CN102165065B - 产生具有高淀粉含量和产量以及高直链淀粉/支链淀粉比例的转基因植物的方法 - Google Patents
产生具有高淀粉含量和产量以及高直链淀粉/支链淀粉比例的转基因植物的方法 Download PDFInfo
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Abstract
一种用于产生具有高淀粉含量和产量以及高直链淀粉/支链淀粉比例的转基因植物的方法。α-1,4-糖原磷酸酶(GP)催化多聚葡聚糖例如淀粉、麦芽糖糊精和糖原非还原性末端的α-1,4键的可逆切割,导致生成葡萄糖-1-磷酸(G1P)。细菌和动物细胞中的GP负责降解糖原。尽管GP活性的提高导致细菌和动物细胞中糖原的细胞内水平的降低,但是本发明公开了通过表达编码GP的基因,产生了具有高淀粉水平和产量以及高直链淀粉/支链淀粉比例的植物。
Description
技术领域
本发明属于遗传工程和植物生理学领域。具体地,本发明包括产生具有高淀粉水平以及高直链淀粉/支链淀粉比例的转基因植物的方法,用于转化细胞的载体,转化的细胞本身,通过该方法获得的转基因植物和它们的用途。
背景技术
植物中的淀粉和细菌以及动物中的糖原都是通过α-1,4和α-1,6型共价键连接的葡萄糖分子的支化均聚物。这些聚合物是重要的糖类和能量储存形式。在植物中,淀粉是在质体中合成的,淀粉大量积聚在诸如种子(小麦、大麦、玉米、豌豆等)和块茎(其中有马铃薯和甘薯)的组织中,并且是人类饮食的基本组成成分。另一方面,淀粉常常用于造纸、化妆品、医药和食品工业中,以及作为基本的原材料用于制造可生物降解的塑料,具有低环境压力的颜料以及生物乙醇。许多淀粉应用是主要基于直链淀粉和支链淀粉的平衡,这决定了淀粉颗粒的结构和它在水悬浮液中的粘度。
ADPG是在植物和细菌中分别用于生物合成淀粉和糖原的通用前体分子。普遍猜测该核苷酸糖的产生仅仅由酶ADPG焦磷酸化酶(AGPase)(EC 2.7.7.27)控制(1-4)。然而,有证据显示蔗糖合成酶(EC 2.4.1.13)(UDP-葡萄糖:D-果糖-2-葡萄糖基转移酶)参与ADPG的合成,ADPG对于淀粉生物合成而言是必要的(5-9)。如果考虑到ADPG降解机制,则在植物(10-13)和细菌(14,15)中都存在ADPG水解酶。
在植物中,淀粉降解是由α-淀粉酶、β-淀粉酶和α-葡糖苷酶催化的水解反应控制的(16,17)。相反,在动物、细菌和酵母中,则是α-1,4-葡聚糖磷酸酶(GP)(EC 2.4.1.1)控制糖原分子在体内的降解(18-22)。GP催化非还原性多聚葡聚糖例如淀粉、麦芽糖糊精和糖原末端的α-1,4键的可逆切割,导致生成葡萄糖-1-磷酸(GlP)。动物、植物或细菌来源的编码具有GP活性的酶的核苷酸序列是保守序列。
植物具有质体内和质体外GP。与在细菌、酵母和动物中所发生的不同,迄今为止植物GP的作用还未知,尤其是质体内的那些。考虑到GP的可逆性,有可能它们在淀粉代谢中的作用依赖于Pi/GlP平衡(21)。因此植物细胞中现有的高Pi/GlP平衡强有力地显示质体GP并不参与淀粉合成,增强了淀粉合成仅仅通过依赖于ADPG产生的方法的理论。
有证据暗示质体GP可能参与Phaseoulus vulgaris和拟南芥(Arabidopsis thaliana)叶子中的淀粉降解(23)。另一方面,已经暗示质体GP具有与非生物胁迫、开花和种子发育相关的功能(24-27)。在考虑到质体GP缺陷的海藻莱茵衣藻(Chlamydomonas reinhardtii)的STA4突变体淀粉水平降低时,关于植物中质体GP的生物功能的讨论进一步复杂化,其暗示质体GP并不参与莱茵衣藻中的淀粉降解(28)。另一方面,有研究显示质体GP在块茎植物例如马铃薯和小麦中不参与淀粉降解(29,30)。这为讨论质体GP可能参与淀粉降解增添了更多困惑,尽管一些研究已经显示了正相关性。
存在一些工作描述了具有低GP活性的植物的产生和表征(24)。这些植物积聚正常水平的淀粉。另一方面,有工作描述了编码GP的大肠杆菌glgP基因的过表达,得到了糖原水平降低或为零的细菌(22)。最后,有一些工作描述了编码哺乳动物GP的基因的过表达得到了糖原水平降低的人细胞(18)。现有技术没有提供在植物GP酶和它的具体功能之间的明确联系。事实上,迄今为止还没有关于具有高GP活性的更高级植物品种的产生和/或鉴定的已知出版物。
本发明精确地集中在该最后一点,描述了具有高GP活性(细胞质的和质体的)的块茎转基因植物,其特征在于,表达编码具有GP活性的蛋白质/酶的基因,获得高GP活性(细胞质的和质体的),并因而得到高淀粉水平和性能,高直链淀粉/支链淀粉平衡。考虑的另一个重要方面是本发明显示编码GP酶的基因的表达,因此提高了GP活性,导致淀粉含量提高。
因此,本发明打破了现有技术中针对动物、细菌和酵母GP活性所建立的偏见,其限定了GP活性倾向于切割葡萄糖聚合物的α-1,4键而产生GlP。这还显示细菌GP在表达编码该酶的基因的转基因植物中促进淀粉的合成。
因此,本发明的目的是产生具有高淀粉含量和产量以及高直链淀粉/支链淀粉平衡的植物,作为GP活性(细胞质中的GP活性和质体中的GP活性)提高的结果以表达编码具有GP活性的蛋白质的基因。
发明内容
如上所述,本发明的目的是产生具有高淀粉含量和产量以及高直链淀粉/支链淀粉平衡的植物,作为GP活性(细胞质中的和质体中的)提高的结果以表达编码具有高GP活性的蛋白质的基因。为了本发明的目的,注解下列术语:
细胞:所有生物能够自主活动的最小单位(形态的和功能的)。细菌和植物细胞对于本发明是特别感兴趣的。
细胞质:构成细胞内基质的液体溶液。
质体:植物细胞特有的细胞器。其负责光合作用真核生物的光合作用。
遗传载体:用于将外源遗传材料转入细胞内部的“媒介”。本领域中已知的任何载体都可以用于本发明中。然而,在本发明中,优选使用根癌土壤杆菌(Agrobacterium tumefaciens)。
同源序列:几乎一致的DNA核苷酸序列,包括能够在严格条件下发生碱基配对。
异位表达:基因的异位表达是指它的产物在通常不发生表达的位置被表达。
转基因植物:基因组被遗传改造的植物为了实现与野生型植物的生物特征不同的生物特征(如本发明的情况)。
GP酶的高活性:当活性是野生型植物中所存在的活性的至少5倍时,成为高GP酶活性。
高淀粉含量:在本文中所使用的,这种表达方式直接指的是比在对照植物中所观察到的数值高的统计学上显著的数值。图7显示在所分析的野生型植物(CR1和CR2)的块茎中平均淀粉含量大约为290微摩尔葡萄糖/g(鲜重),偏差幅度为10%。因此,“高淀粉含量”可以被认为是超过290微摩尔葡萄糖/g(鲜重)数值的至少20%。在本发明中超过该数值,获得了转基因块茎,其积聚了最少400微摩尔葡萄糖/g(鲜重)的淀粉量。
High直链淀粉/支链淀粉比例:如图9中所示,按照淀粉酶的百分比表达的直链淀粉/支链淀粉比例,在所分析的野生型植物(CR)的块茎中为大约22.5%,误差幅度为2.5%(其构成了在CR中观察到的平均数值的10%)。当所分析的转基因植物的直链淀粉百分比数值比在相应的野生型植物中直链淀粉百分比数值大至少10%时,认为存在高直链淀粉/支链淀粉比例(在这种情况中,直链淀粉/支链淀粉比例大于25%被认为是富含直链淀粉)。
附图说明
图1:表达质粒pET15b-glgP的构建阶段。
图2:表达质粒pBIN20-B33-LCA-GP-NOS的构建阶段。.
图3:表达质粒pBIN2035S-GP-NOS的构建阶段。
图4:SDS-PAGE。从用pET15b-glgP(CECT 7071)转化的大肠杆菌BL21(DE3)C43的细胞提取物纯化的重组GP的染色。
图5:未转换马铃薯植物(品系1)块茎的蛋白质印迹和来自转化CECT 7054(品系2)和CECT 7055(品系3和4)的马铃薯植物的不同克隆的块茎的蛋白质印迹。在每个泳道中,上样50g蛋白质,并且进行SDS-PAGE。使用GP大肠杆菌特异性多克隆抗体对大肠杆菌的GP进行免疫标记。注意,只有表达大肠杆菌glgP的品系才产生了大概93kDa的条带。
图6:野生型马铃薯植物和通过分别使用根癌土壤杆菌(A.tumefaciens)CECT 7054和CECT 7055菌株向其基因组中整合35S-glgP-NOS和B33-ChlTP-glgP-NOS构建体之后表达大肠杆菌glgP的块茎中的GP活性。活性指产自糖原的GlP的量比块茎粗提物鲜重(每miliU/g鲜重)。所分析的两种野生型植物被标记为CR1和CR2。转基因植物被标记为B33-7、B33-11、B33-12、B33-13和B33-14(使用CECT 7055获得的那些)以及35S-1、35S-6、35S-11和35S-14(使用CECT 7054获得的那些)。所表示的数值对应每个品系10株不同植物的块茎的标准平均值和偏差。
图7:野生型马铃薯植物和通过分别使用根癌土壤杆菌(A.tumefaciens)CECT 7054和CECT 7055菌株向其基因组中整合35S-glgP-NOS和B33-ChlTP-glgP-NOS构建体之后表达大肠杆菌glgP的块茎中的淀粉含量。所分析的两种野生型植物被标记为CR1和CR2。转基因植物被标记为B33-7、B33-11、B33-12、B33-13和B33-14(使用CECT7055获得的那些)以及35S-1、35S-6、35S-11和35S-14(使用CECT 7054获得的那些)。所表示的数值对应每个品系10株不同植物的块茎的标准平均值和偏差。
图8:野生型马铃薯植物和通过分别使用根癌土壤杆菌(A.tumefaciens)CECT 7054和CECT 7055菌株向其基因组中整合35S-glgP-NOS和B33-ChlTP-glgP-NOS构建体之后表达大肠杆菌glgP的块茎中的蔗糖(A)、葡萄糖(B)和果糖(C)的含量。所分析的两种野生型植物被标记为CR1和CR2。转基因植物被标记为B33-7、B33-11、B33-12、B33-13和B33-14(使用CECT 7055获得的那些)以及35S-1、35S-6、35S-11和35S-14(使用CECT 7054获得的那些)。所表示的数值对应每个品系10株不同植物的块茎的标准平均值和偏差。
图9:野生型马铃薯植物和通过分别使用根癌土壤杆菌(A.tumefaciens)CECT 7054和CECT 7055菌株向其基因组中整合35S-glgP-NOS和B33-ChlTP-glgP-NOS构建体之后表达大肠杆菌glgP的块茎中的直链淀粉/支链淀粉比例,表达为直链淀粉百分比。所分析的两种野生型植物被标记为CR1和CR2。转基因植物被标记为B33-7、B33-11、B33-12、B33-13和B33-14(使用CECT 7055获得的那些)以及35S-1、35S-6、35S-11和35S-14(使用CECT 7054获得的那些)。所表示的数值对应每个品系10株不同植物的块茎的标准平均值和偏差。
发明的详细描述
获得和纯化活性重组GP
大肠杆菌glgP的核苷酸序列是已知的(22),形成两条特异性引物(SEQID NO:1和SEQ ID NO:2),对应基因的5’端和3’端。通过使用这些引物,利用传统的PCR方法从大肠杆菌的基因组DNA扩增大约2460碱基对的DNA片段。将该DNA片段引入质粒pGemT-easy(Promega)中,得到pG-glgP构建体(图1),将其在XL1蓝色宿主细菌中进行扩增。使用限制性酶XhoI和BamHI消化PG-glgP。将所释放的片段(含有glgP)克隆入表达质粒pET-15b(+)(Novagen)的相同限制性位点。将所得到标记为名称pET15b-glgP的质粒(图1)通过电穿孔引入菌株大肠杆菌BL21(DE3)C43(Novagen)中,保藏号为CECT 7071。通过向在37℃下培养的100ml细胞培养物中添加1mM异丙基-硫代半乳糖苷(IPTG)来实现glgP表达。在诱导培养六小时后,收获细菌并重悬浮在4ml结合缓冲液(Novagen,His-结合纯化试剂盒)中,超声处理并在40,000g下离心20分钟。将含有在N-末端具有组氨酸标签的重组GP的上清液通过Novagen″His-结合″蛋白质纯化试剂盒的亲和柱。根据试剂盒的指导,用6ml推荐的洗脱缓冲液对GP进行洗脱,所述推荐的洗脱缓冲液含有200mM咪唑而不是1摩尔。在洗脱后,将蛋白质快速进行透析以除去所有痕量的可能会不可逆地使GP失活的咪唑。
可溶性糖和淀粉含量的确定
使用科学文献中描述的技术提取可溶性糖(34,35)。使用配合PA10CarboPac株、ED50电化学检测仪、GP50 E1梯度泵和E01洗脱剂组织器的DIONEX自动离子层析检测蔗糖、麦芽糖、麦芽三糖、麦芽四糖、麦芽五糖、麦芽六糖、麦芽七糖、果糖和葡萄糖(22)。使用配合Partisil-10-SAX柱的HPLC Waters系统测定ADPG(10)。使用文献中描述的商业试剂盒测量淀粉、糖原和支链淀粉(36)。使用文献中描述的分光光度计法测定直链淀粉/支链淀粉比例(36)。
鉴定具有GP酶活性的产物
通过下列功能特征鉴定鉴定GP酶产物:
-它是α-1,4-糖原磷酸酶(EC 2.4.1.1)。通常,该酶催化葡萄糖分子(支化或非支化)通过α-1,4和α-1,6键共价连接的均聚多糖,例如麦芽糖糊精、淀粉和糖原的非还原端的α-1,4键的可逆磷酸分解,导致GlP的产生。
获得特异性GP大肠杆菌多克隆抗体
在SDS-PAGE上分离两毫克纯化的重组大肠杆菌GP。洗脱后,将纯化的重组GP与弗氏完全佐剂混合(以50/50比值),然后等分成三份相同的部分,将每个部分以两周的周期注射入兔子内。在首次注射后大约两周之后,从兔子中提取含有针对大肠杆菌GP特异性的多克隆抗体的血清。
通过蛋白质印迹技术鉴定产物
在SDS-PAGE上分离野生型植物以及表达编码大肠杆菌GP的glgP基因的转基因植物的块茎的粗提取物。随后,将它们转移至硝酸纤维素膜,并且根据文献中描述的方法使用抗大肠杆菌GP的特异性抗体检测大肠杆菌GP(37)。
获得表达大肠杆菌glgP的转基因植物
a.在块茎的淀粉质体(amyloplasts)中具有高GP活性的马铃薯植物
利用酶NcoI和BamHI消化质粒pG-glgP-NcoI(与pG-glgP相同,只是质粒pG-glgP-NcoI在ATG翻译起始密码子处具有NcoI位点)。将所释放的片段克隆入pSK-B33-SuSy-NOS的NcoI和BamHI位点(B33是编码patatin的基因的启动子区域,patatin基因的表达是块茎特异性的)(38),得到了质粒pSK-B33-glgP-NOS(图2)。
为了产生编码定位在质体中的大肠杆菌的GP的基因构建体,利用NcoI和BamHI消化pG-glgP-NcoI。将所释放的片段克隆在质粒pSK-ChlTP-ASPP的NcoI和BamH位点之间,质粒pSK-ChlTP-ASPP含有编码蛋白质P541的叶绿体转运肽的基因的区域,得到了质粒pSK-ChlTP-glgP(图2)。
为了产生编码特异性积聚在马铃薯块茎的淀粉质体中的大肠杆菌的GP的构建体,连续用SalI、T4-DNA聚合酶和BamHI消化pSK-ChlTP-glgP。在已经连续用NcoI、T4-DNA聚合酶和BamHI消化后,将所释放的片段克隆入pSK-B33-glgP-NOS,得到了质粒pSK-B33-ChlTP-glgP-NOS(图2)。
为了产生对于通过根癌土壤杆菌(A.tumefaciens)转化植物而言必要的含有基因构建体B33-ChlTP-NOS-glgP的双向质粒,依次用NotI、T4 DNA聚合酶和XhoI消化psK-B33-ChlTP-glgP-NOS。将所释放的片段克隆入双向质粒pBIN20(40),所述双向质粒pBIN20之前被依次用HpaI、T4 DNA聚合酶和XhoI消化,得到质粒pBIN20-B33-ClP-GP-NOS(图2)。通过电穿孔将PBIN20-B33-ClP-GP-NOS引入不同的根癌土壤杆菌菌株内,这些根癌土壤杆菌对于转化物种例如马铃薯、拟南芥、番茄、烟草、玉米和水稻而言是必要的(例如,菌株C58:GV2260,保藏号CECT 7055)。将CECT7055用于根据该物种的常规转化规程转化马铃薯植物(41)。
b.组成型表达glgP以及具有高细胞质GP活性的植物
为了产生具有高细胞质GP活性的植物,形成大肠杆菌glgP基因构建体,它们的表达由烟草花叶病毒的组成型启动子35S控制。为此,用XhoI和NcoI消化质粒pSK-35S。将所释放的片段(含有结合至促进区的启动子35S(42))克隆在质粒pSK-B33-glgP-NOS的XhoI和NcoI位点,得到质粒pSK-35S-glgP-NOS(图3)。为了将该构建体通过根癌土壤杆菌转至植物基因组,必须要之前将该构建体克隆在双向质粒。为此,依次用酶NotI,T4DNA聚合酶和XhoI消化pSK-35S-glgP-NOS。将释放的片段克隆入双向质粒pBIN20,其之前已经被连续用酶HpaI,T4 DNA聚合酶和XhoI进行消化。将所获得的质粒标记名称为pBIN2035S-GP-NOS(图3)。通过电穿孔将pBIN2035S-GP-NOS引入不同的根癌土壤杆菌菌株内(例如菌株C58:GV2260,保藏号为CECT 7054),这些根癌土壤杆菌对于转化物种例如马铃薯、拟南芥、番茄、烟草、玉米和水稻而言是必要的。
因此,本发明的第一目的涉及一种获得转基因植物的方法,与未转化的野生型植物相比,所述转基因植物具有高GP活性,高淀粉含量和产量以及高直链淀粉/支链淀粉比例,其特征在于,利用包含动物、植物或细菌来源的核苷酸序列的表达载体转化野生型植物,所述核苷酸序列编码具有高GP活性的蛋白质并且在经转化的植物中表达所述蛋白质。
在优选的实施方式中,本发明方法的特征在于,在所述经转化的植物中编码和表达的蛋白质的GP活性是所述野生型动物的GP活性的至少5倍,所产生的转基因植物的淀粉含量比未转化的野生型植物的淀粉含量高至少20%,所述转基因植物的直链淀粉含量比未转化的野生型植物的直链淀粉含量高至少10%,在所有情况下未经转化的野生型植物与转基因植物在相同的条件下生长。
在另一个优选的实施方式中,本发明方法的特征在于,用于转化所述野生型植物的表达载体中所包含的核苷酸序列选自下列:
a.编码特征为SEQ ID NO:4的氨基酸序列的核苷酸序列;
b.特征为SEQ ID NO:3的核苷酸序列;
c.与“a”或“b”中所限定的核苷酸序列杂交并且编码具有GP活性的酶产物的核苷酸序列;
d.由于遗传密码子简并性而与“a”、“b”或“c”中所限定的核苷酸序列不同的核苷酸序列。
在另一个优选的实施方式中,本发明方法的特征在于,在所述转化的转基因植物内,通过利用包含质粒pBIN2035S-GP-NOS的根癌土壤杆菌CECT 7054转化所述野生型植物能够达到在细胞质水平的高GP活性,以及通过利用包含质粒pBIN20-B33-ClP-GP-NOS的根癌土壤杆菌CECT7055转化所述野生型植物达到在质体水平的高GP活性。
本发明的第二方面涉及表达载体:
-根癌土壤杆菌CECT 7054,其特征在于包含质粒pBIN2035S-GP-NOS
-根癌土壤杆菌CECT 7055,其特征在于包含质粒pBIN20-B33-ClP-GP-NOS。
本发明的第三个目的涉及被上述两种载体转化或者感染的细胞,所述细胞是细菌或植物细胞。
在优选的实施方式中,本发明的细菌细胞的特征在于是大肠杆菌BL21(DE3)C43的细菌细胞,其被利用表达编码重组GP蛋白质的基因的质粒pET15b-glgP转化。
在另一个优选的实施方式中,本发明的植物细胞的特征在于所述植物细胞被包含质粒pBIN2035S-GP-NOS的根癌土壤杆菌CECT 7054转化或者感染,或者被包含质粒pBIN20-B33-ClP-GP-NOS的根癌土壤杆菌CECT 7055转化或者感染,其中属于下列植物物种:马铃薯(Solanumtuberosum),烟草(Nicotiana tabacum),水稻(Oryza sativa),玉米(Zea mays)和拟南芥(Arabidopsis thaliana)。
本发明的第四个目的涉及前述细菌细胞用于生产活性重组GP蛋白质以及生产针对GP的特异性抗体的用途。
本发明的第五个目的涉及被包含质粒pBIN2035S-GP-NOS的根癌土壤杆菌CECT 7054转化或者感染的细菌或植物细胞的用途,或者被包含质粒pBIN20-B33-ClP-GP-NOS的根癌土壤杆菌CECT 7055转化或者感染的细菌或植物细胞用于生产淀粉的用途。
本发明的第六个目的涉及被包含质粒pBIN2035S-GP-NOS的载体根癌土壤杆菌CECT 7054转化的转基因植物,或者被包含质粒pBIN20-B33-ClP-GP-NOS的根癌土壤杆菌CECT 7055转化的转基因植物,其特征在于,相对于野生型植物在细胞质水平和质体水平上均具有高GP活性,因而与未经转化的野生型植物相比具有高淀粉含量和产量以及高直链淀粉/支链淀粉比例,在所有情况中,野生型植物都是在与转基因植物在相同条件和相同的年份生长的。
在优选的实施方式中,本发明的转基因植物的特征在于GP活性为野生型植物的GP活性的至少5倍,它的淀粉含量比野生型的淀粉含量高至少20%,直链淀粉含量的数值比野生型植物的直链淀粉含量的数值高至少10%,在所有情况中,野生型植物都是在相同的条件下进行培育的。
在另一个优选的实施方式中,本发明的转基因植物的特征在于,它表达glgP基因(SEQ ID NO:3)并且编码具有高GP活性的蛋白质,并且进一步所述转基因植物选自包括下列的组:马铃薯(Solanum tuberosum)、烟草(Nicotiana tabacum)、水稻(Oryza sativa)、玉米(Zea mays)和拟南芥(Arabidopsis thaliana)。
本发明的第七个目的涉及前述转基因植物用于生产淀粉的用途。
本发明的第八个目的涉及针对GP酶的多克隆或单克隆抗体。
本发明的第九目的涉及所述抗体用于测量样品中所存在GP浓度的用途。
根据布达佩斯条约进行的微生物保藏
在本发明中所使用的微生物被保藏于西班牙普通微生物保藏中心(Colecciónde Cultivos Tipo-CECT),位于Research Building ofValencia University,Burjassot Campus,Burjassot 46100(Valencia,Spain)。
·转化质粒pET15b-glgP的大肠杆菌BL21(DE3)C43菌株于2005年3月7日被保藏,保藏号为CECT 7071。
·转化质粒pBIN20-B33-ClP-GP-NOS的根癌土壤杆菌C58:GV2260菌株于2005年1月14日被保藏,保藏号为CECT 7055。
·转化质粒pBIN2035S-GP-NOS的根癌土壤杆菌C58:GV2260菌株于2005年1月14日被保藏,保藏号为CECT 7054。
实施例
下面所提出的实施例是为了描述本发明而不限制本发明的范围。
实施例1:在大肠杆菌中产生重组GP
根据编码大肠杆菌GP的glgP基因的核苷酸序列知识形成两条特异性的引物(它们的序列是有义链5’-3’),SEQ ID NO:1和SEQ ID NO:2。通过使用这些引物,利用传统的PCR方法从大肠杆菌的基因组DNA扩增DNA片段;将其引入质粒pGemT-easy(Promega),得到质粒pG-glgP。用限制性酶XhoI和BamHI消化PG-glgP。将所释放的片段(含有glgP)克隆入表达质粒pET-15b(+)(Novagen)的相同限制性位点。通过电穿孔将所得到的质粒(标记名称为pET15b-glgP,图1)引入大肠杆菌BL21(DE3)C43(Novagen)。pET-15b(+)中克隆的核苷酸序列片段是SEQ ID NO:3。在SEQ ID NO:3表达之后得到的氨基酸序列是SEQ ID NO:4。
通过添加1mM IPTG,发生转化pET15b-glgP的细菌BL21(DE3)C43(CECT 7071)中glgP表达的诱导。额外在37摄氏度下培养6小时后,观察到转化pET15b-glgP的细菌积聚了大约95kD的蛋白质,其可以被以简单的方式借助亲和层析使用″His-结合”纯化试剂盒(Novagen)纯化成均一的(图4)。此外,这些细菌的特征在于不含有糖原(22)。
实施例2:酶促检验
在37摄氏度下进行酶促反应。根据所分析的多聚糖原的降解测量GP活性。在第一步骤中,将50微升反应混合物温浴15分钟,该反应混合物由下列构成:50mM HEPES(pH 7.5)、30mM磷酸缓冲液(pH 7.5)、多聚糖原(相当于10mM葡萄糖)和蛋白质提取物。在煮沸2分钟后,停止反应,以30,000g离心20分钟。将上清中存在的释放的GlP通过下列方法任一项进行测定:
·通过分光光度计法。将300微升含有Hepes 50mM pH 7,EDTA 1mM,MgCl2 2mM,KCl 15mM,NAD+0.6mM,1单位磷酸葡萄糖变位酶以及另一种Leuconostoc mesenteroides的葡萄糖-6-磷酸脱氢酶,以及30微升从步骤1得到的上清液温浴20分钟,在340nm处使用Multiskan EX spectrophotometer(Labsystems)监控NADH的产生。在步骤1中不存在糖原时,所有蛋白质提取物所产生的NADH的量都是可以忽略不计的。
·通过层析。将40微升来自步骤1的上清液,使用配有Carbo-Pac PA10柱和电流测定的DX-500Dionex系统进行高亲和力液相层析。
单位(U)被描述为催化每分钟产生1微摩尔产物的酶的量。
实施例3:具有GP活性的产物的鉴定
所获得的具有GP活性的产物满足任何GP相关科学文献中描述的通常特征(22.43-45)。
·大肠杆菌的GP识别5个或以上葡萄糖分子的均聚多糖例如糖原、淀粉、麦芽七糖、麦芽六糖和麦芽五糖。
·它对麦芽四糖、麦芽三糖或麦芽糖不起作用。
·它不被ADP葡萄糖、UDP葡萄糖、核苷单磷酸酯、核苷二磷酸酯、核苷三磷酸酯例如AMP、ADP和ATP、3-磷酸甘油酯、果糖-1,6-二磷酸、果糖-6-磷酸、葡萄糖-6-磷酸或葡萄糖所抑制。
·在变性凝胶中的纯化的蛋白质的表观分子量为约93kDa(图4)。
实施例4:根据下面的异位表达编码GP的基因,制备具有高GP活性的植物(质体高GP活性以及细胞质高GP活性)
使用菌株根癌土壤杆菌CECT 7054(其容纳质粒pBIN2035S-GP-NOS),获得组成型表达glgP的下列:马铃薯植物(Solanumtuberosum),烟草(Nicotiana tabacum),水稻(Oryza sativa),玉米(Zea mays)和拟南芥(Arabidopsis thaliana)。使用菌株根癌土壤杆菌CECT 7055(其容纳质粒pBIN20-B33-LCA-GP-NOS),获得在其块茎中表达glgP的马铃薯植物。使用CECT 7055和CECT 7054转化的的马铃薯植物的块茎聚集由针对大肠杆菌的GP获得的多克隆抗体特异性识别的一种蛋白质(图5),然而,在相同环境和种植条件(灌溉、肥料、杀虫剂处理等)、相同的年份生长的未转化的野生型马铃薯植物的块茎不会表达该酶。
使用CECT 7054(其容纳编码定位于细胞质的GP的构建体)转化的马铃薯植物以及使用CECT7055转化的马铃薯植物(其容纳编码定位于淀粉质体中的GP的构建体)具有下列特征:
1.GP活性是在未转化的块茎中存在的活性的5-8倍(图6)。
2.与未转化的块茎相比为高淀粉含量(在未转化的块茎中为大约300μmol葡萄糖/g鲜重,而在品系35S-glgP-NOS和B33-ChlTP-glgP-NOS中观察到的为450-700μmol葡萄糖/g鲜重)(图7)。因此,令人吃惊的是,与在细菌和哺乳动物细胞中所发生的不同,GP活性的提高导致了淀粉含量的提高。
3.高蔗糖含量以及低葡萄糖和果糖的趋势(图8)。
4.与未转化植物的块茎相比为高直链淀粉/支链淀粉比例(图9)。同样,该结果是令人吃惊的,因为如果GP具有葡萄糖聚合物的降解作用,则异位表达GP的块茎的直链淀粉/支链淀粉比例应该低于未转化的植物的块茎中所观察到的直链淀粉/支链淀粉比例。图7和图9中所表示的结果显示GP的表达引起在贮藏器官中聚集的淀粉量的增加。
5.与未转化的植物相比,异位表达GP的植物的外形没有异常。
6.与未转化的植物相比,异位表达GP的品系中块茎的数目是正常的。每株植物以kg鲜重计的产量不受GP表达的影响,而每株植物的淀粉产量则受GP表达的影响。
表1:大肠杆菌GP的底物特异性。反应混合物(50μl)包括50mMHEPES(pH 7.5),30mM Pi,指定的糖原(5mM葡萄糖)和3U重组GP。在37摄氏度下温浴3小时后,在100摄氏度下加热2分钟停止反应。通过具有电流测定的的HPLC分析产物。
表2:不同化合物对大肠杆菌GP活性的影响
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Claims (13)
1.选自下组的核苷酸序列在用于提高植物的α-1,4-葡聚糖磷酸酶活性、淀粉含量和产量以及直链淀粉/支链淀粉比例中的用途:
(a)编码表征为SEQ ID NO:4的氨基酸序列的核苷酸序列;
(b)表征为SEQ ID NO:3的核苷酸序列;
(c)与“a”或“b”中所限定的核苷酸序列杂交并且编码具有α-1,4-葡聚糖磷酸酶活性的酶产物的核苷酸序列;
(d)由于遗传密码子简并性而与“a”、“b”或“c”中所限定的核苷酸序列不同的核苷酸序列。
2.根据权利要求1所述的用途,其中所述核苷酸序列选自:
(a)编码表征为SEQ ID NO:4的氨基酸序列的核苷酸序列;
(b)表征为SEQ ID NO:3的核苷酸序列;
(c)由于遗传密码子简并性而与“a”或“b”中所限定的核苷酸序列不同的核苷酸序列。
3.根据权利要求1或2所述的用途,其中所述核苷酸序列选自:
(a)表征为SEQ ID NO:3的核苷酸序列;
(b)由于遗传密码子简并性而与SEQ ID NO:3不同的核苷酸序列。
4.根据前述权利要求任一项所述的用途,其中所述核苷酸序列为SEQ ID NO:3。
5.一种质粒,其为如图3所示的质粒pBIN2035S-GP-NOS。
6.一种质粒,其为以CECT 7055保藏的质粒pBIN20-B33-C1P-GP-NOS。
7.根癌土壤杆菌(Agrobacterium tumefaciens)CECT 7054,其特征在于包含如图3所示的质粒pBIN2035S-GP-NOS。
8.根癌土壤杆菌(Agrobacterium tumefaciens)CECT 7055,其特征在于包含质粒pBIN20-B33-C1P-GP-NOS。
9.在转化植物中表达的表达载体在用于提高植物的α-1,4-葡聚糖磷酸酶活性、淀粉含量和产量以及直链淀粉/支链淀粉比例中的用途,其中所述表达载体中所包含的核苷酸序列选自:
(a)编码表征为SEQ ID NO:4的氨基酸序列的核苷酸序列;
(b)表征为SEQ ID NO:3的核苷酸序列;
(c)与“a”或“b”中所限定的核苷酸序列杂交并且编码具有α-1,4-葡聚糖磷酸酶活性的酶产物的核苷酸序列;
(d)由于遗传密码子简并性而与“a”、“b”或“c”中所限定的核苷酸序列不同的核苷酸序列。
10.根据权利要求9所述的表达载体在用于提高植物的α-1,4-葡聚糖磷酸酶活性、淀粉含量和产量以及直链淀粉/支链淀粉比例中的用途,其中所述表达载体中所包含的核苷酸序列选自:
(a)编码表征为SEQ ID NO:4的氨基酸序列的核苷酸序列;
(b)表征为SEQ ID NO:3的核苷酸序列;
(c)由于遗传密码子简并性而与“a”或“b”中所限定的核苷酸序列不同的核苷酸序列。
11.根据权利要求9或10所述的表达载体在用于提高植物的α-1,4-葡聚糖磷酸酶活性、淀粉含量和产量以及直链淀粉/支链淀粉比例中的用途,其中所述表达载体中所包含的核苷酸序列选自:
(a)表征为SEQ ID NO:3的核苷酸序列;
(b)由于遗传密码子简并性而与SEQ ID NO:3不同的核苷酸序列。
12.根据权利要求9-11中任一项所述的表达载体在用于提高植物的α-1,4-葡聚糖磷酸酶活性、淀粉含量和产量以及直链淀粉/支链淀粉比例中的用途,其中所述表达载体中所包含的核苷酸序列为SEQID NO:3。
13.根据权利要求5-6中任一项所述的质粒在用于提高植物的α-1,4-葡聚糖磷酸酶活性、淀粉含量和产量以及直链淀粉/支链淀粉比例中的用途。
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