CN116555332A - 一种免打顶棉花种质的创制方法 - Google Patents
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Abstract
本发明属于植物基因工程技术领域,具体涉及一种免打顶棉花种质的创制方法;本发明提供利用CRISPR‑Cas9系统创造免打顶棉花材料的方法。本发明的方法选取特异针对早花、株型基因GoDG的靶位点sgRNA,与Cas9蛋白在棉花内稳定表达,获得提前终止生长、矮小的棉花(小锦)。再利用小锦与受体棉花(野生型,WT)进行杂交后,获得短果枝、短果枝+自封顶、长果枝+自封顶新株型。该发明的优点在于创造免打顶棉花。
Description
技术领域
本发明属于植物基因工程技术领域,具体涉及一种免打顶棉花种质的创制方法。
背景技术
打顶,亦称摘心,即摘除棉株主茎顶尖一叶一心,能消除植株顶端生长优势,调节营养物质的运输分配;改善棉田透光通风条件,提高光能利用率,减少蕾铃脱落,促使棉株体内营养物质向生殖器官的运输,促进早熟,增加铃重及霜前收花率,改善纤维品质。打顶过早不能充分利用生长季节,且造成赘芽丛生;过迟则无效果,枝多,消耗大量养分,减轻后期的铃重。棉花打顶有人工打顶和化学药剂封顶2种,人工打顶效果较好,但是成本高,每亩费用在45-75元之间,制约了棉花生产全程的机械化。化学打顶是指利用植物生长调节剂氟节胺+甲哌鎓去除棉花的顶端优势,强效抑制棉花顶端生长。化学打顶较人工打顶成本略低,约为每亩30-50元之间,但是化学打顶后期管理上控制不好容易导致棉株营养生长与生殖生长不协调,即出现二次生长、长势偏旺等现象,影响结铃和棉铃生长发育进程,且化学药剂对环境有污染,不符合棉花产业绿色、高效可持续发展的要求。棉花每年打顶的费用估算在15亿元左右,且随着人工成本的提高会同步增加。培育免打顶的棉花品种,不仅可以降低棉农植棉成本,提高棉花生产全程机械化水平,创造巨大的经济效益,还可减少化学药剂使用,具有显著的生态效益,有重大应用价值。
发明内容
本发明的目的在于提供一种植株自己消除植株顶端生长优势的棉花新材料及创制方法,降低棉农植棉成本。
本发明的技术方案如下所述:
一种免打顶棉花种质的创制方法,具体为:
将无标记(无T-DNA)小锦与野生型棉花进行杂交后,筛选获得短果枝+自封顶、长果枝+自封顶的免打顶棉花种质;所述小锦是利用基因编辑技术敲除或敲低GoDG_At基因和GoDG_Dt基因的棉花材料,所述小锦呈现提前终止生长、矮小的表型;所述GoDG_At基因的核苷酸序列如SEQ ID NO:1所示,所述GoDG_Dt基因的核苷酸序列如SEQ ID NO:2所示。
进一步地,所述小锦的创制方法具体为:
(1)创制棉花基因编辑载体;
(2)在陆地棉GoDG保守区域选择如SEQ ID NO:1所示的GoDG_At基因,SEQ ID NO:2所示的GoDG_Dt基因共有的靶位点sgRNA;
(3)将sgRNA构建到基因编辑载体;
(4)利用农杆菌介导的方法转化棉花;其中,编辑GoDG后的植株呈现提前终止生长、矮小的表型即获得小锦。
进一步地,所述步骤(1)中,棉花基因编辑载体为CHGE,CHGE由酶切后的pRGEB32-GhU6.7-NPTⅡ载体与tRNA-CcdB片段连接组成,tRNA-CcdB片段的序列如SEQ ID NO:4所示。
进一步地,所述的sgRNA的序列如SEQ ID NO:3所示,sgRNA:GTCCTAGTGACCCTTACCTG
一种所述创制方法创制获得的免打顶棉花种质的应用,可用于棉花功能基因组学研究。
本发明的有益效果:
(1)本发明的sgRNA具有较高特异性核编辑效率,并可以同时编辑GoDG_At、GoDG_Dt。
(2)本发明高效获得提前终止生长、矮小的表型(小锦)植株,并且可遗传至后代。
(3)本发明获得短果枝、短果枝+自封顶、长果枝+自封顶新株型。
本发明利用基因编辑技术编辑棉花封顶基因(determinacy gene,GoDG),出现多种棉花自封顶材料。从中培育出免打顶的棉花品种,不仅可以降低棉农植棉成本,提高棉花生产全程机械化水平,创造巨大的经济效益,还可减少化学药剂使用,具有显著的生态效益,有重大应用价值。
附图说明
图1:CHGE载体图谱;
图2:是本发明的小锦的基因编辑鉴定以及应用结果图;附图标记说明:A.sgRNA同时靶向GoDG_A07/D07的第二个外显子。B.转基因T0棉花植株的鉴定。WT和T0中Cas9和GoDG片段的PCR分析。C.在T2中分离无Cas9的棉花植株。WT和T2中Cas9和GoDG片段的PCR分析。T2-2和T2-4植物被鉴定为不含Cas9的棉花植株。D.从T0到T4的稳定编辑序列变化。sgRNA目标站点在背景中突出显示。核苷酸缺失或插入标记在右侧。E.T4测序峰图;上半部分显示GoDG_A07中有5个碱基缺失,下半部分显示GoDG_D07中有4个碱基缺失。F.WT和小锦的表型。与WT相比,小锦的主茎和果枝表现出确定的表型。G.WT和小锦的表型方案。三角形代表单轴芽顶端分生组织,线条代表叶子,封闭圆圈代表花朵。H.小锦、拟南芥和烟草盆栽形态比较。比例尺9厘米。I.小锦与野生型棉的纤维比较。比例尺1厘米。J.在通过VIGS沉默GoPGF后,在棉铃(33天)上没有观察到腺体。
图3:小锦室内外栽培表型;附图标记说明:A.培养约30天的室内转基因T1棉花;B.室内转基因T2棉花;其中,WT表示野生型棉花;C.2022年浙江杭州转基因T4棉花植株;D.2021-2022年海南三亚T3棉花植株;E.和F.2022年在新疆阿拉尔种植的T4棉花植株。
图4:盆栽小锦不同发育时期的表型;A.开花期;B.15DPA棉铃;C.吐絮的棉铃。比例尺:5厘米。
图5:杂交后代不同表型;
具体实施方式
若未特别指明,实施例中所用的技术手段为本领域常规手段,所用原料均为市售商品。
实施例1:CRISPR载体构建
以载体PGTR4为模板扩增tRNA,扩增tRNA的引物序列分别为tRNA-S和tRNA-AS。以载体PK2GW7.0为模板扩增CcdB,扩增CcdB的引物序列分别为CcdB-2S和CcdB-AS。利用重叠延伸PCR连接tRNA与CcdB得到tRNA-CcdB(序列如SEQ ID NO:4所示),连接的引物为tRNA-S和CcdB-AS。PCR引物序列见表1。首先将pRGEB32-GhU6.7-NPTⅡ载体BsaⅠ酶切,37℃放置5h,然后利用凝胶回收试剂盒将酶切产物纯化。酶切后的pRGEB32-GhU6.7-NPTⅡ载体与tRNA-CcdB片段通过ClonExpress II One Step Cloning Kit(Vazyme C112-02)进行连接,将连接产物转化到大肠杆菌感受态,测序正确的质粒命名为cotton high-throughput geneediting(CHGE,图1)。根据GoDG_At和GoDG_Dt共有序列,设计保守的sgRNA(SEQ ID NO:3):GTCCTAGTGACCCTTACCTG)以同时敲除两个拷贝(图2A)。并以sgRNAP、CHGE-S1、CHGE-AS1形成双链DNA与BsaI酶切后的CHGE载体进行连接,形成敲除GoDG的载体。PCR体系及条件分别见表2、3。
表1引物序列
表2PCR体系
表3PCR条件
实施例2:利用CRISPR-Cas9系统转化棉花
将上述构建好的载体通过农杆菌介导的转化方法导入棉花宿主细胞。具体转化步骤如下:
(1)挑选饱满、健康的陆地棉棉花种子,剥去种皮后,用0.1wt%的HgCl2浸泡10min,再用无菌水洗涤3次,将灭菌后的棉种,接种于无菌苗萌发培养基上,在黑暗条件下,置于28℃恒温箱中培养4-6d;
(2)取无菌苗下胚轴,切成0.5-0.8cm小段接种于0.5OD值的用农杆菌活化培养基悬浮的农杆菌菌液中,侵染10mim后,用无菌滤纸吸干下胚轴表面的菌液;
(3)将下胚轴接种在含有共培养培养基的培养皿中,在21℃下共培养48h;将下胚轴放入含有500mg/L头孢霉素的无菌水中,冲洗3遍,吸干表面水份后,接种到选择培养基中,每1个月继代培养1次,直到获得胚性愈伤组织;将胚性愈伤组织转入分化培养基,获得大量胚状体;
(4)将获得的胚状体转移到分化培养基,直到获得转基因植株幼苗后转移至生根培养基。
转化用的培养基成分及配制方法:
无菌苗萌发培养基:1/2MS大量元素,15g/L葡萄糖,2.5g/L的植物凝胶;pH=6.1-6.2。
农杆菌活化培养基:胰化蛋白胨5g/L,NaCl 5g/L,MgSO4.7H2O 0.1g/L,KH2PO4,0.25g/L,甘露醇5g/L,甘氨酸1.0g/L;pH=5.85-5.95。
共培养培养基:MSB+2,4-D 0.1mg/L,KT 0.1mg/L,3wt%葡萄糖,0.25wt%植物凝胶,pH=5.8。
选择培养基:MSB,2,4-D 0.1mg/L,KT 0.1mg/L,3wt%葡萄糖,0.3wt%植物凝胶,卡那霉素50mg/L,头孢霉素400mg/L;pH:5.85-5.95。
分化培养基:在MSB培养基中去掉NH4NO3,将KNO3用量加倍,Gln 1.0g/L,Asn 0.5g/L,IBA 0.5mg/L,KT 0.15mg/L,3wt%葡萄糖,0.25wt%植物凝胶;pH=6.1-6.2。
生根培养基:1/2MS无机盐+B5有机物,15g/L葡萄糖,2.5g/L的植物凝胶;pH=5.90-5.95;
MSB:MS培养基+B5维生素。
实施例3:检测CRISPR-Cas9对GoDG基因的编辑效果
(1)再生苗阳性鉴定
利用T-DNA上的Cas9序列进行转基因植株的阳性鉴定(图2B),转基因植株均为阳性。引物序列:正向引物Cas9 F(SEQ ID NO:12):GCTTGTGCGTTTCGATTTGA,反向引物Cas9 R(SEQ ID NO:13):CCGCTCGTGCTTCTTATCCT;PCR条件:95℃5min;95℃30sec;58℃30sec;72℃60sec;共32个循环;72℃5min;15℃保存。
利用GoDG基因GODG S、GODG AS引物进行扩增基因序列,检测是否存在大片段基因缺失。PCR体系和PCR条件同上。引物:正向引物GODG S(SEQ ID NO:14):GTGATGACAGACCCAGAT,反向引物GODG AS(SEQ ID NO:15):GAAGAGGATACTTACCAAA;
利用Cas9引物(同上)鉴定转基因植株是否为阳性植株,本发明共获得再生苗18株,其中阳性植株18株,阳性率为100%(图2B,展示部分单株)。转基因植株均低矮表型,表型率为100%。。在T2代可以筛选到无标记的单株(图2C)。
(3)GoDG基因的测序
通过设计引物扩增得到包含编辑位点序列。引物序列如下所示:
正向引物GODGCX F(SEQ ID NO:16):ggagtgagtacggtgtgcGTGATGACAGACCCAGAT,
反向引物GODGCX R(SEQ ID NO:17):gagttggatgctggatggGAAGAGGATACTTACCAAA;
将基因编辑植株及其后代进行扩增测序,并通过GoDG_At、GoDG_Dt之间的SNP进行区分At、Dt的测序结果(图2D、E)。基因编辑植株在sgRNA位置上发生编辑并遗传给后代。
(4)利用sgRNA潜在脱靶位点的预测及深度测序检测
利用sgRNAcas9_3.0.5.pl和extract_targetSeq.pl程序对sgRNA进行全基因组潜在脱靶位点预测及位点侧翼序列的提取,针对序列利用在线网站batchprimer3(http://probes.pw.usda.gov/batchprimer3/)进行批量设计引物,从基因组中扩增目标序列并等量混合建库,然后利用Illumina Hiseq 3000测序仪对目标序列进行150bp双端测序,最后对测序结果统计(见表4)。
表4潜在脱靶序列检测
实施例4:GoDG编辑棉花的表型及应用
GoDG编辑后的植株呈现提前终止生长、矮小的表型(图2F、G),并且在后代中T1~T4均稳定遗传(图3)。我们将该材料命名为小锦。根据T0和T4代靶位点序列的比对,产生的基因编辑片段是稳定遗传的(图2D和2E)。室内培养观察表明,在生长室(25℃,16h光照,8h黑暗,光照强度=20000Lux,湿度=60%)内,小锦可以在狭小的空间(高×宽=17.9±5.5cm×24.2±7.6cm)中培养,如拟南芥和本氏烟草(图2H)一样,而野生型植物达到79.8±5.8cm×47.8±5.7cm。由于小锦的体积小,在一个三维空间(体积=长×宽×高=1.2米×0.55米×2.0米=1.32立方米)、四层培养架上可以种植288株(9×9×9厘米花盆,图4)。此外,小锦的开花时间明显提前,第一朵花在播种后仅55天开花,而野生型(WT)需要86天,在25℃下节省1个月。最重要的是,与生长室中的WT(127.6±2.1天)相比,小锦的生长期只需要99.8±2.6天。值得一提的是,温度越高,开花时间越快。比如小锦在32℃下36.5±1.1天可开花,其中25%(n=20)35天开花。从这个意义上说,小锦可以在一年内在生长室中繁殖四代,便于进行大规模的室内研究。小锦和WT之间的种子大小和纤维长度差异不显着(图2I)。与WT(每铃17.5±6.6粒种子)相比,小锦每铃含有更少的种子(7.1±3.1)。在通过VIGS沉默GoPGF后,小锦不仅在茎、叶和苞片上显示出无腺体表型,而且在33日龄的棉铃上也显示出无腺体表型(图2J)。在相同的生长时间(88天)下,WT仍处于萌芽期(图2J)。因此,小锦可用于在较短的时间内研究全株生长期几乎所有器官和组织的发育。
实施例5:免打顶棉花种质创制
用无标记(无T-DNA)的小锦与受体棉花(野生型,WT)进行杂交后,在后代中除了小锦表型,还发现了多种表型(图5),主要有正常株型、短果枝、短果枝+自封顶、长果枝+自封顶。正常株型中果枝较长、一般有3~5个铃连续排列;短果枝株型果枝呈零式果枝或I式果枝,其中I式果枝长度在2cm~15cm之间,并且三个铃在一个果枝末端成簇,但是顶端生长点正常;短果枝+自封顶株型果枝呈零式果枝或I式果枝,其中I式果枝长度在2cm~15cm之间,并且三个铃在一个果枝末端成簇,顶端位置铃成簇生长点终止生长;长果枝+自封顶果枝较长、一般有3~5个铃连续排列,但是顶端位置铃成簇生长点终止生长。
免打顶的生长特点不仅可以节省打顶人工费,还能消除植株顶端生长优势,调节营养物质的运输分配;改善棉田透光通风条件,提高光能利用率,减少蕾铃脱落,促使棉株体内营养物质向生殖器官的运输,促进早熟,增加铃重及霜前收花率,改善纤维品质。
显然,上述实施例仅仅是为清楚地说明所作的举例,而并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其他不同形式的变化或变动。这里无需也无法把所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明的保护范围。
Claims (5)
1.一种免打顶棉花种质的创制方法,其特征在于,具体为:
将无T-DNA的小锦与野生型棉花进行杂交后,筛选获得短果枝+自封顶、长果枝+自封顶的免打顶棉花种质;所述小锦是利用基因编辑技术敲除或敲低GoDG_At基因和GoDG_Dt基因的棉花材料,所述小锦呈现提前终止生长、矮小的表型;所述GoDG_At基因的核苷酸序列如SEQ ID NO:1所示,所述GoDG_Dt基因的核苷酸序列如SEQ ID NO:2所示。
2.根据权利要求1所述的方法,其特征在于,所述小锦的创制方法具体为:
(1)创制棉花基因编辑载体;
(2)在陆地棉GoDG保守区域选择如SEQ ID NO:1所示的GoDG_At基因,SEQ ID NO:2所示的GoDG_Dt基因共有的靶位点sgRNA;
(3)将sgRNA构建到基因编辑载体;
(4)利用农杆菌介导的方法转化棉花;其中,编辑GoDG后的植株呈现提前终止生长、矮小的表型即获得小锦。
3.根据权利要求2所述的方法,其特征在于,所述步骤(1)中,棉花基因编辑载体为CHGE由酶切后的pRGEB32-GhU6.7-NPTⅡ载体与tRNA-CcdB片段连接组成,tRNA-CcdB片段的序列如SEQ ID NO:4所示。
4.根据权利要求2所述的方法,其特征在于,所述的sgRNA的序列如SEQ ID NO:3所示。
5.一种权利要求1所述创制方法创制获得的免打顶棉花种质的应用。
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