选到的大麦耐镉胁迫基因型ZN8、镉胁迫敏感基因型W6nk2为主要材料,克隆大麦耐镉胁迫相关基因HvNAT2,对阐明大麦响应镉胁迫的分子机理和育种及生产具有重要意义。
黄金希望(Golden Promise,GP),栽培大麦品种,愈伤组织再生能力相对较强,是目前大麦遗传转化研究的主要材料。
实施例1、HvNAT2基因CDS区的克隆和分析
1、大麦生长条件
耐镉胁迫基因型ZN8、镉胁迫敏感基因型W6nk2为课题组前期筛选到的大麦基因型,发表于Chen F,Wang F,Zhang GP,Wu FB(通讯作者).Identification of barleyvarieties tolerant to cadmium toxicity.Biological Trace Element Research,2008,121(2):171-179.Chen F,Dong J,Wang F,Wu FB(通讯作者),Zhang GP,Li GM,ChenZF,Chen J,Wei K.Identification of barley genotypes with low grain Cdaccumulation and its interaction with four microelements.Chemosphere,2007,67:2082-2088。
大麦ZN8、W6nk2的种子用2%的H2O2消毒30min,随后用蒸馏水冲洗5-6次,将消毒过的种子置于湿润的沙床上,于生长室中进行暗培养(22℃/18℃,白天/夜间),萌发之后进行补光。第7d时,选取长势一致的幼苗转移至1L盛有大麦基本培养液的黑色塑料桶中,用5孔塑料盖覆盖,每孔两株幼苗,使用海绵进行固定,在大麦培养室中进行培养,大麦基本培养液使用1/5Hogland配方。
2、HvNAT2基因CDS区、启动子序列的克隆
2.1HvNAT2基因CDS区序列的克隆
根据TaKaRaMiniBST Plant RNA Extraction Kit(Takara,日本)RNA提取试剂盒的说明书进行ZN8和W6nk2叶片总RNA的提取,并用DNaseI去除总RNA中基因组DNA污染,将提取的总RNA反转录成cDNA。使用NCBI的Primer-BLAST进行特异性引物设计,具体引物序列为:HvNAT2-CDS-F:5’-ATGTCGGAGGTCAAGCCGG-3’(SEQ ID No.4);HvNAT2-CDS-R:5’-TTACGACGGCGGGAAGAAG-3’'(SEQ ID No.5)。
将PCR扩增产物,连接到pMD18-T载体(Takara,日本),转化大肠杆菌DH5α,挑取阳性克隆送公司测序,测序正确的分别进行提质粒和甘油保存,所得质粒命名为pMD18-T-HvNAT2。PCR引物合成工作和基因测序工作均由浙江尚亚生物技术有限公司完成。
大麦ZN8中HvNAT2基因的CDS区核苷酸序列如SEQ ID NO.1所示。ZN8、W6nk2中HvNAT2基因的CDS区核苷酸序列差异如图1所示。
HvNAT2基因的CDS全长为1575bp,编码一个含524个氨基酸残基的蛋白序列,该基因包含14个外显子和13个内含子。通过比对两基因型ZN8和W6nk2中HvNAT2基因的CDS序列,两者在第1368和第1542个碱基存在差异(图1),但是两者的氨基酸序列一致(图2)。
2.2HvNAT2启动子序列的克隆
通过Ensembl数据库得到HvNAT2的5’UTR之前的序列,以此为基础设计引物扩增转录本5’UTR前2kb的DNA序列。扩增所用引物序列为:
HvNAT2-promoter-F:5’-TTTGGCGTTTCTCAAGGGTG-3’(SEQ ID No.6)
HvNAT2-promoter-R:5’-GGCTTGACCTCCGACATTTC-3’(SEQ ID No.7)
浙农8号和W6nk2的DNA参照Shen等(2017)用CTAB法提取,以此为模板进行PCR反应。扩增产物纯化后与pMD18-T载体连接测序。测序完成后,通过PlantCARE(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)网站启动子区顺式作用元件进行预测,使用TBtools软件进行图像绘制。
大麦ZN8和W6nk2中HvNAT2启动子顺式作用元件如图3所示。
3、HvNAT2蛋白序列分析
通过SMART(http://smart.embl-heidelberg.de/)和Protter(http://wlab.ethz.ch/protter/start/)网站对HvNAT2的蛋白功能域进行预测预测分析。在SWISS-MODEL(https://swissmodel.expasy.org/)网站对HvNAT2编码的氨基酸进行蛋白质三级结构预测。
结果显示,HvNAT蛋白含有1个Pfam结构域Xan_ur_permease,含有保守的(Q/E/P)NXGXXXXT(R/K/G)和QH结构域。属于APC(Amino-polyamine-organocation)超家族,转运核酸类物质,预测有11个跨膜结构域(Transmembrane domains,TMDs),与NAT家族10-12个TMDs相符合。蛋白三级结构预测表明具有嘌呤/H+同向转运结构(图4)。
利用oneKP植物转录组数据库分析表明,NAT2在植物和藻类中普遍存在,最早可能起源于酸藻。HvNAT2与小麦TaNAT2和山羊草AetNAT2的关系最近,其次是二穗短柄草的BdNAT2(图5)。HvNAT2和TaNAT2、OsNAT2、ZmNAT2蛋白序列比对结果显示,HvNAT2蛋白序列与TaNAT2蛋白序列有99.05%的相似度,与OsNAT2蛋白序列有95.61%的相似度,与ZmNAT2有91.48%的相似度(图6)。
4、HvNAT2基因的表达模式分析
通过荧光定量PCR技术,对HvNAT2基因在大麦ZN8根系、茎、叶片中的相对表达量进行了分析。
荧光定量PCR检测HvNAT2基因的表达量。用总RNA提取试剂盒(Takara,日本)分别提取不同处理样本的总RNA,并用DNaseI去除总RNA中基因组DNA污染,用PrimeScriptTMRTreagent Kit反转录试剂盒(Takara,日本)将各样本总RNA分别反转录成单链cDNA。用SYBR绿色荧光酶复合物(Takara,日本)和Light Cycler 480PCR仪(Roche,瑞士)对相应样本中HvNAT2基因的表达进行荧光定量PCR分析(qRT-PCR),并用一个内参基因GAPDH对表达值进行矫正处理。qRT-PCR体系见表1:
表1
PCR的具体程序为:95℃30s,(95℃5s,60℃30s)40个循环。溶解曲线程序为:95℃5s,60℃1min,95℃,50℃30s降温。利用2-ΔΔCt相对定量方法计算基因表达值变化。每组实验重复三次。
qRT-PCR引物序列为:
HvNAT2-q-F:5’-GCAGGTAAATGGAATCGGCATC-3’(SEQ ID No.8);
HvNAT2-q-R:5’-TAACCCCACGTTCTCCACTGA-3’(SEQ ID No.9);
GAPDH-F:5’-AAGCATGAAGATACAGGGAGTGTG-3’(SEQ ID No.10);
GAPDH-R:5’-AAATTTATTCTCGGAAGAGGTTGTACA-3’(SEQ ID No.11)。
结果显示,HvNAT2基因在大麦叶片中的表达量高于茎和根系,而且在灌浆期的种子中表达量最高(图7A)。
进一步分析HvNAT2基因对镉胁迫时间的响应变化,分别选取0、1、3、6、12、24、48和72h这8个时间点,测量了耐镉品种ZN 8叶片中HvNAT2基因的相对表达量,结果HvNAT2在10μM Cd胁迫1h后即达到最大值,随后逐渐下降至处理后6h时,与对照持平;随后继续下调表达,到第3d重新与对照持平(图7B)。
采用地高辛(DIG)为标记的原位PCR技术,对HvNAT2基因在ZN8根系和叶片中的细胞定位进行了分析。结果显示,在根系中,该基因在大部分细胞都有信号,其中在中柱部分颜色最深信号最强;在大麦叶片中,只在中柱检测到信号(图7C),表明HvNAT2主要在维管束中表达。HvNAT2的亚细胞定位分析显示,HvNAT2定位于细胞质膜上(图7D)。
实施例2、基因过表达和RNAi沉默在大麦GP中验证HvNAT2基因功能
1、过表达载体和RNAi沉默载体的构建
本实验构建过表达载体的方法为Gateway。分别提取大麦ZN8和大麦品种黄金希望(Golden Promise)的总RNA,将其反转录成cDNA,设计关于HvNAT2基因的过表达和RNAi沉默引物,以ZN8的cDNA为模板进行目的基因CDS区的扩增,以GP的cDNA为模板进行目的基因RNAi片段的扩增。设计引物扩增带有接头的HvNAT2基因的427bp大小片段(530至956bp的序列)(SEQ ID NO.3),用KOD ONE高保真酶扩增。引物序列如下:
其中HvNAT2基因过表达的CDS区引物序列如下:
HvNAT2-OX-F:
5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTATGTCGGAGGTCAAGCCGG-3’(SEQ ID No.12);
HvNAT2-OX-R:
5’-GGGGACCACTTTGTACAAGAAAGCTGGGTTTACGACGGCGGGAAGAAG-3’(SEQ ID No.13)。
其中HvNAT2基因的RNAi引物序列如下:
HvNAT2-RNAi-F:
5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTGAGGATTCCCTGTGGTTGGG-3’(SEQ IDNo.14);
HvNAT2-RNAi-R:
5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCTGCTCAGGATATGGGCTGG-3’(SEQ IDNo.15)。
用1%的琼脂糖凝胶电泳检测PCR扩增产物,对目标产物进行胶回收纯化,测量回收产物浓度。随后将已知浓度的基因回收产物按下表所述进行BP重组反应。BP重组反应体系见表2:
表2
Gateway BP Clonase II enzyme mix | 2μL |
attB-PCR产物 | 150ng |
pDONR(Zeo)入门载体 | 150ng |
TE缓冲液(pH=8.0) | 补足10μL |
25℃反应4h,加入1μL的Proteinase K,37℃继续反应10min,反应产物立即转化大肠杆菌DH5α感受态细胞,涂布博来霉素抗性的LB固体培养基,37℃培养16h左右,挑取单克隆,摇菌进行菌液PCR,将阳性克隆的菌液送往公司测序,测序正确的单克隆进行扩繁,菌液甘油保存和提质粒保存,质粒分别命名为pDONR(Zeo)-HvNAT2-OX和pDONR/Zeo-HvNAT2-RNAi。
将上述已知浓度的质粒按照下表进行LR反应(pBract214载体为过表达载体,pANDA载体为RNAi沉默载体)。LR反应体系见表3:
表3
Gateway LR Clonase II enzyme mix | 2μL |
pDONR(Zeo)-HvNAT2-OX/RNAi重组载体 | 150ng |
pBract214/pANDA目的载体 | 150ng |
TE缓冲液(pH=8.0) | 补足10μL |
25℃反应5h,加入1μl的Proteinase K,37℃继续反应10min,反应产物立即转化大肠杆菌DH5α感受态细胞,涂布卡那霉素抗性的LB固体培养基,37℃培养16h左右,挑取单克隆,摇菌进行菌液PCR,将有目的条带的菌液送往公司测序,测序正确的单克隆进行扩繁,菌液甘油保存和提质粒保存,质粒分别命名为pBract214-HvNAT2和pANDA-HvNAT2(图8)。
将测序正确的质粒转化农杆菌感受态细胞AGL1(pSoup),涂布利福平+卡那霉素抗性的YEB平板,28℃培养40-48h左右,PCR验证阳性克隆。取阳性克隆菌液100μL加入到10mLMG培养液(pH=7.2,含25μg/mL利福平,50μg/mL卡那霉素)中培养,28℃,180rpm,摇菌至OD600=0.6-0.7,加入等体积的30%无菌甘油,混匀后液氮快速冷冻,保存在-80℃备用。
2、大麦幼胚遗传转化
2.1遗传转化材料准备
遗传转化以栽培大麦品种黄金希望(Golden Promise)的幼胚为外植体,一般在大麦开花2-3周后收获籽粒,当幼胚直径在1.5-2mm时收获穗。
2.2幼胚分离及消毒
选取符合标准的大麦未成熟幼胚种子,将其从穗上剥离,去除芒,种子用70%酒精表面消毒30s,用灭菌水洗涤数次。再用10%次氯酸钠浸泡4min,用灭菌水洗涤数次。在无菌滤纸上,进行未成熟胚的分离,除去胚轴,盾板朝上置于愈伤组织诱导培养基上,23-24℃,黑暗培养2-3d。
2.3农杆菌侵染和共培养
将保存的农杆菌菌液400μL加入到10mL不含抗生素的MG液体培养基中,在28℃过夜培养,180rpm,OD600=1.3-1.4,用于幼胚侵染。将准备好的农杆菌侵染液滴加在每一个幼胚上,晾干。用封口膜封住平板,23-24℃,黑暗,共培养2d。
2.4选择培养
与农杆菌共培养2d后,将幼胚转移到新鲜的愈伤组织诱导培养基平板上进行选择培养。此时培养基中含有50mg/L潮霉素以筛选阳性愈伤组织,含有160mg/L特泯菌以抑制农杆菌生长。23-24℃,暗培养56d后(每14d更换一次培养基平板),将由幼胚分离得到的愈伤组织转移到转移培养基上,在24℃弱光下,培养21d,此时将产生绿点。
2.5转基因植株再生
将绿点转移到继代培养基中继续培养,当地上部叶子达到2-3cm时,根开始建成。将小苗转移到生根培养基中,不添加任何生长调节剂,抗生素不变。之后将根系建成的小苗取出,洗掉培养基,转移到含有蛭石和营养基质的小盆中,在人工气候室中生长(22℃/18℃,白天/夜间),直至收获种子。
3、转基因植株的验证
通过预先冷冻和加热干燥的方式破除转基因大麦种子的休眠,种子在湿润的砂床上萌发。出苗7d后,转移到含有基本营养液的水培容器中继续生长。两叶期,选取转基因植株叶片,提取DNA,以野生型GP为阴性对照,pBract214-HvNAT2和pANDA-HvNAT2的质粒为阳性对照,验证携带目的片段的载体是否转入大麦基因组。DNA验证阳性的植株,对其地上部和根系再进行RNA提取和反转录,以野生型GP中HvNAT2基因的表达量为参照,通过荧光定量PCR对HvNAT2基因的表达量进行验证。
过表达植株的DNA验证引物为:
pBract-F:5’-GCATATGCAGCAGCTATATGTG-3’(SEQ ID No.16);
OX-HvNAT2-R:5’-TTACGACGGCGGGAAGAAG-3’(SEQ ID No.17)。
RNAi沉默植株的DNA验证引物为:
pANDA-F:5’-TACGGCGTGGATACGTTAGC-3’(SEQ ID No.18);
RNAi-HvNAT2-R:5’-CTGCTCAGGATATGGGCTGG-3’(SEQ ID No.19)。
用于荧光定量PCR的验证引物与上述SEQ ID No.6和SEQ ID No.7一致。选择三个过表达株系HvNAT2-OX1,HvNAT2-OX2和HvNAT2-OX3验证HvNAT2的功能。它们HvNAT2基因相对表达量分别是GP的31、47和33倍(图10E)。两个干扰株系HvNAT2-RNAi1和HvNAT2-RNAi2,它们HvNAT2基因相对表达量分别是GP的0.34和0.60倍(图11D)。
4、HvNAT2过表达和RNAi沉默株系的表型鉴定
对三叶期的大麦野生型GP、HvNAT2过表达和HvNAT2-RNAi沉默植株进行水培镉胁迫试验,对各株系的长势、株高、根长、生物量进行了观察和测定。
如图10所示,在对照条件下,野生型、过表达植株的长势和生长性状没有显著差异,而10μM Cd处理30d,过表达株系的长势显著优于GP(图10A),镉胁迫下GP地上部和地下部干重分别下降66.7%和48.3%,而过表达株系的地上部和地下部相较于对照分别下降了45.8%和20.1%,下降幅度显著低于GP(图10B);过表达株系的分蘖数平均为4至5个,而GP的分蘖数在2个左右(图10D),且GP枯死的老叶显著多于过表达株系。
如图11所示,在对照条件下,野生型、沉默植株的长势和生长性状没有显著差异,而10μM Cd处理15d,HvNAT2-RNAi植株长势显著低于野生型(GP),与对照相比,镉胁迫使GP的地上部和地下部干重分别下降了22.0%和28.9%,而沉默植株分别下降了40.1%和55.3%(图11B)。
上述结果证明,HvNAT2基因正调控大麦的耐镉性。
综上所述,通过对大麦HvNAT2的分离克隆和分析,结合同源过表达和RNAi技术在大麦GP上对该基因进行功能验证,结果发现,HvNAT2过表达显著增强植株的耐镉性,而HvNAT2-RNAi沉默植株的耐镉性显著降低。本发明为大麦耐镉胁迫育种与生产提供了理论依据和相关基因。
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
序列表
<110> 浙江大学
<120> 大麦HvNAT2基因及其用途
<160> 19
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1575
<212> DNA
<213> 大麦(Hordeum vulgare L.)
<400> 1
atgtcggagg tcaagccgga ggagatcagc cacccggcca tggagcagct gcaggggttc 60
gagtactgca tagactccaa ccctccgtgg ggggaggcca tcatactggg cttccagcac 120
tacatcctgg cgctgggcac ggccgtcatg atcccggcgg tgctggtgcc catgatgggc 180
ggcagcgacg gagacagggt gagggtggtg cagacgctgc tgttcgtcac tggcatcaac 240
acgctgctgc agtcgctctt cgggacgcgg ctgcccacgg tcataggcgg ctcctacgcc 300
ttcgtcgtcc cggtgatggc catcgtgcaa gactcgtcgc tcgcggccat acctgacgac 360
cacgagaggt tcctccagag catgagggcc atacagggag cactgatagt gtcctccagc 420
attcagatca tccttggtta cagccagctc tggggtattt tctccaggtt cttcagtcca 480
ctgggaatgg cgccggtggt tgctctgctc ggttttggcc tcttcgaaag aggattccct 540
gtggttggga gatgcgttga ggttggcctg ccaatgttaa tcctctttgt tgtgctttcc 600
cagtatctga agaatgtaca gataagagag atccccatac tggaacgatt ctcgctgttc 660
atttgcatcg cattggtctg ggcctatgct caaatcctca cttctggtgg cgcgtataat 720
catagcaccg agatcactca gatcaactgc cgcactgacc gtgccaatct catctcttcc 780
gccccatgga tcaagatccc atatcctctg caatgggggg caccaacctt cagcgcaggc 840
caatcgttcg gtatggtatc tgcggttttg gtctcgctaa tagagtccac agcttcttac 900
agcgccgcat ctcggcttgc aagtgcaact ccacctccag cccatatcct gagcagaggc 960
atcgggtggc agggaatcgg catcctcctt tgtgggctct ttggaacagg caccggctcc 1020
actgtctcag tggagaacgt ggggttacta ggatcaacga ggatcgggag ccgccgcgtc 1080
atacaaatct gtgctggctt catgatcttc ttctccatgc tggggaaatt tggagcgctg 1140
ttcgcttcca tcccgttcac catcttcgcc gcggtgtact gcgtcctgtt cggactagtt 1200
gctgcggtgg ggctctcctt cttgcagttc accaacatga actccatgcg caacctgttc 1260
atcgtcggcg tgtccatctt ccttggctta tccgtgccgg agtacttctt ccgctacagc 1320
atggccgccc agcgtggtcc tgcgcacact aaagctggat ggttcaacga ctacatcaac 1380
accatcttct cgtcgccgcc gacggtgggg ctgatggtgg ccgtgttcct ggacaacacg 1440
ctggaggtga aggacgccgg cagggaccgc gggatgccgt ggtgggtgcc cttccgctcc 1500
ttcaaggggg acagcaggaa cgaggagttc tacagcctcc cgttcaacct caaccgcttc 1560
ttcccgccgt cgtaa 1575
<210> 2
<211> 524
<212> PRT
<213> 大麦(Hordeum vulgare L.)
<400> 2
Met Ser Glu Val Lys Pro Glu Glu Ile Ser His Pro Ala Met Glu Gln
1 5 10 15
Leu Gln Gly Phe Glu Tyr Cys Ile Asp Ser Asn Pro Pro Trp Gly Glu
20 25 30
Ala Ile Ile Leu Gly Phe Gln His Tyr Ile Leu Ala Leu Gly Thr Ala
35 40 45
Val Met Ile Pro Ala Val Leu Val Pro Met Met Gly Gly Ser Asp Gly
50 55 60
Asp Arg Val Arg Val Val Gln Thr Leu Leu Phe Val Thr Gly Ile Asn
65 70 75 80
Thr Leu Leu Gln Ser Leu Phe Gly Thr Arg Leu Pro Thr Val Ile Gly
85 90 95
Gly Ser Tyr Ala Phe Val Val Pro Val Met Ala Ile Val Gln Asp Ser
100 105 110
Ser Leu Ala Ala Ile Pro Asp Asp His Glu Arg Phe Leu Gln Ser Met
115 120 125
Arg Ala Ile Gln Gly Ala Leu Ile Val Ser Ser Ser Ile Gln Ile Ile
130 135 140
Leu Gly Tyr Ser Gln Leu Trp Gly Ile Phe Ser Arg Phe Phe Ser Pro
145 150 155 160
Leu Gly Met Ala Pro Val Val Ala Leu Leu Gly Phe Gly Leu Phe Glu
165 170 175
Arg Gly Phe Pro Val Val Gly Arg Cys Val Glu Val Gly Leu Pro Met
180 185 190
Leu Ile Leu Phe Val Val Leu Ser Gln Tyr Leu Lys Asn Val Gln Ile
195 200 205
Arg Glu Ile Pro Ile Leu Glu Arg Phe Ser Leu Phe Ile Cys Ile Ala
210 215 220
Leu Val Trp Ala Tyr Ala Gln Ile Leu Thr Ser Gly Gly Ala Tyr Asn
225 230 235 240
His Ser Thr Glu Ile Thr Gln Ile Asn Cys Arg Thr Asp Arg Ala Asn
245 250 255
Leu Ile Ser Ser Ala Pro Trp Ile Lys Ile Pro Tyr Pro Leu Gln Trp
260 265 270
Gly Ala Pro Thr Phe Ser Ala Gly Gln Ser Phe Gly Met Val Ser Ala
275 280 285
Val Leu Val Ser Leu Ile Glu Ser Thr Ala Ser Tyr Ser Ala Ala Ser
290 295 300
Arg Leu Ala Ser Ala Thr Pro Pro Pro Ala His Ile Leu Ser Arg Gly
305 310 315 320
Ile Gly Trp Gln Gly Ile Gly Ile Leu Leu Cys Gly Leu Phe Gly Thr
325 330 335
Gly Thr Gly Ser Thr Val Ser Val Glu Asn Val Gly Leu Leu Gly Ser
340 345 350
Thr Arg Ile Gly Ser Arg Arg Val Ile Gln Ile Cys Ala Gly Phe Met
355 360 365
Ile Phe Phe Ser Met Leu Gly Lys Phe Gly Ala Leu Phe Ala Ser Ile
370 375 380
Pro Phe Thr Ile Phe Ala Ala Val Tyr Cys Val Leu Phe Gly Leu Val
385 390 395 400
Ala Ala Val Gly Leu Ser Phe Leu Gln Phe Thr Asn Met Asn Ser Met
405 410 415
Arg Asn Leu Phe Ile Val Gly Val Ser Ile Phe Leu Gly Leu Ser Val
420 425 430
Pro Glu Tyr Phe Phe Arg Tyr Ser Met Ala Ala Gln Arg Gly Pro Ala
435 440 445
His Thr Lys Ala Gly Trp Phe Asn Asp Tyr Ile Asn Thr Ile Phe Ser
450 455 460
Ser Pro Pro Thr Val Gly Leu Met Val Ala Val Phe Leu Asp Asn Thr
465 470 475 480
Leu Glu Val Lys Asp Ala Gly Arg Asp Arg Gly Met Pro Trp Trp Val
485 490 495
Pro Phe Arg Ser Phe Lys Gly Asp Ser Arg Asn Glu Glu Phe Tyr Ser
500 505 510
Leu Pro Phe Asn Leu Asn Arg Phe Phe Pro Pro Ser
515 520
<210> 3
<211> 427
<212> DNA
<213> 大麦(Hordeum vulgare L.)
<400> 3
gaggattccc tgtggttggg agatgcgttg aggttggcct gccaatgtta atcctctttg 60
ttgtgctttc ccagtatctg aagaatgtac agataagaga gatccccata ctggaacgat 120
tctcgctgtt catttgcatc gcattggtct gggcctatgc tcaaatcctc acttctggtg 180
gcgcgtataa tcatagcacc gagatcactc agatcaactg ccgcactgac cgtgccaatc 240
tcatctcttc cgccccatgg atcaagatcc catatcctct gcaatggggg gcaccaacct 300
tcagcgcagg ccaatcgttc ggtatggtat ctgcggtttt ggtctcgcta atagagtcca 360
cagcttctta cagcgccgca tctcggcttg caagtgcaac tccacctcca gcccatatcc 420
tgagcag 427
<210> 4
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgtcggagg tcaagccgg 19
<210> 5
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ttacgacggc gggaagaag 19
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tttggcgttt ctcaagggtg 20
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ggcttgacct ccgacatttc 20
<210> 8
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gcaggtaaat ggaatcggca tc 22
<210> 9
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
taaccccacg ttctccactg a 21
<210> 10
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
aagcatgaag atacagggag tgtg 24
<210> 11
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
aaatttattc tcggaagagg ttgtaca 27
<210> 12
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ggggacaagt ttgtacaaaa aagcaggcta tgtcggaggt caagccgg 48
<210> 13
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ggggaccact ttgtacaaga aagctgggtt tacgacggcg ggaagaag 48
<210> 14
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ggggacaagt ttgtacaaaa aagcaggctg aggattccct gtggttggg 49
<210> 15
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
ggggaccact ttgtacaaga aagctgggtc tgctcaggat atgggctgg 49
<210> 16
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gcatatgcag cagctatatg tg 22
<210> 17
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ttacgacggc gggaagaag 19
<210> 18
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
tacggcgtgg atacgttagc 20
<210> 19
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
ctgctcagga tatgggctgg 20
Claims (7)
1.大麦HvNAT2基因在调控大麦对镉胁迫耐受性方面的应用,其特征在于,该基因的CDS区核苷酸序列如SEQ ID NO.1所示。
2.如权利要求1所述的应用,其特征在于,所述大麦HvNAT2基因编码的蛋白质的氨基酸序列如SEQ ID NO.2所示。
3.如权利要求1所述的应用,其特征在于,大麦HvNAT2基因过表达使植株耐镉性增强;大麦HvNAT2基因沉默使植株对镉的耐受性降低。
4.如权利要求3所述的应用,其特征在于,所述应用包括:将所述大麦HvNAT2基因插入过表达载体中构建重组质粒,然后利用农杆菌介导技术将目的片段转入受体大麦中,筛选获得功能性获得的转基因植株。
5.如权利要求4所述的应用,其特征在于,所述过表达载体为pBract214。
6.如权利要求4所述的应用,其特征在于,农杆菌介导遗传转化材料采用大麦幼胚。
7.如权利要求4所述的应用,其特征在于,所述受体大麦的品种为黄金希望。
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