CN107760663A - 油莎草pepc基因的克隆及表达载体的构建和应用 - Google Patents
油莎草pepc基因的克隆及表达载体的构建和应用 Download PDFInfo
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Abstract
本发明公开油莎草pepc基因的克隆及表达载体的构建和应用,通过对设计特异引物、RACE PCR扩增获得全长为3399bp的pepc基因,进而首先设计合成pepc oligo DNA序列,其次构建出具有导向作用的sgRNA载体,最后通过双酶切将包含有启动子区域、pepc oligo DNA序列及sgRNA的片段切开,与含有Cas蛋白的pP1C.1进行连接,成功构建pepc基因的CRISPR/Cas9载体,此载体在对烟草叶片进行浸染后,使烟草含油率提高33.4%,为后续pepc基因载体转化产油植物,获得、培育含油率较高的转基因植株奠定了基础,对于新品种培育、生产推广具有重要作用。
Description
技术领域
本发明主要涉及分子生物学技术领域,具体地说,本发明涉及油莎草基因克隆及表达载体的技术领域。
背景技术
油莎草(Cyperus esculentus L.Var.sativus)是块茎含油类作物,莎草科单子叶草本植物。油莎草的含油量是目前油料作物中最高的,被誉为“油料作物之王”,其油酸的含量占脂肪酸含量的72.7%,与被誉为“最健康的油”—橄榄油接近。而培育含油量更高、各种脂肪酸比例更健康的油料作物新品种是作物育种的重要任务之一。根据“底物竞争假说”,植物种子中的脂肪和蛋白质竞争相同的底物-丙酮酸,其中蛋白质的合成是由PEPC羧化酶催化而脂肪的合成由乙酰CoA羧化酶催化,光合作用生成的碳是流向生成脂肪还是生成蛋白质,取决于PEPC羧化酶(PEPCase)与乙酰CoA羧化酶(ACCase)的相对活性。由此可以看出,在油料作物中,PEPC羧化酶活性的强弱直接影响了作物中油脂的含量。
构建pepc基因表达载体,有利于后续pepc基因载体转化油莎草,获得含油率较高的转基因油莎草植株,为培育含油率较高的转基因油莎草植株奠定了基础,对于新品种培育、生产推广具有重要作用。
发明内容
为了培育含油量更高、各种脂肪酸比例更健康的油莎草新品种,本发明旨在于提供一种油莎草pepc基因的克隆及表达载体构建方法,为后续pepc基因载体转化产油植物,获得含油率较高的转基因植株奠定基础。
本发明通过以下技术方案实现的:
本发明具体提供油莎草pepc基因的克隆方法,具体采用以下技术步骤:
(1)以地上茎叶生长至7~8个时的油莎草地上茎叶为材料,提取总RNA;
(2)设计特异引物:
GSP1(5’RACE):5'-GCTTGTTGAGTATGATGCCCT-3';
GSP2(3’RACE):5'-CCCTTCATGGTGAGGATAAGA-3';
(3)利用正向引物、反向引物进行RACE PCR扩增;
第一步:
扩增体系如下:样本基因组DNA2μl、10*UPM 1.5μl、5’或3’GSP(10μM)1.5μl、La-Taq0.2μl、dNTP 2μl、Mg2+2μl、Buffer 2μl、ddH2O 13.8μl,总计25μl;
扩增程序如下:
94℃预变性5min,94℃30s,56℃30s,72℃3min30s,经34个循环后,72℃延伸10min。
第二步:扩增体系如下:第一步反应物2μl、10*UPM 1.5μl、5’或3’GSP(10μM)1.5μl、La-Taq 0.2μl、dNTP 2μl、Mg2+2μl、Buffer2μl、ddH2O 13.8μl、总计25μl。
优选的,第二步中RACE PCR体系5’和3’RACE温度分别设置为54℃和58℃。
获得PEPCase酶pepc基因全长cDNA,大小为3399bp。
进一步,本发明提供油莎草PEPCase蛋白,PEPCase蛋白氨基酸的数量为965个,理论分子量及等电点分别为110129.27Da和6.07,脂肪系数为90.77,分子式C4920H7797N1353O1446S34;碱性氨基酸(Arg+Lys)为127,酸性氨基酸(Asp+Glu)为140;PEPCase蛋白的GRAVY值为-0.386,该蛋白为亲水性蛋白。PEPC羧化酶蛋白质不包含信号肽,推测油莎草PEPC羧化酶蛋白为非分泌性蛋白;利用蛋白质的二级结构在线软件预测油莎草PEPC羧化酶蛋白质,结果表明无规则卷曲(L):β-折叠(E):α-螺旋(H)为31.92:3.94:64.15,α-螺旋占较大的比例,而β-折叠所占比例较少,三级结构预测结果表明,油莎草PEPCase蛋白为同源四聚体,对其保守结构域预测结果显示油莎草PEPCase蛋白中含有PEPCase超家族保守结构域。
本发明还提供一种油莎草pepc基因的CRISPR/Cas9载体,具体采用以下技术步骤构建获得:
(1)设计Oligo DNA序列:根据pP1C.5载体的图谱,在20bp碱基序列上游加入GCAA酶切位点,下游加入CAAA酶切位点,上下游均为BbsI的酶切位点;
(2)引物退火形成双链:将已合成好的互补单链分别加入ddH2O稀释后配成母液(100μM);然后分别取20μL的互补单链进行混合,首先95℃加热3min,紧接着将温度降到40℃以下,互补单链配对结合成双链DNA;
(3)CRISPER/Cas9载体的构建:首先加入BbsI对pP1C.5载体进行酶切,回收3.2kb的片段;然后将步骤(2)获得的双链DNA与回收纯化后的3.2kb的片段进行连接、转化DH5a,根据插入位点及pP1C.5的碱基序列设计特异性引物,扩增并送生工测序;后插入Oligo DNA序列的载体即为sgRNA载体;加入EcoRI、NheI对sgRNA载体进行双酶切,切胶回收片段520bp,同时对pP1C.1载体进行EcoRI、XbaI双酶切,回收纯化线性化pP1C.1载体片段;然后将520bp的DNA片段与线性化pP1C.1载体进行连接,转化DH5a,得到油莎草pepc基因CRISPR/Cas9载体。
优选的,步骤(3)中设计的特异性引物为:
p5-F:5'-TTATATGGGAAAGAACAATAGTATT-3';
p5-R:5'-TGCAGAATTCGAAGCTTGAG-3'。
同时,本发明还提供上述的油莎草pepc基因的CRISPR/Cas9载体在提高含油量转基因植物中的应用。
优选的,油莎草pepc基因的CRISPR/Cas9载体在提高含油量转基因植物方面的应用在转化时采用农杆菌介导法。
更优选的,油莎草pepc基因的CRISPR/Cas9载体在提高含油量转基因植物方面的应用转化时采用农杆菌菌株EHA105。
进一步的,油莎草pepc基因的CRISPR/Cas9载体在提高含油量转基因植物方面的应用转化时油莎草愈伤组织的诱导方法,具体包括以下技术步骤:
(1)消毒:选取40粒无虫眼,无伤疤的新鲜油莎豆块茎,清洗干净,再水流冲洗5-6h,水浴锅中40℃25min,摇床24h震荡至油莎草块茎表面的土及其他杂质冲洗完全,然后用无菌滤纸吸干油莎草块茎表面水分,75%的乙醇浸泡消毒30s,0.1%的HgCl2浸泡消毒20min。
(2)愈伤组织形成:将消毒过的新鲜油莎草块茎置于灭过菌的无菌滤纸上,将带有芽的块茎切下,切成长度0.5cm大小,快速接种于培养基上,避光培养2周后进行光照培养,培养温度37℃。
优选的,步骤(2)采用培养基的配方为MS+2,4-D 1.6+6-BA 1.0。
通过实施本发明的技术方案,可以达到以下有益效果:
(1)本发明通过提供一种油莎草pepc基因的克隆方法及表达载体,为后续pepc基因载体转化油莎草,获得、培育含油率较高的转基因油莎草植株奠定了基础,对于新品种培育、生产推广具有重要作用。
(2)本发明提供的油莎草pepc基因的CRISPR/Cas9载体在对烟草叶片进行浸染瞬时转化后,烟草含油率可达26.53%,相比于对照组含油率提高33.4%,并进行油莎草愈伤组织的诱导,成功诱导出愈伤组织。
附图说明
图1显示为油莎草pepc基因的CRISPER/Cas9载体的构建流程图。
图2显示为油莎草pepc基因的CRISPR/Cas9载体在对烟草叶片进行浸染后的烟草含油率。
图3显示为油莎草块茎诱导出的愈伤组织。
具体实施方式
下面,举实施例说明本发明,但是,本发明并不限于下述的实施例。
本发明采用的缓冲液、剪切酶、Cas9载体等试剂均可通过公共渠道购买,工艺中所采用的设备和仪器均为本领域常见的设备。
本发明中选用的所有材料、试剂和仪器都为本领域熟知的,但不限制本发明的实施,其他本领域熟知的一些试剂和设备都可适用于本发明以下实施方式的实施。
实施例一:油莎草pepc基因的克隆方法和PEPCase蛋白
本发明具体提供油莎草pepc基因的克隆方法,具体采用以下技术步骤:
(3)以地上茎叶生长至7~8个时的油莎草地上茎叶为材料,提取总RNA;
(4)设计特异引物:
GSP1(5’RACE):5'-GCTTGTTGAGTATGATGCCCT-3';
GSP2(3’RACE):5'-CCCTTCATGGTGAGGATAAGA-3';
(3)利用正向引物、反向引物进行RACE PCR扩增;
第一步:
扩增体系如下:样本基因组DNA2μl、10*UPM 1.5μl、5’或3’GSP(10μM)1.5μl、La-Taq0.2μl、dNTP 2μl、Mg2+2μl、Buffer2μl、ddH2O 13.8μl,总计25μl;
扩增程序如下:
94℃预变性5min,94℃30s,56℃30s,72℃3min30s,经34个循环后,72℃延伸10min。
第二步:扩增体系如下:第一步反应物2μl、10*UPM 1.5μl、5’或3’GSP(10μM)1.5μl、La-Taq 0.2μl、dNTP 2μl、Mg2+2μl、Buffer2μl、ddH2O 13.8μl、总计25μl。
优选的,第二步中RACE PCR体系设置5’和3’RACE温度分别为54℃和58℃。
获得PEPCase酶pepc基因全长cDNA,大小为3399bp。
本发明提供油莎草PEPCase蛋白,PEPCase蛋白氨基酸的数量为965个,理论分子量及等电点分别为110129.27Da和6.07,脂肪系数为90.77,分子式C4920H7797N1353O1446S34;碱性氨基酸(Arg+Lys)为127,酸性氨基酸(Asp+Glu)为140;PEPCase蛋白的GRAVY值为-0.386,该蛋白为亲水性蛋白。PEPC羧化酶蛋白质不包含信号肽,推测油莎草PEPC羧化酶蛋白为非分泌性蛋白;利用蛋白质的二级结构在线软件预测油莎草PEPC羧化酶蛋白质,结果表明无规则卷曲(L):β-折叠(E):α-螺旋(H)为31.92:3.94:64.15,α-螺旋占较大的比例,而β-折叠所占比例较少,三级结构预测结果表明,油莎草PEPCase蛋白为同源四聚体,对其保守结构域预测结果显示油莎草PEPCase蛋白中含有PEPCase超家族保守结构域。
实施例二:油莎草pepc基因CRISPR/Cas9载体的构建方法
本发明提供油莎草pepc基因CRISPR/Cas9载体的构建方法,具体采用以下技术步骤:
(4)设计Oligo DNA序列:根据pP1C.5载体的图谱,在20bp碱基序列上游加入GCAA酶切位点,下游加入CAAA酶切位点,上下游均为BbsI的酶切位点;
(5)引物退火形成双链:将已合成好的互补单链分别加入ddH2O稀释后配成母液(100μM);然后分别取20μL的互补单链进行混合,首先95℃加热3min,紧接着将温度降到40℃以下,互补单链配对结合成双链DNA;
(6)CRISPER/Cas9载体的构建:首先加入BbsI对pP1C.5载体进行酶切,回收3.2kb的片段;然后将步骤(2)获得的双链DNA与回收纯化后的3.2kb的片段进行连接、转化DH5a,根据插入位点及pP1C.5的碱基序列设计特异性引物,扩增并送生工测序;后插入Oligo DNA序列的载体即为sgRNA载体;加入EcoRI、NheI对sgRNA载体进行双酶切,切胶回收片段520bp,同时对pP1C.1载体进行EcoRI、XbaI双酶切,回收纯化线性化pP1C.1载体片段;然后将520bp的DNA片段与线性化pP1C.1载体进行连接,转化DH5a,得到油莎草pepc基因CRISPR/Cas9载体。油莎草pepc基因的CRISPER/Cas9载体的构建流程见图1。
优选的,步骤(3)中设计的特异性引物为:
p5-F:5'-TTATATGGGAAAGAACAATAGTATT-3';
p5-R:5'-TGCAGAATTCGAAGCTTGAG-3'。
实施例三:油莎草pepc基因的CRISPR/Cas9载体在转基因植物方面的应用实验
将构建好的pepc基因CRISPR/Cas9载体通过注射渗透转入农杆菌菌株EHA105,调整EHA105菌液浓度使其OD600值达到0.6,通过注射法对生长7周的烟草叶片进行浸染,25℃培养25天,测定烟草含油率,结果见图2。
由图可知,对照组的烟草含油率为19.89%,试验组的含油率为26.53%,相较于未进行pepc基因CRISPR/Cas9载体转化的烟草叶,实验组的含油率提高33.4%,可知本发明提供的油莎草pepc基因CRISPR/Cas9载体对提高植株含油率有明显效果。
实施例四:油莎草愈伤组织的诱导
油莎草pepc基因的CRISPR/Cas9载体在提高含油量转基因植物方面的应用转化时进行油莎草愈伤组织的诱导,具体包括以下技术步骤:
(1)消毒:选取40粒无虫眼,无伤疤的新鲜油莎豆块茎,清洗干净,再水流冲洗5-6h,水浴锅中40℃25min,摇床24h震荡至油莎草块茎表面的土及其他杂质冲洗完全,然后用无菌滤纸吸干油莎草块茎表面水分,75%的乙醇浸泡消毒30s,0.1%的HgCl2浸泡消毒20min。
(2)愈伤组织形成:将消毒过的新鲜油莎草块茎置于灭过菌的无菌滤纸上,将带有芽的块茎切下,切成长度0.5cm大小,快速接种于配方为MS+2,4-D 1.6+6-BA 1.0的培养基上,避光培养2周后进行光照培养,培养温度37℃。
油莎豆块茎在MS+6-BA 1.0+2,4-D 1.6的诱导培养基上5天后逐渐开始膨大,20天后成功诱导出愈伤组织,如附图3所示。
综上所述,本发明通过提供一种油莎草pepc基因的克隆方法及表达载体,为后续pepc基因载体转化油莎草,获得、培育含油率较高的转基因油莎草植株奠定了基础,对于新品种培育、生产推广具有重要作用;此外,本发明提供的油莎草pepc基因的CRISPR/Cas9载体在对烟草叶片进行浸染后,烟草含油率可达26.53%,相比于对照组含油率提高33.4%,并进行油莎草愈伤组织的诱导,成功诱导出愈伤组织。
如上所述,即可较好地实现本发明,上述的实施例仅仅是对本发明的优选实施方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种改变和改进,均应落入本发明确定的保护范围内。
Claims (10)
1.油莎草pepc基因的克隆方法,其特征在于,具体采用以下技术步骤:
(1)以地上茎叶生长至7~8个时的油莎草地上茎叶为材料,提取总RNA;
(2)设计特异引物:
GSP1(5’RACE):5'-GCTTGTTGAGTATGATGCCCT-3';
GSP2(3’RACE):5'-CCCTTCATGGTGAGGATAAGA-3';
(3)利用正向引物、反向引物进行RACE PCR扩增;
第一步:
扩增体系如下:样本基因组DNA2μl、10*UPM 1.5μl、5’或3’GSP(10μM)1.5μl、La-Taq0.2μl、dNTP 2μl、Mg2+2μl、Buffer 2μl、ddH2O 13.8μl,总计25μl;
扩增程序如下:
94℃预变性5min,94℃30s,56℃30s,72℃3min30s,经34个循环后,72℃延伸10min。
第二步:扩增体系如下:第一步反应物2μl、10*UPM 1.5μl、5’或3’GSP(10μM)1.5μl、La-Taq 0.2μl、dNTP 2μl、Mg2+2μl、Buffer2μl、ddH2O 13.8μl、总计25μl。
2.如权利要求1所述的油莎草pepc基因的克隆方法,其特征在于,所述的第二步扩增体系的5’和3’RACE温度分别设置为54℃和58℃。
3.油莎草PEPCase蛋白,其特征在于,PEPCase蛋白氨基酸的数量为965个,理论分子量及等电点分别为110129.27Da和6.07,脂肪系数为90.77,分子式C4920H7797N1353O1446S34;碱性氨基酸(Arg+Lys)为127,酸性氨基酸(Asp+Glu)为140;PEPCase蛋白的GRAVY值为-0.386,该蛋白为亲水性蛋白。PEPC羧化酶蛋白质不包含信号肽,推测油莎草PEPC羧化酶蛋白为非分泌性蛋白;利用蛋白质的二级结构在线软件预测油莎草PEPC羧化酶蛋白质,结果表明无规则卷曲(L):β-折叠(E):α-螺旋(H)为31.92:3.94:64.15,α-螺旋占较大的比例,而β-折叠所占比例较少,三级结构预测结果表明,油莎草PEPCase蛋白为同源四聚体,对其保守结构域预测结果显示油莎草PEPCase蛋白中含有PEPCase超家族保守结构域。
4.油莎草pepc基因的CRISPR/Cas9载体,其特征在于,具体采用以下技术步骤构建:
(1)设计Oligo DNA序列:根据pP1C.5载体的图谱,在20bp碱基序列上游加入GCAA酶切位点,下游加入CAAA酶切位点,上下游均为BbsI的酶切位点;
(2)引物退火形成双链:将已合成好的互补单链分别加入ddH2O稀释后配成母液(100μM);然后分别取20μL的互补单链进行混合,首先95℃加热3min,紧接着将温度降到40℃以下,互补单链配对结合成双链DNA;
(3)CRISPER/Cas9载体的构建:首先加入BbsI对pP1C.5载体进行酶切,回收3.2kb的片段;然后将步骤(2)获得的双链DNA与回收纯化后的3.2kb的片段进行连接、转化DH5a,根据插入位点及pP1C.5的碱基序列设计特异性引物,扩增并送生工测序;后插入Oligo DNA序列的载体即为sgRNA载体;加入EcoRI、NheI对sgRNA载体进行双酶切,切胶回收片段520bp,同时对pP1C.1载体进行EcoRI、XbaI双酶切,回收纯化线性化pP1C.1载体片段;然后将520bp的DNA片段与线性化pP1C.1载体进行连接,转化DH5a,得到油莎草pepc基因CRISPR/Cas9载体。
5.如权利要求4所述的油莎草pepc基因的CRISPR/Cas9载体,其特征在于,所述的步骤(3)中设计的特异性引物为:
p5-F:5'-TTATATGGGAAAGAACAATAGTATT-3';
p5-R:5'-TGCAGAATTCGAAGCTTGAG-3'。
6.如权利要求4或5所述的油莎草pepc基因的CRISPR/Cas9载体在提高含油量转基因植物中的应用。
7.如权利要求6所述的油莎草pepc基因的CRISPR/Cas9载体在提高含油量转基因植物中的应用,其特征在于,所述的应用在转化时采用农杆菌介导法。
8.如权利要求6所述的油莎草pepc基因的CRISPR/Cas9载体在提高含油量转基因植物中的应用,其特征在于,所述的应用转化时采用农杆菌菌株EHA105。
9.如权利要求6所述的油莎草pepc基因的CRISPR/Cas9载体在提高含油量转基因植物方面的应用转化时油莎草愈伤组织的诱导方法,其特征在于,所述的油莎草愈伤组织的诱导方法具体包括以下步骤:
(1)消毒:选取40粒无虫眼,无伤疤的新鲜油莎豆块茎,清洗干净,再水流冲洗5-6h,水浴锅中40℃25min,摇床24h震荡至油莎草块茎表面的土及其他杂质冲洗完全,然后用无菌滤纸吸干油莎草块茎表面水分,75%的乙醇浸泡消毒30s,0.1%的HgCl2浸泡消毒20min;
(2)愈伤组织形成:将消毒过的新鲜油莎草块茎置于灭过菌的无菌滤纸上,将带有芽的块茎切下,切成长度0.5cm大小,快速接种于培养基上,避光培养2周后进行光照培养,培养温度37℃。
10.如权利要求9所述的油莎草pepc基因的CRISPR/Cas9载体在提高含油量转基因植物方面的应用转化时油莎草愈伤组织的诱导方法,其特征在于,步骤(2)所述的培养基的配方为MS+2,4-D 1.6+6-BA 1.0。
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