CN1639338A - 修饰的Cry3A毒素及编码它的核酸序列 - Google Patents
修饰的Cry3A毒素及编码它的核酸序列 Download PDFInfo
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Abstract
本发明公开了控制植物害虫的组合物和方法。特别地，提供了新的编码对玉米根虫具有增强毒性的修饰Cry3A毒素的核酸分子。通过在Cry3A毒素的至少一个位置插入可被目标昆虫的肠蛋白酶识别的蛋白酶识别位点，例如组织蛋白酶G，设计具有显著增强的毒性的，特别是针对西部和北部玉米根虫具有显著增强的毒性的修饰Cry3A毒素。此外，还公开了制备修饰的Cry3A毒素的方法和利用修饰的Cry3A核酸序列的方法，例如在微生物中用来控制昆虫，或者在转基因植物用来赋予免受昆虫毁坏的保护，以及利用修饰的Cry3A毒素、以及包含该修饰的Cry3A毒素的组合物和制剂的方法，例如向昆虫滋生地区施用修饰的Cry3A毒素或组合物或制剂、或者预防性地处理易感地区或植物，以赋予防备昆虫害虫的保护。
Description
本发明涉及蛋白工程、植物分子生物学和病虫害防治领域。更具体地，本发明涉及新的修饰的Cry3A毒素和其表达产生修饰的Cry3A毒素的核酸序列，以及制备修饰的Cry3A毒素和相应的核酸序列的方法和使用它们控制昆虫的方法。
玉米根虫物种被认为是最有破坏性的玉米害虫。在美国的三个重要物种是西部玉米根虫Diabrotica virgifera virgifera；北部玉米根虫D.longicornis barberi和南部玉米根虫D.undecimpunctatahowardi。只有西部和北部玉米根虫被被认为是美国玉米种植地带的主要害虫。玉米根虫幼虫通过几乎专门摄食玉米根系而导致最重要的植物损伤。这种损伤已证明增加植物倒伏、降低谷物产量和植物产量以及改变谷物营养含量。幼虫摄食还通过打开细菌和真菌感染穿过根系的途径，引起根茎腐烂病而对玉米造成间接影响。成年玉米根虫在晚夏活跃于玉米田，在那里它们摄食谷穗、穗丝和花粉，干扰正常的授粉。
玉米根虫主要是通过密集施用化学农药控制的，其通过抑制昆虫生长、阻止昆虫摄食或繁殖或导致死亡起作用。这样可以实现不错的玉米根虫控制，但这些化学制品往往也能够影响其它的有益生物。广泛应用化学杀虫剂所引起的另一个问题是抗性昆虫种类的出现。而另外一个问题是由于玉米根虫在地下摄食的事实而引起的，从而难于应用杀虫剂拯救处理。所以大多数杀虫剂施用是在种植的时候预防性地进行的。这种做法造成大量的环境负担。这通过多种农场管理惯例已经部分地得到缓和，但可替代害虫控制机制的需求渐增。
生物学害虫控制制剂，例如表达杀虫毒素象δ-内毒素等的苏云金芽孢杆菌(Bacillus thuringiensis)(Bt)菌株，也已经被施用于作物植物，取得了主要针对鳞翅目昆虫害虫的令人满意的结果。δ-内毒素是已知当被某些昆虫摄入后拥有杀虫活性的保持在晶体基质中的蛋白。各种δ-内毒素已经根据其活性谱和序列同源性予以分类。在1990年之前，主要类别是根据其活性谱确定的，Cry1蛋白对鳞翅目(Lepidoptera)(蛾和蝴蝶)有活性，Cry2蛋白对鳞翅目和双翅目(Diptera)(苍蝇和蚊子)都有活性，Cry3蛋白对鞘翅目(Coleoptera)(甲虫)有活性，而Cry4蛋白对双翅目有活性(Hofte and Whitely，1989，Microbiol.Rev.53：242-255)。最近发展了新的命名法，它以氨基酸序列同源性而非昆虫靶标特异性为基础，对Cry蛋白系统地进行分类(Crickmore等1998，Microbiol.Molec.Biol.Rev.62：807-813)。
单个Btδ-内毒素的杀虫活性谱相当窄，给定的δ-内毒素只对一定目内少数物种有活性。举例来说，Cry3A蛋白已知对科罗拉多土豆甲虫马铃薯甲虫(Leptinotarsa decemlineata)非常有毒性，但对Diabrotica属的相关甲虫有非常小的或者没有毒性(Johnson等，1993，J.Econ.Entomol.86：330-333)。根据Slaney等(1992，InsectBiochem.Molec.Biol.22：9-18)，Cry3A蛋白对南部玉米根虫幼虫的毒性比对科罗拉多土豆甲虫小至少2000倍。还已知Cry3A蛋白对西部玉米根虫具有很少或没有毒性。
δ-内毒素的特异性是产生活性毒素蛋白所涉及的各个步骤的有效性及其随后与昆虫中肠的上皮细胞相互作用的结果。为能够杀虫，大多数已知的δ-内毒素必需首先被昆虫摄入并通过蛋白水解活化形成活性毒素。杀虫晶体蛋白的活化是一个多步骤的过程。摄入后，晶体必需首先在昆虫肠中溶解。一旦溶解，δ-内毒素即通过特定的蛋白水解切割被活化。昆虫肠中的蛋白酶可以通过决定在何处加工δ-内毒素而在特异性中起作用。一旦δ-内毒素被溶解并加工，它与昆虫中肠上皮细胞表面的特定受体结合，并随之整合进刷状缘膜的脂双层中。于是形成离子通道，破坏中肠的正常功能，最终导致昆虫死亡。
在鳞翅目中，肠蛋白酶将δ-内毒素从130-140kDa的原毒素(Protoxin)加工成大约60-70kDa的毒性蛋白。原毒素到毒素的加工据报道是通过除去N-和C-末端氨基酸进行的，而加工的准确定位取决于参与的特定昆虫肠液(Ogiwara等，1992，J.Invert.Pathol.60：121-126)。δ-内毒素的蛋白水解活化可以在决定其特异性方面起重要作用。举例来说，来自Bt Var.aizawa的δ-内毒素，被称作IC1，已经基于它与其它已知的Cry1Ab蛋白的序列同源性而被归类为Cry1Ab蛋白。Cry1Ab蛋白典型地有抗鳞翅目昆虫的活性。不过，该IC1蛋白对鳞翅目和双翅目都有活性，取决于该蛋白怎样被加工(Haider等1986，Euro.J.Biochem.156：531-540)。在双翅目肠中获得53kDa的活化IC1毒素，而在鳞翅目肠中获得55kDa的活化IC1毒素。IC1与全型(holotype)HD-1 Cry1Ab蛋白仅有4个氨基酸的差别，因此受体结合区域的总变化看起来不能解释活性的差别。两种不同昆虫肠中的不同蛋白水解切割很可能允许活化的分子不同地折叠，从而暴露能够结合不同受体的不同区域。所以这种特异性似乎存在于不同昆虫的肠蛋白酶中。
鞘翅目昆虫具有更加中性至偏酸的肠，而鞘翅目特异的δ-内毒素与活化的鳞翅目特异的毒素大小相似。所以，鞘翅目特异的δ-内毒素的加工以前被认为是毒性非必需的。然而，最近的数据提示鞘翅目活性δ-内毒素被溶解，并被蛋白水解为较小的毒性多肽。苏云金芽孢杆菌粉虫变种(B.thuringiensis var.tenebrionis)产生的73kDaCry3Aδ-内毒素蛋白在该细菌中在N-末端迅速加工，在晶体形成期间或之后丢失49-57个残基，产生通常所分离到的67kDa的形式(Carroll等，1989，Biochem.J.261：99-105)。McPherson等，1988(Biotechnology 6：61-66)也证明天然的cry3A基因在同一阅读框内含有两个功能翻译起始密码子，一个编码73kDa的蛋白，而另一个编码67kDa的蛋白，分别起始于推断的氨基酸序列(参见SEQ ID NO：2)的Met-1和Met-48。两个蛋白因而都可是被认为是天然存在的全长Cry3A蛋白。用胰蛋白酶或者昆虫肠提取物处理可溶性67kDa Cry3A蛋白产生55kDa的切割产物，在N-末端具有推断的氨基酸序列的Asn-159。发现这个多肽象天然的67kDa Cry3A毒素一样对易感鞘翅目昆虫具有毒性(Carroll等Ibid)。因此，天然胰蛋白酶识别位点存在于天然Cry3A毒素(SEQ ID NO：2)推断的氨基酸序列的Arg-158和Asn-159之间。Cry3A还可以被胰凝乳蛋白酶切割，产生49、11和6kDa的3个多肽。49和6kDa组份的N-末端分析表明第一个氨基酸分别为Ser-162和Tyr-588(Carroll等，1997 J.Invert.Biol.70：41-49)。因此，天然胰凝乳蛋白酶识别位点存在于Cry3A推断的氨基酸序列(SEQ ID NO：2)的His-161和Ser-162之间以及Tyr-587和Tyr-588之间。49kDa胰凝乳蛋白酶产物在中性pH时似乎比天然的67kDa蛋白或55kDa的胰蛋白酶产物更大程度地溶解，并保留了对Cry3A-易感昆虫科罗拉多土豆甲虫和芥菜甲虫(Phaedon cochleariae)的完全的杀虫活性。
昆虫肠蛋白酶典型地起辅助昆虫从饮食的蛋白中获得所需的氨基酸的作用。了解得最清楚的昆虫消化蛋白酶是最为常见的丝氨酸蛋白酶(Englemann and Geraerts，1980，J.Insect Physiol.261：703-710)，特别是在鳞翅目物种中。大多数鞘翅目幼虫和成虫，例如科罗拉多土豆甲虫，具有微酸的中肠，而半胱氨酸蛋白酶提供了主要的蛋白水解活性(Wolfson and Mudock，1990，J.Chem.Ecol.16：1089-1102)。更精确地，Thie和Houseman(1990，Insect Biochem.20：313-318)鉴定并表征了科罗拉多土豆甲虫的半胱氨酸蛋白酶组织蛋白酶B和H，以及天冬氨酸蛋白酶组织蛋白酶D。Gillikin等(1992，Arch.Insect Biochem.Physiol.19：285-298)表征了西部玉米根虫幼虫肠中的蛋白水解活性，并发现了15种主要是半胱氨酸型蛋白酶。在本发明公开之前，没有报道指出丝氨酸蛋白酶组织蛋白酶G存在于西部玉米根虫中。昆虫肠蛋白酶的多样性和不同活性水平可能影响昆虫对特定Bt毒素的敏感性。
在过去的5年中，已经描述了许多新的和新奇的具有改善的或者新的生物活性的Bt菌株以及δ-内毒素，包括对线虫有活性的菌株(EP0517367 A1)。不过，相对而言，这些菌株和毒素中很少对鞘翅目昆虫具有活性。此外，没有任何一种已知的鞘翅目活性δ-内毒素，例如Cry3A，Cry3B，Cry3C，Cry7A，Cry8A，Cry8B和Cry8C，具有足够的抗玉米根虫的口服毒性，以便在例如通过微生物或转基因植物递送时提供足够的田间控制。所以，需要开发制备对玉米根虫有活性的新毒素的其它方法。
由于已经取得了关于δ-内毒素如何起作用的更多知识，改造δ-内毒素使之具有新活性的尝试增加了。由于1991年Cry3A三维结构的解决，使得工程改造δ-内毒素成为可能(Li等，1991，Nature 353：815-821)。该蛋白具有3个结构域：N-末端结构域I，从残基1至290，由7个α螺旋组成，结构域II，从残基291至500，含有3个β-片层，以及C-末端结构域III，从残基501至644，为β-夹层(β-Sandwich)。基于这个结构，形成了关于δ-内毒素结构/功能关系的假说。一般认为结构域I主要是负责在昆虫肠膜中的孔形成(Gazit and Shai，1993，Appl.Environ.Microbiol.57：2816-2820)，结构域II主要负责与肠受体相互作用(Ge等，1991，J.Biol.Chem.32：3429-3436)，而结构域III最可能与蛋白稳定性有关(Li等1991，同上)，而且对离子通道活性具有调控影响(Chen等，1993，PNAS 90：9041-9045)。
鳞翅目活性δ-内毒素已经被工程改造，试图提高比活或者拓宽杀虫活性谱。例如，将Cry1Aa的蚕蛾(Bombyx mori)特异性结构域移到Cry1Ac中，从而赋予所得的嵌合蛋白新的杀虫活性(Ge等1989，PNAS 86：4037-4041)。同样，Bosch等1998(美国专利5,736,131)通过以Cry1C的结构域III取代Cry1E的结构域III，从而制备了具有更广鳞翅目活性谱的Cry1E-Cry1C杂合毒素，建立了新的鳞翅目活性毒素。
已经报道了许多工程改造鞘翅目活性δ-内毒素的尝试。Van Rie等1997(美国专利No.5,659,123)通过用氨基酸丙氨酸随机取代结构域II中据认为对于溶剂接近重要的氨基酸来工程改造Cry3A。许多限于受体结合结构域II的这些随机取代据报道与增加的西部玉米根虫毒性有关。可是，他人已经证明Cry3A结构域II中的有些丙氨酸取代导致受体结合破坏或结构不稳定(Wu and Dean，1996，J.Mol.Biol.255：628-640)。English等1999(国际专利申请公开号WO99/31248)报道了导致对南部和西部玉米根虫毒性增加的Cry3Bb中的氨基酸取代。不过，在报道的35个Cry3Bb突变体中，只有3个，突变主要在结构域II以及结构域II-结构域I界面处，有抗西部玉米根虫的活性。此外，在相同的测定中，野生型Cry3Bb对西部玉米根虫的毒性差别，要大于突变的Cry3Bb毒素和野生型Cry3Bb之间的任何差别。所以，Cry3Bb突变体毒性的提高似乎主要局限于南部玉米根虫。
仍然需要设计新的和有效的为农民提供经济效益并且为环境所接受的害虫控制剂。特别需要的是控制对现存昆虫控制剂具有或者能够变得具有抗性的西部玉米根虫，美国主要玉米害虫的修饰的Cry3A毒素。此外，人们期望其施用将环境负担最小化，象通过转基因植物而施用的制剂。
考虑到这些需求，本发明的一个目的是提供编码对玉米根虫具有增加的毒性的修饰的Cry3A毒素的新核酸序列。根据本发明，通过在Cry3A毒素的至少一个位置插入被靶标昆虫肠蛋白酶所识别的蛋白酶识别位点，设计了具有显著增大的毒性、特别是对西部和北部玉米根虫具有显著增大的毒性的修饰的Cry3A毒素。本发明进一步涉及由于该核酸序列表达而产生的新的修饰的Cry3A毒素，以及含有该修饰的Cry3A毒素的组合物和制剂，其能够抑制昆虫害虫存活、生长和繁殖的能力，或者能够限制作物植物与昆虫相关的损伤或损失。本发明进一步涉及制备修饰的Cry3A毒素的方法，以及利用修饰的cry3A核酸序列的方法，例如在微生物中用来控制昆虫，或者在转基因植物用来赋予免受昆虫毁坏的保护，以及利用修饰的Cry3A毒素、以及包含该修饰的Cry3A毒素的组合物和制剂的方法，例如向昆虫滋生地区施用修饰的Cry3A毒素或组合物或配方，或者预防性地处理昆虫易感地区或植物，以赋予抵抗昆虫害虫的保护。
本文所述的新的修饰的Cry3A毒素对昆虫具有高活性。例如，本发明修饰的Cry3A毒素可以被用来控制经济上重要的昆虫害虫例如西部玉米根虫(Diabrotica virgifera virgifera)和北部玉米根虫(D.longicornis barberi)。修饰的Cry3A毒素可以单独或者与其它植物控制策略结合使用，以用最小的环境影响产生最大的害虫控制效率。
根据一方面，本发明提供了分离的包括编码修饰的Cry3A毒素的核苷酸序列的核酸分子，其中修饰的Cry3A毒素包括至少一个额外的不是天然存在于Cry3A毒素中的蛋白酶识别位点。这个被靶标昆虫的肠蛋白酶(gut protease)所识别的额外的蛋白酶识别位点，被插入到Cry3A毒素中与天然存在的蛋白酶识别位点大致相同的位置。修饰的Cry3A毒素导致的靶标昆虫的死亡率比Cry3A毒素所导致的相同靶标昆虫的死亡率更高。优选地，修饰的Cry3A毒素导致靶标昆虫至少约50％的死亡率，而Cry3A毒素只能导致该昆虫不超过约30％的死亡率。
在这方面的一个实施方式中，靶标昆虫的肠蛋白酶是从由丝氨酸蛋白酶、半胱氨酸蛋白酶和天冬氨酸蛋白酶组成的组中选择的。根据这个实施方式优选的丝氨酸蛋白酶包括组织蛋白酶G、胰蛋白酶、胰凝乳蛋白酶、羧肽酶、内肽酶和弹性蛋白酶，最优选组织蛋白酶G。
在这方面的另一个实施方式中，所述额外的蛋白酶识别位点被插入到Cry3A毒素结构域I中或者结构域III中或者结构域I和结构域III中。优选地，额外的蛋白酶识别位点被插入到结构域I中或者结构域III中或者结构域I和结构域III中取代、邻近天然存在的蛋白酶识别位点或者在天然存在的蛋白酶识别位点之内的位置。
而在另一个实施方式中，额外的蛋白酶识别位点被插入到结构域I中与SEQ ID NO：2氨基酸位154和162相应的氨基酸之间。优选地，额外的蛋白酶识别位点被插入到SEQ ID NO：2氨基酸位154和162之间或者SEQ ID NO：4氨基酸位107和115之间。
在另一个实施方式中，额外的蛋白酶识别位点被插入到与SEQ IDNO：2氨基酸位154和160相应的氨基酸之间。优选地，额外的蛋白酶识别位点被插入到SEQ ID NO：2氨基酸位154和160之间或者SEQID NO：4氨基酸位107和113之间。
在进一步的实施方式中，额外的蛋白酶识别位点被插入到结构域I中与SEQ ID NO：2氨基酸位154和158相应的氨基酸之间。优选地，额外的蛋白酶识别位点被插入到结构域I中SEQ ID NO：2氨基酸位154和158之间或者SEQ ID NO：4氨基酸位107和111之间。
在另一个实施方式中，额外的蛋白酶识别位点被插入到结构域III中与SEQ ID NO：2氨基酸位583和589相应的氨基酸之间。优选地，额外的蛋白酶位点被插入到结构域III中SEQ ID NO：2氨基酸位583和589之间或者SEQ ID NO：4氨基酸位536和542之间。
还有另一个实施方式中，额外的蛋白酶识别位点被插入到结构域III中与SEQ ID NO：2氨基酸位583和588相应的氨基酸之间。优选地，额外的蛋白酶识别位点被插入到结构域III中SEQ ID NO：2氨基酸位583和588之间或者SEQ ID NO：4氨基酸位536和541之间。
而在另一个实施方式中，额外的蛋白酶识别位点被插入到结构域III中与SEQ ID NO：2氨基酸位587和588相应的氨基酸之间。优选地，额外的蛋白酶识别位点被插入到结构域III中SEQ ID NO：2氨基酸位587和588之间或者SEQ ID NO：4氨基酸位540和541之间。
在一个实施方式中，额外的蛋白酶识别位点被插入到未修饰的Cry3A毒素的结构域I和结构域III中。优选地，额外的蛋白酶识别位点被插入到结构域I中取代或者邻近天然存在的蛋白酶识别位点的位置和结构域III中在天然存在的蛋白酶识别位点之内的、取代或者邻近天然存在的蛋白酶识别位点的位置。
在另一个实施方式中，额外的蛋白酶识别位点被插入到结构域I中与SEQ ID NO：2的氨基酸位154和160相应的氨基酸之间以及结构域III中与SEQ ID NO：2的氨基酸位587以及588相应的氨基酸之间。优选地，额外的蛋白酶识别位点被插入到结构域I中SEQ ID NO：2的氨基酸位154和160之间以及结构域III中SEQ ID NO：2的氨基酸位587和588之间，或者结构域I中SEQ ID NO：4的氨基酸位107和113之间以及结构域III中SEQ ID NO：4的氨基酸位540和541之间。
而在另一个实施方式中，额外的蛋白酶识别位点位于结构域I中与SEQ ID NO：2的氨基酸位154和158相应的氨基酸之间以及结构域III中与SEQ ID NO：2的氨基酸位587以及588相应的氨基酸之间。优选地，额外的蛋白酶识别位点被插入到结构域I中SEQ ID NO：2的氨基酸位154和158之间以及结构域III中SEQ ID NO：2的氨基酸位587和588之间，或者结构域I中SEQ ID NO：4的氨基酸位107和111之间以及结构域III中SEQ ID NO：4的氨基酸位540和541之间。
在另一个实施方式中，额外的蛋白酶识别位点位于结构域I中与SEQ ID NO：2的氨基酸位154和158相应的氨基酸之间以及结构域III中与SEQ ID NO：2的氨基酸位583以及588相应的氨基酸之间。优选地，额外的蛋白酶识别位点被插入到结构域I中SEQ ID NO：2的氨基酸位154和158之间以及结构域III中SEQ ID NO：2的氨基酸位583和588之间，或者结构域I中SEQ ID NO：4的氨基酸位107和111之间以及结构域III中SEQ ID NO：4的氨基酸位536和541之间。
在优选的实施方式中，本发明的分离的核酸分子包括SEQ ID NO：6的核苷酸1-1791、SEQ ID NO：8的核苷酸1-1806、SEQ ID NO：10的核苷酸1-1818、SEQ ID NO：12的核苷酸1-1794、SEQ ID NO：14的核苷酸1-1812、SEQ ID NO：16的核苷酸1-1812、SEQ ID NO：18的核苷酸1-1818、或者SEQ ID NO：20的核苷酸1-1791。
而在另一个优选的实施方式中，本发明的分离的核酸分子编码包括SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11、SEQ ID NO：13、SEQ ID NO：15、SEQ ID NO：17、SEQ ID NO：19或SEQ ID NO：21中所示的氨基酸序列的修饰的Cry3A毒素。
根据本发明的一个实施方式，分离的核酸分子编码具有抗鞘翅目昆虫活性的修饰的Cry3A毒素。优选地，修饰的Cry3A毒素具有抗西部玉米根虫的活性。
本发明提供了包括可操作地连接于本发明的核酸分子的异源启动子序列的嵌合基因。本发明还提供了包含这样的嵌合基因的重组载体。进一步地，本发明提供了包含这样的嵌合基因的转基因非人宿主细胞。根据本发明这方面的转基因宿主细胞可以是细菌细胞或植物细胞，优选植物细胞。本发明进一步提供了包含这样的植物细胞的转基因植物根据本发明这方面的转基因植物可以是高粱、小麦、向日葵、番茄、马铃薯、油菜作物(cole crops)、棉花、稻、大豆、甜菜、甘蔗、烟草、大麦、油料种子芸苔(oilseed rape)或者玉米，优选玉米。本发明还提供了来自由高粱、小麦、向日葵、番茄、马铃薯、油菜作物、棉花、稻、大豆、甜菜、甘蔗、烟草、大麦、油料种子芸苔和玉米组成的转基因植物组的种子。在一个特别优选的实施方式中，种子来自转基因玉米植物。
在另一方面，本发明提供了通过表达本发明的核酸分子产生的毒素。在优选的实施方式中，毒素通过表达包括SEQ ID NO：6的核苷酸1-1791、SEQ ID NO：8的核苷酸1-1806、SEQ ID NO：10的核苷酸1-1818、SEQ ID NO：12的核苷酸1-1794、SEQ ID NO：14的核苷酸1-1812、SEQ ID NO：16的核苷酸1-1812、SEQ ID NO：18的核苷酸1-1818、或者SEQ ID NO：20的核苷酸1-1791的核酸分子而产生。
在另一个实施方式中，本发明的毒素具有抗鞘翅目昆虫，优选抗西部玉米根虫的活性。
在一个实施方式中，本发明的毒素包括SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11、SEQ ID NO：13、SEQ ID NO：15、SEQ ID NO：17、SEQ ID NO：19或SEQ ID NO：21所示的氨基酸序列。
本发明还提供了包括有效昆虫控制量的根据本发明的毒素的组合物。
在另一方面，本发明提供了制备具有抗昆虫活性的毒素的方法，包括：(a)获得包含嵌合基因的宿主细胞，该基因本身包括可操作地连接于本发明的核酸分子的异源启动子序列；和(b)在该转基因宿主细胞中表达核酸分子，这产生至少一个具有抗昆虫活性的毒素。
在进一步的方面，本发明提供了制备昆虫抗性转基因植物的方法，包括向转基因植物引入本发明的核酸分子，其中该核酸分子可以以控制昆虫有效量的水平在转基因植物中表达。在优选的实施方式中，昆虫是鞘翅目昆虫，优选西部玉米根虫。
而在进一步的方面，本发明提供了控制昆虫的方法，包括向昆虫递送有效量的本发明的毒素。根据一个实施方式，昆虫是鞘翅目昆虫，优选西部玉米根虫。优选地，毒素经口递送给昆虫。在一个优选的实施方式中，毒素通过包含表达本发明毒素的核酸序列的转基因植物口服递送。
同时本发明还提供制备修饰的Cry3A毒素的方法，包括：(a)获得编码Cry3A毒素的cry3A毒素基因；(b)鉴定靶标昆虫的肠蛋白酶；(c)获得编码所述肠蛋白酶识别序列的核苷酸序列；(d)将(c)中核苷酸序列插入到cry3A毒素基因的结构域I中或者结构域III中或者结构域I和结构域III中取代编码天然存在的蛋白酶识别位点的核苷酸序列、在编码天然存在的蛋白酶识别位点的核苷酸序列之内、或者邻近编码天然存在的蛋白酶识别位点的核苷酸序列的位置，从而建立修饰的cry3A毒素基因；(e)将修饰的cry3A毒素基因插入到表达盒中；(f)在非人宿主细胞中表达修饰的cry3A毒素基因，导致宿主细胞产生修饰的Cry3A毒素；和(g)对修饰的Cry3A毒素进行抗靶标昆虫的生物测定，由此，修饰的Cry3A毒素导致比Cry3A毒素所致的靶标昆虫死亡率更高的靶标昆虫死亡率。在优选的实施方式中，当Cry3A毒素导致不超过约30％的死亡率时，修饰的Cry3A毒素导致靶标昆虫至少大约50％的死亡率。
本发明进一步提供了控制昆虫的方法，其中所述转基因植物进一步包括编码第二杀虫素(pesticidal principle)的第二核酸序列或核酸序列组。特别优选的第二核酸序列是编码δ-内毒素的序列，编码植物杀虫蛋白毒素的序列，公开在美国专利5,849,870和5,877,012中，兹并入作为参考，或者编码产生非蛋白性杀虫素的途径的序列。
而本发明的另一个方面提供的是突变根据本发明的核酸分子的方法，其中核酸分子被切割成期望大小的双链随机片段群，包括：(a)向双链随机片段群中添加一种或多种单链或双链寡核苷酸，其中寡核苷酸各自都包含与双链模板多核苷酸相同的区域和异源的区域；(b)将所得的双链随机片段和寡核苷酸的混合物变性为单链片段；(c)将所得的单链片段群与聚合酶在导致单链片段在相同区域退火形成退火片段对的条件下温育，相同区域足以使该对中的一个成员引发另一成员的复制，藉此形成突变的双链多核苷酸；和(d)再重复第二和第三步至少两个循环，其中在下一个循环第二步中所得的混合物包括来自前一个循环第三步的突变的双链多核苷酸，并且其中该下一个循环形成了进一步突变的双链多核苷酸。
通过学习本发明的下列说明和非限制性实施例，本发明的其他方面和优点对所属技术领域熟练技术人员来说将会是显而易见的。
SEQ ID NO：1是天然cry3A编码区。
SEQ ID NO：2是由天然cry3A基因编码的Cry3A毒素的氨基酸序列。
SEQ ID NO：3是起始于天然cry3A编码区核苷酸144的玉米优化cry3A编码区。
SEQ ID NO：4是由玉米优化cry3A基因编码的Cry3A毒素的氨基酸序列。
SEQ ID NO：5是pCIB6850的核苷酸序列。
SEQ ID NO：6是玉米优化的修饰的cry3A054编码序列。
SEQ ID NO：7是由SEQ ID NO：6的核苷酸序列编码的氨基酸序列。
SEQ ID NO：8是玉米优化的修饰的cry3A055编码序列。
SEQ ID NO：9是由SEQ ID NO：8的核苷酸序列编码的氨基酸序列。
SEQ ID NO：10是玉米优化的修饰的cry3A085编码序列。
SEQ ID NO：11是由SEQ ID NO：10的核苷酸序列编码的氨基酸序列。
SEQ ID NO：12是玉米优化的修饰的cry3A082编码序列。
SEQ ID NO：13是由SEQ ID NO：12的核苷酸序列编码的氨基酸序列。
SEQ ID NO：14是玉米优化的修饰的cry3A058编码序列。
SEQ ID NO：15是由SEQ ID NO：14的核苷酸序列编码的氨基酸序列。
SEQ ID NO：16是玉米优化的修饰的cry3A057编码序列。
SEQ ID NO：17是由SEQ ID NO：16的核苷酸序列编码的氨基酸序列。
SEQ ID NO：18是玉米优化的修饰的cry3A056编码序列。
SEQ ID NO：19是由SEQ ID NO：18的核苷酸序列编码的氨基酸序列。
SEQ ID NO：20是玉米优化的修饰的cry3A083编码序列。
SEQ ID NO：21是由SEQ ID NO：20的核苷酸序列编码的氨基酸序列。
SEQ ID NOS：22-34是本发明所用的PCR引物。
SEQ ID NO：35是包括组织蛋白酶G识别位点的氨基酸序列。
SEQ ID NO：36是包括组织蛋白酶G识别位点的氨基酸序列。
SEQ ID NO：37是包括组织蛋白酶G识别位点的氨基酸序列。
SEQ ID NO：38是包括组织蛋白酶G识别位点的氨基酸序列。
为清楚起见，说明书中所用的某些术语定义并介绍如下：本发明修饰的Cry3A毒素的“活性”是指修饰的Cry3A毒素起口服活性昆虫控制剂的作用，具有毒性效果，或者能够干扰或阻止昆虫摄食，它可能或者可能不导致昆虫死亡。当本发明修饰的Cry3A毒素递送给昆虫时，结果典型地是昆虫的死亡，或者昆虫不再以使昆虫可以获得修饰的Cry3A毒素的来源为食。
“邻近”-根据本发明，当额外的蛋白酶识别位点在天然存在的蛋白酶识别位点的4个残基之内，优选3个残基之内，更优选2个残基之内，最优选1个残基之内的时候，该额外的蛋白酶识别位点与天然存在的蛋白酶识别位点“邻近”。例如，插入在Cry3A毒素推断的氨基酸序列(SEQ ID NO：2)Pro-154和Arg-158之间的额外的蛋白酶识别位点与位于Cry3A毒素推断的氨基酸序列(SEQ ID NO：2)Arg-158和Asn-159之间的天然存在的胰蛋白酶识别位点“邻近”。
如本文所用的描述额外的蛋白酶识别位点在Cry3A毒素中相对于天然存在的蛋白酶识别位点插入的位置的短语“大致相同的位置”，是指这个位置距离天然存在的蛋白酶识别位点至多4个残基。这个位置还可以距离天然存在的蛋白酶识别位点3到2个残基。这个位置还可以距离天然存在的蛋白酶识别位点1个残基。“大致相同的位置”还可以指额外的蛋白酶识别位点被插入在天然存在的蛋白酶识别位点之内。
“联合/可操作地连接”是指物理或功能上关联的两个核酸序列。例如，启动子或调控DNA序列被表述为与编码RNA或蛋白的DNA序列“联合”，如果这两个序列可操作地连接，或者如此安置以至于调控序列将会影响编码或结构DNA序列的表达水平的话。
“嵌合基因”或者“嵌合构建体”是重组的核酸序列，其中启动子或调控核酸序列可操作地连接或联合于编码mRNA或表达为蛋白的核酸序列，从而调控核酸序列能够调控该联合的核酸编码序列的转录或表达。在天然情况下，嵌合基因的调控核酸序列通常地并不与所联合的核酸序列可操作地连接。
“编码序列”是被转录成RNA例如mRNA、rRNA、tRNA、snRNA、正义RNA或反义RNA的核酸序列。优选该RNA随后在生物体内被翻译产生蛋白。
“控制”昆虫是指通过毒性效果，抑制昆虫害虫存活、生长、摄食、和/或繁殖的能力，或者限制作物植物与昆虫相关的损伤或损失。“控制”昆虫可以或可以不指杀死昆虫，尽管优选是指杀死昆虫。
相应：在本发明的上下文中，“相应”是指当变体Cry3Aδ-内毒素的氨基酸序列彼此对齐时，与本发明某些列举的位置“相应”的氨基酸是与Cry3A毒素(SEQ ID NO：2)中这些位置对准，但未必是相对于本发明的特定Cry3A氨基酸序列的这些确切数字位置的氨基酸。例如，本发明玉米优化的cry3A基因(SEQ ID NO：3)编码起始于由天然cry3A基因(SEQ ID NO：1)编码的Cry3A毒素(SEQ ID NO：2)的Met-48的Cry3A毒素(SEQ ID NO：4)。所以，根据本发明，SEQ IDNO：4的氨基酸位107-115，包括其间的所有位，以及536-541，包括其间的所有位，分别与SEQ ID NO：2的氨基酸位154-163，包括其间的所有位，以及583-588，包括其间的所有位相应。
如本文所用，“Cry3A毒素”是指大约73kDa的苏云金芽孢杆菌粉虫变种(Bacillus thuringiensis var.tenebrionis)(Kreig等，1983，Z.Angew.Entomol.96：500-5 08)(Bt)鞘翅目活性蛋白(Sekar等，1987，Proc.Nalt.Acad.Sci.84：7036-7040)，例如SEQ ID NO：2，以及来源于Cry3A毒素的任何截短的较低分子量变体，例如SEQ ID NO：4，并保留与Cry3A毒素基本上相同的毒性。较低分子量变体可通过蛋白酶切割Cry3A毒素天然存在的蛋白酶识别位点，或者通过和编码该73kDa Cry3A毒素的翻译起始密码子相同读框内的第二翻译起始密码子获得。Cry3A毒素及其较低分子量变体的氨基酸序列可以在Bt天然存在的毒素中找到。Cry3A毒素可由如SEQ ID NO：1的天然Bt基因，或者由如SEQ ID NO：3的合成编码序列编码。“Cry3A毒素”除了天然存在于Cry3A毒素中的蛋白酶识别位点之外，没有任何额外的蛋白酶识别位点。Cry3A毒素可以被分离、纯化或者在异源系统中表达。
如本文所用，“cry3A基因”，是指SEQ ID NO：1或SEQ ID NO：3的核苷酸序列。cry3A基因(Sekar等，1987，Proc.Natl.Acad.Sci.84：7036-7040)可以是天然存在的，如在苏云金芽孢杆菌粉虫变种(Bacillus thuringiensisvar.tenebrionis)中所发现的(Kreig等，1983，Z.Angew.Entomol.96：500-508)，或者是合成的，并编码Cry3A毒素。本发明的cry3A基因可以指SEQ ID NO：1中的天然cry3A基因，或者如SEQ ID NO：3中的玉米优化cry3A基因。
“递送”毒素是指毒素与昆虫接触，产生毒性效果并控制昆虫。毒素可以通过许多公认的方式递送，如通过昆虫摄入口服，或者通过转基因植物表达、配制的蛋白组合物、可喷洒蛋白组合物、饵料基质或者任何其它本领域公认的毒素递送系统与昆虫接触。
“有效昆虫控制量”是指通过毒性效果，抑制昆虫害虫存活、生长、摄食和/或繁殖的能力，或者限制作物植物与昆虫相关的损伤或损失的毒素浓度。“有效昆虫控制量”可以或可以不指杀死昆虫，尽管优选是指杀死昆虫。
如本文所用，“表达盒”是指能够指导特定核苷酸序列在合适的宿主细胞中表达的核酸序列，包括可操作地连接于感兴趣核苷酸序列的启动子，该核苷酸序列可操作地连接于终止信号。它还典型地包括该核苷酸序列正确翻译所需的序列。包括感兴趣核苷酸序列的表达盒可以是嵌合的，这是指它的至少一个组份相对于它的至少一个其它组份而言是异源的。表达盒还可以是天然存的，但是以可用于异源表达的重组形式获得。
不过，典型地，表达盒相对于宿主是异源的，即，表达盒的特定核酸序列不天然存在于宿主细胞中，因而必需通过转化事件引入宿主细胞或者宿主细胞的先祖细胞。核苷酸序列在表达盒中的表达可以受组成型启动子，或者仅在宿主细胞暴露于某些特定外部刺激时起始转录的诱导型启动子的调控。在多细胞生物，例如植物的情况下，启动子还可以是特定组织或器官、或发育阶段特异性的。
“基因”是位于基因组内的限定区域，其除了前述的编码核酸序列之外，还包括其它的，主要是调控性的，负责控制编码部分表达，也就是说转录和翻译的核酸序列。基因还可以包括其它5′和3′非翻译序列和终止序列。可以存在的进一步元件是例如内含子。
“感兴趣基因”是指任何当转移到植物以后，赋予植物所期望的特征的基因，所述特征例如抗生素抗性、病毒抗性、昆虫抗性、疾病抗性或者其它害虫抗性、杀虫剂耐受性、改善的营养价值、工业操作中改善的性能或者改变的繁殖能力。“感兴趣基因”还可以是被转移到植物中用于在植物中产生有商业价值的酶或代谢物的基因。
“肠蛋白酶”是在昆虫消化道中天然发现的蛋白酶。该蛋白酶通常参与摄入蛋白的消化。
“异源”核酸序列是与其被引入的宿主细胞非天然相关的核酸序列，包括非天然存在的多拷贝天然存在的核酸序列。
“同源”核酸序列是与其被引入的宿主细胞天然相关的核酸序列。
“同源重组”是同源核酸分子之间的核酸片段的彼此互换。
“杀虫”定义为能够控制昆虫，优选杀死它们的毒性生物活性。
当核酸序列编码与参照核酸序列所编码的多肽具有相同氨基酸序列的多肽时，该核酸序列与参照核酸序列“同类编码”。
“分离的”核酸分子或分离的毒素是通过人工，脱离天然环境存在，并因而不是天然产物的核酸分子或毒素。分离的核酸分子或毒素可能以纯化的形式存在，或者可能存在于非天然环境，例如重组宿主细胞中。
本发明的“修饰的Cry3A毒素”是指Cry3A来源的毒素，具有至少一个额外的可被靶标昆虫肠蛋白酶识别的、不天然存在于Cry3A毒素中的蛋白酶识别位点。修饰的Cry3A毒素不是天然存在的，并且通过人工方式，包括与苏云金芽孢杆菌中所发现的天然毒素不同的氨基酸序列。修饰的Cry3A毒素导致靶标昆虫的死亡率比Cry3A毒素所致的相同靶标昆虫的死亡率更高。
根据本发明的“修饰的cry3A基因”是指cry3A来源的基因，包括至少一个额外的不天然存在于未修饰的cry3A基因中的蛋白酶识别位点的编码序列。修饰的cry3A基因可以来自于天然cry3A基因，或者来自于合成cry3A基因。
“天然存在的蛋白酶识别位点”是Cry3A毒素内被非昆虫来源的蛋白酶或被来自Cry3A毒素敏感昆虫物种的蛋白酶或肠提取物切割的位置。例如，被胰蛋白酶和在敏感昆虫肠提取物中发现的蛋白酶识别的天然存在的蛋白酶识别位点，存在于推断的Cry3A毒素氨基酸序列(SEQ ID NO：2)的Arg-158和Asn-159之间。被胰凝乳蛋白酶识别的天然存在的蛋白酶识别位点，存在于推断的Cry3A毒素氨基酸序列(SEQ ID NO：2)的His-161和Ser-162之间以及Tyr-587和Tyr-588之间。
“核酸分子”或“核酸序列”是能够从任何来源分离的单链或双链DNA或RNA线性区段。在本发明的上下文中，核酸分子优选是DNA区段。
“植物”是处于任何发育阶段的任何植物，特别是种子植物。
“植物细胞”是植物的结构和生理单位，包括原生质体和细胞壁。植物细胞可能是分离的单细胞或者培养细胞的形式，或者作为更高级组织单位，例如，举例来说，植物组织、植物器官或全植物的一部分。
“植物细胞培养”是指培养植物单位例如，举例来说，原生质体、细胞培养细胞、植物组织中的细胞、花粉、花粉管、胚珠、胚囊、接合子以及处于各种发育阶段的胚。
“植物材料”是指叶、茎、根、花或花的一部分、果实、花粉、卵细胞、接合子、种子、插条、细胞或组织培养物，或者植物任何其它部分或产品。
“植物器官”是植物的有清楚和明显构造和分化的部分，例如根、茎、叶、花芽或胚。
如本文所用，“植物组织”是指组织成结构和功能单位的植物细胞群。包括任何种植(in planta)或培养的植物的组织。这个术语包括，但不限于，全植物、植物器官、植物种子、组织培养物以及任何组织成结构和/或功能单位的植物细胞群。该术语与或不与如上文所列的、或者以其它方式被这个定义所涵盖的任何特定植物组织类型结合使用，并不旨在排除任何其它类型的植物组织。
“启动子”是编码区上游的非翻译DNA序列，含RNA聚合酶结合位点并起始DNA转录。启动子区还可以包括其它起基因表达调节剂作用的元件。
“原生质体”是分离的没有细胞壁或仅有部分细胞壁的植物细胞。
“调控元件”是指参与控制核苷酸序列表达的序列。调控元件包括可操作地连接于感兴趣核苷酸序列的启动子和终止信号。它们典型地还包含核苷酸序列正确翻译所需的序列。
“取代”天然存在的蛋白酶识别位点-根据本发明，当额外的蛋白酶识别位点的插入消除了天然存在的蛋白酶识别位点的时候，则该额外的蛋白酶识别位点“取代”了天然存在的蛋白酶识别位点。例如，插入在Cry3A毒素推断的氨基酸序列(SEQ ID NO：2)Pro-154和Pro-160之间、消除了Arg-158和Asn-159残基的额外的蛋白酶识别位点“取代”了位于Cry3A毒素推断的氨基酸序列(SEQ ID NO：2)Arg-158和Asn-159之间的天然存在的胰蛋白酶识别位点。
“丝氨酸蛋白酶”描述了利用基于丝氨酸对靶标肽键亲核攻击的机制催化共价肽键水解的同组酶。丝氨酸蛋白酶是序列特异性的。也就是说，每个丝氨酸蛋白酶识别蛋白质内酶识别所发生的特异亚序列。
“靶标昆虫”是对Cry3A毒素几乎不或不敏感、并鉴定为用本发明技术控制的候选者的昆虫害虫物种。这种控制可通过许多手段实现，但最优选通过在转基因植物中表达本发明的核酸分子而实现。
“靶标昆虫肠蛋白酶”是在靶标昆虫中发现的蛋白酶，其识别位点可以被插入到Cry3A毒素中建立本发明的修饰的Cry3A毒素。
“转化”是向宿主细胞或生物引入异源核酸的方法。特别地，“转化”是指DNA分子稳定整合到感兴趣生物体的基因组内。
“转化的/转基因的/重组的”是指其中引入了异源核酸分子的宿主生物例如细菌或植物。核酸分子可以被稳定地整合到宿主的基因组中，或者核酸分子还可以作为染色体外分子存在。这种染色体外分子可以是自主复制的。转化的细胞、组织或植物被理解为不仅涵盖转化操作的终产物，而且涵盖了其转基因后代。“未转化的”、“未转基因的”、“未重组的”宿主是指野生型生物，例如不含异源核酸分子的细菌或植物。
“在”天然存在的蛋白酶识别位点“之内”-根据本发明，当额外的蛋白酶识别位点存在于位于天然存在的蛋白酶识别位点之前的氨基酸残基和之后的氨基酸残基之间的时候，则该额外的蛋白酶识别位点“在”天然存在的蛋白酶识别位点“之内”。例如，插入在Cry3A毒素推断的氨基酸序列(SEQ ID NO：2)Tyr-587和Tyr-588之间的额外的蛋白酶识别位点“在”位于Cry3A毒素推断的氨基酸序列(SEQ IDNO：2)Tyr-587和Tyr-588之间的天然存在的胰凝乳蛋白酶识别位点“之内”。在天然存在的蛋白酶识别位点之内插入额外的蛋白酶识别位点可以或可以不改变蛋白酶对天然存在的蛋白酶识别位点的识别。
核苷酸通过其碱基以下列标准缩写表示：腺嘌呤(A)、胞嘧啶(C)、胸腺嘧啶(T)和鸟嘌呤(G)。氨基酸类似地以下列标准缩写表示：丙氨酸(Ala；A)、精氨酸(Arg；R)、天冬酰胺(Asn；N)、天冬氨酸(Asp；D)、半胱氨酸(Cys；C)、谷氨酰胺(Gln；Q)、谷氨酸(Glu；E)、甘氨酸(Gly；G)、组氨酸(His；H)、异亮氨酸(Ile；I)、亮氨酸(Leu；L)、赖氨酸(Lys；K)、甲硫氨酸(Met；M)、苯丙氨酸(Phe；F)、脯氨酸(Pro；P)、丝氨酸(Ser；S)、苏氨酸(Thr；T)、色氨酸(Trp；W)、酪氨酸(Tyr；Y)和缬氨酸(Val；V)。
本发明涉及其表达产生修饰的Cry3A毒素的修饰的cry3A核酸序列，以及制备并利用修饰的Cry3A毒素控制昆虫害虫。修饰的cry3A核酸序列的表达产生可用来控制鞘翅目昆虫(coleopteran insects)例如西部玉米根虫和北部玉米根虫的修饰的Cry3A毒素。本发明的修饰的Cry3A毒素包括至少一个额外的不天然存在于Cry3A毒素中的蛋白酶识别位点。被靶标昆虫的肠蛋白酶识别的该额外的蛋白酶识别位点，被插入到Cry3A毒素中与天然存在的蛋白酶识别位点大致相同的位置。修饰的Cry3A毒素导致靶标昆虫的死亡率比Cry3A毒素所致的相同靶标昆虫的死亡率更高。优选地，对于Cry3A毒素导致不超过约30％的死亡率的靶标昆虫修饰的Cry3A毒素导致至少约50％的死亡率。
在一个优选的实施方式中，本发明涵盖了编码修饰的Cry3A毒素的分离的核酸分子，其中额外的蛋白酶识别位点被靶标昆虫的肠蛋白酶组织蛋白酶G识别。组织蛋白酶G活性被确定存在于靶标昆虫西部玉米根虫的肠中，如实施例2所述。优选地，用来确定组织蛋白酶G活性存在的底物氨基酸序列AAPF(SEQ ID NO：35)，被插入到根将本发明的Cry3A毒素中。根据本发明，还可以利用其它组织蛋白酶G识别位点，例如AAPM(SEQ ID NO：36)、AVPF(SEQ ID NO：37)、PFLF(SEQ ID NO：38)或其它如Tanaka等1985(Biochemistry 24：2040-2047)的方法所确定的组织蛋白酶G识别位点，该文献兹并入作为参考。可以利用靶标昆虫肠中鉴定的其它蛋白酶的蛋白酶识别位点，例如其它丝氨酸蛋白酶、半胱氨酸蛋白酶和天冬氨酸蛋白酶所识别的蛋白酶识别位点。这个实施方式中涵盖的优选丝氨酸蛋白酶包括胰蛋白酶、胰凝乳蛋白酶、羧肽酶、内肽酶和弹性蛋白酶。
在另一个优选的实施方式中，本发明涵盖了编码修饰的Cry3A毒素的分离的核酸分子，其中额外的蛋白酶识别位点被插入到Cry3A毒素结构域I中或者结构域III中或者结构域I和结构域III中。优选地，额外的蛋白酶识别位点被插入到Cry3A毒素结构域I、或者结构域III、或者结构域I和结构域III中取代天然存在的蛋白酶识别位点、邻近天然存在的蛋白酶识别位点或者在天然存在的蛋白酶识别位点之内的位置。这里具体示例的是编码包括组织蛋白酶G识别位点的修饰的Cry3A毒素的核酸分子，该位点被插入到未修饰的Cry3A毒素结构域I、或者结构域III、或者结构域I和结构域III中取代、邻近或者在天然存在的蛋白酶识别位点之内的位置。
制备编码修饰的Cry3A毒素的修饰的cry3A核酸分子的方法的具体示例的教导见实施例3。所属领域熟练技术人员将会意识到其它本领域已知的方法也可以用来将额外的蛋白酶识别位点插入到根据本发明的Cry3A毒素中。
在另一个优选的实施方式中，本发明涵盖了编码修饰的Cry3A毒素的分离的核酸分子，其中额外的蛋白酶识别位点被插入到结构域I中与SEQ ID NO：2氨基酸位154和162相应的氨基酸之间。优选地，额外的蛋白酶识别位点被插入到SEQ ID NO：2氨基酸位154和162之间或者SBQ ID NO：4氨基酸位107和115之间。在一个优选的实施方式中，额外的蛋白酶识别位点被插入到与SEQ ID NO：2氨基酸位154和160相应的氨基酸之间。优选地，额外的蛋白酶识别位点被插入到SEQ ID NO：2氨基酸位154和160之间或者SEQ ID NO：4氨基酸位107和113之间。本文具体示例的是命名为cry3A054(SEQ ID NO：6)的核酸分子，它编码修饰的Cry3A054毒素(SEQ ID NO：7)，该毒素包含被插入到结构域I中SEQ ID NO：4氨基酸位107和113之间的组织蛋白酶G识别位点。该组织蛋白酶G识别位点取代了天然存在的胰蛋白酶识别位点，并邻近天然存在的胰凝乳蛋白酶识别位点。当在异源宿主中表达时，SEQ ID NO：6的核酸分子产生抗西部玉米根虫和北部玉米根虫的昆虫控制活性，证明SEQ ID NO：6所示的核酸序列对于这样的昆虫控制活性是足够的。
在另一个优选的实施方式中，额外的蛋白酶识别位点被插入到结构域I中与SEQ ID NO：2氨基酸位154和158相应的氨基酸之间。优选地，额外的蛋白酶识别位点被插入到结构域I中SEQ ID NO：2的氨基酸位154和158之间或者SEQ ID NO：4的氨基酸位107和111之间。本文具体示例的是编码修饰的Cry3A055毒素(SEQ ID NO：9)、命名为cry3A055(SEQ ID NO：8)以及编码修饰的Cry3A085毒素(SEQ ID NO：11)、命名为cry3A085(SEQ ID NO：10)的核酸分子，包含被插入到结构域I中SEQ ID NO：4氨基酸位107和111之间的组织蛋白酶G识别位点。该组织蛋白酶G识别位点邻近天然存在的胰蛋白酶和胰凝乳蛋白酶识别位点。当在异源宿主中表达时，SEQ ID NO：8或SEQ IDNO：10的核酸分子产生抗西部玉米根虫和北部玉米根虫的昆虫控制活性，证明SEQ ID NO：8或SEQ ID NO：10所示的核酸序列对于这样的昆虫控制活性是足够的。
在优选的实施方式中，本发明涵盖了编码修饰的Cry3A毒素的分离的核酸分子，其中额外的蛋白酶识别位点被插入到结构域III中与SEQ ID NO：2氨基酸位583和589相应的氨基酸之间。优选地，额外的蛋白酶识别位点被插入到结构域III中SEQ ID NO：2氨基酸位583和589之间或者SEQ ID NO：4氨基酸位536和542之间。
在另一个优选的实施方式中，本发明涵盖了编码修饰的Cry3A毒素的分离的核酸分子，其中额外的蛋白酶识别位点被插入到结构域III中与SEQ ID NO：2氨基酸位583和588相应的氨基酸之间。优选地，额外的蛋白酶识别位点被插入到结构域III中SEQ ID NO：2氨基酸位583和588之间或者SEQ ID NO：4氨基酸位536和541之间。本文具体示例的是命名为cry3A082(SEQ ID NO：12)的核酸分子，它编码修饰的Cry3A082毒素(SEQ ID NO：13)，包含被插入到结构域III中SEQ ID NO：4氨基酸位536和541之间的组织蛋白酶G识别位点。该组织蛋白酶G识别位点取代了天然存在的胰凝乳蛋白酶识别位点。当在异源宿主中表达时，SEQ ID NO：12的核酸分子产生抗西部玉米根虫和北部玉米根虫的昆虫控制活性，证明SEQ ID NO：12所示的核酸序列对于这样的昆虫控制活性是足够的。
在另一个优选的实施方式中，额外的蛋白酶识别位点被插入到结构域III中与SEQ ID NO：2氨基酸位587和588相应的氨基酸之间。优选地，额外的蛋白酶识别位点被插入到结构域III中SEQ ID NO：2氨基酸位587和588之间或者SEQ ID NO：4氨基酸位540和541之间。本文具体示例的是命名为cry3A058(SEQ ID NO：14)的核酸分子，它编码修饰的Cry3A058毒素(SEQ ID NO：15)，包含被插入到结构域III中SEQ ID NO：4氨基酸位540和541之间的组织蛋白酶G识别位点。该组织蛋白酶G识别位点位于天然存在的胰凝乳蛋白酶识别位点之内。当在异源宿主中表达时，SEQ ID NO：14的核酸分子产生抗西部玉米根虫和北部玉米根虫的昆虫控制活性，证明SEQ ID NO：14所示的核酸序列对于这样的昆虫控制活性是足够的。
而在另一个优选的实施方式中，本发明涵盖了编码修饰的Cry3A毒素的分离的核酸分子，其中额外的蛋白酶识别位点被插入到结构域I中与SEQ ID NO：2的氨基酸位154和160相应的氨基酸之间以及结构域III中与SEQ ID NO：2的氨基酸位587和588相应的氨基酸之间。优选地，额外的蛋白酶识别位点被插入到结构域I中SEQ ID NO：2的氨基酸位154和160之间以及结构域III中SEQ ID NO：2的氨基酸位587和588之间或者结构域I中SEQ ID NO：4的氨基酸位107和113之间以及结构域III中SEQ ID NO：4的氨基酸位540和541之间。本文具体示例的是命名为cry3A057(SEQ ID NO：16)的核酸分子，它编码修饰的Cry3A057毒素(SEQ ID NO：17)，包含被插入到SEQ ID NO：4的结构域I中氨基酸位107和113之间以及结构域III中氨基酸位540和541之间的组织蛋白酶G识别位点。所述组织蛋白酶G识别位点取代了结构域I中天然存在的胰蛋白酶识别位点，并与其中天然存在的胰凝乳蛋白酶识别位点邻近，而且位于结构域III中天然存在的胰凝乳蛋白酶识别位点之内。当在异源宿主中表达时，SEQ ID NO：16的核酸分子产生抗西部玉米根虫和北部玉米根虫的昆虫控制活性，证明SEQ ID NO：16所示的核酸序列对于这样的昆虫控制活性是足够的。
而在另一个优选的实施方式中，额外的蛋白酶识别位点位于结构域I中与SEQ ID NO：2的氨基酸位154和158相应的氨基酸之间以及结构域III中与SEQ ID NO：2的氨基酸位587和588相应的氨基酸之间。优选地，额外的蛋白酶识别位点被插入到SEQ ID NO：2的结构域I中氨基酸位154和158之间以及结构域III中氨基酸位587和588之间或者SEQ ID NO：4的结构域I中氨基酸位107和111之间以及结构域III中氨基酸位540和541之间。本文具体示例的是命名为cry3A056(SEQ ID NO：18)的核酸分子，它编码修饰的Cry3A056毒素(SEQ ID NO：19)，包含被插入到SEQ ID NO：4的结构域I中氨基酸位107和111之间以及结构域III中氨基酸位540和541之间的组织蛋白酶G识别位点。所述组织蛋白酶G识别位点邻近结构域I中天然存在的胰蛋白酶和胰凝乳蛋白酶识别位点，并且位于结构域III中天然存在的胰凝乳蛋白酶识别位点之内。当在异源宿主中表达时，SEQID NO：18的核酸分子产生抗西部玉米根虫和北部玉米根虫的昆虫控制活性，证明SEQ ID NO：18所示的核酸序列对于这样的昆虫控制活性是足够的。
还有另一个优选的实施方式中，额外的蛋白酶识别位点位于结构域I中与SEQ ID NO：2的氨基酸位154和158相应的氨基酸之间以及结构域III中与SEQ ID NO：2的氨基酸位583和588相应的氨基酸之间。优选地，额外的蛋白酶识别位点被插入到SEQ ID NO：2的结构域I中氨基酸位154和158之间以及结构域III中氨基酸位583和588之间或者SEQ ID NO：4的结构域I中氨基酸位107和111之间以及结构域III中氨基酸位536和541之间。本文具体示例的是命名为cry3A083(SEQ ID NO：20)的核酸分子，它编码修饰的Cry3A083毒素(SEQ ID NO：21)，包含被插入到SEQ ID NO：4的结构域I中氨基酸编码107和111之间以及结构域III中氨基酸位536和541之间的组织蛋白酶G识别位点。所述组织蛋白酶G识别位点邻近结构域I中天然存在的胰蛋白酶和胰凝乳蛋白酶识别位点，并取代了结构域III中天然存在的胰凝乳蛋白酶识别位点。当在异源宿主中表达时，SEQ IDNO：20的核酸分子产生抗西部玉米根虫和北部玉米根虫的昆虫控制活性，证明SEQ ID NO：20所示的核酸序列对于这样的昆虫控制活性是足够的。
在优选的实施方式中，本发明的分离的核酸分子包含SEQ ID NO：6的核苷酸1-1791、SEQ ID NO：8的核苷酸1-1806、SEQ ID NO：10的核苷酸1-1812、SEQ ID NO：12的核苷酸1-1794、SEQ ID NO：14的核苷酸1-1818、SEQ ID NO：16的核苷酸1-1812、SEQ ID NO：18的核苷酸1-1791、以及SEQ ID NO：20的核苷酸1-1818。
在另一个优选的实施方式中，本发明涵盖了编码包含SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11、SEQ ID NO：13、SEQ ID NO：15、SEQ ID NO：17、SEQ ID NO：19或SEQ ID NO：21中所示的氨基酸序列的修饰的Cry3A毒素的分离的核酸分子。
本发明还涵盖了包含本发明的核酸序列的重组载体。在这样的载体中，核酸序列优选包含在包括该核苷酸序列在能够表达该核苷酸序列的宿主细胞中表达所用的调控元件的表达盒中。这种调控元件通常包括启动子和终止信号，并优选还包括允许本发明的核酸序列所编码的多肽有效翻译的元件。包含核酸序列的载体通常能够在特定宿主细胞中复制，优选作为染色体外分子，并因而被用来在宿主细胞中扩增本发明的核酸序列。在一个实施方式中，用于这种载体的宿主细胞是微生物，例如细菌，特别是苏云金芽孢杆菌(Bacillus thuringiensis)或大肠杆菌(E.coli)。在另一个实施方式中，用于这样的重组载体的宿主细胞是内寄生菌(endophytes)或体表寄生菌(epiphytes)。优选的用于这种载体的宿主细胞是真核细胞，例如植物细胞。植物细胞例如玉米细胞是最优选的宿主细胞。在另一个优选的实施方式中，这样的载体是病毒载体，并用来在特定的宿主细胞，例如昆虫细胞或植物细胞中复制核苷酸序列。重组载体还可用来将本发明的核苷酸序列转化到宿主细胞中，从而核苷酸序列被稳定地整合到这样的宿主细胞的DNA中。在一个实施方式中，这样的宿主细胞是原核细胞。在一个优选的实施方式中，这样的宿主细胞是真核细胞，如植物细胞。在一个最优选的实施方式中，宿主细胞是植物细胞，例如玉米细胞。
在另一个方面，本发明涵盖了通过表达本发明的核酸分子所产生的修饰的Cry3A毒素。
在优选的实施方式中，本发明的修饰的Cry3A毒素包括由本发明的核苷酸序列编码的多肽。在进一步优选的实施方式中，修饰的Cry3A毒素通过表达包含SEQ ID NO：6的核苷酸1-1791、SEQ ID NO：8的核苷酸1-1806、SEQ ID NO：10的核苷酸1-1812、SEQ ID NO：12的核苷酸1-1794、SEQ ID NO：14的核苷酸1-1818、SEQ ID NO：16的核苷酸1-1812、SEQ ID NO：18的核苷酸1-1791、和SEQ ID NO：20的核苷酸1-1818的核酸分子产生。
在优选的实施方式中，本发明的修饰的Cry3A毒素包括SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11、SEQ ID NO：13、SEQ ID NO：15、SEQ ID NO：17、SEQ ID NO：19或SEQ ID NO：21中所示的氨基酸序列。
本发明修饰的Cry3A毒素在生物测定中进行抗昆虫害虫测试的时候具有昆虫控制活性。在另一个优选的实施方式中，本发明修饰的Cry3A毒素具有抗鞘翅目昆虫，优选抗西部玉米根虫和北部玉米根虫的活性。本发明修饰的Cry3A毒素的昆虫控制特性进一步在实施例4和6中举例说明。
本发明还涵盖了包含有效昆虫控制量的根据本发明的修饰的Cry3A毒素的组合物。
在另一个优选的实施方式中，本发明涵盖了制备具有抗昆虫活性的修饰的Cry3A毒素的方法，包括：(a)获得包含嵌合基因的宿主细胞，该基因本身包括可操作地连接于本发明的核酸分子的异源启动子序列；和(b)在该转基因宿主细胞中表达核酸分子，这产生至少一个具有抗昆虫活性的修饰的Cry3A毒素。
在进一步优选的实施方式中，本发明涵盖了制备昆虫抗性转基因植物的方法，包括向转基因植物引入本发明的核酸分子，其中核酸分子可以以控制昆虫有效的量在转基因植物中表达。在优选的实施方式中，昆虫是鞘翅目昆虫，优选西部玉米根虫和北部玉米根虫。
而在进一步优选的实施方式中，本发明涵盖了控制昆虫的方法，包括向昆虫递送有效量的本发明的修饰的Cry3A毒素。根据这个实施方式，昆虫是鞘翅目昆虫，优选西部玉米根虫和北部玉米根虫。优选地，修饰的Cry3A毒素通过口服递送给昆虫。在一个优选的方面，毒素通过包含表达本发明的修饰的Cry3A毒素的核酸序列的转基因植物口服递送。
本发明还涵盖了制作修饰的Cry3A毒素的方法，包括：(a)获得编码Cry3A毒素的cry3A基因；(b)鉴定靶标昆虫的肠蛋白酶；(c)获得编码所述肠蛋白酶识别位点的核苷酸序列；(d)将(c)中核苷酸序列插入到所述cry3A毒素基因的结构域I中或者结构域III中或者结构域I和结构域III中取代编码天然存在的蛋白酶识别位点的核苷酸序列、在编码天然存在的蛋白酶识别位点的核苷酸序列之内、或者与编码天然存在的蛋白酶识别位点的核苷酸序列邻近的位置，从而建立修饰的cry3A毒素基因；(e)将修饰的cry3A毒素基因插入到表达盒中；(f)在非人宿主细胞中表达修饰的cry3A毒素基因，导致宿主细胞产生修饰的Cry3A毒素；和(g)对修饰的Cry3A毒素进行抗靶标昆虫的生物测定，其导致靶标昆虫比Cry3A毒素所致的死亡率更高的死亡率。在优选的实施方式中，当Cry3A毒素导致不超过大约30％的死亡率时，修饰的Cry3A毒素导致靶标昆虫至少大约50％的死亡率。
本发明进一步涵盖了控制昆虫的方法，其中转基因植物进一步包括编码第二杀虫素的第二核酸序列或核酸序列组。特别优选的第二核酸序列是编码δ-内毒素的序列，编码植物杀虫蛋白毒素的序列(公开在美国专利5,849,870和5,877,012中，兹并入作为参考)，或者编码产生非蛋白性杀虫素的途径的序列。
在进一步的实施方式中，本发明的核苷酸序列可以通过在称作体外重组或者DNA改组的技术中掺入随机突变而被进一步修饰。这个技术描述在Stemmer等Nature 370：389-391(1994)以及美国专利5,605,793中，兹被并入作为参考。基于本发明原始的核苷酸序列，制备了核苷酸序列的数百万突变拷贝，并收集具有改善的特性的变体，例如增加的杀虫活性，增强的稳定性，或者不同的特异性或靶标昆虫害虫范围。该方法包括从包括本发明的核苷酸序列的模板双链多核苷酸形成突变的双链多核苷酸，其中模板双链多核苷酸被切割成希望大小的双链随机片段群，并且包括步骤：向所得的双链随机片段群中添加一种或多种单链或双链寡核苷酸，其中所述寡核苷酸包含与双链模板多核苷酸相同的区域和异源的区域(area of heterology)；将所得的双链随机片段和寡核苷酸的混合物变性为单链片段；将所得的单链片段群与聚合酶在导致所述单链片段在所述相同区域退火形成退火片段对的条件下温育，所述相同区域足以使一对中的一个成员引发另一个成员的复制，藉此形成突变的双链多核苷酸；以及再重复第二和第三步至少两个更多的循环，其中在下一个循环第二步中所得的混合物包括来自前一个循环第三步的突变的双链多核苷酸，并且该下一个循环形成了进一步突变的双链多核苷酸。在优选的实施方式中，双链随机片段群中单种双链随机片段的浓度小于总DNA的1％重量。在进一步优选的实施方式中，模板双链多核苷酸包括至少大约100种多核苷酸。在另一个优选的实施方式中，双链随机片段的大小从大约5bp到5kb。在进一步优选的实施方式中，该方法的第四步包括重复第二和第三步至少10个循环。
核苷酸序列在异源微生物宿主中的表达
作为生物学昆虫控制剂，杀虫的修饰的Cry3A毒素通过在能够表达该核苷酸序列的异源宿主细胞中表达核苷酸序列而产生。在第一个实施方式中，制备了包含本发明的核苷酸序列的修饰的苏云金芽孢杆菌(B.thringiensis)细胞。这种修饰包括现有调控元件的突变或缺失，从而导致核苷酸序列改变的表达，或者掺入控制核苷酸序列表达的新的调控元件。在另一个实施方式中，通过插入到染色体中，或者通过引入含核苷酸序列的染色体外复制分子向苏云金芽孢杆菌(Bacillus thuringiensis)细胞中添加额外拷贝的一种或多种所述核苷酸序列。
在另一个实施方式中，至少一个本发明的核苷酸序列被插入到包含启动子和终止信号的合适的表达盒中。
核苷酸序列的表达是组成型的，或者利用响应各种类型的刺激引发转录的诱导型启动子。在优选的实施方式中，表达毒素的细胞是微生物，例如病毒、细菌或真菌。在优选的实施方式中，病毒，例如杆状病毒，在基因组中含有本发明的核苷酸序列，并在感染合适的适于病毒复制和核苷酸表达的真核细胞之后表达大量相应的杀虫毒素。这样制备的杀虫毒素被用作为杀虫剂。可替代地，经工程改造包括所述核苷酸序列的杆状病毒被用于体内感染昆虫，并通过表达杀虫蛋白或者通过病毒感染联合杀虫蛋白表达而杀死它们。
细菌细胞也是表达本发明的核苷酸序列的宿主。在优选的实施方式中，利用能够在植物组织内生活和复制的非致病共生细菌，即所谓的内寄生菌，或者能够在叶际或根际建群的非致病共生细菌，即所谓的体表寄生菌。这样的细菌包括土壤杆菌属(Agrobacterium)、产碱菌属(Alcaligenes)、固氮螺菌属(Azospirillum)、固氮菌属(Azotobacter)、芽孢杆菌属(Bacillus)、棍状杆菌属(Clavibacter)、肠杆菌属(Enterobacter)、欧文菌属(Erwinia)、黄杆菌属(Flavobacter)、克雷伯氏菌属(Klebsiella)、假单胞菌属(Pseudomonas)、根瘤菌属(Rhizobium)、沙雷菌属(Serratia)、链霉菌属(Streptomyces)和黄单胞菌属(Xanthomonas)的细菌。共生真菌，例如木霉属(Trichoderma)和粘帚霉属(Gliocladium)也是用于相同目的而表达本发明核苷酸序列的可能的宿主。
这些基因操作技术是不同宿主特异的，并且是本领域公知的。例如，表达载体pKK223-3和pKK223-2可用来在大肠杆菌中(E.coli)表达以或者是转录融合或者是翻译融合的方式，在tac或trc启动子后面的异源基因。为表达编码多个ORFs(开放读框)的操纵子，最简单的操作是将操纵子以转录融合的方式插入到载体例如pKK223-3中，允许利用异源基因的同源核糖体结合位点。
在革兰氏阳性物种例如芽孢杆菌(Bacillus)中过表达的技术也是本领域公知的，并可用于本发明的上下文中(Quax等In：Industrial Microorganisms：Basic and Applied MolecularGenetics，编者Baltz等，American Society for Microbiology，Washington(1993))。可替代的过表达系统有赖于，例如酵母载体，并包括毕持酵母属(Pichia)、酵母属(Saccharomyces)和克鲁维酵母属(Kluyveromyces)的利用(Sreekrishna，In：Industrialmicroorganisms：basic and applied molecular genetics，Baltz，Hegeman，和Skatrud编，American Society for Microbiology，Washington(1993)；Dequin和Barre，Biotechnology L2：173-177(1994)；van den Berg等，Biotechnology 8：135-139(1990))。
植物转化
在特别优选的实施方式中，至少一个本发明杀虫性修饰的Cry3A毒素在高等生物，例如植物中表达。在这种情况下，转基因植物表达有效量的修饰的Cry3A毒素，保护自身免受昆虫害虫侵害。当昆虫开始摄食这种转基因植物时，它还摄入了表达的修饰的Cry3A毒素。这将制止昆虫进一步噬咬植物组织或者可能甚至伤害或杀死昆虫。本发明的核苷酸序列被插入到表达盒中，其接着优选稳定地被整合到所述植物的基因组中。在另一个优选的实施方式中，核苷酸序列被包含在非致病性自主复制的病毒中。根据本发明转化的植物可以是单子叶或双子叶的，并且包括但不限于，玉米、小麦、大麦、黑麦、甘薯、菜豆、豌豆、菊苣、莴苣、卷心菜、花椰菜、嫩茎花椰菜、芜菁、萝卜、菠菜、芦笋、洋葱、大蒜、胡椒、芹菜、倭瓜、南瓜、大麻、夏南瓜、苹果、梨、温柏、甜瓜、李、樱桃、桃、油桃、杏、草莓、葡萄、覆盆子、黑莓、凤梨、鳄梨、番木瓜、芒果、香蕉、大豆、番茄、高粱、甘蔗、甜菜、向日葵、菜籽油菜、苜蓿、烟草、胡萝卜、棉花、紫花苜蓿、稻、马铃薯、茄子、黄瓜、鼠耳芥属(Arabidopsis)以及木本植物例如针叶树和落叶树。
一旦预期的核苷酸序列被转化到特定的植物物种中，利用传统的育种技术，它可以在该物种内繁殖或者转移到相同物种的其它品种中，特别包括商业品种。
本发明的核苷酸序列优选在转基因植物中表达，从而导致在转基因植物中生物合成相应的修饰的Cry3A毒素。
这样产生了对昆虫具有增强的抗性的转基因植物。为了其在转基因植物中表达，本发明的核苷酸序列可能需要其它修饰和优化。虽然在许多情况下，来自微生物生物的基因能够高水平地在植物中表达而无需修饰，转基因植物中的低水平表达可能由于微生物核苷酸序列具有不是植物优选的密码子而产生。
本领域公知所有的生物体具有特定的密码子利用偏好，而且本发明所述的核苷酸序列的密码子可以被改变以符合植物的偏好，同时维持其编码的氨基酸。此外，植物中的高表达最能从具有至少大约35％，优选大于大约45％，更优选大于大约50％，并最优选大于大约60％的GC含量的编码序列取得。具有低GC含量的微生物核苷酸序列在植物中弱表达，可能归因于存在可能使信息不稳定的ATTTA基序，以及可导致不恰当多聚腺苷酰化的AATAAA基序。虽然优选的基因序列可能在单子叶植物和双子叶植物的物种中都能充分地表达，序列可以被修饰以解决单子叶植物或双子叶植物特定的密码子偏好和GC含量偏好，因为已经证明这些偏好是不同的(Murray等Nucl.Acids Res.17：477-498(1989))。另外，核苷酸序列被进行可能导致信息截断的不正常剪接位点存在的筛选。所有需要在核苷酸序列内进行的变化，例如上文所述的那些，可利用公开的专利申请EP 0385962(to Monsanto)、EP 0359472(to Lubrizol)以及WO 93/07278(to Ciba-Geigy)中描述的方法，利用定点突变、PCR以及合成基因构建的公知技术进行。
在本发明的一个实施方式中，根据美国专利5,625,136中公开的程序制备了cry3A基因，该文献兹并入作为参考。在这个程序中，使用了玉米偏好的密码子，即玉米中最频繁地编码某氨基酸的单一密码子。特定氨基酸的玉米偏好密码子可以从例如得自于玉米的已知基因序列获得。在Murray等，Nucleic Acids Research 17：477-498(1989)中建立了来自玉米植物的28个基因的玉米密码子使用，兹将其公开的内容并入作为参考。用玉米优化密码子制得的合成序列示于SEQ ID NO：3中。
以这种方式，核苷酸序列可以被优化在任何植物中表达。基因序列的全部或任何一部分公知可以被优化或合成。也就是说，还可以利用合成或部分优化的序列。
为有效起始翻译，与起始甲硫氨酸邻近的序列可能需要修饰。例如，它们可以通过包含已知在植物中有效的序列加以修饰。Joshi已提出一种合适的植物共有序列(NAR 15：6643-6653(1987))而Clonetech提出了另一个共有序列翻译起始子(1993/1994 catalog，page 210)。这些共有序列适用于本发明的核苷酸序列。该序列被掺入包含核苷酸序列的构建物中，一直到并包括ATG(同时保留第二个氨基酸未修饰)，或者替代地，一直到并包括ATG之后的GTC(具有修饰转基因的第二个氨基酸的可能性)。
核苷酸序列在转基因植物中的表达通过在植物中起作用的启动子驱动。启动子的选择将视表达的时间和空间需求而变，并且还取决于靶标物种。从而，优选本发明的核苷酸序列在叶中、在茎或杆中、在穗中、在花序中(如穗状花序、圆锥花序、穗轴等)、在根和/或幼苗中表达。不过，在许多情况下，寻求防备不止一种类型的昆虫害虫的保护，因而期望在多个组织中表达。虽然来自双子叶植物的许多启动子已经证明能够在单子叶植物中有效，并且反之亦然，理想地，选择双子叶植物启动子用于双子叶植物的表达，而单子叶植物启动子用于单子叶植物的表达。不过，对于所选的启动子的起源没有限制；只要它们可有效驱动核苷酸序列在期望细胞中表达就足够了。
优选的组成型表达的启动子包括来自编码肌动蛋白或泛素的基因的启动子和CaMV 35S和19S启动子。本发明的核苷酸序列还可以在化学调节的启动子的调控下表达。这使得杀虫的修饰的Cry3A毒素仅仅在作物植物用诱导化学制品处理的时候合成。优选的基因表达化学诱导技术在公开的专利申请EP 0 332 104(to Ciba-Geigy)和美国专利5,614,395中有详细描述。优选的化学诱导启动子是烟草PR-1a启动子。
优选的启动子种类是创伤可诱导的。已经描述了数目众多的在创伤部位以及在植物病原体感染部位表达的启动子。理想地，这样的启动子应当仅在感染部位局部有活性，并且通过这种方式，杀虫的修饰的Cry3A毒素仅在需要合成杀虫的修饰的Cry3A毒素以杀死入侵的昆虫害虫的细胞中积累。优选的这类启动子包括那些由Stanford等Mol.Gen.Genet.215：200-208(1989)、Xu等Plant Molec.Biol.22：573-588(1993)、Logemann等Plant Cell 1：151-158(1989)、Rohrmeier & Lehle，Plant Molec.Biol.22：783-792(1993)、Firek等Plant Molec.Biol.22：129-142(1993)以及Warner等Plant J.3：191-201(1993)等描述的启动子。
可用于在植物，优选玉米中表达修饰的Cry3A毒素基因的组织特异性或组织偏好性启动子，是那些指导在根、髓、叶或花粉，特别是在根中表达的启动子。这样的启动子，例如那些从PEPC或trpA分离的启动子，公开在美国专利No.5,625,136中，或者从MTL分离的启动子，公开在美国专利No.5,466,785中。兹将两个美国专利都全文并入作为参考。
进一步优选的实施方式是以创伤可诱导或病原体感染可诱导方式表达核苷酸序列的转基因植物。
除了启动子，还存在许多转录终止子可被用来使用本发明修饰的Cry3A毒素基因构建嵌合基因。转录终止子负责转基因之外的转录终止和它正确的多聚腺苷酰化。合适的转录终止子以及那些公知在植物中起作用的终止子包括CaMV 35S终止子、tm1终止子、胭脂碱合成酶终止子、豌豆rbcS E9终止子及其它本领域公知的终止子。它们可用于单子叶植物和双子叶植物中。任何已知在植物中起作用的可用启动子都可用于本发明的上下文中。
无数其它序列可以被掺入本发明所述的表达盒中。这包括已经被证明增强表达的序列例如内含子序列(如来自Adhl和bronzel的)以及病毒前导序列(如来自TMV、MCMV和AMV的)。
可能优选将本发明的核苷酸序列的表达靶向植物中不同的细胞定位。在一些情况下，可能期望定位在细胞溶质中，而在其它情况下，可能优选定位于某些亚细胞细胞器。转基因编码的酶的亚细胞定位是利用本领域公知的技术进行的。典型地，对编码来自已知的细胞器官靶向基因产物的靶向肽的DNA进行操作，并融合到核苷酸序列的上游。已知许多这样的叶绿体靶向序列，并已经证明了它们在异源构建物中起作用。本发明的核苷酸序列的表达还可被靶向宿主细胞的内质网膜或者液泡。实现这点的技术是本领域公知的。
适于植物转化的载体在本说明书别处描述了。对于土壤农杆菌介导的转移，双元载体或者携带至少一个T-DNA边界序列的载体是适当的，而对于直接基因转移，任何载体都适宜，并且可能优选仅含感兴趣构建物的线性DNA。对于直接基因转移，可以利用单个DNA种类的转化或者共转化(Schocher等Biotechnology 4：1093-1096(1986))。对于直接基因转移和土壤农杆菌介导的转移，转化通常(但不是必需)用可能提供抗生素(卡那霉素、潮霉素或氨甲蝶呤)或者除草剂(basta)抗性的选择性标记进行。包含本发明的修饰的Cry3A毒素的植物转化载体还可能如美国专利5,767,378和5,994,629中所述，包含提供转基因植物正向选择的基因(如磷酸甘露糖异构酶；PMI)，该文献兹并入作为参考。不过，选择性标记的选择对于本发明不是关键性的。
在另一个实施方式中，本发明的核苷酸序列被直接转化到质体基因组中。质体转化的主要优势在于质体一般无需大量的密码子优化就能够表达细菌基因，而且质体能够在单个启动子的控制下表达多个开放读框。质体转化技术在美国专利No.5,451,513、5,545,817和5,545,818、PCT申请no.WO 95/16783以及McBride等(1994)Proc.Nati.Acad.Sci.USA 91，7301-7305中被广泛地描述。叶绿体转化的基本技术包括将位于选择性标记连同感兴趣基因两侧的克隆的质体DNA区一起引入到合适的靶组织中，这通过例如biolistics(生物轰击)或原生质体转化(如氯化钙或PEG介导的转化)来进行。1到1.5kb的侧翼区，命名为靶向序列，促进与质体基因组的同源重组并因而允许取代或修饰质体基因组的特定区域。最初利用赋予壮观霉素和/或链霉素抗性的叶绿体16SrRNA和rps12基因点突变作为选择性标记(Svab，Z，Hajdukiewicz，P.，和Maliga，P.(1990)Proc.Nati.Acad.Sci.USA 87，8526-8530；Staub，J.M.，and Maliga，P.(1992)Plant Cell 4，39-45)。这以大约每100次靶叶轰击1个的频率产生稳定的同质转化株。这些标记之间克隆位点的存在允许建立引入外来基因的质体靶向载体(Staub，J.M.，and Maliga，P.(1993)EMBO J.12，601-606)。转化频率的大幅增长通过用显性选择标记，编码壮观霉素-cletoxifying酶氨基糖苷-3′-腺苷酰转移酶的细菌aadA基因替代隐性rRNA或者r-蛋白抗生素抗性基因获得(Svab，Z.，and Maliga，P.(1993)Proc.Natl.Acad.Sci.USA 90，913-917)。以前，这个标记被成功地用于绿藻莱因哈德衣藻(Chlamydomonas reinhardtii)质体基因组的高频转化(Goldschmidt-Clermont，M.(1991)Nucl.Acids Res.19：4083-4089)。其它可用于质体转化的选择性标记是本领域公知的，并涵盖在本发明的范围之内。典型地，转化后大约需要15-20个细胞分裂循环达到同质状态。质体表达，其中基因通过同源重组被插入到每个植物细胞中存在的所有数千个环状质体基因组拷贝中，利用了超越核表达的基因的庞大的拷贝数目优势，使得表达水平能够容易地超越总可溶性植物蛋白的10％。在优选的实施方式中，本发明的核苷酸序列被插入到质体靶向载体中并转化到期望的植物宿主质体基因组中。获得含有本发明的核苷酸序列的质体基因组同质的植物，并且优选能够高表达所述核苷酸序列。
昆虫控制素的联合
本发明修饰的Cry3A毒素可以与Btδ-内毒素或其它杀虫素(pesticidal principles)联合使用以增加害虫靶标范围。此外，本发明修饰的Cry3A毒素与Btδ-内毒素或其它具有不同性质的杀虫素联合使用具有预防和/或管理昆虫抗性的独有的实用性。
其它杀虫素包括，例如，凝集素、α-淀粉酶、过氧化物酶和胆固醇氧化酶。植物杀虫蛋白基因，例如象美国专利No.5,889,174中公开的vip1A(a)和vip2A(a)(兹并入作为参考)，也可用于本发明。
这种不止一个杀虫素在同一个转基因植物中的共表达可通过基因工程改造植物，使之含有并表达所有的必要基因而实现。可替代地，植物亲本1，可被基因工程改造表达本发明的基因。第二植物亲本2，可被基因工程改造表达补充的昆虫控制素。通过将亲本1和亲本2杂交，获得表达所有引入亲本1和2的基因的子代植物。
本发明的转基因种子还可以用如美国专利Nos.5,849,320和5,876,739中所述的杀虫种子包衣处理，该文献兹并入作为参考。当杀虫种子包衣和本发明的转基因种子具有抗相同靶标昆虫的活性时，这种组合可用于(i)增强本发明的修饰的Cry3A毒素抗靶标昆虫的活性的方法以及(ii)通过提供抗靶标昆虫的第二作用机制，防止对本发明修饰的Cry3A毒素形成抗性的方法之中。从而，本发明提供了增强抗靶标昆虫(例如玉米根虫)活性或防止靶标昆虫形成抗性的方法，包括向包含一种或多种本发明的修饰的Cry3A毒素的转基因植物种子施用杀虫种子包衣。
即使杀虫种子包衣具有抗不同昆虫的活性，该杀虫种子衣料可用来扩大昆虫控制范围，例如通过向本发明具有抗鞘翅目昆虫活性的转基因种子添加具有抗鳞翅目昆虫活性的杀虫种子包衣，产生的被包衣的转基因种可控制鳞翅目和鞘翅目两种昆虫害虫。
实施例
本发明将通过参考下列详细的实施例进一步加以说明。这些实施例仅仅是为了举例说明的目的而提供的，而且并不旨在限制，除非另外指明。这里所用的标准重组DNA和分子克隆技术是本领域公知的，并由J.Sambrook，等，Molecular Cloning：A Laboratory Manual，3d Ed.，Cold Spring Harbor，NY：Cold Spring Harbor LaboratoryPress(2001)、由T.J.Silhavy，M.L.Berman，和L.W.Enquist，Experiments with Gene Fusions，Cold Spring Harbor Laboratory，Cold Spring Harbor，NY(1984)以及由Ausubel，F.M.等，CurrentProtocols in Molecular Biology，New York，John Wiley and SonsInc.，(1988)、Reiter，等，Methods in Arabidopsis Research，World Scientific Press(1992)、以及Schultz等，Plant MolecularBiology Manual，Kluwer Academic Publishers(1998)描述。
实施例1：玉米优化cry3A基因的构建
玉米优化cry3A基因根据美国专利5,625,136所公开的程序制备。在这个程序中，使用了玉米偏好的密码子，即玉米中最频繁地编码某氨基酸的单一密码子。特定氨基酸的玉米偏好密码子得自于玉米中的已知基因序列。在Murray等，Nucleic Acids Research 17：477-498(1989)中建立了来自玉米植物的28个基因的玉米密码子使用。用玉米优化密码子制备的合成序列示于SEQ ID NO：3中。
实施例2：西部玉米根虫肠中组织蛋白酶G酶活性的鉴定
西部玉米根虫肠中组织蛋白酶G-样(丝氨酸蛋白酶)和组织蛋白酶B-样(半胱氨酸蛋白酶)酶活性利用比色底物测量。每1ml反应物含有5个匀浆的西部玉米根虫第3龄期的中肠和1mg溶于反应缓冲液中的底物(10mM Tris，5mM NaCl，0.01M DTT，pH7.5)。测试的组织蛋白酶G底物是Ala-Ala-Pro-Phe(SEQ ID NO：35)-pNA和组织蛋白酶B底物是Arg-Arg-pNA。反应物于28℃温育1小时。比较处理组与对照组中可指示蛋白酶识别合适底物效率的黄色形成的强度。如果没有颜色或者检测到轻微的本底颜色，则反应被评定为阴性(-)。高于本底25％、50％、75％或100％的反应分别评定为+、++、+++或++++。酶分析结果示于下表。
表1
| 反应 | 产物颜色强度 |
| 仅有WCR(西部玉米根虫)肠 | - |
| 仅有组织蛋白酶B底物 | - |
| 仅有组织蛋白酶G底物 | - |
| WCR肠+组织蛋白酶B底物 | + |
| WCR肠+组织蛋白酶G底物 | +++ |
这是首次在西部玉米根虫肠中鉴定到丝氨酸蛋白酶组织蛋白酶G活性。较之于组织蛋白酶B半胱氨酸蛋白酶活性，西部玉米根虫肠明显具有较强的组织蛋白酶G丝氨酸蛋白酶活性。选择AAPF序列(SEQ IDNO：35)作为组织蛋白酶G蛋白酶的识别位点，用于建立本发明的修饰的Cry3A毒素。
实施例3：修饰的cry3A基因的构建
在结构域I中、结构域III中或者结构域I和结构域III中包含编码组织蛋白酶G识别位点的核苷酸序列的修饰的cry3A基因利用重叠PCR制备。包含在质粒pCIB6850(SEQ ID NO：5)中的玉米优化cry3A基因(SEQ ID NO：2)被用作为起始模板。制备了8个编码修饰的Cry3A毒素的经修饰cry3A基因构建物：cry3A054、cry3A055和cry3A085在结构域I包含组织蛋白酶G识别位点编码序列；cry3A058、cry3A082在结构域III包含组织蛋白酶G识别位点编码序列；而cry3A056、cry3A057、cry3A083在结构域I和结构域III中包含组织蛋白酶G识别位点编码序列。这8个修饰的cry3A基因以及它们编码的修饰的Cry3A毒素描述如下：
包含在pCMS054中的cry3A054
cry3A054(SEQ ID NO：6)包括编码修饰的Cry3A毒素的核苷酸序列。利用3个重叠PCR引物对将编码组织蛋白酶G识别位点的核苷酸序列插入到未修饰的玉米优化cry3A中：
1.BamExt1-5’-GGATCCACCATGACGGCCGAC-3’ (SEQ ID NO：22)
AAPFtail3-5’-GAACGGTGCAGCGGGGTTCTTCTGCCAGC-3’ (SEQ ID NO：23)
2.Tail5mod-5’-GCTGCACCGTTCCCCCACAGCCAGGGCCG-3’ (SEQ ID NO：24)
XbaIExt2-5’-TCTAGACCCACGTTGTACCAC-3’ (SEQ ID NO：25)
3.BamExt1-5’-GGATCCACCATGACGGCCGAC-3’ (SEQ ID NO：22)
XbaIExt2-5’-TCTAGACCCACGTTGTACCAC-3’ (SEQ ID NO：25)
引物对1和引物对2产生两个独特的PCR产物。然后将这些产物等份混合，并利用引物对3连接该产物以产生一个PCR片断，它被克隆回初始的pCIB6850模板中。然后将该修饰的cry3A054基因转移至pBluescript(Stratagene)中。所得的质粒被命名为pCMS054且包含cry3A054基因(SEQ ID NO：6)。
修饰的Cry3A054毒素(SEQ ID NO：7)由包含在pCMS054中的修饰的cry3A基因编码，具有组织蛋白酶G识别位点，后者包含氨基酸序列AAPF(SEQ ID NO：35)，插入在未修饰的Cry3A毒素(SEQ ID NO：4)氨基酸107和113之间的结构域I中。该组织蛋白酶G识别位点取代了天然存在的胰蛋白酶识别位点，并且邻近天然存在的胰凝乳蛋白酶识别位点。
包含在pCMS055中的cry3A055
cry3A055(SEQ ID NO：8)包括编码修饰的Cry3A毒素的核苷酸序列。利用3个重叠PCR引物对将编码组织蛋白酶G识别位点的核苷酸序列插入到未修饰的玉米优化cry3A中：
1.BamExt1-5’-GGATCCACCATGACGGCCGAC-3’ (SEQ ID NO：22)
AAPFtail3-5’-GAACGGTGCAGCGGGGTTCTTCTGCCAGC-3’ (SEQ ID NO：23)
2.AAPFtail4-5’-GCTGCACCGTTCCGCAACCCCCACAGCCA-3’ (SEQ ID NO：26)
XbaIExt2-5’-TCTAGACCCACGTTGTACCAC-3’ (SEQ ID NO：25)
3.BamExt1-5’-GGATCCACCATGACGGCCGAC-3’ (SEQ ID NO：22)
XbaIExt2-5’-TCTAGACCCACGTTGTACCAC-3’ (SEQ ID NO：25)
引物对1和引物对2产生两个独特的PCR产物。然后将这些产物等份混合，并利用引物对3连接该产物以产生一个PCR片断，它被克隆回初始的pCIB6850模板中。然后将该修饰的cry3A055基因转移至pBluescript(Stratagene)中。所得的质粒被命名为pCMS055且包含cry3A055基因(SEQ ID NO：8)。
修饰的Cry3A055毒素(SEQ ID NO：9)由包含在pCMS055中的修饰的cry3A基因编码，具有组织蛋白酶G识别位点，后者包含氨基酸序列AAPF(SEQ ID NO：35)，插入在未修饰的Cry3A毒素(SEQ ID NO：4)氨基酸107和111之间的结构域I中。该组织蛋白酶G识别位点邻近天然存在的胰蛋白酶和胰凝乳蛋白酶识别位点。
包含在pCMS058中的cry3A058
cry3A058(SEQ ID NO：14)包括编码修饰的Cry3A毒素的核苷酸序列。利用3个重叠PCR引物对将编码组织蛋白酶G识别位点的核苷酸序列插入到未修饰的玉米优化cry3A中：
1.SalExt-5’-GAGCGTCGACTTCTTCAAC-3’ (SEQ ID NO：27)
AAPF-Y2-5’-GAACGGGTGCAGCGTATTGGTTGAAGGGGGC-3’ (SEQ ID NO：28)
2.AAPF-Y1-5’-GCTGCACCGTTCTACTTCGACAAGACCATC-3’ (SEQ ID NO：29)
SacExt-5’-GAGCTCAGATCTAGTTCACGG-3’ (SEQ ID NO：30)
3.SalExt-5’-GAGCGTCGACTTCTTCAAC-3’ (SEQ ID NO：27)
SacExt-5’-GAGCTCAGATCTAGTTCACGG-3’ (SEQ ID NO：30)
引物对1和引物对2产生两个独特的PCR产物。然后将这些产物等份混合，并利用引物对3连接该产物以产生一个PCR片断，它被克隆回初始的pCIB6850模板中。然后将该修饰的cry3A058基因转移至pBluescript(Stratagene)中。所得的质粒被命名为pCMS058且包含cry3A058基因(SEQ ID NO：14)。
修饰的Cry3A058毒素(SEQ ID NO：15)由该修饰的cry3A基因编码，具有组织蛋白酶G识别位点，后者包含氨基酸序列AAPF(SEQ IDNO：35)，插入在未修饰的Cry3A毒素(SEQ ID NO：4)氨基酸540和541之间的结构域III中。该组织蛋白酶G识别位点在天然存在的胰凝乳蛋白酶识别位点之内。
包含cry3A082的pCMS082
cry3A082(SEQ ID NO：12)包括编码修饰的Cry3A毒素的核苷酸序列。利用1个快速变化定点突变(QuickChange Site DirectedMutagenesis)PCR引物对将编码组织蛋白酶G识别位点的核苷酸序列插入到未修饰的玉米优化cry3A中：
BBmod1-5’-CGGGGCCCCCGCTGCACCGTTCTACTTCGACA-3’ (SEQ ID NO：31)
BBmod2-5’-TGTCGAAGTAGAACGGTGCAGCGGGGGCCCCG-3’ (SEQ ID NO：32)
该引物对产生一种独特的PCR产物。该产物被克隆回初始的pCIB6850模板中。然后将该修饰的cry3A082基因转移至pBluescript(Stratagene)中。所得的质粒被命名为pCMS082且包含cry3A082基因(SEQ ID NO：12)。
修饰的Cry3A082毒素(SEQ ID NO：13)由该修饰的cry3A基因编码，具有组织蛋白酶G识别位点，后者包含氨基酸序列AAPF(SEQ IDNO：35)，插入在未修饰的Cry3A毒素(SEQ ID NO：4)氨基酸539和542之间的结构域III中。该组织蛋白酶G识别位点取代了天然存在的胰凝乳蛋白酶识别位点。
包含在pCMS056中的cry3A056
cry3A056(SEQ ID NO：18)包括编码修饰的Cry3A毒素的核苷酸序列。利用6个重叠PCR引物对将2个组织蛋白酶G识别位点插入到未修饰的cry3A中：
1.BamExt1-5’-GGATCCACCATGACGGCCGAC-3’ (SEQ ID NO：22)
AAPFtail3-5’-GAACGGTGCAGCGGGGTTCTTCTGCCAGC-3’ (SEQ ID NO：23)
2.AAPFtail4-5’-GCTGCACCGTTCCGCAACCCCCACAGCCA-3’ (SEQ ID NO：26)
XbaIExt2-5’-TCTAGACCCACGTTGTACCAC-3’ (SEQ ID NO：25)
3.BamExt1-5’-GGATCCACCATGACGGCCGAC-3’ (SEQ ID NO：22)
XbaIExt2-5’-TCTAGACCCACGTTGTACCAC-3’ (SEQ ID NO：25)
4.SalExt-5’-GAGCGTCGACTTCTTCAAC-3’ (SEQ ID NO：27)
AAPF-Y2-5’-GAACGGTGCAGCGTATTGGTTGAAGGGGGC-3’ (SEQ ID NO：28)
5.AAPF-Y1-5’-GCTGCACCGTTCTACTTCGACAAGACCATC-3’ (SEQ ID NO：29)
SacExt-5’-GAGCTCAGATCTAGTTCACGG-3’ (SEQ ID NO：30)
6.SalExt-5’-GAGCGTCGACTTCTTCAAC-3’ (SEQ ID NO：27)
SacExt-5’-GAGCTCAGATCTAGTTCACGG-3’ (SEQ ID NO：30)
引物对1和引物对2产生两个独特的PCR产物。然后将这些产物等份混合，并利用引物对3连接该产物以产生一个PCR片断，它被克隆回初始的pCIB6850质粒中。然后将该修饰的cry3A055基因转移至pBluescript(Stratagene)中。所得的质粒被命名为pCMS055。引物对4和引物对5产生另一组独特的片断，它们由引物6通过另一个PCR连接。该片断被克隆到包含在pCMS055中的修饰cry3A055基因的结构域III中。所得的质粒被命名为pCMS056且包含cry3A056基因(SEQID NO：18)。
修饰的Cry3A056毒素(SEQ ID NO：19)由该修饰的cry3A基因编码，具有组织蛋白酶G识别位点，后者包含氨基酸序列AAPF(SEQ IDNO：35)，插入在未修饰的Cry3A毒素(SEQ ID NO：4)氨基酸107和111之间的结构域I中以及氨基酸540和541之间的结构域III中。该组织蛋白酶G识别位点邻近结构域I中天然存在的胰蛋白酶和胰凝乳蛋白酶识别位点，并且在结构域III中天然存在的胰凝乳蛋白酶识别位点之内。
包含在pCMS057中的cry3A057
cry3A057(SEQ ID NO：16)包括编码修饰的Cry3A毒素的核苷酸序列。利用6个重叠PCR引物对将2个组织蛋白酶G识别位点插入到未修饰的cry3A中：
1.BamExt1-5’-GGATCCACCATGACGGCCGAC-3’ (SEQ ID NO：22)
AAPFtail3-5’-GAACGGTGCAGCGGGGTTCTTCTGCCAGC-3’ (SEQ ID NO：23)
2.Tail5mod-5’-GCTGCACCGTTCCCCCACAGCCAGGGCCG-3’ (SEQ ID NO：24)
XbaIExt2-5’-TCTAGACCCACGTTGTACCAC-3’ (SEQ ID NO：25)
3.BamExt1-5’-GGATCCACCATGACGGCCGAC-3’ (SEQ ID NO：22)
XbaIExt2-5’-TCTAGACCCACGTTGTACCAC-3’ (SEQ ID NO：25)
4.SalExt-5’-GAGCGTCGACTTCTTCAAC-3’ (SEQ ID NO：27)
AAPF-Y2-5’-GAACGGTGCAGCGTATTGGTTGAAGGGGGC-3’ (SEQ ID NO：28)
5.AAPF-Y1-5’-GCTGCACCGTTCTACTTCGACAAGACCATC-3’ (SEQ ID NO：29)
SacExt-5’-GAGCTCAGATCTAGTTCACGG-3’ (SEQ ID NO：30)
6.SalExt-5’-GAGCGTCGACTTCTTCAAC-3’ (SEQ ID NO：27)
SacExt-5’-GAGCTCAGATCTAGTTCACGG-3’ (SEQ ID NO：30)
引物对1和引物对2产生两个独特的PCR产物。然后将这些产物等份混合，并利用引物对3连接该产物以产生一个PCR片断，它被克隆回初始的pCIB6850质粒中。然后将该修饰的cry3A054基因转移至pBluescript(Stratagene)中。所得的质粒被命名为pCMS054。引物对4和引物对5产生另一组独特的片断，它们由引物6通过另一个PCR连接。该片断被克隆到包含在pCMS054中的修饰cry3A054基因的结构域III中。所得的质粒被命名为pCMS057且包含cry3A057基因(SEQID NO：16)。
修饰的Cry3A057毒素(SEQ ID NO：17)由该修饰的cry3A基因编码，具有组织蛋白酶G识别位点，后者包含氨基酸序列AAPF(SEQ IDNO：35)，插入在未修饰的Cry3A毒素(SEQ ID NO：4)氨基酸107和113之间的结构域I中以及氨基酸540和541之间的结构域III中。该组织蛋白酶G识别位点取代了结构域I中天然存在的胰蛋白酶识别位点并邻近胰凝乳蛋白酶识别位点，而且在结构域III中天然存在的胰凝乳蛋白酶识别位点之内。
包含在pCMS083中的cry3A083
cry3A083(SEQ ID NO：20)包括编码修饰的Cry3A毒素的核苷酸序列。利用3个重叠PCR引物对和1个快速变化定点突变PCR引物对将2个组织蛋白酶G识别位点插入到未修饰的cry3A中：
1.BamExt1-5’-GGATCCACCATGACGGCCGAC-3’ (SEQ ID NO：22)
AAPFtail3-5’-GAACGGTGCAGCGGGGTTCTTCTGCCAGC-3’ (SEQ ID NO：23)
2.AAPFtail4-5’-GCTGCACCGTTCCGCAACCCCCACAGCCA-3’(SEQ ID NO：26)
XbaIExt2-5’-TCTAGACCCACGTTGTACCAC-3’ (SEQ ID NO：25)
3.BamExt1-5’-GGATCCACCATGACGGCCGAC-3’ (SEQ ID NO：22)
XbaIExt2-5’-TCTAGACCCACGTTGTACCAC-3’ (SEQ ID NO：25)
BBmod1-5’-CGGGGCCCCCGCTGCACCGTTCTACTTCGACA-3 (SEQ ID NO：31)
BBmod2-5’-TGTCGAAGTAGAACGGTGCAGCGGGGGCCCCG-3’ (SEQ ID NO：32)
引物对1和引物对2产生两个独特的PCR产物。然后将这些产物等份混合，并利用引物对3连接该产物以产生一个PCR片断，它被克隆回初始的pCIB6850质粒中。然后将该修饰的cry3A055基因转移至pBluescript(Stratagene)中。所得的质粒被命名为pCMS055。引物对4产生另一个独特的片断，它被克隆到包含在pCMS055中的修饰cry3A的结构域III中。所得的质粒被命名为pCMS083且包含cry3A083基因(SEQ ID NO：20)。
修饰的Cry3A083毒素(SEQ ID NO：21)由该修饰的cry3A基因编码，具有组织蛋白酶G识别位点，后者包含氨基酸序列AAPF(SEQ IDNO：35)，插入在未修饰的Cry3A毒素(SEQ ID NO：4)氨基酸107和111之间的结构域I中以及氨基酸539和542之间。该组织蛋白酶G识别位点邻近结构域I中天然存在的胰蛋白酶和胰凝乳蛋白酶识别位点，并取代了结构域III中天然存在的胰凝乳蛋白酶识别位点。
包含在pCMS085中的cry3A085
cry3A085基因(SEQ ID NO：10)在与上文所述的cry3A055基因相同的位置包括组织蛋白酶G编码序列。该cry3A085基因在5′端额外插入了编码SEQ ID NO：2所示的推断的氨基酸序列的氨基酸41-47和附加的甲硫氨酸的24个核苷酸。这些额外的核苷酸是利用下列PCR引物对插入在cry3A055基因5′端的：
mo3Aext-5’-GGATCCACCATGAACTACAAGGAGTTCCTCCGC-
ATGACCGCCGACAAC-3’ (SEQ ID NO：33)
CMSI6-5’-CCTCCACCTGCTCCATGAAG-3’(SEQ ID NO：34)
修饰的Cry3A085毒素(SEQ ID NO：11)由该修饰的cry3A基因编码，具有组织蛋白酶G识别位点，后者包含氨基酸序列AAPF(SEQ IDNO：35)，插入在与未修饰的Cry3A毒素(SEQ ID NO：4)107和111相应的氨基酸之间的结构域I中，并该毒素在N-末端额外具有8个氨基酸残基，其中第2个残基与SEQ ID NO：2中所示的氨基酸序列的氨基酸编码41相应。
实施例4：修饰的Cry3A毒素的杀虫剂活性
在昆虫生物测定中测试修饰的Cry3A毒素抗西部玉米根虫、北部玉米根虫和南部玉米根虫的杀虫剂活性。生物测定是使用饵料掺入法进行的。过夜培养表达本发明的修饰的Cry3A毒素之一的大肠杆菌克隆。超声破碎500μl过夜培养物，然后与500μl熔融的人工饵料(artificial diet)(Marrone等(1985)
J.of Economic Entomology78：290-293)混合。一旦饵料凝固，其被分配于培养皿中，并在该饵料上面放置20只新生的玉米根虫。培养皿维持在30℃。6天后记录死亡率。所有修饰的Cry3A毒素导致西部和北部玉米根虫50％-100％的死亡率，而未修饰的Cry3A毒素导致0％-30％的死亡率。没有任何一个修饰的Cry3A毒素具有抗南部玉米根虫的活性。
实施例5：包含修饰的cry3A编码序列的转基因玉米植物的建立
选择3个修饰的cry3A基因，代表结构域I修饰的cry3A055、代表结构域III修饰的cry3A058、以及代表结构域I和结构域III修饰的cry3A056，用来转化进入玉米植物。包括修饰的cry3A编码序列的表达盒被转移至合适的载体中用于农杆菌(Agrobacterium)-介导的玉米转化。对于本实施例，除了修饰的cry3A基因之外，表达盒还包括MTL启动子(美国专利No.5,466,785)和本领域公知的nos终止子。
未成熟玉米胚的转化基本上如Negrotto等，2000，Plant CellReports 19：798-803中所描述的进行。对于本实施例，所有的培养基组分都如Negrotto等(同上)所述。不过，可以替换为本领域公知的各种培养基组分。
用于转化的基因被克隆进合适于玉米转化的载体中。本实施例所用的载体含有用于选择转基因系的磷酸甘露糖异构酶(PMI)(Negrotto等(2000)Plant Cell Reports 19：798-803)。
含有植物转化质粒的农杆菌(Agrobacterium)菌株LBA4404(pSB1)在YEP(酵母膏(5g/L)，蛋白胨(10g/L)，NaCl(5g/L)，15g/l琼脂，pH 6.8)固体培养基上于28℃培养2-4天。大约0.8×109农杆菌(Agrobacterium)悬浮在补充有100μM As的LS-inf培养基中(Negrotto等，(2000)Plant Cell Rep 19：798-803)。细菌在这种培养基中预诱导30-60分钟。
来自A188和其它合适的基因型的未成熟胚从8-12日龄的穗中切离放进液体LS-inf+100μM As中。胚用新鲜感染培养基冲洗1次。然后加入农杆菌(Agrobacterium)溶液，并将胚震荡30秒，使之与细菌一起沉淀5分钟。然后将胚盾片朝上转移到LSAs培养基中，并暗培养2到3天。随后，将每只皮氏培养板上的20到25个之间的胚转移到补充有氨噻肟头孢霉素(250mg/l)和硝酸银(1.6mg/l)的LSDc培养基中，并于28℃暗培养10天。
将产生胚胎发生性愈伤组织的未成熟胚转移到LSD1M0.5S培养基中。培养物在这种培养基上选择6周，并于第3周进行继代培养步骤。将存活的愈伤组织转移到补充有甘露糖的Reg1培养基中。经光培养后(16小时光照/8小时暗培养编制)，将绿色组织转移到不含生长调节剂的Reg2培养基中，并孵育1-2周。将幼苗转移到含有Reg3培养基的Magenta GA-7盒(Magenta Corp，Chicago III.)中，并光照培养。2-3周后，通过PCR测试植物中PMI基因和修饰的cry3A基因的存在。将PCR试验中的阳性植物转移到温室中，并测试其对玉米根虫的抗性。
实施例6：转基因玉米植物的分析
玉米根虫效力
根切除生物测定
当植物从Magenta GA-7盒中移植到土壤中时取样。这允许从相对于土壤环境而言的适度无菌环境中对根取样。取样包括切下小块的根(大约2-4cm长)，并将其置于小培养皿中强化phytagar(phytagar12g，蔗糖9g，MS盐3ml，MS维生素3ml，制霉菌素(25mg/ml)3ml，氨噻肟头孢霉素(50mg/ml)7ml，金霉素(50mg/ml)7ml，链霉素(50mg/ml)7ml，dH2O，600ml)之上。阴性对照或者是来自同一实验的修饰的cry3A基因PCR阴性的转基因植物，或者来自在人工气候室培养的非转基因植物(与测试植物大小相似)。如果对土壤中的对照根取样，则将根样品用水洗涤以除去土壤残渣，浸泡在制霉菌素溶液中(5mg/ml)，从浸泡液中取出，用纸巾吸干，并放置在phytagar皿中。
通过将10只第一龄期幼虫放置在每个phytagar皿盖的内表面，然后将盖紧紧地重新密封，对根样品接种西部玉米根虫。幼虫用画笔的细尖来处理。在所有的皿都接种之后，将皿碟室温下置于暗处直到采集数据。
接种后3-4天采集数据。幼虫百分死亡率和根的可见损伤等级一起计算。摄食损伤定级为高、中、低和无，并分别给出数值3、2、1和0。导致至少40％死亡率并具有2和更低的损伤等级的根样品被认为是阳性的。
下表的结果显示，表达修饰的Cry3A毒素的植物导致西部玉米根虫40-100％的死亡率，而对照植物导致0-30％的死亡率。同样，表达修饰的Cry3A毒素的植物遭受比对照植物显著低的摄食损伤(feedingdamage)。
表2
| T0事件 | 表达的修饰Cry3A毒素 | 百分死亡率/植物A B C D E | 每个事件的平均损伤等级 |
| 240A7240B2240B9240B10240A15240A5240A9 | Cry3A055Cry3A055Cry3A055Cry3A055Cry3A055Cry3A055Cry3A055 | 80 40 80 6060 60 60 8040 60 60 10080 40 60 6080 60 50 70 7060 80 6050 60 60 70 70 | 0.81.25110.60.331.6 |
| 244A4244A7244A5244B7244B6 | Cry3A058Cry3A058Cry3A058Cry3A058Cry3A058 | 5040 40 60509050 40 60 | 11.3111 |
| 243A3243A4243B1243B4245B2 | Cry3A056Cry3A056Cry3A056Cry3A056Cry3A056 | 50 90 80 6050 80 6080 9070 60 50 8090 50 70 60 | 1.251.70.51.51 |
| WT1WT2 | -- | 0 10 20 10 00 30 0 0 20 | 2.62.8 |
全植物生物测定
某些利用上文所述的根切除生物测定鉴定的阳性植物利用全植物生物测定评价对西部玉米根虫的抗性。植物一般在根切除试验完成后3天内进行感染。
西部玉米根虫卵被预孵育，使得可在植物接种后2-3天孵化。卵在0.2％琼脂中悬浮，并以大约200只卵/植物施于测试植物周围的土壤中。
卵孵化后2周，评价植物由于西部玉米根虫幼虫所导致的损伤。植物所达高度、倒伏以及根群(root mass)是用来确定植物对西部玉米根虫摄食损伤是否具有抗性的标准。在评价时，对照植物典型地比修饰的Cry3A植物小。同样，非转基因对照植物和表达由玉米优化cry3A基因编码的未修饰Cry3A毒素的植物在这段时期内由于大部分根的严重削减(pruning)，导致没有根群积累而倒伏。在评价时，表达本发明的修饰Cry3A毒素的植物比对照植物高，没有倒伏，并且由于修饰的Cry3A毒素的杀虫剂活性而具有大的完整根群。
酶联免疫吸附测定(ELISA)
根据美国专利No.5,625,136公开的方法的ELISA分析被用来定量测定转基因植物中修饰和未修饰的Cry 3A蛋白的水平。
表3：全植物生物测定结果和蛋白质水平
| 转基因玉米植物 | 表达的Cry3A毒素类型 | 根中的Cry3A蛋白水平(ng/mg) | 倒伏的植物 | 完整根群 |
| 240A2E | 修饰的Cry3A055 | 224 | - | + |
| 240A9C | 修饰的Cry3A055 | 71 | - | + |
| 240B9D | 修饰的Cry3A055 | 204 | - | + |
| 240B9E | 修饰的Cry3A055 | 186 | - | + |
| 240B10D | 修饰的Cry3A055 | 104 | - | + |
| 240B10E | 修饰的Cry3A055 | 70 | - | + |
| 240A15E | 修饰的Cry3A055 | 122 | - | + |
| 240B4D | 修饰的Cry3A055 | 97 | - | + |
| 243B5A | 修饰的Cry3A056 | 41 | - | + |
| 244A7A | 修饰的Cry3A058 | 191 | - | + |
| 710-2-51 | 玉米优化的 | 39 | + | - |
| 710-2-54 | 玉米优化的 | 857 | + | - |
| 710-2-61 | 玉米优化的 | 241 | + | - |
| 710-2-67 | 玉米优化的 | 1169 | + | - |
| 710-2-68 | 玉米优化的 | 531 | + | - |
| 710-2-79 | 玉米优化的 | 497 | + | - |
| 710-2-79 | 玉米优化的 | 268 | + | - |
| WT1对照 | - | 0 | + | - |
| WT2对照 | - | 0 | + | - |
序列表
<110>Syngenta Participations AG
<120>修饰的Cry3A毒素及编码它的核酸序列
<130>60065/PCT
<140>
<141>
<150>US 60/316421
<151>2001-08-31
<160>34
<170>PatentIn Ver.3.0
<210>1
<211>1932
<212>DNA
<213>苏云金芽孢杆菌
<220>
<221>CDS
<222>(1)..(1932)
<223>根据Sekar等1987，Proc.Natl.Acad.Sci.84：7036-7040的天然cry3A编码序列
<400>1
atg aat ccg aac aat cga agt gaa cat gat aca ata aaa act act gaa 48
Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Thr Thr Glu
1 5 10 15
aat aat gag gtg cca act aac cat gtt caa tat cct tta gcg gaa act 96
Asn Asn Glu Val Pro Thr Asn His Val Gln Tyr Pro Leu Ala Glu Thr
20 25 30
cca aat cca aca cta gaa gat tta aat tat aaa gag ttt tta aga atg 144
Pro Asn Pro Thr Leu Glu Asp Leu Asn Tyr Lys Glu Phe Leu Arg Met
35 40 45
act gca gat aat aat acg gaa gca cta gat agc tct aca aca aaa gat 192
Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys Asp
50 55 60
gtc att caa aaa ggc att tcc gta gta ggt gat ctc cta ggc gta gta 240
Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val Val
65 70 75 80
ggt ttc ccg ttt ggt gga gcg ctt gtt tcg ttt tat aca aac ttt tta 288
Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu
85 90 95
aat act att tgg cca agt gaa gac ccg tgg aag gct ttt atg gaa caa 336
Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu Gln
100 105 110
gta gaa gca ttg atg gat cag aaa ata gct gat tat gca aaa aat aaa 384
Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn Lys
115 120 125
gct ctt gca gag tta cag ggc ctt caa aat aat gtc gaa gat tat gtg 432
Ala Leu Ala Glu Leu Gln Gly Leu Gln Ash Asn Val Glu Asp Tyr Val
130 135 140
agt gca ttg agt tca tgg caa aaa aat cct gtg agt tca cga aat cca 480
Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Val Ser Ser Arg Asn Pro
145 150 155 160
cat agc cag ggg cgg ata aga gag ctg ttt tct caa gca gaa agt cat 528
His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His
165 170 175
ttt cgt aat tca atg cct tcg ttt gca att tct gga tac gag gtt cta 576
Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val Leu
180 185 190
ttt cta aca aca tat gca caa gct gcc aac aca cat tta ttt tta cta 624
Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu Leu
195 200 205
aaa gac gct caa att tat gga gaa gaa tgg gga tac gaa aaa gaa gat 672
Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp
210 215 220
att gct gaa ttt tat aaa aga caa cta aaa ctt acg caa gaa tat act 720
Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr
225 230 235 240
gac cat tgt gtc aaa tgg tat aat gtt gga tta gat aaa tta aga ggt 768
Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly
245 250 255
tca tct tat gaa tct tgg gta aac ttt aac cgt tat cgc aga gag atg 816
Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met
260 265 270
aca tta aca gta tta gat tta att gca cta ttt cca ttg tat gat gtt 864
Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val
275 280 285
cgg cta tac cca aaa gaa gtt aaa acc gaa tta aca aga gac gtt tta 912
Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu
290 295 300
aca gat cca att gtc gga gtc aac aac ctt agg ggc tat gga aca acc 960
Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr
305 310 315 320
ttc tct aat ata gaa aat tat att cga aaa cca cat cta ttt gac tat 1008
Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr
325 330 335
ctg cat aga att caa ttt cac acg cgg ttc caa cca gga tat tat gga 1056
Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly
340 345 350
aat gac tct ttc aat tat tgg tcc ggt aat tat gtt tca act aga cca 1104
Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro
355 360 365
agc ata gga tca aat gat ata atc aca tct cca ttc tat gga aat aaa 1152
Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys
370 375 380
tcc agt gaa cct gta caa aat tta gaa ttt aat gga gaa aaa gtc tat 1200
Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr
385 390 395 400
aga gcc gta gca aat aca aat ctt gcg gtc tgg ccg tcc gct gta tat 1248
Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val Tyr
405 410 415
tca ggt gtt aca aaa gtg gaa ttt agc caa tat aat gat caa aca gat 1296
Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp
420 425 430
gaa gca agt aca caa acg tac gac tca aaa aga aat gtt ggc gcg gtc 1344
Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val
435 440 445
agc tgg gat tct atc gat caa ttg cct cca gaa aca aca gat gaa cct 1392
Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro
450 455 460
cta gaa aag gga tat agc cat caa ctc aat tat gta atg tgc ttt tta 1440
Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu
465 470 475 480
atg cag ggt agt aga gga aca atc cca gtg tta act tgg aca cat aaa 1488
Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His Lys
485 490 495
agt gta gac ttt ttt aac atg att gat tcg aaa aaa att aca caa ctt 1536
Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln Leu
500 505 510
ccg tta gta aag gca tat aag tta caa tct ggt gct tcc gtt gtc gca 1584
Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val Val Ala
515 520 525
ggt cct agg ttt aca gga gga gat atc att caa tgc aca gaa aat gga 1632
Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn Gly
530 535 540
agt gcg gca act att tac gtt aca ccg gat gtg tcg tac tct caa aaa 1680
Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser Gln Lys
545 550 555 560
tat cga gct aga att cat tat gct tct aca tct cag ata aca ttt aca 1728
Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr Phe Thr
565 570 575
ctc agt tta gac ggg gca cca ttt aat caa tac tat ttc gat aaa acg 1776
Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe Asp Lys Thr
580 585 590
ata aat aaa gga gac aca tta acg tat aat tca ttt aat tta gca agt 1824
Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu Ala Ser
595 600 605
ttc agc aca cca ttc gaa tta tca ggg aat aac tta caa ata ggc gtc 1872
Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile Gly Val
610 615 620
aca gga tta agt gct gga gat aaa gtt tat ata gac aaa att gaa ttt 1920
Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys Ile Glu Phe
625 630 635 640
att cca gtg aat 1932
Ile Pro Val Asn
<210>2
<211>644
<212>PRT
<213>苏云金芽孢杆菌
<400>2
Met Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Thr Thr Glu
1 5 10 15
Asn Asn Glu Val Pro Thr Asn His Val Gln Tyr Pro Leu Ala Glu Thr
20 25 30
Pro Asn Pro Thr Leu Glu Asp Leu Asn Tyr Lys Glu Phe Leu Arg Met
35 40 45
Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys Asp
50 55 60
Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val Val
65 70 75 80
Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu
85 90 95
Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu Gln
100 105 110
Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn Lys
115 120 125
Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr Val
130 135 140
Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Val Ser Ser Arg Asn Pro
145 150 155 160
His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His
165 170 175
Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val Leu
180 185 190
Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu Leu
195 200 205
Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp
210 215 220
Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr
225 230 235 240
Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly
245 250 255
Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met
260 265 270
Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val
275 280 285
Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu
290 295 300
Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr
305 310 315 320
Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr
325 330 335
Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly
340 345 350
Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro
355 360 365
Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys
370 375 380
Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr
385 390 395 400
Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val Tyr
405 410 415
Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp
420 425 430
Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val
435 440 445
Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro
450 455 460
Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu
465 470 475 480
Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His Lys
485 490 495
Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln Leu
500 505 510
Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val Val Ala
515 520 525
Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn Gly
530 535 540
Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser Gln Lys
545 550 555 560
Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr Phe Thr
565 570 575
Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe Asp Lys Thr
580 585 590
Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu Ala Ser
595 600 605
Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile Gly Val
610 615 620
Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys Ile Glu Phe
625 630 635 640
Ile Pro Val Asn
<210>3
<211>1803
<212>DNA
<213>人工序列
<220>
<221>CDS
<222>(1)..(1794)
<223>玉米优化的cry3A编码序列
<400>3
atg acg gcc gac aac aac acc gag gcc ctg gac agc agc acc acc aag 48
Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys
1 5 10 15
gac gtg atc cag aag ggc atc agc gtg gtg ggc gac ctg ctg ggc gtg 96
Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
20 25 30
gtg ggc ttc ccc ttc ggc ggc gcc ctg gtg agc ttc tac acc aac ttc 144
Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe
35 40 45
ctg aac acc atc tgg ccc agc gag gac ccc tgg aag gcc ttc atg gag 192
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu
50 55 60
cag gtg gag gcc ctg atg gac cag aag atc gcc gac tac gcc aag aac 240
Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn
65 70 75 80
aag gca ctg gcc gag cta cag ggc ctc cag aac aac gtg gag gac tat 288
Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr
85 90 95
gtg agc gcc ctg agc agc tgg cag aag aac ccc gtc tcg agc cgc aac 336
Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Val Ser Ser Arg Asn
100 105 110
ccc cac agc cag ggc cgc atc cgc gag ctg ttc agc cag gcc gag agc 384
Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser
115 120 125
cac ttc cgc aac agc atg ccc agc ttc gcc atc agc ggc tac gag gtg 432
His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val
130 135 140
ctg ttc ctg acc acc tac gcc cag gcc gcc aac acc cac ctg ttc ctg 480
Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu
145 150 155 160
ctg aag gac gcc caa atc tac gga gag gag tgg ggc tac gag aag gag 528
Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu
165 170 175
gac atc gcc gag ttc tac aag cgc cag ctg aag ctg acc cag gag tac 576
Asp Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr
180 185 190
acc gac cac tgc gtg aag tgg tac aac gtg ggt cta gac aag ctc cgc 624
Thr Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg
195 200 205
ggc agc agc tac gag agc tgg gtg aac ttc aac cgc tac cgc cgc gag 672
Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu
210 215 220
atg acc ctg acc gtg ctg gac ctg atc gcc ctg ttc ccc ctg tac gac 720
Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp
225 230 235 240
gtg cgc ctg tac ccc aag gag gtg aag acc gag ctg acc cgc gac gtg 768
Val Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val
245 250 255
ctg acc gac ccc atc gtg ggc gtg aac aac ctg cgc ggc tac ggc acc 816
Leu Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr
260 265 270
acc ttc agc aac atc gag aac tac atc cgc aag ccc cac ctg ttc gac 864
Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp
275 280 285
tac ctg cac cgc atc cag ttc cac acg cgt ttc cag ccc ggc tac tac 912
Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr
290 295 300
ggc aac gac agc ttc aac tac tgg agc ggc aac tac gtg agc acc cgc 960
Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg
305 310 315 320
ccc agc atc ggc agc aac gac atc atc acc agc ccc ttc tac ggc aac 1008
Pro Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn
325 330 335
aag agc agc gag ccc gtg cag aac ctt gag ttc aac ggc gag aag gtg 1056
Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val
340 345 350
tac cgc gcc gtg gct aac acc aac ctg gcc gtg tgg ccc tct gca gtg 1104
Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val
355 360 365
tac agc ggc gtg acc aag gtg gag ttc agc cag tac aac gac cag acc 1152
Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr
370 375 380
gac gag gcc agc acc cag acc tac gac agc aag cgc aac gtg ggc gcc 1200
Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala
385 390 395 400
gtg agc tgg gac agc atc gac cag ctg ccc ccc gag acc acc gac gag 1248
Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu
405 410 415
ccc ctg gag aag ggc tac agc cac cag ctg aac tac gtg atg tgc ttc 1296
Pro Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe
420 425 430
ctg atg cag ggc agc cgc ggc acc atc ccc gtg ctg acc tgg acc cac 1344
Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His
435 440 445
aag agc gtc gac ttc ttc aac atg atc gac agc aag aag atc acc cag 1392
Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln
450 455 460
ctg ccc ctg gtg aag gcc tac aag ctc cag agc ggc gcc agc gtg gtg 1440
Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val Val
465 470 475 480
gca ggc ccc cgc ttc acc ggc ggc gac atc atc cag tgc acc gag aac 1488
Ala Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn
485 490 495
ggc agc gcc gcc acc atc tac gtg acc ccc gac gtg agc tac agc cag 1536
Gly Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser Gln
500 505 510
aag tac cgc gcc cgc atc cac tac gcc agc acc agc cag atc acc ttc 1584
Lys Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr Phe
515 520 525
acc ctg agc ctg gac ggg gcc ccc ttc aac caa tac tac ttc gac aag 1632
Thr Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe Asp Lys
530 535 540
acc atc aac aag ggc gac acc ctg acc tac aac agc ttc aac ctg gcc 1680
Thr Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu Ala
545 550 555 560
agc ttc agc acc cct ttc gag ctg agc ggc aac aac ctc cag atc ggc 1728
Ser Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile Gly
565 570 575
gtg acc ggc ctg agc gcc ggc gac aag gtg tac atc gac aag atc gag 1776
Val Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys Ile Glu
580 585 590
ttc atc ccc gtg aac tag atctgagct 1803
Phe Ile Pro Val Asn
595
<210>4
<211>597
<212>PRT
<213>由SEQ ID NO：3编码的Cry3A
<400>4
Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys
1 5 10 15
Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
20 25 30
Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe
35 40 45
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu
50 55 60
Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn
65 70 75 80
Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr
85 90 95
Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Val Ser Ser Arg Asn
100 105 110
Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser
115 120 125
His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val
130 135 140
Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu
145 150 155 160
Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu
165 170 175
Asp Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr
180 185 190
Thr Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg
195 200 205
Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu
210 215 220
Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp
225 230 235 240
Val Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val
245 250 255
Leu Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr
260 265 270
Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp
275 280 285
Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr
290 295 300
Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg
305 310 315 320
Pro Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn
325 330 335
Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val
340 345 350
Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val
355 360 365
Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr
370 375 380
Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala
385 390 395 400
Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu
405 410 415
Pro Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe
420 425 430
Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His
435 440 445
Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln
450 455 460
Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val Val
465 470 475 480
Ala Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn
485 490 495
Gly Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser Gln
500 505 510
Lys Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr Phe
515 520 525
Thr Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe Asp Lys
530 535 540
Thr Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu Ala
545 550 555 560
Ser Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile Gly
565 570 575
Val Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys Ile Glu
580 585 590
Phe Ile Pro Val Asn
595
<210>5
<211>7208
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>pCIB6850
<400>5
gatccaccat gacggccgac aacaacaccg aggccctgga cagcagcacc accaaggacg 60
tgatccagaa gggcatcagc gtggtgggcg acctgctggg cgtggtgggc ttccccttcg 120
gcggcgccct ggtgagcttc tacaccaact tcctgaacac catctggccc agcgaggacc 180
cctggaaggc cttcatggag caggtggagg ccctgatgga ccagaagatc gccgactacg 240
ccaagaacaa ggcactggcc gagctacagg gcctccagaa caacgtggag gactatgtga 300
gcgccctgag cagctggcag aagaaccccg tctcgagccg caacccccac agccagggcc 360
gcatccgcga gctgttcagc caggccgaga gccacttccg caacagcatg cccagcttcg 420
ccatcagcgg ctacgaggtg ctgttcctga ccacctacgc ccaggccgcc aacacccacc 480
tgttcctgct gaaggacgcc caaatctacg gagaggagtg gggctacgag aaggaggaca 540
tcgccgagtt ctacaagcgc cagctgaagc tgacccagga gtacaccgac cactgcgtga 600
agtggtacaa cgtgggtcta gacaagctcc gcggcagcag ctacgagagc tgggtgaact 660
tcaaccgcta ccgccgcgag atgaccctga ccgtgctgga cctgatcgcc ctgttccccc 720
tstacgacgt gcgcctgtac cccaaggagg tgaagaccga gctgacccgc gacgtgctga 780
ccgaccccat cgtgggcgtg aacaacctgc gcggctacgg caccaccttc agcaacatcg 840
agaactacat ccgcaagccc cacctgttcg actacctgca ccgcatccag ttccacacgc 900
gtttccagcc cggctactac ggcaacgaca gcttcaacta ctggagcggc aactacgtga 960
gcacccgccc cagcatcggc agcaacgaca tcatcaccag ccccttctac ggcaacaaga 1020
gcagcgagcc cgtgcagaac cttgagttca acggcgagaa ggtgtaccgc gccgtggcta 1080
acaccaacct ggccgtgtgg ccctctgcag tgtacagcgg cgtgaccaag gtggagttca 1140
gccagtacaa cgaccagacc gacgaggcca gcacccagac ctacgacagc aagcgcaacg 1200
tgggcgccgt gagctgggac agcatcgacc agctgccccc cgagaccacc gacgagcccc 1260
tggagaaggg ctacagccac cagctgaact acgtgatgtg cttcctgatg cagggcagcc 1320
gcggcaccat ccccgtgctg acctggaccc acaagagcgt cgacttcttc aacatgatcg 1380
acagcaagaa gatcacccag ctgcccctgg tgaaggccta caagctccag agcggcgcca 1440
gcgtggtggc aggcccccgc ttcaccggcg gcgacatcat ccagtgcacc gagaacggca 1500
gcgccgccac catctacgtg acccccgacg tgagctacag ccagaagtac cgcgcccgca 1560
tccactacgc cagcaccagc cagatcacct tcaccctgag cctggacggg gcccccttca 1620
accaatacta cttcgacaag accatcaaca agggcgacac cctgacctac aacagcttca 1680
acctggccag cttcagcacc cctttcgagc tgagcggcaa caacctccag atcggcgtga 1740
ccggcctgag cgccggcgac aaggtgtaca tcgacaagat cgagttcatc cccgtgaact 1800
agatctgagc tcaagatctg ttgtacaaaa accagcaact cactgcactg cacttcactt 1860
cacttcactg tatgaataaa agtctggtgt ctggttcctg atcgatgact gactactcca 1920
ctttgtgcag aacttagtat gtatttgtat ttgtaaaata cttctatcaa taaaatttct 1980
aattcctaaa accaaaatcc agtgggtacc gaattcactg gccgtcgttt tacaacgtcg 2040
tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc 2100
cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct 2160
gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca 2220
ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg 2280
acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta 2340
cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc 2400
gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat 2460
aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat 2520
ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata 2580
aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct 2640
tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa 2700
agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa 2760
cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt 2820
taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg 2880
tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca 2940
tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa 3000
cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt 3060
gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 3120
cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa 3180
actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga 3240
ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc 3300
tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga 3360
tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga 3420
acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga 3480
ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat 3540
ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt 3600
ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct 3660
gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 3720
ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc 3780
aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 3840
gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc 3900
gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 3960
aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 4020
cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 4080
tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 4140
ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 4200
atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt 4260
cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt 4320
ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga 4380
gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc 4440
cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg 4500
cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca 4560
ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg 4620
aaacagctat gaccatgatt acgccaagct tgcacatgac aacaattgta agaggatgga 4680
gaccacaacg atccaacaat acttctgcga cgggctgtga agtatagaga agttaaacgc 4740
ccaaaagcca ttgtgtttgg aatttttagt tattctattt ttcatgatgt atcttcctct 4800
aacatgcctt aatttgcaaa tttggtataa ctactgattg aaaatatatg tatgtaaaaa 4860
aatactaagc atatttgtga agctaaacat gatgttattt aagaaaatat gttgttaaca 4920
gaataagatt aatatcgaaa tggaaacatc tgtaaattag aatcatctta caagctaaga 4980
gatgttcacg ctttgagaaa cttcttcaga tcatgaccgt agaagtagct ctccaagact 5040
caacgaaggc tgctgcaatt ccacaaatgc atgacatgca tccttgtaac cgtcgtcgcc 5100
gctataaaca cggataactc aattccctgc tccatcaatt tagaaatgag caagcaagca 5160
cccgatcgct caccccatat gcaccaatct gactcccaag tctctgtttc gcattagtac 5220
cgccagcact ccacctatag ctaccaattg agacctttcc agcctaagca gatcgattga 5280
tcgttagagt caaagagttg gtggtacggg tactttaact accatggaat gatggggcgt 5340
gatgtagagc ggaaagcgcc tccctacgcg gaacaacacc ctcgccatgc cgctcgacta 5400
cagcctcctc ctcgtcggcc gcccacaacg agggagcccg tggtcgcagc caccgaccag 5460
catgtctctg tgtcctcgtc cgacctcgac atgtcatggc aaacagtcgg acgccagcac 5520
cagactgacg acatgagtct ctgaagagcc cgccacctag aaagatccga gccctgctgc 5580
tggtagtggt aaccattttc gtcgcgctga cgcggagagc gagaggccag aaatttatag 5640
cgactgacgc tgtggcaggc acgctatcgg aggttacgac gtggcgggtc actcgacgcg 5700
gagttcacag gtcctatcct tgcatcgctc gggccggagt ttacgggact tatccttacg 5760
acgtgctcta aggttgcgat aacgggcgga ggaaggcgtg tggcgtgcgg agacggttta 5820
tacacgtagt gtgcgggagt gtgtttcgta gacgcgggaa agcacgacga cttacgaagg 5880
ttagtggagg aggaggacac actaaaatca ggacgcaaga aactcttcta ttatagtagt 5940
agagaagaga ttataggagt gtgggttgat tctaaagaaa atcgacgcag gacaaccgtc 6000
aaaacgggtg ctttaatata gtagatatat atatatagag agagagagaa agtacaaagg 6060
atgcatttgt gtctgcatat gatcggagta ttactaacgg ccgtcgtaag aaggtccatc 6120
atgcgtggag cgagcccatt tggttggttg tcaggccgca gttaaggcct ccatatatga 6180
ttgtcgtcgg gcccataaca gcatctcctc caccagttta ttgtaagaat aaattaagta 6240
gagatatttg tcgtcgggca gaagaaactt ggacaagaag aagaagcaag ctaggccaat 6300
ttcttgccgg caagaggaag atagtggcct ctagtttata tatcggcgtg atgatgatgc 6360
tcctagctag aaatgagaga agaaaaacgg acgcgtgttt ggtgtgtgtc aatggcgtcc 6420
atccttccat cagatcagaa cgatgaaaaa gtcaagcacg gcatgcatag tatatgtata 6480
gcttgtttta gtgtggcttt gctgagacga atgaaagcaa cggcgggcat atttttcagt 6540
ggctgtagct ttcaggctga aagagacgtg gcatgcaata attcagggaa ttcgtcagcc 6600
aattgaggta gctagtcaac ttgtacattg gtgcgagcaa ttttccgcac tcaggagggc 6660
tagtttgaga gtccaaaaac tataggagat taaagaggct aaaatcctct ccttatttaa 6720
ttttaaataa gtagtgtatt tgtattttaa ctcctccaac ccttccgatt ttatggctct 6780
caaactagca ttcagtctaa tgcatgcatg cttggctaga ggtcgtatgg ggttgttaat 6840
agcatagcta gctacaagtt aaccgggtct tttatattta ataaggacag gcaaagtatt 6900
acttacaaat aaagaataaa gctaggacga actcgtggat tattactaaa tcgaaatgga 6960
cgtaatattc caggcaagaa taattgttcg atcaggagac aagtggggca ttggaccggt 7020
tcttgcaagc aagagcctat ggcgtggtga cacggcgcgt tgcccataca tcatgcctcc 7080
atcgatgatc catcctcact tgctataaaa agaggtgtcc atggtgctca agctcagcca 7140
agcaaataag acgacttgtt tcattgattc ttcaagagat cgagcttctt ttgcaccaca 7200
aggtcgag 7208
<210>6
<211>1801
<212>DNA
<213>人工序列
<220>
<221>CDS
<222>(1)..(1791)
<223>玉米优化的修饰的cry3A054编码序列.
<220>
<221>misc_feature
<222>(322)..(333)
<223>组织蛋白酶G识别位点编码序列
<400>6
atg acg gcc gac aac aac acc gag gcc ctg gac agc agc acc acc aag 48
Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys
1 5 10 15
gac gtg atc cag aag ggc atc agc gtg gtg ggc gac ctg ctg ggc gtg 96
Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
20 25 30
gtg ggc ttc ccc ttc ggc ggc gcc ctg gtg agc ttc tac acc aac ttc 144
Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe
35 40 45
ctg aac acc atc tgg ccc agc gag gac ccc tgg aag gcc ttc atg gag 192
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu
50 55 60
cag gtg gag gcc ctg atg gac cag aag atc gcc gac tac gcc aag aac 240
Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn
65 70 75 80
aag gca ctg gcc gag cta cag ggc ctc cag aac aac gtg gag gac tat 288
Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr
85 90 95
gtg agc gcc ctg agc agc tgg cag aag aac ccc gct gca ccg ttc ccc 336
Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Pro
100 105 110
cac agc cag ggc cgc atc cgc gag ctg ttc agc cag gcc gag agc cac 384
His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His
115 120 125
ttc cgc aac agc atg ccc agc ttc gcc atc agc ggc tac gag gtg ctg 432
Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val Leu
130 135 140
ttc ctg acc acc tac gcc cag gcc gcc aac acc cac ctg ttc ctg ctg 480
Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu Leu
145 150 155 160
aag gac gcc caa atc tac gga gag gag tgg ggc tac gag aag gag gac 528
Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp
165 170 175
atc gcc gag ttc tac aag cgc cag ctg aag ctg acc cag gag tac acc 576
Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr
180 185 190
gac cac tgc gtg aag tgg tac aac gtg ggt cta gac aag ctc cgc ggc 624
Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly
195 200 205
agc agc tac gag agc tgg gtg aac ttc aac cgc tac cgc cgc gag atg 672
Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met
210 215 220
acc ctg acc gtg ctg gac ctg atc gcc ctg ttc ccc ctg tac gac gtg 720
Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val
225 230 235 240
cgc ctg tac ccc aag gag gtg aag acc gag ctg acc cgc gac gtg ctg 768
Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu
245 250 255
acc gac ccc atc gtg ggc gtg aac aac ctg cgc ggc tac ggc acc acc 816
Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr
260 265 270
ttc agc aac atc gag aac tac atc cgc aag ccc cac ctg ttc gac tac 864
Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr
275 280 285
ctg cac cgc atc cag ttc cac acg cgt ttc cag ccc ggc tac tac ggc 912
Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly
290 295 300
aac gac agc ttc aac tac tgg agc ggc aac tac gtg agc acc cgc ccc 960
Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro
305 310 315 320
agc atc ggc agc aac gac atc atc acc agc ccc ttc tac ggc aac aag 1008
Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys
325 330 335
agc agc gag ccc gtg cag aac ctt gag ttc aac ggc gag aag gtg tac 1056
Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr
340 345 350
cgc gcc gtg gct aac acc aac ctg gcc gtg tgg ccc tct gca gtg tac 1104
Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val Tyr
355 360 365
agc ggc gtg acc aag gtg gag ttc agc cag tac aac gac cag acc gac 1152
Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp
370 375 380
gag gcc agc acc cag acc tac gac agc aag cgc aac gtg ggc gcc gtg 1200
Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val
385 390 395 400
agc tgg gac agc atc gac cag ctg ccc ccc gag acc acc gac gag ccc 1248
Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro
405 410 415
ctg gag aag ggc tac agc cac cag ctg aac tac gtg atg tgc ttc ctg 1296
Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu
420 425 430
atg cag ggc agc cgc ggc acc atc ccc gtg ctg acc tgg acc cac aag 1344
Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His Lys
435 440 445
agc gtc gac ttc ttc aac atg atc gac agc aag aag atc acc cag ctg 1392
Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln Leu
450 455 460
ccc ctg gtg aag gcc tac aag ctc cag agc ggc gcc agc gtg gtg gca 1440
Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val Val Ala
465 470 475 480
ggc ccc cgc ttc acc ggc ggc gac atc atc cag tgc acc gag aac ggc 1488
Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn Gly
485 490 495
agc gcc gcc acc atc tac gtg acc ccc gac gtg agc tac agc cag aag 1536
Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser Gln Lys
500 505 510
tac cgc gcc cgc atc cac tac gcc agc acc agc cag atc acc ttc acc 1584
Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr Phe Thr
515 520 525
ctg agc ctg gac ggg gcc ccc ttc aac caa tac tac ttc gac aag acc 1632
Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe Asp Lys Thr
530 535 540
atc aac aag ggc gac acc ctg acc tac aac agc ttc aac ctg gcc agc 1680
Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu Ala Ser
545 550 555 560
ttc agc acc cct ttc gag ctg agc ggc aac aac ctc cag atc ggc gtg 1728
Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile Gly Val
565 570 575
acc ggc ctg agc gcc ggc gac aag gtg tac atc gac aag atc gag ttc 1776
Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys Ile Glu Phe
580 585 590
atc ccc gtg aac tag atctgagctc 1801
Ile Pro Val Asn
595
<210>7
<211>596
<212>PRT
<213>人工序列
<220>
<221>misc_feature
<222>(322)..(333)
<223>组织蛋白酶G识别位点编码序列
<400>7
Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys
1 5 10 15
Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
20 25 30
Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe
35 40 45
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu
50 55 60
Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn
65 70 75 80
Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr
85 90 95
Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Pro
100 105 110
His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His
115 120 125
Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val Leu
130 135 140
Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu Leu
145 150 155 160
Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp
165 170 175
Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr
180 185 190
Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly
195 200 205
Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met
210 215 220
Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val
225 230 235 240
Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu
245 250 255
Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr
260 265 270
Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr
275 280 285
Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly
290 295 300
Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro
305 310 315 320
Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys
325 330 335
Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr
340 345 350
Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val Tyr
355 360 365
Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp
370 375 380
Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val
385 390 395 400
Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro
405 410 415
Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu
420 425 430
Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His Lys
435 440 445
Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln Leu
450 455 460
Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val Val Ala
465 470 475 480
Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn Gly
485 490 495
Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser Gln Lys
500 505 510
Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr Phe Thr
515 520 525
Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe Asp Lys Thr
530 535 540
Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu Ala Ser
545 550 555 560
Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile Gly Val
565 570 575
Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys Ile Glu Phe
580 585 590
Ile Pro Val Asn
595
<210>8
<211>1807
<212>DNA
<213>人工序列
<220>
<221>CDS
<222>(1)..(1806)
<223>玉米优化的修饰的cry3A055编码序列.
<220>
<221>misc_feature
<222>(322)..(333)
<223>组织蛋白酶G识别位点编码序列.
<400>8
atg acg gcc gac aac aac acc gag gcc ctg gac agc agc acc acc aag 48
Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys
1 5 10 15
gac gtg atc cag aag ggc atc agc gtg gtg ggc gac ctg ctg ggc gtg 96
Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
20 25 30
gtg ggc ttc ccc ttc ggc ggc gcc ctg gtg agc ttc tac acc aac ttc 144
Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe
35 40 45
ctg aac acc atc tgg ccc agc gag gac ccc tgg aag gcc ttc atg gag 192
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu
50 55 60
cag gtg gag gcc ctg atg gac cag aag atc gcc gac tac gcc aag aac 240
Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn
65 70 75 80
aag gca ctg gcc gag cta cag ggc ctc cag aac aac gtg gag gac tat 288
Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr
85 90 95
gtg agc gcc ctg agc agc tgg cag aag aac ccc gct gca ccg ttc cgc 336
Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg
100 105 110
aac ccc cac agc cag ggc cgc atc cgc gag ctg ttc agc cag gcc gag 384
Asn Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu
115 120 125
agc cac ttc cgc aac agc atg ccc agc ttc gcc atc agc ggc tac gag 432
Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu
130 135 140
gtg ctg ttc ctg acc acc tac gcc cag gcc gcc aac acc cac ctg ttc 480
Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe
145 150 155 160
ctg ctg aag gac gcc caa atc tac gga gag gag tgg ggc tac gag aag 528
Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys
165 170 175
gag gac atc gcc gag ttc tac aag cgc cag ctg aag ctg acc cag gag 576
Glu Asp Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu
180 185 190
tac acc gac cac tgc gtg aag tgg tac aac gtg ggt cta gac aag ctc 624
Tyr Thr Asp His Cys Val Lys Trp Tyr Ash Val Gly Leu Asp Lys Leu
195 200 205
cgc ggc agc agc tac gag agc tgg gtg aac ttc aac cgc tac cgc cgc 672
Arg Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg
210 215 220
gag atg acc ctg acc gtg ctg gac ctg atc gcc ctg ttc ccc ctg tac 720
Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr
225 230 235 240
gac gtg cgc ctg tac ccc aag gag gtg aag acc gag ctg acc cgc gac 768
Asp Val Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp
245 250 255
gtg ctg acc gac ccc atc gtg ggc gtg aac aac ctg cgc ggc tac ggc 816
Val Leu Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly
260 265 270
acc acc ttc agc aac atc gag aac tac atc cgc aag ccc cac ctg ttc 864
Thr Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe
275 280 285
gac tac ctg cac cgc atc cag ttc cac acg cgt ttc cag ccc ggc tac 912
Asp Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr
290 295 300
tac ggc aac gac agc ttc aac tac tgg agc ggc aac tac gtg agc acc 960
Tyr Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr
305 310 315 320
cgc ccc agc atc ggc agc aac gac atc atc acc agc ccc ttc tac ggc 1008
Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly
325 330 335
aac aag agc agc gag ccc gtg cag aac ctt gag ttc aac ggc gag aag 1056
Asn Lys Ser Ser Glu Pro val Gln Asn Leu Glu Phe Asn Gly Glu Lys
340 345 350
gtg tac cgc gcc gtg gct aac acc aac ctg gcc gtg tgg ccc tct gca 1104
Val Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala
355 360 365
gtg tac agc ggc gtg acc aag gtg gag ttc agc cag tac aac gac cag 1152
Val Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln
370 375 380
acc gac gag gcc agc acc cag acc tac gac agc aag cgc aac gtg ggc 1200
Thr Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly
385 390 395 400
gcc gtg agc tgg gac agc atc gac cag ctg ccc ccc gag acc acc gac 1248
Ala Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp
405 410 415
gag ccc ctg gag aag ggc tac agc cac cag ctg aac tac gtg atg tgc 1296
Glu Pro Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys
420 425 430
ttc ctg atg cag ggc agc cgc ggc acc atc ccc gtg ctg acc tgg acc 1344
Phe Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr
435 440 445
cac aag agc gtc gac ttc ttc aac atg atc gac agc aag aag atc acc 1392
His Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr
450 455 460
cag ctg ccc ctg gtg aag gcc tac aag ctc cag agc ggc gcc agc gtg 1440
Gln Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val
465 470 475 480
gtg gca ggc ccc cgc ttc acc ggc ggc gac atc atc cag tgc acc gag 1488
Val Ala Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu
485 490 495
aac ggc agc gcc gcc acc atc tac gtg acc ccc gac gtg agc tac agc 1536
Asn Gly Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser
500 505 510
cag aag tac cgc gcc cgc atc cac tac gcc agc acc agc cag atc acc 1584
Gln Lys Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr
515 520 525
ttc acc ctg agc ctg gac ggg gcc ccc ttc aac caa tac tac ttc gac 1632
Phe Thr Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe Asp
530 535 540
aag acc atc aac aag ggc gac acc ctg acc tac aac agc ttc aac ctg 1680
Lys Thr Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu
545 550 555 560
gcc agc ttc agc acc cct ttc gag ctg agc ggc aac aac ctc cag atc 1728
Ala Ser Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile
565 570 575
ggc gtg acc ggc ctg agc gcc ggc gac aag gtg tac atc gac aag atc 1776
Gly Val Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys Ile
580 585 590
gag ttc atc ccc gtg aac tag atc tga gct c 1807
Glu Phe Ile Pro Val Asn Ile Ala
595 600
<210>9
<211>598
<212>PRT
<213>人工序列
<220>
<221>misc_feature
<222>(322)..(333)
<223>组织蛋白酶G识别位点编码序列.
<400>9
Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys
1 5 10 15
Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
20 25 30
Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe
35 40 45
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu
50 55 60
Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn
65 70 75 80
Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr
85 90 95
Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg
100 105 110
Asn Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu
115 120 125
Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu
130 135 140
Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe
145 150 155 160
Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys
165 170 175
Glu Asp Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu
180 185 190
Tyr Thr Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu
195 200 205
Arg Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg
210 215 220
Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr
225 230 235 240
Asp Val Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp
245 250 255
Val Leu Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly
260 265 270
Thr Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe
275 280 285
Asp Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr
290 295 300
Tyr Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr
305 310 315 320
Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly
325 330 335
Asn Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys
340 345 350
Val Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala
355 360 365
Val Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln
370 375 380
Thr Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly
385 390 395 400
Ala Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp
405 410 415
Glu Pro Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys
420 425 430
Phe Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr
435 440 445
His Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr
450 455 460
Gln Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val
465 470 475 480
Val Ala Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu
485 490 495
Asn Gly Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser
500 505 510
Gln Lys Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr
515 520 525
Phe Thr Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe Asp
530 535 540
Lys Thr Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu
545 550 555 560
Ala Ser Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile
565 570 575
Gly Val Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys Ile
580 585 590
Glu Phe Ile Pro Val Asn
595
<210>10
<211>1818
<212>DNA
<213>人工序列
<220>
<221>CDS
<222>(1)..(1818)
<223>玉米优化的修饰的cry3A085编码序列.
<220>
<221>misc_feature
<222>(346)..(357)
<223>组织蛋白酶G识别位点编码序列.
<400>10
atg aac tac aag gag ttc ctc cgc atg acc gcc gac aac aac acc gag 48
Met Asn Tyr Lys Glu Phe Leu Arg Met Thr Ala Asp Asn Asn Thr Glu
1 5 10 15
gcc ctg gac agc agc acc acc aag gac gtg atc cag aag ggc atc agc 96
Ala Leu Asp Ser Ser Thr Thr Lys Asp Val Ile Gln Lys Gly Ile Ser
20 25 30
gtg gtg ggc gac ctg ctg ggc gtg gtg ggc ttc ccc ttc ggc ggc gcc 144
Val Val Gly Asp Leu Leu Gly Val Val Gly Phe Pro Phe Gly Gly Ala
35 40 45
ctg gtg agc ttc tac acc aac ttc ctg aac acc atc tgg ccc agc gag 192
Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn Thr Ile Trp Pro Ser Glu
50 55 60
gac ccc tgg aag gcc ttc atg gag cag gtg gag gcc ctg atg gac cag 240
Asp Pro Trp Lys Ala Phe Met Glu Gln Val Glu Ala Leu Met Asp Gln
65 70 75 80
aag atc gcc gac tac gcc aag aac aag gca ctg gcc gag cta cag ggc 288
Lys Ile Ala Asp Tyr Ala Lys Asn Lys Ala Leu Ala Glu Leu Gln Gly
85 90 95
ctc cag aac aac gtg gag gac tat gtg agc gcc ctg agc agc tgg cag 336
Leu Gln Asn Asn Val Glu Asp Tyr Val Ser Ala Leu Ser Ser Trp Gln
100 105 110
aag aac ccc gct gca ccg ttc cgc aac ccc cac agc cag ggc cgc atc 384
Lys Asn Pro Ala Ala Pro Phe Arg Asn Pro His Ser Gln Gly Arg Ile
115 120 125
cgc gag ctg ttc agc cag gcc gag agc cac ttc cgc aac agc atg ccc 432
Arg Glu Leu Phe Ser Gln Ala Glu Ser His Phe Arg Asn Ser Met Pro
130 135 140
agc ttc gcc atc agc ggc tac gag gtg ctg ttc ctg acc acc tac gcc 480
Ser Phe Ala Ile Ser Gly Tyr Glu Val Leu Phe Leu Thr Thr Tyr Ala
145 150 155 160
cag gcc gcc aac acc cac ctg ttc ctg ctg aag gac gcc caa atc tac 528
Gln Ala Ala Asn Thr His Leu Phe Leu Leu Lys Asp Ala Gln Ile Tyr
165 170 175
gga gag gag tgg ggc tac gag aag gag gac atc gcc gag ttc tac aag 576
Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp Ile Ala Glu Phe Tyr Lys
180 185 190
cgc cag ctg aag ctg acc cag gag tac acc gac cac tgc gtg aag tgg 624
Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr Asp His Cys Val Lys Trp
195 200 205
tac aac gtg ggt cta gac aag ctc cgc ggc agc agc tac gag agc tgg 672
Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly Ser Ser Tyr Glu Ser Trp
210 215 220
gtg aac ttc aac cgc tac cgc cgc gag atg acc ctg acc gtg ctg gac 720
Val Asn Phe Asn Arg Tyr Arg Arg Glu Met Thr Leu Thr Val Leu Asp
225 230 235 240
ctg atc gcc ctg ttc ccc ctg tac gac gtg cgc ctg tac ccc aag gag 768
Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val Arg Leu Tyr Pro Lys Glu
245 250 255
gtg aag acc gag ctg acc cgc gac gtg ctg acc gac ccc atc gtg ggc 816
Val Lys Thr Glu Leu Thr Arg Asp Val Leu Thr Asp Pro Ile Val Gly
260 265 270
gtg aac aac ctg cgc ggc tac ggc acc acc ttc agc aac atc gag aac 864
Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr Phe Ser Asn Ile Glu Asn
275 280 285
tac atc cgc aag ccc cac ctg ttc gac tac ctg cac cgc atc cag ttc 912
Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr Leu His Arg Ile Gln Phe
290 295 300
cac acg cgt ttc cag ccc ggc tac tac ggc aac gac agc ttc aac tac 960
His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly Asn Asp Ser Phe Asn Tyr
305 310 315 320
tgg agc ggc aac tac gtg agc acc cgc ccc agc atc ggc agc aac gac 1008
Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro Ser Ile Gly Ser Asn Asp
325 330 335
atc atc acc agc ccc ttc tac ggc aac aag agc agc gag ccc gtg cag 1056
Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys Ser Ser Glu Pro Val Gln
340 345 350
aac ctt gag ttc aac ggc gag aag gtg tac cgc gcc gtg gct aac acc 1104
Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr Arg Ala Val Ala Asn Thr
355 360 365
aac ctg gcc gtg tgg ccc tct gca gtg tac agc ggc gtg acc aag gtg 1152
Asn Leu Ala Val Trp Pro Ser Ala Val Tyr Ser Gly Val Thr Lys Val
370 375 380
gag ttc agc cag tac aac gac cag acc gac gag gcc agc acc cag acc 1200
Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp Glu Ala Ser Thr Gln Thr
385 390 395 400
tac gac agc aag cgc aac gtg ggc gcc gtg agc tgg gac agc atc gac 1248
Tyr Asp Ser Lys Arg Asn Val Gly Ala Val Ser Trp Asp Ser Ile Asp
405 410 415
cag ctg ccc ccc gag acc acc gac gag ccc ctg gag aag ggc tac agc 1296
Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu Glu Lys Gly Tyr Ser
420 425 430
cac cag ctg aac tac gtg atg tgc ttc ctg atg cag ggc agc cgc ggc 1344
His Gln Leu Asn Tyr Val Met Cys Phe Leu Met Gln Gly Ser Arg Gly
435 440 445
acc atc ccc gtg ctg acc tgg acc cac aag agc gtc gac ttc ttc aac 1392
Thr Ile Pro Val Leu Thr Trp Thr His Lys Ser Val Asp Phe Phe Asn
450 455 460
atg atc gac agc aag aag atc acc cag ctg ccc ctg gtg aag gcc tac 1440
Met Ile Asp Ser Lys Lys Ile Thr Gln Leu Pro Leu Val Lys Ala Tyr
465 470 475 480
aag ctc cag agc ggc gcc agc gtg gtg gca ggc ccc cgc ttc acc ggc 1488
Lys Leu Gln Ser Gly Ala Ser Val Val Ala Gly Pro Arg Phe Thr Gly
485 490 495
ggc gac atc atc cag tgc acc gag aac ggc agc gcc gcc acc atc tac 1536
Gly Asp Ile Ile Gln Cys Thr Glu Asn Gly Ser Ala Ala Thr Ile Tyr
500 505 510
gtg acc ccc gac gtg agc tac agc cag aag tac cgc gcc cgc atc cac 1584
Val Thr Pro Asp Val Ser Tyr Ser Gln Lys Tyr Arg Ala Arg Ile His
515 520 525
tac gcc agc acc agc cag atc acc ttc acc ctg agc ctg gac ggg gcc 1632
Tyr Ala Ser Thr Ser Gln Ile Thr Phe Thr Leu Ser Leu Asp Gly Ala
530 535 540
ccc ttc aac caa tac tac ttc gac aag acc atc aac aag ggc gac acc 1680
Pro Phe Asn Gln Tyr Tyr Phe Asp Lys Thr Ile Asn Lys Gly Asp Thr
545 550 555 560
ctg acc tac aac agc ttc aac ctg gcc agc ttc agc acc cct ttc gag 1728
Leu Thr Tyr Asn Ser Phe Asn Leu Ala Ser Phe Ser Thr Pro Phe Glu
565 570 575
ctg agc ggc aac aac ctc cag atc ggc gtg acc ggc ctg agc gcc ggc 1776
Leu Ser Gly Asn Asn Leu Gln Ile Gly Val Thr Gly Leu Ser Ala Gly
580 585 590
gac aag gtg tac atc gac aag atc gag ttc atc ccc gtg aac 1818
Asp Lys Val Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Asn
595 600 605
<210>11
<211>606
<212>PRT
<213>人工序列
<220>
<221>misc_feature
<222>(346)..(357)
<223>组织蛋白酶G识别位点编码序列.
<400>11
Met Asn Tyr Lys Glu Phe Leu Arg Met Thr Ala Asp Asn Asn Thr Glu
1 5 10 15
Ala Leu Asp Ser Ser Thr Thr Lys Asp Val Ile Gln Lys Gly Ile Ser
20 25 30
Val Val Gly Asp Leu Leu Gly Val Val Gly Phe Pro Phe Gly Gly Ala
35 40 45
Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn Thr Ile Trp Pro Ser Glu
50 55 60
Asp Pro Trp Lys Ala Phe Met Glu Gln Val Glu Ala Leu Net Asp Gln
65 70 75 80
Lys Ile Ala Asp Tyr Ala Lys Asn Lys Ala Leu Ala Glu Leu Gln Gly
85 90 95
Leu Gln Asn Asn Val Glu Asp Tyr Val Ser Ala Leu Ser Ser Trp Gln
100 105 110
Lys Asn Pro Ala Ala Pro Phe Arg Asn Pro His Ser Gln Gly Arg Ile
115 120 125
Arg Glu Leu Phe Ser Gln Ala Glu Ser His Phe Arg Ash Ser Met Pro
130 135 140
Ser Phe Ala Ile Ser Gly Tyr Glu Val Leu Phe Leu Thr Thr Tyr Ala
145 150 155 160
Gln Ala Ala Asn Thr His Leu Phe Leu Leu Lys Asp Ala Gln Ile Tyr
165 170 175
Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp Ile Ala Glu Phe Tyr Lys
180 185 190
Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr Asp His Cys Val Lys Trp
195 200 205
Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly Ser Ser Tyr Glu Ser Trp
210 215 220
Val Asn Phe Asn Arg Tyr Arg Arg Glu Met Thr Leu Thr Val Leu Asp
225 230 235 240
Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val Arg Leu Tyr Pro Lys Glu
245 250 255
Val Lys Thr Glu Leu Thr Arg Asp Val Leu Thr Asp Pro Ile Val Gly
260 265 270
Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr Phe Ser Asn Ile Glu Asn
275 280 285
Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr Leu His Arg Ile Gln Phe
290 295 300
His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly Asn Asp Ser Phe Asn Tyr
305 310 315 320
Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro Ser Ile Gly Ser Asn Asp
325 330 335
Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys Ser Ser Glu Pro Val Gln
340 345 350
Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr Arg Ala Val Ala Asn Thr
355 360 365
Asn Leu Ala Val Trp Pro Ser Ala Val Tyr Ser Gly Val Thr Lys Val
370 375 380
Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp Glu Ala Ser Thr Gln Thr
385 390 395 400
Tyr Asp Ser Lys Arg Asn Val Gly Ala Val Ser Trp Asp Ser Ile Asp
405 410 415
Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu Glu Lys Gly Tyr Ser
420 425 430
His Gln Leu Asn Tyr Val Met Cys Phe Leu Met Gln Gly Ser Arg Gly
435 440 445
Thr Ile Pro Val Leu Thr Trp Thr His Lys Ser Val Asp Phe Phe Asn
450 455 460
Met Ile Asp Ser Lys Lys Ile Thr Gln Leu Pro Leu Val Lys Ala Tyr
465 470 475 480
Lys Leu Gln Ser Gly Ala Ser Val Val Ala Gly Pro Arg Phe Thr Gly
485 490 495
Gly Asp Ile Ile Gln Cys Thr Glu Asn Gly Ser Ala Ala Thr Ile Tyr
500 505 510
Val Thr Pro Asp Val Ser Tyr Ser Gln Lys Tyr Arg Ala Arg Ile His
515 520 525
Tyr Ala Ser Thr Ser Gln Ile Thr Phe Thr Leu Ser Leu Asp Gly Ala
530 535 540
Pro Phe Asn Gln Tyr Tyr Phe Asp Lys Thr Ile Asn Lys Gly Asp Thr
545 550 555 560
Leu Thr Tyr Asn Ser Phe Asn Leu Ala Ser Phe Ser Thr Pro Phe Glu
565 570 575
Leu Ser Gly Asn Asn Leu Gln Ile Gly Val Thr Gly Leu Ser Ala Gly
580 585 590
Asp Lys Val Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Asn
595 600 605
<210>12
<211>1794
<212>DNA
<213>人工序列
<220>
<221>CDS
<222>(1)..(1794)
<223>玉米优化的修饰的cry3A082编码序列.
<220>
<221>misc_feature
<222>(1609)..(1620)
<223>组织蛋白酶G识别位点编码序列
<400>12
atg acg gcc gac aac aac acc gag gcc ctg gac agc agc acc acc aag 48
Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys
1 5 10 15
gsc gtg atc cag aag ggc atc agc gtg gtg ggc gac ctg ctg ggc gtg 96
Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
20 25 30
gtg ggc ttc ccc ttc ggc ggc gcc ctg gtg agc ttc tac acc aac ttc 144
Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe
35 40 45
ctg aac acc atc tgg ccc agc gag gac ccc tgg aag gcc ttc atg gag 192
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu
50 55 60
cag gtg gag gcc ctg atg gac cag aag atc gcc gac tac gcc aag aac 240
Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn
65 70 75 80
aag gca ctg gcc gag cta cag ggc ctc cag aac aac gtg gag gac tat 288
Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr
85 90 95
gtg agc gcc ctg agc agc tgg cag aag aac ccc gtc tcg agc cgc aac 336
Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Val Ser Ser Arg Asn
100 105 110
ccc cac agc cag ggc cgc atc cgc gag ctg ttc agc cag gcc gag agc 384
Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser
115 120 125
cac ttc cgc aac agc atg ccc agc ttc gcc atc agc ggc tac gag gtg 432
His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val
130 135 140
ctg ttc ctg acc acc tac gcc cag gcc gcc aac acc cac ctg ttc ctg 480
Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu
145 150 155 160
ctg aag gac gcc caa atc tac gga gag gag tgg ggc tac gag aag gag 528
Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu
165 170 175
gac atc gcc gag ttc tac aag cgc cag ctg aag ctg acc cag gag tac 576
Asp Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr
180 185 190
acc gac cac tgc gtg aag tgg tac aac gtg ggt cta gac aag ctc cgc 624
Thr Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg
195 200 205
ggc agc agc tac gag agc tgg gtg aac ttc aac cgc tac cgc cgc gag 672
Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu
210 215 220
atg acc ctg acc gtg ctg gac ctg atc gcc ctg ttc ccc ctg tac gac 720
Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp
225 230 235 240
gtg cgc ctg tac ccc aag gag gtg aag acc gag ctg acc cgc gac gtg 768
Val Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val
245 250 255
ctg acc gac ccc atc gtg ggc gtg aac aac ctg cgc ggc tac ggc acc 816
Leu Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr
260 265 270
acc ttc agc aac atc gag aac tac atc cgc aag ccc cac ctg ttc gac 864
Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp
275 280 285
tac ctg cac cgc atc cag ttc cac acg cgt ttc cag ccc ggc tac tac 912
Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr
290 295 300
ggc aac gac agc ttc aac tac tgg agc ggc aac tac gtg agc acc cgc 960
Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg
305 310 315 320
ccc agc atc ggc agc aac gac atc atc acc agc ccc ttc tac ggc aac 1008
Pro Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn
325 330 335
aag agc agc gag ccc gtg cag aac ctt gag ttc aac ggc gag aag gtg 1056
Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val
340 345 350
tac cgc gcc gtg gct aac acc aac ctg gcc gtg tgg ccc tct gca gtg 1104
Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val
355 360 365
tac agc ggc gtg acc aag gtg gag ttc agc cag tac aac gac cag acc 1152
Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr
370 375 380
gac gag gcc agc acc cag acc tac gac agc aag cgc aac gtg ggc gcc 1200
Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala
385 390 395 400
gtg agc tgg gac agc atc gac cag ctg ccc ccc gag acc acc gac gag 1248
Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu
405 410 415
ccc ctg gag aag ggc tac agc cac cag ctg aac tac gtg atg tgc ttc 1296
Pro Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe
420 425 430
ctg atg cag ggc agc cgc ggc acc atc ccc gtg ctg acc tgg acc cac 1344
Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His
435 440 445
aag agc gtc gac ttc ttc aac atg atc gac agc aag aag atc acc cag 1392
Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln
450 455 460
ctg ccc ctg gtg aag gcc tac aag ctc cag agc ggc gcc agc gtg gtg 1440
Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val Val
465 470 475 480
gca ggc ccc cgc ttc acc ggc ggc gac atc atc cag tgc acc gag aac 1488
Ala Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn
485 490 495
ggc agc gcc gcc acc atc tac gtg acc ccc gac gtg agc tac agc cag 1536
Gly Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser Gln
500 505 510
aag tac cgc gcc cgc atc cac tac gcc agc acc agc cag atc acc ttc 1584
Lys Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr Phe
515 520 525
acc ctg agc ctg gac ggg gcc ccc gct gca ccg ttc tac ttc gac aag 1632
Thr Leu Ser Leu Asp Gly Ala Pro Ala Ala Pro Phe Tyr Phe Asp Lys
530 535 540
acc atc aac aag ggc gac acc ctg acc tac aac agc ttc aac ctg gcc 1680
Thr Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu Ala
545 550 555 560
agc ttc agc acc cct ttc gag ctg agc ggc aac aac ctc cag atc ggc 1728
Ser Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile Gly
565 570 575
gtg acc ggc ctg agc gcc ggc gac aag gtg tac atc gac aag atc gag 1776
Val Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys Ile Glu
580 585 590
ttc atc ccc gtg aac tag 1794
Phe Ile Pro Val Asn
595
<210>13
<211>597
<212>PRT
<213>人工序列
<220>
<221>misc_feature
<222>(1609)..(1620)
<223>组织蛋白酶G识别位点编码序列
<400>13
Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys
1 5 10 15
Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
20 25 30
Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe
35 40 45
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu
50 55 60
Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn
65 70 75 80
Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr
85 90 95
Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Val Ser Ser Arg Asn
100 105 110
Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser
115 120 125
His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val
130 135 140
Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu
145 150 155 160
Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu
165 170 175
Asp Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr
180 185 190
Thr Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg
195 200 205
Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu
210 215 220
Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp
225 230 235 240
Val Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val
245 250 255
Leu Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr
260 265 270
Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp
275 280 285
Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr
290 295 300
Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg
305 310 315 320
Pro Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn
325 330 335
Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val
340 345 350
Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val
355 360 365
Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr
370 375 380
Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala
385 390 395 400
Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu
405 410 415
Pro Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe
420 425 430
Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His
435 440 445
Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln
450 455 460
Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val Val
465 470 475 480
Ala Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn
485 490 495
Gly Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser Gln
500 505 510
Lys Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr Phe
515 520 525
Thr Leu Ser Leu Asp Gly Ala Pro Ala Ala Pro Phe Tyr Phe Asp Lys
530 535 540
Thr Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu Ala
545 550 555 560
Ser Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile Gly
565 570 575
Val Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys Ile Glu
580 585 590
Phe Ile Pro Val Asn
595
<210>14
<211>1816
<212>DNA
<213>人工序列
<220>
<221>CDS
<222>(1)..(1812)
<223>玉米优化的修饰的cry3A058编码序列.
<220>
<221>misc_feature
<222>(1621)..(1632)
<223>组织蛋白酶G识别位点编码序列
<400>14
atg acg gcc gac aac aac acc gag gcc ctg gac agc agc acc acc aag 48
Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys
1 5 10 15
gac gtg atc cag aag ggc atc agc gtg gtg ggc gac ctg ctg ggc gtg 96
Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
20 25 30
gtg ggc ttc ccc ttc ggc ggc gcc ctg gtg agc ttc tac acc aac ttc 144
Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe
35 40 45
ctg aac acc atc tgg ccc agc gag gac ccc tgg aag gcc ttc atg gag 192
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu
50 55 60
cag gtg gag gcc ctg atg gac cag aag atc gcc gac tac gcc aag aac 240
Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn
65 70 75 80
aag gca ctg gcc gag cta cag ggc ctc cag aac aac gtg gag gac tat 288
Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr
85 90 95
gtg agc gcc ctg agc agc tgg cag aag aac ccc gtc tcg agc cgc aac 336
Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Val Ser Ser Arg Asn
100 105 110
ccc cac agc cag ggc cgc atc cgc gag ctg ttc agc cag gcc gag agc 384
Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser
115 120 125
cac ttc cgc aac agc atg ccc agc ttc gcc atc agc ggc tac gag gtg 432
His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val
130 135 140
ctg ttc ctg acc acc tac gcc cag gcc gcc aac acc cac ctg ttc ctg 480
Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu
145 150 155 160
ctg aag gac gcc caa atc tac gga gag gag tgg ggc tac gag aag gag 528
Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu
165 170 175
gac atc gcc gag ttc tac aag cgc cag ctg aag ctg acc cag gag tac 576
Asp Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr
180 185 190
acc gac cac tgc gtg aag tgg tac aac gtg ggt cta gac aag ctc cgc 624
Thr Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg
195 200 205
ggc agc agc tac gag agc tgg gtg aac ttc aac cgc tac cgc cgc gag 672
Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu
210 215 220
atg acc ctg acc gtg ctg gac ctg atc gcc ctg ttc ccc ctg tac gac 720
Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp
225 230 235 240
gtg cgc ctg tac ccc aag gag gtg aag acc gag ctg acc cgc gac gtg 768
Val Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val
245 250 255
ctg acc gac ccc atc gtg ggc gtg aac aac ctg cgc ggc tac ggc acc 816
Leu Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr
260 265 270
acc ttc agc aac atc gag aac tac atc cgc aag ccc cac ctg ttc gac 864
Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp
275 280 285
tac ctg cac cgc atc cag ttc cac acg cgt ttc cag ccc ggc tac tac 912
Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr
290 295 300
ggc aac gac agc ttc aac tac tgg agc ggc aac tac gtg agc acc cgc 960
Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg
305 310 315 320
ccc agc atc ggc agc aac gac atc atc acc agc ccc ttc tac ggc aac 1008
Pro Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn
325 330 335
aag agc agc gag ccc gtg cag aac ctt gag ttc aac ggc gag aag gtg 1056
Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val
340 345 350
tac cgc gcc gtg gct aac acc aac ctg gcc gtg tgg ccc tct gca gtg 1104
Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val
355 360 365
tac agc ggc gtg acc aag gtg gag ttc agc cag tac aac gac cag acc 1152
Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr
370 375 380
gac gag gcc agc acc cag acc tac gac agc aag cgc aac gtg ggc gcc 1200
Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala
385 390 395 400
gtg agc tgg gac agc atc gac cag ctg ccc ccc gag acc acc gac gag 1248
Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu
405 410 415
ccc ctg gag aag ggc tac agc cac cag ctg aac tac gtg atg tgc ttc 1296
Pro Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe
420 425 430
ctg atg cag ggc agc cgc ggc acc atc ccc gtg ctg acc tgg acc cac 1344
Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His
435 440 445
aag agc gtc gac ttc ttc aac atg atc gac agc aag aag atc acc cag 1392
Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln
450 455 460
ctg ccc ctg gtg aag gcc tac aag ctc cag agc ggc gcc agc gtg gtg 1440
Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val Val
465 470 475 480
gca ggc ccc cgc ttc acc ggc ggc gac atc atc cag tgc acc gag aac 1488
Ala Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn
485 490 495
ggc agc gcc gcc acc atc tac gtg acc ccc gac gtg agc tac agc cag 1536
Gly Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser Gln
500 505 510
aag tac cgc gcc cgc atc cac tac gcc agc acc agc cag atc acc ttc 1584
Lys Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr Phe
515 520 525
acc ctg agc ctg gac ggg gcc ccc ttc aac caa tac gct gca ccg ttc 1632
Thr Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Ala Ala Pro Phe
530 535 540
tac ttc gac aag acc atc aac aag ggc gac acc ctg acc tac aac agc 1680
Tyr Phe Asp Lys Thr Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser
545 550 555 560
ttc aac ctg gcc agc ttc agc acc cct ttc gag ctg agc ggc aac aac 1728
Phe Asn Leu Ala Ser Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn
565 570 575
ctc cag atc ggc gtg acc ggc ctg agc gcc ggc gac aag gtg tac atc 1776
Leu Gln Ile Gly Val Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile
580 585 590
gac aag atc gag ttc atc ccc gtg aac tag atc tga gctc 1816
Asp Lys Ile Glu Phe Ile Pro Val Asn Ile
595 600
<210>15
<211>601
<212>PRT
<213>人工序列
<220>
<221>misc_feature
<222>(1621)..(1632)
<223>组织蛋白酶G识别位点编码序列
<400>15
Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys
1 5 10 15
Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
20 25 30
Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe
35 40 45
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu
50 55 60
Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn
65 70 75 80
Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr
85 90 95
Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Val Ser Ser Arg Asn
100 105 110
Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser
115 120 125
His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val
130 135 140
Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu
145 150 155 160
Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu
165 170 175
Asp Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr
180 185 190
Thr Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg
195 200 205
Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu
210 215 220
Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp
225 230 235 240
Val Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val
245 250 255
Leu Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr
260 265 270
Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp
275 280 285
Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr
290 295 300
Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg
305 310 315 320
Pro Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn
325 330 335
Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val
340 345 350
Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val
355 360 365
Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr
370 375 380
Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala
385 390 395 400
Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu
405 410 415
Pro Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe
420 425 430
Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His
435 440 445
Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln
450 455 460
Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val Val
465 470 475 480
Ala Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn
485 490 495
Gly Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser Gln
500 505 510
Lys Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr Phe
515 520 525
Thr Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Ala Ala Pro Phe
530 535 540
Tyr Phe Asp Lys Thr Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser
545 550 555 560
Phe Asn Leu Ala Ser Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn
565 570 575
Leu Gln Ile Gly Val Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile
580 585 590
Asp Lys Ile Glu Phe Ile Pro Val Asn
595 600
<210>16
<211>1813
<212>DNA
<213>人工序列
<220>
<221>CDS
<222>(1)..(1812)
<223>玉米优化的修饰的cry3A057编码序列.
<220>
<221>misc_feature
<222>(322)..(333)
<223>组织蛋白酶G识别位点编码序列
<220>
<221>misc_feature
<222>(1618)..(1629)
<223>组织蛋白酶G识别位点编码序列
<400>16
atg acg gcc gac aac aac acc gag gcc ctg gac agc agc acc acc aag 48
Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr nys
1 5 10 15
gac gtg atc cag aag ggc atc agc gtg gtg ggc gac ctg ctg ggc gtg 96
Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
20 25 30
gtg ggc ttc ccc ttc ggc ggc gcc ctg gtg agc ttc tac acc aac ttc 144
Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe
35 40 45
ctg aac acc atc tgg ccc agc gag gac ccc tgg aag gcc ttc atg gag 192
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu
50 55 60
cag gtg gag gcc ctg atg gac cag aag atc gcc gac tac gcc aag aac 240
Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn
65 70 75 80
aag gca ctg gcc gag cta cag ggc ctc cag aac aac gtg gag gac tat 288
Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr
85 90 95
gtg agc gcc ctg agc agc tgg cag aag aac ccc gct gca ccg ttc ccc 336
Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Pro
100 105 110
cac agc cag ggc cgc atc cgc gag ctg ttc agc cag gcc gag agc cac 384
His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His
115 120 125
ttc cgc aac agc atg ccc agc ttc gcc atc agc ggc tac gag gtg ctg 432
Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val Leu
130 135 140
ttc ctg acc acc tac gcc cag gcc gcc aac acc cac ctg ttc ctg ctg 480
Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu Leu
145 150 155 160
aag gac gcc caa atc tac gga gag gag tgg ggc tac gag aag gag gac 528
Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp
165 170 175
atc gcc gag ttc tac aag cgc cag ctg aag ctg acc cag gag tac acc 576
Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr
180 185 190
gac cac tgc gtg aag tgg tac aac gtg ggt cta gac aag ctc cgc ggc 624
Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly
195 200 205
agc agc tac gag agc tgg gtg aac ttc aac cgc tac cgc cgc gag atg 672
Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met
210 215 220
acc ctg acc gtg ctg gac ctg atc gcc ctg ttc ccc ctg tac gac gtg 720
Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val
225 230 235 240
cgc ctg tac ccc aag gag gtg aag acc gag ctg acc cgc gac gtg ctg 768
Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu
245 250 255
acc gac ccc atc gtg ggc gtg aac aac ctg cgc ggc tac ggc acc acc 816
Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr
260 265 270
ttc agc aac atc gag aac tac atc cgc aag ccc cac ctg ttc gac tac 864
Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr
275 280 285
ctg cac cgc atc cag ttc cac acg cgt ttc cag ccc ggc tac tac ggc 912
Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly
290 295 300
aac gac agc ttc aac tac tgg agc ggc aac tac gtg agc acc cgc ccc 960
Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro
305 310 315 320
agc atc ggc agc aac gac atc atc acc agc ccc ttc tac ggc aac aag 1008
Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys
325 330 335
agc agc gag ccc gtg cag aac ctt gag ttc aac ggc gag aag gtg tac 1056
Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr
340 345 350
cgc gcc gtg gct aac acc aac ctg gcc gtg tgg ccc tct gca gtg tac 1104
Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val Tyr
355 360 365
agc ggc gtg acc aag gtg gag ttc agc cag tac aac gac cag acc gac 1152
Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp
370 375 380
gag gcc agc acc cag acc tac gac agc aag cgc aac gtg ggc gcc gtg 1200
Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val
385 390 395 400
agc tgg gac agc atc gac cag ctg ccc ccc gag acc acc gac gag ccc 1248
Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro
405 410 415
ctg gag aag ggc tac agc cac cag ctg aac tac gtg atg tgc ttc ctg 1296
Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu
420 425 430
atg cag ggc agc cgc ggc acc atc ccc gtg ctg acc tgg acc cac aag 1344
Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His Lys
435 440 445
agc gtc gac ttc ttc aac atg atc gac agc aag aag atc acc cag ctg 1392
Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln Leu
450 455 460
ccc ctg gtg aag gcc tac aag ctc cag agc ggc gcc agc gtg gtg gca 1440
Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val Val Ala
465 470 475 480
ggc ccc cgc ttc acc ggc ggc gac atc atc cag tgc acc gag aac ggc 1488
Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn Gly
485 490 495
agc gcc gcc acc atc tac gtg acc ccc gac gtg agc tac agc cag aag 1536
Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser Gln Lys
500 505 510
tac cgc gcc cgc atc cac tac gcc agc acc agc cag atc acc ttc acc 1584
Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr Phe Thr
515 520 525
ctg agc ctg gac ggg gcc ccc ttc aac caa tac gct gca ccg ttc tac 1632
Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Ala Ala Pro Phe Tyr
530 535 540
ttc gac aag acc atc aac aag ggc gac acc ctg acc tac aac agc ttc 1680
Phe Asp Lys Thr Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe
545 550 555 560
aac ctg gcc agc ttc agc acc cct ttc gag ctg agc ggc aac aac ctc 1728
Asn Leu Ala Ser Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu
565 570 575
cag atc ggc gtg acc ggc ctg agc gcc ggc gac aag gtg tac atc gac 1776
Gln Ile Gly Val Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp
580 585 590
aag atc gag ttc atc ccc gtg aac tag atc tga gct c 1813
Lys Ile Glu Phe Ile Pro Val Asn Ile Ala
595 600
<210>17
<211>600
<212>PRT
<213>人工序列
<220>
<221>misc_feature
<222>(322)..(333)
<223>组织蛋白酶G识别位点编码序列
<220>
<221>misc_feature
<222>(1618)..(1629)
<223>组织蛋白酶G识别位点编码序列
<400>17
Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys
1 5 10 15
Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
20 25 30
Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe
35 40 45
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu
50 55 60
Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn
65 70 75 80
Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr
85 90 95
Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Pro
100 105 110
His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His
115 120 125
Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val Leu
130 135 140
Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu Leu
145 150 155 160
Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp
165 170 175
Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr
180 185 190
Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly
195 200 205
Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met
210 215 220
Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val
225 230 235 240
Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu
245 250 255
Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr
260 265 270
Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr
275 280 285
Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly
290 295 300
Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro
305 310 315 320
Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys
325 330 335
Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr
340 345 350
Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val Tyr
355 360 365
Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp
370 375 380
Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val
385 390 395 400
Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro
405 410 415
Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu
420 425 430
Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His Lys
435 440 445
Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln Leu
450 455 460
Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val Val Ala
465 470 475 480
Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu Asn Gly
485 490 495
Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser Gln Lys
500 505 510
Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr Phe Thr
515 520 525
Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Ala Ala Pro Phe Tyr
530 535 540
Phe Asp Lys Thr Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe
545 550 555 560
Asn Leu Ala Ser Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu
565 570 575
Gln Ile Gly Val Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp
580 585 590
Lys Ile Glu Phe Ile Pro Val Asn
595 600
<210>18
<211>1819
<212>DNA
<213>人工序列
<220>
<221>CDS
<222>(1)..(1818)
<223>玉米优化的修饰的cry3A056编码序列.
<220>
<221>misc_feature
<222>(322)..(333)
<223>组织蛋白酶G识别位点编码序列.
<220>
<221>misc_feature
<222>(1624)..(1635)
<223>组织蛋白酶G识别位点编码序列.
<400>18
atg acg gcc gac aac aac acc gag gcc ctg gac agc agc acc acc aag 48
Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys
1 5 10 15
gac gtg atc cag aag ggc atc agc gtg gtg ggc gac ctg ctg ggc gtg 96
Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
20 25 30
gtg ggc ttc ccc ttc ggc ggc gcc ctg gtg agc ttc tac acc aac ttc 144
Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe
35 40 45
ctg aac acc atc tgg ccc agc gag gac ccc tgg aag gcc ttc atg gag 192
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu
50 55 60
cag gtg gag gcc ctg atg gac cag aag atc gcc gac tac gcc aag aac 240
Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn
65 70 75 80
aag gca ctg gcc gag cta cag ggc ctc cag aac aac gtg gag gac tat 288
Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr
85 90 95
gtg agc gcc ctg agc agc tgg cag aag aac ccc gct gca ccg ttc cgc 336
Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg
100 105 110
aac ccc cac agc cag ggc cgc atc cgc gag ctg ttc agc cag gcc gag 384
Asn Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu
115 120 125
agc cac ttc cgc aac agc atg ccc agc ttc gcc atc agc ggc tac gag 432
Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu
130 135 140
gtg ctg ttc ctg acc acc tac gcc cag gcc gcc aac acc cac ctg ttc 480
Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe
145 150 155 160
ctg ctg aag gac gcc caa atc tac gga gag gag tgg ggc tac gag aag 528
Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys
165 170 175
gag gac atc gcc gag ttc tac aag cgc cag ctg aag ctg acc cag gag 576
Glu Asp Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu
180 185 190
tac acc gac cac tgc gtg aag tgg tac aac gtg ggt cta gac aag ctc 624
Tyr Thr Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu
195 200 205
cgc ggc agc agc tac gag agc tgg gtg aac ttc aac cgc tac cgc cgc 672
Arg Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg
210 215 220
gag atg acc ctg acc gtg ctg gac ctg atc gcc ctg ttc ccc ctg tac 720
Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr
225 230 235 240
gac gtg cgc ctg tac ccc aag gag gtg aag acc gag ctg acc cgc gac 768
Asp Val Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp
245 250 255
gtg ctg acc gac ccc atc gtg ggc gtg aac aac ctg cgc ggc tac ggc 816
Val Leu Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly
260 265 270
acc acc ttc agc aac atc gag aac tac atc cgc aag ccc cac ctg ttc 864
Thr Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe
275 280 285
gac tac ctg cac cgc atc cag ttc cac acg cgt ttc cag ccc ggc tac 912
Asp Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr
290 295 300
tac ggc aac gac agc ttc aac tac tgg agc ggc aac tac gtg agc acc 960
Tyr Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr
305 310 315 320
cgc ccc agc atc ggc agc aac gac atc atc acc agc ccc ttc tac ggc 1008
Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly
325 330 335
aac aag agc agc gag ccc gtg cag aac ctt gag ttc aac ggc gag aag 1056
Asn Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys
340 345 350
gtg tac cgc gcc gtg gct aac acc aac ctg gcc gtg tgg ccc tct gca 1104
Val Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala
355 360 365
gtg tac agc ggc gtg acc aag gtg gag ttc agc cag tac aac gac cag 1152
Val Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln
370 375 380
acc gac gag gcc agc acc cag acc tac gac agc aag cgc aac gtg ggc 1200
Thr Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly
385 390 395 400
gcc gtg agc tgg gac agc atc gac cag ctg ccc ccc gag acc acc gac 1248
Ala Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp
405 410 415
gag ccc ctg gag aag ggc tac agc cac cag ctg aac tac gtg atg tgc 1296
Glu Pro Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys
420 425 430
ttc ctg atg cag ggc agc cgc ggc acc atc ccc gtg ctg acc tgg acc 1344
Phe Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr
435 440 445
cac aag agc gtc gac ttc ttc aac atg atc gac agc aag aag atc acc 1392
His Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr
450 455 460
cag ctg ccc ctg gtg aag gcc tac aag ctc cag agc ggc gcc agc gtg 1440
Gln Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val
465 470 475 480
gtg gca ggc ccc cgc ttc acc ggc ggc gac atc atc cag tgc acc gag 1488
Val Ala Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu
485 490 495
aac ggc agc gcc gcc acc atc tac gtg acc ccc gac gtg agc tac agc 1536
Asn Gly Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser
500 505 510
cag aag tac cgc gcc cgc atc cac tac gcc agc acc agc cag atc acc 1584
Gln Lys Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr
515 520 525
ttc acc ctg agc ctg gac ggg gcc ccc ttc aac caa tac gct gca ccg 1632
Phe Thr Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Ala Ala Pro
530 535 540
ttc tac ttc gac aag acc atc aac aag ggc gac acc ctg acc tac aac 1680
Phe Tyr Phe Asp Lys Thr Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn
545 550 555 560
agc ttc aac ctg gcc agc ttc agc acc cct ttc gag ctg agc ggc aac 1728
Ser Phe Asn Leu Ala Ser Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn
565 570 575
aac ctc cag atc ggc gtg acc ggc ctg agc gcc ggc gac aag gtg tac 1776
Asn Leu Gln Ile Gly Val Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr
580 585 590
atc gac aag atc gag ttc atc ccc gtg aac tag atc tga gct c 1819
Ile Asp Lys Ile Glu Phe Ile Pro Val Asn Ile Ala
595 600
<210>19
<211>602
<212>PRT
<213>人工序列
<220>
<221>misc_feature
<222>(322)..(333)
<223>组织蛋白酶G识别位点编码序列.
<220>
<221>misc_feature
<222>(1624)..(1635)
<223>组织蛋白酶G识别位点编码序列.
<400>19
Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys
1 5 10 15
Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
20 25 30
Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe
35 40 45
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu
50 55 60
Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn
65 70 75 80
Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr
85 90 95
Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg
100 105 110
Asn Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu
115 120 125
Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu
130 135 140
Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe
145 150 155 160
Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys
165 170 175
Glu Asp Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu
180 185 190
Tyr Thr Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu
195 200 205
Arg Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg
210 215 220
Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr
225 230 235 240
Asp Val Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp
245 250 255
Val Leu Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly
260 265 270
Thr Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe
275 280 285
Asp Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr
290 295 300
Tyr Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr
305 310 315 320
Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly
325 330 335
Asn Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys
340 345 350
Val Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala
355 360 365
Val Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln
370 375 380
Thr Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly
385 390 395 400
Ala Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp
405 410 415
Glu Pro Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys
420 425 430
Phe Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr
435 440 445
His Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr
450 455 460
Gln Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val
465 470 475 480
Val Ala Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu
485 490 495
Asn Gly Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser
500 505 510
Gln Lys Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr
515 520 525
Phe Thr Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Ala Ala Pro
530 535 540
Phe Tyr Phe Asp Lys Thr Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn
545 550 555 560
Ser Phe Asn Leu Ala Ser Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn
565 570 575
Asn Leu Gln Ile Gly Val Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr
580 585 590
Ile Asp Lys Ile Glu Phe Ile Pro Val Asn
595 600
<210>20
<211>1797
<212>DNA
<213>人工序列
<220>
<221>CDS
<222>(1)..(1791)
<223>玉米优化的修饰的cry3A083编码序列.
<220>
<221>misc_feature
<222>(322)..(333)
<223>组织蛋白酶G识别位点编码序列.
<220>
<221>misc_feature
<222>(1612)..(1623)
<223>组织蛋白酶G识别位点编码序列
<400>20
atg acg gcc gac aac aac acc gag gcc ctg gac agc agc acc acc aag 48
Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys
1 5 10 15
gac gtg atc cag aag ggc atc agc gtg gtg ggc gac ctg ctg ggc gtg 96
Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
20 25 30
gtg ggc ttc ccc ttc ggc ggc gcc ctg gtg agc ttc tac acc aac ttc 144
Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe
35 40 45
ctg aac acc atc tgg ccc agc gag gac ccc tgg aag gcc ttc atg gag 192
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu
50 55 60
cag gtg gag gcc ctg atg gac cag aag atc gcc gac tac gcc aag aac 240
Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn
65 70 75 80
aag gca ctg gcc gag cta cag ggc ctc cag aac aac gtg gag gac tat 288
Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr
85 90 95
gtg agc gcc ctg agc agc tgg cag aag aac ccc gct gca ccg ttc cgc 336
Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg
100 105 110
aac ccc cac agc cag ggc cgc atc cgc gag ctg ttc agc cag gcc gag 384
Asn Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu
115 120 125
agc cac ttc cgc aac agc atg ccc agc ttc gcc atc agc ggc tac gag 432
Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu
130 135 140
gtg ctg ttc ctg acc acc tac gcc cag gcc gcc aac acc cac ctg ttc 480
Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe
145 150 155 160
ctg ctg aag gac gcc caa atc tac gga gag gag tgg ggc tac gag aag 528
Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys
165 170 175
gag gac atc gcc gag ttc tac aag cgc cag ctg aag ctg acc cag gag 576
Glu Asp Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu
180 185 190
tac acc gac cac tgc gtg aag tgg tac aac gtg ggt cta gac aag ctc 624
Tyr Thr Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu
195 200 205
cgc ggc agc agc tac gag agc tgg gtg aac ttc aac cgc tac cgc cgc 672
Arg Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg
210 215 220
gag atg acc ctg acc gtg ctg gac ctg atc gcc ctg ttc ccc ctg tac 720
Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr
225 230 235 240
gac gtg cgc ctg tac ccc aag gag gtg aag acc gag ctg acc cgc gac 768
Asp Val Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp
245 250 255
gtg ctg acc gac ccc atc gtg ggc gtg aac aac ctg cgc ggc tac ggc 816
Val Leu Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly
260 265 270
acc acc ttc agc aac atc gag aac tac atc cgc aag ccc cac ctg ttc 864
Thr Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe
275 280 285
gac tac ctg cac cgc atc cag ttc cac acg cgt ttc cag ccc ggc tac 912
Asp Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr
290 295 300
tac ggc aac gac agc ttc aac tac tgg agc ggc aac tac gtg agc acc 960
Tyr Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr
305 310 315 320
cgc ccc agc atc ggc agc aac gac atc atc acc agc ccc ttc tac ggc 1008
Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly
325 330 335
aac aag agc agc gag ccc gtg cag aac ctt gag ttc aac ggc gag aag 1056
Asn Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys
340 345 350
gtg tac cgc gcc gtg gct aac acc aac ctg gcc gtg tgg ccc tct gca 1104
Val Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala
355 360 365
gtg tac agc ggc gtg acc aag gtg gag ttc agc cag tac aac gac cag 1152
Val Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln
370 375 380
acc gac gag gcc agc acc cag acc tac gac agc aag cgc aac gtg ggc 1200
Thr Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly
385 390 395 400
gcc gtg agc tgg gac agc atc gac cag ctg ccc ccc gag acc acc gac 1248
Ala Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp
405 410 415
gag ccc ctg gag aag ggc tac agc cac cag ctg aac tac gtg atg tgc 1296
Glu Pro Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys
420 425 430
ttc ctg atg cag ggc agc cgc ggc acc atc ccc gtg ctg acc tgg acc 1344
Phe Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr
435 440 445
cac aag agc gtc gac ttc ttc aac atg atc gac agc aag aag atc acc 1392
His Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr
450 455 460
cag ctg ccc ctg gtg aag gcc tac aag ctc cag agc ggc gcc agc gtg 1440
Gln Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val
465 470 475 480
gtg gca ggc ccc cgc ttc acc ggc ggc gac atc atc cag tgc acc gag 1488
Val Ala Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu
485 490 495
aac ggc agc gcc gcc acc atc tac gtg acc ccc gac gtg agc tac agc 1536
Asn Gly Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser
500 505 510
cag aag tac cgc gcc cgc atc cac tac gcc agc acc agc cag atc acc 1584
Gln Lys Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr
515 520 525
ttc acc ctg agc ctg gac ggg gcc ccc gct gca ccg ttc tac ttc gac 1632
Phe Thr Leu Ser Leu Asp Gly Ala Pro Ala Ala Pro Phe Tyr Phe Asp
530 535 540
aag acc atc aac aag ggc gac acc ctg acc tac aac agc ttc aac ctg 1680
Lys Thr Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu
545 550 555 560
gcc agc ttc agc acc cct ttc gag ctg agc ggc aac aac ctc cag atc 1728
Ala Ser Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile
565 570 575
ggc gtg acc ggc ctg agc gcc ggc gac aag gtg tac atc gac aag atc 1776
Gly Val Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys Ile
580 585 590
gag ttc atc ccc gtg aactag 1797
Glu Phe Ile Pro Val
595
<210>21
<211>597
<212>PRT
<213>人工序列
<220>
<221>misc_feature
<222>(322)..(333)
<223>组织蛋白酶G识别位点编码序列.
<220>
<221>misc_feature
<222>(1612)..(1623)
<223>组织蛋白酶G识别位点编码序列
<400>21
Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys
1 5 10 15
Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val
20 25 30
Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe
35 40 45
Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu
50 55 60
Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn
65 70 75 80
Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr
85 90 95
Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg
100 105 110
Asn Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu
115 120 125
Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu
130 135 140
Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe
145 150 155 160
Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys
165 170 175
Glu Asp Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu
180 185 190
Tyr Thr Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu
195 200 205
Arg Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg
210 215 220
Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr
225 230 235 240
Asp Val Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp
245 250 255
Val Leu Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly
260 265 270
Thr Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe
275 280 285
Asp Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr
290 295 300
Tyr Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr
305 310 315 320
Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly
325 330 335
Asn Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys
340 345 350
Val Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala
355 360 365
Val Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln
370 375 380
Thr Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly
385 390 395 400
Ala Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp
405 410 415
Glu Pro Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys
420 425 430
Phe Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr
435 440 445
His Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr
450 455 460
Gln Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val
465 470 475 480
Val Ala Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu
485 490 495
Asn Gly Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser
500 505 510
Gln Lys Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr
515 520 525
Phe Thr Leu Ser Leu Asp Gly Ala Pro Ala Ala Pro Phe Tyr Phe Asp
530 535 540
Lys Thr Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu
545 550 555 560
Ala Ser Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile
565 570 575
Gly Val Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys Ile
580 585 590
Glu Phe Ile Pro Val
595
<210>22
<211>21
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(21)
<223>BamExt1引物
<400>22
ggatccacca tgacggccga c 21
<210>23
<211>29
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(29)
<223>AAPFtail3引物
<400>23
gaacggtgca gcggggttct tctgccagc 29
<210>24
<211>29
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(29)
<223>AAPFtail4引物
<400>24
gctgcaccgt tcccccacag ccagggccg 29
<210>25
<211>21
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(21)
<223>XbaIExt2引物
<400>25
tctagaccca cgttgtacca c 21
<210>26
<211>29
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(29)
<223>Tail5mod引物
<400>26
gctgcaccgt tccgcaaccc ccacagcca 29
<210>27
<211>19
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(19)
<223>SalExt引物
<400>27
gagcgtcgac ttcttcaac 19
<210>28
<211>30
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(30)
<223>AAPF-Y2引物
<400>28
gaacggtgca gcgtattggt tgaagggggc 30
<210>29
<211>30
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(30)
<223>AAPF-Y1引物
<400>29
gctgcaccgt tctacttcga caagaccatc 30
<210>30
<211>21
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(21)
<223>SacExt引物
<400>30
gagctcagat ctagttcacg g 21
<210>31
<211>32
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(32)
<223>BBmod1引物
<400>31
cggggccccc gctgcaccgt tctacttcga ca 32
<210>32
<211>32
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(32)
<223>BBmod2引物
<400>32
tgtcgaagta gaacggtgca gcgggggccc cg 32
<210>33
<211>48
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(48)
<223>mo3Aext引物
<400>33
ggatccacca tgaactacaa ggagttcctc cgcatgaccg ccgacaac 48
<210>34
<211>20
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<222>(1)..(20)
<223>CMS16引物
<400>34
cctccacctg ctccatgaag 20
Claims (105)
1.分离的核酸分子，其包括编码含非天然存在的蛋白酶识别位点的修饰的Cry3A毒素的核苷酸序列，其中所述蛋白酶识别位点修饰Cry3A毒素，且位于从由以下位置所组成的组中选择的位置：
a)与SEQ ID NO：4氨基酸位107和115相应的氨基酸之间的位置；
b)与SEQ ID NO：4氨基酸位536和542相应的氨基酸之间的位置；和
c)与SEQ ID NO：4氨基酸位107和115相应的氨基酸之间以及与SEQ ID NO：4氨基酸位536和542相应的氨基酸之间的位置，
其中所述蛋白酶识别位点可被西部玉米根虫的肠蛋白酶识别，并且其中在人工饵料生物测定中所述修饰的Cry3A毒素导致的西部玉米根虫的死亡率比所述Cry3A毒素所导致的西部玉米根虫的死亡率高。
2.根据权利要求1的分离的核酸分子，其中所述肠蛋白酶是组织蛋白酶G。
3.根据权利要求1的分离的核酸分子，其中所述蛋白酶识别位点位于SEQ ID NO：4氨基酸位107和115之间。
4.根据权利要求1的分离的核酸分子，其中所述蛋白酶识别位点位于与SEQ ID NO：4氨基酸位107和113相应的氨基酸之间。
5.根据权利要求4的分离的核酸分子，其中所述蛋白酶识别位点位于SEQ ID NO：4氨基酸位107和113之间。
6.根据权利要求1的分离的核酸分子，其中所述蛋白酶识别位点位于与SEQ ID NO：4氨基酸位107和111相应的氨基酸之间。
7.根据权利要求6的分离的核酸分子，其中所述蛋白酶识别位点位于SEQ ID NO：4氨基酸位107和111之间。
8.根据权利要求1的分离的核酸分子，其中所述蛋白酶识别位点位于SEQ ID NO：4氨基酸位536和542之间。
9.根据权利要求1的分离的核酸分子，其中所述蛋白酶识别位点位于与SEQ ID NO：4氨基酸位536和541相应的氨基酸之间。
10.根据权利要求9的分离的核酸分子，其中所述额外蛋白酶位点位于SEQ ID NO：4氨基酸位536和541之间。
11.根据权利要求1的分离的核酸分子，其中所述蛋白酶识别位点位于与SEQ ID NO：4氨基酸位540和541相应的氨基酸之间。
12.根据权利要求11的分离的核酸分子，其中所述蛋白酶识别位点位于SEQ ID NO：4氨基酸位540和541之间。
13.根据权利要求1的分离的核酸分子，其中所述蛋白酶识别位点位于SEQ ID NO：4氨基酸位107和115之间以及SEQ ID NO：4氨基酸位536和542之间。
14.根据权利要求1的分离的核酸分子，其中所述蛋白酶识别位点位于与SEQ ID NO：4氨基酸位107和113相应的氨基酸之间以及与SEQ ID NO：4氨基酸位540和541相应的氨基酸之间。
15.根据权利要求14的分离的核酸分子，其中所述蛋白酶识别位点位于SEQ ID NO：4氨基酸位107和113之间以及SEQ ID NO：4氨基酸位540和541之间。
16.根据权利要求1的分离的核酸分子，其中所述蛋白酶识别位点位于与SEQ ID NO：4氨基酸位107和111相应的氨基酸之间以及与SEQ ID NO：4氨基酸位536和541相应的氨基酸之间。
17.根据权利要求16的分离的核酸分子，其中所述蛋白酶识别位点位于SEQ ID NO：4氨基酸位107和111之间以及SEQ ID NO：4氨基酸位536和541之间。
18.根据权利要求1的分离的核酸分子，其中所述蛋白酶识别位点位于与SEQ ID NO：4氨基酸位107和111相应的氨基酸之间以及与SEQ ID NO：4氨基酸位540和541相应的氨基酸之间。
19.根据权利要求18的分离的核酸分子，其中所述蛋白酶识别位点位于SEQ ID NO：4氨基酸位107和111之间以及SEQ ID NO：4氨基酸位540和541之间。
20.根据权利要求1的分离的核酸分子，其中对于所述Cry3A毒素导致不超过30％的死亡率的西部玉米根虫，所述修饰的Cry3A毒素导致至少50％的死亡率。
21.根据权利要求1的分离的核酸分子，其中所述核苷酸序列包括SEQ ID NO：6的核苷酸1-1791、SEQ ID NO：8的核苷酸1-1806、SEQ ID NO：10的核苷酸1-1812、SEQ ID NO：12的核苷酸1-1794、SEQ ID NO：14的核苷酸1-1818、SEQ ID NO：16的核苷酸1-1812、SEQ ID NO：18的核苷酸1-1791、或者SEQ ID NO：20的核苷酸1-1818。
22.根据权利要求1的分离的核酸分子，其中所述修饰的Cry3A毒素包括SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11、SEQ ID NO：13、SEQ ID NO：15、SEQ ID NO：17、SEQ ID NO：19或SEQ ID NO：21所示的氨基酸序列。
23.根据权利要求1的分离的核酸分子，其中所述修饰的Cry3A毒素具有抗北部玉米根虫的活性。
24.包括可操作地连接于权利要求1的核酸分子的异源启动子序列的嵌合构建体。
25.包括权利要求24的嵌合构建体的重组载体。
26.包括权利要求24的嵌合构建体的转基因非人宿主细胞。
27.根据权利要求26的转基因宿主细胞，它是细菌细胞。
28.根据权利要求26的转基因宿主细胞，它是植物细胞。
29.包括权利要求28的转基因植物细胞的转基因植物。
30.根据权利要求29的转基因植物，其中所述植物是玉米植物。
31.来自权利要求29的转基因植物的转基因种子。
32.来自权利要求30的玉米植物的转基因种子。
33.通过表达根据权利要求1的核酸分子产生的分离的毒素。
34.根据权利要求33的分离的毒素，其中所述毒素通过表达包括SEQ ID NO：6的核苷酸1-1791、SEQ ID NO：8的核苷酸1-1806、SEQID NO：10的核苷酸1-1812、SEQ ID NO：12的核苷酸1-1794、SEQ IDNO：14的核苷酸1-1818、SEQ ID NO：16的核苷酸1-1812、SEQ ID NO：18的核苷酸1-1791、或者SEQ ID NO：20的核苷酸1-1818的核酸分子而产生。
35.根据权利要求33的分离的毒素，其中所述毒素具有抗北部玉米根虫的活性。
36.根据权利要求33的分离的毒素，其中所述毒素包括SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11、SEQ ID NO：13、SEQ ID NO：15、SEQ ID NO：17、SEQ ID NO：19或SEQ ID NO：21所示的氨基酸序列。
37.包括导致西部玉米根虫死亡有效量的权利要求33的毒素的组合物。
38.包含编码含非天然存在的蛋白酶识别位点的修饰的Cry3A毒素的核苷酸序列的转基因玉米植物，其中所述蛋白酶识别位点修饰Cry3A毒素，且位于从由以下位置所组成的组中选择的位置：
a)与SEQ ID NO：4氨基酸位107和115相应的氨基酸之间的位置；
b)与SEQ ID NO：4氨基酸位536和542相应的氨基酸之间的位置；和
c)与SEQ ID NO：4氨基酸位107和115相应的氨基酸之间以及与SEQ ID NO：4氨基酸位536和542相应的氨基酸之间的位置，
其中所述蛋白酶识别位点可被西部玉米根虫的肠蛋白酶识别，并且其中所述转基因植物在根组织中以导致西部玉米根虫死亡的水平表达所述修饰Cry3A毒素。
39.根据权利要求38的转基因玉米植物，其中所述蛋白酶识别位点位于SEQ ID NO：4氨基酸位107和111之间。
40.根据权利要求38的转基因玉米植物，其中所述蛋白酶识别位点位于SEQ ID NO：4氨基酸位540和541之间。
41.根据权利要求38的转基因玉米植物，其中所述蛋白酶识别位点位于SEQ ID NO：4氨基酸位107和111之间以及SEQ ID NO：4氨基酸位540和541之间。
42.根据权利要求38的转基因玉米植物，其中所述核苷酸序列包括SEQ ID NO：8的核苷酸1-1806、SEQ ID NO：14的核苷酸1-1818、或者SEQ ID NO：18的核苷酸1-1791。
43.根据权利要求38的转基因玉米植物，其中所述修饰的Cry3A毒素包括sEQ ID NO：9、SEQ ID NO：15或SEQ ID NO：19所示的氨基酸序列。
44.根据权利要求38的转基因玉米植物，其中所述根组织导致西部玉米根虫100％的死亡率。
45.根据权利要求38的转基因玉米植物，其中所述根组织导致西部玉米根虫90％的死亡率。
46.根据权利要求38的转基因玉米植物，其中所述根组织导致西部玉米根虫80％的死亡率。
47.根据权利要求38的转基因玉米植物，其中所述根组织导致西部玉米根虫70％的死亡率。
48.根据权利要求38的转基因玉米植物，其中所述根组织导致西部玉米根虫60％的死亡率。
49.根据权利要求38的转基因玉米植物，其中所述根组织导致西部玉米根虫50％的死亡率。
50.根据权利要求38的转基因玉米植物，其中所述根组织导致西部玉米根虫40％的死亡率。
51.根据权利要求38的转基因玉米植物，其中所述转基因植物以足以防止西部玉米根虫严重削减该转基因植物的根的水平表达所述的修饰Cry3A毒素。
52.根据权利要求38的转基因玉米植物，其中所述转基因植物以足以防止西部玉米根虫摄食损伤导致植物倒伏的水平表达所述的修饰Cry3A毒素。
53.根据权利要求38-52任何一项的转基因玉米植物，它是近交植物。
54.根据权利要求38-52任何一项的转基因玉米植物，它是杂交植物。
55.来自权利要求53的植物的转基因种子。
56.来自权利要求54的植物的转基因种子。
57.包括非天然存在的蛋白酶识别位点的修饰的Cry3A毒素，其中所述蛋白酶识别位点修饰Cry3A毒素，且位于从由以下位置所组成的组中选择的位置：
a)与SEQ ID NO：4氨基酸位107和115相应的氨基酸之间的位置；
b)与SEQ ID NO：4氨基酸位536和542相应的氨基酸之间的位置；和
c)与SEQ ID NO：4氨基酸位107和115相应的氨基酸之间以及与SEQ ID NO：4氨基酸位536和542相应的氨基酸之间的位置，
其中所述蛋白酶识别位点可被西部玉米根虫的肠蛋白酶识别，并且其中在人工饵料生物测定中所述修饰的Cry3A毒素导致的西部玉米根虫的死亡率比所述Cry3A毒素所导致的西部玉米根虫的死亡率高。
58.根据权利要求57的修饰的Cry3A毒素，其中所述肠蛋白酶是组织蛋白酶G。
59.根据权利要求57的修饰的Cry3A毒素，其中所述蛋白酶识别位点位于SEQ ID NO：4氨基酸位107和115之间。
60.根据权利要求57的修饰的Cry3A毒素，其中所述蛋白酶识别位点位于与SEQ ID NO：4氨基酸位107和113相应的氨基酸之间。
61.根据权利要求60的修饰的Cry3A毒素，其中所述蛋白酶识别位点位于SEQ ID NO：4氨基酸位107和113之间。
62.根据权利要求57的修饰的Cry3A毒素，其中所述蛋白酶识别位点位于与SEQ ID NO：4氨基酸位107和111相应的氨基酸之间。
63.根据权利要求62的修饰的Cry3A毒素，其中所述蛋白酶识别位点位于SEQ ID NO：4氨基酸位107和111之间。
64.根据权利要求57的修饰的Cry3A毒素，其中所述蛋白酶识别位点位于SEQ ID NO：4氨基酸位536和542之间。
65.根据权利要求57的修饰的Cry3A毒素，其中所述蛋白酶识别位点位于与SEQ ID NO：4氨基酸位536和541相应的氨基酸之间。
66.根据权利要求65的修饰的Cry3A毒素，其中所述额外蛋白酶位点位于SEQ ID NO：4氨基酸位536和541之间。
67.根据权利要求57的修饰的Cry3A毒素，其中所述蛋白酶识别位点位于与SEQ ID NO：4氨基酸位540和541相应的氨基酸之间。
68.根据权利要求67的修饰的Cry3A毒素，其中所述蛋白酶位点位于SEQ ID NO：4氨基酸位540和541之间。
69.根据权利要求57的修饰的Cry3A毒素，其中所述蛋白酶识别位点位于SEQ ID NO：4氨基酸位107和115之间以及SEQ ID NO：4氨基酸位536和542之间。
70.根据权利要求57的修饰的Cry3A毒素，其中所述蛋白酶识别位点位于与SEQ ID NO：4氨基酸位107和113相应的氨基酸之间以及与SEQ ID NO：4氨基酸位540和541相应的氨基酸之间。
71.根据权利要求70的修饰的Cry3A毒素，其中所述蛋白酶识别位点位于SEQ ID NO：4氨基酸位107和113之间以及SEQ ID NO：4氨基酸位541和541之间。
72.根据权利要求57的修饰的Cry3A毒素，其中所述蛋白酶识别位点位于与SEQ ID NO：4氨基酸位107和111相应的氨基酸之间以及与SEQ ID NO：4氨基酸位536和541相应的氨基酸之间。
73.根据权利要求72的修饰的Cry3A毒素，其中所述蛋白酶识别位点位于SEQ ID NO：4氨基酸位107和111之间以及SEQ ID NO：4氨基酸位536和541之间。
74.根据权利要求57的修饰的Cry3A毒素，其中所述蛋白酶识别位点位于与SEQ ID NO：4氨基酸位107和111相应的氨基酸之间以及与SEQ ID NO：4氨基酸位540和541相应的氨基酸之间。
75.根据权利要求74的修饰的Cry3A毒素，其中所述蛋白酶识别位点位于SEQ ID NO：4氨基酸位107和111之间以及SEQ ID NO：4氨基酸位540和541之间。
76.根据权利要求57的修饰的Cry3A毒素，其中对于所述Cry3A毒素导致不超过30％的死亡率的西部玉米根虫，所述修饰的Cry3A毒素导致至少50％的死亡率。
77.根据权利要求57的修饰的Cry3A毒素，它由SEQ ID NO：6的核苷酸1-1791、SEQ ID NO：8的核苷酸1-1806、SEQ ID NO：10的核苷酸1-1812、SEQ ID NO：12的核苷酸1-1794、SEQ ID NO：14的核苷酸1-1818、SEQ ID NO：16的核苷酸1-1812、SEQ ID NO：18的核苷酸1-1791、或者SEQ ID NO：20的核苷酸1-1818编码。
78.根据权利要求57的修饰的Cry3A毒素，包括SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11、SEQ ID NO：13、SEQ ID NO：15、SEQID NO：17、SEQ ID NO：19或SEQ ID NO：21所示的氨基酸序列。
79.根据权利要求57的修饰的Cry3A毒素，它具有抗北部玉米根虫的活性。
80.控制西部玉米根虫侵染玉米植物的方法，该方法包括：
(a)提供根据权利要求38的转基因玉米植物；和
(b)使所述西部玉米根虫与该植物接触。
81.产生修饰的Cry3A毒素的方法，包括：
(a)获得根据权利要求27的转基因宿主细胞；
(b)在允许修饰的Cry3A毒素表达的条件下培养该转基因宿主细胞；和
(c)回收表达的修饰Cry3A毒素。
82.产生抗昆虫植物的方法，包括
(a)将根据权利要求1的核酸分子稳定地整合到植物细胞的基因组中；和
(b)从所述的转化植物细胞再生稳定转化的植物，其中所述稳定转化的植物表达使所述转化植物抗至少西部玉米根虫有效量的修饰的Cry3A毒素。
83.控制至少西部玉米根虫的方法，包括向西部玉米根虫口服递送有效量的根据权利要求33的毒素。
84.制造修饰的Cry3A毒素的方法，包括：
(a)获得编码Cry3A毒素的cry3A基因；
(b)鉴定西部玉米根虫的肠蛋白酶；
(c)获得编码所述肠蛋白酶识别位点的核苷酸序列；
(d)将所述核苷酸序列插入到所述cry3A基因中，使得所述识别位点位于所述Cry3A毒素中与SEQ ID NO：4氨基酸位107和115相应的氨基酸之间的位置、或者与SEQ ID NO：4氨基酸位536和542相应的氨基酸之间的位置、或者与SEQ ID NO：4氨基酸位107和115相应的氨基酸之间以及与SEQ ID NO：4氨基酸位536和542相应的氨基酸之间的位置，从而建立修饰的cry3A基因；
(e)将所述修饰的cry3A基因插入到表达盒中；和
(f)将所述表达盒转化到非人宿主细胞中，其中所述宿主细胞产生修饰的Cry3A毒素。
85.修饰的cry3A基因，其包括编码含非天然存在的蛋白酶识别位点的修饰的Cry3A毒素的核苷酸序列，其中所述修饰的cry3A基因包括编码所述蛋白酶识别位点的编码序列，其中所述编码序列修饰cry3A基因，且插入在从由以下位置所组成的组中选择的位置：
a)编码与SEQ ID NO：4氨基酸位107和115相应的氨基酸的密码子之间的位置；
b)编码与SEQ ID NO：4氨基酸位536和542相应的氨基酸的密码子之间的位置；和
c)编码与SEQ ID NO：4氨基酸位107和115相应的氨基酸的密码子之间以及编码与SEQ ID NO：4氨基酸位536和542相应的氨基酸的密码子之间的位置，
其中所述蛋白酶识别位点可被西部玉米根虫的肠蛋白酶识别，并且其中在人工饵料生物测定中所述修饰的Cry3A毒素导致的西部玉米根虫的死亡率比所述Cry3A毒素所导致的西部玉米根虫的死亡率高。
86.根据权利要求85的修饰的cry3A基因，其中所述肠蛋白酶是组织蛋白酶G。
87.根据权利要求85的修饰的cry3A基因，其中所述核苷酸序列包括SEQ ID NO：6的核苷酸1-1791、SEQ ID NO：8的核苷酸1-1806、SEQ ID NO：10的核苷酸1-1812、SEQ ID NO：12的核苷酸1-1794、SEQ ID NO：14的核苷酸1-1818、SEQ ID NO：16的核苷酸1-1812、SEQ ID NO：18的核苷酸1-1791、或者SEQ ID NO：20的核苷酸1-1818。
88.根据权利要求85的修饰的cry3A基因，其中所述修饰的Cry3A毒素包括SEQ ID NO：7、SEQ ID NO：9、SEQ ID NO：11、SEQ ID NO：13、SEQ ID NO：15、SEQ ID NO：17、SEQ ID NO：19或SEQ ID NO：21所示的氨基酸序列。
89.根据权利要求85的修饰的cry3A基因，其中所述修饰的Cry3A毒素具有抗北部玉米根虫的活性。
90.包括可操作地连接于权利要求85的修饰的cry3A基因的异源启动子序列的嵌合构建体。
91.包括权利要求90的嵌合构建体的重组载体。
92.包括权利要求90的嵌合构建体的转基因非人宿主细胞。
93.根据权利要求92的转基因宿主细胞，它是细菌细胞。
94.根据权利要求92的转基因宿主细胞，它是植物细胞。
95.包括权利要求94的转基因植物细胞的转基因植物。
96.根据权利要求95的转基因植物，其中所述植物是玉米植物。
97.来自权利要求95的转基因植物的转基因种子。
98.来自权利要求96的玉米植物的转基因种子。
99.包含修饰的cry3A基因的转基因玉米植物，该基因包括编码含非天然存在的蛋白酶识别位点的修饰的Cry3A毒素的核苷酸序列，其中所述修饰的cry3A基因包括编码所述蛋白酶识别位点的编码序列，其中所述编码序列修饰cry3A基因，且插入在从由以下位置所组成的组中选择的位置：
a)编码与SEQ ID NO：4氨基酸位107和115相应的氨基酸的密码子之间的位置；
b)编码与SEQ ID NO：4氨基酸位536和542相应的氨基酸的密码子之间的位置；和
c)编码与SEQ ID NO：4氨基酸位107和115相应的氨基酸的密码子之间以及编码与SEQ ID NO：4氨基酸位536和542相应的氨基酸的密码子之间的位置，
其中所述蛋白酶识别位点可被西部玉米根虫的肠蛋白酶识别，并且其中所述转基因植物在根组织中以导致西部玉米根虫死亡的水平表达所述修饰Cry3A毒素。
100.根据权利要求99的转基因玉米植物，其中所述核苷酸序列包括SEQ ID NO：8的核苷酸1-1806、SEQ ID NO：14的核苷酸1-1818、或者SEQ ID NO：18的核苷酸1-1791。
101.根据权利要求99的转基因玉米植物，其中所述修饰的Cry3A毒素包括SEQ ID NO：9、SEQ ID NO：15或SEQ ID NO：19所示的氨基酸序列。
102.根据权利要求99-101任何一项的转基因玉米植物，它是近交植物。
103.根据权利要求99-101任何一项的转基因玉米植物，它是杂交植物。
104.来自权利要求102的植物的转基因种子。
105.来自权利要求103的植物的转基因种子。
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| CN111574599A (zh) * | 2020-05-18 | 2020-08-25 | 福建农林大学 | 一种解决杀虫毒素被昆虫肠道消化酶过度酶解的毒素改造方法 |
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| BR0312064A (pt) * | 2002-06-26 | 2005-08-09 | Du Pont | Genes que codificam proteìnas com atividade pesticida |
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| US7309785B1 (en) * | 2003-10-03 | 2007-12-18 | Dow Agrosciences Llc | Modified chimeric Cry35 proteins |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102369286B (zh) * | 2009-02-05 | 2014-12-10 | 阿森尼克斯公司 | 变体axmi-r1δ-内毒素基因和使用它们的方法 |
| CN113735950A (zh) * | 2015-06-24 | 2021-12-03 | 先正达参股股份有限公司 | 用于在植物中赋予杀昆虫特性的核酸分子 |
| CN113735950B (zh) * | 2015-06-24 | 2024-03-19 | 先正达参股股份有限公司 | 用于在植物中赋予杀昆虫特性的核酸分子 |
| CN111574599A (zh) * | 2020-05-18 | 2020-08-25 | 福建农林大学 | 一种解决杀虫毒素被昆虫肠道消化酶过度酶解的毒素改造方法 |
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