CN114317592B

CN114317592B - 一种定点突变植物纤维素合酶CESA的方法及cesa突变体在制备纳米晶中的应用

Info

Publication number: CN114317592B
Application number: CN202111551615.XA
Authority: CN
Inventors: 彭良才; 张冉; 胡振; 鲁俊; 王艳婷; 夏涛; 王友梅
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2021-12-17
Filing date: 2021-12-17
Publication date: 2024-02-20
Anticipated expiration: 2041-12-17
Also published as: CN114317592A

Abstract

本发明公开了一种定点突变植物纤维素合酶CESA的方法及cesa突变体在制备纳米晶中的应用，属于植物基因工程和纳米材料领域。该方法包括以下步骤：(1)根据纤维素合成酶CESA基因的CRISPR/Cas9编辑靶位点，设计引物并扩增构建靶序列；(2)将靶序列构建到载体上，得到重组载体；(3)将重组载体转化植株，获取次生细胞壁三个CESA基因的点突变体和移码突变体。含有点突变体或移码突变体的转基因植物纤维素结构和性质发生改变，将转基因植物秸秆经酸水解法水解可以制备更小尺寸的纤维素纳米晶。本发明利用基因编辑技术改良水稻纤维素纳米结构，为高效制备小尺寸纤维素纳米晶颗粒提供了低廉且优质的纤维素原材料。

Description

一种定点突变植物纤维素合酶CESA的方法及cesa突变体在制备纳米晶中的应用

技术领域

本发明涉及植物基因工程和纳米材料领域，特别是涉及一种定点突变植物纤维素合酶CESA的方法及cesa突变体在制备纳米晶中的应用。

背景技术

纤维素纳米晶是一种高比表面积、低密度、力学性能优异的一维棒状纳米颗粒。它具有纳米效应、可定制的表面化学特性、各向异性以及光学特性，同时还继承了原生纤维素的可再生性、低毒性、生物相容性、易得性和低成本等优良性质，使其成为各种应用领域的理想纳米材料。纤维素纳米晶的几何尺寸和结构参数因材料来源、提取方法和制造条件的不同而不同，宽度为5-20nm，长度为100-500nm，结晶度指数可达90％。更小尺寸的纤维素纳米晶具备更高比表面积和纳米效应，更利于在微观场景下的负载和运输。目前，酸水解、机械处理、氧化、酶解和离子液的处理都被报道为生产纤维素纳米晶的方法。制备更小尺寸的纤维素纳米晶往往需要更强的处理条件或者特殊的生物质材料。

纤维素纳米晶的生产有赖于植物天然的木质纤维素的结构。其生产的关键在于消除天然纤维素微纤丝的非结晶区从而完全释放晶体单元，由于空间位阻减小和动力学因素，沿纤维上间隔排列的无序区域极易于水解作用，而对水解过程具有较高抗性的晶体区域则保持完整，于是微纤丝从非晶体区被间隔切断产生短的纤维素纳米晶。天然的纤维素是植物通过纤维素合酶复合体CSC进行合成的，其主要的催化单元是纤维素合酶CESA。大多数植物表达几种不同的CESA亚型，不同的亚型对初生和次生细胞壁的形成是必需的。通常，三个CESA亚型(如水稻中CESA4、CESA7、CESA9)构成了核心组件的次生细胞壁CSC。分子遗传证据证明，CESA单一亚基的功能缺失会造成纤维素的合成受阻，改变纤维素的结构和性质。

基于以上研究背景，我们发现迄今为止尚无通过定点突变纤维素合酶改变天然纤维素微纤丝结构，从而直接减小所制备纤维素纳米晶尺寸的报道。因此，遗传改良纤维素超微结构获得更小尺寸纤维素纳米晶的方法，对于扩宽和改进其应用场景、减小工业化处理成本具有重要意义。

发明内容

本发明的目的是提供一种定点突变植物纤维素合酶CESA的方法及cesa突变体在制备纳米晶中的应用，以解决上述现有技术存在的问题，该方法获取的突变体能在水稻废弃秸秆的纤维素微纤丝中引入更多的非晶体间隔区域，通过常规酸水解的方法可将其水解成更小尺度的纤维素纳米晶颗粒，可极大的增加了优质原生纤维素资源，同时也减少了化工能源投入。

为实现上述目的，本发明提供了如下方案：

本发明提供一种利用CRISPR/Cas9系统定点突变植物纤维素合酶CESA的方法，包括以下步骤：

(1)根据纤维素合成酶CESA基因的CRISPR/Cas9编辑靶位点，设计引物并扩增构建靶序列；所述CESA基因包括CESA4、CESA7和CESA9基因；

(2)将所述靶序列构建到载体上，得到重组载体；

(3)将所述重组载体转化植株，获取次生细胞壁三个CESA基因的点突变体和移码突变体。

优选的是，针对不同靶位点设计扩增引物，以获取靶序列，扩增引物具体包括：

SEQ ID NO：1-2所示序列为CESA4靶序列的扩增引物，SEQ ID NO：3-4所示序列为cesa4-KO突变体筛选特异性引物；

SEQ ID NO：5-6所示序列为CESA7靶序列的扩增引物，,SEQ ID NO：7-8所示序列为cesa7-KO突变体筛选特异性引物；

SEQID NO：9-10所示序列为CESA9靶序列的扩增引物，SEQ ID NO：11-12所示序列为cesa9-KO突变体筛选特异性引物；

SEQ ID NO：13-14所示序列为CESA4靶序列的扩增引物，SEQ ID NO：15-16所示序列为cesa4-Q突变体筛选特异性引物；

SEQ ID NO：17-18所示序列为CESA7靶序列的扩增引物，SEQ ID NO：19-20所示序列为cesa7-PCR突变体筛选特异性引物；

SEQ ID NO：21-24所示序列为CESA9靶序列的扩增引物，SEQ ID NO：25-26所示序列为cesa9-TMD突变体筛选特异性引物；

SEQ ID NO：27-28所示序列为CESA9靶序列的扩增引物，SEQ ID NO：29-30所示序列为cesa9-ZnF突变体筛选特异性引物。

本发明还提供一种所述的方法制备的cesa突变体，所述CESA突变体包括点突变体和移码突变体。

优选的是，所述点突变体包括对CESA4核心催化位点基序、CESA7植物保守结构域、CESA9跨膜结构域或CESA9锌指结构域的突变。

优选的是，所述移码突变包括对CESA4、CESA7或CESA9基因碱基的缺失。

本发明还提供一种转基因植物的构建方法，包括使所述植物包含所述的cesa突变体或其编码基因的步骤。

优选的是，所述cesa突变体或其编码基因使所述转基因植物纤维素纳米长度减小。

本发明还提供一种含有所述的cesa突变体的转基因植物茎秆在生产纤维素纳米晶上的应用。

优选的是，所述cesa突变体使所制备的纤维素纳米晶聚合度和尺寸均减小。

优选的是，所述转基因植物茎秆采用超声辅助硫酸水解法制备所述纤维素纳米晶。

本发明公开了以下技术效果：

(1)本发明利用基因编辑技术对纤维素合酶CESA定点突变产生2种类型水稻cesa突变体，即点突变体(株型正常发育、仅茎秆表现出脆性)和移码突变体(株型发育不良、同时茎秆表现出脆性)。

(2)本发明利用基因编辑得到的cesa突变体使水稻原生纤维素性质和结构发生改变，点突变体聚合度和纳米纤维长度适度减小，而移码突变体聚合度和纳米纤维长度剧烈减小。

(3)本发明利用基因编辑得到了包含cesa突变体的转基因植物，利用其茎秆所制备的纤维素纳米晶尺寸减小，点突变体使纳米晶尺寸适度减小，而移码突变体使纳米晶尺寸剧烈减小。

(4)本发明可直接获得改良纤维素结构的水稻秸秆生物质，可多年、大量、低廉的获得优质原材料，且适用于所有纤维素纳米晶颗粒的制备方法。

附图说明

为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。

图1为水稻突变体的突变方式和生物学性状；A.野生型水稻NPB和cesa突变体的植株照片；B.CRISPR/Cas9基因编辑位点以及蛋白突变位点示意图，ZnF为锌指结构域，TMD为跨膜结构域，P-CR为植物保守结构域，CSR为类特异区，QxxRW为核心催化位点基序；

图2为水稻突变体茎秆原生纤维素结构与性质；A.原子力显微镜原位观察纤维素微纤丝照片；上图标尺为400nm，下图为上图黑色方框部分的放大图片，标尺为100nm；B.野生型与cesa突变体纤维素微纤丝长度统计，n＝100；C.野生型与cesa突变体粗纤维素聚合度；

图3为水稻突变体茎秆所制备纤维素纳米晶的尺寸观测；A.原子力显微镜原位观察纤维素纳米晶照片；上图标尺为800nm，下图为上图黑色方框部分的放大图片，标尺为200nm；B.野生型与cesa突变体纤维素纳米晶长度统计，n＝100；C.野生型与cesa突变体纤维素纳米晶直径统计，n＝100。

具体实施方式

现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。

应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。

除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。

在不背离本发明的范围或精神的情况下，可对本发明说明书的具体实施方式做多种改进和变化，这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见的。本申请说明书和实施例仅是示例性的。

关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等，均为开放性的用语，即意指包含但不限于。

实施例1利用基因编辑技术定点突变纤维素合酶

(1)查找基因编辑位点和合成spacer序列

从水稻基因组数据库(http://rice.uga.edu/)获得OsCESA4/7/9基因序列。

通过网站(http://www.genome.arizona.edu/crispr/CRISPRsearch.html)查找针对OsCESA4/7/9基因的CRISPR/Cas9编辑靶位点；设计引物后扩增靶位点序列(Space序列)构建到载体pRGEB32或者pCSGAPO1上。

扩增引物如表1：

(2)CRISPR/Cas9载体构建

1)cesa4-KO、cesa7-KO、cesa9-KO、cesa4-Q、cesa7-PCR、cesa9-ZnF 6个载体构建

取浓度为100μM的正向和反向序列各1μL，依次加入装有3μL ddH₂O的PE管中。将PE管置于37℃水浴锅中60min，再取出置于95℃沸水中10min，之后以0.1℃/sec的速度降温至25℃。用BsaI酶切pRGEB32或者pCSGAPO1质粒，酶切体系包括pRGEB32或者pCSGAPO1质粒2μL、10×NEB Smart-cut Buffer 2μL、10×BSA 2μL、Bsa I酶液1μL和ddH2O 13μL；酶切反应温度和时间分别为37℃和4h。酶切反应结束后加入0.5μL CIAP去磷酸化酶继续反应30min。用PCR回收试剂盒回收酶切后质粒。利用T4 DNA连接酶连接退火后DNA双链和酶切后质粒，连接体系包括酶切后质粒2μL、退火后DNA双链1μL、10×T4 DNA连接酶Buffer 0.50μL、T4DNA连接酶1μL和ddH₂O 0.50μL；连接反应温度和时间分别为4℃和16h。取1μL连接液转入大肠杆菌DH5a感受态，挑选单菌落进行菌落PCR。挑取阳性菌落摇菌并提取质粒，再将质粒进行DNA测序。将测序正确后的质粒转入农杆菌EHA105，通过PCR验证筛选农杆菌阳性菌落，保存菌种待用。

2)cesa9-TMD载体构建

利用“多顺反子tRNA-gRNA多靶位点基因编辑法”构建OsCESA9基因双位点突变体，所用载体包括pRGEB32和pGTR，其中双元载体pRGEB32用于表达tRNA-gRNA和Cas9蛋白，pGTR用于构建多顺反子tRNA-gRNA片段。具体实施方法如下：

①基因编辑位点查找

利用http://www.genome.arizona.edu/crispr/CRISPRsearch.html网站查找OsCESA9的基因编辑靶位点，本发明中突变体所选两个靶位点位于OsCESA9基因3’端，序列分别为：

target1：CCATCAACAACGGCTCCGAGGCG

target2：TCCCTTCACCATCAAGGCCAGGG

其中下划线为PAM序列(NGG或其反相互补序列CCN)，其余20bp为spacer序列。

②根据spacer序列设计PCR扩增引物：

target1-F：TAGGTCTCCAGCCGTTGTTGAGTTTTAGAGCTAGAA

target1-R：ATGGTCTCAGGCTCCGAGGCGTGCACCAGCCGGGAA

target2-F：TAGGTCTCCCCATCAAGGCCAGTTTTAGAGCTAGAA

target2-R：ATGGTCTCAATGGTGAAGGGATGCACCAGCCGGGAA

另外，设计扩增tRNA-gRNA小片段的引物L5F/L3R和扩增tRNA-gRNA全长的引物S5F/S3R，引物序列如下：

L5F:5’-CGGGTCTCAGGCAGGATGGGCAGTCTGGGCAACAAAGCACCAGTGG-3’

L3R:5’-TAGGTCTCCAAACGGATGAGCGACAGCAAACAAAAAAAAAAGCACCGACTCG-3’

S5F：5’-CGGGTCTCAGGCAGGATGGGCAGTCTGGGCA-3’

S5R：5’-TAGGTCTCCAAACGGATGAGCGACAGCAAAC-3’

③PCR扩增tRNA-gRNA各片段(Fragment 1-3)，PCR扩增体系如下：pGTR(200ng/μL)1μL、10×Easy Taq buffer 5μL、dNTPs(10mM)1μL、Primer F(10μmol/L)2.5μL、Primer R(10μmol/L)2.5μL、Easy Taq(5U/μL,TRANSGEN)0.5μL和ddH₂O 37.5μL。

PCR反应程序如下：95℃5min；95℃30sec、50℃30sec、72℃30sec，35个循环；72℃5min；20℃保持。

Fragment 1-3的扩增引物分别如下：

Fragment 1：L5-F+target 1-R；Fragment 2：target 1-F+target 2-R；Fragment3：target 2-F+L3-R；

④利用PCR产物试剂盒(上海生工)纯化Fragment 1-3片段；

⑤GG assembly组装多顺反子tRNA-gRNA序列，反应体系如下：

Fragment 1 50ng、Fragment 2 50ng、Fragment 3 50ng、2×T7 DNA ligaseBuffer(NEB)10μL、T7 DNA Ligase(3000U/μl,NEB)0.5μL、Bovine Serum Albumin(1mg/ml)、2μL、Bsa I(10U/μl,NEB)0.反应时间和温度：(37℃，5min；20℃10min)40cycles；20℃，60min。反应结束后加180μL ddH₂O稀释10倍；

⑥PCR扩增GG assembly反应产物，PCR反应体系如下：

Diluted product 1μL、10×Easy Taq buffer 5μL、dNTPs(10mM)1μL、S5-F(10μmol/L)2.5μL、S5-R(10μmol/L)2.5μL、Easy Taq(5U/μL,TRANSGEN)0.5μL和ddH₂O 37.5μL。PCR反应程序如下：

95℃5min；95℃30sec、58℃30sec、72℃1min，35个循环；72℃5min；20℃保持。

⑦PCR产物纯化试剂盒纯化多顺反子tRNA-gRNA片段；

⑧FokΙ酶切tRNA-gRNA片段，酶切体系如下：tRNA-gRNA fragment 17μL、10×CutSmart Buffer 2μL和FokΙ(5U/μL,NEB)1μL。37℃酶切4h。

⑨BsaΙ酶切pRGEB32载体，酶切体系如下：pRGEB32 17μL、10×CutSmart Buffer 2μL和BsaΙ(5U/μL,NEB)1μL。37℃酶切4h。

⑩胶回收试剂盒纯化回收(上海生工)酶切后的tRNA-gRNA片段和pRGEB32载体；

连接多顺反子tRNA-gRNA片段和pRGEB32载体，连接体系如下：tRNA-gRNAfragment 3μL、pRGEB32 1μL、T4 DNA ligase(5U/μL,Takara)0.5μL、10×ligation buffer1μL和ddH₂O 4.5μL。

取1μL连接液转入大肠杆菌DH5a感受态，挑选单菌落进行菌落PCR。挑取阳性菌落摇菌并提取质粒，再将质粒进行DNA测序。将测序正确后的质粒转入农杆菌EHA105，通过PCR验证筛选农杆菌阳性菌落，保存菌种待用。

(3)水稻愈伤侵染和组织培养

选取饱满、无霉斑的成熟水稻种子(日本晴(Oryza sativa Nipponbare))去壳，依次用无菌水冲洗3遍、70％的乙醇处理1分钟、无菌水冲洗3遍，最后用0.1％升汞溶液消毒12min。用无菌水将消毒后种子冲洗6遍，将种子放在灭菌滤纸上吸干水分，然后用无菌镊子夹到诱导培养基上，放置于28℃，暗培养4周。挑选颜色鲜亮、紧实且干燥的胚性愈伤，放于继代培养基上，放置于28℃，暗培养2周。在带有相应抗性选择的LB固体培养基上于28℃预培养农杆菌两天。将农杆菌转移至装有悬浮培养基的离心管里，涡旋振荡重悬农杆菌，调节农杆菌的悬浮液至OD₆₀₀值0.8-1.0。挑选颜色鲜亮、紧实且干燥的胚性愈伤转移至灭菌好的三角瓶内，加入农杆菌悬浮液，浸泡愈伤30min。转移愈伤至灭菌好的滤纸上，在超净工作台上吹干，然后将愈伤放置在已铺有一层滤纸的共培养基上培养3天，温度为20℃。将共培养的愈伤转移至灭菌好的三角瓶内，用灭菌水洗涤愈伤10次。将愈伤浸泡在含400ppm羧苄青霉素的灭菌水中30分钟，每2min摇动一次。转移愈伤至灭菌滤纸上吸干水分，并在超净工作台上吹4h。转移愈伤至选择培养基上选择培养2次，每次2周左右，第一次选择培养基中羧苄青霉素筛选浓度为400ppm，第二次选择培养基中羧苄青霉素筛选浓度为250ppm。将抗性愈伤转移至分化培养基上，26℃光照下培养。待苗高10cm左右，剪掉分化时产生的根，然后将其转移至生根培养基中，26℃光培养2周。打开瓶盖，向瓶内注入无菌水，炼苗2天后，洗掉根上的残留培养基，移栽至土壤中。

上述所涉及的培养基组成成分如下：

诱导培养基：N6max(20×)50mL、N6min(100×)10mL、Fe²⁺EDTA(100×)10mL、维生素E(400×)2.50mL、2,4-D(1mg/mL)2.50mL、酪蛋白0.30g、L-脯氨酸2.878g、蔗糖30g和Phyta4.00g，调节PH至5.8。

继代培养基：N6max(20×)50mL、N6min(100×)10mL、Fe²⁺EDTA(100×)

10mL、维生素E(400×)2.50mL、2,4-D(1mg/mL)2.00mL、酪蛋白0.30g、L-脯氨酸2.878g、蔗糖30g和琼脂8.00g，调节PH至5.8。

共培养基：N6max(20×)25mL、N6min(100×)5mL、Fe²⁺EDTA(100×)10mL、维生素E(400×)2.50mL、2,4-D(1mg/mL)3.00mL、酪蛋白0.80g、蔗糖20g、葡萄糖10g和琼脂8.00g，调节PH至5.8。

悬浮培养基：N6max(20×)25mL、N6min(100×)5mL、Fe²⁺EDTA(100×)5mL、维生素E(400×)2.50mL、2,4-D(1mg/mL)2.00mL、酪蛋白0.80g、蔗糖20g和葡萄糖5.00g，调节PH至5.4。选择培养：N6max(20×)50mL、N6min(100×)10mL、Fe²⁺EDTA(100×)10mL、维生素E(400×)2.50mL、2,4-D(1mg/mL)2.50mL、酪蛋白0.80g、蔗糖30g和琼脂8g、潮霉素(50mg/mL)1000μL、羧苄青霉素(200mg/mL)2000/1250μL(一选/二选)，调节PH至6。

分化培养基：N6max(20×)50mL、N6min(100×)10mL、Fe²⁺EDTA(100×)10mL、维生素E(400×)2.5mL、6-BA(1mg/mL)2mL、KT(1mg/mL)2mL、NAA(1mg/mL)200μL、IAA(1mg/mL)200μL、酪蛋白1g、蔗糖30g和琼脂8g，调节PH至6。

生根培养基：MSmax(20×)25mL、MSmin(100×)5mL、Fe²⁺EDTA(100×)5mL、维生素E(400×)1.25mL、蔗糖30g和琼脂8g，调节PH至6。

LB培养基：NaCl 10g、蛋白胨10g、酵母提取物5g和琼脂15g，121℃灭菌20分钟，冷到55℃左右，加抗生素Kana+(50mg/mL)。

(4)筛选突变体

待组培苗于分裂期时摘取约10cm长叶片，用CTAB法提取DNA。利用Cas9基因特异性引物进行PCR扩增以检测转基因阳性苗。

所用Cas9基因扩增引物(通用)序列为：

Cas9-F：5'-GACTCCTCCCACCACCCA-3'

Cas9-R：5'-AACCAACATCCAATCCCA-3'

PCR扩增体系：DNA(500ng/μL)1μL、10×Easy Taq buffer 5μL、dNTPs(10mM)1μL、Cas9-F(10μmol/L)2.5μL、Cas9-R(10μmol/L)2.5μL、Easy Taq(5U/μL,TRANSGEN)0.5μL和ddH₂O 37.5μL。

PCR反应程序如下：95℃5min；95℃30sec、50℃30sec、72℃1min，30个循环；72℃5min；20℃10min。

再次利用突变测序引物(特异：见表1的最后一列，每个载体用的不一样)扩增阳性苗中靶位点上下游共600bp长度基因片段，通过测序检测转基因阳性苗的突变情况。筛选T₀代中具有目标突变方式的突变体，收获突变体种子，种植得到T₁代群体。提取T₁代分蘖期水稻叶片DNA，利用SeqF/R引物测序筛选纯合突变体。

如图1所示，含有基因编辑技术得到的点突变体的水稻株型正常发育，且仅茎秆表现出脆性；含有移码突变体的水稻株型发育不良、同时茎秆表现出脆性(图1A)。其中，cesa4-KO突变体的核苷酸序列如SEQ ID NO:31所示，氨基酸序列如SEQ ID NO:32所示；cesa7-KO突变体的核苷酸序列如SEQ ID NO:33所示，氨基酸序列如SEQ ID NO:34所示；cesa9-KO突变体的核苷酸序列如SEQ ID NO:35所示，氨基酸序列如SEQ ID NO:36所示；cesa4-Q突变体的核苷酸序列如SEQ ID NO:37所示，氨基酸序列如SEQ ID NO:38所示；cesa7-PCR突变体的核苷酸序列如SEQ ID NO:39所示，氨基酸序列如SEQ ID NO:40所示；cesa4-TMD突变体的核苷酸序列如SEQ ID NO:41所示，氨基酸序列如SEQ ID NO:42所示；cesa4-ZnF突变体的核苷酸序列如SEQ ID NO:43所示，氨基酸序列如SEQ ID NO:44所示。

(5)取水稻抽穗早期约3cm长的倒2节茎秆，用琼脂糖包埋震动切片技术(VT1000S，Leica)制备50-100μm厚度的半横切片；用16％酸性亚氯酸钠(含体积分数为1.5％的乙酸)孵育切片以去除木质素，孵育条件为50℃、48h，中间更换一次试剂，将处理后的切片用纯水冲洗5次以上。用含1％HCl的70％乙醇进行超声洗涤玻片，然后用poly-L-lysine solution(P8920；Sigma；1:10)包裹玻片表面5min，然后60℃烘烤1h。在处理好的玻片表面固定住水稻茎秆切片，放置在真空罐中，等待AFM上机操作。利用PicoPlus分子成像系统和PicoScan3000控制器，采用开环Agilent多功能AFM扫描仪进行成像。

(6)纤维素聚合度测定

纤维素聚合度(DP)采用粘度法测定，粘度计型号为0.7mm–0.8mm乌氏粘度计，根据美国药典中粘度表查找(USP.25/NF20，2002)相对粘度η与特性粘度[η]的对应关系，聚合度按照Grobe修正公式DP＝190[η]计算得到。

如图2所示，CESA突变体使水稻原生纤维素性质和结构发生改变，点突变体聚合度和纳米纤维长度适度减小，而移码突变体聚合度和纳米纤维长度剧烈减小。

(7)分离制备纤维素纳米晶

该纤维素纳米晶的制备采用超声波辅助硫酸水解法，具体步骤如下：

收获转基因水稻秸秆，烘干后粉碎过40目筛。称取1g水稻秸秆粉末，用8％NaClO₂(含1.5％乙酸)50℃150r/min处理2-3天，每隔12h换一次反应液，直至粉末变白，以去除木质素；单蒸水洗至中性；继续用5％NaOH提取半纤维素，共计5次即可抽提充分，上清液由黄变澄清；单蒸水洗至中性；甲醇洗一次，丙酮洗一次，通风橱过夜，次日50℃烘箱处理若干小时获得干燥纯白色粉末，过筛即制得粗纤维。取0.1g粗纤维，加入2mL 64％H₂SO₄(w/w)，45℃和40KHz条件下超声处理80-100min(优选90min)；加入十倍体积蒸馏水终止反应，第一次离心弃上清；再加入蒸馏水混匀离心，第二次或第三次上清变乳白色即为纤维素纳米晶；收集乳白色纤维素纳米晶，8000-14000D透析2-3天至中性。

(8)将(7)所制备的纤维素纳米晶的浓度稀释到0.1％，滴定到新剥开的云母片上，自然干燥后，利用原子力显微镜PicoPlus分子成像系统和PicoScan 3000控制器，采用开环Agilent多功能扫描仪进行成像。选择100个纳米晶颗粒进行统计长度和直径。

如图3所示，点突变体使纳米晶尺寸适度减小，而移码突变体使纳米晶尺寸剧烈减小。

以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。

序列表

<110> 华中农业大学

<120> 一种定点突变植物纤维素合酶CESA的方法及cesa突变体在制备纳米晶中的应用

<160> 44

<170> SIPOSequenceListing 1.0

<210> 1

<211> 25

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 1

tgtgtgtgcg cgtcgtcccc gcacg 25

<210> 2

<211> 25

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 2

aaaccgtgcg gggacgacgc gcaca 25

<210> 3

<211> 21

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 3

gttccatgtc cgctcaacct t 21

<210> 4

<211> 20

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 4

tgcttaccac tcccaactcc 20

<210> 5

<211> 26

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 5

tgtgtgccac ctcctcgccg cacacc 26

<210> 6

<211> 26

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 6

aaacggtgtg cggcgaggag gtggca 26

<210> 7

<211> 23

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 7

cctgcatctg atcatcaacc agc 23

<210> 8

<211> 21

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 8

cttggtgggg ctcttgatct g 21

<210> 9

<211> 24

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 9

ggcatgaact tcgagaagcg gttc 24

<210> 10

<211> 24

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 10

aaacgaaccg cttctcgaag ttca 24

<210> 11

<211> 21

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 11

cgctgtacgg atacaaccca c 21

<210> 12

<211> 20

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 12

gacccgtaga tccaccccag 20

<210> 13

<211> 25

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 13

tgtgtgagcc caccggagaa cctgg 25

<210> 14

<211> 25

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 14

aaacccaggt tctccggtgg gctca 25

<210> 15

<211> 20

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 15

tccgcaaacc catctacact 20

<210> 16

<211> 20

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 16

caccgatgac ccagaactgc 20

<210> 17

<211> 24

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 17

ggcatgcatc acccaccctt cctc 24

<210> 18

<211> 24

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 18

aaacgaggaa gggtgggtga tgca 24

<210> 19

<211> 21

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 19

ttggagcatc tcgtgatcag t 21

<210> 20

<211> 20

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 20

atcgttagct cacgtcacgg 20

<210> 21

<211> 36

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 21

taggtctcca gccgttgttg agttttagag ctagaa 36

<210> 22

<211> 36

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 22

atggtctcag gctccgaggc gtgcaccagc cgggaa 36

<210> 23

<211> 36

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 23

taggtctccc catcaaggcc agttttagag ctagaa 36

<210> 24

<211> 36

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 24

atggtctcaa tggtgaaggg atgcaccagc cgggaa 36

<210> 25

<211> 20

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 25

cgggatcgac accaacttca 20

<210> 26

<211> 20

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 26

atggccatct gtccattccc 20

<210> 27

<211> 25

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 27

tgtgtggcgc tcgtactcgt agcag 25

<210> 28

<211> 25

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 28

aaacctgcta cgagtacgag cgcca 25

<210> 29

<211> 20

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 29

ccatatagcc cctgccacaa 20

<210> 30

<211> 20

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 30

cctcccgtag ctcatcttgc 20

<210> 31

<211> 276

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 31

atgatggagt cgggggtccc gccctgcgcc gcgtgcgggg acgcacgccg cctgccgcgc 60

ctgcagctac gcgctctgca aggcctgcct cgacgaggac gccgccgagg gacgcaccac 120

ctgcgcgcgc tgcggcgggg agtacggcgc gcccgaccca gcgcatggcc agggggcggt 180

cgtggaggag gaagtggaag agagccacga gccggcggcc ggcggtgttc gcgagagggt 240

caccatggcc agccaactca gcgatcacca ggatga 276

<210> 32

<211> 91

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 32

Met Met Glu Ser Gly Val Pro Pro Cys Ala Ala Cys Gly Asp Ala Arg

1 5 10 15

Arg Leu Pro Arg Leu Gln Leu Arg Ala Leu Gln Gly Leu Pro Arg Arg

20 25 30

Gly Arg Arg Arg Gly Thr His His Leu Arg Ala Leu Arg Arg Gly Val

35 40 45

Arg Arg Ala Arg Pro Ser Ala Trp Pro Gly Gly Gly Arg Gly Gly Gly

50 55 60

Ser Gly Arg Glu Pro Arg Ala Gly Gly Arg Arg Cys Ser Arg Glu Gly

65 70 75 80

His His Gly Gln Pro Thr Gln Arg Ser Pro Gly

85 90

<210> 33

<211> 1179

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 33

atggacaccg cctccgtcac cggtggcgag cacaagggga aggagaagac gtgccgggtg 60

tgcggcgagg gaggacggga agccgttcgt ggcgtgcgcc gagtgcggct tcccggtgtg 120

caagccctgc tacgagtacg agcgcagcga gggcacccag tgctgccccc agtgcaacac 180

ccgctacaag cgccacaaag ggtgcccacg ggtggaaggc gacgaggacg acggcggcga 240

catggacgac ttcgaggagg agttccagat caagagcccc accaagcaga aaccccccca 300

cgagcccgtc aacttcgacg tctactcgga gaacggcgag cagccggcac agaagtggcg 360

ccctggaggc ccggcgctct cttccttcac cggaagcgtg gctgggaagg atctggagca 420

ggagagggag atggagggtg gcatggagtg gaaggacagg atcgacaagt ggaagacgaa 480

gcaggagaag cggggcaagc tcaaccgcga cgacagcgac gacgacgacg acaagaacga 540

cgacgagtac atgctgctcg cggaggcgag gcagccgctg tggaggaagg tgccgatccc 600

gtcgagcaag atcaacccgt accggatcgt gatcgtgctc cggctggtgg tgctctgctt 660

cttcctcaag ttccggatca cgacgccggc gatggacgcg gtgccgctgt ggctggcctc 720

ggtgatctgc gagctgtggt tcgcgctgtc gtggatcctc gaccagctgc ccaagtggtc 780

gccggtgacg agggagacgt acctggaccg gctggccctc cggtacgagc gcgacggcga 840

gccgtgccgc ctggccccga tcgatttctt cgtcagcacg gtggacccgc tcaaggagcc 900

gcccatcatc accgccaaca ccgtgctgtc catcctcgcc gtcgactacc ccgtcgaccg 960

cgtctcctgc tacgtctccg acgacggcgc gtccatgctg ctcttcgaca cgctctccga 1020

gaccgccgag ttcgcccgcc ggtgggtccc cttctgcaag aagttcacca tcgagccccg 1080

cgcccccgag ttctacttct cccagaagat cgactacctc aaggacaagg tccagcccac 1140

cttcgtcaaa gaacgccgcg ccatgaagag agagtatga 1179

<210> 34

<211> 392

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 34

Met Asp Thr Ala Ser Val Thr Gly Gly Glu His Lys Gly Lys Glu Lys

1 5 10 15

Thr Cys Arg Val Cys Gly Glu Gly Gly Arg Glu Ala Val Arg Gly Val

20 25 30

Arg Arg Val Arg Leu Pro Gly Val Gln Ala Leu Leu Arg Val Arg Ala

35 40 45

Gln Arg Gly His Pro Val Leu Pro Pro Val Gln His Pro Leu Gln Ala

50 55 60

Pro Gln Arg Val Pro Thr Gly Gly Arg Arg Arg Gly Arg Arg Arg Arg

65 70 75 80

His Gly Arg Leu Arg Gly Gly Val Pro Asp Gln Glu Pro His Gln Ala

85 90 95

Glu Thr Pro Pro Arg Ala Arg Gln Leu Arg Arg Leu Leu Gly Glu Arg

100 105 110

Arg Ala Ala Gly Thr Glu Val Ala Pro Trp Arg Pro Gly Ala Leu Phe

115 120 125

Leu His Arg Lys Arg Gly Trp Glu Gly Ser Gly Ala Gly Glu Gly Asp

130 135 140

Gly Gly Trp His Gly Val Glu Gly Gln Asp Arg Gln Val Glu Asp Glu

145 150 155 160

Ala Gly Glu Ala Gly Gln Ala Gln Pro Arg Arg Gln Arg Arg Arg Arg

165 170 175

Arg Gln Glu Arg Arg Arg Val His Ala Ala Arg Gly Gly Glu Ala Ala

180 185 190

Ala Val Glu Glu Gly Ala Asp Pro Val Glu Gln Asp Gln Pro Val Pro

195 200 205

Asp Arg Asp Arg Ala Pro Ala Gly Gly Ala Leu Leu Leu Pro Gln Val

210 215 220

Pro Asp His Asp Ala Gly Asp Gly Arg Gly Ala Ala Val Ala Gly Leu

225 230 235 240

Gly Asp Leu Arg Ala Val Val Arg Ala Val Val Asp Pro Arg Pro Ala

245 250 255

Ala Gln Val Val Ala Gly Asp Glu Gly Asp Val Pro Gly Pro Ala Gly

260 265 270

Pro Pro Val Arg Ala Arg Arg Arg Ala Val Pro Pro Gly Pro Asp Arg

275 280 285

Phe Leu Arg Gln His Gly Gly Pro Ala Gln Gly Ala Ala His His His

290 295 300

Arg Gln His Arg Ala Val His Pro Arg Arg Arg Leu Pro Arg Arg Pro

305 310 315 320

Arg Leu Leu Leu Arg Leu Arg Arg Arg Arg Val His Ala Ala Leu Arg

325 330 335

His Ala Leu Arg Asp Arg Arg Val Arg Pro Pro Val Gly Pro Leu Leu

340 345 350

Gln Glu Val His His Arg Ala Pro Arg Pro Arg Val Leu Leu Leu Pro

355 360 365

Glu Asp Arg Leu Pro Gln Gly Gln Gly Pro Ala His Leu Arg Gln Arg

370 375 380

Thr Pro Arg His Glu Glu Arg Val

385 390

<210> 35

<211> 2358

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 35

atggaggcga gcgccgggct ggtggccggg tcgcacaacc ggaacgagct ggtgctgatc 60

cgggggcacg aggagcccaa gccgctgcgg gcgctgagcg ggcaggtgtg cgagatatgc 120

ggcgacgagg tcggccgcac cgtcgacggc gacctcttcg tcgcctgcaa cgagtgcggc 180

ttcccggtgt gccgcccctg ctacgagtac gagcgccgcg agggcaccca gaactgcccc 240

cagtgcaaga cccgctacaa gcgcctcaag gggagcccga gggtgcccgg ggacgaggac 300

gaggaggaca ttgacgacct ggagcacgag ttcaacatcg acgacgagaa gcagaagcag 360

ctgcagcagg atcaggatgg catgcagaac agccacatca ccgaggcgat gctgcacggc 420

aagatgagct acgggagggg ccccgacgac ggcgacggca acagcacccc gctcccgccg 480

atcatcaccg gcgctcgctc cgtcccggtg agcggggagt tcccgatatc gaacagccat 540

ggccatggcg agttctcctc ttccctgcac aagcgcatcc acccctaccc ggtgtctgag 600

ccagggagtg caaagtggga cgagaagaaa gaggtgagct ggaaggagag gatggacgac 660

tggaaatcca agcagggcat cgtcgccggc ggcgcccccg atcccgacga ctacgacgcc 720

gacgtcccac tgaacgacga ggcgaggcag ccgctgtcga ggaaggtgtc gatcgcgtcg 780

agcaaggtga acccgtaccg gatggtgatc atcctccgtc tcgtggtgct cggcttcttc 840

ctccggtacc gcatcctcca cccggtgccc gacgccatcc cgctgtggct cacctccatc 900

atctgcgaga tctggttcgc cgtgtcgtgg atcctcgacc agttccccaa gtggtacccg 960

atcgacaggg agacctacct cgaccgcctc tccctccgct acgagcgcga gggggagccg 1020

tcgctgctgt cggcggtgga cctgttcgtc agcacggtgg atccgctcaa ggagccgccg 1080

ctggtcacgg ccaacaccgt gctgtccatc ctcgccgtcg actaccccgt cgacaaggtg 1140

tcctgctacg tctccgacga cggcgcgtcc atgctcacgt tcgagtcgct gtcggagacg 1200

gcggagttcg cccgcaagtg ggtgccattc tgcaagaagt tcagcatcga gccccgcgcc 1260

ccggagttct acttctccca gaaggtcgac tacctcaagg acaaggtcca tcccaacttc 1320

gtccaggagc gccgcgccat gaagagagag tacgaggagt tcaaggtgag gatcaacgcg 1380

ctggtggcga aggcgcagaa ggtgccggcg gaagggtgga tcatgaagga cgggacgcca 1440

tggccgggga acaacacccg cgaccacccg ggcatgatcc aggtgttcct cggccacagc 1500

ggcggccacg acaccgaggg caacgagctc ccccgcctcg tctacgtctc ccgtgagaag 1560

cgccccggct tccagcacca caagaaggcc ggcgccatga acgccctcat tcgtgtgtcg 1620

gccgtgctga cgaacgcgcc gttcatgctc aacttggatt gcgatcacta catcaacaac 1680

agcaaggcca tcagggaggc gatgtgcttc ctcatggatc cgcaggtcgg acggaaggtt 1740

tgctacgtgc agttcccgca gaggttcgac ggcatcgacg tccacgaccg atacgccaac 1800

cgcaacaccg tcttcttcga catcaacatg aaggggcttg atgggatcca gggcccggtg 1860

tacgtcggga cagggtgcgt gttcaggcgg caggcgctgt acggatacaa cccacccaag 1920

ggacccaaga ggcccaagat ggtgacctgc gactgctgcc cttgcttcgg gaggaagaag 1980

cggaagcacg gcaaggacgg cctcccggag gccgtcgccg ccgacggcgg gatggacagc 2040

gacaaggaga tgctcatgtc gcagatgaac ttcgagaagc gtcgggcagt cggcggcgtt 2100

cgtgacgtcg acgctgatgg aggaaggcgg cgtcccgccg tcgtccagcc ccgccgcgct 2160

cctcaaggag gccatccatg tcatcagctg cggctacgag gacaagaccg actggggtct 2220

cgagctgggg tggatctacg ggtcgatcac ggaggacatc ctaacggggt tcaagatgca 2280

ctgccgcggg tggaggtcgg tgtactgcat gccgaagagg gcggcgttca aggggtcagc 2340

gccgatcaac ctatctga 2358

<210> 36

<211> 785

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 36

Met Glu Ala Ser Ala Gly Leu Val Ala Gly Ser His Asn Arg Asn Glu

1 5 10 15

Leu Val Leu Ile Arg Gly His Glu Glu Pro Lys Pro Leu Arg Ala Leu

20 25 30

Ser Gly Gln Val Cys Glu Ile Cys Gly Asp Glu Val Gly Arg Thr Val

35 40 45

Asp Gly Asp Leu Phe Val Ala Cys Asn Glu Cys Gly Phe Pro Val Cys

50 55 60

Arg Pro Cys Tyr Glu Tyr Glu Arg Arg Glu Gly Thr Gln Asn Cys Pro

65 70 75 80

Gln Cys Lys Thr Arg Tyr Lys Arg Leu Lys Gly Ser Pro Arg Val Pro

85 90 95

Gly Asp Glu Asp Glu Glu Asp Ile Asp Asp Leu Glu His Glu Phe Asn

100 105 110

Ile Asp Asp Glu Lys Gln Lys Gln Leu Gln Gln Asp Gln Asp Gly Met

115 120 125

Gln Asn Ser His Ile Thr Glu Ala Met Leu His Gly Lys Met Ser Tyr

130 135 140

Gly Arg Gly Pro Asp Asp Gly Asp Gly Asn Ser Thr Pro Leu Pro Pro

145 150 155 160

Ile Ile Thr Gly Ala Arg Ser Val Pro Val Ser Gly Glu Phe Pro Ile

165 170 175

Ser Asn Ser His Gly His Gly Glu Phe Ser Ser Ser Leu His Lys Arg

180 185 190

Ile His Pro Tyr Pro Val Ser Glu Pro Gly Ser Ala Lys Trp Asp Glu

195 200 205

Lys Lys Glu Val Ser Trp Lys Glu Arg Met Asp Asp Trp Lys Ser Lys

210 215 220

Gln Gly Ile Val Ala Gly Gly Ala Pro Asp Pro Asp Asp Tyr Asp Ala

225 230 235 240

Asp Val Pro Leu Asn Asp Glu Ala Arg Gln Pro Leu Ser Arg Lys Val

245 250 255

Ser Ile Ala Ser Ser Lys Val Asn Pro Tyr Arg Met Val Ile Ile Leu

260 265 270

Arg Leu Val Val Leu Gly Phe Phe Leu Arg Tyr Arg Ile Leu His Pro

275 280 285

Val Pro Asp Ala Ile Pro Leu Trp Leu Thr Ser Ile Ile Cys Glu Ile

290 295 300

Trp Phe Ala Val Ser Trp Ile Leu Asp Gln Phe Pro Lys Trp Tyr Pro

305 310 315 320

Ile Asp Arg Glu Thr Tyr Leu Asp Arg Leu Ser Leu Arg Tyr Glu Arg

325 330 335

Glu Gly Glu Pro Ser Leu Leu Ser Ala Val Asp Leu Phe Val Ser Thr

340 345 350

Val Asp Pro Leu Lys Glu Pro Pro Leu Val Thr Ala Asn Thr Val Leu

355 360 365

Ser Ile Leu Ala Val Asp Tyr Pro Val Asp Lys Val Ser Cys Tyr Val

370 375 380

Ser Asp Asp Gly Ala Ser Met Leu Thr Phe Glu Ser Leu Ser Glu Thr

385 390 395 400

Ala Glu Phe Ala Arg Lys Trp Val Pro Phe Cys Lys Lys Phe Ser Ile

405 410 415

Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ser Gln Lys Val Asp Tyr Leu

420 425 430

Lys Asp Lys Val His Pro Asn Phe Val Gln Glu Arg Arg Ala Met Lys

435 440 445

Arg Glu Tyr Glu Glu Phe Lys Val Arg Ile Asn Ala Leu Val Ala Lys

450 455 460

Ala Gln Lys Val Pro Ala Glu Gly Trp Ile Met Lys Asp Gly Thr Pro

465 470 475 480

Trp Pro Gly Asn Asn Thr Arg Asp His Pro Gly Met Ile Gln Val Phe

485 490 495

Leu Gly His Ser Gly Gly His Asp Thr Glu Gly Asn Glu Leu Pro Arg

500 505 510

Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Phe Gln His His Lys

515 520 525

Lys Ala Gly Ala Met Asn Ala Leu Ile Arg Val Ser Ala Val Leu Thr

530 535 540

Asn Ala Pro Phe Met Leu Asn Leu Asp Cys Asp His Tyr Ile Asn Asn

545 550 555 560

Ser Lys Ala Ile Arg Glu Ala Met Cys Phe Leu Met Asp Pro Gln Val

565 570 575

Gly Arg Lys Val Cys Tyr Val Gln Phe Pro Gln Arg Phe Asp Gly Ile

580 585 590

Asp Val His Asp Arg Tyr Ala Asn Arg Asn Thr Val Phe Phe Asp Ile

595 600 605

Asn Met Lys Gly Leu Asp Gly Ile Gln Gly Pro Val Tyr Val Gly Thr

610 615 620

Gly Cys Val Phe Arg Arg Gln Ala Leu Tyr Gly Tyr Asn Pro Pro Lys

625 630 635 640

Gly Pro Lys Arg Pro Lys Met Val Thr Cys Asp Cys Cys Pro Cys Phe

645 650 655

Gly Arg Lys Lys Arg Lys His Gly Lys Asp Gly Leu Pro Glu Ala Val

660 665 670

Ala Ala Asp Gly Gly Met Asp Ser Asp Lys Glu Met Leu Met Ser Gln

675 680 685

Met Asn Phe Glu Lys Arg Arg Ala Val Gly Gly Val Arg Asp Val Asp

690 695 700

Ala Asp Gly Gly Arg Arg Arg Pro Ala Val Val Gln Pro Arg Arg Ala

705 710 715 720

Pro Gln Gly Gly His Pro Cys His Gln Leu Arg Leu Arg Gly Gln Asp

725 730 735

Arg Leu Gly Ser Arg Ala Gly Val Asp Leu Arg Val Asp His Gly Gly

740 745 750

His Pro Asn Gly Val Gln Asp Ala Leu Pro Arg Val Glu Val Gly Val

755 760 765

Leu His Ala Glu Glu Gly Gly Val Gln Gly Val Ser Ala Asp Gln Pro

770 775 780

Ile

785

<210> 37

<211> 2970

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 37

atgatggagt cgggggtccc gccctgcgcc gcgtgcgggg acgacgcgca cgccgcctgc 60

cgcgcctgca gctacgcgct ctgcaaggcc tgcctcgacg aggacgccgc cgagggacgc 120

accacctgcg cgcgctgcgg cggggagtac ggcgcgcccg acccagcgca tggccagggg 180

gcggtcgtgg aggaggaagt ggaagagagc cacgagccgg cggccggcgg tgttcgcgag 240

agggtcacca tggccagcca actcagcgat caccaggatg aaggagttca tgccaggact 300

atgagcaccc atgccaggac aattagcagt gtctctggag ttgggagtga attgaatgat 360

gaatctggga agcccatctg gaagaacagg gtggagagct ggaaggagaa aaagaaagag 420

aagaaagcct cagcaaagaa agctgcagca aaagctcaag ctccacctgt tgaagaacag 480

attatggatg aaaaagactt gacagatgca tatgagccac tttctcggat cattcctata 540

tcgaagaaca agctcacacc ttacagggca gtaatcatta tgcgtctggt agttctgggg 600

ctcttctttc actaccgtat caccaaccct gtttacagcg cctttggtct ctggatgaca 660

tcagtcatat gtgagatctg gtttggattc tcctggatcc tggatcagtt ccccaagtgg 720

tgtcctatca atcgcgaaac ttatgttgat aggctgattg cacgatatgg agatggagaa 780

gattctgggt tggcacctgt agatttcttt gtcagtacag tggatccatt gaaagagcct 840

cctctaatca ctgcgaatac tgttctgtcc attcttgccg tggactatcc agttgagaag 900

atttcgtgtt acgtgtcaga cgatggttcg gctatgctca catttgaatc acttgcggaa 960

acagcagaat ttgctaggag atgggttcca ttttgcaaga agtactccat tgagccacgt 1020

gcccctgagt tctacttctc acagaagatt gactacttga aggacaagat acacccatct 1080

ttcgtcaagg agcgtcgggc tatgaagagg gattacgaag aatacaaggt gaggataaat 1140

gctttggttg ctaaggctca gaagacacct gaggaaggct ggattatgca agatgggaca 1200

ccatggcctg ggaacaaccc tcgtgaccac cctggaatga tccaggtttt ccttggtgag 1260

accggtgctc gggactttga cggaaatgag cttcctcggc tagtctatgt ttcgagagag 1320

aaaagaccag ggtaccaaca ccacaagaaa gcaggggcca tgaacgcttt ggttcgagta 1380

tccgcagttc tgacaaatgc cccttacatt cttaatcttg actgtgatca ctatgttaac 1440

aatagcaaag ctgttcgtga agcgatgtgt ttcatgatgg acccttctgt tggtagagat 1500

gtctgctatg tccaattccc acaaaggttc gatggcattg atcgcagcga tcgttatgcc 1560

aataggaatg ttgtgttctt tgacgttaac atgaaagggc ttgatggcct ccaaggtccg 1620

gtttatgtgg gaactggttg ttgtttctat aggcaagcgc tctatggtta tggaccacca 1680

tcactgcctg cgcttccaaa gtcttctgtc tgttcatggt gctgctgctg ctgtcctaag 1740

aaaaaagctg aaaaaagtga gaaagaaatg catagagact ctcgacgtga agaccttgag 1800

tccgccattt tcaatcttcg ggaaattgac aactatgatg agtatgaacg ctcgatgctt 1860

atctcccaga tgagctttga gaagtcgttt ggactgtcat cagtgttcat agaatcaact 1920

cttatggaga atggaggcgt tcctgagtcc gcaaacccat ctacactgat caaagaggcc 1980

attcatgtca ttagctgtgg atatgaagag aaaactgaat ggggaaaaga gattggctgg 2040

atctacggtt cagtcacaga ggatatcctg actgggttta agatgcactg ccgtggctgg 2100

cgatccatct actgcatgcc gataagaccc gcgttcaagg gatcagcgcc aatcaacctt 2160

tctgatcgtc tccaccaggt tctccggtat gctctcggtt ccgttgagat cttcctcagc 2220

cggcactgcc cactctggta cggctacggt ggtggtcgtc tgaaatggct ccagagattg 2280

tcctacatca acaccatcgt ctacccgttc acctctcttc ctcttattgc atactgttgc 2340

ctgcccgcca tttgcctgct cacaggcaaa ttcatcattc ccacgctctc caacgctgca 2400

accatatggt ttcttggcct cttcatctcc atcatcgtga cgagcgtcct ggagctgcgg 2460

tggagtggca tcggcatcga ggactggtgg cgcaacgagc agttctgggt catcggtggc 2520

gtctccgccc acctcttcgc cgtgttccag ggtatcctca agatgattgc tgggctggac 2580

accaacttca ccgttacggc caaggcgacg gacgacaccg agttcggcga gctgtacgtg 2640

ttcaagtgga cgacggtgct gatcccgccg acctccatcc tggtgctcaa cctcgtcggc 2700

gtggtggccg ggttctccga cgcgctcaac agcggctacg agtcctgggg cccgctcttc 2760

ggcaaggtgt tcttcgccat gtgggtgatc atgcacttgt accccttcct caagggtctc 2820

atgggtcgcc agaaccggac gcccaccatc gtcgtgctct ggtctgtcct gctcgcctcc 2880

gtcttctccc tcctctgggt caagatcgac ccgttcatcg gcagctccga gaccaccacc 2940

accaacagct gcgccaactt cgactgctga 2970

<210> 38

<211> 989

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 38

Met Met Glu Ser Gly Val Pro Pro Cys Ala Ala Cys Gly Asp Asp Ala

1 5 10 15

His Ala Ala Cys Arg Ala Cys Ser Tyr Ala Leu Cys Lys Ala Cys Leu

20 25 30

Asp Glu Asp Ala Ala Glu Gly Arg Thr Thr Cys Ala Arg Cys Gly Gly

35 40 45

Glu Tyr Gly Ala Pro Asp Pro Ala His Gly Gln Gly Ala Val Val Glu

50 55 60

Glu Glu Val Glu Glu Ser His Glu Pro Ala Ala Gly Gly Val Arg Glu

65 70 75 80

Arg Val Thr Met Ala Ser Gln Leu Ser Asp His Gln Asp Glu Gly Val

85 90 95

His Ala Arg Thr Met Ser Thr His Ala Arg Thr Ile Ser Ser Val Ser

100 105 110

Gly Val Gly Ser Glu Leu Asn Asp Glu Ser Gly Lys Pro Ile Trp Lys

115 120 125

Asn Arg Val Glu Ser Trp Lys Glu Lys Lys Lys Glu Lys Lys Ala Ser

130 135 140

Ala Lys Lys Ala Ala Ala Lys Ala Gln Ala Pro Pro Val Glu Glu Gln

145 150 155 160

Ile Met Asp Glu Lys Asp Leu Thr Asp Ala Tyr Glu Pro Leu Ser Arg

165 170 175

Ile Ile Pro Ile Ser Lys Asn Lys Leu Thr Pro Tyr Arg Ala Val Ile

180 185 190

Ile Met Arg Leu Val Val Leu Gly Leu Phe Phe His Tyr Arg Ile Thr

195 200 205

Asn Pro Val Tyr Ser Ala Phe Gly Leu Trp Met Thr Ser Val Ile Cys

210 215 220

Glu Ile Trp Phe Gly Phe Ser Trp Ile Leu Asp Gln Phe Pro Lys Trp

225 230 235 240

Cys Pro Ile Asn Arg Glu Thr Tyr Val Asp Arg Leu Ile Ala Arg Tyr

245 250 255

Gly Asp Gly Glu Asp Ser Gly Leu Ala Pro Val Asp Phe Phe Val Ser

260 265 270

Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Ile Thr Ala Asn Thr Val

275 280 285

Leu Ser Ile Leu Ala Val Asp Tyr Pro Val Glu Lys Ile Ser Cys Tyr

290 295 300

Val Ser Asp Asp Gly Ser Ala Met Leu Thr Phe Glu Ser Leu Ala Glu

305 310 315 320

Thr Ala Glu Phe Ala Arg Arg Trp Val Pro Phe Cys Lys Lys Tyr Ser

325 330 335

Ile Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ser Gln Lys Ile Asp Tyr

340 345 350

Leu Lys Asp Lys Ile His Pro Ser Phe Val Lys Glu Arg Arg Ala Met

355 360 365

Lys Arg Asp Tyr Glu Glu Tyr Lys Val Arg Ile Asn Ala Leu Val Ala

370 375 380

Lys Ala Gln Lys Thr Pro Glu Glu Gly Trp Ile Met Gln Asp Gly Thr

385 390 395 400

Pro Trp Pro Gly Asn Asn Pro Arg Asp His Pro Gly Met Ile Gln Val

405 410 415

Phe Leu Gly Glu Thr Gly Ala Arg Asp Phe Asp Gly Asn Glu Leu Pro

420 425 430

Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Tyr Gln His His

435 440 445

Lys Lys Ala Gly Ala Met Asn Ala Leu Val Arg Val Ser Ala Val Leu

450 455 460

Thr Asn Ala Pro Tyr Ile Leu Asn Leu Asp Cys Asp His Tyr Val Asn

465 470 475 480

Asn Ser Lys Ala Val Arg Glu Ala Met Cys Phe Met Met Asp Pro Ser

485 490 495

Val Gly Arg Asp Val Cys Tyr Val Gln Phe Pro Gln Arg Phe Asp Gly

500 505 510

Ile Asp Arg Ser Asp Arg Tyr Ala Asn Arg Asn Val Val Phe Phe Asp

515 520 525

Val Asn Met Lys Gly Leu Asp Gly Leu Gln Gly Pro Val Tyr Val Gly

530 535 540

Thr Gly Cys Cys Phe Tyr Arg Gln Ala Leu Tyr Gly Tyr Gly Pro Pro

545 550 555 560

Ser Leu Pro Ala Leu Pro Lys Ser Ser Val Cys Ser Trp Cys Cys Cys

565 570 575

Cys Cys Pro Lys Lys Lys Ala Glu Lys Ser Glu Lys Glu Met His Arg

580 585 590

Asp Ser Arg Arg Glu Asp Leu Glu Ser Ala Ile Phe Asn Leu Arg Glu

595 600 605

Ile Asp Asn Tyr Asp Glu Tyr Glu Arg Ser Met Leu Ile Ser Gln Met

610 615 620

Ser Phe Glu Lys Ser Phe Gly Leu Ser Ser Val Phe Ile Glu Ser Thr

625 630 635 640

Leu Met Glu Asn Gly Gly Val Pro Glu Ser Ala Asn Pro Ser Thr Leu

645 650 655

Ile Lys Glu Ala Ile His Val Ile Ser Cys Gly Tyr Glu Glu Lys Thr

660 665 670

Glu Trp Gly Lys Glu Ile Gly Trp Ile Tyr Gly Ser Val Thr Glu Asp

675 680 685

Ile Leu Thr Gly Phe Lys Met His Cys Arg Gly Trp Arg Ser Ile Tyr

690 695 700

Cys Met Pro Ile Arg Pro Ala Phe Lys Gly Ser Ala Pro Ile Asn Leu

705 710 715 720

Ser Asp Arg Leu His Gln Val Leu Arg Tyr Ala Leu Gly Ser Val Glu

725 730 735

Ile Phe Leu Ser Arg His Cys Pro Leu Trp Tyr Gly Tyr Gly Gly Gly

740 745 750

Arg Leu Lys Trp Leu Gln Arg Leu Ser Tyr Ile Asn Thr Ile Val Tyr

755 760 765

Pro Phe Thr Ser Leu Pro Leu Ile Ala Tyr Cys Cys Leu Pro Ala Ile

770 775 780

Cys Leu Leu Thr Gly Lys Phe Ile Ile Pro Thr Leu Ser Asn Ala Ala

785 790 795 800

Thr Ile Trp Phe Leu Gly Leu Phe Ile Ser Ile Ile Val Thr Ser Val

805 810 815

Leu Glu Leu Arg Trp Ser Gly Ile Gly Ile Glu Asp Trp Trp Arg Asn

820 825 830

Glu Gln Phe Trp Val Ile Gly Gly Val Ser Ala His Leu Phe Ala Val

835 840 845

Phe Gln Gly Ile Leu Lys Met Ile Ala Gly Leu Asp Thr Asn Phe Thr

850 855 860

Val Thr Ala Lys Ala Thr Asp Asp Thr Glu Phe Gly Glu Leu Tyr Val

865 870 875 880

Phe Lys Trp Thr Thr Val Leu Ile Pro Pro Thr Ser Ile Leu Val Leu

885 890 895

Asn Leu Val Gly Val Val Ala Gly Phe Ser Asp Ala Leu Asn Ser Gly

900 905 910

Tyr Glu Ser Trp Gly Pro Leu Phe Gly Lys Val Phe Phe Ala Met Trp

915 920 925

Val Ile Met His Leu Tyr Pro Phe Leu Lys Gly Leu Met Gly Arg Gln

930 935 940

Asn Arg Thr Pro Thr Ile Val Val Leu Trp Ser Val Leu Leu Ala Ser

945 950 955 960

Val Phe Ser Leu Leu Trp Val Lys Ile Asp Pro Phe Ile Gly Ser Ser

965 970 975

Glu Thr Thr Thr Thr Asn Ser Cys Ala Asn Phe Asp Cys

980 985

<210> 39

<211> 3171

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 39

atggacaccg cctccgtcac cggtggcgag cacaagggga aggagaagac gtgccgggtg 60

tgcggcgagg aggtggcggc gagggaggac gggaagccgt tcgtggcgtg cgccgagtgc 120

ggcttcccgg tgtgcaagcc ctgctacgag tacgagcgca gcgagggcac ccagtgctgc 180

ccccagtgca acacccgcta caagcgccac aaagggtgcc cacgggtgga aggcgacgag 240

gacgacggcg gcgacatgga cgacttcgag gaggagttcc agatcaagag ccccaccaag 300

cagaaacccc cccacgagcc cgtcaacttc gacgtctact cggagaacgg cgagcagccg 360

gcacagaagt ggcgccctgg aggcccggcg ctctcttcct tcaccggaag cgtggctggg 420

aaggatctgg agcaggagag ggagatggag ggtggcatgg agtggaagga caggatcgac 480

aagtggaaga cgaagcagga gaagcggggc aagctcaacc gcgacgacag cgacgacgac 540

gacgacaaga acgacgacga gtacatgctg ctcgcggagg cgaggcagcc gctgtggagg 600

aaggtgccga tcccgtcgag caagatcaac ccgtaccgga tcgtgatcgt gctccggctg 660

gtggtgctct gcttcttcct caagttccgg atcacgacgc cggcgatgga cgcggtgccg 720

ctgtggctgg cctcggtgat ctgcgagctg tggttcgcgc tgtcgtggat cctcgaccag 780

ctgcccaagt ggtcgccggt gacgagggag acgtacctgg accggctggc cctccggtac 840

gagcgcgacg gcgagccgtg ccgcctggcc ccgatcgatt tcttcgtcag cacggtggac 900

ccgctcaagg agccgcccat catcaccgcc aacaccgtgc tgtccatcct cgccgtcgac 960

taccccgtcg accgcgtctc ctgctacgtc tccgacgacg gcgcgtccat gctgctcttc 1020

gacacgctct ccgagaccgc cgagttcgcc cgccggtggg tccccttctg caagaagttc 1080

accatcgagc cccgcgcccc cgagttctac ttctcccaga agatcgacta cctcaaggac 1140

aaggtccagc ccaccttcgt caaagaacgc cgcgccatga agagagagta tgaggagttc 1200

aaggtgagga taaacgcgct ggtggcgaag gcgcagaaga agccggacgg gacgccatgg 1260

ccggggaaca acacgaggga ccacccgggg atgatccagg tgtacctggg cagccagggc 1320

gcgctcgacg tcgagggcag cgagctgccg cggctggtgt acgtgtcccg cgagaagcgg 1380

cccggctaca accaccacaa gaaggccggc gccatgaact ccctcgttcg cgtctccgcc 1440

gtgcttacca acgccccctt catcctcaac ctcgactgcg accactacgt caacaacagc 1500

aaggccgtcc gcgaggccat gtgcttcctc atggacaagc agctcggcaa gaagctgtgc 1560

tacgtccagt tcccccagcg cttcgacggc atcgaccgcc acgatcgcta cgccaaccgc 1620

aacaccgtct tcttcgacat caacatgaag gggctggacg ggatacaggg gccggtgtac 1680

gtggggacgg ggacggtgtt caacaggcag gcgctgtacg gatacgaccc gccgcggccg 1740

gagaagaggc cgaagatgac gtgcgactgc tggccgtcgt ggtgctgctg ctgctgctgc 1800

ttcggcgggg ggaagcgcgg caagtcgcac aagaacaaga agggcggcgg cggcggcgag 1860

ggcggcggcc tcgacgagcc gcgccgcggg ctgctcgggt tctacaagaa gaggagcaag 1920

aaggacaagc tcggcggcgg cgcggcgtcg ctcgccggag ggaagaaagg gtaccggaag 1980

caccagcgcg ggttcgagct ggaggagatc gaggagggcc tcgaggggta cgacgagctg 2040

gagcgctcgt cgctcatgtc gcagaagagc ttcgagaagc ggttcggcca gtcgccggtg 2100

ttcatcgcct ccaccctcgt cgaggacggc ggcctccccc agggcgccgc cgccgacccc 2160

gccgccctca tcaaggaggc catccacgtc atcagctgcg gctacgagga gaagaccgag 2220

tggggcaagg agattgggtg gatctacggg tcggtgacgg aggacatctt aacggggttc 2280

aagatgcatt gccgtgggtg gaagtcggtg tactgcacgc cggcgagggc ggcattcaag 2340

gggtcggcgc ccatcaacct gtcggatcgt ctgcaccagg tgctccggtg ggcgctcggc 2400

tccgtcgaga tcttcatgag ccgccattgc ccgctctggt acgcctatgg cggccgcctc 2460

aagtggctcg agcgcttcgc ctacaccaac accatcgtct accccttcac ctccattccc 2520

ctcctcgcct actgcaccat ccccgccgtc tgcctcctca ccggcaagtt catcatcccc 2580

acgcttaaca atttggcgag catatggttc atagcgcttt tcctgtcgat catcgcgacg 2640

ggggtgctgg agctgcggtg gagcggggtg agcatcgagg actggtggag gaacgagcag 2700

ttctgggtga tcggcggcgt gtcggcgcac ctgttcgccg tgttccaggg cctcctcaag 2760

gtgctcggcg gcgtggacac caacttcacg gtgacgtcca aggccgccgc cgacgagacc 2820

gacgcgttcg gcgagctcta cctgttcaag tggacgacgc tgctggtgcc gccgacgacg 2880

ctgatcatca tcaacatggt ggggatcgtc gccggcgtgt cggacgccgt gaacaacggg 2940

tacgggtcgt ggggcccgct gttcgggaag ctcttcttct ccttctgggt catcctccac 3000

ctctacccct tcctcaaggg gctcatgggg aggcagaacc ggacgcccac cattgtcgtg 3060

ctctggtcca tcctcctcgc ctccatcttc tccctcgtct gggtcaggat cgaccccttc 3120

atccccaagc ccaagggccc cgtcctcaag ccatgcgggg tctcgtgctg a 3171

<210> 40

<211> 1056

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 40

Met Asp Thr Ala Ser Val Thr Gly Gly Glu His Lys Gly Lys Glu Lys

1 5 10 15

Thr Cys Arg Val Cys Gly Glu Glu Val Ala Ala Arg Glu Asp Gly Lys

20 25 30

Pro Phe Val Ala Cys Ala Glu Cys Gly Phe Pro Val Cys Lys Pro Cys

35 40 45

Tyr Glu Tyr Glu Arg Ser Glu Gly Thr Gln Cys Cys Pro Gln Cys Asn

50 55 60

Thr Arg Tyr Lys Arg His Lys Gly Cys Pro Arg Val Glu Gly Asp Glu

65 70 75 80

Asp Asp Gly Gly Asp Met Asp Asp Phe Glu Glu Glu Phe Gln Ile Lys

85 90 95

Ser Pro Thr Lys Gln Lys Pro Pro His Glu Pro Val Asn Phe Asp Val

100 105 110

Tyr Ser Glu Asn Gly Glu Gln Pro Ala Gln Lys Trp Arg Pro Gly Gly

115 120 125

Pro Ala Leu Ser Ser Phe Thr Gly Ser Val Ala Gly Lys Asp Leu Glu

130 135 140

Gln Glu Arg Glu Met Glu Gly Gly Met Glu Trp Lys Asp Arg Ile Asp

145 150 155 160

Lys Trp Lys Thr Lys Gln Glu Lys Arg Gly Lys Leu Asn Arg Asp Asp

165 170 175

Ser Asp Asp Asp Asp Asp Lys Asn Asp Asp Glu Tyr Met Leu Leu Ala

180 185 190

Glu Ala Arg Gln Pro Leu Trp Arg Lys Val Pro Ile Pro Ser Ser Lys

195 200 205

Ile Asn Pro Tyr Arg Ile Val Ile Val Leu Arg Leu Val Val Leu Cys

210 215 220

Phe Phe Leu Lys Phe Arg Ile Thr Thr Pro Ala Met Asp Ala Val Pro

225 230 235 240

Leu Trp Leu Ala Ser Val Ile Cys Glu Leu Trp Phe Ala Leu Ser Trp

245 250 255

Ile Leu Asp Gln Leu Pro Lys Trp Ser Pro Val Thr Arg Glu Thr Tyr

260 265 270

Leu Asp Arg Leu Ala Leu Arg Tyr Glu Arg Asp Gly Glu Pro Cys Arg

275 280 285

Leu Ala Pro Ile Asp Phe Phe Val Ser Thr Val Asp Pro Leu Lys Glu

290 295 300

Pro Pro Ile Ile Thr Ala Asn Thr Val Leu Ser Ile Leu Ala Val Asp

305 310 315 320

Tyr Pro Val Asp Arg Val Ser Cys Tyr Val Ser Asp Asp Gly Ala Ser

325 330 335

Met Leu Leu Phe Asp Thr Leu Ser Glu Thr Ala Glu Phe Ala Arg Arg

340 345 350

Trp Val Pro Phe Cys Lys Lys Phe Thr Ile Glu Pro Arg Ala Pro Glu

355 360 365

Phe Tyr Phe Ser Gln Lys Ile Asp Tyr Leu Lys Asp Lys Val Gln Pro

370 375 380

Thr Phe Val Lys Glu Arg Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe

385 390 395 400

Lys Val Arg Ile Asn Ala Leu Val Ala Lys Ala Gln Lys Lys Pro Asp

405 410 415

Gly Thr Pro Trp Pro Gly Asn Asn Thr Arg Asp His Pro Gly Met Ile

420 425 430

Gln Val Tyr Leu Gly Ser Gln Gly Ala Leu Asp Val Glu Gly Ser Glu

435 440 445

Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Tyr Asn

450 455 460

His His Lys Lys Ala Gly Ala Met Asn Ser Leu Val Arg Val Ser Ala

465 470 475 480

Val Leu Thr Asn Ala Pro Phe Ile Leu Asn Leu Asp Cys Asp His Tyr

485 490 495

Val Asn Asn Ser Lys Ala Val Arg Glu Ala Met Cys Phe Leu Met Asp

500 505 510

Lys Gln Leu Gly Lys Lys Leu Cys Tyr Val Gln Phe Pro Gln Arg Phe

515 520 525

Asp Gly Ile Asp Arg His Asp Arg Tyr Ala Asn Arg Asn Thr Val Phe

530 535 540

Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln Gly Pro Val Tyr

545 550 555 560

Val Gly Thr Gly Thr Val Phe Asn Arg Gln Ala Leu Tyr Gly Tyr Asp

565 570 575

Pro Pro Arg Pro Glu Lys Arg Pro Lys Met Thr Cys Asp Cys Trp Pro

580 585 590

Ser Trp Cys Cys Cys Cys Cys Cys Phe Gly Gly Gly Lys Arg Gly Lys

595 600 605

Ser His Lys Asn Lys Lys Gly Gly Gly Gly Gly Glu Gly Gly Gly Leu

610 615 620

Asp Glu Pro Arg Arg Gly Leu Leu Gly Phe Tyr Lys Lys Arg Ser Lys

625 630 635 640

Lys Asp Lys Leu Gly Gly Gly Ala Ala Ser Leu Ala Gly Gly Lys Lys

645 650 655

Gly Tyr Arg Lys His Gln Arg Gly Phe Glu Leu Glu Glu Ile Glu Glu

660 665 670

Gly Leu Glu Gly Tyr Asp Glu Leu Glu Arg Ser Ser Leu Met Ser Gln

675 680 685

Lys Ser Phe Glu Lys Arg Phe Gly Gln Ser Pro Val Phe Ile Ala Ser

690 695 700

Thr Leu Val Glu Asp Gly Gly Leu Pro Gln Gly Ala Ala Ala Asp Pro

705 710 715 720

Ala Ala Leu Ile Lys Glu Ala Ile His Val Ile Ser Cys Gly Tyr Glu

725 730 735

Glu Lys Thr Glu Trp Gly Lys Glu Ile Gly Trp Ile Tyr Gly Ser Val

740 745 750

Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His Cys Arg Gly Trp Lys

755 760 765

Ser Val Tyr Cys Thr Pro Ala Arg Ala Ala Phe Lys Gly Ser Ala Pro

770 775 780

Ile Asn Leu Ser Asp Arg Leu His Gln Val Leu Arg Trp Ala Leu Gly

785 790 795 800

Ser Val Glu Ile Phe Met Ser Arg His Cys Pro Leu Trp Tyr Ala Tyr

805 810 815

Gly Gly Arg Leu Lys Trp Leu Glu Arg Phe Ala Tyr Thr Asn Thr Ile

820 825 830

Val Tyr Pro Phe Thr Ser Ile Pro Leu Leu Ala Tyr Cys Thr Ile Pro

835 840 845

Ala Val Cys Leu Leu Thr Gly Lys Phe Ile Ile Pro Thr Leu Asn Asn

850 855 860

Leu Ala Ser Ile Trp Phe Ile Ala Leu Phe Leu Ser Ile Ile Ala Thr

865 870 875 880

Gly Val Leu Glu Leu Arg Trp Ser Gly Val Ser Ile Glu Asp Trp Trp

885 890 895

Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Val Ser Ala His Leu Phe

900 905 910

Ala Val Phe Gln Gly Leu Leu Lys Val Leu Gly Gly Val Asp Thr Asn

915 920 925

Phe Thr Val Thr Ser Lys Ala Ala Ala Asp Glu Thr Asp Ala Phe Gly

930 935 940

Glu Leu Tyr Leu Phe Lys Trp Thr Thr Leu Leu Val Pro Pro Thr Thr

945 950 955 960

Leu Ile Ile Ile Asn Met Val Gly Ile Val Ala Gly Val Ser Asp Ala

965 970 975

Val Asn Asn Gly Tyr Gly Ser Trp Gly Pro Leu Phe Gly Lys Leu Phe

980 985 990

Phe Ser Phe Trp Val Ile Leu His Leu Tyr Pro Phe Leu Lys Gly Leu

995 1000 1005

Met Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Val Leu Trp Ser Ile

1010 1015 1020

Leu Leu Ala Ser Ile Phe Ser Leu Val Trp Val Arg Ile Asp Pro Phe

1025 1030 1035 1040

Ile Pro Lys Pro Lys Gly Pro Val Leu Lys Pro Cys Gly Val Ser Cys

1045 1050 1055

<210> 41

<211> 3135

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 41

atggaggcga gcgccgggct ggtggccggg tcgcacaacc ggaacgagct ggtgctgatc 60

cgggggcacg aggagcccaa gccgctgcgg gcgctgagcg ggcaggtgtg cgagatatgc 120

ggcgacgagg tcggccgcac cgtcgacggc gacctcttcg tcgcctgcaa cgagtgcggc 180

ttcccggtgt gccgcccctg ctacgagtac gagcgccgcg agggcaccca gaactgcccc 240

cagtgcaaga cccgctacaa gcgcctcaag gggagcccga gggtgcccgg ggacgaggac 300

gaggaggaca ttgacgacct ggagcacgag ttcaacatcg acgacgagaa gcagaagcag 360

ctgcagcagg atcaggatgg catgcagaac agccacatca ccgaggcgat gctgcacggc 420

aagatgagct acgggagggg ccccgacgac ggcgacggca acagcacccc gctcccgccg 480

atcatcaccg gcgctcgctc cgtcccggtg agcggggagt tcccgatatc gaacagccat 540

ggccatggcg agttctcctc ttccctgcac aagcgcatcc acccctaccc ggtgtctgag 600

ccagggagtg caaagtggga cgagaagaaa gaggtgagct ggaaggagag gatggacgac 660

tggaaatcca agcagggcat cgtcgccggc ggcgcccccg atcccgacga ctacgacgcc 720

gacgtcccac tgaacgacga ggcgaggcag ccgctgtcga ggaaggtgtc gatcgcgtcg 780

agcaaggtga acccgtaccg gatggtgatc atcctccgtc tcgtggtgct cggcttcttc 840

ctccggtacc gcatcctcca cccggtgccc gacgccatcc cgctgtggct cacctccatc 900

atctgcgaga tctggttcgc cgtgtcgtgg atcctcgacc agttccccaa gtggtacccg 960

atcgacaggg agacctacct cgaccgcctc tccctccgct acgagcgcga gggggagccg 1020

tcgctgctgt cggcggtgga cctgttcgtc agcacggtgg atccgctcaa ggagccgccg 1080

ctggtcacgg ccaacaccgt gctgtccatc ctcgccgtcg actaccccgt cgacaaggtg 1140

tcctgctacg tctccgacga cggcgcgtcc atgctcacgt tcgagtcgct gtcggagacg 1200

gcggagttcg cccgcaagtg ggtgccattc tgcaagaagt tcagcatcga gccccgcgcc 1260

ccggagttct acttctccca gaaggtcgac tacctcaagg acaaggtcca tcccaacttc 1320

gtccaggagc gccgcgccat gaagagagag tacgaggagt tcaaggtgag gatcaacgcg 1380

ctggtggcga aggcgcagaa ggtgccggcg gaagggtgga tcatgaagga cgggacgcca 1440

tggccgggga acaacacccg cgaccacccg ggcatgatcc aggtgttcct cggccacagc 1500

ggcggccacg acaccgaggg caacgagctc ccccgcctcg tctacgtctc ccgtgagaag 1560

cgccccggct tccagcacca caagaaggcc ggcgccatga acgccctcat tcgtgtgtcg 1620

gccgtgctga cgaacgcgcc gttcatgctc aacttggatt gcgatcacta catcaacaac 1680

agcaaggcca tcagggaggc gatgtgcttc ctcatggatc cgcaggtcgg acggaaggtt 1740

tgctacgtgc agttcccgca gaggttcgac ggcatcgacg tccacgaccg atacgccaac 1800

cgcaacaccg tcttcttcga catcaacatg aaggggcttg atgggatcca gggcccggtg 1860

tacgtcggga cagggtgcgt gttcaggcgg caggcgctgt acggatacaa cccacccaag 1920

ggacccaaga ggcccaagat ggtgacctgc gactgctgcc cttgcttcgg gaggaagaag 1980

cggaagcacg gcaaggacgg cctcccggag gccgtcgccg ccgacggcgg gatggacagc 2040

gacaaggaga tgctcatgtc gcagatgaac ttcgagaagc ggttcgggca gtcggcggcg 2100

ttcgtgacgt cgacgctgat ggaggaaggc ggcgtcccgc cgtcgtccag ccccgccgcg 2160

ctcctcaagg aggccatcca tgtcatcagc tgcggctacg aggacaagac cgactggggt 2220

ctcgagctgg ggtggatcta cgggtcgatc acggaggaca tcctaacggg gttcaagatg 2280

cactgccgcg ggtggaggtc ggtgtactgc atgccgaaga gggcggcgtt caaggggtca 2340

gcgccgatca acctatctga ccgtctcaac caggtgctcc ggtgggcgct cggctccgtc 2400

gagatcttct tcagccggca cagcccgctc ctctacggct acaagaacgg caacctcaag 2460

tggctcgagc gcttctccta catcaacacc accatctacc ccttcacttc tctccccctc 2520

ctcgcctact gcaccctacc cgccgtctgc ctcctcaccg gcaagttcat catgcctccg 2580

attagcacgt ttgcgagttt gttcttcatc gcgctcttca tctccatctt cgcgacgggc 2640

atcctggaga tgaggtggag cggggtgagc atcgaggagt ggtggaggaa cgagcagttc 2700

tgggtcatcg gcggcgtgtc ggcgcacctg ttcgcggtgg tgcagggcct gctcaaggtg 2760

ctggccggga tcgacaccaa cttcaccgtc acgtccaagg ccaccggaga cgaggacgac 2820

gagttcgcgg agctctacgc cttcaagtgg accaccctcc tcatcccgcc caccacgctg 2880

ctcatcctca acatcatcgg cgtcgtcgcc ggcgtctccg acgccatcag ctccgaggcg 2940

tggggcccgc tcttcgggaa gctcttcttc gccttctggg tcatcgtcca cctctacccc 3000

ttcctcaagg ggctcatggg gaggcagaac cggacgccca ccattgttgt catctggtcc 3060

gtgctgctcg cctccatctt ttccttgctc tgggtcagga ttgatccctt caccatcaag 3120

gctcaggggc cctga 3135

<210> 42

<211> 1044

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 42

Met Glu Ala Ser Ala Gly Leu Val Ala Gly Ser His Asn Arg Asn Glu

1 5 10 15

Leu Val Leu Ile Arg Gly His Glu Glu Pro Lys Pro Leu Arg Ala Leu

20 25 30

Ser Gly Gln Val Cys Glu Ile Cys Gly Asp Glu Val Gly Arg Thr Val

35 40 45

Asp Gly Asp Leu Phe Val Ala Cys Asn Glu Cys Gly Phe Pro Val Cys

50 55 60

Arg Pro Cys Tyr Glu Tyr Glu Arg Arg Glu Gly Thr Gln Asn Cys Pro

65 70 75 80

Gln Cys Lys Thr Arg Tyr Lys Arg Leu Lys Gly Ser Pro Arg Val Pro

85 90 95

Gly Asp Glu Asp Glu Glu Asp Ile Asp Asp Leu Glu His Glu Phe Asn

100 105 110

Ile Asp Asp Glu Lys Gln Lys Gln Leu Gln Gln Asp Gln Asp Gly Met

115 120 125

Gln Asn Ser His Ile Thr Glu Ala Met Leu His Gly Lys Met Ser Tyr

130 135 140

Gly Arg Gly Pro Asp Asp Gly Asp Gly Asn Ser Thr Pro Leu Pro Pro

145 150 155 160

Ile Ile Thr Gly Ala Arg Ser Val Pro Val Ser Gly Glu Phe Pro Ile

165 170 175

Ser Asn Ser His Gly His Gly Glu Phe Ser Ser Ser Leu His Lys Arg

180 185 190

Ile His Pro Tyr Pro Val Ser Glu Pro Gly Ser Ala Lys Trp Asp Glu

195 200 205

Lys Lys Glu Val Ser Trp Lys Glu Arg Met Asp Asp Trp Lys Ser Lys

210 215 220

Gln Gly Ile Val Ala Gly Gly Ala Pro Asp Pro Asp Asp Tyr Asp Ala

225 230 235 240

Asp Val Pro Leu Asn Asp Glu Ala Arg Gln Pro Leu Ser Arg Lys Val

245 250 255

Ser Ile Ala Ser Ser Lys Val Asn Pro Tyr Arg Met Val Ile Ile Leu

260 265 270

Arg Leu Val Val Leu Gly Phe Phe Leu Arg Tyr Arg Ile Leu His Pro

275 280 285

Val Pro Asp Ala Ile Pro Leu Trp Leu Thr Ser Ile Ile Cys Glu Ile

290 295 300

Trp Phe Ala Val Ser Trp Ile Leu Asp Gln Phe Pro Lys Trp Tyr Pro

305 310 315 320

Ile Asp Arg Glu Thr Tyr Leu Asp Arg Leu Ser Leu Arg Tyr Glu Arg

325 330 335

Glu Gly Glu Pro Ser Leu Leu Ser Ala Val Asp Leu Phe Val Ser Thr

340 345 350

Val Asp Pro Leu Lys Glu Pro Pro Leu Val Thr Ala Asn Thr Val Leu

355 360 365

Ser Ile Leu Ala Val Asp Tyr Pro Val Asp Lys Val Ser Cys Tyr Val

370 375 380

Ser Asp Asp Gly Ala Ser Met Leu Thr Phe Glu Ser Leu Ser Glu Thr

385 390 395 400

Ala Glu Phe Ala Arg Lys Trp Val Pro Phe Cys Lys Lys Phe Ser Ile

405 410 415

Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ser Gln Lys Val Asp Tyr Leu

420 425 430

Lys Asp Lys Val His Pro Asn Phe Val Gln Glu Arg Arg Ala Met Lys

435 440 445

Arg Glu Tyr Glu Glu Phe Lys Val Arg Ile Asn Ala Leu Val Ala Lys

450 455 460

Ala Gln Lys Val Pro Ala Glu Gly Trp Ile Met Lys Asp Gly Thr Pro

465 470 475 480

Trp Pro Gly Asn Asn Thr Arg Asp His Pro Gly Met Ile Gln Val Phe

485 490 495

Leu Gly His Ser Gly Gly His Asp Thr Glu Gly Asn Glu Leu Pro Arg

500 505 510

Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Phe Gln His His Lys

515 520 525

Lys Ala Gly Ala Met Asn Ala Leu Ile Arg Val Ser Ala Val Leu Thr

530 535 540

Asn Ala Pro Phe Met Leu Asn Leu Asp Cys Asp His Tyr Ile Asn Asn

545 550 555 560

Ser Lys Ala Ile Arg Glu Ala Met Cys Phe Leu Met Asp Pro Gln Val

565 570 575

Gly Arg Lys Val Cys Tyr Val Gln Phe Pro Gln Arg Phe Asp Gly Ile

580 585 590

Asp Val His Asp Arg Tyr Ala Asn Arg Asn Thr Val Phe Phe Asp Ile

595 600 605

Asn Met Lys Gly Leu Asp Gly Ile Gln Gly Pro Val Tyr Val Gly Thr

610 615 620

Gly Cys Val Phe Arg Arg Gln Ala Leu Tyr Gly Tyr Asn Pro Pro Lys

625 630 635 640

Gly Pro Lys Arg Pro Lys Met Val Thr Cys Asp Cys Cys Pro Cys Phe

645 650 655

Gly Arg Lys Lys Arg Lys His Gly Lys Asp Gly Leu Pro Glu Ala Val

660 665 670

Ala Ala Asp Gly Gly Met Asp Ser Asp Lys Glu Met Leu Met Ser Gln

675 680 685

Met Asn Phe Glu Lys Arg Phe Gly Gln Ser Ala Ala Phe Val Thr Ser

690 695 700

Thr Leu Met Glu Glu Gly Gly Val Pro Pro Ser Ser Ser Pro Ala Ala

705 710 715 720

Leu Leu Lys Glu Ala Ile His Val Ile Ser Cys Gly Tyr Glu Asp Lys

725 730 735

Thr Asp Trp Gly Leu Glu Leu Gly Trp Ile Tyr Gly Ser Ile Thr Glu

740 745 750

Asp Ile Leu Thr Gly Phe Lys Met His Cys Arg Gly Trp Arg Ser Val

755 760 765

Tyr Cys Met Pro Lys Arg Ala Ala Phe Lys Gly Ser Ala Pro Ile Asn

770 775 780

Leu Ser Asp Arg Leu Asn Gln Val Leu Arg Trp Ala Leu Gly Ser Val

785 790 795 800

Glu Ile Phe Phe Ser Arg His Ser Pro Leu Leu Tyr Gly Tyr Lys Asn

805 810 815

Gly Asn Leu Lys Trp Leu Glu Arg Phe Ser Tyr Ile Asn Thr Thr Ile

820 825 830

Tyr Pro Phe Thr Ser Leu Pro Leu Leu Ala Tyr Cys Thr Leu Pro Ala

835 840 845

Val Cys Leu Leu Thr Gly Lys Phe Ile Met Pro Pro Ile Ser Thr Phe

850 855 860

Ala Ser Leu Phe Phe Ile Ala Leu Phe Ile Ser Ile Phe Ala Thr Gly

865 870 875 880

Ile Leu Glu Met Arg Trp Ser Gly Val Ser Ile Glu Glu Trp Trp Arg

885 890 895

Asn Glu Gln Phe Trp Val Ile Gly Gly Val Ser Ala His Leu Phe Ala

900 905 910

Val Val Gln Gly Leu Leu Lys Val Leu Ala Gly Ile Asp Thr Asn Phe

915 920 925

Thr Val Thr Ser Lys Ala Thr Gly Asp Glu Asp Asp Glu Phe Ala Glu

930 935 940

Leu Tyr Ala Phe Lys Trp Thr Thr Leu Leu Ile Pro Pro Thr Thr Leu

945 950 955 960

Leu Ile Leu Asn Ile Ile Gly Val Val Ala Gly Val Ser Asp Ala Ile

965 970 975

Ser Ser Glu Ala Trp Gly Pro Leu Phe Gly Lys Leu Phe Phe Ala Phe

980 985 990

Trp Val Ile Val His Leu Tyr Pro Phe Leu Lys Gly Leu Met Gly Arg

995 1000 1005

Gln Asn Arg Thr Pro Thr Ile Val Val Ile Trp Ser Val Leu Leu Ala

1010 1015 1020

Ser Ile Phe Ser Leu Leu Trp Val Arg Ile Asp Pro Phe Thr Ile Lys

1025 1030 1035 1040

Ala Gln Gly Pro

<210> 43

<211> 3168

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 43

atggaggcga gcgccgggct ggtggccggg tcgcacaacc ggaacgagct ggtgctgatc 60

cgggggcacg aggagcccaa gccgctgcgg gcgctgagcg ggcaggtgtg cgagatatgc 120

ggcgacgagg tcggccgcac cgtcgacggc gacctcttcg tcgcctgcaa cgagtgcggc 180

ttcccggtgt gccgcccctg ctacgagtac aaccgccgcg agggcaccca gaactgcccc 240

cagtgcaaga cccgctacaa gcgcctcaag gggagcccga gggtgcccgg ggacgaggac 300

gaggaggaca ttgacgacct ggagcacgag ttcaacatcg acgacgagaa gcagaagcag 360

ctgcagcagg atcaggatgg catgcagaac agccacatca ccgaggcgat gctgcacggc 420

aagatgagct acgggagggg ccccgacgac ggcgacggca acagcacccc gctcccgccg 480

atcatcaccg gcgctcgctc cgtcccggtg agcggggagt tcccgatatc gaacagccat 540

ggccatggcg agttctcctc ttccctgcac aagcgcatcc acccctaccc ggtgtctgag 600

ccagggagtg caaagtggga cgagaagaaa gaggtgagct ggaaggagag gatggacgac 660

tggaaatcca agcagggcat cgtcgccggc ggcgcccccg atcccgacga ctacgacgcc 720

gacgtcccac tgaacgacga ggcgaggcag ccgctgtcga ggaaggtgtc gatcgcgtcg 780

agcaaggtga acccgtaccg gatggtgatc atcctccgtc tcgtggtgct cggcttcttc 840

ctccggtacc gcatcctcca cccggtgccc gacgccatcc cgctgtggct cacctccatc 900

atctgcgaga tctggttcgc cgtgtcgtgg atcctcgacc agttccccaa gtggtacccg 960

atcgacaggg agacctacct cgaccgcctc tccctccgct acgagcgcga gggggagccg 1020

tcgctgctgt cggcggtgga cctgttcgtc agcacggtgg atccgctcaa ggagccgccg 1080

ctggtcacgg ccaacaccgt gctgtccatc ctcgccgtcg actaccccgt cgacaaggtg 1140

tcctgctacg tctccgacga cggcgcgtcc atgctcacgt tcgagtcgct gtcggagacg 1200

gcggagttcg cccgcaagtg ggtgccattc tgcaagaagt tcagcatcga gccccgcgcc 1260

ccggagttct acttctccca gaaggtcgac tacctcaagg acaaggtcca tcccaacttc 1320

gtccaggagc gccgcgccat gaagagagag tacgaggagt tcaaggtgag gatcaacgcg 1380

ctggtggcga aggcgcagaa ggtgccggcg gaagggtgga tcatgaagga cgggacgcca 1440

tggccgggga acaacacccg cgaccacccg ggcatgatcc aggtgttcct cggccacagc 1500

ggcggccacg acaccgaggg caacgagctc ccccgcctcg tctacgtctc ccgtgagaag 1560

cgccccggct tccagcacca caagaaggcc ggcgccatga acgccctcat tcgtgtgtcg 1620

gccgtgctga cgaacgcgcc gttcatgctc aacttggatt gcgatcacta catcaacaac 1680

agcaaggcca tcagggaggc gatgtgcttc ctcatggatc cgcaggtcgg acggaaggtt 1740

tgctacgtgc agttcccgca gaggttcgac ggcatcgacg tccacgaccg atacgccaac 1800

cgcaacaccg tcttcttcga catcaacatg aaggggcttg atgggatcca gggcccggtg 1860

tacgtcggga cagggtgcgt gttcaggcgg caggcgctgt acggatacaa cccacccaag 1920

ggacccaaga ggcccaagat ggtgacctgc gactgctgcc cttgcttcgg gaggaagaag 1980

cggaagcacg gcaaggacgg cctcccggag gccgtcgccg ccgacggcgg gatggacagc 2040

gacaaggaga tgctcatgtc gcagatgaac ttcgagaagc ggttcgggca gtcggcggcg 2100

ttcgtgacgt cgacgctgat ggaggaaggc ggcgtcccgc cgtcgtccag ccccgccgcg 2160

ctcctcaagg aggccatcca tgtcatcagc tgcggctacg aggacaagac cgactggggt 2220

ctcgagctgg ggtggatcta cgggtcgatc acggaggaca tcctaacggg gttcaagatg 2280

cactgccgcg ggtggaggtc ggtgtactgc atgccgaaga gggcggcgtt caaggggtca 2340

gcgccgatca acctatctga ccgtctcaac caggtgctcc ggtgggcgct cggctccgtc 2400

gagatcttct tcagccggca cagcccgctc ctctacggct acaagaacgg caacctcaag 2460

tggctcgagc gcttctccta catcaacacc accatctacc ccttcacttc tctccccctc 2520

ctcgcctact gcaccctacc cgccgtctgc ctcctcaccg gcaagttcat catgcctccg 2580

attagcacgt ttgcgagttt gttcttcatc gcgctcttca tctccatctt cgcgacgggc 2640

atcctggaga tgaggtggag cggggtgagc atcgaggagt ggtggaggaa cgagcagttc 2700

tgggtcatcg gcggcgtgtc ggcgcacctg ttcgcggtgg tgcagggcct gctcaaggtg 2760

ctggccggga tcgacaccaa cttcaccgtc acgtccaagg ccaccggaga cgaggacgac 2820

gagttcgcgg agctctacgc cttcaagtgg accaccctcc tcatcccgcc caccacgctg 2880

ctcatcctca acatcatcgg cgtcgtcgcc ggcgtctccg acgccatcaa caacggctcc 2940

gaggcgtggg gcccgctctt cgggaagctc ttcttcgcct tctgggtcat cgtccacctc 3000

taccccttcc tcaaggggct catggggagg cagaaccgga cgcccaccat tgttgtcatc 3060

tggtccgtgc tgctcgcctc catcttttcc ttgctctggg tcaggattga tcccttcacc 3120

atcaaggcca ggggccctga cgtcaggcag tgcggcatca actgctga 3168

<210> 44

<211> 1055

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 44

Met Glu Ala Ser Ala Gly Leu Val Ala Gly Ser His Asn Arg Asn Glu

1 5 10 15

Leu Val Leu Ile Arg Gly His Glu Glu Pro Lys Pro Leu Arg Ala Leu

20 25 30

Ser Gly Gln Val Cys Glu Ile Cys Gly Asp Glu Val Gly Arg Thr Val

35 40 45

Asp Gly Asp Leu Phe Val Ala Cys Asn Glu Cys Gly Phe Pro Val Cys

50 55 60

Arg Pro Cys Tyr Glu Tyr Asn Arg Arg Glu Gly Thr Gln Asn Cys Pro

65 70 75 80

Gln Cys Lys Thr Arg Tyr Lys Arg Leu Lys Gly Ser Pro Arg Val Pro

85 90 95

Gly Asp Glu Asp Glu Glu Asp Ile Asp Asp Leu Glu His Glu Phe Asn

100 105 110

Ile Asp Asp Glu Lys Gln Lys Gln Leu Gln Gln Asp Gln Asp Gly Met

115 120 125

Gln Asn Ser His Ile Thr Glu Ala Met Leu His Gly Lys Met Ser Tyr

130 135 140

Gly Arg Gly Pro Asp Asp Gly Asp Gly Asn Ser Thr Pro Leu Pro Pro

145 150 155 160

Ile Ile Thr Gly Ala Arg Ser Val Pro Val Ser Gly Glu Phe Pro Ile

165 170 175

Ser Asn Ser His Gly His Gly Glu Phe Ser Ser Ser Leu His Lys Arg

180 185 190

Ile His Pro Tyr Pro Val Ser Glu Pro Gly Ser Ala Lys Trp Asp Glu

195 200 205

Lys Lys Glu Val Ser Trp Lys Glu Arg Met Asp Asp Trp Lys Ser Lys

210 215 220

Gln Gly Ile Val Ala Gly Gly Ala Pro Asp Pro Asp Asp Tyr Asp Ala

225 230 235 240

Asp Val Pro Leu Asn Asp Glu Ala Arg Gln Pro Leu Ser Arg Lys Val

245 250 255

Ser Ile Ala Ser Ser Lys Val Asn Pro Tyr Arg Met Val Ile Ile Leu

260 265 270

Arg Leu Val Val Leu Gly Phe Phe Leu Arg Tyr Arg Ile Leu His Pro

275 280 285

Val Pro Asp Ala Ile Pro Leu Trp Leu Thr Ser Ile Ile Cys Glu Ile

290 295 300

Trp Phe Ala Val Ser Trp Ile Leu Asp Gln Phe Pro Lys Trp Tyr Pro

305 310 315 320

Ile Asp Arg Glu Thr Tyr Leu Asp Arg Leu Ser Leu Arg Tyr Glu Arg

325 330 335

Glu Gly Glu Pro Ser Leu Leu Ser Ala Val Asp Leu Phe Val Ser Thr

340 345 350

Val Asp Pro Leu Lys Glu Pro Pro Leu Val Thr Ala Asn Thr Val Leu

355 360 365

Ser Ile Leu Ala Val Asp Tyr Pro Val Asp Lys Val Ser Cys Tyr Val

370 375 380

Ser Asp Asp Gly Ala Ser Met Leu Thr Phe Glu Ser Leu Ser Glu Thr

385 390 395 400

Ala Glu Phe Ala Arg Lys Trp Val Pro Phe Cys Lys Lys Phe Ser Ile

405 410 415

Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ser Gln Lys Val Asp Tyr Leu

420 425 430

Lys Asp Lys Val His Pro Asn Phe Val Gln Glu Arg Arg Ala Met Lys

435 440 445

Arg Glu Tyr Glu Glu Phe Lys Val Arg Ile Asn Ala Leu Val Ala Lys

450 455 460

Ala Gln Lys Val Pro Ala Glu Gly Trp Ile Met Lys Asp Gly Thr Pro

465 470 475 480

Trp Pro Gly Asn Asn Thr Arg Asp His Pro Gly Met Ile Gln Val Phe

485 490 495

Leu Gly His Ser Gly Gly His Asp Thr Glu Gly Asn Glu Leu Pro Arg

500 505 510

Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Phe Gln His His Lys

515 520 525

Lys Ala Gly Ala Met Asn Ala Leu Ile Arg Val Ser Ala Val Leu Thr

530 535 540

Asn Ala Pro Phe Met Leu Asn Leu Asp Cys Asp His Tyr Ile Asn Asn

545 550 555 560

Ser Lys Ala Ile Arg Glu Ala Met Cys Phe Leu Met Asp Pro Gln Val

565 570 575

Gly Arg Lys Val Cys Tyr Val Gln Phe Pro Gln Arg Phe Asp Gly Ile

580 585 590

Asp Val His Asp Arg Tyr Ala Asn Arg Asn Thr Val Phe Phe Asp Ile

595 600 605

Asn Met Lys Gly Leu Asp Gly Ile Gln Gly Pro Val Tyr Val Gly Thr

610 615 620

Gly Cys Val Phe Arg Arg Gln Ala Leu Tyr Gly Tyr Asn Pro Pro Lys

625 630 635 640

Gly Pro Lys Arg Pro Lys Met Val Thr Cys Asp Cys Cys Pro Cys Phe

645 650 655

Gly Arg Lys Lys Arg Lys His Gly Lys Asp Gly Leu Pro Glu Ala Val

660 665 670

Ala Ala Asp Gly Gly Met Asp Ser Asp Lys Glu Met Leu Met Ser Gln

675 680 685

Met Asn Phe Glu Lys Arg Phe Gly Gln Ser Ala Ala Phe Val Thr Ser

690 695 700

Thr Leu Met Glu Glu Gly Gly Val Pro Pro Ser Ser Ser Pro Ala Ala

705 710 715 720

Leu Leu Lys Glu Ala Ile His Val Ile Ser Cys Gly Tyr Glu Asp Lys

725 730 735

Thr Asp Trp Gly Leu Glu Leu Gly Trp Ile Tyr Gly Ser Ile Thr Glu

740 745 750

Asp Ile Leu Thr Gly Phe Lys Met His Cys Arg Gly Trp Arg Ser Val

755 760 765

Tyr Cys Met Pro Lys Arg Ala Ala Phe Lys Gly Ser Ala Pro Ile Asn

770 775 780

Leu Ser Asp Arg Leu Asn Gln Val Leu Arg Trp Ala Leu Gly Ser Val

785 790 795 800

Glu Ile Phe Phe Ser Arg His Ser Pro Leu Leu Tyr Gly Tyr Lys Asn

805 810 815

Gly Asn Leu Lys Trp Leu Glu Arg Phe Ser Tyr Ile Asn Thr Thr Ile

820 825 830

Tyr Pro Phe Thr Ser Leu Pro Leu Leu Ala Tyr Cys Thr Leu Pro Ala

835 840 845

Val Cys Leu Leu Thr Gly Lys Phe Ile Met Pro Pro Ile Ser Thr Phe

850 855 860

Ala Ser Leu Phe Phe Ile Ala Leu Phe Ile Ser Ile Phe Ala Thr Gly

865 870 875 880

Ile Leu Glu Met Arg Trp Ser Gly Val Ser Ile Glu Glu Trp Trp Arg

885 890 895

Asn Glu Gln Phe Trp Val Ile Gly Gly Val Ser Ala His Leu Phe Ala

900 905 910

Val Val Gln Gly Leu Leu Lys Val Leu Ala Gly Ile Asp Thr Asn Phe

915 920 925

Thr Val Thr Ser Lys Ala Thr Gly Asp Glu Asp Asp Glu Phe Ala Glu

930 935 940

Leu Tyr Ala Phe Lys Trp Thr Thr Leu Leu Ile Pro Pro Thr Thr Leu

945 950 955 960

Leu Ile Leu Asn Ile Ile Gly Val Val Ala Gly Val Ser Asp Ala Ile

965 970 975

Asn Asn Gly Ser Glu Ala Trp Gly Pro Leu Phe Gly Lys Leu Phe Phe

980 985 990

Ala Phe Trp Val Ile Val His Leu Tyr Pro Phe Leu Lys Gly Leu Met

995 1000 1005

Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Val Ile Trp Ser Val Leu

1010 1015 1020

Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Arg Ile Asp Pro Phe Thr

1025 1030 1035 1040

Ile Lys Ala Arg Gly Pro Asp Val Arg Gln Cys Gly Ile Asn Cys

1045 1050 1055

Claims

1.含有cesa突变体的转基因水稻茎秆在生产纤维素纳米晶上的应用，其特征在于，所述cesa突变体包括CESA4、CESA7和CESA9突变体，所述cesa突变体包括点突变体和移码突变体；

所述cesa突变体使所制备的纤维素纳米晶聚合度和尺寸均减小，所述尺寸包括长度和直径的尺寸；

所述cesa突变体的氨基酸序列如SEQ ID NO：32、SEQ ID NO：34、SEQ ID NO：36、SEQ IDNO：38、SEQ ID NO：40、SEQ ID NO：42或SEQ ID NO：44所示。

2.根据权利要求1所述的应用，其特征在于，所述转基因水稻茎秆采用超声辅助硫酸水解法制备所述纤维素纳米晶。

3.一种转基因水稻的构建方法，其特征在于，包括使水稻包含cesa突变体或其编码基因的步骤；所述cesa突变体包括CESA4、CESA7和CESA9突变体，所述cesa突变体包括点突变体和移码突变体；

所述cesa突变体或其编码基因使所述转基因水稻纤维素纳米长度减小；