CN112210563B - 大麦转录因子HvbZIP10基因及其在小麦抗条锈病、叶锈病中的应用 - Google Patents
大麦转录因子HvbZIP10基因及其在小麦抗条锈病、叶锈病中的应用 Download PDFInfo
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Abstract
本发明提供了大麦转录因子HvbZIP10基因及其在小麦抗条锈病、叶锈病中的应用。本发明涉及基因序列，具体公开了大麦转录因子HvbZIP10，其核苷酸序列如SEQ ID No.1所示，以及过表达HvbZIP10小麦转基因材料的制备过程。本发明通过实验验证HvbZIP10基因可显著提高小麦对小麦条锈病和小麦叶锈病的抗性水平。
Description
技术领域
本发明属于生物基因工程技术领域，涉及大麦转录因子HvbZIP10基因及其在小麦抗条锈病、叶锈病中的应用。
背景技术
小麦作为主要粮食作物，其质量与产量严重影响着我国的粮食安全与社会稳定，小麦的高产与稳产对我国的农业发展有重要意义。由条形柄锈菌小麦专化型(Pucciniastriiformis f.sp.tritici)和小麦叶锈菌(Puccinia triticina,Pt)分别引起的小麦条锈病和小麦叶锈病，是严重影响我国小麦生产的重要真菌病害。近年来由于种植密度的增加以及农业耕作制度的改变，小麦条锈病和叶锈病的发生日趋严重，严重影响着我国的粮食产量与质量。小麦条锈病和叶锈病病原菌均具有变异频率高的特点，其生理小种在短时间内就能完成多次变异，这使得单一抗性的小麦品种在短期内容易丧失抗性。因此，发掘新的抗病种质资源显得尤为必要。
转录因子也称反式作用因子，是指能够与真核基因启动子区域中的顺式作用元件发生特异性相互作用、并对转录有激活或抑制作用的DNA结合蛋白。本世纪以来，研究人员相继分离了一系列植物转录因子，并通过研究证明转录因子不仅调控植物生长发育和生理过程中相关基因的表达，在植物对外界环境包括病原物的入侵、低温、高盐、干旱、激素等胁迫反应方面也起着非常重要的作用。
碱性亮氨酸拉链bZIP转录因子是植物中最丰富、最多样的转录因子，参与许多生物过程，在植物的生物、非生物胁迫应答和发育等生理过程中发挥重要作用。在所有真核生物中都存在含有bZIP结构域的蛋白，bZIP蛋白通常由一个bZIP结构域组成，具有两个基本特征：碱性区域和二聚区。碱性区域与DNA结合，二聚区域形成同源和异源二聚体。bZIP分为13组(记为A-M)。其中，D组包括TGA转录因子，根据它们的保守DNA结合基序TGACG命名。大多数D组的bZIP转录因子提高了植物对病原菌和非生物胁迫的生存能力。烟草TGA1a是第一个从植物中发现的bZIP转录因子，与病程相关基因PR1的启动子区域结合调控后者表达。TGA转录因子蛋白N端具有STDXDT磷酸化位点，C端具有谷氨酰胺结合位点，这些位点共同参与了TGA转录因子应对不同胁迫的抗逆反应。
发明内容
本发明的目的在于提供大麦转录因子HvbZIP10基因及其在小麦抗条锈病、叶锈病中的应用。
本发明提供了大麦转录因子HvbZIP10基因，其核苷酸序列如SEQ ID No.1所示，或该序列经取代、缺失和/或添加一个或几个核苷酸形成的编码同等功能氨基酸序列的由SEQID No.1衍生的核苷酸序列。
本发明提供了前述基因序列的克隆方法，具体为：以大麦cDNA为模板，利用引物采用聚合酶链式反应PCR法克隆得到所述基因序列；所述引物的核苷酸序列如SEQ ID No.2-3。
本发明提供了含有前述基因序列的表达载体。作为优选，所述表达载体为真核表达载体，进一步优选为pLGY-02载体。例如，所述真核表达载体可以为将前述基因序列克隆进pLGY-02载体获得。
本发明提供了含有前述表达载体的宿主。可选的，所述宿主可以为大肠杆菌、农杆菌、小麦等。例如，所述小麦可以为普通小麦春麦品种JW1。
本发明提供了前述基因序列在调控植物对小麦条锈病和小麦叶锈病的抗病性方面的应用。作为优选，所述植物为小麦。更为优选，所述普通小麦为普通小麦春麦品种JW1。
本发明的有益效果在于：本发明提供了大麦HvbZIP10基因序列，以及过表达HvbZIP10小麦转基因材料的制备过程。并通过实验验证了所述小麦转基因材料对小麦条锈病和小麦叶锈病的抗病性显著提高。
附图说明
图1为本发明所述过表达HvbZIP10基因的小麦转基因材料接种小麦条锈菌CYR32的抗病水平显著提高。
图2为本发明所述过表达HvbZIP10基因的小麦转基因材料接种小麦叶锈菌THTT的抗病水平显著提高。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
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。
植物材料：大麦品种“Golden Promise”，普通小麦春麦品种“JW1”。
菌株与载体：大肠杆菌TOP10感受态细胞(CB104)购自天根生化科技(北京)有限公司。T克隆载体pGEM-Teasy购自北京全式金生物技术有限公司。农杆菌GV3101和小麦转基因载体pLGY-02由本研究室保存。小麦条锈菌毒性生理小种CYR32、小麦叶锈病毒性生理小种THTT由河北农业大学保存。
主要试剂：琼脂糖购自SIGMA公司；2×Premix Taq酶购自北京康为世纪生物科技有限公司、限制性内切酶KpnⅠ、SpeⅠ(宝生物TaKaRa工程有限公司)；蔗糖、葡萄糖、胰蛋白胨、琼脂粉、Tween-20、异丙醇、甘油、β-巯基乙醇、氯化钠、氢氧化钠、无水乙醇、硼酸、Tris-HCl等试剂均购自保定市万科化学试剂经营部。AL2000 DNA Marker、质粒小提试剂盒、胶回收及纯化试剂盒购自生工生物工程股份有限公司、QIAGEN植物总RNA提取试剂盒购自天根生化科技有限公司，SYBR Premix Dimer Eraser荧光定量试剂盒和反转录试剂盒购自北京全式金生物技术有限公司。
主要仪器：Applied Biosystems Veriti Thermal Cycler PCR扩增仪(ThermoFisher)、WH-861旋涡混合器(太仓市科教器械厂)、高速冷冻离心机Centrifuge 5810R(Eppendorf公司)、小型高速台式离心机Centrifuge 5415D(Eppendorf公司)、超净工作台(AIR TECH公司)、恒温震荡培养箱(上海苏坤实业有限公司)、SX-500灭菌锅(TOMY公司)、制冰机(SCOTSMAN公司)、摩尔元素型超纯水机(上海摩勒科学仪器有限公司)、微波炉(Galanz公司)、水浴锅(北京市长风仪器仪表公司)、微量移液器(Eppendorf公司)、SONY CarlZeiss Vario Sonnar照相机(SONY)、LightCycler96实时荧光定量PCR仪(Roche公司)、样品研磨机等。
实施例1、大麦HvbZIP10基因的克隆
大麦叶片RNA提取：使用RNA Extraction Kit试剂盒(QIAGEN,Hilden,Germany)进行RNA提取。将大麦材料“Golden Promise”苗期第二叶叶片样品在灭菌的研钵中用液氮迅速研磨成粉末状，待用。配制Buffer RLT混合液，每毫升Buffer RLT加入10μLβ-巯基乙醇，现配现用，混匀后冰上放置。从液氮中取出研磨好的RNA样品，迅速加入500μL Buffer RLT混合液，充分振荡，10000g离心2min。用移液枪吸取上清液转入至紫色离心柱中，10000g离心1min。将收集到的液体转入到粉色离心柱内，加入提前预冷的无水乙醇(加入量为收集液的1/2)，颠倒混匀，静置使核酸析出。瞬离30s，倒掉收集液。加入700μL RW1(洗除蛋白质)，瞬离30s，倒掉收集液。加入500μL的Buffer RPE(使用前需加入44mL无水乙醇)，瞬离30s，倒掉收集液。重复上述步骤一次，10000g离心2min。将离心柱换到一个新2mL收集管中，空离1min。离心结束后，将粉色离心柱放入到试剂盒自带的1.5mL离心管中，用移液枪在吸附膜中央加入30μL RNase-free water，离心1min。保存收集到的RNA样品，用Nanodrop超微量分光光度计测RNA浓度。
大麦cDNA反转录：提取得到的RNA样品用
First-Strand cDNASynthesis SuperMix反转录试剂盒(全式金)反转生成cDNA。将所有RNA样品均一化算为1000ng，用RNase free ddH2O补足至8μL，再加入1μL Oligo(dT)12-18Primer(50μM)，吹吸混匀，放入PCR仪中65℃/5min，4℃/2min。然后加入10μL 2×ES Reaction Mix，1μL
RT/RI Enzyme Mix，混匀，再次放到PCR仪中42℃/15min，85℃加热5s使
RT/RI失去活性，4℃保存。得到的cDNA模板用无菌水以1：5的比例进行稀释，-20℃保存待用。
PCR扩增：以大麦cDNA为模板，利用HvbZIP10基因扩增引物进行PCR扩增。引物信息如SEQ ID No.2-3。PCR扩增体系：cDNA：1μL，F/R引物各0.5μL，2×Premix Taq 12.5μL，ddH2O补至25μL。PCR反应条件为：94℃预变性5min，94℃变性30s，退火：60℃/30s，延伸：72℃/1min，35个循环，最后72℃延伸7min。
PCR产物电泳及目的片段回收纯化：配置1％的琼脂糖凝胶，0.5×TBE电泳液，电泳条件设置为：U＝110V、I＝100mA、P＝90W、Time＝30min。将目的片段在切胶仪下切胶，利用凝胶回收试剂盒(生工公司)回收。
克隆载体构建：将回收产物连接到pGEM-Teasy载体，反应体系为：5μL的2×Buffer缓冲液、3.0μL PCR胶回收产物、1.0μL T4 DNA连接酶、1.0μL pGEM T-easy载体，离心使各试剂充分混匀，22℃连接至少1h或4℃过夜连接。
重组质粒的转化：取DH5α感受态细胞在冰上融化5min；加入全部的连接液混匀，冰浴20min；金属浴热激60s；冰浴5min后加LB液体培养基150μL；37℃并且200rpm摇50min；超净工作台涂板(Amp抗性固体培养基)，晾干后用封口膜封口，于37℃培养箱过夜培养。
重组质粒的筛选：挑取8个斑(每个斑留一半)，做好阳性及阴性对照，用通用引物T7-F/SP6-R或交叉引物进行PCR鉴定；阳性菌落挑斑摇菌，接种于10mL灭菌离心管中的6mLAmp抗性液体LB培养基中，于37℃摇床中200rpm震荡过夜培养。
质粒的提取：
Sangon Biotech公司的DNA质粒小提试剂盒提取质粒。将培养好的菌液吸出500μL到2mL的离心管中，再加入500μL 50％的甘油，-20℃保存甘油菌。将剩余的菌液8000g离心2min收集菌体，倒掉上清。加入250μL Buffer P1(使用前加入RNase A，4℃保存)，振荡彻底悬浮菌体。加入250μL Buffer P2(使用前加入显色剂，28℃保存)，颠倒混匀，静置2min。再用移液枪加入350μL Buffer P3，上下颠倒，使蓝色彻底消失直至出现白色絮状物。12000g离心8min，杂质沉入管底，将上清转入到吸附柱中，瞬离，弃废液。再向吸附柱中加入500μL Wash solution，瞬离，弃废液。重复Wash solution步骤一次。空吸附柱离心1min。将吸附柱转到一个灭菌的1.5mL离心管中，加入40μL提前60℃预热的Elution buffer，室温静置1min，离心1min。保存收集到的DNA溶液。将提取好的质粒吸取5μL送北京华大基因有限公司测序，测序成功的质粒放-20℃保存。测序结果表明，连接得到的T载体含有663bp的DNA插入片段，包含HvbZIP10基因的ORF区段，如SEQ ID No.1。
实施例2、HvbZIP10基因小麦转基因载体pLGY-02的构建
小麦转基因载体pLGY-02的构建：提取测序正确的HvbZIP10-T重组质粒和pLGY-02载体的质粒，利用限制性内切酶KpnI+SpeI进行双酶切。具体酶切体系为：质粒1.0μg，KpnI(15U/μL)1.0μL，SpeI(10U/μL)1.0μL，10×Buffer 2.0μL，ddH2O补至20μL。酶切混合液于37℃金属浴中酶切3-5h。酶切产物电泳检测后，胶回收目的基因片段和pLGY-02载体片段，进行连接。连接体系：目的片段12μL，pLGY-02载体片段5μL，T4 DNA连接酶1.0μL，T4 DNALigase buffer 2.0μL，ddH2O补至20μL。离心使各试剂充分混匀，22℃连接至少1h或于4℃冰箱过夜连接。连接产物转化至大肠杆菌，PCR检测，挑取阳性菌落摇菌，提取质粒进行双酶切检测，阳性质粒送公司进行测序，筛选获得HvbZIP10-pLYG-02重组载体。
实施例3、HvbZIP10过表达小麦转基因植株的制备
农杆菌介导的小麦转基因材料制备由山东济南邦地生物有限公司完成，转化背景材料为普通小麦春麦材料JW1，采用农杆菌介导的小麦幼胚转化方法。
SDS法提取基因组DNA：取样并标记；每管加入1个打磨珠，用液氮预冷后配平放入打样机内，1100g打磨1min，取出后放入600μL Extraction buffer(100mL 0.1M Tris-HClpH＝7.5、100mL 0.5M EDTA pH＝8.0、125mL 10％SDS)震荡后放入65℃水浴锅水浴30min；取出放冰上15min冷却至室温再加入300μL的6M Ammonium Acetate(醋酸铵)混匀后放入4℃冰箱15min，12000g离心15min；取600μL上清液于已放入360μL异丙醇的1.5mL的离心管中，混匀置于4℃冰箱沉淀15min。取出后12000g离心15min，将上清液倒掉；加入400μL的75％乙醇，12000g离心15min，将上清液倒掉；重复上述步骤一次；将含有DNA的管放入超净工作台上，打开盖子，吹干；用100μL无菌水回溶DNA常温放置约半天。利用转基因载体检测引物对转基因材料基因组DNA进行PCR检测，确定转基因阳性植株。
实施例4、小麦转基因材料HvbZIP10-OE的抗条锈病、叶锈病抗性鉴定
小麦条锈菌和小麦叶锈菌的纯化和扩繁：接种前先将低温储存的小麦条锈菌毒性生理小种CYR32和小麦叶锈菌毒性生理小种THTT的夏孢子在42℃温水中活化30min，然后水化，加入0.1％Tween-20，用涂抹法将活化好的菌种均匀地接种到一心一叶的小麦感条锈菌、叶锈菌材料JW1的叶片上，接种条锈菌后喷水雾置于15±5℃黑暗条件下保湿12-18h后转入温室培养，接种叶锈菌后喷水雾置于20±5℃黑暗条件下保湿12-18h后转入温室培养。套好玻璃罩子并蒙好纱布，接种后12d左右叶片表面出现大量孢子堆，在干燥条件下收集条锈菌的夏孢子，新鲜的锈菌孢子再扫描接种到一心一叶的小麦材料上，即可大量扩繁锈菌用于试验，在干燥条件下收集锈菌孢子备用，锈菌孢子近期使用则可以保存于4℃硅胶盒中，长期保存则抽真空处理于-20℃保存。
小麦转基因材料抗条锈病、叶锈病抗性鉴定：小麦转基因材料与其野生型种植一周后得到一叶一心期幼苗。第一片叶片完全展开时，利用抖接法接种纯化完成的小麦条锈菌CYR32和小麦叶锈菌THTT。将接种完成的麦苗放置于合适温度的培养室内，直至发病。取小麦转基因材料的第一叶进行转基因鉴定；取小麦转基因与野生型小麦的第二叶进行拍照，利用植物病害表型统计ASSESS软件进行产孢面积百分比的数据分析。结果表明，在小麦中过表达大麦转录因子HvbZIP10基因可显著提高植株对小麦条锈病(图1)和小麦叶锈病(图2)的抗性水平。
<110> 河北农业大学
<120> 大麦转录因子HvbZIP10基因及其在小麦抗条锈病、叶锈病中的应用
<130> Novel07221
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 663
<212> DNA
<213> 大麦（Hordeum vulgare L.）
<400> 1
atggacgaca acggggacat agatttcacc aatccggaga cgtacctgtg cccagccatg 60
ggcggcgatc cccacgacag ttgctccatg tcaatggaca gctacttcga cgacatcctg 120
aaagacccgg agcacctcgc atgtactcgg atcctgctgg cggagtcgga tgatgtcgcc 180
gagacctctg agtcgccgca agaggacgga cccaagaaga agcgcccgcc cggtaaccgg 240
gcagccgtga ggaggtaccg tgagaagaag aaggcccaca cgacgctgct ggaggaagag 300
gtggctcgcc tcaaggctct aaacaagcag ctcgtgagga ggcttcagag tcagtcgtcc 360
ctcgaggctg aggcctcgag gctccgctgc ctgcttgtcg acattagagg gaggatcgaa 420
ggggagctcg gtgctttccc ttaccaacgg ccagtgaaga acaaggattt ggctgaccag 480
ggaagttccc taggtatagg tggtgcccag aaggttaggc tcagatgcaa caatccggtt 540
tactgcagtc cagagatgcc ggccacgaca atggatgacg atggtgttat cagtggcgaa 600
ctgttgggtc aaggtgcaaa tgacaagtgg ctcccaggtt tgccagatga tgtaaagagg 660
tga 663
<210> 2
<211> 22
<212> DNA
<213> 人工序列-正向引物
<400> 2
atggacgaca acggggacat ag 22
<210> 3
<211> 25
<212> DNA
<213> 人工序列-反向引物
<400> 3
tcacctcttt acatcatctg gcaaa 25
Claims (1)
1.过表达SEQ ID NO.1所示基因在提高普通小麦对小麦条锈病和小麦叶锈病的抗病性方面的应用。
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