CN114395581A - 一种通过基因编辑技术创制西瓜全缘叶种质的方法 - Google Patents
一种通过基因编辑技术创制西瓜全缘叶种质的方法 Download PDFInfo
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Abstract
本发明公开了一种通过基因编辑技术创制西瓜全缘叶种质的方法,属于植物基因工程技术领域。本发明首次利用CRISPR/Cas9基因编辑***,对HD‑ZIP转录因子Cla97C04G076510或其保守结构域进行基因编辑敲除,使得该基因功能缺失或者出现突变,从而形成全缘叶片表型西瓜种质。通过本方法创制的西瓜叶片全缘系与正常的材料相比,除了叶片形状不同外,其他组织如根、茎、卷须、雌花、雄花、生长势等都无可见表型变化;如果将创制的西瓜全缘种质用于杂交种生产或群体改良,将对西瓜商品杂交种纯度的鉴定及改良西瓜叶型具有重要意义。
Description
技术领域
本发明涉及植物基因工程技术领域,更具体的说是涉及一种通过基因编辑技术创制西瓜全缘叶种质的方法。
背景技术
西瓜(Citrullus lanatus),为葫芦科(Cucurbitaceae)西瓜属(Citrullus),是世界上种植比较广泛的一种重要的葫芦科作物,更是一种水果型经济作物,因其种植面积和年消费量而被评为世界第五大水果。目前,商品种杂交纯度的鉴定一直是我国西瓜产业中亟待解决的技术难题,若无法及时地把母本自交苗从杂交苗中去除,将会给西瓜生产带来严重损失。
西瓜叶形变异作为一种理想形态标记,可用于直观、快速、准确的鉴定杂交种的纯度,降低伪杂种在作物生产中带来的损失。目前,在其他作物中叶缘裂刻基因功能有相关报道,包括油菜叶缘裂刻基因(BnLL1);羽衣甘蓝控制裂叶关键基因(Bo9g184610);拟南芥叶缘裂刻相关基因(LMI1)等。西瓜的裂叶基因还未见基因功能鉴定的报道。
因此,提供一种通过基因编辑技术创制西瓜全缘叶种质的方法是本领域技术人员亟需解决的问题。
发明内容
有鉴于此,本发明提供了一种通过基因编辑技术创制西瓜全缘叶种质的方法,利用CRISPR/Cas9编辑***对西瓜HD-ZIP转录因子Cla97C04G076510进行编辑,创制全缘叶种质。
为了实现上述目的,本发明采用如下技术方案:
一种通过基因编辑技术创制西瓜全缘叶种质的方法,具体步骤如下:
(1)根据Cla97C04G076510的CDS序列设计基因编辑的靶位点,包含两个靶点Target1和Target2;
Target1序列:5’-GTGGAAGGTGAACAAAGTA-3’;SEQ ID NO.2;
Target2序列:5’-GCTAAGAGAACAAGCAACG-3’;SEQ ID NO.3;
(2)构建CRISPR/Cas9编辑载体
①以中间载体pCBC-DT1T2为模板,利用引物Target1F/Target2R进行PCR扩增,回收目的片段;
②利用限制性内切酶BsaI-HF对CRISPR/Cas9载体pBSE402进行酶切,回收酶切后的载体pBSE402;
③将步骤①回收的目的片段与步骤②酶切后的载体pBSE402进行同源重组连接,获得连接产物;
④将连接产物转化大肠杆菌感受态DH5α,经测序正确,抽提重组质粒,转化至农杆菌感受态细胞EHA105;
(3)西瓜遗传转化
经验证正确后,用于西瓜遗传转化,获得基因编辑植株Cla97C04G076510-CR。
进一步,步骤(2)①所述引物Target1F/Target2R序列如下:
Target1F:5’-TTCTAGCTCTAAAACTACTTTGTTCACCTTCCACCAATCTCTTAGTCGACTCTAC-3’;SEQ ID NO.4;
Target2R:5’-TCGAAGTAGTGATTGGCTAAGAGAACAAGCAACGGTTTTAGAGCTAGAAATAGC-3’;SEQ ID NO.5。
进一步,创制的西瓜全缘叶种质在杂交种生产或群体改良中的应用。
本发明采用CRISPR/Cas9基因编辑技术,对基因Cla97C04G076510(HD-ZIP转录因子)或其保守结构域序列进行定点编辑或敲除,从而使得该基因蛋白功能缺失或者发生突变,创制全缘叶片表型的西瓜种质。本发明创制的西瓜全缘叶在生产上的应用,为西瓜商品杂交种纯度的鉴定提供基因资源,同时为西瓜叶形改良提供理论基础。
经由上述的技术方案可知,与现有技术相比,本发明公开提供了一种通过基因编辑技术创制西瓜全缘叶种质的方法,该方法首次利用CRISPR/Cas9基因编辑***,对HD-ZIP转录因子Cla97C04G076510或其保守结构域进行基因编辑敲除,使得该基因功能缺失或者出现突变,从而形成全缘叶片表型西瓜种质。通过本方法创制的西瓜叶片全缘系与正常的材料相比,除了叶片形状不同外,其他组织如根、茎、卷须、雌花、雄花、生长势等都无可见表型变化;如果将创制的西瓜全缘种质用于杂交种生产或群体改良,将对西瓜商品杂交种纯度的鉴定及改良西瓜叶型具有重要意义。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1附图为本发明Cla97C04G076510基因结构示意图;
图2附图为本发明pBSE402载体图谱;
图3附图为本发明重组质粒sgRNA表达元件;
其中,U6-26p、U6-29p为启动子,U6-26t为终止子,gRNA-Sc为gRNA骨架,Target1、Target2为靶位点;
图4附图为本发明YL与Cla97C04G076510-CR在2个靶点的比对结果;
其中,-表示缺失;红色标记的碱基为CRISPR/Cas9***靶点的PAM结构;
图5附图为本发明未编辑植株和编辑植株的叶片表型;
其中,A:YL(未编辑)叶片呈现裂刻;B:编辑植株(Cla97C04G076510-CR)叶片呈现全缘。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1 HD-ZIP转录因子Cla97C04G076510的编辑位点选择
根据Cla97C04G076510的CDS序列(SEQIDNO.1)和靶位点在线设计网站CRISPR-P(http://crispr.hzau.edu.cn/CRISPR2/news.php,V2)设计基因编辑的靶位点,设计一套编辑位点,包含两个靶点(Target1和Target2)。
HD-ZIP转录因子Cla97C04G076510的CDS序列如下:
ATGGAGTGGAATAATGGCAAAACTTTCACTCCTAACTTTGTTTCTCCACCACCACATTCTTCTTCCTCTGCTTTGCTCTACAACTGCAACAACTACAACTGCAATTTCGATCACTTTCCTGAAGTGATTGATATTCTTAACCTTTCAGTTCAATCAGTGATGGAGACGACGACGACAAAGAAAGTGGCGGTGGAAGGTGAACAAAGTATGGTTGGAGGAGTGGATCAAAAGAAGAAGAGATTGAGTCAGGATCAGTTGGAGGCATTAGAGAGAAGCTTTCAAGAGGAAGTGAAGCTTGATCCTGATAGAAAAATGAAGCTATCAAAGGAACTTGGGCTCCAACCAAGACAAATAGCTGTGTGGTTTCAAAATAGAAGAGCTCGCTGGAAAACTAAGCAGCTTGAGCATCTCTATGATACTCTCAAACAACAATTTGATAATATCTCTAAGGAAAAACATAACCTTCAACAAGAGGTGATAAAACTAAGAAGCATGCTAAGAGAACAAGCAACGAGGAATCAAGGGTCGATAGGTTGCACGGATGTCTCCGGGGAAGAGACGACGGTGGAATGCACATCGGTTGAGATTCTGAGCTGTAACAATTACATCTACAATAATGTGGAAGATTTCAACCAAATATCAGCTTCAGCTCCTCCATTCTGGTGGGGTGCTGAGGCTACTCACCTGCCTTCTTATCCTTAG;SEQ ID NO.1。
Cla97C04G076510基因结构示意图见图1。其中,靶点Target1在基因Cla97C04G076510的非保守结构域,而Target2则在LZmotif保守结构域内。
靶点序列:
Target1序列:5’-GTGGAAGGTGAACAAAGTA-3’;SEQ ID NO.2;
Target2序列:5’-GCTAAGAGAACAAGCAACG-3’;SEQ ID NO.3。
实施例2 CRISPR/Cas9编辑载体构建
(1)PCR扩增
根据上述靶点序列合成接头引物,具体如下:
Target1序列引物:
Target1F:5’-TTCTAGCTCTAAAACTACTTTGTTCACCTTCCACCAATCTCTTAGTCGACTCTAC-3’;SEQ ID NO.4;
Target2序列引物:
Target2R:5’-TCGAAGTAGTGATTGGCTAAGAGAACAAGCAACGGTTTTAGAGCTAGAAATAGC-3’;SEQ ID NO.5;
划下划线部分为靶点序列。
以中间载体pCBC-DT1T2为模板,使用高保真酶PrimeStar Max Premix(TaKaRa)进行PCR扩增,扩增体系为:PrimeStar Max Premix(2×)25μL,模板(100ng/μL)2.5μL,上下游引物各2.5μL,ddH2O 17.5μL。PCR反应程序为:98℃10s,58℃5s,72℃15s,38个循环;72℃5min。PCR产物经电泳检测后(片段大小为626bp),进行胶回收。
(2)载体酶切和连接
利用限制性内切酶BsaI-HF(NEW ENGLAND BioLabs)对CRISPR/Cas9载体pBSE402(载体图谱见图2)进行酶切。酶切体系为:pBSE4022μg,CutSmart buffer 5μL,BsaI-HF 1μL,ddH2O补至50μL。37℃酶切2h,经电泳检测后,进行胶回收。
PCR产物与载体连接:将PCR扩增的产物与酶切后的载体pBSE402进行同源重组连接。连接体系为:载体与***片段摩尔比约为1:2;5×反应缓冲液4μL;NovoRecPlus重组酶1μL;ddH2O补齐至20μL。50℃,连接10min。
(3)重组质粒转化
取5μl的连接产物热激转化至大肠杆菌感受态DH5a中,涂于含有50mg/L卡那霉素的LB固体培养平板中,37℃过夜培养。利用引物U626-IDF和U629-IDR对菌落进行阳性检测,用引物EDIT进行菌液测序。
其中,U626-IDF和U629-IDR的具体引物序列如下:
U626-IDF:5’-TGTCCCAGGATTAGAATGATTAGGC-3’;SEQ ID NO.6;
U629-IDR:5’-AGCCCTCTTCTTTCGATCCATCAAC-3’;SEQ ID NO.7;
其中,EDIT的具体引物序列如下:
EDIT:5’-GGGAATCTGAAAGAAGAGAAGCAG-3’;SEQ ID NO.8;
经测序正确后,抽提重组质粒,并将其转化至农杆菌感受态细胞EHA105中,PCR验证(利用引物U626-IDF和U629-IDR)正确后用于西瓜遗传转化。
其中,重组质粒sgRNA表达元件见图3。
农杆菌转化步骤如下:
取1μl重组质粒加入农杆菌EHA105感受态细胞中,依次于冰上静置5min,液氮中快速冷冻5min,37℃水浴5min,最后水浴5分钟,每管加入400μl不含抗生素的LB液体培养基进行复苏,将其转移到200rpm,28℃摇床上进行振荡培养2~3小时。取100μl经过复苏的菌液于含抗生素(Kan)的LB固体培养基上涂板,于室温下晾干,等菌液充分吸收后,将培养皿倒置于28℃培养箱中培养2-3d。
实施例3西瓜遗传转化
取饱满的西瓜种质‘YL’(利用发根农杆菌体系检测西瓜CRISPR/Cas9***的靶位点,张月乔等,中国瓜菜,2020,33(4):7-11)种子,于50~55℃蒸馏水浸泡约30min后,剥去种壳。在超净工作台中将剥取的种仁用75%的酒精消毒约30s,用无菌水清洗两遍之后再用3%次氯酸钠浸泡消毒15min,再无菌水清洗5~7次,之后晾干,平铺于BM固体培养基(Agar4.43g/L)中,置于25℃黑暗培养3d。
待种子发芽后将其取出,切去子叶的两端,将剩余子叶部分均分成8块便于侵染。期间,将PCR验证正确的EHA105单菌落挑至含有50mg/L卡那霉素和20mg/L利福平的LB液体培养基中,待菌液浓度摇至OD600=0.8时,用MS培养液(MS519(Phyto Tech Labs)4.43g/L,蔗糖30g/L,6-BA 1.5mg/L)重悬菌液使其终浓度OD600=0.2。将切好的子叶在菌液重悬液中浸泡15min,取出晾干,然后在垫有滤纸的共培养培养基CM(MS519 4.43g/L,蔗糖30g/L;G3251(Phyto Tech Labs)3g/L;6-BA 1.5mg/L)中进行共培养,25℃黑暗培养3d。
共培养3d后,将子叶块取出并用无菌水洗去表面多余的农杆菌菌液(清洗约5-7次)至无菌水清澈,取出晾干后放置于恢复培养基RM(MS519 4.43g/L,蔗糖30g/L;G32513g/L;6-BA 1.5mg/L,200mg/L Timentin(Scientific Research Special))上进行恢复培养,28℃培养。
恢复培养5-7d后,将恢复培养的子叶转移至选择培养基SM(MS519 4.43g/L,蔗糖30g/L;G3251(Phyto Tech Labs)3g/L、1.5mg/L6-BA、200mg/L Timentin、1.4mg/L Basta)上进行选择培养,28℃继代培养3-4周,每7d更换培养基继代一次。之后将有明显芽点的外植体转移至芽伸长培养基中(MS524(Phyto Tech Labs)4.43g/L、蔗糖30g/L、G32513g/L、肌醇1g/L、SH有机溶液500μL/L、NAA 0.01mg/L、6-BA 0.1mg/L、Timentin 200mg/L、Basta1.4mg/L),培养条件为28℃,黑暗8h/d,光照16h/d,光照强度为8000Lx。将筛选出来的芽切下(注意切口不能含有愈伤),转移至生根培养基(MS519 4.43g/L,蔗糖30g/L,6-BA 1.5mg/L,G3251 3g/L,0.5mg/L IBA和200mg/L Timentin)中进行生根培养,28℃培养至生根。其中,SH有机溶液的配制方法如下:5g烟酸、5g VB1、0.5g VB6,定容至500ml。
待再生苗生根长至4-5片真叶时从培养瓶中取出,用清水缓慢冲洗掉根部的培养基,移栽至预先经过高温高压灭菌的基质中,浇透水后保温保湿培养,3-4天后见穴盘盖上有水珠,逐渐开盖炼苗。
实施例4西瓜转基因植株的检测
利用CTAB法抽提具有GFP荧光(载体pBSE402上带有GFP)的西瓜再生苗的DNA,步骤:取少部分幼嫩叶片在液氮中快速研磨成粉末状,放于1.5ml的离心管中;加入预热的800μlCTAB提取缓冲液,65℃水浴30min;加入等体积氯仿异戊醇,其中氯仿与异戊醇的体积比为24:1,混匀后8000r/min离心10min;将上清液转入新的离心管,加2/3体积的异丙醇,上下颠倒轻轻混匀;10000r/min离心10min;倒去上清液,用体积分数为75%的乙醇冲洗沉淀两次,倒掉后吸干剩余液体,干燥3min后,用100μl ddH2O(含0.1%RNA酶)溶解,4℃保存备用。
以抽提的各DNA为模板,利用引物CR-F和CR-R对两个靶位点的序列进行PCR扩增,阳性对照为重组质粒,阴性对照为非转基因植株DNA。
其中,CR-F和CR-R的具体引物序列如下:
CR-F:5’-GCGAAAACATTGTTAGAGTGGTAG-3’;SEQ ID NO.9;
CR-R:5’-CTAAGGATAAGAAGGCAGGTGA-3’;SEQ ID NO.10。
扩增体系为:2×Taq PCR StarMix with loading Dye10μL,模板1μL,引物各1μL,ddH2O 7μL。PCR反应程序为:94℃3min;94℃30s,58℃30s,72℃1min,30个循环;72℃5min。根据条带大小回收PCR产物,并进行TA克隆(pClone007 Versatile Simple Vector Kit,TSINGKE),菌液PCR(利用引物CR-F、CR-R)阳性检测后,挑选单克隆,送测进行编辑确认。
其中,编辑植株Cla97C04G076510-CR中对应Target1的靶点测序结果如下:
5’-GTGGAAGGTGAACAA-3’;SEQ ID NO.11;
编辑植株Cla97C04G076510-CR中对应Target2的靶点测序结果如下:
5’-AACGAGG-3’;SEQ ID NO.12。
比对结果见图4,该套靶点获得一株基因编辑植株Cla97C04G076510-CR,在Target1和Target2靶点区域之间出现缺失。
实施例5西瓜转基因植株的表型观察
将编辑植株Cla97C04G076510-CR种植在日光温室,正常管理,观察其表型,结果见图5。图5结果显示:相比于未编辑植株YL的裂刻叶,编辑植株呈现叶片全缘、有叶缘褶皱。
综上所述,本发明提供了一种通过基因编辑技术创制全缘叶西瓜新种质的方法,通过对西瓜HD-ZIP转录因子Cla97C04G076510进行序列或保守结构域HD-ZIP的编辑,可快速获得隐性全缘叶西瓜种质,在西瓜群体改良和商品杂交种纯度的鉴定具有重要意义。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
序列表
<110> 西北农林科技大学
<120> 一种通过基因编辑技术创制西瓜全缘叶种质的方法
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atggagtgga ataatggcaa aactttcact cctaactttg tttctccacc accacattct 60
tcttcctctg ctttgctcta caactgcaac aactacaact gcaatttcga tcactttcct 120
gaagtgattg atattcttaa cctttcagtt caatcagtga tggagacgac gacgacaaag 180
aaagtggcgg tggaaggtga acaaagtatg gttggaggag tggatcaaaa gaagaagaga 240
ttgagtcagg atcagttgga ggcattagag agaagctttc aagaggaagt gaagcttgat 300
cctgatagaa aaatgaagct atcaaaggaa cttgggctcc aaccaagaca aatagctgtg 360
tggtttcaaa atagaagagc tcgctggaaa actaagcagc ttgagcatct ctatgatact 420
ctcaaacaac aatttgataa tatctctaag gaaaaacata accttcaaca agaggtgata 480
aaactaagaa gcatgctaag agaacaagca acgaggaatc aagggtcgat aggttgcacg 540
gatgtctccg gggaagagac gacggtggaa tgcacatcgg ttgagattct gagctgtaac 600
aattacatct acaataatgt ggaagatttc aaccaaatat cagcttcagc tcctccattc 660
tggtggggtg ctgaggctac tcacctgcct tcttatcctt ag 702
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Claims (3)
1.一种通过基因编辑技术创制西瓜全缘叶种质的方法,其特征在于,具体步骤如下:
(1)根据Cla97C04G076510的CDS序列设计基因编辑的靶位点,包含两个靶点Target1和Target2;
Target1序列:5’-GTGGAAGGTGAACAAAGTA-3’;SEQ ID NO.2;
Target2序列:5’-GCTAAGAGAACAAGCAACG-3’;SEQ ID NO.3;
(2)构建CRISPR/Cas9编辑载体
①以中间载体pCBC-DT1T2为模板,利用引物Target1F/Target2R进行PCR扩增,回收目的片段;
②利用限制性内切酶BsaI-HF对CRISPR/Cas9载体pBSE402进行酶切,回收酶切后的载体pBSE402;
③将步骤①回收的目的片段与步骤②酶切后的载体pBSE402进行同源重组连接,获得连接产物;
④将连接产物转化大肠杆菌感受态DH5α,经测序正确,抽提重组质粒,转化至农杆菌感受态细胞EHA105;
(3)西瓜遗传转化
经验证正确后,用于西瓜遗传转化,获得基因编辑植株Cla97C04G076510-CR。
2.根据权利要求1所述的一种通过基因编辑技术创制西瓜全缘叶种质的方法,其特征在于,步骤(2)①所述引物Target1F/Target2R序列如下:
Target1F:5’-TTCTAGCTCTAAAACTACTTTGTTCACCTTCCACCAATCTCTTAGTCGACTCTAC-3’;SEQ ID NO.4;
Target2R:5’-TCGAAGTAGTGATTGGCTAAGAGAACAAGCAACGGTTTTAGAGCTAGAAATAGC-3’;SEQID NO.5。
3.根据权利要求1所述的一种通过基因编辑技术创制西瓜全缘叶种质的方法,其特征在于,创制的西瓜全缘叶种质在杂交种生产或群体改良中的应用。
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