CN116479035B - 一种定点突变创制草莓耐弱光种质的方法及其应用 - Google Patents
一种定点突变创制草莓耐弱光种质的方法及其应用 Download PDFInfo
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Abstract
本发明专利申请公开了一种定点突变创制耐弱光草莓种质的方法及其应用。本申请涉及基因工程领域,尤其是涉及草莓育种领域。本发明的培育耐弱光草莓的方法,包括向目的草莓中导入草莓基因组编辑的载体,得到叶色黄化的草莓突变体,突变体与目的草莓相比,在弱光下光合能力提高。采用本发明的方法能够实现对草莓的基因编辑,获得具有耐弱光能力的草莓,具有很高的育种和栽培价值。
Description
技术领域
本发明属于植物基因工程技术领域,具体涉及通过定点突变创制草莓种质的方法及其应用。
背景技术
草莓是世界上最受欢迎的的水果之一,温室草莓种植业发展迅速,规模不断扩大。光合作用是植物生产的物质基础,受温度、湿度、CO2浓度、光照强度等多种环境因素的影响,光照强度对北半球中高纬度地区设施栽培的影响尤为重要,尤其是冬春季和一些雨水较多的地区,由于低温、弱光和高湿度的环境,使光合作用的效率比较低,导致草莓生长周期变长、产量低下、病虫害增多。因此,培育耐弱光的草莓种质,提高草莓的光合作用效率,对提高草莓的生产效率十分重要。
叶绿素分子可以吸收光能、向光合作用电子传递链输入电子,在反应中心驱动电荷分离,因此,叶绿素分子对光合作用至关重要。Mg2+插入到原卟啉IX(Proto IX)是公认的叶绿素合成的第一步,由镁螯合酶催化完成。因此,镁螯合酶是调控叶绿素合成的关键酶之一,由三个亚基CHLH,CHLD和CHLI组成,其中CHLI负责ATP酶的水解。作为叶绿素合成的关键基因,CHLI基因功能缺失导致叶片黄化。。
CRISPR/Cas9是基于细菌或古细菌规律成簇的间隔短回文重复CRISPR(clusteredregularly interspaced short palindromic repeats)介导的获得性免疫系统衍生而来的基因编辑技术。该技术首先设计的一段sgRNA(单分子引导RNA,single guiding RNA),该基因转录的RNA可以通过碱基互补配对识别目标DNA序列,指导Cas 9核酸酶切割识别的双链DNA,诱发同源重组(H DR,homologous directed repair)或非同源末端链接(NHEJ,non-homologous end-joining),进而实现目的DNA编辑。基于细菌Ⅱ型免疫机制开发的基因编辑技术在植物特别是作物遗传改良上具有重大的应用前景。该技术的基本要求之一就是受体细胞内表达sgRNA,该分子负责识别特异性的基因编辑位点,然后介导结合Cas9蛋白行使DNA酶切活性,在设计的位点引入DNA双链断裂损伤,通过胞内的NHEJ或HDR修复途径引入突变。因此,sgRNA的表达是该技术的重要组成部分。
发明内容
本申请所要解决的技术问题是如何培育耐弱光的草莓。
本申请的发明人惊讶地发现,通过突变草莓叶绿素合成的关键基因CHLI,使得CHLI基因功能缺失获得的chli杂合突变体表现出更优异的耐弱光性,能够在弱光下(50μmol m-2s-1),具有更强的光合作用效率。
在第一个方面中,本申请提供了一种培育耐弱光草莓的方法,所述方法包括突变叶绿素合成基因CHLI。
在第二个方面中,本申请提供了一种培育耐弱光草莓的方法,所述方法包括向目的草莓基因组中导入含有Cas9蛋白基因和sgRNA的草莓基因组编辑载体,其中所述sgRNA在草莓中识别的靶标DNA为编码草莓CHLI蛋白的DNA片段。优选地,所述sgRNA识别的靶标位点为SEQ ID NO:3所示的DNA序列。
在第二方面的一个实施方案中,所述CHLI蛋白为含有SEQ ID NO:1所示的氨基酸序列的蛋白质。
MASVLGTCST ATLAARPLSS PTSRTSIPSL SLTQGQSCGT KFYGGLRIHG KKSRARFHVASVATEVNPSE QAQRLAAKES QRPVYPFAAI VGQDEMKLCL LLNVIDPKIG GVMIMGDRGT GKSTTVRSLTDLLPEIKIVA GDPYNSDPED PEAMGPEVRE SIIKGVQLPV ATTKINMVDL PLGATEDRVC GTIDIEKALTEGVKAFEPGL LAKANRGILY VDEVNLLDDH LVDVLLDSAA SGWNTVEREG ISISHPARFI LIGSGNPEEGELRPQLLDRF GMHAQVGTVR DAELRVKIVE ERARFDKNPK EFRVSYEAEQ DKLQQQITSA RSGLSSVQIDQDLKVKISRV CSDLNVDGLR GDIVTNRAAK ALAALKGRDK VTPEDIATVI PNCLRHRLRK DPLESIDSGLLVIEKFYEIF S(SEQ ID NO:1)
在第二方面的一个实施方案中,所述CHLI蛋白为其DNA编码序列含有本申请所述sgRNA序列或其互补序列的CHLI蛋白。
上述方法中,编码所述的CHLI蛋白质的chli基因具有如SEQ ID NO:2所示的核苷酸序列。
ATGGCATCAGTACTCGGAACTTGCTCCACCGCAACCTTGGCCGCTCGTCCTCTCTCATCTCCCACTTCCAGGACTTCAATTCCCTCCCTCTCTTTGACCCAAGGGCAGAGTTGTGGGACTAAGTTTTATGGAGGGTTAAGGATTCATGGGAAGAAGAGCAGGGCTCGGTTCCATGTTGCCAGTGTTGCCACTGAAGTCAACCCTTCTGAACAGGCACAGAGGCTTGCTGCTAAGGAGAGCCAGAGGCCGGTGTATCCATTTGCTGCTATTGTAGGACAGGATGAGATGAAACTGTGTCTTTTACTGAATGTGATTGACCCCAAGATTGGGGGTGTCATGATCATGGGTGATAGGGGAACTGGGAAATCCACAACTGTTAGGTCCTTGACTGATTTGCTTCCCGAAATTAAGATAGTTGCTGGTGATCCCTACAATTCAGACCCAGAAGATCCGGAGGCCATGGGCCCGGAAGTGAGGGAGAGCATTATTAAAGGAGTGCAACTTCCTGTGGCGACGACTAAGATCAACATGGTTGATTTACCTCTGGGTGCTACAGAAGATAGAGTGTGTGGGACAATTGACATTGAGAAAGCTCTGACTGAGGGTGTGAAGGCATTTGAACCGGGACTTCTTGCAAAAGCTAATAGAGGCATTCTTTATGTGGATGAAGTTAATCTGTTGGATGATCATTTAGTGGATGTTCTATTGGATTCTGCTGCCTCTGGATGGAACACAGTGGAGAGGGAGGGTATTTCGATTTCTCATCCAGCAAGATTCATTTTGATTGGTTCTGGCAATCCAGAAGAAGGGGAGCTCAGGCCGCAGTTGCTTGATCGTTTTGGTATGCATGCTCAAGTTGGGACTGTGAGGGATGCAGAACTGAGAGTGAAGATTGTCGAGGAGAGAGCTCGGTTTGACAAAAACCCAAAAGAATTTCGGGTTTCTTACGAAGCTGAGCAAGATAAGCTTCAGCAACAAATTACTTCAGCTAGGAGTGGTCTTTCATCTGTACAGATTGACCAGGATCTCAAGGTGAAAATCTCCCGGGTTTGTTCAGATTTGAATGTCGACGGATTGAGAGGAGACATAGTGACTAACAGGGCTGCAAAAGCTTTGGCTGCTCTAAAGGGGAGGGATAAGGTGACTCCAGAAGATATTGCTACTGTCATCCCTAACTGCTTAAGACATCGTCTTCGGAAGGATCCTTTAGAGTCGATTGACTCTGGTTTACTTGTCATTGAGAAATTTTACGAAATCTTTAGCTGA(SEQ ID NO:2)
在一个实施方案中,所述sgRNA识别的靶位点序列为SEQ ID NO:3或SEQ ID NO:4所示所示的DNA分子。
CCT CTG GGT GCT ACA GAA GAT AG(SEQ ID NO:3)
在另一个实施方案中,所述sgRNA是SEQ ID NO:3或SEQ ID NO:4所示的DNA分子。
CTG GGT GCT A CA GAA GAT AG(SEQ ID NO:4)
本领域普通技术人员可以很容易地采用已知的方法,例如定向进化和点突变的方
法,对本发明的编码所述蛋白质CHLI的核苷酸序列进行突变。那些经过人工修饰的,具有与本发明分离得到的所述蛋白质CHLI的核苷酸序列75%或者更高同一性的核苷酸,只要编码所述蛋白质CHLI,均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
本申请的第四个方面涉及一种产生耐弱光的转基因草莓植株的方法,其特征在于将本申请所述的含有Cas9蛋白基因和DNA序列为SEQ ID NO:3sgRNA的基因组编辑载体导入到草莓植株中并稳定表达。
因此,本申请的第五个方面涉及一种提高草莓植株弱光下光合能力的方法,所述方法包括向目的草莓基因组中导入含有Cas9蛋白基因和sgRNA的草莓基因组编辑载体并稳定表达,其中所述sgRNA在草莓中识别的靶标DNA为编码草莓CHLI蛋白的DNA片段。
在一个实施方案中,所述CHLI蛋白为含有SEQ ID NO:1所示的氨基酸序列的蛋白质。
在另一个实施方案中,所述CHLI蛋白为其DNA编码序列含有本申请所述sgRNA序列或其互补序列的CHLI蛋白。
在进一步的实施方案中,所述sgRNA的核苷酸序列如SEQ ID NO:3所示。
本申请采用含有Cas9蛋白基因和sgRNA的基因组编辑载体通过重组农杆菌转化森林草莓‘Rugen’的叶片,能够对CHLI基因进行编辑,CHLI基因通过CRISPR/Cas9核酸内切酶编辑之后,可造成CHLI的编码基因突变,产生chli基因杂合的植株,杂合突变的株系具有在正常光下叶片黄化,弱光下叶片浅绿色的表型,而且弱光下净光合速率和气孔导度与野生型相比显著提高,叶绿体发育正常,与野生型相比,更适宜在弱光下生长。采用本发明的方法能够实现对草莓的基因编辑,获得耐弱光的草莓,具有很高的育种和栽培价值。
附图说明
图1为杂合草莓突变体植株在不同光照强度下的外观形态。其中chli为杂合突变体。
图2是杂合草莓突变体植株在不同光照强度下的叶绿素含量。图中标注了不同株系的编号,其中chli为杂合株系。平均值和标准偏差来自于三次生物学重复和三次技术重复,*表示与野生型相比有显著差异,**表示与野生型相比有极显著差异(Student’st-test,*P<0.05,**P<0.01)。
图3是杂合草莓突变体植株在不同光照强度下的光合能力参数。其中A为净光合速率,B为气孔导度。
图4是杂合突变体在不同光照强度下光合作用基因的表达量检测。*表示与野生型相比有显著差异,**表示与野生型相比有极显著差异(Student’s t-test,*P<0.05,**P<0.01)。
图5是杂合草莓突变体植株在不同光照强度下叶绿体超微结构。其中右图为左图叶绿体的放大结构。TM:类囊体膜;SG:淀粉粒
具体实施方式
以下结合实施例及附图对本发明做进一步详细描述:
实施例1:草莓CRISPR/Cas9基因编辑载体的构建
本实施例中,sgRNA的靶点序列为SEQ ID No:3的序列,对应靶基因为chli基因。本实施例所用的母载体pKSE401带有GFP标签。
1、根据靶点设计并合成引物
FveCHLI-t-F:5’-ATTGCTGGGTGCTACAGAAGATAG-3’,和
FveCHLI-t-R:5’-AAACCTATCTTCTGTAGCACCCAG-3’。
2、将FveCHLI-t-F和FveCHLI-t-R稀释后进行退火,得到带粘性末端的双链DNA片段gRNA。
3、用限制性内切酶BsaI酶切载体pKSE401,回收载体骨架。
4、将步骤3回收的载体骨架和步骤2得到的带粘性末端的双链DNA片段进行连接,得到重组载体pKSE401-CHLI。将重组载体pKSE401-CHLI进行测序。测序引物为:
U6-26P-F:5’-TGTCCCAGGATTAGAATGATTAGGC-3’,和
U6-26P-R:5’-CCCCAGAAATTGAACGCCGAAGAAC-3’。
测序结果表明,得到的重组质粒pKSE410-CHLI含有SEQ ID No:3所示的DNA双链分子,所述SEQ ID No:3所示的DNA双链分子插入到了载体pKSE401的限制性内切酶BsaI的识别位点中。
实施例2:chli突变体的获得及验证
一、重组农杆菌的构建
将实施例1得到的重组载体pKSE401-CHLI利用电转法转化农杆菌GV3101感受态,得到重组农杆菌。将重组农杆菌挑单克隆进行摇菌跑PCR进行扩增检测,检测引物为U6-26P-F和U6-26P-R。检测结果表明条带大小正确,重组载体pKSE401-CHLI成功转入农杆菌GV3101感受态细胞中,重组农杆菌构建正确。
二、培育草莓耐弱光突变体
将步骤一构建好的重组农杆菌通过叶盘法侵染进入森林草莓‘Ruegen’的叶片经过延迟筛选和抗性筛选,获得完整的植株。具体步骤如下:
1、预培养:
以新继代20d左右的森林草莓‘Ruegen’的组培苗为材料,在超净工作台上用灭菌剪刀剪下长势良好的叶片
切成大约5mm×5mm的叶条,叶片背面朝上接到预培养培养基中,培养基配方为:MS+30g/L蔗糖+0.8mg/LIBA+2mg/LTDZ+8g/L琼脂,置于25℃组培室进行暗培养。
2、侵染
在-80℃保存的带有目的基因质粒的农杆菌在LB固体平板上划线,挑取新长出的单菌落摇菌至菌液OD为0.4-0.6,离心收集菌体,用MS侵染液(MS+30g/L蔗糖+500mg/L乙酰丁香酮,pH5.8)重悬菌体并调节菌液OD为0.4-0.6,将切好的的叶条轻轻放入侵染液中,在侧摆摇床上(20rpm/min)共培养10-15min,将叶片吸干多余的菌液后接回原来的预培养培养基在黑暗中共培养3d。
3、脱菌
在叶片与农杆菌共培养3d左右,叶片周围会出现拉丝状农杆菌,将叶片在超净工作台上用镊子轻轻夹到无菌组培瓶中,用无菌水清洗4-5遍,每次5min,期间摇晃组培瓶,使农杆菌充分溶解到无菌水中,待清洗完毕,将叶片夹到无菌玻璃皿中,用无菌滤纸吸干叶片表面水分。
4、延迟培养
将步骤3中的叶片转接到延迟筛选培养基(MS+20g/L蔗糖
+2mg/LTDZ+0.8mg/LIBA+8g/L琼脂+200mg/L特美汀+300mg/L头孢)上,叶片背面朝上,置于25℃组培室进行暗培养15d。
5、抗性筛选
将步骤4中延迟筛选15d左右的叶片转移到抗性筛选培养基,抗性筛选培养基配方在延迟筛选培养基的基础上多加15mg/L卡那霉素和200g/L土豆,置于25℃组培室进行培养,设置培养光周期为16h。培养期间每15d更换一次培养基,直至长出的不定芽叶片展开。
6、生根培养
对叶片有明显黄化和白化表型的经过步骤5培养的植株,转接到生根培养基上进行生根培养,培养基配方为MS+20g/L蔗糖+200mg/L特美汀+300mg/L头孢+8g/L琼脂,待植株长到瓶口,拧松瓶盖进行炼苗3d后,取出植株洗干净根部后,移栽到灭菌的基质中,在培养箱进行培养,培养箱设置16h光周期,温度为白天25℃,夜晚22℃,光照强度为300μmol m-2s-1。
三、草莓基因编辑突变体的鉴定
利用GFP绿色荧光和PCR检测技术对T0代阳性植株进行检测。在体式显微镜下利用GFP绿色荧光通道观察培养皿中长出的不定芽,在发绿色荧光的植株上做标记,并将其转移到生根培养基上进行生根培养。待植株生根并长到组培瓶瓶口时,移栽并进行PCR检测。首先利用CTAB法(Allen et al.,2006)提取T0代植株基因组DNA,利用pKSE401载体上包含sgRNA的一对引物U6-26-P和U6-26-T扩增基因组DNA来检测sgRNA是否存在,如若存在,说明该植株是阳性植株。在CHLI基因包含靶位点区域设计一对引物gCHLI-F:GCCAGAGGCCGGTGTATCC和gCHLI-R:TCAGCACCTCCCTCAGCTAAAGA对阳性植株DNA进行扩增并送测序(擎科生物)检测靶位点是否编辑,测序结果利用网站(http://dsdecode.scgene.com/home/)进行解码分析。对于测序结果复杂无法进行解码的情况,对PCR产物进行纯化后连接商业化载体pMD19-T(takara),转化大肠杆菌DH5α,随机挑选15个单克隆进行测序,对测序结果进行分析,计算编辑效率(编辑效率=编辑株数/总阳性株数)。
共获得重组农杆菌转化的草莓植株114株,根据阳性植株的叶片表型,最终确定编辑植株56株(编辑效率48.7%),其中纯合编辑7株,均为替换突变,叶片均表现出白化表型,由于白化表型无法合成叶绿素,因此不作为本实施例中的研究材料;10株嵌合编辑,测序结果中野生型序列超过编辑序列,叶片中有白色斑点;有39株杂合编辑,均为移码突变,但突变类型不一样,叶片均表现为黄化。
四、chli杂合突变体的耐弱光性鉴定
供试材料:chli突变体T0代杂合体和森林草莓‘Ruegen’移出组培瓶20d左右的幼苗。
1.对供试植株进行不同光照强度处理,设置两个光照强度,调节光照培养箱的光照强度至300μmol m-2s-1作为高光照强度,用小型遮阳网遮挡部分光照,调节光照强度低至50μmol m-2s-1,设置光周期为16小时,培养7d。设置三次重复实验,每次重复试验3盆。
2.完成步骤1后,取叶片对叶绿素含量进行测定。
结果表明,chli在50μmol m-2s-1光照强度下叶片颜色明显深于300μmol m-2s-1,但仍与‘Ruegen’存在差异,‘Ruegen’叶片在处理前后差异不大(图1)。对chli及‘Ruegen’在不同光照强度下的叶绿素含量进行测定,发现在高光照强度下,chli突变体叶绿素a(Chla)和叶绿素b(Chlb)含量显著低于野生型,但在低光照强度下,尽管chli突变体Chla含量与野生型存在显著差异,但Chlb的含量与野生型差异不显著(图2)。
3.完成步骤1后,利用便携式光合仪CIRAS-3(PPSYSTEMS,美国)测定光合作用相关参数,内部参数的设置参照使用说明书完成。
结果表明在300μmol m-2s-1光强下,chli突变体的净光合速率显著低于野生型,气孔导度低于野生型;相反,在50μmol m-2s-1光强下,chli突变体的净光合速率和气孔导度均显著高于野生型(图3)。因此,chli突变体比野生型更耐弱光。
4.完成步骤1后,对核基因编码的光合相关基因(补光色素复合体LHCⅠ和LHCⅡ)的表达水平进行了检测。根据各基因序列设计如下实时荧光定量PCR检测引物如下:
FveCHP1-qF(内参基因):5’-TGCATATATCAAGCAACTTTACACTGA-3’
FveCHP1-qR(内参基因):5’-ATAGCTGAGATGGATCTTCCTGTGA-3’
Lhca1-qF:5’-TGGGTAAAGGCACAAGAATGG-3’
Lhca1-qR:5’-TGCTCTACGAAGGCAATGGA-3’
Lhca2-qF:5’-GCAGGCAAAGGGGTGTCA-3’
Lhca2-qR:5’-GGGGTCGAAGCCAAAATCA-3’
Lhcb1-qF1:5’-AAGCAGTCAAGCTCAGTCCC
Lhcb1-qR1:5’-CAGTGTCCCAACCGTAGTCC
Lhcb1-qF2:5’-ATCCGTCAGCCCGTGGTA
Lhcb1-qR2:5’-GGTTCTTGGCAAAGGTCTCAG
Lhcb2-qF:AACAAGTATGGCGCAGACCG
Lhcb2-qR:TGAGACCCAGCCTTGAACCA
Lhcb3-qF:GGCGACTACGGATGGGACA
Lhcb3-qR:GCATGAACAAGGTTGGGGTT
利用TaKaRa的实时荧光定量PCR试剂盒在Bio-Rad IQ5实时荧光定量PCR仪上进行RT-qPCR试验。反应体系为:SYBR Premix Ex Taq II 10.5μL,cDNA模板1.0μL,Forward-引物0.8μL,Reverse-引物0.8μL,ddH2O 7.4μL。PCR扩增程序:95℃预变性3min,40个循环(95℃30s,58℃30s)。PCR循环后,50℃保持1min,然后以每10秒逐渐增加0.5℃,进行熔解曲线分析。用IQ5软件标准化表达方法分析基因的相对表达水平。每个处理分别进行3次生物学重复和3次技术重复。
结果表明,与‘Ruegen’在高光照强度下,chli突变体中大部分的FveLhca和FveLhcb基因呈下调趋势,但在弱光下,结果恰好相反,大部分的光合相关基因呈上调趋势(图4),证明chli突变体在弱光下光合能力比‘Ruegen’更高。
完成步骤1后,对叶片超微结构进行观察,选取不同光照强度下黄化突变体chli和‘Ruegen’植株同一叶片位置的叶片为材料,通过制备超薄切片并进行透射电子显微镜观察,比较分析其叶肉细胞结构上的差异,具体操作步骤如下:
(1)取样:选取黄化突变体chli和‘Ruegen’的叶片,避开叶脉,切成2mm×5mm长条。
(2)前固定:将叶条浸泡在2.5%戊二醛(0.1M磷酸缓冲液,pH7.2-7.4)中,抽真空使叶条完全浸没在戊二醛中,在4℃条件下至少固定叶片5h。用0.1M磷酸缓冲液漂洗,3-4次,每次15min。
(3)后固定:用1%锇酸(0.1M磷酸缓冲液,pH 7.2-7.4)固定2-3h,用用0.1M磷酸缓冲液漂洗,3-4次,每次15min。
(4)梯度脱水:将样品置于不同浓度的乙醇(30%,50%,70%,80%,90%,100%)中依次进行脱水,每个浓度重复两次,每次10min。
(5)树脂渗透:将样品放置在25%的包埋剂中渗透2h,更换包埋剂,用50%包埋剂渗透8h,第二次更换包埋剂(75%),可在包埋剂中加醋酸钠,过夜渗透12h,最后用纯树脂包埋剂在室温条件下渗透2次,每次24h。
(6)包埋固化:用包埋模具进行包埋,55℃烘箱固化48h。
(7)切片:先用全自动修片机切掉多余的树脂胶,暴露出样品,再用半薄切片机(Leica RM2265。德国)切取8μm样品置于载玻片上,甲苯胺蓝染色后观察,确定目标位置后,利用超薄切片机(徕卡UC7)切取70nm的样品装到200目铜网上。
(8)染色:采用铀铅双染色,用2%醋酸双氧铀染色10 -30min,用蒸馏水冲洗干净,用滤纸吸干多余的水分,再用柠檬酸铅染色5-15min,用蒸馏水冲洗干净,吸干水分。
(9)电镜观察:将染色好的样品放置在透射电子显微镜(TECNAI G2 SpiritBioTWIN,FEI,美国)进行观察拍照。
结果表明,在高光照强度下,chli突变体类囊体垛叠结构较少,但在低光照强度下,其叶绿体的发育正常,与野生型无差异,证明chli突变体在低光照强度下,叶绿体发育正常(图5),而叶绿体是植物光合作用的场所,因此进一步说明chli突变体适宜在弱光下生长。
参考文献
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Claims (4)
1.培育耐弱光草莓的方法,包括向目的草莓导入草莓基因组编辑的载体,筛选黄化叶片草莓植株得到耐弱光草莓,所述耐弱光草莓与所述目的草莓相比,光合能力更强,适应弱光能力更强;
所述草莓基因组编辑载体含有Cas9蛋白基因和sgRNA,
所述sgRNA在草莓中识别的靶标DNA为编码草莓中CHLI蛋白的DNA片段,
其中所述sgRNA识别的靶位点为SEQ ID NO:3所示的DNA分子,所述sgRNA是SEQ ID NO:4所示的DNA分子。
2. 根据权利要求1所述的方法,其中所述CHLI蛋白的氨基酸序列是SEQ ID NO:1。
3.草莓基因组编辑的载体,其含有Cas9蛋白基因和sgRNA,其中所述sgRNA在草莓中识别的靶标DNA为编码CHLI蛋白的DNA片段,所述sgRNA识别的靶位点为SEQ ID NO:3所示的DNA分子,其中所述sgRNA是SEQ ID NO:4所示的DNA分子。
4.根据权利要求3所述的载体在培育耐弱光草莓的应用。
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