CN105671074B - 一种提高植物甲硫氨酸含量的载体及其构建方法和用途 - Google Patents
一种提高植物甲硫氨酸含量的载体及其构建方法和用途 Download PDFInfo
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Abstract
本发明提供了一种可提高玉米等植物中甲硫氨酸和/或半胱胺酸含量的表达载体,该载体通过采用Rbcs启动子介导的拟南芥丝氨酸乙酰转移酶基因在作物中的过量表达,使丝氨酸乙酰转移酶完成了维管束鞘细胞特异性表达,从而提高了玉米籽粒中总甲硫氨酸和半胱胺酸的含量,并提高了富含甲硫氨酸的10 kDa‑zein含量和植株中的谷胱甘肽含量。本发明还提供了该载体的构建方法及用途,以及使用该载体培育具有高甲硫氨酸和半胱胺酸含量的转基因玉米的方法。
Description
技术领域:本发明属于植物分子育种领域,具体提供了一种表达载体及其用途和构建方法,该载体可提高植物甲硫氨酸和谷氨酰胺含量。
背景技术:
蛋白短缺包括蛋白含量短缺和营养品质低下,是21世纪全球性的严重问题,在尽快提高作物;品种的蛋白质含量的同时,积极改善蛋白质的氨基酸组成,提高营养价值,具有重要意义。
硫是仅次于氮、磷、钾的植物必需的第4大营养元素,缺硫时蛋白质合成受阻,导致非蛋白氮积累,不仅影响作物生长,而且降低农产品;品质;植物含硫量为0.1%-0.5%,其变幅明显受植物种类、品种、器官和生育期影响。十字花科植物需硫最多,豆科、百合科植物次之,禾本科植物较少。含硫氨基酸如甲硫氨酸(也称蛋氨酸,methionine,Met)、胱氨酸、半胱氨酸(cysteine,Cys)等是合成蛋白质的重要氨基酸;蛋白质中含硫氨基酸的含量与种子萌发、生长和作物品种均有密切关系。其中,蛋氨酸作为人体和单瘤胃动物的必需氨基酸之一,在水稻、小麦、大麦、荞麦、玉米,尤其是豆类和玉米等主要的粮食作物和经济作物中含量极低,是主要的限制性氨基酸,因此提高这些作物蛋氨酸含量显得尤为重要。
蛋氨酸是动物生长所必需的氨基酸之一,在动物生长发育和新陈代谢中需要大量的蛋氨酸参与,而其又是大豆饼粕等饲料原料中最易缺乏的一种氨基酸。作为禽类的第一限制性氨基酸,蛋氨酸可以起到促进家禽生长、改善胴体品质、提高机体免疫力和抗氧化功能等效果。
在以大豆饼粕为饲料时,必需添加人工合成的甲硫氨酸。目前市场供应的饲料级蛋氨酸采取化学合成的方法生产,石油深加工产品是其生产的主要前体原料,国际原油价格的跌涨在一定程度上影响蛋氨酸的价格起伏。随着蛋氨酸应用技术的成熟,添加比例增加,蛋氨酸需求逐年增长。截止2015年1月,全球蛋氨酸供应厂家达到7家,生产工厂达到13座,拟建工厂2座,停产工厂1座。特别是亚洲地区,蛋氨酸厂家供应虽然增加,但在集中度较高的前提下,重大突发性事件仍会造成供应短期缺口,从而导致价格上涨。2014年,蛋氨酸主要由欧洲和北美洲供应。
蛋氨酸人工合成需要量较大,而且由于化学工艺复杂,对环境保护造成威胁,且生产成本较高以及市场需求量大,供不应求,导致价格虚高,提高了饲料养殖家禽的成本。
蛋氨酸作为植物含硫氨基酸,在植物生长发育过程中起重要作用。植物和微生物通过吸收土壤中的无机硫成分,合成仅有的两个含硫氨基酸半胱氨酸和蛋氨酸。植物将吸收的+6硫酸盐还原后,形成半胱胺酸含有-2的硫。植物中,从硫的吸收还原到半胱胺酸的合成需要5种酶来完成,如下所示:
蛋氨酸代谢途径
首先,ATP硫酸化酶生成5’磷酸腺苷酸硫酸(APS)。然后以谷胱甘肽为底
物,在5’-磷酸腺苷酸硫酸还原酶(5'-adenylylsulfate reductase,APR)的作用下,由APS生成亚硫酸盐(SO3 2-)。
植物中PAPS是其它硫化物的底物,而以PAPS为底物进行硫的还原,就意味着在具有3’磷酸腺苷酰硫酸还原酶(3'-phospho-5'-adenylylsulfate reductase,PAPR)的同时还需要有APS激酶(APK)来完成硫的还原。然后在亚硫酸还原酶作用下生成硫化物(S2-),这个过程在大肠杆菌中比较普遍。
在OAS-TL的作用下,硫化物和OAS一起生成半胱氨酸。OAS是由丝氨酸乙酰转移酶(serine acetyltransferase,SAT)合成的。半胱氨酸除直接生成蛋白质外,大部分用来合成甲硫氨酸和谷胱甘肽。
提高重要经济作物中的蛋氨酸含量也为食品和工业原料方面节省成本。在提高经济作物中蛋氨酸的含量方面,前人已有研究,在拟南芥中过量表达拟南芥CGS(AtCGS)使叶片中的可溶性蛋氨酸提高了6.2倍,在烟草中提高12.8倍,窄叶羽扇豆中过量表达拟南芥丝氨酸转移酶提高了豆荚的自由半胱氨酸含量。在玉米中过量表达富含蛋氨酸的10-kDamRNA,提高了籽粒蛋氨酸含量,同时过量表达蛋氨酸合成途径中关键酶基因3’磷酸腺苷酰硫酸还原酶(PAPR)和5’磷酸腺苷酸硫酸还原酶(APR)提高了玉米中含硫氨基酸的含量,但植株表现不正常。丝氨酸转移酶作为蛋氨酸合成前体半胱氨酸的合成途径中的关键酶,在蛋氨酸的合成中起到关键作用。在马铃薯中过量表达大肠杆菌丝氨酸转移酶,使其半胱氨酸和谷胱甘肽的含量提高;在水稻中过量表达大肠杆菌丝氨酸转移酶使水稻中的蛋氨酸和半胱氨酸含量提高。
美国专利US 7560623使用农杆菌介导玉米丝氨酸转移酶和ATP硫化酶分别在玉米泛素启动子ubiquitin启动下,以pin 11为终止子完成在玉米HiII中的表达,采用除草剂为筛选标记,农杆菌浸染玉米HiII幼胚,获得转基因株系中玉米干物质中半胱氨酸提高了19%,蛋氨酸提高了63%。其缺点是,该发明用组成型启动子,表达产物在其它组织积累可能导致植株畸形,生长缓慢,叶片发黄,接受率低等缺点。该方法主要提高了玉米干物质中的蛋氨酸含量,而本发明采用组织特异性启动子,植株能够正常生长,结实率高,并提高了谷胱甘肽的含量,间接提高植株的抗逆抗氧化能力。
由于在人工驯化的过程中,甲硫氨酸的含量被忽略。甲硫氨酸在谷类和豆类作物中都严重缺乏。由于缺乏甲硫氨酸,这些作物的营养价值降低了50%-70%。这些必需氨基酸的缺乏,会导致人们产生一系列蛋白缺乏症,比如抵抗力下降,血压降低,智力迟钝,儿童早熟。这些并发症以下简称蛋白质能量营养不良(PEM),世界卫生组织(WHO)估计发展中国家总人口的大概30%都有这个并发症。
随着人们生活水平的提高,在育种中不但要获得高产稳产抗病的新品种,品质的提高也显得越来越重要。传统育种提高玉米蛋氨酸水平未能取得成功。正如黄金大米等功能型食品的问世,也为育种提供了新思路和新方向。
因此,为了能使玉米中甲硫氨酸水平达到世界卫生组织公布的5%,提高玉米籽粒中富含甲硫氨酸的蛋白(18kDa和10kDaδ-zein)的含量,本发明技术方案可有效实现高甲硫氨酸玉米育种的相关工作,获得高品质的玉米。
发明内容
本研究以蛋氨酸代谢途径入手,以源流库为理论基础,采用基因工程的操作手段,从拟南芥中克隆了丝氨酸转移酶基因(AtSAT1),以1,5-二磷酸核酮糖羧化酶小亚基(Rbcs)为启动子,通过农杆菌介导玉米HiII幼胚遗传转化,首次获得了甲硫氨酸含量提高的转基因玉米HiII,并以B73为父本,进行两次回交,获得了甲硫氨酸提高的B73材料。达到了通过调控甲硫氨酸的生物合成量来提高玉米中总体的甲硫氨酸水平的目的。籽粒中自由甲硫氨酸含量提高,为富含甲硫氨酸的储藏蛋白18kDaδ-zein和10kDaδ-zein提供更多的自由甲硫氨酸,使籽粒的整体甲硫氨酸含量提高,从而获得高甲硫氨酸突变体材料。由于启动子的组织特异性,避免了含硫产物在其它器官的过多积累而使植株中毒。籽粒中没有外源蛋白的积累,保证了转基因的生物安全性,从而获得具有高甲硫氨酸玉米新材料,为玉米品质育种提供理论依据。
本发明提供了一种载体,也称表达载体或人工序列,其核苷酸序列为:将序列表SEQ ID NO.14所示的核苷酸序列的第8621位至11200位的核苷酸替换为序列表SEQ IDNO.12所示的核苷酸后的核苷酸序列。该载体可命名为pTF102/RbcS1P:AtSAT1。
本发明还提供了上述两种载体提高植物甲硫氨酸和/或半胱胺酸含量的用途;进一步的,该载体为通过转染农杆菌、导入植物;所述农杆菌优选为根癌农杆菌;所述植物优选为玉米。
本发明提供了上述载体的构建方法,包括以下步骤:
(1)克隆启动子RbcS1,将启动子RbcS1克隆至载体pMD19-T上,得载体pMD19-T:RbcS1,其核苷酸序列如序列表SEQ ID NO.9所示;
(2)从拟南芥中克隆丝氨酸转移酶基因AtSAT1,将AtSAT1克隆到pMD19-T载体上,得载体pMD19-T:AtSAT1,其核苷酸序列如序列表SEQ ID NO.10所示;
(3)取表达载体pTF102,限制性内切酶BamH I和Sac I酶切载体pTF102,用限制性内切酶BamH I和Sac I酶切载体pMD19-T:AtSAT1;用T4Ligase将表达载体pTF102的酶切产物与载体pMD19-T:AtSAT1的酶切产物连接,得载体pTF102:AtSAT1,其核苷酸序列如序列表SEQ ID NO.11所示;
(4)用限制性内切酶EcoR I和Bam HI酶切载体pTF102:AtSAT1,用限制性内切酶Mfe I和Bcl I酶切载体pMD19-T:RbcS1,用T4Ligase将载体pTF102:AtSAT1的酶切产物与载体pMD19-T:RbcS1的酶切产物连接,得载体pTF102/RbcS1P:AtSAT1,其核苷酸序列如序列表SEQ ID NO.12所示。
进一步的,
步骤(1)所述克隆启动子RbcS1所用引物的核苷酸序列为序列表SEQ ID NO.3、SEQID NO.4所示;;
步骤(2)所述从拟南芥中克隆丝氨酸转移酶基因AtSAT1所用引物的核苷酸序列为序列表SEQ ID NO.1、SEQ ID NO.2所示。
本发明还提供了一种转基因玉米的培育方法,包括以下步骤:
(1)将上述载体导入农杆菌菌株中,活化,备用;
(2)取授粉13天左右的玉米HiII杂交种幼胚(1.5–2.0mm),用预先活化的农杆菌浸染幼胚;
(3)吸去菌液,把幼胚转移到共培养基上;20℃共培养三天后转到恢复培养基上,28℃黑暗培养7天之后把有生长能力的幼胚转到第一次筛选培养基上,28℃黑暗培养2周。再转到第二次筛选培养基上,黑暗条件下培养2周;把生长良好的愈伤组织转到第一次分化培养基上进行分化,28℃黑暗分化3周,再转到24℃光照分化,得转基因玉米植株。
具体的,步骤(3)所述的浸染液和培养基配方如下:
YEP培养基配方:5g L-1酵母提取物,10g L-1蛋白胨,5g L-1氯化钠,15g L-1琼脂粉,pH 6.8,培养基冷到50℃后加抗生素;
浸染液配方:N6培养基盐和维生素,1.5mg L-1的2,4-D-二氯苯氧乙酸丁酯,0.7gL-1L-脯氨酸,68.4g L-1蔗糖,36g L-1葡萄糖,pH 5.2,乙酰丁香酮(AS)终浓度为100μmol L-1;
100μmol L-1乙酰丁香酮(AS):先用二甲基亚砜溶解乙酰丁香酮(AS)配成200mmolL-1后用水1:1稀释成100mmol L-1;
共培养培养基配方:N6盐和维生素,1.5mg L-1 2,4-D-二氯苯氧乙酸丁酯,0.7g L-1L-脯氨酸,30g L-1蔗糖,3g L-1植物凝胶(gelrite),pH 5.8,0.85mg L-1硝酸银,100μmolL-1乙酰丁香酮(AS),400mg L-1半胱氨酸;
恢复培养基配方:N6培养基盐和维生素,1.5mg L-1 2,4-D-二氯苯氧乙酸丁酯,0.7g L-1L-脯氨酸,30g L-1蔗糖,0.5g 2-(4-吗啉)-乙烷磺酸(MES),3g L-1植物凝胶(gelrite),pH 5.8,0.85mg L-1硝酸银,250mg L-1噻孢霉素;
第一次筛选培养基配方:N6培养基盐和维生素,1.5mg L-1 2,4-二氯苯氧乙酸丁酯(2,4-D),0.7g L-1L-脯氨酸,30g L-1蔗糖,0.5g 2-(4-吗啉)-乙烷磺酸,3g L-1植物凝胶(gelrite),pH 5.8。0.85mg L-1硝酸银,250mg L-1噻孢霉素,1.5mg L-1双丙氨膦(Bialaphos);
第二次筛选培养基配方:N6培养基盐和维生素,1.5mg L-1 2,4-D二氯苯氧乙酸丁酯,0.7g L-1L-脯氨酸,30g L-1蔗糖,0.5g 2-(4-吗啉)-乙烷磺酸(MES),3g L-1植物凝胶(gelrite),pH 5.8,0.85mg L-1硝酸银,250mg L-1噻孢霉素,3.0mg L-1双丙氨膦(Bialaphos);
第一次分化培养基配方:乙酰丁香酮(MS)盐和维生素,60g L-1蔗糖,100mg L-1肌醇,3g L-1植物凝胶(gelrite),pH 5.8。250mg L-1噻孢霉素和3mg L-1双丙氨膦(Bialaphos)灭菌后加;
第二次分化培养基配方:乙酰丁香酮(MS)盐和维生素,100mg L-1肌醇,30g L-1蔗糖,3g L-1植物凝胶(gelrite),pH 5.8。
本发明技术路线图如说明书附图1所示。
甲硫氨酸是含硫必需氨基酸,与生物体内各种含硫化合物的代谢密切相关。本发明方案获得了具有HiII遗传背景的高蛋氨酸材料;通过杂交转育获得了B73遗传背景的高蛋氨酸材料,提高了玉米籽粒蛋白质品质。以此材料为基础,可以将其用于改良其他骨干自交系的蛋氨酸水平。通过将改良后的高蛋氨酸骨干自交系组配优良组合,获得高蛋氨酸玉米新品种。高蛋氨酸品种具有蛋白品质高,在用于作为家禽等饲料时,减少了人工合成蛋氨酸的需求量,减少了工业生产蛋氨酸的成本及所造成的环境污染。在以玉米为主食的非洲国家,由于蛋氨酸的摄入不足,普遍存在蛋氨酸缺乏症,如食欲减退、生长减缓或体重减轻、肾脏水肿和肝脏铁积累等现象,最后导致肝坏死或纤维化。本发明专利中的高蛋氨酸玉米为解决这一问题提供了创造性的新方法。
本发明通过采用Rbcs启动子介导的拟南芥丝氨酸乙酰转移酶基因在作物中的过量表达,使丝氨酸乙酰转移酶完成了维管束鞘细胞特异性表达。通过该方法提高了玉米籽粒中总甲硫氨酸和半胱胺酸的含量,并提高了富含蛋氨酸的10kDa-zein含量,以及提高了植株中谷胱甘肽含量。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想的前提下,还可以做出其他多种形式的修改、替换或者变更。
以下以实施例形式的具体实施方法,对本发明的上述内容在做进一步的详细说明,但不应理解为下述实施例用于限定本发明的保护范围。
附图说明
图1载体构建的技术路线图。
图2载体构建流程图。
图3外源基因拷贝数绝对定量分析。
其中control/对照组:含有目标片段的质粒,1,3,8为转基因T1代株系。
图4过量表达AtSAT1的玉米T2蛋白印迹分析;
其中,Ladder:蛋白标准分子量,1到10分别为转基因株系1-10,WT为B73。
图5酶活性测定。
图6转基因T2谷胱甘肽含量。
图7过量表达AtSAT1玉米T2籽粒醇溶蛋白SDS-PAGE分析。
图8 RbcS1启动子PCR扩增产物的凝胶电泳分析图谱;
其中,M:1kb plus DNA ladder,1、2:1030bp全长RbcS1启动子核酸序列。
图9菌落PCR检测目的基因RbcS1;
其中,M:1kb plus DNA ladder,1-5:含有目的片段的抗性克隆,6:阴性对照。
图10目的基因AtSAT1的克隆;
其中,M:1kb plus DNA ladder,1-2:1、2:840bp全长AtSAT1核酸序列
图11菌落PCR检测目的基因AtSAT1;
其中,M:1kb plus DNA ladder,1-5:含有目的片段的抗性克隆,6:阴性对照。选3,6号克隆测序。
图12 pTF102:AtSAT1的构建;
其中,M:1kb plus DNA ladder,1:pMD19-T-AtSAT1质粒用Bam H I和Sac I酶切,2:用Bam H I和Sac I酶切pTF102质粒。
图13.pTF102/RbcS1P:AtSAT1酶切实验结果;
其中,M:1kb plus DNA ladder,1:用EcoR I和BamH I酶切pTF102:AtSAT1,2:用Mfe I和Bcl I酶切pMD19-T-RbcS1。
图14转入AtSAT1基因后的阳性转基因植株电泳鉴定图谱;
其中,M:1kb plus DNA ladder,1-12:转基因株系,WT:野生型玉米B73。
以下为具体实施方式
实施例一目的基因克隆及载体pTF102/RbcS1P:AtSAT1的构建
1、载体pMD19-T:RbcS1的构建
所用启动子Bundle sheath-specific Rubisco small subunit 1(RBCS1gene)简称RbcS1(GenBank登录号:AH005359.3,序列见http://www.ncbi.nlm.nih.gov/nuccore/339635306),该启动子来自玉米,长1030bp,(Sattarzadeh A,Fuller J,Moguel S,etal.Transgenic maize lines with cell-type specific expression of fluorescentproteins in plastids[J].Plant biotechnology journal,2010,8(2):112-125.)。该启动子后面有约20bp的信号肽,使表达的蛋白靶向维管束鞘细胞,实现组织细胞特异表达,避免了外源蛋白在非功能组织积累对植株的毒害。使用引物(引物核苷酸序列为序列表SEQID NO.3和SEQ ID NO.4的核苷酸序列所示,引物前端已分别添加了酶切位点Mfe I和BclI),按照本领域常用的方法,以玉米cDNA为模板,PCR反应体系如下:94℃2min,94℃20sec,58℃30sec and72℃60sec,32cycles;72℃6min,10℃till end;取6μl做琼脂糖凝胶电泳。对RbcS1启动子分别进行两次PCR扩增,扩增产物1、2的凝胶电泳分析结果如说明书附图8所示;实验结果表明扩增得到了启动子RbcS1。使用转化甲基转移酶缺失的E.coli感受态细胞(适用于无dam和dcm甲基化),涂布在含有50mg/L链霉素的LB平板上。用单克隆RbcS1,做菌液PCR,并送阳性克隆测序,实验结果见说明书附图9所示。所有使用到Mfe I限制性内切酶的连接产物都只能转化甲基转移酶缺失的E.coli感受态细胞。
将启动子RbcS1通过T-A克隆至pMD19-T载体(pMD19-T载体的核苷酸序列如序列表SEQ ID NO.13的核苷酸序列所示,购买公司及货号为:TaKaRa,D102A),克隆使用的连接体系如下表1所示,16℃连接过夜。构建好的载体命名为pMD19-T:RbcS1,经测序,其核苷酸序列为将序列表SEQ ID NO.9所示的核苷酸插入pMD19-T载体的第431位形成的核苷酸序列。pMD19-T是通过T-Aclone连入新片段,不需要酶切,该质粒商品本身在430bp处有缺口,末端为碱基T,可以和PCR产物两端的A碱基配对,从而在T4连接酶的作用下连接,完成插入新片段。
表1 T4连接酶连接体系
2、载体pMD19-T:AtSAT1的构建
(1)目的基因AtSAT1的克隆
Arabidoposis thaliana SAT1,简称AtSAT1(Serat2;1,At1g55920,从拟南芥中克隆,全名为Arabidopsis thaliana serine acetyltransferase,其序列见http:// www.arabidopsis.org/servlets/TairObject?type=locus&name=At1g55920)。以拟南芥cDNA为模板,引物为SEQ ID NO.1和SEQ ID NO.2,CDS区长约828bp,N端加上Rbcs信号肽,以使目的基因在维管束鞘中特异表达。PCR反应体系如下:94℃2min,94℃20sec,58℃30secand 72℃60sec,32cycles;72℃6min,10℃till end;取6μl做琼脂糖凝胶电泳。对克隆产物进行菌落PCR检测,检测结果如说明书附图11所示
(2)克隆AtSAT1(828bp)的5’端引物(SEQ ID NO.1)已加BamH I酶切位点,3’端引物(SEQ ID NO.2)已加Sac I酶切,用于后期酶切连入pTF102,首先通过T-A克隆到pMD19-T载体(TaKaRa Code:D102A)上,命名为pMD19-T:AtSAT1,如图10,并送genewiz公司测序(http://www.genewiz.com/),经测序,其核苷酸序列为将序列表SEQ ID NO.10所示的核苷酸插入pMD19-T载体的第431位形成的核苷酸序列。
3、载体pTF102:AtSAT1的构建
将pTF102表达载体(pTF102表达载体的核苷酸序列如序列表SEQ ID NO.14所示)和pMD19-T:AtSAT1载体分别用BamH I和Sac I酶切,T4Ligase连接,命名为pTF102:AtSAT1,其核苷酸序列为将pTF102载体的第8621位至第11179位的核苷酸序列直接替换为序列表SEQ ID NO.11所示的核苷酸后,形成的核苷酸序列。所示。经酶切鉴定实验,实验结果如说明书附图12.
酶切体系如表2所示
表2 BamH I和Sac I酶切酶切体系
注:37℃酶切3小时。
T4Ligase连接酶体系如表3所示:
表3 T4连接酶连接体系
注:16℃连接过夜。
4、新载体pTF102/RbcS1P:AtSAT1的构建
用EcoR I和Bam H I酶切步骤3载体pTF102:AtSAT1(酶切体系如下表4所示),用Mfe I和Bcl I酶切步骤1制备载体pMD19-T:RbcS1,T4Ligase连接构建好的载体命名为pTF102/RbcS1P:AtSAT1。经酶切方法鉴定和测序,该载体的核苷酸序列为将pTF102:AtSAT1载体的第9449位至第10372位的核苷酸直接替换为序列表SEQ ID NO.9所示的核苷酸后形成的核苷酸序列,也即是将序列表SEQ ID NO.14所示的核苷酸序列的第8621位至11200位的核苷酸替换为序列表SEQ ID NO.12所示的核苷酸序列后的核苷酸序列后的核苷酸序列。
表明经过本发明的构建方法得到所述载体人工序列。酶切鉴定的实验结果见说明书附图13所示。
表4 Eco R I和Sac I酶切酶切体系
37℃酶切3小时。
Mfe I*和Bcl I酶切酶切体系
37℃酶切3小时,加限制性内切酶Bcl I(20,000units/ml)0.5μl,50℃
酶切3小时。连入启动子RbcS1,同3.1.2.2第3步。构建好的载体命名为pTF102/RbcS1P:AtSAT1。
具体构建流程图见图2。
实施例二转基因株系的制备
1、转基因株系的获得
将实施例一制备的表达载体pTF102/RbcS1P:AtSAT1,导入常用的根癌农杆菌菌株;取授粉13天左右的玉米HiII杂交种幼胚(1.5–2.0mm),用预先活化的农杆菌浸染幼胚。吸去菌液,把幼胚转移到共培养基上。20度共培养三天后转到恢复培养基上。28度黑暗培养7天之后把有生长能力的幼胚转到第一次筛选培养基上,28度黑暗培养2周。再转到第二次筛选培养基上,黑暗条件下培养2周。把生长良好的愈伤组织转到第一次分化培养基上进行分化,28度黑暗分化3周。再转到24度光照分化成植株。
2、外源DNA检测
(1)植物基因组DNA提取
DNA提取缓冲液:100mM Tris-HCl pH 8.5,100mM NaCl,20mM EDTA pH 8.0,1%sarkosyl(N-Lauroylsarcosine)
步骤如下:
a)取1cm×2cm大小的叶片于2ml离心管中,加入两颗钢珠。
b)将其在液氮中速冻。
c)用植物组织研磨仪将叶片磨成细粉。
d)加入600μl提取液,混合充分。
e)加入600μl酚/氯仿(v/v 1:1),混合均匀。
f)13,000r/min离心10min。
g)将200μl上清液转入另外一个干净的1.5μl离心管。
h)加入20μl醋酸钠(3M,pH5.2),140μl异丙醇,混匀后冰上放置10min。
i)13,000r/min离心12min,倒掉上清液。
j)用1ml 70%的酒精清洗DNA,13,000r/min离心5min,倒掉酒精。
k)将离心管倒置于实验台上20min,风干DNA。
l)将DNA溶于100μl灭菌蒸馏水中。
(2)PCR检测转基因植株
PCR反应液的配制(25μl):12.5μl SIGMA Red Taq+1μl Primers mix+10.5μl无菌水。最后加入1μl上述DNA。所用引物为SEQ ID NO.5,SEQ ID NO.6。
PCR反应体系如下:
94℃2min,94℃20sec,58℃30sec and 72℃35sec,32cycles;72℃1min,10℃tillend;取6μl做琼脂糖凝胶电泳。
凝胶电泳的实验结果见说明书附图14所示电泳图如下:
转化效率见表5。
表5转化效率统计
a转化效率为:(PCR检测阳性的转化事件数/所浸染的幼胚数)*100%。
所述的浸染液和培养基配方如下:
YEP培养基配方:5g L-1酵母提取物,10g L-1蛋白胨,5g L-1氯化钠,15g L-1琼脂粉,pH 6.8,培养基冷到50℃后加抗生素;
浸染液配方:N6培养基盐和维生素,1.5mg L-1的2,4-D-二氯苯氧乙酸丁酯,0.7gL-1L-脯氨酸,68.4g L-1蔗糖,36g L-1葡萄糖,pH 5.2,乙酰丁香酮(AS)终浓度为100μmol L-1;
100μmol L-1乙酰丁香酮(AS):先用二甲基亚砜溶解乙酰丁香酮(AS)配成200mmolL-1后用水1:1稀释成100mmol L-1;
共培养培养基配方:N6培养基盐和维生素,1.5mg L-1 2,4-D-二氯苯氧乙酸丁酯,0.7g L-1L-脯氨酸,30g L-1蔗糖,3g L-1植物凝胶(gelrite),pH 5.8,0.85mg L-1硝酸银,100μmol L-1乙酰丁香酮(AS),400mg L-1半胱氨酸;
恢复培养基配方:N6培养基盐和维生素,1.5mg L-1 2,4-D-二氯苯氧乙酸丁酯,0.7g L-1L-脯氨酸,30g L-1蔗糖,0.5g 2-(4-吗啉)-乙烷磺酸(MES),3g L-1植物凝胶(gelrite),pH 5.8,0.85mg L-1硝酸银,250mg L-1噻孢霉素;
第一次筛选培养基配方:N6培养基盐和维生素,1.5mg L-1 2,4-二氯苯氧乙酸丁酯(2,4-D),0.7g L-1L-脯氨酸,30g L-1蔗糖,0.5g 2-(4-吗啉)-乙烷磺酸,3g L-1植物凝胶(gelrite),pH 5.8。0.85mg L-1硝酸银,250mg L-1噻孢霉素,1.5mg L-1双丙氨膦(Bialaphos);
第二次筛选培养基配方:N6培养基盐和维生素,1.5mg L-1 2,4-D二氯苯氧乙酸丁酯,0.7g L-1L-脯氨酸,30g L-1蔗糖,0.5g 2-(4-吗啉)-乙烷磺酸(MES),3g L-1植物凝胶(gelrite),pH 5.8,0.85mg L-1硝酸银,250mg L-1噻孢霉素,3.0mg L-1双丙氨膦(Bialaphos);
第一次分化培养基配方:乙酰丁香酮(AS)盐和维生素,60g L-1蔗糖,100mg L-1肌醇,3g L-1植物凝胶(gelrite),pH 5.8。250mg L-1噻孢霉素和3mg L-1双丙氨膦(Bialaphos)灭菌后加;
第二次分化培养基配方:乙酰丁香酮(AS)盐和维生素,100mg L-1肌醇,30g L-1蔗糖,3g L-1植物凝胶(gelrite),pH 5.8。
(2)获得转基因T0代株系38个,将表达酶活性高的株系的T1代种子播种后以B73为父本测交,收获种子为T2代,将T2代播种后,以B73为父本测交,获得的T3代种子用于测量蛋氨酸含量。
2、实验结果与分析
(1)外源基因拷贝数分析-绝对定量PCR(QPCR)
选取表现良好的转基因T1代株系1,3,8,PCR引物见表1,采用美国应用生物系统公司7300/7500实时定量PCR仪,方法参照7300/7500实时定量PCR仪的操作指南;PCR循环如下:94℃2min;94℃20sec,58℃30sec,72℃35sec,32cycles;72℃1min。株系1为单拷贝,株系3和8为多拷贝,见说明书附图3所示。所用引物为序列表SEQ ID NO.5、SEQ ID NO.6、SEQID NO.7、SEQ ID NO.8所示。
(2)外源蛋白含量分析
随机挑选转基因T2代AtSAT1过量表达株系若干,待植株长到一个月大小取叶片进行分析,每个样品都用20μg蛋白,用7.5%SDS-PAGE电泳,一抗稀释比例为1:200,具体操作方法参照本领域常规方法进行。
实验分析结论,见说明书附图4所示。
转基因株系中目的蛋白有不同量的积累,大小约为29.8kDa,而在野生型B73中,目的蛋白处有植物本身蛋白的表达,表达量较小。由此可见,导入目的基因AtSAT1已成功翻译成相应蛋白质。经检测,AtSAT1基因编码蛋白的氨基酸序列为序列表SEQ ID NO.15的氨基酸序列所示。
(3)外源基因酶活性测定
按照Nguyen HC方法(Nguyen HC,Hoefgen R,Hesse H.Improving the nutritivevalue of rice seeds:elevation of cysteine and methionine contents in riceplants by ectopic expression of a bacterial serine acetyltransferase[J].Journal of experimental botany,2012,63(16):5991-6001.)、Blaszczyk A方法(Blaszczyk A,Brodzik R,Sirko A.Increased resistance to oxidative stress intransgenic tobacco plants overexpressing bacterial serine acetyltransferase[J].The Plant journal:for cell and molecular biology,1999,20(2):237-243.)、分别测T1和T2中随机几个株系的SAT酶活性,测得的酶活性见说明书附图5,每个转化事件取三个植株测定。由图5可知,野生型对照B73的酶活性为3.29nmol/min/mg。而所以含AtSAT1的转基因株系其SAT酶活性基本都比B73高。此处测定的SAT酶活性,包括玉米中内源的SAT酶活性和转入的AtSAT1的酶活性,所以转基因株系中的SAT酶活性是相对于野生型的酶活性。
(4)叶片中谷胱甘肽含量测定
在植物中,谷胱甘肽是硫酸盐还原的主要方式之一。对过量表达AtSAT1基因的玉米叶片的谷胱甘肽含量进行了测定发现:和非转基因野生型玉米相比,转基因玉米叶片中的谷胱甘肽提高到2.5倍。如说明书附图6所示,T1代株系1(658.7±159.9nmol g-1FW),株系8(543.1±17.4nmol g-1FW)均比野生型B73植物中的谷胱甘肽水平(322.2±7.4nmol g-1FW)高。
(5)籽粒醇溶蛋白分析
对收获的过量表达AtSAT11的转基因株系,1,3,4,5,6,7,8以及野生型HiII hybid(B×A),进行了醇溶蛋白(zein)SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析。大多数玉米自交系都含有较低水平的10kDa-zein,而富含蛋氨酸的10kDa-zein和自由蛋氨酸共同决定了籽粒蛋氨酸水平。以不同10kDa-zein的玉米自交系(BSSS53,Mo17,B73,A188和A654)为对照,如说明书附图7可见,在其它醇溶蛋白水平基本一致的情况下,以含量稳定的19kDazein为内参,转基因株系中10kDa-zein明显比左边自交系含量高。
M:Protein ladder,B×A,1,3,4,5,6,7,8分别为野生型HiII,过量表达AtSAT1的玉米转基因株系T2代,每个样品加入的蛋白来自1000mg成熟干籽粒(Wu Y,Wang W,MessingJ.Balancing of sulfur storage in maize seed[J].BMC plant biology,2012,12:77.)。
(6)籽粒总氨基酸含量测定
选取种质资源野生型B73,转基因株系1(TG line 1)两个样品各20g籽粒,送到NJFL(New Jersey Feed Lab)http://www.njfl.com/,测籽粒的总氨基酸含量及甲硫氨酸和半胱氨酸含量(测定方法见Wu Y,Wang W,Messing J.Balancing of sulfur storage inmaize seed[J].BMC plant biology,2012,12:77.)。由表6可知,在非转基因野生型B73种子中,赖氨酸,甲硫氨酸和半胱氨酸相对含量分别是2.61%,1.92%和1.92%;而本发明过量表达AtSAT1的转基因籽粒中,分别为2.80%,3.06%和2.45%。表明和野生型相比,转基因玉米成熟籽粒中甲硫氨酸和半胱胺酸含量显著,而赖氨酸确没有变化。和B73相比,在甲硫氨酸和半胱胺酸的含量提高的同时,过量表达AtSAT1或EcPAPR的玉米籽粒中其它氨基酸的组成没有发生明显变化。
表6籽粒的总氨基酸含量及甲硫氨酸和半胱氨酸含量
备注:AAab为氨基酸绝对含量,AArel为氨基酸相对含量。
Claims (3)
1.一种载体在提高玉米甲硫氨酸和/或半胱胺酸含量的用途,所述载体的核苷酸序列为:将序列表SEQ ID NO.14所示的核苷酸序列的第8621位至11200位的核苷酸替换为序列表SEQ ID NO.12所示的核苷酸后的核苷酸序列。
2.一种转基因玉米的培育方法,其特征在于,包括以下步骤:
(1)将载体导入农杆菌菌株中,活化,备用;所述载体的核苷酸序列为:将序列表SEQ IDNO.14所示的核苷酸序列的第8621位至11200位的核苷酸替换为序列表SEQ ID NO.12所示的核苷酸后的核苷酸序列;
(2)取授粉13天的玉米HiII杂交种幼胚,用预先活化的农杆菌浸染幼胚;
(3)吸去菌液,把幼胚转移到共培养基上;20℃共培养三天后转到恢复培养基上,28℃黑暗培养7天之后把有生长能力的幼胚转到第一次筛选培养基上,28℃黑暗培养2周,再转到第二次筛选培养基上,黑暗条件下培养2周;把生长良好的愈伤组织转到第一次分化培养基上进行分化,28℃黑暗分化3周,再转到第二次分化培养基上在24℃光照分化,得转基因玉米植株。
3.根据权利要求2所述的一种转基因玉米的培育方法,其特征在于,
步骤(1)中活化采用YEP培养基,配方:5g L-1酵母提取物,10g L-1蛋白胨,5g L-1氯化钠,15g L-1琼脂粉,pH 6.8,培养基冷到50℃后加抗生素;
步骤(2)中浸染采用的浸染液配方:N6培养基盐和维生素,1.5mg L-1的2,4-D-二氯苯氧乙酸丁酯,0.7g L-1L-脯氨酸,68.4g L-1蔗糖,36g L-1葡萄糖,pH 5.2,乙酰丁香酮100μmolL-1;
步骤(3)中:共培养培养基配方:N6培养基盐和维生素,1.5mg L-1 2,4-D-二氯苯氧乙酸丁酯,0.7g L-1L-脯氨酸,30g L-1蔗糖,3g L-1植物凝胶,pH 5.8,0.85mg L-1硝酸银,100μmolL-1乙酰丁香酮,400mg L-1半胱氨酸;
恢复培养基配方:N6培养基盐和维生素,1.5mg L-1 2,4-D-二氯苯氧乙酸丁酯,0.7g L-1L-脯氨酸,30g L-1蔗糖,0.5g 2-(4-吗啉)-乙烷磺酸,3g L-1植物凝胶,pH 5.8,0.85mg L-1硝酸银,250mg L-1噻孢霉素;
第一次筛选培养基配方:N6培养基盐和维生素,1.5mg L-1 2,4-二氯苯氧乙酸丁酯,0.7g L-1 L-脯氨酸,30g L-1蔗糖,0.5g 2-(4-吗啉)-乙烷磺酸,3g L-1植物凝胶,pH 5.8,0.85mg L-1硝酸银,250mg L-1噻孢霉素,1.5mg L-1双丙氨膦;
第二次筛选培养基配方:N6培养基盐和维生素,1.5mg L-1 2,4-D二氯苯氧乙酸丁酯,0.7g L-1 L-脯氨酸,30g L-1蔗糖,0.5g 2-(4-吗啉)-乙烷磺酸,3g L-1植物凝胶,pH 5.8,0.85mg L-1硝酸银,250mg L-1噻孢霉素,3.0mg L-1双丙氨膦;
第一次分化培养基配方:MS盐和维生素,60g L-1蔗糖,100mg L-1肌醇,3g L-1植物凝胶,pH 5.8,250mg L-1噻孢霉素和3mg L-1,双丙氨膦灭菌后加;
第二次分化培养基配方:MS盐和维生素,100mg L-1肌醇,30g L-1蔗糖,3g L-1植物凝胶,pH 5.8。
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