CN114836467B - 一种植物高效遗传转化和筛选体系及其方法与应用 - Google Patents
一种植物高效遗传转化和筛选体系及其方法与应用 Download PDFInfo
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Abstract
本发明公开了一种植物高效遗传转化和筛选体系及其方法与应用,属于植物转基因工程技术领域,所述植物高效遗传转化和筛选体系包括双元载体pCWBG和双元载体pBOE/pBC,所述双元载体pCWBG至少包含第一筛选标记基因、Bbm蛋白编码基因、Wus2蛋白编码基因和CRE蛋白编码基因的表达盒。本发明通过构建玉米Bbm和Wus2表达盒切除载体,建立了双元载体共转、双标记筛选和早期Bbm和Wus2基因切除的高效遗传转化方法,提高了玉米转化效率,显著降低了转化苗和转化植株畸形不育率,节约了转化成本,可在玉米转基因和基因编辑育种上广泛开发应用。
Description
技术领域
本发明涉及的是植物转基因工程技术领域,尤其涉及的是一种植物高效遗传转化和筛选体系及其方法与应用。
背景技术
自从上世纪80年代转基因植物诞生以来,植物转基因工程作为提高作物品质、产量、抗胁迫能力等的有效手段之一,其发展势头始终不减,现已跃入大规模商业化生产阶段,特别是近年来基因编辑技术的快速发展为植物基因功能研究和育种提供了更加高效广阔的定向遗传改良新技术。
然而植物转基因和基因编辑其遗传转化是基础。植物遗传转化的方法有多种,农杆菌介导法是目前植物转基因研究中应用最为广泛的遗传转化技术,其优点是操作简单、转化效率较高、成本低,只将位于同一T-DNA区内的外源目的基因和标记基因导入植物,并且紧密相连,目前主要应用于双子叶植物和少数单子叶植物的遗传转化。
玉米是重要的粮食、饲料和工业原料作物,在解决粮食安全和资源短缺以及促进国民经济发展等方面具有重要作用。转基因技术已成为玉米基因功能研究和遗传改良中的一种重要手段。玉米作为单子叶植物农杆菌遗传转化基因型依赖性强,遗传转化效率低,目前只有A188、KN5585、B104等少数自交系能诱导产生TypeⅡ型胚性愈伤组织并分化成再生植株实现遗传转化,大多自交系受体材料难以实现遗传转化,极大地限制了玉米转基因及基因编辑技术的发展和规模化育种应用。
Baby boom(Bbm)和Wuschel2(Wus2)是植物干细胞发育中的关键调控因子。Bbm编码一个AP2/ERF转录因子,在维持干细胞不分化状态中发挥重要作用;Wus2编码同源异型结构域蛋白,能使周围的细胞具有干细胞特征。一些研究者将Bbm和Wus2基因应用到植物遗传转化方法中,将转化效率从2%提升到25%及以上,虽然不同受体的转化效率依然有差别,但可使更多的基因型实现遗传转化(Mookkan M,et al.,Selectable marker independenttransformation of recalcitrant maize inBared B73 and sorghum P898012mediatedby morphogenic regulators Baby boom和Wuschel2.Plant Cell Rep,201736:1477,LoweK,et al.,Morphogenic regulators Baby boom and Wuschel improve monocottransformation.Plant Cell,2016,28:1998)。目前公开的将形态调节基因Bbm和Wus应用于玉米遗传转化中,虽然可以明显提高转化效率,但是转化苗畸形率高,转化植株中畸形不育的比率高,且Bbm和Wus2基因表达单元较大,难以用单一载体进行外源基因转化和基因编辑,限制了Bbm和Wus2基因在玉米遗传转化育种中规模化应用。
发明内容
本发明提供了一种植物高效遗传转化和筛选体系及其方法与应用,目的在于:解决利用Bbm和Wus2基因提高玉米遗传转化效率中单一载体应用于外源基因转化和基因编辑负载过大,转化苗和转化植株畸形不育率高,筛选效率低,难以在规模化育种中应用的问题,提供一种双元载体双向筛选提高玉米优良遗传转化率和育种应用方法。
本发明是通过以下技术方案实现的:
本发明提供了一种植物高效遗传转化和筛选体系,包括双元载体pCWBG和双元载体pBOE/pBC,其中:
双元载体pCWBG至少包含第一筛选标记基因、Bbm蛋白编码基因、Wus2蛋白编码基因和CRE蛋白编码基因的表达盒,所述CRE蛋白编码基因的表达盒由热激启动子启动表达,所述第一筛选标记基因、Bbm蛋白编码基因、Wus2蛋白编码基因和CRE蛋白编码基因的表达盒的LB和RB边界连接有CRE蛋白切割靶标序列;
双元载体pBOE/pBC为含有第二筛选标记基因表达盒的植物过量表达载体pBOE或基因编辑载体pBC,所述第二筛选标记与第一筛选标记为不同类型的筛选标记。
作为本发明的进一步优化方案,第一筛选标记为荧光筛选标记,第二筛选标记为化学筛选标记。
作为本发明的进一步优化方案,第一筛选标记为GFP或RFP,第二筛选标记为Bar或Htp。
作为本发明的进一步优化方案,所述植物为玉米,所述热激启动子为玉米pZmHSR1启动子,所述CRE蛋白切割靶标序列为loxP序列。
作为本发明的进一步优化方案,所述过量表达载体包含用于插入待转化的外源目的基因的MCS多酶切位点,所述基因编辑载体包含用于插入待编辑的目的基因靶标sgRNA的多酶切位点和Cas9基因表达盒。
本发明还提供了一种利用上述植物高效遗传转化和筛选体系对植物进行高效遗传转化和筛选的方法,包括以下步骤:
(1)将待表达的外源目的基因插入植物过量表达载体中,或将待编辑的目的基因靶标sgRNA插入基因编辑载体中,构建双元载体pBOE/pBC;
(2)将双元载体pCWBG和双元载体pBOE/pBC共转化植物组织细胞,选择含有第一筛选标记和第二筛选标记的植物愈伤组织细胞为双元载体pCWBG和双元载体pBOE/pBC的共转组织;
(3)将共转组织进行高温热激处理,启动热激启动子,表达CRE蛋白,将Bbm、Wus2、GFP和CRE表达盒切除。
(4)继续培养植物愈伤组织,培养过程中利用第一筛选标记和第二筛选标记进行筛选,选择仅含有第二筛选标记的植物组织,获得转基因植株。
作为本发明的进一步优化方案,所述第一筛选标记为GFP,所述第二筛选标记为Bar,所述步骤(2)的筛选方法包括:在共培养和静息阶段用GFP荧光标记筛选转化胚和转化愈伤组织,取绿色荧光的愈伤组织转移到含有双丙氨膦筛选剂的培养基上,选择双筛存活下来的愈伤组织为双元载体pCWBG和双元载体pBOE/pBC的共转组织。
作为本发明的进一步优化方案,所述植物为玉米,所述热激启动子为pZmHSR1,所述步骤(3)的高温热激处理条件为42℃下高温处理2h。
本发明还提供了一种上述植物高效遗传转化和筛选体系在植物遗传转化和筛选中的应用,进一步地,所述植物为玉米。
本发明相比现有技术具有以下优点:本发明通过构建玉米Bbm和Wus2表达盒切除载体,建立了双元载体共转、双标记筛选和早期Bbm和Wus2基因切除的高效遗传转化方法,提高了玉米转化效率,显著降低了转化苗和转化植株畸形不育率,节约了转化成本,可在玉米转基因和基因编辑育种上广泛开发应用。
附图说明
图1为双元载体pCWBG质粒图谱;
图2为相关载体T-DNA区域组成图;
图3为玉米遗传转化各分化阶段图片;
图4为未切除(左)与切除(右)Bbm和Wus2基因分化时获得正常植株的情况;
图5为非荧光(左)和荧光(右)愈伤组织在Bar筛选条件下的转化筛选效率图。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解,下面结合具体图示,进一步阐述本发明。
实施例1
本发明提供的双元载体pCWBG是一种蛋白组合表达载体,所述蛋白组合至少包含第一筛选标记、Bbm、Wus2和CRE四种蛋白,其中,如图1所示为本实施例的一种用于玉米高效遗传转化和筛选体系的双元载体pCWBG,该双元载体pCWBG的第一筛选标记为GFP蛋白,所述GFP蛋白由含有CaMV35s启动子连接编码GFP蛋白的核酸分子连接nos终止子的表达盒表达;所述Bbm蛋白由含有pZmPLTP启动子连接编码Bbm蛋白的核酸分子连接tT28终止子的表达盒表达;所述Wus2蛋白由含有pZmAXIG1启动子连接编码Wus2蛋白的核酸分子连接tIN2-1终止子的表达盒表达;所述CRE蛋白由含有玉米热激启动子pZmHSR1连接编码CRE蛋白的核酸分子连接nos终止子的表达盒表达,并在T-DNA的LB和RB边界处连有CRE蛋白切割靶标序列loxP。
本发明提供的双元载体pBOE,为过表达载体,其特征是MCS和筛选标记的组合,MCS为多酶切位点,可以插入任何需要转化的外源目的基因。本实施例的双元载体pBOE插入的外源目的基因由pZmUbi启动子启动,Bar为筛选标记,Bar也可以替换成其他筛选基因如Htp,本实施例的Bar由CaMV35s启动子启动。
本发明提供的双元载体pBC,为基因编辑载体,其特征是sgRNA、Cas9和筛选标记的组合。本实施例的双元载体pBC,由pZmU6启动子连接sgRNA表达盒一个或两个,由pZmUbi启动子连接Cas9基因连接nos终止子,由35S启动子连接Bar基因连接PolyA终止子组成的筛选标记表达盒表达,sgRNA可以替换成任何待编辑的靶标sgRNA,Bar也可以替换成其他筛选基因如Htp。
本实施例的宿主细胞包含上述的双元载体pCWBG、pBOE或pBC,宿主细胞为农杆菌细胞,农杆菌菌株为EHA105。
本实施例的pZmHSR1启动子序列如SEQ ID NO.1所示,编码CRE蛋白的核酸分子序列如SEQ ID NO.2所示;pZmPLTP启动子序列如SEQ ID NO.3所示,编码Bbm蛋白的核酸分子来自玉米Bbm基因,序列如SEQ ID NO.4所示,tT28终止子序列如SEQ ID NO.5所示;pZmAXIG1启动子序列如SEQ ID NO.6所示,编码Wus2蛋白的核酸分子来自玉米Wus2基因,序列如SEQ ID NO.7所示,tIN2-1终止子序列如SEQ ID NO.8所示;loxP序列如SEQ ID NO.9所示。
本实施例相关载体T-DNA区域组成如图2所示。
本实施例还提供一种双元载体玉米遗传转化高效筛选方法,用上述双元载体pCWBG与双元载体pBOE/pBC共转化玉米幼胚受体细胞,通过GFP荧光和Bar抗性高效筛选共转化组织细胞及获得不含GFP、Bbm、Wus2和CRE四种蛋白转化植株,提高转化率和正常转化植株率。
上述方法中包括:用含有pCWBG和pBOE载体农杆菌混合浸染共转化,前期使用GFP绿色荧光和Bar抗性筛选共转化的组织细胞,提高胚状体发生和共转化细胞率,同时节省人力物力成本,提高工作效率;然后在筛选阶段热激处理并用Bar抗性筛选无绿色荧光分化组织,获得剔除GFP、Bbm、Wus2和CRE四种蛋白表达盒结构,获得只含有pBOE载体T-DNA结构的过表达植株;
本实施例采用了pZmHSR1启动子启动CRE-loxP系统,在筛选阶段进行热激处理,切除形态调节Bbm、Wus2基因和GFP、CRE基因蛋白表达盒结构,降低了畸形苗的比例,获得大量玉米正常转化苗和转化株。
本实施例在筛选阶段培养基中加入了除草剂,在热激处理后,选择没有绿色荧光且能在抗性培养基中生长的愈伤组织进行分化出苗。
上述方法中还包括:用双元载体pCWBG和pBC共转化,用于基因编辑,其筛选方法同上,获得剔除GFP、Bbm、Wus2和CRE四种蛋白表达盒结构,只含有pBC载体Cas9-sgRNA的T-DNA结构的转化植株,进行分子检测获得基因编辑植株,后代可通过分离去除pBC载体T-DNA结构。
为验证本发明体系和方法的有益效果,本发明使用了多个优良自交系进行测试,包括玉米CM1、CM25、B104、B73自交系,(CM1、CM25见Zhao et al.BMC Plant Biology,2019,19:273),结果显示,不同基因型转化效率有所差异。
下面对本发明的具体实验过程进行详细阐述。
试验例1、表达Bbm、Wus2提高玉米转化率
在玉米中组成型表达玉米形态发生转录因子Bbm和Wus2可以大大提高转化效率,但畸形苗比例高,也会诱导玉米植株的表型异常和不育,因此很难进行商业化应用。pZmAXIG1是具有生长素诱导特性的启动子,而pZmPLTP是磷脂转移酶蛋白基因启动子,在愈伤、胚中特异性表达,用pZmAXIG1和pZmPLTP启动子驱动Wus2和Bbm的时空特异性表达,可以在一定程度上克服Wus2和Bbm基因带来的负面效应。
本发明首先测试了pZmAXIG1和pZmPLTP启动子驱动Wus2和Bbm对一些优良玉米自交系的转化促进效果。测试时使用的表达载体是pWBG和pGBE,载体示意图见图2。其中,pWBG载体T-DNA区包含六个表达盒,分别是促进转化的两个表达盒:pZmAXIG1启动子连接ZmWus2基因连接tPINII终止子,以及pZmPLTP启动子连接ZmBbm基因连接tT28终止子;另外还含有两个筛选标记GFP及Bar;pGBE相比较pWBG,只包含两个筛选标记GFP及Bar,不含Wus2和Bbm表达盒。
pWBG载体转化玉米方法,用CM1、CM25、B104、B73这4个优良自交系幼胚作为转化受体材料,具体的转化步骤如下:
1)将含有pWBG的EHA105和含有pGBE的EHA105农杆菌菌株在活化培养基上划线,28℃黑暗培养48h。
2)将玉米自交系授粉后9-12天,玉米幼胚长至0.5-2.0mm时从玉米穗上剥取获得的幼胚浸入悬浮培养基,悬浮浸泡10-30min,完成幼胚收集后移走液体,热激3min,冰浴1min,再分别加入含有pWBG的EHA105和含有pGBE的EHA105农杆菌菌株中的侵染培养基中侵染5min,期间不停的摇动。
3)将幼胚移至共培养基上,23℃黑暗培养24-48h。
4)将幼胚移至静息培养基上,26-34℃黑暗培养1-2周。
5)将带有绿色荧光的幼胚移至选择培养基上培养,选择培养基中含双丙胺膦诱导抗性愈伤组织;将抗性愈伤组织转移至分化培养基中,25℃,5000lx,光照培养3周,分化形成再生小苗。
6)再生小苗在生根培养基上生根后炼苗、移栽,得到转化植株。
转化过程中所用到的培养基配方如下:
农杆菌活化培养基:酵母提取物5g/L+蛋白胨10g/L+氯化钠5g/L+琼脂15g/L;
悬浮培养基:1/2MS+蔗糖68.5g/L+葡萄糖36g/L+L-脯氨酸0.115g/L+MES 0.5g/L;
侵染培养基:1/2MS+蔗糖68.5g/L+葡萄糖36g/L+L-脯氨酸0.115g/L+MES 0.5g/L+乙酰丁香酮200mM+半胱氨酸200mg/L;
共培养基:1/2MS+蔗糖20g/L+葡萄糖10g/L+脯氨酸0.115g/L+盐酸硫胺素0.5mg/L+AgNO320mM+L-半胱氨酸200mg/L+2,4-D 0.5mg/L+毒莠定2.2mg/L+乙酰丁香酮200mM;
静息培养基:MS+蔗糖30g/L+脯氨酸1.38g/L+盐酸硫胺素0.5mg/L+AgNO320mM+水解酪蛋白0.5g/L+2,4-D 0.5mg/L+毒莠定2.2mg/L+特美汀200mg/L;
筛选培养基:MS+蔗糖30g/L+脯氨酸1.38g/L+盐酸硫胺素0.5mg/L+AgNO320mM+水解酪蛋白0.5g/L+2,4-D 0.5mg/L+毒莠定2.2mg/L+特美汀200mg/L+双丙胺膦3mg/L;
分化培养基:MS+蔗糖30g/L+6-BA 0.1mg/L+KT 1mg/L+特美汀200mg/L+双丙胺膦3mg/L
生根培养基:MS+蔗糖20g/L+MES 0.5g/L+NAA 0.2mg/L。
pWBG转化时发现农杆菌侵染后3天的愈伤组织上就能够观察到由于Bbm和Wus2的表达引起的胚状突起,7天后就可观察到明显的成簇胚状体及丰富的胚状突起,这些愈伤组织可以成功在培养基上分化成苗(图3)。玉米转化效率的统计结果(表1)显示,含Bbm和Wus2表达盒pWBG对不同自交系的转化效率在16.7%~30.7%之间,平均可达23.2%。
而不含Bbm和Wus2表达盒pGBE幼胚转化频率很低,B73无法获得转化苗。这些结果显示,Bbm和Wus2的表达显著提高了供试玉米自交系的转化效率,但转化苗多丛生,无主茎,畸形率高,正常转化苗比率较低,只有3.3%~7.3%。同时在获得的所有T0代转化植株中,均含有Bbm和Wus2基因,这两个基因在转化成功后不再有利用价值,而且他们的表达会使植株表现出不同程度的异常,因此,T0代转化株必须剔除Bbm和Wus2基因表达盒。所以该载体不适合在玉米转基因和基因编辑育种上应用。
表1 pWBG载体对四种玉米优良自交系的转化效率
试验例2、双元载体转化玉米剔除Bbm和Wus2基因提高正常转化苗频率
虽然表达Bbm和Wus2可以显著提高玉米遗传转化效率,但是获得的后代玉米植株中仍存在发育障碍等各种不利农艺性状,并且获得的植株中含有Bbm和Wus2基因,限制了该技术在转基因和基因编辑育种中推广应用。
为解决上述问题,利用本发明的双元载体进行共转化,其中载体pCWBG含有Bbm和Wus2,同时具有GFP的筛选标记和CRE-loxP切除系统,主要用于早期利用Bbm和Wus2提高转化频率,并通过热激启动CRE-loxP切除系统剔除含有Bbm和Wus2等基因的T-DNA单元,以提高正常苗转化频率;载体pBOE为过表达载体,载体pBC为基因编辑载体,都含有Bar基因,通过共转化和GFP及Bar双向筛选策略,高效获得不含Bbm和Wus2基因的正常转基因植株。
具体的测试工作以CM1、CM25、B104、B73四个自交系幼胚为受体开展。
利用双元载体pCWBG和pBOE共转化获得不含Bbm、Wus2和GFP表达盒的T-DNA的过表达转化株,具体转化方法包括以下步骤:
1)将含有pCWBG的EHA105和含有pBOE的EHA105农杆菌菌株在活化培养基上划线,28℃黑暗培养48h。
2)将玉米自交系授粉后9-12天,玉米幼胚长至0.5-2.0mm时从玉米穗上剥取获得的幼胚浸入悬浮培养基,悬浮浸泡10-30min,完成幼胚收集后移走液体,热激3min,冰浴1min再加入含有pCWBG的EHA105和含有pBOE的EHA105农杆菌菌株的侵染培养基中共侵染5min,期间不停的摇动。
3)将幼胚移至共培养基上,23℃黑暗共培养24-48h,在共培养阶段用GFP荧光标记进行筛选转化胚和转化愈伤组织。
4)将步骤3)筛选的转化胚和转化愈伤组织移至静息培养基上,26-34℃黑暗培养1-2周,在静息培养阶段继续用GFP荧光标记进行筛选转化胚和转化愈伤组织。
5)将带有GFP绿色荧光的幼胚移至选择培养基上培养,选择培养基中含双丙胺膦诱导抗性愈伤组织,进行3周的pBOE载体筛选,这样双筛存活下来的愈伤组织为双元载体pCWBG和pBOE共转愈伤组织;
对存活下来的共转愈伤组织进行42℃2h高温处理,启动pZmHSR1启动子,表达CRE蛋白将Bbm、Wus2、GFP和CRE表达盒切除,同时设置未经高温处理的共转愈伤组织作为对照组;
切除效果可以通过观察绿色荧光,切除了将不激发绿色荧光;将还具有绿色荧光的愈伤组织恢复一段时间后再进行二次高温处理;
选择无绿色荧光Bar抗性愈伤组织转移至分化培养基中,25℃,5000lx,光照培养3周,分化形成再生小苗。
6)再生小苗在生根培养基上生根后炼苗、移栽,生根阶段也可以再对植株进行PCR检测,再次确定植株中不含有Bbm、Wus2、GFP和CRE表达盒,得到剔除Bbm、Wus2、GFP和CRE表达盒的正常过表达转基因植株,结果如下表2和图4所示,图4为未切除(左)与切除(右)Bbm和Wus2分化时获得正常植株的情况。
表2 pCWBG与pBOE共转剔除Bbm、Wus2基因获得正常过表达植株的效果
从表2的实验结果可以看出,过表达植株中,四个自交系未高温处理(fgw)的正常苗转化率在3.6%-7.1%;而经过高温处理(gw),切除Bbm、Wus2的正常苗转化率为15.6%-27.6%。正常转化苗占转化苗95%以上。
试验例3、GFP报告和Bar抗性双向筛选提高玉米正常苗转化效率
农杆菌侵染后在共培养及静息阶段利用可视化的GFP荧光标记进行筛选转化胚和转化愈伤组织,可显著提高转化组织的筛选效率(表3)。采用荧光手电筒,小巧方便,为后期的筛选工作节省了大量时间成本和人力物力成本。
表3 pWBG载体利用GFP绿色荧光报告筛选效率
用本发明的双元载体pCWBG和pBC(基因编辑载体)共转化,同时设置阴性对照,利用不含GFP的双元载体pCWBG(pCWB)和pBC共转化作为对比,测试4个自交系通过GFP报告和Bar抗性双向筛选提高玉米转化效率和正常苗转化率的影响(表4、图5),由表4可见,利用GFP荧光在共培和静息阶段早期选择较非GFP选择显著提高了转化率,在供试自交系中大约提高7%左右,这可能是通过GFP选择了Bbm、Wus2基因,通过Bbm、Wus2表达刺激了分化,提高了转化率。再经高温处理,通过无荧光选择和Bar筛选可获得更高的切除Bbm、Wus2基因的正常苗转化率,同时分化阶段通过无荧光选择和Bar筛选还可减少单一转化pBC株的逃逸,其正常苗转化率达15.5%-26.1%。展示了可视化GFP荧光和Bar抗性两段式双向选择对提高玉米正常苗转化效率的优势,可以用于玉米大规模商业化转基因和基因编辑育种。
表4 pCWBG与pBC共转通过GFP和Bar双向筛选提高正常转化苗频率
试验例4、不同热激启动子对提高玉米遗传转化效率的影响
为验证热激启动子的种类对CRE驱动表达和剪切效率的影响,本实施例使用多个热激启动子pZmHSR1、pZmHSR2、pZmHSR3,构建了不同的pCWBG载体,将含有不同热激启动子的pCWBG载体与pBOE共转化玉米B104自交系,pZmHSR2具有如SEQ ID NO.10所示核苷酸序列,pZmHSR3具有如SEQ ID NO.11所示核苷酸序列,具体转化方法同试验例2,统计正常苗转化率和正常转化苗占转化苗的比率。
表5不同热激启动子对玉米遗传转化效率的影响
本实施例使用经多个热激启动子比较筛选获得了玉米pZmHSR1启动子驱动CRE表达对玉米遗传转化效果最好,pZmHSR1启动子在玉米中34℃以下驱动基因表达量极低,42℃热激驱动基因高表达。
结论:
本发明测试了不同优良自交系用双元载体转化玉米过程中,双元载体对转化效率、正常苗转化效率和报告基因与抗性基因双向选择对转化筛选效率的影响。结果表明加入Bbm和Wus2基因转化效率显著提高,筛选阶段热激切除Bbm和Wus2基因,显著降低植株畸形率,得到更多正常苗转化株,利用双向筛选可显著提高转化效率和正常苗转化率。总之,利用本发明的双元载体双向选择进行玉米转化,可显著提升转化效率和正常苗转化效率,减少基因型限制,缩短转化成株时间,节省人力物力,获得剔除Bbm和Wus2基因的转基因植株没有不利的农艺性状,可以应用于玉米规模化转基因和基因编辑研究与育种。
以上为本发明一种详细的实施方式和具体的操作过程,是以本发明技术方案为前提下进行实施,但本发明的保护范围不限于上述的实施例。
序列表
<110> 安徽农业大学
<120> 一种植物高效遗传转化和筛选体系及其方法与应用
<141> 2022-05-25
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2001
<212> DNA
<213> 玉米(Zea mays)
<400> 1
cttggagccc cctgggaaaa ctggtgatcc tgtgtcaatg taaagagacg tatgatctga 60
aacgcctctg tctaacgcct ggacagagac taatgggtat ttaacttccc attcggtcga 120
cataagcact ctatctactt tctcatatgt tggattgggt aaagaatttg cccaagtaaa 180
ttgtcttcca tttaactcaa tctctcgtaa atcaaagtta tctattatcg cgttaaaaag 240
gaacagccat ctcatattaa aattattttg gtttttttcc ttcctgctcc tcatcatatt 300
aaaatctccc ccaattaagg ttgacagata attttgctgc caagttctta ccagctctgt 360
caaaaaggct attttgaagt cctcctgagc agggccatag actgccatta aaatccattt 420
gaaattatcc accctattgt tgagatgaaa tttaacaaaa aattcccctt caaccattag 480
tgttaaatcg aatctgtctc tccttactcc caaaagaatc cctcccgatc ttcctcgggg 540
tgggagacaa tgccacacaa attcagcacc tccggtcagt cgtgacaaat tcgattgtga 600
catgtcttgt ttacctgttt ccattacccc tataaaatct agattaaatt cattgactgt 660
atctgaaatg taccgaaatt tagccaagtc tgaaagacct atgctattcc aaaataggct 720
tctcataatg aacaattatt acatattttt ctgttttttc gttttttgtg tgatttacga 780
gatgaggcca ttaggtcaca taatttagtg tctagatcct ctgtcaccaa atccacctca 840
gaaacatcct taatcaagtg ggcgagtaag gcaccatcac catctatggc ttcgtcgtca 900
cttaattcaa aaggagaaat cgatgggtca ggcctaataa tttgattctc cttgcatctg 960
tctctatcct tcgtaatgca tttagcgaaa agttaaccct cgatgtatcc ttttccatct 1020
taaaatctag attacttaaa ttacgctcgc aaagctcatc ggacaacgac tcaaaagaat 1080
aagagctagg ttgtgttgac ataccagtat tttcagcctc tgacatacta aagcctttga 1140
tggacagggt cctttcctga cgttttccat caatatccat attgtgttct gctattcggc 1200
gcattgtctt gttcagaaca ggctcatctg tcaaatacaa accatcaatg gttttggtat 1260
tattgtgcaa gctaggtcgt aacctgaatg gaaattctca tggagcgtac cttttatact 1320
agcaagtgga tttgtagcct tgtattttcc aatagcacgg tcgcacatgg tcaggtgagg 1380
tgaccgcacc gttccaatcc aatatggcaa tatccaacgg ccaggcgcca ggctgcggta 1440
ccgagctgtc cggctgcaaa ccaggctgtt tgcagctaga agacgaagcg gctagaagac 1500
gaggaaaaaa atttgttgca gtccggctgc aaaccaggct gtttgcagcc cctcacaaaa 1560
aggaggatag gccccacccg caggtaaata atacaactaa aacgttatct ggtggacctg 1620
caggtggggc ctatccacct ttttgtgagg ggctgcaaac agcctaattt gcagccgggc 1680
tgcaacaaat ttttttcctc gtcttctagc cgcttcgtgg agttgtcggt ccacgctggc 1740
gcgtccccgt ctcgccgcgt gccccgccct cgcgccacgt ccggctgctt ccagcgccac 1800
ctcgctcccg ccggttccag acgcttcttc cctttccgca taaaagccac caccacgaga 1860
gctcgctctc cttcacctct gaccaccctg agccatcaca gagaagcgtc cgctgcgcac 1920
acccaggtcc ttttcatcta tctgtgagcc gtcacaactc gtactgtcgt acagtatact 1980
tgcgcgccga accagtcgcc c 2001
<210> 2
<211> 1029
<212> DNA
<213> 噬菌体P1(Bacteriophage P1)
<400> 2
agcaacctcc tgaccgtgca ccagaacctc ccagccctcc cggtcgatgc cacctccgac 60
gaggtgagga agaacctgat ggacatgttc cgcgaccgcc aggccttcag cgagcacacc 120
tggaagatgc tgctctccgt gtgcaggtcc tgggccgcct ggtgcaagct caacaaccgc 180
aagtggttcc cagccgagcc ggaggacgtc agggactacc tgctctacct ccaggccagg 240
ggcctcgccg tcaagaccat ccagcagcac ctcggccagc tgaacatgct ccacaggagg 300
agcggcctcc cgaggccaag cgactccaac gccgtctccc tggtcatgcg caggatcagg 360
aaggagaacg tcgatgccgg cgagcgcgcc aagcaggctc tcgctttcga gcgcaccgac 420
ttcgaccagg tcaggtccct catggagaac tccgaccgct gccaggacat ccgcaacctg 480
gccttcctgg gcatcgccta caacaccctc ctcaggatcg ccgagatcgc caggatcagg 540
gtcaaggaca tcagcaggac cgacggcggc aggatgctca tccacatcgg ccgcaccaag 600
accctggtga gcaccgccgg cgtcgagaag gccctcagcc tgggtgtgac caagctcgtg 660
gagcgctgga tctccgtcag cggcgtcgcc gacgacccaa acaactacct cttctgccgc 720
gtgaggaaga atggcgtcgc cgccccgtcc gccacctctc agctttccac ccgcgccctc 780
gaaggcatct tcgaggccac ccacaggctc atctacggcg ccaaggacga ctccggccag 840
aggtacttgg cctggtccgg ccactccgcc cgcgttggtg ctgctaggga catggccagg 900
gccggcgtgt ccatcccgga gatcatgcag gccggcggct ggaccaacgt gaacatcgtg 960
atgaactaca tcaggaacct cgacagcgag accggcgcga tggttcgcct gctggaggac 1020
ggcgactaa 1029
<210> 4
<211> 1065
<212> DNA
<213> 玉米(Zea mays)
<400> 4
ttaccctagc tccctgcggc tgttacgcgg tcccccatca atcttctgtt cttgcggttg 60
tagcctgtgt aacagtgcta gagtatgtat gataaatagg ttttaagtct gcttacatga 120
cattttttat tgtggaagag acatataaaa attagagaga gtggttctca tgcaacggcg 180
gacggcccgg tgctaaaaga gcttcaagac aaaataatga aacaggaaga gagtagattt 240
atctaagagc caactttatt atatgaatgt gtttattgtt ggctttagat gatatggtaa 300
ggagttagag ctaataatat ataggctcta ttattattat tattaattaa actcgctcta 360
aggaggaaag tgggaggaag ggacgaggac gaagactact ggaagcatcg tccatggatg 420
atggatgtgg tgtctcttaa tgtaggtggc cggaggatgt acgtgttaat tgcgcgataa 480
gcactcagat ccaaccgcaa actacctcca cactgacaca ctgatagaga gaaagagaga 540
cctccgacga ctgccgccgc agatgagcca cgtacgtata cgacgtctgc cggccggctc 600
aggctgccgc catcaccctg ctcgaaagtc gcgttaggcg gcgccagcta cataggagta 660
tctagtctag ccagttagta tactactact gcgctgatga tgaattaact ctgcatatat 720
actgtacatg cctccctcca acacccaacc acctcctgct cggctcttaa taacttggac 780
acggatcgat gccatccaag gaagaagacg acgacgacga cggaacatcc accatgcaag 840
cttgcatcca tacgccgata cgcgtgcatc catccatcca ccattatttc cattttccac 900
cgatcacacg tacacaggcc tatttaagga gcgacatccc actgcaactc tcctcaccac 960
tcatcaccag ctagctctag caaagcactt gccatctacc gaccgccgca ttccaaacag 1020
cccgacgagc tagcagagcg gcaggcacct ccctcctcaa ggaac 1065
<210> 4
<211> 2130
<212> DNA
<213> 玉米(Zea mays)
<400> 4
atggccactg tgaacaactg gctcgctttc tccctctccc cgcaggagct gccgccctcc 60
cagacgacgg actccacgct catctcggcc gccaccgccg accatgtctc cggcgatgtc 120
tgcttcaaca tcccccaaga ttggagcatg aggggatcag agctttcggc gctcgtcgcg 180
gagccgaagc tggaggactt cctcggcggc atctccttct ccgagcagca tcacaagtcc 240
aactgcaact tgatacccag cactagcagc acagtttgct acgcgagctc agctgctagc 300
accggctacc atcaccagct gtaccagccc accagctccg cgctccactt cgcggactcc 360
gtcatggtgg cctcctcggc cggtgtccac gacggcggtt ccatgctcag cgcggccgcc 420
gctaacggtg tcgctggcgc tgccagtgcc aacggcggcg gcatcgggct gtccatgatc 480
aagaactggc tgcggagcca accggcgccc atgcagccga gggcggcggc ggctgagggc 540
gcgcaggggc tctctttgtc catgaacatg gcggggacga cccaaggcgc tgctggcatg 600
ccacttctcg ctggagagcg cgcacgggcg cccgagagtg tatcgacgtc agcacagggt 660
ggtgccgtcg tcgtcacggc gccgaaggag gatagcggtg gcagcggtgt tgccggtgct 720
ctagtagccg tgagcacgga cacgggtggc agcggcggcg cgtcggctga caacacggca 780
aggaagacgg tggacacgtt cgggcagcgc acgtcgattt accgtggcgt gacaaggcat 840
agatggactg ggagatatga ggcacatctt tgggataaca gttgcagaag ggaaggacaa 900
actcgtaagg gtcgtcaagt ctatttaggt ggctatgata aagaggagaa agctgctagg 960
gcttatgatc ttgctgctct gaagtactgg ggtgccacaa caacaacaaa ttttccagtg 1020
agtaactacg aaaaggagct cgaggacatg aagcacatga caaggcagga gtttgtagcg 1080
tctctgagaa ggaagagcag tggtttctcc agaggtgcat ccatttacag gggagtgact 1140
aggcatcacc aacatggaag atggcaagca cggattggac gagttgcagg gaacaaggat 1200
ctttacttgg gcaccttcag cacccaggag gaggcagcgg aggcgtacga catcgcggcg 1260
atcaagttcc gcggcctcaa cgccgtcacc aacttcgaca tgagccgcta cgacgtgaag 1320
agcatcctgg acagcagcgc cctccccatc ggcagcgccg ccaagcgtct caaggaggcc 1380
gaggccgcag cgtccgcgca gcaccaccac gccggcgtgg tgagctacga cgtcggccgc 1440
atcgcctcgc agctcggcga cggcggagcc ctagcggcgg cgtacggcgc gcactaccac 1500
ggcgccgcct ggccgaccat cgcgttccag ccgggcgccg ccaccacagg cctgtaccac 1560
ccgtacgcgc agcagccaat gcgcggcggc gggtggtgca agcaggagca ggaccacgcg 1620
gtgatcgcgg ccgcgcacag cctgcaggac ctccaccact tgaacctggg cgcggccggc 1680
gcgcacgact ttttctcggc agggcagcag gccgccgccg cagctgcgat gcacggcctg 1740
gctagcatcg acagtgcgtc gctcgagcac agcaccggct ccaactccgt cgtctacaac 1800
ggcggggtcg gcgatagcaa cggcgccagc gccgttggca gcggcggtgg ctacatgatg 1860
ccgatgagcg ctgccggagc aaccactaca tcggcaatgg tgagccacga gcagatgcat 1920
gcacgggcct acgacgaagc caagcaggct gctcagatgg ggtacgagag ctacctggtg 1980
aacgcggaga acaatggtgg cggaaggatg tctgcatggg ggaccgtcgt ctctgcagcc 2040
gcggcggcag cagcaagcag caacgacaac attgccgccg acgtcggcca tggcggcgcg 2100
cagctcttca gtgtctggaa cgacacttaa 2130
<210> 5
<211> 1762
<212> DNA
<213> 人工序列()
<400> 5
tgacatctta tagtctgcaa cctctcgtgt ctgaattcct atctttatca agtgttattg 60
cttccacgac tataggacag ctttcgtcga aagcttttgc tcatgtgatc tcgaaggatt 120
catctagtct gatttttcgt gacttgtatc ggttttattg gattcatcca acatatatca 180
ataaaaaatg agttgtgttt cctttcttcc tagttcagtt aaaattattt ccctcctgcg 240
cttgtgctgt aattgtctgt gtacctgttg tttgtgactg tgttagttcc cttggatatg 300
atttcgtatt tgatatgtac atggagatag cttagcttca ttattggagt atgaagttag 360
tatgacatag tcactctcct ggaaaattga cactgcaaac catattttta ttctgaacca 420
caaatcctag tcagtccgct ggcatatgcc gtccgtttgc tgaatccaga acgtgggttt 480
ggagatgtac ggctgagatg cctctatgcg aaggggatct cgtggtgaaa cgagatggga 540
gtagagcaac gcccgtggaa gatgcttcaa acttccacac ttttgagcaa cgatcggcag 600
tagtaaggta gacgatttca agatcaaagc atatgaagat aaacaacatc aacaacaaaa 660
tttgttgggg ttctatagag agaaacagag ctacatacat acactgtttt gtatctacca 720
tctgagatga tgaaaagatg aaaaactaaa gaatgccccg gcgccaacgc caggacacgc 780
cgcgcgcgcg tcacccgagc catctcttga cccagccggc gctgtatatt tacacacgtt 840
gcagcatcga tcaccacctg ttcgatcgcg tcgccgtcac ctgacatctt atagtctgca 900
acctctcgtg tctgaattcc tatctttatc aagtgttatt gcttccacga ctataggaca 960
gctttcgtcg aaagcttttg ctcatgtgat ctcgaaggat tcatctagtc tgatttttcg 1020
tgacttgtat cggttttatt ggattcatcc aacatatatc aataaaaaat gagttgtgtt 1080
tcctttcttc ctagttcagt taaaattatt tccctcctgc gcttgtgctg taattgtctg 1140
tgtacctgtt gtttgtgact gtgttagttc ccttggatat gatttcgtat ttgatatgta 1200
catggagata gcttagcttc attattggag tatgaagtta gtatgacata gtcactctcc 1260
tggaaaattg acactgcaaa ccatattttt attctgaacc acaaatccta gtcagtccgc 1320
tggcatatgc cgtccgtttg ctgaatccag aacgtgggtt tggagatgta cggctgagat 1380
gcctctatgc gaaggggatc tcgtggtgaa acgagatggg agtagagcaa cgcccgtgga 1440
agatgcttca aacttccaca cttttgagca acgatcggca gtagtaaggt agacgatttc 1500
aagatcaaag catatgaaga taaacaacat caacaacaaa atttgttggg gttctataga 1560
gagaaacaga gctacataca tacactgttt tgtatctacc atctgagatg atgaaaagat 1620
gaaaaactaa agaatgcccc ggcgccaacg ccaggacacg ccgcgcgcgc gtcacccgag 1680
ccatctcttg acccagccgg cgctgtatat ttacacacgt tgcagcatcg atcaccacct 1740
gttcgatcgc gtcgccgtca cc 1762
<210> 6
<211> 1317
<212> DNA
<213> 玉米(Zea mays)
<400> 6
cgacccatcg ctgctttgtc tacatcatgt tcttcatcat cctccccagg cgacgcgtgc 60
tgctgttctt attcagacta ccgttcgagt gactgcatgg cgtacatctt tctgcatcga 120
ctttgtacgg ctacatcgaa catatacacg agatgtctcg tgtgaataga gtcactaatg 180
ccttaagcat cggttactcc gtagggtaca ttctgttctt cttatttgtg catattttta 240
ttgttgttta ctgattatac gagtagttat acatacatgc acatacatat catcacatat 300
atcacaatat ttttctaaat taaattaaaa ctaaaaatga ctaaatttct aacaccaacg 360
acattgtaat gttttctcca acaactttac ctattctaca ttgttctatt tcgaatttca 420
ctctataaac aacatagtct acaatggaaa acagtgcttt gtacgactat atacgcgatg 480
tgtggctaca acataagaca atatagtcgt ttgaagattg aacctatata tcggtacggt 540
taatccgtct atgtacgtgg gcatgacgaa cacccgtgat aacgaaggat taacgtgcac 600
aatcataaat ccaaagtagg agcggtgcat gatgagaatc gctctcagta ctcgacataa 660
tgaaccttac gaggtacaac aggcaggcag gcagggacca ggggccgcct ttatttcagg 720
ctcgctggcc ccacgggcgt gctgcgtgca cgaagggcac taccccaacc tctcaccgaa 780
aaaccgcgct ggatcggcaa atcaaacgag gtggtgcccc gtgcccactc tccacgtcca 840
cggcaccatc cctctgcagc cgctcaccag ccatgccgtg tcgcggaacg gcacaaccac 900
ccccaaccca ctcacgaaac cccgtcccgg ccgtgcccgt gtcggtccgc gctcggcaac 960
gaggcggccc gcgctgctga gtcccctgga cacccgacac cctgtcggcc ctttgtttat 1020
tcatcccgaa atctcatctg cccccacggc cgactgcgct gcgccgcccg gatatatata 1080
cccatcgtta tcgatcgatc gatcgcgtca ctcacgggta gctcatggtc gagcgtagca 1140
tgcaggaact tatttgccgt gcgctcccag gtctccgctc gcgtgccttc cagtctgtct 1200
cacactagct gctgtgggac gatcgaagtg ggtgtgtcag ctagctagct gcgccgtgac 1260
cacgcacatg accgcagtgc gcgcggggct gatcaaggga aagtgatcgg atggcgg 1317
<210> 7
<211> 971
<212> DNA
<213> 玉米(Zea mays)
<400> 7
ccaatgcggg cggcggtgga gcgggaggag gcagcggcag cggcagcgtg gctgcgccgg 60
cggtgtgccg ccccagcggc tcgcggtgga cgccgacgcc ggagcagatc aggatgctga 120
aggagctcta ctacggctgc ggcatccggt cgcccagctc ggagcagatc cagcgcatca 180
ccgccatgct gcggcagcac ggcaagatcg agggcaagaa cgtcttctac tggttccaga 240
accacaaggc ccgcgagcgc cagaagcgcc gcctcaccag cctcgacgtc aacgtgcccg 300
ccgccggcgc ggccgacgcc accaccagcc aactcggcgt cctctcgctg tcgtcgccgc 360
cttcaggcgc ggcgcctccc tcgcccaccc tcggcttcta cgccgccggc aatggcggcg 420
gatcggctgg gctgctggac acgagttccg actggggcag cagcggcgct gccatggcca 480
ccgagacatg cttcctgcag gactacatgg gcgtgacgga cacgggcagc tcgtcgcagt 540
ggccatgctt ctcgtcgtcg gacacgataa tggcggcggc ggcggccgcg gcgcgggtgg 600
cgacgacgcg ggcgcccgag acactccctc tcttcccgac ctgcggcgac gacgacgacg 660
acgacagcca gcccccgccg cggccgcggc acgcagtccc agtcccggca ggcgagacca 720
tccgcggcgg cggcggcagc agcagcagct acttgccgtt ctggggtgcc ggtgccgcgt 780
ccacaactgc cggcgccact tcttccgttg cgatccagca gcaacaccag ctgcaggagc 840
agtacagctt ttacagcaac agcacccagc tggccggcac cggcagccaa gacgtatcgg 900
cttcagcggc cgccctggag ctgagcctca gctcatggtg ctccccttac cctgctgcag 960
ggagcatgtg a 971
<210> 8
<211> 331
<212> DNA
<213> 人工序列()
<400> 8
cctcggtgac gtggggcaac ctagacttgt ccatcttctg gattggccaa cttaattaat 60
gtatgaaata aaaggatgca cacatagtga catgctaatc actataatgt gggcatcaaa 120
gttgtgtgtt atgtgtaatt actagttatc tgaataaaag agaaagagat catccatatt 180
tcttatccta aatgaatgtc acgtgtcttt ataattcttt gatgaaccag atgcatttca 240
ttaaccaaat ccatatacat ataaatatta atcatatata attaatatca attgggttag 300
caaaacaaat ctagtctagg tgtgttttgc g 331
<210> 9
<211> 34
<212> DNA
<213> 噬菌体P1(Bacteriophage P1)
<400> 9
ataacttcgt atagcataca ttatacgaag ttat 34
<210> 10
<211> 2001
<212> DNA
<213> 玉米(Zea mays)
<400> 10
gtggcggtct gtgccctagg cagggcgacg tgggggttcc tccgaagccg aggttgagtc 60
tgccttccgt tgccgtggcc gagcccgagc caaggggtcg ggcgaggcgg aagtcgttcg 120
gccgaggcca gggcggagtc cgagccctgg ggtcgggcga agcggagttt cgtcgtcttc 180
caggtgttag cctgagtccg agccctgggg tcgggcgagg cggagttcgc cgtcttccgg 240
gtcttagcct gagtccgagc cctggggtcg ggcgaggcgg agttcgccgt cttccgggtc 300
ttagcccgag tccgagccct ggggtcgggc ggagcggagt tcgccgtctt ccgggtctta 360
gcccgagtcc gagccctggg gtcgggcggg gcggagttcg ccgtcttccg ggtcttagcc 420
cgagtccgag ccctggggtc gggcggagcg gagttcgccg tcttccgggt tttagcccga 480
gtccgagccc tggggtcggg cggagcggag ttcgccgtct tccgggtctt agcccgagtc 540
cgagccctgg ggtcgggcgg agcggagttc gccgtggcgc ctttgacaag gcctgactgc 600
ctgtcagact cactctgtcg agtggcactg cagtcggagt ggcgcaggcg gcgctgtcct 660
tctgtcagac tggccagtgg agcggtggag tgacggcggt cacttcggct ctgccggggg 720
cgcgtgtcag gataaaggtg tcaggccacc tttgcattaa atgcccctgc aatttggtca 780
gtcggtgtgg cgatttagtc aaggttgctt ctgagcgaag ccaaggcctc gggcgagccg 840
gtgatgtgtc cgccataaaa agggggcctc gggcgagacg gaagtctctc gaggtcggct 900
gcccttggcc gaggctaggc tcgggtgaag cgtgatcgag tcactcgtgt ggactgatcc 960
ctgacttaat cgtacccatc aggcctttgc agctttatgc tgatgggggt taccagctga 1020
gaattaggcg tcttgagggt acccctaatt atggtccccg acaatgtata tgttctatat 1080
atataggttt ataatcatct atatatatga atactgatat aaaaatctat agctaaaacg 1140
attgctattt tgagatgttg agagtaattt ttaaaggagt ctaagaaata aaaggattcg 1200
gcacatttat gacggaagag acggagatat ggagtactcg gagtaatcag attcgatccc 1260
tacgcggaag cggaagggta ggtttgtgtg tgtggtggcc tcaagcaaga tatccgcacc 1320
gaagccgagc gcgaggagga aatggaaacc ctgcctccac atccggacaa cccacccacc 1380
gcacgcatag tgccagcatc cgccgtccag ctccctcgac caacggcccc gatcctctaa 1440
cttggagccc aacacgcggc ccgactgcgc accacgcacc tgccctcgcg ccgcgccgtt 1500
catgcccata tattctgccc gcctttcgtc cttttcgtcc ccatttccat tcctcgctca 1560
ccaagccaac aaccaaatcg aattcgccga cgactctctt tattcctccc gctcgccgcc 1620
gcggccgccg cctgagggca agaagcgagc gagcgcaccg cggatctccc tcccggcccc 1680
gccgcgcgca ccggtgaaga cgacgagccc ctcgtgaatg gtgggagccc gcgcgacgtc 1740
cgtcgcctcg cagccgcagc cggaggaggc ctccttccgc ggcttcggtt actgcatgca 1800
gggctggtac caccgctgca tcggtctcga cccctgaacc ccgccgccac ttgcttcttt 1860
gatttgattt cattcggttc gttcggtgcc cctcccgtcc tggatctgtt gcgcgagagc 1920
gcggcagcgc gcgagattcg cccccttctc ttctcatagg aagcaggaag caagccgatc 1980
gctcgctgag cgccgccgcc g 2001
<210> 12
<211> 2001
<212> DNA
<213> 玉米(Zea mays)
<400> 12
ccaaagcgcg aaccaagcag tcaacccaac atagttcaag caccaagaaa ttgaacccca 60
gattcaagca tcgcagtcga caaccgtgag gggaaatgca ataaggatgc ggggaggtca 120
tggcgcacta cctctcgcag ctgctcgtcg gtgagcgcgt cctcaaaggc atcctcctct 180
tcttccggcg ccctcgacgc cttccctcca gcggctgcct cctcctcctc gggcgccgct 240
gaagacgagg ggcgcgccgc gtccccggcg accgcctgct cctccgccac cggcaacgag 300
gggcgcgtcg cctcctcggg ctccagctcg atcagcacca tgtctttccc ccgagccgct 360
acctcgccga gcgcttccac ctagccgtgg tgatggcggt ggaggaggga acgtagcggc 420
tgtggcggct gcgtcgccag tggatattgc tgcccgagga tacctctggc gacgagggga 480
atcacctcga ggagatcagg gtctatggat ctactgcgta gacatccagc ggcggcggtg 540
gtaagctgga gacggagagg cggcagtgga ggcgagagag atgggattac gggattcgca 600
gagaagagga gaccgaaagg tggacgcgag gagtctcgca ggttcggggg tttgggcgga 660
cgtgaggttc ggttatgcca gagtggagta tttttttttc gggtacgcga atttcttctt 720
cgcctttgtc agcaaggtag atattacaga cgcgtgtcaa gcattttatt actatattaa 780
ccgcggcagc atagaaggct agaagctcag ctgaggtggt cacgccattt cttttaactg 840
caattgtacg atctttatcc aacggccagt taaagcggac tgatccctaa gcagcacgac 900
aggaaagttc cagagaaatc ttcatagctc cgaagcaggt ctctcagcag cagcacaaca 960
ggatcgttac agataaatct tcgtcgctct catgatccca tccctcccca catttcagcg 1020
tccacctttt attattacta tattaaccgc gggagcatag aagctcagct gaggtggcca 1080
cgccatttct tttaattgca atcatacgat ctttatccaa cggccagtta aagcggacag 1140
atccctcagc agcacgacag aaagatccag agaaatcttc atcgctccga agcaggtctc 1200
tcagtagcag cacaacggga tcgttccaga taaatcttcg tcgctctcat gatctcatcc 1260
ctccccacat ttcagcgtcc acctcaaata gccgaacgga atcccagata actaagctga 1320
ggtggccacg ccatttcttt taattgcaat cgtacaatct ttatccaacg gccagttaaa 1380
gcggacagat ccctcagcag cacgacagga aagatccaga gaaatcttca tcgctccgaa 1440
gcaggtctcc cagcagcagc acaacgggat cgttccagat aaatcttcgt tgctctcatg 1500
atcccatccc tccccacatt tcagcgtcca cctcaaatag ccgaacggaa tcccagataa 1560
ctaagattat tactatatta accgggggag catagaagct caactgaggt ggccacgcca 1620
tttcttttaa ctgcaatcgt acgatcttta tccaacggcc agttaaagcg gacagatccc 1680
tcagcaacac gacaggaaag ttccagagaa atcttcatcg ctccgaagca ggtctctcag 1740
cagcacaacg ggatcgttcc agataaatct tcgtcgctct catgatccca tccctcccac 1800
atttcagcgt ccacctcaaa tagccgaacg gaatcccaga taactaagaa atggccaccg 1860
aaaaaggaac agagcgagct atccctcacc aaattgtcaa ccaagcctgc tccccctgcc 1920
taggccacag gccagtatga taatagagaa aaagcaagca ggagggatgg gcgagtgaga 1980
tgcttcgttc acgttccagt g 2001
Claims (8)
1.植物高效遗传转化载体,所述植物为玉米,其特征在于,包括双元载体pCWBG和双元载体pBOE/pBC,其中:
双元载体pCWBG至少包含第一筛选标记基因、Bbm蛋白编码基因、Wus2蛋白编码基因和CRE蛋白编码基因的表达盒,所述CRE蛋白编码基因的表达盒由热激启动子启动表达,所述第一筛选标记基因、Bbm蛋白编码基因、Wus2蛋白编码基因和CRE蛋白编码基因的表达盒的LB和RB边界连接有CRE蛋白切割靶标序列;
双元载体pBOE/pBC为含有第二筛选标记基因表达盒的植物过量表达载体pBOE或基因编辑载体pBC,所述第二筛选标记与第一筛选标记为不同类型的筛选标记,第一筛选标记为荧光筛选标记,第二筛选标记为化学筛选标记。
2.根据权利要求1所述植物高效遗传转化载体,其特征在于,第一筛选标记为GFP或RFP,第二筛选标记为Bar或Htp。
3.根据权利要求1所述的植物高效遗传转化载体,其特征在于,所述热激启动子为玉米pZmHSR1启动子,所述CRE蛋白切割靶标序列为loxP序列。
4.根据权利要求1所述的植物高效遗传转化载体,其特征在于,所述过量表达载体包含用于插入待转化的外源目的基因的MCS多酶切位点,所述基因编辑载体包含用于插入待编辑的目的基因靶标sgRNA的多酶切位点和Cas9基因表达盒。
5.一种利用如权利要求1所述的植物高效遗传转化载体对植物进行高效遗传转化和筛选的方法,其特征在于,包括以下步骤:
(1)将待转化的外源目的基因插入植物过量表达载体中,或将待编辑的目的基因靶标sgRNA插入基因编辑载体中,构建双元载体pBOE/pBC;
(2)将双元载体pCWBG和双元载体pBOE/pBC共转化植物组织细胞,选择含有第一筛选标记和第二筛选标记的植物愈伤组织细胞为双元载体pCWBG和双元载体pBOE/pBC的共转组织;
(3)将共转组织进行高温热激处理,启动热激启动子,表达CRE蛋白,将Bbm、Wus2、GFP和CRE表达盒切除;
(4)继续培养植物愈伤组织,培养过程中利用第一筛选标记和第二筛选标记进行筛选,选择仅含有第二筛选标记的植物组织,获得转基因植株。
6.根据权利要求5所述的一种对植物进行高效遗传转化和筛选的方法,其特征在于,所述第一筛选标记为GFP,所述第二筛选标记为Bar,所述步骤(2)包括:在共培养和静息阶段用GFP荧光标记筛选转化胚和转化愈伤组织,取绿色荧光的愈伤组织转移到含有双丙氨膦筛选剂的培养基上,选择双筛存活下来的愈伤组织为双元载体pCWBG和双元载体pBOE/pBC的共转组织。
7.根据权利要求5所述的一种对植物进行高效遗传转化和筛选的方法,其特征在于,所述植物为玉米,所述热激启动子为pZmHSR1,所述步骤(3)的高温热激处理条件为42℃下高温处理2h。
8.一种如权利要求1所述的植物高效遗传转化载体在植物遗传转化和筛选中的应用,所述植物为玉米。
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