CN102229662B - 一种莲膜联蛋白及其表达载体和应用 - Google Patents
一种莲膜联蛋白及其表达载体和应用 Download PDFInfo
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- CN102229662B CN102229662B CN201110158933XA CN201110158933A CN102229662B CN 102229662 B CN102229662 B CN 102229662B CN 201110158933X A CN201110158933X A CN 201110158933XA CN 201110158933 A CN201110158933 A CN 201110158933A CN 102229662 B CN102229662 B CN 102229662B
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Abstract
本发明公开了一种莲膜联蛋白及其表达载体和应用,属于植物基因工程技术领域。本发明的莲膜联蛋白具有SEQ ID NO:1所示的氨基酸序列,其编码基因的核苷酸序列如SEQ ID NO:2所示。本发明还公开了该莲膜联蛋白的表达载体,是由其编码基因***到原核或真核表达载体中构建而成的。本发明的莲膜联蛋白能够有效提高植物种子耐热性能,增强种子的萌发活性,在植物品种选育和农业生产领域具有广泛的应用前景。
Description
技术领域
本发明涉及分子生物学与植物基因工程技术领域,具体涉及一种提高植物种子耐热性和种子活力的莲膜联蛋白及其应用。
背景技术
高温是影响植物生长、发育和繁殖的重要环境因素之一。由于温室效应的影响,全球暖化的趋势日益明显,高温导致的种子活力和存活率下降对作物品质和产量造成的危害也日益加剧,因此,研究种子耐热机制,寻找提高种子耐热性和活力的关键基因,具有重要的科学意义和经济价值。
莲(Nelumbo nucifera Gaertn.)种子是迄今为止被证实的最长寿和最耐高温的种子之一(; Shen-Miller, et al., 1995, Exceptional seed longevity and robust growth: ancient sacred lotus from China. Am J Bot 82: 1367-1380;),表明莲种子具有非常高效的防御和修复***,为我们挖掘种子耐热性和种子活力相关的新基因提供了极佳的材料。
膜联蛋白(Annexins)是一类Ca2+及磷脂结合蛋白,在除酵母以外的生物界普遍存在(Mortimer, et al., 2008, Annexins: multifunctional components of growth and adaptation. J Exp Bot 59: 533-544)。植物膜联蛋白参与胞外分泌,细胞伸长,细胞壁合成,根瘤形成和果实成熟等生理过程。越来越多的研究表明植物膜联蛋白具有过氧化物酶的活性,参与植物中各种逆境应答反应,包括低温胁迫,干旱胁迫,盐胁迫,重金属胁迫和氧化胁迫等(Breton, G., et al., 2000, Two novel intrinsic annexins accumulate in wheat membranes in response to low temperature. Plant Cell Physiol 41: 177-184;Lee, S., et al., 2004, Proteomic identification of annexins, calcium-dependent membrane binding proteins that mediate osmotic stress and abscisic acid signal transduction in Arabidopsis. Plant Cell 16: 1378-1391;Jami, S.K.,et al., 2008, Ectopic expression of an annexin from Brassica juncea confers tolerance to abiotic and biotic stress treatments in transgenic tobacco. Plant Physiol Bioch 46: 1019-1030; Konopka-Postupolska, D., et al., 2009, The role of annexin 1 in drought stress in Arabidopsis. Plant Physiol 150: 1394-1410)。由于活性氧(ROS)的大量产生导致的氧化胁迫被认为是热伤害的最主要的组成之一(Kim, K.H.,et al., 2010, Enhanced tolerance of transgenic tall fescue plants overexpressing 2-Cys peroxiredoxin against methyl viologen and heat stresses. Biotechnol Lett 32: 571-576),膜联蛋白在热适应中的作用不容忽视。Rhee等 (Rhee, H.J., et al., 2000, Annexin I is a stress protein induced by heat, oxidative stress and a sulfhydryl-reactive agent. Eur J Biochem 267: 3220-3225) 对动物细胞进行热处理时发现高温提高了annexin I的表达,表明其可能是热胁迫响应蛋白,但目前在植物中尚无膜联蛋白与种子耐热性相关的报道。
发明内容
本发明的目的在于根据现有技术中的上述不足,提供一种与植物种子耐热性和活力相关的蛋白莲膜联蛋白。
本发明的另一目的是提供上述莲膜联蛋白的表达载体。
本发明的又一目的是提供上述莲膜联蛋白的应用。
本发明通过以下技术方案实现上述目的:
一种莲膜联蛋白,命名为NnANN1(Nelumbo nucifera Annexin1),来源于中国莲(Nelumbo nucifera Gaertn.),是如下(1)或(2)的蛋白质:
(1)该蛋白的氨基酸序列如SEQ ID NO:1所示;
(2)通过将序列SEQ ID NO:1经过一个或几个氨基酸残基的取代、缺失或添加而衍生的蛋白质,且该蛋白与(1)的蛋白具有相同的生物活性。
上述莲膜联蛋白的编码基因(NnANN1基因),NnANN1开放读码框为948 bp,5′-UTR 为63 bp,3′-UTR为312 bp,编码315个氨基酸。该编码基因的核苷酸序列如SEQ ID NO:2所示。或者与SEQ ID NO:2所示序列有90%以上同源性,且该DNA分子编码的蛋白与(1)的蛋白具有相同生物活性。
一种莲膜联蛋白表达载体,包含莲膜联蛋白的编码基因。即将编码基因***到出发载体中得到,出发载体可以选用本领域已知的的各种载体。
上述出发载体优选为原核表达载体pET14b、pET32a或真核表达载体pBI121等。
一种莲膜联蛋白表达载体的制备方法,步骤如下:
(1)以莲cDNA文库为模板,以SEQ ID NO:3~4为引物进行PCR,得到莲膜联蛋白的编码基因;
(2)将步骤(1)的PCR产物与pGEMT-Easy载体进行连接,得到中间载体;
(3)中间载体转化大肠杆菌DH5α感受态,培养后进行PCR、酶切和测序鉴定;
(4)鉴定正确的菌体进行双酶切后与出发载体连接,连接产物再次转化大肠杆菌DH5α感受态,培养后进行PCR、酶切和测序鉴定,鉴定正确的即为莲膜联蛋白表达载体。
一种根癌农杆菌,含有上述的表达载体,优选的出发菌株为根癌农杆菌EHA105。
上述莲膜联蛋白或其编码基因在提高种子耐热性能或/和促进种子萌发活性中的应用。
与现有技术相比,本发明具有以下有益效果:
通过植物表达载体将本发明的莲膜联蛋白转入植物中,可以提高植物种子在热胁迫和人工老化条件下的萌发活力。将转入表达质粒的种子和野生型种子用50℃高温处理6h后进行萌发实验,结果显示处理过的种子萌发速度和最终萌发率都明显高于对照的野生型种子,表明NnANN1能提高植物种子在热胁迫下的萌发活力。人工老化处理后萌发实验结果显示处理过的种子的萌发速度和最终萌发率都远高于野生型种子,表明野生型种子大部分都丧失了活力,而处理的种子仍然保持较高的活力。上述结果表明NnANN1具有提高植物种子耐热性和活力的能力。该蛋白对于种子耐热分子机制的研究,高活力和耐热性的品种的选育具有重要的理论意义和经济价值,本发明在农业领域具有广阔的应用空间和市场前景。
附图说明
图1.植物表达载体pBI121-NnANN1的示图,RB为右边界,LB为左边界。
图2.转基因拟南芥种子的Q-PCR和Western Blot检测结果,A为Q-PCR的表达分析结果,WT表示野生型,OE1、OE2、OE3分别是3个不同的转基因株系;B为Western Blot的检测结果,35kD是分子量大小,CBB:考马斯亮蓝。
图3.野生型和转基因拟南芥种子热处理后的萌发结果分析图,WT表示野生型,OE1、OE2、OE3表示三个不同的转基因株系。
图4.野生型和转基因拟南芥种子热处理后的幼苗生长图,WT表示野生型,OE1、OE2、OE3表示三个不同的转基因株系。
图5.野生型和转基因拟南芥种子人工老化处理后的萌发结果分析图,WT表示野生型,OE1、OE2、OE3表示三个不同的转基因株系。
图6.人工老化处理野生型和转基因拟南芥种子后的幼苗生长图,WT表示野生型,OE1、OE2、OE3表示三个不同的转基因株系。
具体实施方式
下面结合具体实施例对本发明做进一步说明。以下实施例中,凡未注明具体实验条件的,均为按照本领域技术人员熟知的常规条件进行。
实施例1 莲膜联蛋白基因NnANN1的克隆
(1)莲种子胚轴的准备:收获发育成熟后期的莲种子,剥取胚轴;
(2)总RNA 的提取:采用Invitrogen公司的Trizol产品提取总RNA;
(3) cDNA文库构建:采用Clontech公司的SMART cDNA library construction kit构建文库;
(4)EST测序:以获得的阳性单克隆菌液为测序样品,委托上海博亚生物技术公司,用3730测序仪对PCR验证后的阳性单克隆进行序列测定;
(5)同源检索:使用BLAST工具搜索所得EST序列在 GenBank中的同源基因,确认得到的基因为莲膜联蛋白基因。
实施例2 植物表达载体的构建
(1)基因片段克隆:以实施例1所述的莲cDNA文库为模板,通过PCR克隆得到添加了酶切位点SmaI和SacI的PCR片段。PCR引物如下,下划线部分为酶切位点:
正向引物:SEQ ID NO:3:5′-TCCCCCGGG ATGGCTACCATCACAGTCCCTG -3′;
反向引物:SEQ ID NO:4:5′-CTGGAGCTCTCACAGCTCTTCGCACCCCAGT -3′;
PCR反应体系为:2 μl莲cDNA文库,6 μl dNTP(2.5 mM),1.5 μl正向引物(10 μM),1.5 μl反向引物(10 μM),5 μl 10×PCR缓冲液,1 μl EX Taq酶(Takara公司产品),最后补充去离子水,使总体系为50 μl。PCR的反应条件为:94℃ 3分钟;然后进入如下循环:94℃ 30 秒,58℃ 30 秒,72℃ 30 秒,共30个循环;最后72℃延伸7分钟。
(2)T载体连接:取2 μl PCR产物与pGEMT-Easy载体进行连接,按照Promega公司的说明书步骤进行操作,构建中间载体pGEMT-MT2a。
(3)大肠杆菌转化:将中间载体pGEMT-MT2a转化大肠杆菌DH5α感受态(天根公司产品),按照天根公司的产品说明书进行操作。在含有IPTG、X-gal和氨苄青霉素(100mg/l)的LB固体培养基上涂板,37℃过夜培养后,挑取白色单菌落,在含有氨苄青霉素(100mg/l)的LB液体培养基中生长,取少量菌液进行PCR鉴定。
(4)细菌质粒DNA 的制备:收集上述LB液体培养基中的菌体,按照天根公司的质粒小提试剂盒说明书制备细菌质粒DNA,酶切和测序鉴定。测序由广州Invitrogen公司完成。
(5)表达载体的构建:提取测序正确的克隆的质粒,按照Takara公司的产品说明书,用SmaI和SacI进行双酶切,然后回收小片段,连接入用同样酶消化的表达载体pBI121中,构建植物表达载体pBI121-NnANN1,示图如图1所示。然后将连接产物转化大肠杆菌DH5α感受态,进行PCR、酶切和测序鉴定。
(6)根癌农杆菌的转化:提取测序正确的克隆的质粒,通过电击法转化根癌农杆菌EHA105。
实施例3 拟南芥的遗传转化
1. 拟南芥转化前处理
当其主苔长至5-6cm时,在花序基部剪掉整个花序,去其顶端优势,1周后在腋芽部位长出4-6个新生侧苔,待其侧苔花序形成花蕾并部分开花或形成1-2个角果时,即可用于转化,转化前需剪去已长成的角果。转化的前一天给植物浇足水分,并罩一个塑料袋以保持高湿环境。
2. 浸染培养基的准备
用于浸泡拟南芥花絮的浸染培养基成分为含5%的蔗糖的1/2MS培养基,pH=5.8 (用KOH调节),高压灭菌。用时添加0.02%-0.05%表面活性剂Silwet L-77或表面活性剂吐温-20(tween-20),操作需轻柔。
3. 农杆菌准备及拟南芥转化
(1)菌种的活化:将储存的已转化根癌农杆菌EHA105液或平板保存的菌在含卡那霉素(Km,100mg/L )和利福平(Rif,30mg/L)的YEB固体培养基上划板活化,并进行PCR检测;
(2)挑取活化的含目的基因的单克隆阳性农杆菌至5m1新鲜含卡那霉素和利福平的YEB液体培基中,28℃,180rpm摇培24小时;
(3)取上述菌液5ml(1%-2%)接至500 m1新鲜含卡那霉素和利福平的YEB液体培养基中,28℃,180rpm培养18-24小时,使OD值达到0.8左右(用YEB+Rif+Km作为空白对照);
(4)将上述菌液分装到100 ml离心管中,室温,5000转离心20分钟收集菌体;
(5)将细菌悬浮在浸染培养基中,使最终菌体适宜浓度OD600值大约为0.8-1(用浸染培养基作对照);
(6)将上述浸染液倒在烧杯中,将待转化的植物迅速倒扣在浸染液中,使花序浸没60秒钟后取出。操作时尽量小心,不要让土屑等掉入到浸染培养基中;
(7)转化后用吸水纸吸去过多的菌液,但不需要吸得太干。将植株平放,并用黑色塑料袋罩住拟南芥地上部分。保湿暗培养16-24h后,小心去掉塑料袋,并浇足水,恢复正常光照;
(8)为了提高转化率,可以在一周以后重复一次侵染过程;
(9)正常管理,收获成熟种子。收获后的种子在含有50mg/l的卡那霉素和150mg/l的羧苄青霉素的MS固体培养基上筛选转基因阳性植株。
实施例4 转基因种子的Q-PCR检测
(1)总RNA的提取:按照百泰克公司的植物通用RNA提取试剂盒说明书步骤提取野生型和不同的转基因株系的拟南芥干种子总RNA;
(2)第一链cDNA的合成:按照Takara公司的PrimeScript 1st Strand cDNA Synthesis Kit的说明书步骤操作;
(3)Q-PCR反应:
NnANN1基因引物:
正向引物:SEQ ID NO: 5:5′- TGCGAAGTCTGACAATCGGA -3′;
反向引物:SEQ ID NO: 6:5′-GCTCACATCTACCTCATCACCAT -3′。
内参Actin2基因引物:
正向引物:SEQ ID NO: 7:5′- ATTACCCGATGGGCAAGTCA-3′;
反向引物:SEQ ID NO: 8:5′- TGCTCATACGGTCAGCGATA-3′。
NnANN1和Actin2的Q-PCR反应体系为:1 μl稀释50倍的第一链cDNA模板,12.5μl SYBR荧光实时定量PCR Master Mix (Toyobo公司产品),0.5 μl正向引物(10 μM),0.5 μl反向引物(10 μM),最后补充去离子水,使总体系为25 μl。PCR反应条件为:95℃ 10秒,58℃ 15秒,72℃ 20秒。反应循环数为40。Q-PCR的结果如图2中A所示,在野生型拟南芥种子中没有检测到莲膜联蛋白基因NnANN1的表达,而在三个不同的转基因株系中都能检测到不同程度的表达。
实施例5 转基因种子的Western Blot检测
(1)原核表达:以实施例2所述的中间载体pGEMT-MT2a为模板,通过PCR克隆得到添加了酶切位点EcoRI和 XhoI的PCR片段。PCR引物如下,下划线部分为酶切位点:
正向引物:SEQ ID NO: 9:
5′-CCGGAATTC ATGGCTACCATCACAGTCCCTG -3′;
反向引物:SEQ ID NO: 10:
5′- CCGCTCGAGTCACAGCTCTTCGCACCCCAGT-3′;
PCR反应体系为:1 μl模板,4 μl dNTP(2.5 mM),2 μl正向引物(10 μM),2 μl反向引物(10 μM),5 μl 10×PCR缓冲液,0.3 μl EX Taq酶(Takara公司产品),最后补充去离子水,使总体系为50 μl。PCR反应程序:94℃ 3分钟;然后进入如下循环:94℃ 30 秒,58℃ 30 秒,72℃ 30 秒,共30个循环;最后72℃延伸7分钟。纯化后的PCR产物用EcoRI和 XhoI酶切后,回收,连接入用同样酶消化的表达载体pET-14b(Novagen公司产品)中,构建原核表达载体6His-NnANN1。然后转化大肠杆菌BL21(DE3),进行PCR、酶切和测序鉴定,方法与实施例2相同。蛋白的纯化按照Novagen公司的His bind purification kit说明书进行。
(2)抗体制备:上述纯化后的蛋白用于注射兔子制备特异性抗体anti-NnANN1。
(3)蛋白提取:野生型和转基因拟南芥种子的蛋白提取参照Fan 等(1997, Antisense suppression of phospholipase D [alpha] retards abscisic acid [mdash] and ethylene-promoted senescence of postharvest arabidopsis Leaves. The Plant Cell Online 9: 2183)的方法。
(4)Western Blot:免疫印记参照Mizzen(Mizzen, C.A.,et al., 1996, Sensitive detection of metallothioneins-1, -2 and -3 in tissue homogenates by immunoblotting: a method for enhanced membrane transfer and retention. J. Biochem. Bioph. Meth. 32: 77-83)的方法。Western Blot的结果如图2中B所示,在野生型拟南芥种子没有检测到莲膜联蛋白NnANN1的积累,而在三个不同的转基因株系中都能检测到不同程度的莲膜联蛋白NnANN1的积累。
实施例6 转基因种子的热胁迫萌发实验
野生型和转基因拟南芥种子在消毒灭菌后,播种于1/2 MS固体培养基上放置于4℃层积处理两天后,50℃培养箱中处理6 h,高温处理后的种子在正常条件下进行萌发。统计每一天种子的萌发率,共计7天,萌发情况如图3所示,转基因种子的萌发速度和最终的萌发率都远高于野生型的种子。在播种后第七天,野生型种子的萌发率仅为16%,而转基因株系的萌发率高达50-64%,表明过量表达基因NnANN1能提高种子在热胁迫下萌发的活力。热胁迫处理的种子幼苗生长情况见图4,野生型幼苗长势明显弱于三个转基因株系。
实施例7 转基因种子经人工老化处理后的萌发实验
(1)人工老化是在干燥器中完成的。老化前先用10%市售的漂白剂(bleach)处理干燥器等器皿30分钟,然后用大量清水冲洗干燥器,最后用灭菌水再冲洗一次,晾干,防止人工老化期间真菌的生长。
(2)在干燥器底部注入灭菌水,将干燥器密封后放置于隔水式恒温培养箱中,温度设为43℃,平衡两天,让干燥器中的湿度达到100%。
(3)将野生型和转基因的拟南芥种子用1.5 ml的去掉盖子的离心管装好后(每管大约100粒)放置于离心管架上后迅速放入已经平衡好的干燥器中,密封好干燥器,处理72小时。处理期间尽量不要打开恒温培养箱的门。
(4)72小时后,将处理好的拟南芥种子取出,平衡至室温后马上进行消毒灭菌,4℃层积处理两天后进行萌发实验,统计萌发率及对幼苗的生长情况进行拍照。人工老化后的种子萌发结果如图5所示,转基因种子的萌发速度和最终的萌发率都远高于野生型的种子。在播种后第十天,野生型种子的萌发率仅为20%,而转基因株系的萌发率高达58-70%。处理后的幼苗生长情况见图6,野生型经过老化处理基本上没有生长,而三个转基因株系的幼苗能够正常生长,表明过量表达基因NnANN1能提高种子的活力。
SEQUENCE LISTING
<110> 中山大学
<120> 一种莲膜联蛋白及其表达载体和应用
<130>
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 315
<212> PRT
<213> 人工序列
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Met Ala Thr Ile Thr Val Pro Asp His Thr Ser Pro Val Glu Asp Ala
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Glu Asn Leu Arg Lys Ala Cys Glu Gly Trp Gly Thr Asn Glu Asn Thr
20 25 30
Ile Val Ser Ile Leu Gly His Arg Asn Ala Val Gln Arg Lys Gln Ile
35 40 45
Arg Gln Ala Tyr Glu Glu Ile Tyr Gln Glu Asp Leu Ile Lys Arg Leu
50 55 60
Glu Ser Glu Leu Lys Gly Glu Phe Glu Lys Ala Val Tyr Arg Trp Ile
65 70 75 80
Leu Asp Pro Ala Asp Arg Asp Ala Ile Leu Ala His Val Ala Ala Arg
85 90 95
Asn Ala Lys Ser Asp Asn Arg Thr Ile Ile Glu Ile Ala Cys Ile Arg
100 105 110
Ser Pro Glu Glu Leu Leu Ala Ala Lys Arg Ala Tyr His Phe Arg Tyr
115 120 125
Lys His Ser Leu Glu Glu Asp Val Ala Ser Arg Thr Thr Gly Asp Phe
130 135 140
Arg Lys Leu Leu Val Ala Leu Val Ser Thr Tyr Arg Tyr Asp Gly Asp
145 150 155 160
Glu Val Asp Val Ser Leu Ala Gly Ser Glu Ala Lys Ile Leu His Asn
165 170 175
Met Ile Glu Gly Lys Ser Phe Asn His Glu Glu Val Ile Arg Ile Leu
180 185 190
Ser Thr Arg Ser Lys Ala Gln Leu Asn Ala Thr Phe Asn Arg Tyr Lys
195 200 205
Asp Thr His Gly Ala Ser Ile Thr Lys Ser Leu Ser Gly Asn Pro Ala
210 215 220
Asp Glu Phe Ser Glu Ala Leu Cys Ile Ala Ile Gln Cys Ile Arg Ser
225 230 235 240
Pro Gln Lys Tyr Phe Glu Lys Val Leu Arg Asn Ala Ile Asn Lys Val
245 250 255
Gly Thr Asp Glu Asp Ala Ile Thr Arg Val Ile Val Thr Arg Ala Glu
260 265 270
Lys Asp Leu Lys Asp Ile Lys Asp Leu Tyr His Lys Arg Asn Asn Ala
275 280 285
Ser Leu Glu His Ala Ile Ser Lys Asp Thr Ser Gly Asp Tyr Lys Ile
290 295 300
Phe Leu Leu Thr Leu Leu Gly Cys Glu Glu Leu
305 310 315
<210> 2
<211> 1323
<212> DNA
<213> 人工序列
<400> 2
tgtgcaggct gtagagtgta gaagagaaag aaacagtaag gggttttaag tgattgctca 60
acaatggcta ccatcacagt ccctgatcac acttctcctg ttgaagatgc agagaacctc 120
agaaaggctt gcgagggatg ggggaccaat gagaatacca ttgtttcaat actcgggcat 180
agaaatgcag ttcaaaggaa acaaatcagg caggcttatg aggagattta tcaagaagat 240
ctaattaagc gacttgaatc cgaactcaaa ggggaatttg agaaagctgt atatcgctgg 300
atactagatc ctgcagacag agatgcaatc ttggctcatg tagctgctag aaatgcaaag 360
tctgacaatc ggacgatcat tgaaattgct tgcattcgat ctcccgagga actcttggcg 420
gcgaaacggg cctaccattt ccgctacaag cattctctgg aggaagatgt cgcctctcgt 480
actacaggcg acttccgtaa gcttctagtt gccttagtga gcacttacag gtatgatggt 540
gatgaggtag atgtgagcct agctggatca gaagccaaaa ttcttcataa tatgattgaa 600
gggaaatcct ttaaccatga agaggtaatc agaatcttaa gcacaaggag caaggcacag 660
ctcaatgcaa ccttcaaccg ctacaaggac acacatggag cttccattac caagagttta 720
tcaggtaatc ctgctgatga gttctctgag gcactatgta tagctatcca atgcatcaga 780
tcacctcaaa agtactttga aaaggttctg cgtaatgcaa ttaataaggt aggaactgat 840
gaagatgcaa ttacgcgtgt gattgtaaca cgtgcagaga aggacttgaa ggacatcaag 900
gatttatatc acaagagaaa caatgcatct cttgagcatg caatcagcaa ggatacttca 960
ggcgactaca aaattttcct ccttacttta ctggggtgcg aagagctgta actctgttgt 1020
ccaaatgtgt ggaagaaatt gttaaatctg gattctgtgg tttgagaatt ggtgtctggt 1080
ttgtggggtt tagtttctag gctactggga aagcattttg ctttctgggt tgttaaatgg 1140
taacgagtgc tgaactttgg tgtgcaaata atcctatgat ttccattact cctgctctgt 1200
ttcttcccta gtttctgagt gtcttagatc agaatcttgc ttgatgttta ccggttggaa 1260
ctcagctttc taagaaattt ttatgatgtt gatgaaaaaa aaaaaaaaaa aaaaaaaaaa 1320
aaa 1323
<210> 3
<211> 31
<212> DNA
<213> 人工序列
<400> 3
tcccccggga tggctaccat cacagtccct g 31
<210> 4
<211> 31
<212> DNA
<213> 人工序列
<400> 4
ctggagctct cacagctctt cgcaccccag t 31
<210> 5
<211> 20
<212> DNA
<213> 人工序列
<400> 5
tgcgaagtct gacaatcgga 20
<210> 6
<211> 23
<212> DNA
<213> 人工序列
<400> 6
gctcacatct acctcatcac cat 23
<210> 7
<211> 20
<212> DNA
<213> 人工序列
<400> 7
attacccgat gggcaagtca 20
<210> 8
<211> 20
<212> DNA
<213> 人工序列
<400> 8
tgctcatacg gtcagcgata 20
<210> 9
<211> 31
<212> DNA
<213> 人工序列
<400> 9
ccggaattca tggctaccat cacagtccct g 31
<210> 10
<211> 31
<212> DNA
<213> 人工序列
<400> 10
ccgctcgagt cacagctctt cgcaccccag t 31
Claims (8)
1.一种莲膜联蛋白,其特征在于该蛋白的氨基酸序列如SEQ ID NO:1所示。
2.权利要求1所述莲膜联蛋白的编码基因,其特征在于核苷酸序列如SEQ ID NO:2所示。
3.一种莲膜联蛋白表达载体,其特征在于包含权利要求2所述的编码基因。
4.根据权利要求3所述的表达载体,其特征在于出发载体为原核表达载体pET14b、pET32a或真核表达载体pBI121。
5.权利要求3或4所述表达载体的制备方法,其特征在于步骤如下:
(1)以莲cDNA文库为模板,以SEQ ID NO:3~4为引物进行PCR,得到莲膜联蛋白的编码基因;
(2)将步骤(1)的PCR产物与pGEMT-Easy载体进行连接,得到中间载体;
(3)中间载体转化大肠杆菌DH5α感受态,培养后进行PCR、酶切和测序鉴定;
(4)鉴定正确的菌体进行双酶切后与出发载体连接,连接产物再次转化大肠杆菌DH5α感受态,培养后进行PCR、酶切和测序鉴定,鉴定正确的即为莲膜联蛋白表达载体。
6.一种根癌农杆菌,其特征在于含有权利要求3或4所述的表达载体。
7.根据权利要求6所述的根癌农杆菌,其特征在于以根癌农杆菌EHA105作为出发菌。
8.权利要求2所述莲膜联蛋白的编码基因在提高种子耐热性能或促进种子萌发活性中的应用。
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CN108610402B (zh) * | 2018-04-13 | 2021-05-25 | 中山大学 | 花生膜联蛋白基因AhANN6在提高植物及微生物抗高温和抗氧化胁迫中的应用 |
CN112680457A (zh) * | 2021-02-02 | 2021-04-20 | 河北师范大学 | 一种膜联蛋白基因在植物热胁迫中的应用 |
CN113444161A (zh) * | 2021-06-29 | 2021-09-28 | 南京农业大学 | 萝卜膜联蛋白RsANN1a及其编码基因和应用 |
CN116555322B (zh) * | 2023-01-17 | 2023-10-27 | 中国科学院华南植物园 | TtANXNL基因及其编码蛋白的应用 |
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