CN112813098B - 利用人工突变创制玉米bhlh51雄性不育系 - Google Patents
利用人工突变创制玉米bhlh51雄性不育系 Download PDFInfo
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Abstract
本发明公开了利用人工突变创制玉米bhlh51雄性不育系。本发明利用CRISPR/Cas9基因组编辑技术对玉米育性基因ZmbHLH51进行编辑,然后通过杂交和自交在F2代获得纯合且不含Cas9转基因的雄性不育突变体,从而创制出稳定的玉米bhlh51雄性不育系。bhlh51雄性不育系与目标材料进行杂交和回交,可以使目标材料获得bhlh51雄性不育的性状和基因突变,对促进玉米不育化杂交育种和制种具有重要意义。本发明还针对获得的bhlh51雄性不育突变体设计了功能分子标记,在玉米雄性不育系培育、不育化杂交育种、制种和分子标记辅助选择中具有重要的应用价值。
Description
技术领域
本发明属于植物生物技术育种领域,具体涉及利用人工突变创制玉米bhlh51雄性不育系。
背景技术
玉米是我国第一大粮食作物,常年播种面积在5.5亿亩以上,玉米种业健康发展对保障国家粮食安全有着重大的战略意义。同时,玉米种业也是全球市值最大、商业化程度最高、科技含量最高的种业领域,是全球种业竞争的战略要地。我国玉米种业与国际领先水平相比,在科技创新和产业模式等方面,仍然存在巨大差距。一是受限于玉米雄性不育基础研究尚未取得根本性突破等因素,导致玉米自交系知识产权保护困难,造成近年来我国玉米种业长期存在跟随性、模仿性育种现象,重大新品种选育效率缓慢。二是玉米制种产业仍然处于依靠人工去雄为主的劳动密集型阶段,成本高,资源消耗巨大,种子质量难以保障。
玉米是杂种优势利用最成功的作物之一,雄性不育系是作物杂种优势利用和杂交制种的重要材料,主要包括细胞质雄性不育(CMS)和细胞核雄性不育(GMS)。CMS 是由线粒体基因和核基因共同控制的,虽然已应用于玉米育种和杂交种生产中,但存在资源利用率低、不育系胞质单一、易感病等问题。 GMS 由核基因单独控制,可以克服 CMS缺陷,但很难通过常规育种方法大量繁殖纯合不育系。近年来,随着生物技术的进步,通过基因工程和分子设计育种相结合创制的玉米多控不育技术以及植物通用型显性不育技术,可以有效地解决玉米隐性核雄性不育系的保持和繁殖问题。实现上述技术应用的重要前提是获得大量功能明确的控制玉米雄性发育的GMS基因和对应的雄性不育材料。
与模式植物拟南芥和模式作物水稻相比,玉米中已克隆和鉴定的GMS基因和创制的雄性不育材料均相对较少。CRISPR/Cas9(Clustered, Regularly Interspaced, ShortPalindromicRepeats-associated Endonuclease 9)基因编辑技术由于具有成本低廉、操作简易和突变诱导率高等特点,被越来越广泛地应用于植物基因功能研究,作物遗传改良和育种等多个方面,应用前景十分广阔。利用CRISPR/Cas9技术挖掘鉴定玉米雄性不育候选基因和创制雄性不育材料,可以快速丰富玉米GMS基因和不育材料资源,从而促进玉米不育化育种和制种的推广和应用,最终可以有效解决我国玉米种业行业长期面临缺乏稳定的玉米不育系和突破性大品种的瓶颈问题。
发明内容
针对现有技术的不足,本发明的目的是提供利用人工突变创制玉米bhlh51雄性不育系,可以用来创制玉米雄性不育系,从而应用于玉米杂交育种和制种。
为实现上述目的,本发明提供了一种创制玉米雄性不育系的方法,其特征在于,抑制玉米基因组中的育性基因编码蛋白的表达和/或活性,进而使所述育性基因功能丧失,得到玉米雄性不育系;所述育性基因为ZmbHLH51(Zm00001d053895);所述ZmbHLH51(Zm00001d053895)的核酸序列如SEQ ID NO.1所示;所述ZmbHLH51(Zm00001d053895)编码蛋白的氨基酸序列如SEQ ID NO.2所示。
在一些实施方案中,上述抑制蛋白表达和/或活性的方法包括基因编辑、RNA干扰、T-DNA***中任一种。
在一些实施方案中,上述基因编辑采用CRISPR/Cas9方法。
在一些实施方案中,在该基因第一外显子处设计CRISPR/Cas9载体靶点,所述靶点的DNA序列如SEQ ID NO.3或SEQ ID NO.4所示。
在另一方面,本发明还提供一种获得bhlh51雄性不育系的方法,将通过上述方法获得的bhlh51雄性不育系与目标材料进行杂交和回交,从而使目标材料获得bhlh51雄性不育的性状和基因突变。
本发明还包括通过上述任一方法获得的bhlh51雄性不育系在杂交育种和制种中的应用。所述在杂交育种和制种中的应用是指将bhlh51雄性不育系作为母本与其他父本进行杂交,或是将获得的bhlh51雄性不育系与其他目标材料进行杂交和回交,从而使目标材料获得bhlh51雄性不育的性状和基因突变。
更进一步地,本发明还提供了一种玉米雄性不育系bhlh51的分子标记引物,引物ZmbHLH51-F和ZmbHLH51-R序列分别如SEQ ID NO.5和SEQ ID NO.6所示。
本发明的优点及有益效果如下:本发明利用CRISPR/Cas9基因编辑技术,通过遗传转化,将育性基因bHLH51(Zm00001d053895)在玉米中特异敲除,经一系列实验获得了三种具有新的突变位点的bhlh51雄性不育系。这些不育系育性稳定,呈现完全败育,可以创制不同遗传背景的玉米雄性不育系,从而可以应用于玉米杂交育种和制种。针对三种blhh51的雄性不育系开发共分离分子标记,可用于植株的育性等位基因鉴定、分子标记辅助育种中目标单株的筛选和种子纯度鉴定等。
附图说明
图1为ZmbHLH51基因在玉米不同发育时期花药中的表达模式分析
S5,造孢细胞时期;S6, 小孢子母细胞时期;S7,减数***开始时期;S8a,减数***Ⅰ,二分体时期;S8b,减数***Ⅱ,四分体时期;S8b-9,四分体-单核小孢子时期;S9,单核小孢子时期;S9-10,单核小孢子-小孢子空泡化时期;S10,小孢子空泡化时期;S11,小孢子第一次不均等有丝***,二核小孢子时期;S12,小孢子第二次有丝***,三核小孢子时期。
图2为pCas9-ZmbHLH51定点突变表达载体的物理图谱
pCas9-ZmbHLH51:从T-DNA的左边界到右边界分别是除草剂抗性基因Bar的表达盒,核酸酶编码基因Cas9的表达盒,ZmbHLH51基因靶标2(MT2)的表达盒;靶标1(MT1)的表达盒。
图3为野生型ZmbHLH51与其不育突变体的基因结构和DNA序列分析
野生型ZmbHLH51(WT- ZmbHLH51):基因全长2734 bp,包括7个外显子和6个内含子;bhlh51突变体ZmbHLH51-Cas9-1:在第1个外显子300 bp-342 bp之间缺失43个碱基;突变体ZmbHLH51-Cas9-2:在第1个外显子301 bp-344 bp之间缺失44个碱基;突变体ZmbHLH51-Cas9-3:在第1个外显子292 bp-314 bp之间缺失23个碱基,在342 bp位置***1个碱基。
图4为野生型与bhlh51纯合突变体的雄穗、花药和花粉粒表型分析
上排为玉米野生型(WT)及ZmbHLH51-Cas9-1、ZmbHLH51-Cas9-2、ZmbHLH51-Cas9-3突变体雄穗的表型比较;第二排为WT及ZmbHLH51-Cas9-1、ZmbHLH51-Cas9-2、ZmbHLH51- Cas9-3突变体花药的表型比较;下排为WT及ZmbHLH51-Cas9-1、ZmbHLH51-Cas9-2、ZmbHLH51-Cas9-3突变体花粉粒的I2-KI染色比较。
图5为野生型与bhlh51纯合突变体的花药扫描电镜(SEM)分析
从左至右依次为:野生型(WT)花药整体;bhlh51花药整体;剥开后的WT(上)和bhlh51(下)花药;WT的成熟花粉粒(上)和bhlh51无法扫描到花粉粒(下);WT(上)和bhlh51(下)花药的外表皮角质层;WT(上)和bhlh51(下)花药的内表皮乌氏体。
图6为利用共分离标记对ZmbHLH51-Cas9-1不育系的F2代植株进行基因分型
共分离标记ZmbHLH51-F/R对6株ZmbHLH51-Cas9-1不育系F2代植株的PCR和琼脂糖凝胶电泳鉴定结果:在纯合野生型(AA)植株中扩增出399 bp条带;在bHLH51/ bhlh51杂合型(Aa)植株中扩增出399 bp和356 bp两条带;在bhlh51/ bhlh51纯合突变型(aa)植株中扩增出356 bp条带。
图7为利用共分离标记对ZmbHLH51-Cas9-2不育系的F2代植株进行基因分型
共分离标记ZmbHLH51-F/R对5株ZmbHLH51-Cas9-2不育系F2代植株的PCR和琼脂糖凝胶电泳鉴定结果:在纯合野生型(AA)植株中扩增出399 bp条带;在bHLH51/ bhlh51杂合型(Aa)植株中扩增出399 bp和355 bp两条带;在bhlh51/ bhlh51纯合突变型(aa)植株中扩增出355 bp条带。
图8为利用共分离标记对ZmbHLH51-Cas9-3不育系的F2代植株进行基因分型
共分离标记ZmbHLH51-F/R对5株ZmbHLH51-Cas9-3不育系F2代植株的PCR和琼脂糖凝胶电泳鉴定结果:在纯合野生型(AA)植株中扩增出399 bp条带;在bhlh51/ bhlh51杂合型(Aa)植株中扩增出399 bp和377 bp两条带;在bhlh51/ bhlh51纯合突变型(aa)植株中扩增出377 bp条带。
具体实施方式
下述实施例用来说明本发明,但不限制本发明的范围。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。如无特殊说明,实施例中所用引物及基因的合成和测序均由生工生物工程(上海)股份有限公司完成。其他生化试剂非特别注明外均为常规市售试剂,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例一玉米ZmbHLH51(Zm00001d053895)基因序列和表达模式分析
在maizeGDB库(https://www.maizegdb.org/)中,查询到玉米bHLH51(Zm00001d053895,GRMZM2G139372)基因,在B73中的核酸序列见SEQ ID NO.1所示,基因功能注释为bHLH51转录因子(bHLH-transcription factor 51,bHLH51),其编码蛋白包含625个氨基酸,序列如SEQ ID NO.2所示。
bHLH转录因子参与植物中众多生理过程的调节,虽然ZmbHLH51基因已经有研究表明在玉米中与雄性生殖发育有关,但为了进一步研究该基因与玉米雄性生殖发育的关系以及快速创制雄性不育系,本发明首先利用qRT-PCR分析了该基因在玉米花药发育不同阶段的表达模式。具体步骤如下:
1、玉米花药的取样与发育时期鉴定
从玉米自交系B73处于不同发育阶段的雄穗中,按照花药的长度,收集不同长度的花药样品;每份样品采集20个长度相近的新鲜花药,其中3个固定在FAA溶液中(Coolaber,中国)通过树脂半薄切片实验确定具体的发育阶段,其余17个花药立即冷冻在液氮中,用于RNA的提取。
利用梯度乙醇(50%、70%、90%、100%)对用于树脂切片的固定花药进行脱水,每步15-30分钟。脱水过程中花药可置于70%乙醇中长期保存;为便于后期包埋,可在90%乙醇中加入0.1%的伊红对材料进行染色;为保证脱水彻底,材料须在无水乙醇中脱水2-3次。随后进行树脂替换,将花药依次置于乙醇与Spurr树脂体积比为3:1、1:1、1:3液体中2-4小时,最后置于纯树脂中过夜。树脂置换完成后,将花药置于模具中,加入200 µL Spurr树脂,置于烘箱中,70℃聚合过夜。随后进行修块,然后可利用德国莱卡切片机进行切片,切片厚度为2µm;用镊子夹取切好的片子,置于载玻片中央的无菌水中,42℃条件下展片过夜。将固定有样品的载玻片浸入0.1%的甲苯胺蓝染液中,染色1分钟,然后用去离子水冲洗,再置于展片台上,烘干后即可用于显微观察;也可以封片后长期保存。分析树脂切片的结果,根据玉米14个不同发育时期(Stage1-Stage14:S1-S14)的细胞学特点,确定每份样品的具体发育时期。
2、qRT-PCR分析
用Trizol试剂(Invitrogen,美国)提取上述鉴定的处于不同发育时期(S5-S12)的玉米花药总RNA;然后使用5X All-in-One RT Master Mix(ABM,加拿大)合成cDNA;采用TBGreen™PreMix Ex Taq™(TaKaRa,日本)在QuantStudio5QuantStudio 5 Real-Time PCRSystem (ABI,美国)上进行定量逆转录聚合酶链反应检测,扩增引物为:qbHLH51-F(5’-ACATGAAGCTCGATGGCATCAT-3’)和qbHLH51-R(5’-CTTTCAACATGAACGAACGCA-3’);ZmActin1为参照基因,其扩增引物为:Actin1-F(5’- AAATGACGCAGATTATGTTTGA-3’)和Actin1-R(5’-GCTCGTAGTGAGGGAGTACC-3’);每个发育时期包括三个生物学重复,每个样品有三个技术重复;数据用2-ΔΔCt方法进行分析,并以均值±标准差(Means±SD)的形式给出定量结果。
ZmbHLH51基因呈现花药发育时期特异表达的模式:在玉米花药发育的中期比如S7时期有较高表达,随后开始减弱,然后到花药发育的中后期(S8b)表达又逐渐开始上升,S9时期开始减弱(图1)。
实施例二玉米ZmbHLH51(Zm00001d053895)基因的功能以及利用CRISPR/Cas9方法创制玉米雄性不育系
为了明确玉米ZmbHLH51(Zm00001d053895)在玉米中的功能,本发明采用CRISPR/Cas9基因编辑方法突变Zm00001d053895基因序列,敲除该基因在玉米中的功能。本发明选取玉米杂交种HiII 作为基因编辑的受体材料。本发明分别选取基因保守区的SEQ ID NO.3和SEQ ID NO.4所示序列作为CRISPR/Cas9基因编辑的靶标区域。
1、ZmbHLH51的CRISPR/Cas9基因编辑载体的构建
本发明的基因编辑载体为pBUE411-MT1T2-Cas9,该载体的基础载体为pBUE411- Cas9,中间载体为pCBCmT1T2,提供gRNA。本发明通过在引物上设计靶点然后通过PCR获得MT-sgRNA进而通过酶切连接到基础载体中,具体的构建流程如下:
(1)靶标gRNA的设计。将ZmbHLH51(Zm00001d053895)的基因序列输入http://crispr.hzau.edu.cn/cgi-bin/CRISPR2/CRISPR进行靶标设计。本发明选择的两个靶标区域的DNA序列如SEQ ID NO.3和SEQ ID NO.4所示。本发明的sgRNA骨架序列从中间载体pCBCmT1T2直接扩增获得。
(2)通过在引物上设计靶点然后PCR扩增获得MT-sgRNA。引物ZmbHLH51-MT1-F和引物ZmbHLH51-MT2-R扩增中间载体pCBCmT1T2,用于获得包含第一个和第二个靶标的sgRNA的片段,产物长度为891bp。PCR 体系和条件如下:模板DNA (中间载体pCBCmT1T2≥30ng/μL)1.2μL;Primer F/R:各1.2μL;灭菌ddH2O:11.4μL;2X MCLAB 酶(产品编号:I5HM-200):15μL。PCR的温度程序如下:①98℃ 2分钟;②98℃ 10秒;③58℃ 30秒;④72℃ 30秒;⑤ 从②-④循环34次;⑥72℃ 5分钟;⑦25℃ 10分钟。最后回收PCR产物。载体构建所需的引物序列如下:
ZmbHLH51-MT1-F:5’-ATATATGGTCTCTGGCGAGGACTACTGCGTCTACTGGGTTTTAGAGCTAGAAATAGCAA-3’
ZmbHLH51-MT2-R:5’-ATTATTGGTCTCTAAACCGTTGAGGCCTCTTGTCGGTGCTTCTTGGTGCCGC -3’
(3)通过酶切连接构建到骨架载体。将pBUE411-Cas9载体和回收的带有靶标的sgRNA片段用BsaI消化,同时加入T4连接酶将载体和sgRNA片段连接。15μL的酶切连接体系如下,sgRNA片段:2μL,pBUE411-Cas9载体(≥60ng/μL) :2μL,10x NEB Buffer:1.5μL,BsaI内切酶(产品编号:#R3733S ):1μL,T4 连接酶(产品编号:#M0202M ):1μL,灭菌ddH2O:6μL。
图2所示为目的基因ZmbHLH51(Zm00001d053895)的双靶标(对应第一个靶标和第二个靶标),标记基因Cas9和bar与骨架载体pBUE411-Cas9构建的表达载体pCas9- ZmbHLH51。
2、农杆菌介导的玉米遗传转化
将上述构建的pCas9-ZmbHLH51载体分别通过热激法转入农杆菌EHA105中,PCR进行鉴定;然后加甘油于-80℃保存菌液。以新鲜剥离的1.5mm左右的玉米杂交种HiII的幼胚作为受体材料,将剥离的玉米胚放入含有1.8mL悬浮液的2mL塑料离心管中,放置时间不超过1小时,每个离心管中大约放入100个幼胚;吸去悬浮液,并用新的悬浮液将幼胚清洗2遍,管底保留少量可没过幼胚的悬浮液,然后43℃热激2分钟,之后再冰浴1分钟,用移液枪吸尽管底残留的洗液,并加入1.0mL的农杆菌侵染液,轻摇30秒,然后黑暗静置8分钟。接下来将离心管中的幼胚和侵染液倒入共培养基上,晃匀后用移液枪吸出多余的侵染液,所有幼胚的盾片朝上,于23℃黑暗共培养3天。共培养结束后,用无菌的镊子将幼胚转移至恢复培养基,于28℃培养7-14天,中间过程需留意及时去掉幼胚上长出的幼芽。恢复培养结束后将幼胚放至含1.5 mg/L Bialaphos筛选培养基上筛选培养3轮,每轮筛选2周,然后转到2mg/LBialaphos筛选培养基上筛选培养2轮,每轮筛选2周。将抗性愈伤组织转至扩繁培养基,28℃,暗培养2周。随后将扩繁好的抗性愈伤组织转移至诱导培养基,于28℃黑暗环境下培养2周。然后转到分化培养基中,25℃,5000lx,光照培养2周。培养结束后将分化出的苗簇分离出单个幼苗放置于生根培养基中,25℃,5000lx,光照培养直到生根;将小苗转入小营养钵中生长,生长成活后移栽于温室中,3-4个月后收获后代种子。
3、T0代植株CRISPR/Cas9突变结果检测
为确定T0代植株CRISPR/Cas9突变结果,采取如下步骤进行:
本发明首先采用CTAB法提取玉米叶片DNA,具体方法如下:剪取2厘米左右长度的幼苗叶片,放入装有钢珠的2mL离心管;将装有叶片的离心管放入液氮浸没5分钟,然后利用研磨仪打碎叶片样品;向离心管中加入700μL CTAB提取缓冲液(其中含有1%的β-巯基乙醇),并用力震荡混匀, 65 ℃恒温水浴中预热20-30 min,(期间取出颠倒1-2次,并注意实验样品编号的对应);离心管冷却至室温后加入700μL氯仿:异戊醇(24:1)萃取液,用力摇晃30s之后在室温条件下静置片刻;在4℃条件下,以12000rpm速度离心5 min,取离心完成后的500 μl上清液于新的1.5mL离心管中;将等体积的异丙醇加入到盛有上清液的离心管中轻轻震荡混匀,室温条件下静置10min左右;随后将盛有样品的离心管放入4℃离心机中,以12000rpm速度离心10min之后,轻轻吸取上清液,弃上清,保留沉淀;加入800μL 75 %乙醇,洗涤沉淀两次,以10000rpm速度离心5min,弃掉上清;将样品放置在室温下自然干燥2-4小时,得到DNA沉淀,加入适量无菌水溶解,轻微震荡,充分溶解DNA。DNA样品存于-20℃保存。用Nanodrop检测DNA浓度,稀释至10ng/L用作PCR模板。
然后根据ZmbHLH51(Zm00001d053895)基因序列设计PCR引物。
(1)检测靶标:MT1和MT2;产物大小:399 bp;引物序列如下:
ZmbHLH51-T-F:5’-CATGGCTGCTGCTGCTTAT-3’;
ZmbHLH51-T-R:5’-TGCAGCAGAACCCAGCCATCTC-3’。
提取基因组DNA,按照以下PCR参数扩增:
反应体系:15μL MIX常规PCR体系,0.5μL forward primer,0.5μL reverseprimer,1μL DNA,5.5μL灭菌ddH2O,7.5μL 2x taq mix(产品编号:10103ES )。
反应程序:常规PCR:58℃退火、延伸1分钟、32轮循环。
接着将PCR产物回收并连接T载体测序,通过测序多个T0代独立阳性转化事件靶标区域的DNA序列,明确靶标区域是否发生基因编辑,最终发现3个T0转化事件靶标区域序列发生了变化,且均为同源突变,编辑前后的序列如图3所示,对应3个bhlh51同源突变体:ZmbHLH51-Cas9-1、ZmbHLH51-Cas9-2、ZmbHLH51-Cas9-3。与野生型序列比对显示ZmbHLH51- Cas9-1在靶标1和2处以及间隔的DNA片段发生了缺失突变,ZmbHLH51-Cas9-2在靶标1和2处以及间隔的DNA片段同样发生了缺失突变,而ZmbHLH51-Cas9-3在在靶标1和2处发生了缺失和***突变。
对3个bhlh51突变体中氨基酸序列进行比对分析发现,与未编辑的WT相比,突变后的品系ZmbHLH51-Cas9-1和ZmbHLH51-Cas9-2,其编码的的核苷酸在靶标1和2处的缺失使其氨基酸发生了移码突变,且随后的氨基酸发生提前终止。另外ZmbHLH51-Cas9-3品系中编码的核苷酸在分别在靶标1和2处的***和缺失也使得氨基酸发生移码并导致氨基酸翻译提前终止。因此这些转化体中Zm00001d053895蛋白的功能均表现缺失。
4、F1代植株的基因分型
由于在温室生长的玉米T0代植株,经常雌穗和雄穗发育不协调,同时当编辑的基因与雄性发育相关时也会影响育性,因此为了繁殖T0代植株,并使获得的基因编辑类型遗传下去,本发明使用玉米自交系郑58的野生型花粉为上述获得的ZmbHLH51-Cas9-1、ZmbHLH51-Cas9-2、ZmbHLH51-Cas9-3的T0代植株授粉,进而获得F1代种子,生长的植株为F1代植株。
F1代植株包括2种分离类型,一种为Cas9-阳性植株(转基因植株),另一种为Cas9-阴性植株(非转基因植株),为了避免sgRNA和Cas9对杂交授粉引入的郑58野生型等位基因进行持续编辑,从而造成突变类型的复杂性,我们需要通过基因分型从F1代植株中挑选出不含有Cas9基因,但含有T0代突变类型的植株,这类植株自交后可以得到非转基因的F2代。F1代植株的基因分型步骤如下:
按照上述的CTAB法提取叶片DNA后,首先利用Cas9基因的特异引物Cas9-F(5’-CCCGGACAATAGCGATGT-3’)和Cas9-R(5’- GAGTGGGCCGACGTAGTA-3’)进行PCR扩增。PCR反应体系同上;反应程序:常规PCR:58℃退火、延伸1分钟、32轮循环。PCR产物进行琼脂糖凝胶电泳后,根据结果区分出Cas9-阳性植株和Cas9-阴性植株。
进一步针对Cas9-阴性植株,采用上述的检测MT1和MT2靶标的引物ZmbHLH51-T-F和ZmbHLH51-T-R进行PCR扩增;PCR产物纯化后,连接T载体,进行测序;根据测序结果分析确定F1代突变类型的遗传情况。
实施例三bhlh51不育系的表型分析
上述实施例二鉴定的不含有Cas9基因的F1代植株自交后获得F2代种子,三种bhlh51突变类型(ZmbHLH51-Cas9-1、ZmbHLH51-Cas9-2和ZmbHLH51-Cas9-3)各取1个自交单穗进行穗行播种,在成熟期进行表型的调查。三种F2株系中,可育株与不育株的比例,均符合3:1分离,进一步表明bhlh51不育系的不育性状由单个隐性基因控制,然后针对F2代获得的稳定非转基因bhlh51不育系与野生型进行详细的表型比较。
1、雄穗、花药和花粉活力的观察
在营养生长和雌穗的发育方面, bhlh51不育系(ZmbHLH51-Cas9-1、ZmbHLH51- Cas9-2和ZmbHLH51-Cas9-3)的植株与野生型相比基本无差异;在雄穗发育方面,而bhlh51不育系虽能正常抽雄,但是不能正常开花,花药颖壳完全不开裂,花药明显皱缩干瘪,并且发白不外露(图4);进一步对野生型和突变体的花粉进行I2-KI染色,发现野生型花粉发育正常,花粉粒染色后呈黑色,但突变体无花粉粒形成(图4)。这表明ZmbHLH51(Zm00001d053895)基因控制玉米的雄性发育,通过基因编辑方法创制的bhlh51不育系为无花粉型不育系,呈现完全败育的特点。
2、花药的扫描电镜(SEM)观察
为了深入解析bhlh51的细胞学特征,对野生型和突变体花药内外壁进行扫描电镜(SEM)分析。剥取处于成熟期(S13)的野生型和突变体花药,然后立即固定在FAA(Coolaber,中国)溶液中,固定液的体积不少于所取研究材料体积的20倍;对于突变体花药,可用解剖针在花药壁穿孔以提高固定液的渗透效果,或者反复抽真空至花药沉入固定液底部;室温固定2小时后,将材料置于4℃保存,或依次置于50%、60%、70%、80%、90%、100%的乙醇中进行脱水,每个梯度保持15分钟;材料可置于70%的乙醇中过夜或保存。脱水后的样品进行二氧化碳临界点干燥,然后镀金即可进行观察。发现bhlh51突变体的花药外表皮光滑,始终不能形成网状的角质层结构,而野生型则形成了致密的网状角质层结构;同样bhlh51突变体花药的内表皮也表现光滑,没有致密的颗粒状的乌氏小体形成(图5)。花药角质层是覆盖在花药表面的细胞外脂质层,保护花药免受外部非生物胁迫、内部组织失水和病原体的侵袭,位于花药内壁的乌氏体,被认为是孢粉素前体从绒毡层细胞到小孢子的转运载体。上述结果表明ZmbHLH51(Zm00001d053895)基因突变后,可以影响花药角质层的形成和阻断绒毡层内孢子花粉素前体物质的合成。
实施例四bhlh51不育系鉴定的共分离分子标记开发和应用
1、共分离分子标记的开发
在本发明中,针对获得的三种bhlh51不育系的突变位点,利用Primer5.0软件进行引物设计,开发出一对共分离分子标记:ZmbHLH51-F/R,结合PCR与琼脂糖凝胶电泳检测的方法,根据获得的条带及大小即可分离出突变体的基因型。
共分离分子标记ZmbHLH51-F/R包括第一引物ZmbHLH51-F和第二引物ZmbHLH51-R;该标记能够特异性检测玉米ZmbHLH51-Cas9-1、-2、-3突变体及由其转育的玉米不育材料中的突变基因bhlh51,并能同时区分野生型bHLH51基因和突变型bhlh51基因;针对突变基因bhlh51中分别能扩增出356bp、355bp和377bp的条带,而对野生型bHLH51基因扩增出399bp的条带。引物序列如下:
ZmbHLH51-F:5’-CATGGCTGCTGCTGCTTAT-3’
ZmbHLH51-R:5’-TGCAGCAGAACCCAGCCATCTC-3’
2、共分离分子标记的应用
为了验证上述标记的有效性,以实施例三中获得的F2株系为材料,进行bHLH51等位基因的检测。DNA提取方法、PCR扩增体系和条件同实施例二,PCR产物进行PAGE或琼脂糖凝胶电泳分离。
理论上,ZmbHLH51-F/R在bHLH51/bHLH51纯合野生型(AA)DNA中能扩增出399 bp的条带,在bhlh51/bhlh51纯合突变型材料(aa)DNA中分别扩增出356bp、355bp和377 bp的条带,而在bHLH51/bhlh51杂合型(Aa)材料中,则能分别同时扩增出对应的两条条带。ZmbHLH51-F/R分子标记的验证结果如图6、图7和图8所示,结果显示设计的3个功能性分子标记对F2植株的检测结果完全符合预期,在bHLH51/bHLH51纯合野生型(AA)、bHLH51/ bhlh51杂合型(Aa)和bhlh51/bhlh51突变型材料(aa)中分别扩增出对应大小的条带,可以作为bHLH51,bhlh51等位基因检测的理想标记。
这些分子标记有助于在开花和授粉前确定突变基因型,以便在不同遗传背景下进行杂交和回交选育雄性不育系,具有重要的应用价值。
序列表
<110> 北京科技大学
北京中智生物农业国际研究院
北京首佳利华科技有限公司
<120> 利用人工突变创制ZmbHLH51雄性不育突变体
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2734
<212> DNA
<213> 玉米(Zea mays)
<400> 1
atgacatcgg ccggcgatcc gatctctatc ctgattcctg acacacaagc acggccacgg 60
aaccctcgcg catgcatgct gcctgctgac gcttaccttc gtttcgtttt catggctgct 120
gctgcttatt gctgcgagtg cgatgtgcag gcggcagcgg gcacagtcct ccagtcctcg 180
ggtgaggcga tcgtcgccgg cgccatggga gggggagtcc accaccacca cccgtgcgtg 240
gctgctgatg gagatggggc cggggccggg cccgggccgg ccagcgtgga ggccgcgttg 300
aggcctcttg tcggcgtcga cgcctgggac tactgcgtct actggaggct gtctcctgat 360
cagaggtcgg cgccgacgcc tgggacttgt tcctttttgt gtgtgttctt ttatggagct 420
cggaaatcag aaccaggcga atgcgtgtga tggcgtgtgt gtgccacaaa ctgcttgcag 480
gttcttggag atggctgggt tctgctgcag cagtcagttc gaggcacagc ttccagcgct 540
gggcgacctg cctccatcaa tccagctcga ctcctcgtct gcagggtatg atctccgtca 600
gctccgctat cactcggccg agttctaatc cctgcatgct cgatctgacg gtcggccagg 660
atgcacgccg aggcaatggt gtccaaccag ccgatctggc agagcagccg cgtgccagag 720
ctccaaacag gttactccag tgtacgtaag cagagcacat cgatcgatca tcggctcttg 780
tcatcttcaa cctccctccc tccgtccgat ccagtccact gcctaagaat ccatccaact 840
gcatgtctga tcatcgtctt gtatccatga cagggcatgg tgcaggagcc cgggtccagc 900
ggcggcccga ggacgcggct gctggtgccc gtcgccggcg gcctcgtcga gctcttcgcg 960
gcgagatacg tgcgtaagcc actgcagctg gacctgcgcc gtttcttttt cgttcttatt 1020
attttcttct gtttgccgcc atcacgtgag aaattccatt tccaggattg ttagtttcta 1080
gagtacggct acgggtagtt agctagagag acctgccaga accagaacca gaagcgcacg 1140
gcttctgcgt gagccagcgt cgccgccccc ggcgaagatg tggcggtgaa ccgctcgcaa 1200
cagccacggc ctttgcattt gcatgccatg cgcacacaaa cacatgacat gacatgacat 1260
gacacaccag acttgacgct ctcctgtctt tatatgccgg cgcggcgcgc gggcgtgcgt 1320
gcgcgcgcag atggcggagg aggagcagat ggcggagctg gtgatggcgc agtgcggggt 1380
gccgagcggc ggtgaggggg gcgcgtggcc gccgggattc gcgtgggacg gcggcgcctc 1440
ggacgcgtcg cgtgggatgt acggcgatgc ggtgccgccg tcgctcagcc tgttcgacgc 1500
cgccggcagc gtcgcggcgg acccgttcca ggcggtgcag caggcgccgg gcgccggtgg 1560
tggtggggtg gacgacgtcg ccgggtggca gtatgctgct gcggctggga gcgagctgga 1620
ggcggtgcag ctgcagcagg agcagcagcc gcgcgatgcg gactcggggt ccgaggtcag 1680
cgacatgcag ggggacccag aggacgacgg cgacggcgac gcgcaggggc gtggcggcgg 1740
caagggcggc gggaagcggc agcagtgcaa gaacctcgag gcggagcgga agcggcggaa 1800
gaagctcaac gagcggctct acaagctcag gtcgctcgtc ccgaacatct ccaaggtcgg 1860
tgtccgtcgg ccccaccgtc gccatccatg gaaacgcttg attccttgct tctctgatga 1920
tgccatcgat gatattcttc agatggaccg cgcggcgatc ctcggggacg ccatcgacta 1980
catcgtgggc ctgcagaacc aggtgaaggc gctgcaggac gagctggagg acccggcgga 2040
cggcgccggc gcccccgacg tcctcctgga ccacccgccg ccggcgagcc tggtggggct 2100
ggagaacgac gagtcgccgc ccacgagcca ccagcacccg ctcgccggga ccaagagggc 2160
ccgtgcggcg gcggaggagg aggaggagga gaaggggaat gacatggagc cgcaggtgga 2220
ggtgcggcag gtggaggcca acgagttctt cctgcagatg ctgtgcgagc gccggcccgg 2280
gcgcttcgtc cagatcatgg actccatcgc cgacctggga ctggaggtca ccaacgtcaa 2340
cgtcacctcc cacgagagcc tcgtcctcaa cgtcttccgc gccgccgtac gtacaatgac 2400
accgccgcca ttattgacgc atgcaattcg tagcgcacgt gctcattctc tctctctccc 2460
atggtggtgc tggcagaggc gggacaatga ggtggcagtg caggcggaca gactgaggga 2520
ctcgctgctg gaggtgatgc gggagccgta cggcgtatgg tcgtcgtcgg cgccgcccgt 2580
ggggatgagc ggcagcggca tcgccgacgt gaagcatgac agcgtggaca tgaagctcga 2640
tggcatcatc gacgggcagg cggcaccgag cgtcgcagtg ggggtttcag aggatcacta 2700
cggcggctac aaccatctcc tccaatacct cgct 2734
<210> 2
<211> 625
<212> PRT
<213> 玉米(Zea mays)
<400> 2
Met Thr Ser Ala Gly Asp Pro Ile Ser Ile Leu Ile Pro Asp Thr Gln
1 5 10 15
Ala Arg Pro Arg Asn Pro Arg Ala Cys Met Leu Pro Ala Asp Ala Tyr
20 25 30
Leu Arg Phe Val Phe Met Ala Ala Ala Ala Tyr Cys Cys Glu Cys Asp
35 40 45
Val Gln Ala Ala Ala Gly Thr Val Leu Gln Ser Ser Gly Glu Ala Ile
50 55 60
Val Ala Gly Ala Met Gly Gly Gly Val His His His His Pro Cys Val
65 70 75 80
Ala Ala Asp Gly Asp Gly Ala Gly Ala Gly Pro Gly Pro Ala Ser Val
85 90 95
Glu Ala Ala Leu Arg Pro Leu Val Gly Val Asp Ala Trp Asp Tyr Cys
100 105 110
Val Tyr Trp Arg Leu Ser Pro Asp Gln Arg Phe Leu Glu Met Ala Gly
115 120 125
Phe Cys Cys Ser Ser Gln Phe Glu Ala Gln Leu Pro Ala Leu Gly Asp
130 135 140
Leu Pro Pro Ser Ile Gln Leu Asp Ser Ser Ser Ala Gly Met His Ala
145 150 155 160
Glu Ala Met Val Ser Asn Gln Pro Ile Trp Gln Ser Ser Arg Val Pro
165 170 175
Glu Leu Gln Thr Gly Tyr Ser Ser Gly Met Val Gln Glu Pro Gly Ser
180 185 190
Ser Gly Gly Pro Arg Thr Arg Leu Leu Val Pro Val Ala Gly Gly Leu
195 200 205
Val Glu Leu Phe Ala Ala Arg Tyr Met Ala Glu Glu Glu Gln Met Ala
210 215 220
Glu Leu Val Met Ala Gln Cys Gly Val Pro Ser Gly Gly Glu Gly Gly
225 230 235 240
Ala Trp Pro Pro Gly Phe Ala Trp Asp Gly Gly Ala Ser Asp Ala Ser
245 250 255
Arg Gly Met Tyr Gly Asp Ala Val Pro Pro Ser Leu Ser Leu Phe Asp
260 265 270
Ala Ala Gly Ser Val Ala Ala Asp Pro Phe Gln Ala Val Gln Gln Ala
275 280 285
Pro Gly Ala Gly Gly Gly Gly Val Asp Asp Val Ala Gly Trp Gln Tyr
290 295 300
Ala Ala Ala Ala Gly Ser Glu Leu Glu Ala Val Gln Leu Gln Gln Glu
305 310 315 320
Gln Gln Pro Arg Asp Ala Asp Ser Gly Ser Glu Val Ser Asp Met Gln
325 330 335
Gly Asp Pro Glu Asp Asp Gly Asp Gly Asp Ala Gln Gly Arg Gly Gly
340 345 350
Gly Lys Gly Gly Gly Lys Arg Gln Gln Cys Lys Asn Leu Glu Ala Glu
355 360 365
Arg Lys Arg Arg Lys Lys Leu Asn Glu Arg Leu Tyr Lys Leu Arg Ser
370 375 380
Leu Val Pro Asn Ile Ser Lys Met Asp Arg Ala Ala Ile Leu Gly Asp
385 390 395 400
Ala Ile Asp Tyr Ile Val Gly Leu Gln Asn Gln Val Lys Ala Leu Gln
405 410 415
Asp Glu Leu Glu Asp Pro Ala Asp Gly Ala Gly Ala Pro Asp Val Leu
420 425 430
Leu Asp His Pro Pro Pro Ala Ser Leu Val Gly Leu Glu Asn Asp Glu
435 440 445
Ser Pro Pro Thr Ser His Gln His Pro Leu Ala Gly Thr Lys Arg Ala
450 455 460
Arg Ala Ala Ala Glu Glu Glu Glu Glu Glu Lys Gly Asn Asp Met Glu
465 470 475 480
Pro Gln Val Glu Val Arg Gln Val Glu Ala Asn Glu Phe Phe Leu Gln
485 490 495
Met Leu Cys Glu Arg Arg Pro Gly Arg Phe Val Gln Ile Met Asp Ser
500 505 510
Ile Ala Asp Leu Gly Leu Glu Val Thr Asn Val Asn Val Thr Ser His
515 520 525
Glu Ser Leu Val Leu Asn Val Phe Arg Ala Ala Arg Arg Asp Asn Glu
530 535 540
Val Ala Val Gln Ala Asp Arg Leu Arg Asp Ser Leu Leu Glu Val Met
545 550 555 560
Arg Glu Pro Tyr Gly Val Trp Ser Ser Ser Ala Pro Pro Val Gly Met
565 570 575
Ser Gly Ser Gly Ile Ala Asp Val Lys His Asp Ser Val Asp Met Lys
580 585 590
Leu Asp Gly Ile Ile Asp Gly Gln Ala Ala Pro Ser Val Ala Val Gly
595 600 605
Val Ser Glu Asp His Tyr Gly Gly Tyr Asn His Leu Leu Gln Tyr Leu
610 615 620
Ala
625
<210> 3
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ggactactgc gtctactgg 19
<210> 4
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cgttgaggcc tcttgtcgg 19
<210> 5
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
catggctgct gctgcttat 19
<210> 6
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tgcagcagaa cccagccatc tc 22
Claims (6)
1.一种针对玉米雄性育性基因ZmbHLH51创制等位雄性不育突变体的方法,其特征在于,采用基于CRISPR/Cas9的基因编辑方法,对玉米基因组中的ZmbHLH51基因进行定点突变,进而使其育性功能丧失,得到不同突变类型的玉米雄性不育系;
所述育性基因为编码ZmbHLH51蛋白的基因;
所述CRISPR/Cas9***中包括两个sgRNA,位于ZmbHLH51基因的第一外显子处,分别命名为MT1-gRNA和MT2-gRNA;
所述MT1-gRNA识别的靶序列为SEQ ID NO.3所示;
所述MT2-gRNA识别的靶序列为SEQ ID NO.4所示;
所述编辑的方法为向所述受体玉米中导入玉米基因组编辑的载体;
所述玉米基因组编辑的载体含有所述MT1-gRNA的编码基因、所述MT2-gRNA的编码基因、RNA聚合酶Ⅲ识别的启动子、Cas9蛋白表达盒和筛选标记基因;
用于启动所述MT1-gRNA的编码基因转录的RNA聚合酶Ⅲ识别的启动子为启动子OsU3;
用于启动所述MT2-gRNA的编码基因转录的RNA聚合酶Ⅲ识别的启动子为启动子TaU3。
2.如权利要求1所述的方法,其特征在于:所述ZmbHLH51等位雄性不育突变体包括ZmbHLH51-Cas9-1、ZmbHLH51-Cas9-2和ZmbHLH51-Cas9-3;其中,ZmbHLH51-Cas9-1在第1个外显子300bp-342bp之间缺失43个碱基;ZmbHLH51-Cas9-2在第1个外显子301bp-344bp之间缺失44个碱基;ZmbHLH51-Cas9-3在第1个外显子292bp-314bp之间缺失23个碱基,在342bp位置***1个碱基。
3.一种获得bhlh51雄性不育系的方法,其特征在于通过权利要求1-2之任一所述方法获得的bhlh51雄性不育系与目标材料进行杂交和回交,从而使目标材料获得bhlh51雄性不育的性状和基因突变。
4.通过权利要求3所述的方法获得的bhlh51雄性不育系在杂交育种和制种中的应用。
5.如权利要求4所述的应用,其中所述的杂交育种和制种是指将所述bhlh51雄性不育系作为母本与其他父本进行杂交。
6.如权利要求5所述的应用,包括将获得的bhlh51雄性不育系与其他目标材料进行杂交和回交,从而使目标材料获得 bhlh51雄性不育的性状和基因突变。
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