CN111320679A - 玉米开花期相关的ZmPHYCs突变型蛋白、其编码基因、重组载体和应用 - Google Patents

玉米开花期相关的ZmPHYCs突变型蛋白、其编码基因、重组载体和应用 Download PDF

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CN111320679A
CN111320679A CN202010092489.5A CN202010092489A CN111320679A CN 111320679 A CN111320679 A CN 111320679A CN 202010092489 A CN202010092489 A CN 202010092489A CN 111320679 A CN111320679 A CN 111320679A
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王海洋
李全权
吴广霞
赵永平
王宝宝
谢钰容
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Biotechnology Research Institute of CAAS
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Abstract

本发明公开了玉米开花期相关的ZmPHYCs突变型蛋白、其编码基因、重组载体和应用。本发明利用CRISPR/Cas9基因编辑技术，将玉米ZmPHYC1和ZmPHYC2基因一起突变后，创制新的早花玉米等位基因并剔除CRISPR载体外源序列获得表型稳定遗传的株系，其功能缺失双突变体在长日照条件下表现出早花表型，由此确定玉米ZmPHYC1和ZmPHYC2基因同时突变后产生的ZmPHYCs突变型蛋白在长日照条件下具有促进玉米开花的作用。本发明不仅对调控玉米开花期具有重要意义，还可将其应用于玉米自交系改良和杂交育种，相比常规育种，具有精准、效率高等优势。

Description

玉米开花期相关的ZmPHYCs突变型蛋白、其编码基因、重组载 体和应用
技术领域
本发明涉及ZmPHYCs突变型蛋白及其编码基因，本发明进一步涉及它们在植物开花期调控中的应用，属于玉米ZmPHYCs突变型蛋白及其应用领域。
背景技术
玉米是全球产量最高的作物，约9000年前由其祖先墨西哥西南部的大刍草驯化而来(Matsuoka et al.,2002)。大刍草是一个热带物种，表现出严格的光周期敏感性，需要在短日照条件下才可以开花。开花期是玉米适应当地种植环境的一个复杂性状(Buckler etal.2009)，它是决定玉米生态分布的一个关键因素。现代玉米生长在广泛的纬度范围内，通常被认为是日中性植物。热带玉米种植在长日照条件下表现为开花期延迟(Hung andHolland 2012)。而温带玉米则是通过降低对光周期的敏感性优化开花时间来适应长日照环境(Yang et al.2013)。
为了调节开花时间，植物将白天的长度视为触发花期转变的关键环境信号。有几种类型的光感受器参与白昼感知，其中，最重要的是光敏色素。光敏色素(phy)是一种感受红光和远红光的色素蛋白家族，在植物的整个生命周期中调控诸多发育过程。玉米中共含有6个光敏色素成员，即ZmPHYA1，ZmPHYA2，ZmPHYB1，ZmPHYB2，ZmPHYC1和ZmPHYC2。研究发现光敏色素C(PHYC)能够感知光周期的长短，进而调节植物开花时间。
ZmPHYC1和ZmPHYC2基因突变后对于玉米开花具有何种功能，尚未见任何报道。通过基因编辑技术研究ZmPHYC1和ZmPHYC2基因突变后对于玉米开花的具体功能，不仅对调控玉米开花期具有重要意义，还可将其应用于玉米自交系改良和杂交育种。
发明内容
本发明的目的之一是提供玉米ZmPHYCs突变型蛋白及其编码基因；
本发明的目的之二是提供ZmPHYCs基因编辑载体；
本发明的目的之三是将ZmPHYCs突变型蛋白及其编码基因以及所构建的ZmPHYCs基因编辑载体应用于玉米分子改良育种。
本发明的上述目的是通过以下技术方案来实现的：
本发明利用CRISPR/Cas9基因编辑技术，将玉米ZmPHYC1和ZmPHYC2基因一起突变后，其功能缺失双突变体在长日照条件下表现出早花表型，由此确定玉米ZmPHYC1和ZmPHYC2基因同时突变后产生的ZmPHYCs突变型蛋白在长日照条件下具有促进玉米开花的作用。
本发明首先提供了玉米ZmPHYCs突变型蛋白，该突变型蛋白由ZmPHYC1突变型蛋白和ZmPHYC2突变型蛋白组成；其中，所述ZmPHYC1突变型蛋白的氨基酸序列为SEQ ID No.27-SEQ ID No.29示，其编码基因的核苷酸序列为SEQ ID No.30-SEQ ID No.32所示；所述ZmPHYC2突变型蛋白的氨基酸序列为SEQ ID No.33-SEQ ID No.35所示，其编码基因的核苷酸序列为SEQ ID No.36-SEQ ID No.38所示。
本发明还提供了含有所述编码基因的重组植物表达载体以及含有该重组植物表达载体的宿主细胞；将所述编码基因可操作的与表达调控元件相连接得到可以在植物中表达该编码基因的重组植物表达载体。
所述重组植物表达载体还可含有用于选择转化细胞的选择性标记基因；所述选择性标记基因用于选择经转化的细胞或组织；所述标记基因包括：编码抗生素抗性的基因以及赋予除草剂抗性的基因等。此外，所述的标记基因还包括表型标记，例如β-半乳糖苷酶和荧光蛋白等。
本发明中所述的转化方案以及将所述多核苷酸或多肽引入植物的方案可视用于转化的植物(单子叶植物或双子叶植物)或植物细胞的类型而变化。将所述多核苷酸或多肽引入植物细胞的合适方法包括：显微注射、电穿孔、农杆菌介导的转化、直接基因转移以及高速弹道轰击等。利用常规方法可使已转化的细胞再生稳定转化植株(McCormick etal.Plant Cell Reports.1986.5:81-84)。
本发明可用于转化任何植物种类，包括但不限于：单子叶植物或双子叶植物，优选是玉米。
本发明进一步提供了一种ZmPHYCs基因编辑载体，其构建方法包括：
(Ⅰ)获取玉米U6-1和U6-2启动子片段；
(Ⅱ)制备sgRNA表达盒：
(Ⅲ)将步骤(Ⅱ)获得的2个sgRNA表达盒依次连接CPB-Ubi-hspcas9载体，即得。
其中，步骤(Ⅱ)中所述的制备sgRNA表达盒包括以下步骤：
(A)采用重叠PCR的方法将带有接头的靶点序列和sgRNA骨架序列融合在一起获得2个PCR产物；其中，PCR的上游引物分别为SEQ ID No.11-SEQ ID No.12所示；下游引物为SEQ ID No.17所示，模板序列为SEQ ID No.18所示；
该重叠PCR反应程序如下：(1)94℃2min；(2)98℃，10s；46℃，30s；68℃10s；共35个循环；(3)68℃5min。
(B)再次用重叠PCR的方法将步骤(A)获得的2个融合PCR片段分别与U6-1启动子片段和U6-2启动子片段连接获得2个PCR产物，即为sgRNA连接产物；其中，该重叠PCR中所用到的上、下游引物序列为SEQ ID No.19/SEQ ID No.17和SEQ ID No.19/SEQ ID No.20所示；所用到的模板是U6-1或U6-2启动子片段以及步骤(A)扩增得到的2个PCR产物；该重叠PCR反应程序如下：(1)94℃2min；(2)98℃，10s；52-55℃，30s；68℃40s；共35个循环；(3)68℃5min。
本发明通过CRISPR/Cas9基因编辑技术对ZmPHYCs基因进行敲除突变，最终获得突变后的ZmphyCs蛋白，属于功能缺失型突变；因此，只要是通过CRISPR/Cas9技术导致的ZmPHYCs基因的突变(突变后的序列可能多样化)都会导致该表型的出现：本发明选取背景材料为ZC01，通过所构建的CRISPR/Cas9基因编辑载体同时对玉米中ZmPHYC1和ZmPHYC2两个目标基因进行打靶进行定点编辑，经过筛选从中获得了相对于野生型材料具有开花提前的且已经剔除外源标记基因的ZmphyC1ZmphyC2#1、ZmphyC1ZmphyC2#3和ZmphyC1ZmphyC2#7突变体材料；所筛选得到的ZmphyC1ZmphyC2#1、ZmphyC1ZmphyC2#3和ZmphyC1ZmphyC2#7突变体材料因基因ZmPHYCs功能异常导致雄穗的开花期比野生型材料提前4～6天，这表明ZmPHYCs蛋白的缺失对玉米的雄穗开花期调控起着至关重要的作用。
因此，本发明进一步将获得的ZmPHYCs突变蛋白或所构建的ZmPHYCs基因编辑载体应用于玉米雄穗开花期改良的育种中；具体的，可以将ZmPHYCs突变蛋白或ZmPHYCs基因编辑载体应用于培育植物新品种；其中，所述的植物新品种是早花的玉米新品种。
本发明提供了一种使植物雄穗开花期提前的方法，包括：
(1)将所筛选到的编码ZmPHYCs突变蛋白的编码基因可操作的与调控元件连接后构建得到重组植物表达载体；(2)将所构建的重组植物表达载体转化到植物中筛选得到开花提前的植物新品种；或者，将构建的ZmPHYCs基因编辑载体转化到植物中筛选得到开花提前的植物新品种。
所述重组植物表达载体还可含有用于选择转化细胞的选择性标记基因。选择性标记基因用于选择经转化的细胞或组织。标记基因包括：编码抗生素抗性的基因以及赋予除草剂抗性的基因等。此外，所述的标记基因还包括表型标记，例如β-半乳糖苷酶和荧光蛋白等。
本发明中所述的转化方案以及将所述多核苷酸或多肽引入植物的方案可视用于转化的植物(譬如单子叶植物)，利用常规方法可使已转化的细胞再生稳定转化植株(McCormick et al.Plant Cell Reports.1986.5:81-84)。
本发明可用于转化任何植物种类，包括但不限于：单子叶植物或双子叶植物，优选是玉米。
本发明首次利用基因编辑技术对玉米ZmPHYCs基因进行定点突变创制新的早花玉米新等位基因并剔除CRISPR载体外源序列，获得表型稳定遗传的株系；本发明不仅对调控玉米开花期具有重要意义，还可将其应用于玉米自交系改良和杂交育种，具有精准、效率高等常规育种所不具备的优势，具有广泛的应用前景。
附图说明
图1ZmPHYC1和ZmPHYC2基因的CRISPR/Cas9载体的构建及突变序列比对结果；A.ZmPHYC1和ZmPHYC2的基因结构及靶点位置；B.ZmPHYC1和ZmPHYC2基因的CRISPR/Cas9载体构建模式图；C.玉米ZmphyC1ZmphyC2双突变体中ZmPHYC1和ZmPHYC2基因的测序比对结果。
图2为ZmphyC1ZmphyC2双敲除突变体早花(雄穗散粉期提前)表型；A.ZmphyC1ZmphyC2双突变体在长日照条件下比野生型(WT)散粉提前的植株表型。B.雄穗开花(散粉)表型的局部特写图；C.长日照条件下散粉期统计数据图；D.短日照条件下散粉期统计数据图。
图3玉米开花相关基因ZCN8和CONZ1在ZmphyC1ZmphyC2双敲除突变体和野生型玉米中的表达量检测；灰色阴影部分表示黑暗周期。
具体实施方式
以下结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中的野生型ZC01为中国种子集团(武汉)的玉米自交系转化材料。
实施例1 ZmphyC1ZmphyC2突变体的构建
1、玉米ZmPHYC1和ZmPHYC2基因的靶位点设计
首先从玉米Gramene数据库中获取ZmPHYC1(GRMZM2G057935-T01)(其核苷酸序列为SEQ ID No.1所示，氨基酸序列为SEQ ID No.2所示)和ZmPHYC2(GRMZM2G129889-T01)(其核苷酸序列为SEQ ID No.3所示，其氨基酸序列为SEQ ID No.4所示)的基因组序列，然后用Snap Gene Viewer 3.2软件设计2个靶点，这2个靶点在ZmPHYC1和ZmPHYC2中是高度保守的，即ZmPHYC1和ZmPHYC2可共用这2个靶点。靶点序列分别是：
靶点1(Guide 1)：GTTCTGGAGGTAGGTGGAGACGG(SEQ ID No.5)
靶点2(Guide 2)：GCACATCGCACAGCAACGACAGG(SEQ ID No.6)
其中下划线序列为PAM序列。
然后利用CTAB的方法提取玉米野生型自交系C01的基因组DNA。
ZmPHYC1-F1:5'GCCGCTGCCCCGCGCGGGCC3'(SEQ ID No.7)，
ZmPHYC1-R1:5'ACCTTAGTGGGAAAGGGACG3'(SEQ ID No.8)；
ZmPHYC2-F1:5'GCCCAAGAGTATCGTCGCTG3'(SEQ ID No.9)，
ZmPHYC2-R1:5'CTTAGCGGGAAAGGGACAAA3'(SEQ ID No.10)；
分别用上述引物(SEQ ID No.7-SEQ ID No.10)对所提的基因组DNA进行常规PCR扩增，将扩增产物送测序公司测序。测序结果与玉米Gramene数据库中的ZmPHYC1和ZmPHYC2的参考序列进行Blast比对分析，发现转化所用受体玉米材料ZC01的ZmPHYC1和ZmPHYC2的所设计的靶点序列与参考基因组B73相同,以确认转基因受体材料ZC01的靶点序列与按照B73基因组序列设计的靶点序列完全一致。
最后合成带有接头的靶点序列引物，用于后续的载体构建。引物序列如下：
ZmPHYC-1F:
5'GAGCCGCAAGCACCGAATTGTTCTGGAGGTAGGTGGAGAGTTTTAGAGC TAGAAATAGCAAGTT3'(SEQ ID No.11)
ZmPHYC-2F:
5'GAGCCGCAAGCACCGAATTGCACATCGCACAGCAACGACGTTTTAGAGC TAGAAATAGCAAGTT3'(SEQ ID No.12)
其中，黑色加粗字体部分为连接U6-1启动子(ZmPHYC-1F引物加粗部分)或U6-2启动子(ZmPHYC-2F引物加粗部分)片段的接头引物序列，下划线序列为设计的靶点序列，黑色加粗斜体部分为连接sgR骨架片段的接头引物序列。
2、CRISPR/Cas9基因编辑载体构建
基因编辑载体构建参考Li C等(Li C，et al.,Plant Biotechnol J,2017,15:1566-1576)的报道方法，按以下步骤进行：
(1)玉米U6-1启动子片段的制备
引物序列为MU61-1F：5'TGCTTTTTTTAAGCTGCTGTTTTTGTTAGCCCCATCG3'(SEQ IDNo.13)
MU61-1R：5'AATTCGGTGCTTGCGGCTC3'(SEQ ID No.14)；
模板为B73基因组DNA，
PCR扩增体系为表1
表1 PCR扩增体系
成分 体积
2×PCR Buffer for KOD Fx 25μL
2mM dNTPs 10μL
MU61-1F 1.5μL
MU61-1R 1.5μL
KOD Fx 1μL
模板：B73gDNA 1μL
Add ddH<sub>2</sub>O Up to 50μL
PCR反应程序如下：
Figure BDA0002384173070000041
获得的PCR产物即为玉米U6-1启动子片段。
(2)玉米U6-2启动子片段的制备
引物序列为MU62-1F:5'GGATCCCTAATTGGCCCTTACA 3'(SEQ ID No.15)
MU62-1R:5'GGAGCGGTGGTCGCAGCTGAA 3'(SEQ ID No.16)
模板为B73基因组DNA，
PCR扩增体系为表2。
表2 PCR扩增体系
成分 体积
2×PCR Buffer for KOD Fx 25μL
2mM dNTPs 10μL
MU62-1F 1.5μL
MU62-1R 1.5μL
KOD Fx 1μL
模板：B73gDNA 1μL
Add ddH<sub>2</sub>O Up to 50μL
PCR反应程序如下：
Figure BDA0002384173070000051
获得的PCR产物即为玉米U6-2启动子片段。
(3)sgRNA表达盒的制备
首先用重叠PCR的方法将带有接头的靶点序列和sgR骨架序列融合在一起获得的2段PCR产物，分别命名为ZmPHYC-1片段，ZmPHYC-2片段。PCR扩增体系为表3。
表3 PCR扩增体系
成分 体积
2×PCR Buffer for KOD Fx 25μL
2mM dNTPs 10μL
上游引物 1.5μL
下游引物 1.5μL
KOD Fx 1μL
模板 1μL
Add ddH<sub>2</sub>O Up to 50μL
其中上游引物分别是ZmPHYC-1F，ZmPHYC-2F，下游引物为MUsgR-1R：TGTAAGGGCCAATTAGGGATCCAAAAAAAGCACCGACTCG(SEQ ID No.17)；
模板序列为sgR骨架序列，人工合成sgR骨架片段序列如下：
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT TGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ ID No.18)
PCR反应程序如下：
Figure BDA0002384173070000052
然后再次用重叠PCR的方法将上一步获得的融合PCR片段分别和U6-1启动子，U6-2启动子片段连接上，获得的PCR产物即为sgRNA连接产物，分别命名为U61-ZmPHYC-1片段，U62-ZmPHYC-2片段。
引物序列为：
MU61-2F:TGCACTGCACAAGCTGCTGTTTTTGTTAGCCCCATCG(SEQ ID No.19)MUsgR-2R:5'GGCCAGTGCCAAGCTTAAAAAAAGCACCGACTCG 3'(SEQ ID No.20)
PCR扩增体系见表4。
表4 PCR扩增体系
成分 体积
2×PCR Buffer for KOD Fx 25μL
2mM dNTPs 10μL
上游引物 1.5μL
下游引物 1.5μL
KOD Fx 1μL
片段1 1μL
片段2 1μL
Add ddH<sub>2</sub>O Up to 50μL
其中在连接获得U61-ZmPHYC-1片段时，上游引物用MU61-2F，下游引物用MusgR-1R，片段1为U6-1启动子片段，片段2为ZmPHYC-1片段；在连接获得U62-ZmPHYC-2片段时，上游引物用MU62-1F，下游引物用MusgR-2R，片段1为U6-2启动子片段，片段2为ZmPHYC-2片段。
PCR反应程序如下：
Figure BDA0002384173070000061
(4)sgRNA表达盒连接CPB-Ubi-hspcas9载体
按以下反应体系和过程，将每个sgRNA表达盒依次连接CPB-Ubi-hspcas9载体，获得连接产物。由于ZmPHYC1和ZmPHYC2基因可以共用2个靶点，所以共需要做两次连接反应才能把2个靶点连接到CPB-Ubi-hspcas9载体中。反应体系及过程见表5。
表5 反应体系及过程
成分 体积
sgRNA连接产物 1μL
CPB-pubi-hspcas9载体片段 1μL
重组酶 0.5μL
重组酶为Clontech公司的In-Fusion酶，反应条件为50℃,30min。
其中CPB-pubi-hspcas9载体片段是通过CPB-pubi-hspcas9质粒经HindIII单酶切后获得。sgRNA连接产物为U61-ZmPHYC-1片段，U62-ZmPHYC-2片段,但需要注意的是，每次只能连接1个片段，每次连接完一个片段后需经测序核实后再用HindIII单酶切连接后的质粒载体，用于后续的连接构建。
(4)转化大肠杆菌DH5α及验证
将连接产物用热激法42℃转化大肠杆菌DH5α，菌液涂布于含有50mg/L卡那霉素的平板上,37℃培养约12h。挑取平板上长出的单菌落，摇菌扩繁。以菌液为模板进行PCR验证。PCR扩增体系见表6。
表6 PCR扩增体系
成分 体积
10×Taq Buffer 2.5μL
dNTPs(2.5mM each) 2μL
上游引物 0.5μL
下游引物 0.5μL
rTaq(2.5U/μL) 0.5μL
菌液 1μL
Add ddH<sub>2</sub>O Up to 25μL
其中根据载体设计的引物如下：
上游引物为Ubi-4F:5’CTTAGACATGCAATGCTCATTATCTC3’(SEQ ID No.21)，
下游引物为PstI-R:5’CTGGCGAAAGGGGGATGT3’(SEQ ID No.22)，
用于检测阳性克隆，PCR反应程序如下：
Figure BDA0002384173070000071
提取检测含目的条带的菌液的质粒送公司测序。测序结果正确的质粒经HindIII单酶切后获得第一次的连接产物载体片段，然后再重复步骤(3)和(4)，再连接第二次，将U62-ZmPHYC-2片段连接到CPB-pubi-hspcas9载体中。
3、纯合的去除外源标记基因的ZmphyC1ZmphyC2突变体的获得
将上述阳性质粒转入农杆菌EHA105。采用常规农杆菌介导法转化玉米自交系ZC01获得T0代转基因植株。将T0代植株以单株收种，收获的种子播于大田中，在玉米长至两叶一心时，用Basta试剂(1/1000,V/V)涂抹叶尖，筛选去除载体的(Basta涂抹显示阴性)T1代植株。然后取样提取T1代植株的基因组DNA为模板，鉴定编辑位点的删除片段大小。PCR扩增体系如下见表7。
表7 PCR扩增体系
成分 体积
2×Msater Mix 7.5μL
上游引物 0.3μL
下游引物 0.3μL
模板DNA 1μL
Add ddH<sub>2</sub>O Up to 15μL
其中每个ZmPHYC基因检测引物分别用
ZmPHYC1-F2:5’GTGTATTCCCTCTTCTCCCCCC 3’(SEQ ID No.23)
ZmPHYC1-R2:5’GGCTTGTATGATGGCAGACGAC 3’(SEQ ID No.24)
ZmPHYC2-F2:5’CTCGCTGAAATTCCCTCTTCTT 3’(SEQ ID No.25)
ZmPHYC2-R2:5’GTTGGTCACTGTCGGTATCCC 3’(SEQ ID No.26)
模板DNA分别为野生型植株ZC01和突变体植株的基因组DNA。PCR反应程序如下：
Figure BDA0002384173070000072
Figure BDA0002384173070000081
由于每个ZmPHYC基因设计了两个靶点，故理论上如果Cas9同时在两个靶点准确切割，则每个ZmphyC1ZmphyC2纯合突变体经PCR检测后应该获得单一的小带片段。野生型植株则PCR扩增后应该获得没有切割的单一的大带片段(表8)。
表8 PCR扩增结果
大带bp 小带bp 检测基因
1353bp 914bp ZmPHYC1
1312bp 855bp ZmPHYC2
经初步PCR筛选鉴定后，选取单一的突变条带连接到克隆载体中进行测序鉴定，测序比对结果如图1C所示。在ZmphyC1ZmphyC2突变体材料中，ZmPHYC1和ZmPHYC2基因经Cas9在所设计的两靶位点之间切割后导致删除突变，而且删除序列导致移码突变，提前出现终止密码子。在ZmphyC1ZmphyC2-KO#1材料中，ZmPHYC1突变后的CDS长为198bp，共编码66个氨基酸；ZmPHYC2突变后的CDS长为255bp，共编码85个氨基酸。在ZmphyC1ZmphyC2-KO#3材料中，ZmPHYC1突变后的CDS长为2982bp，共编码994个氨基酸；ZmPHYC2突变后的CDS长为240bp，共编码80个氨基酸。在ZmphyC1ZmphyC2-KO#7材料中，ZmPHYC1突变后的CDS长为258bp，共编码86个氨基酸；ZmPHYC2突变后的CDS长为246bp，共编码82个氨基酸。
从图中可以看出：玉米ZmphyC1ZmphyC2双突变体中ZmPHYC1和ZmPHYC2基因在编辑后都产生碱基的删除或个别碱基的插入，导致编码区移码突变，最终产生突变蛋白。
实施例2 ZmphyC1ZmphyC2突变体的表型分析
1、将野生型与取实施例1中鉴定到的阴性的纯合ZmphyC1ZmphyC2突变体(#1，#3，#7)分别种植于海南省三亚市乐东黎族自治县尖峰镇翁毛村万钟公司产学研示范园和河北省廊坊市广阳区万庄镇伊指挥营村国际高新技术产业园基地，野生型与突变体种植在同一小区，每行15株苗，三次重复。对大田中野生型和突变体植株的开花期调查发现,在长日照条件下，ZmphyC1ZmphyC2突变体的散粉期显著早于野生型(图2A,B,C)，而在短日照条件下，突变体和野生型植株的散粉期无显著差异，这一结果表明，玉米ZmPHYCs突变蛋白能够促进玉米开花(图2D)。
(2)将野生型与实施例1中鉴定到的阴性纯合ZmphyC1ZmphyC2突变体(KO#7)种植于人工培养箱中，并设定长日照和短日照两个光照条件，在V6期取叶片样品，提取RNA，然后反转录cDNA并检测样品中的开花相关基因的表达情况。从图3可看出，在V6期，开花的基因ZCN8(FT基因的同源基因)在ZmphyC1ZmphyC2突变体中表达量在LD条件下明显受到抑制，低于在野生型中的表达量；而在SD条件下ZCN8基因的表达在突变体和野生型中的表达无显著差异，故突变体材料在LD条件下早花，而在SD条件下无开花提前的表型。同样地，正调控FT基因的直接上游的转录因子CO基因的表达(在玉米中的同源基因为CONZ1基因)与ZCN8基因的表达模式能很好的吻合，即在LD条件下CONZ1基因在突变体中的表达高于在野生型中的表达。
SEQUENCE LISTING
<110> 华南农业大学 中国农业科学院生物技术研究所
<120> 玉米开花期相关的ZmPHYCs突变型蛋白、其编码基因、重组载体和应用
<130> BJ-2002-191208A
<160> 38
<170> PatentIn version 3.5
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atgtcgttgc cgtcgaacaa ccggaggacg tgctcccgga gcagctctgc gcggtccaag 60
cacagcgcgc gggtggtggc acagacgccc gtggacgcgc agctgcacgc cgagttcgag 120
ggctcccagc gccacttcga ctactcctcg tcggtgggcg ccgccaaccg cccgtcggca 180
agcaccagca ccgtctccac ctacctccag aacatgcagc ggggccgcta catccagccc 240
ttcggctgcc tgctcgccgt ccacccggac accttcgcgc tgctcgccta cagcgagaac 300
gcgcccgaga tgctcgacct cacgccacac gccgtcccca ccatcgacca gcgggatgcg 360
ctcggcatcg gcgtcgatgt gcgcacgctc ttccgctcgc agagctccgt cgcgcttcac 420
aaggccgccg ccttcgggga ggtcaaccta ctcaacccca tcctcgtgca cgccaggacg 480
tcggggaagc ccttctacgc catattgcac cggatcgacg tcggccttgt catcgacctt 540
gagccggtca atccagccga cgtgccagtc accgccgcgg gcgcgctcaa gtcgtacaag 600
ctcgctgcca aggccatctc caggctgcag tcgctgccca gcgggaacct gtcgttgctg 660
tgcgatgtgc ttgtccgtga ggtgagcgaa ctcacgggct atgaccgggt catggcctac 720
aagttctatg aggatgagca tggcgaggtc atttccgaat gcaggaggtc cgatctggag 780
ccgtatcttg gactgcacta cccagccacc gacatcccgc aggcgtccag gttcctgttt 840
atgaagaaca aagtgcggat gatatgtgat tgctgtgcca ctccagtgaa ggtcattcag 900
gatgatagcc tagcacaacc tctcagcctc tgtggttcca cactcagggc ttcccatggt 960
tgccatgcac agtacatggc aaacatgggt tctgttgcat cacttgcgat gtcagtcact 1020
ataaacgagg atgaggagga agatggggat accgggagtg accaacaacc gaaaggcagg 1080
aagctgtggg ggcttgtcgt ctgccatcat acaagcccga ggttcgtccc tttcccacta 1140
aggtatgctt gtgagtttct cttgcaagta tttggcatac agctcaacaa ggaggtggaa 1200
ttggctgctc aggcaaagga gaggcacatc ctcagaacgc aaacccttct ttgtgatatg 1260
ctcctgcggg atgctcctgt tgggatattt actcggtcac ctaatgtgat ggatctagta 1320
aagtgcgatg gagctgcatt gtattaccag aaccagcttt tggtgcttgg atcaacaccc 1380
tctgagtcag agataaagag cattgcaaca tggctgcagg ataatcatga tggttcaact 1440
gggctgagta ctgacagctt agtggaagcg ggttatcctg gtgctgttgc acttcgtgaa 1500
gttgtgtgtg gcatggcggc cataaagatc tcttccaaag attttatctt ctggttccga 1560
tcgcacacaa caaaggagat caagtggggt ggggctaagc atgaaccggt tgatgcagat 1620
gacgatggca ggaggatgca tccacgatct tcattcaagg ccttcttgga ggtggttaaa 1680
tggagaagtg ttccctggga agatgttgaa atggatgcta tccattcttt gcagttaata 1740
ttacgtggct ccctgccaga tgaagatgcc aacagaaaca atgtaaggtc cattgtaaaa 1800
gctccatctg atgatatgaa gaagatacag gggctacttg aactgagaac agttacaaat 1860
gagatggtcc gcttaattga gacagcaact gcccctgtct tggctgtcga cattgccggt 1920
aacataaatg gatggaacaa taaagctgca gaactaacag gtttacctgt aatggaagcc 1980
atagggaggc ccctgataga tcttgttgtt actgattcta tagaagtggt taagcagatt 2040
ttggactcag ctttacaagg aattgaagag caaaatatgg aaatcaagct taaaacattc 2100
catgaacatg aatgcaatgg tccagtaatc ttgaaggtta actcctgctg tagtcgggac 2160
ctttcagaga aagtcattgg agtttgcttt gtagcacaag atttgaccag gcagaagatg 2220
attatggata agtatactag gatacaagga gactatgttg ccatagtaaa gaaccccact 2280
gagctcatcc ctcccatatt tatgatcaat gatcttggtt cctgcttaga gtggaataaa 2340
gctatgcaga agattaccgg tataaagagg gaagatgcga taaacaaatt gttaattggg 2400
gaggtcttca cgcttcatga ttatggctgt agggtgaaag atcatgcaac tctaacgaaa 2460
cttagcatac tgatgaatgc agtgatttct ggtcaggatc ctgagaagct cttttttggt 2520
ttcttcgaca cagatgggaa gtatattgaa tccttgctga cagtgaacaa gaggacagat 2580
gctgagggta agatcactgg tgctctttgc tttctgcatg tggccagtcc agagcttcag 2640
catgctctcc aggtgcagaa aatgtcggaa caagctgcga caaacagctt taaggaatta 2700
acttacattc gtcaagaatt aaggaaccca ctcaatggta tgcaatttac ttgtaactta 2760
ttgaagcctt ctgaattgac agaggaacag cggcaacttc tttcatctaa tgttctctgt 2820
caggaccagc tgaaaaagat tttacatgac actgatcttg aaagcattga acagtgctat 2880
atggagatga acacagtaga gttcaacctt gagcaagctc tgaatacggt tctgatgcaa 2940
ggcattcctt tgggcaagga aaaacagatt tcaattgaac gtaattggcc tgtggaagta 3000
tcatgcatgt acctttatgg ggacaattta aggcttcagc agatcctagc agactatcta 3060
gcatgcgccc ttcaattcac acaaactgct gaaggaccta tcgtgctcca ggtcatgtct 3120
aagaaggaaa acattggatc tggcatgcag attgctcatt tggagttcag gattgtccat 3180
ccagctccag gcgttccaga ggccctgata caggagatgt tccagcacaa cccaggggtg 3240
tccagggagg gcctcggcct gtacataagc cagaagctgg tgaaaacgat gagcggcacg 3300
gtacagtacc ttcgagaagc cgacacctcg tcgttcatca tcctgatgga gttcccggtc 3360
gcccagctca gcagcaagcg gtccaagcct tcgacgagta aattctga 3408
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Val Ser Thr Tyr Leu Gln Asn Met Gln Arg Gly Arg Tyr Ile Gln Pro
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Phe Gly Cys Leu Leu Ala Val His Pro Asp Thr Phe Ala Leu Leu Ala
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Thr Leu Phe Arg Ser Gln Ser Ser Val Ala Leu His Lys Ala Ala Ala
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Phe Gly Glu Val Asn Leu Leu Asn Pro Ile Leu Val His Ala Arg Thr
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Ser Gly Lys Pro Phe Tyr Ala Ile Leu His Arg Ile Asp Val Gly Leu
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Ser Asp Leu Glu Pro Tyr Leu Gly Leu His Tyr Pro Ala Thr Asp Ile
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Pro Gln Ala Ser Arg Phe Leu Phe Met Lys Asn Lys Val Arg Met Ile
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Cys Asp Cys Cys Ala Thr Pro Val Lys Val Ile Gln Asp Asp Ser Leu
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Ala Gln Pro Leu Ser Leu Cys Gly Ser Thr Leu Arg Ala Ser His Gly
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Cys His Ala Gln Tyr Met Ala Asn Met Gly Ser Val Ala Ser Leu Ala
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Met Ser Val Thr Ile Asn Glu Asp Glu Glu Glu Asp Gly Asp Thr Gly
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Ser Asp Gln Gln Pro Lys Gly Arg Lys Leu Trp Gly Leu Val Val Cys
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His His Thr Ser Pro Arg Phe Val Pro Phe Pro Leu Arg Tyr Ala Cys
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Glu Phe Leu Leu Gln Val Phe Gly Ile Gln Leu Asn Lys Glu Val Glu
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Leu Ala Ala Gln Ala Lys Glu Arg His Ile Leu Arg Thr Gln Thr Leu
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Leu Cys Asp Met Leu Leu Arg Asp Ala Pro Val Gly Ile Phe Thr Arg
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Ser Pro Asn Val Met Asp Leu Val Lys Cys Asp Gly Ala Ala Leu Tyr
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Tyr Gln Asn Gln Leu Leu Val Leu Gly Ser Thr Pro Ser Glu Ser Glu
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Gly Leu Ser Thr Asp Ser Leu Val Glu Ala Gly Tyr Pro Gly Ala Val
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Ala Leu Arg Glu Val Val Cys Gly Met Ala Ala Ile Lys Ile Ser Ser
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Lys Asp Phe Ile Phe Trp Phe Arg Ser His Thr Thr Lys Glu Ile Lys
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Trp Gly Gly Ala Lys His Glu Pro Val Asp Ala Asp Asp Asp Gly Arg
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Arg Met His Pro Arg Ser Ser Phe Lys Ala Phe Leu Glu Val Val Lys
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Trp Arg Ser Val Pro Trp Glu Asp Val Glu Met Asp Ala Ile His Ser
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Leu Gln Leu Ile Leu Arg Gly Ser Leu Pro Asp Glu Asp Ala Asn Arg
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Asn Asn Val Arg Ser Ile Val Lys Ala Pro Ser Asp Asp Met Lys Lys
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Ile Gln Gly Leu Leu Glu Leu Arg Thr Val Thr Asn Glu Met Val Arg
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Leu Ile Glu Thr Ala Thr Ala Pro Val Leu Ala Val Asp Ile Ala Gly
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Asn Ile Asn Gly Trp Asn Asn Lys Ala Ala Glu Leu Thr Gly Leu Pro
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Val Met Glu Ala Ile Gly Arg Pro Leu Ile Asp Leu Val Val Thr Asp
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Ser Ile Glu Val Val Lys Gln Ile Leu Asp Ser Ala Leu Gln Gly Ile
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Glu Glu Gln Asn Met Glu Ile Lys Leu Lys Thr Phe His Glu His Glu
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Cys Asn Gly Pro Val Ile Leu Lys Val Asn Ser Cys Cys Ser Arg Asp
705 710 715 720
Leu Ser Glu Lys Val Ile Gly Val Cys Phe Val Ala Gln Asp Leu Thr
725 730 735
Arg Gln Lys Met Ile Met Asp Lys Tyr Thr Arg Ile Gln Gly Asp Tyr
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Val Ala Ile Val Lys Asn Pro Thr Glu Leu Ile Pro Pro Ile Phe Met
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Ile Asn Asp Leu Gly Ser Cys Leu Glu Trp Asn Lys Ala Met Gln Lys
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Ile Thr Gly Ile Lys Arg Glu Asp Ala Ile Asn Lys Leu Leu Ile Gly
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Glu Val Phe Thr Leu His Asp Tyr Gly Cys Arg Val Lys Asp His Ala
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Thr Leu Thr Lys Leu Ser Ile Leu Met Asn Ala Val Ile Ser Gly Gln
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Asp Pro Glu Lys Leu Phe Phe Gly Phe Phe Asp Thr Asp Gly Lys Tyr
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Ile Glu Ser Leu Leu Thr Val Asn Lys Arg Thr Asp Ala Glu Gly Lys
850 855 860
Ile Thr Gly Ala Leu Cys Phe Leu His Val Ala Ser Pro Glu Leu Gln
865 870 875 880
His Ala Leu Gln Val Gln Lys Met Ser Glu Gln Ala Ala Thr Asn Ser
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Phe Lys Glu Leu Thr Tyr Ile Arg Gln Glu Leu Arg Asn Pro Leu Asn
900 905 910
Gly Met Gln Phe Thr Cys Asn Leu Leu Lys Pro Ser Glu Leu Thr Glu
915 920 925
Glu Gln Arg Gln Leu Leu Ser Ser Asn Val Leu Cys Gln Asp Gln Leu
930 935 940
Lys Lys Ile Leu His Asp Thr Asp Leu Glu Ser Ile Glu Gln Cys Tyr
945 950 955 960
Met Glu Met Asn Thr Val Glu Phe Asn Leu Glu Gln Ala Leu Asn Thr
965 970 975
Val Leu Met Gln Gly Ile Pro Leu Gly Lys Glu Lys Gln Ile Ser Ile
980 985 990
Glu Arg Asn Trp Pro Val Glu Val Ser Cys Met Tyr Leu Tyr Gly Asp
995 1000 1005
Asn Leu Arg Leu Gln Gln Ile Leu Ala Asp Tyr Leu Ala Cys Ala
1010 1015 1020
Leu Gln Phe Thr Gln Thr Ala Glu Gly Pro Ile Val Leu Gln Val
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Met Ser Lys Lys Glu Asn Ile Gly Ser Gly Met Gln Ile Ala His
1040 1045 1050
Leu Glu Phe Arg Ile Val His Pro Ala Pro Gly Val Pro Glu Ala
1055 1060 1065
Leu Ile Gln Glu Met Phe Gln His Asn Pro Gly Val Ser Arg Glu
1070 1075 1080
Gly Leu Gly Leu Tyr Ile Ser Gln Lys Leu Val Lys Thr Met Ser
1085 1090 1095
Gly Thr Val Gln Tyr Leu Arg Glu Ala Asp Thr Ser Ser Phe Ile
1100 1105 1110
Ile Leu Met Glu Phe Pro Val Ala Gln Leu Ser Ser Lys Arg Ser
1115 1120 1125
Lys Pro Ser Thr Ser Lys Phe
1130 1135
<210> 3
<211> 3408
<212> DNA
<213> Zea mays L
<400> 3
atgtcgtcgc cgtcgaacaa ccgtgggacg tgctcccgga gcagctctgc gcggtccaag 60
cacagcgcgc gggtggtggc gcagacgccc gtggacgcgc agctgcacgc cgatttcgag 120
ggctcccagc gccacttcga ctactcatcc tcggtgggcg ccgccaaccg cccgtcggcc 180
agcacgagca ccgtctccac ctacctccag aacatgcagc ggggccgcta catccagccc 240
ttcggctgcc tgctcgccgt ccacccggac accttcgcgc tgctcgccta cagcgagaac 300
gcgccggaga tgctcgacct cacgccacac gcggtcccaa ccatcgacca gcgggacgcg 360
ctcaccatcg gcgccgacgt gcgcacgctc ttccgctcgc agagctccgt cgcgcttcac 420
aaggccgcca ccttcgggga ggtcaacctg ctcaacccca tcctcgtgca cgccaggacg 480
tcggggaagc ccttctacgc catattgcac cggatcgacg tcggccttgt catcgacctt 540
gagccgttca acccagcaga cgtgccagtc acggccgcgg gcgcgcttaa gtcgtacaag 600
ctcgccgcca aggccatctc caggctgcag tcgctgccca gcgggaacct gtcgttgctg 660
tgcgatgtgc ttgtccgtga ggtgagcgag ctcacgggct atgaccgggt catggcgtac 720
aagttccatg aggatgagca cggtgaggtc atttctgagt gcaggaggtc cgatctggag 780
ccgtatcttg gcctgcacta cccagccacc gacatcccgc aggcgtccag gtttctgttt 840
atgaagaaca aaatgcggat gatatgtgat ttctctgcca ctccagtgct gatcattcag 900
gatggcagcc ttgcacagcc cgtcagcctc tgtggttcta ccctcagggc ttcccatggt 960
tgccatgcac agtacatggc aaacatgggt tctgttgcat cgcttgtgat gtcagtcact 1020
ataaacgacg atgaggagga agatggggat accgacagtg accaacaacc gaaaggcagg 1080
aagctgtggg ggctggtcgt ctgccatcat acaagcccga ggtttgtccc tttcccgcta 1140
aggtacgctt gcgagtttct cttgcaagta tttggcatac agctcagcaa ggaggtggaa 1200
ctggctgctc aggcaaagga gaggcacatc ctcagaacgc aaacccttct ttgtgatatg 1260
ctcctgcggg atgctcttgt tgggatattt acccagtcac ctaatgtgat ggatctagta 1320
aagtgcgatg gagctgcatt gtattatcag aaccaggttt tggtgctcgg atcaacaccg 1380
tccgagtcag agattaagag cattgccaca tggctgcagg agaaccatga tggttcaact 1440
gggctgagta ctgacagctt agtggaagcg ggttatcctg gtgctgctgc actccgtgaa 1500
gtcgtgtgtg gcatggtggc cattaagatc tcttccaaaa attttatctt ctggttccga 1560
tcacacacaa caaaggagat caagtggagt ggggctaagc atgaaccgtt tgacgcagat 1620
gacaatggca ggaagatgca tccacgatct tcattcaagg ccttcttgga ggtggttaaa 1680
tggagaagtg ttccctggga ggatgttgaa atggatgcta tccattcttt gcagttaata 1740
ttacgtgact ccctgcaagg tgaagatgcc aacagaaaca acatcaggtc cattgtaaaa 1800
gctccatctg atgatatgaa gaagttacag gggctacttg aactaagaac agttacaaac 1860
gagatggtcc gcttaattga gacagcaact gcccctgtct tggctgttga cattgccggt 1920
aacataaatg gatggaacaa aaaagctgca gaactaacag ggttacctgt aatggaagcc 1980
atagggaggc ctctgataga tcttgttgtt gctgattctg ttgaagtggt taagcagatt 2040
ttggactcag ctttacaagg aattgaagag caaaatctgg aaatcaagct taaaacattc 2100
catgaacagg agtgttgtgg tccagttatc ttgatgatta actcctgctg tagtcgggat 2160
ctttcagaga aagtcattgg agtttgcttt gtagcacaag atttgaccag gcagaagatg 2220
attatggata agtatactag gatacaagga gactatgttg ccataataaa gaaccccagt 2280
gagctcatcc ctcccatatt tatgatcaat gatcttggtt cctgcttaga gtggaataaa 2340
gctatgcaga agattactgg tatgaagagg gaagacgcga taaataagtt gttaattggg 2400
gaggtcttca cgctccatga ttatggctgt agggtgaaag atcatgctac tctaacgaaa 2460
cttagcatac tgatgaatgc agtgatttct ggtcaggatc cagagaagct cctgtttggt 2520
ttcttcggca caggtgggaa gtatattgaa tccttgctga cagtgaacaa gagaacaaat 2580
gctgagggta aaatcactgg cgctctttgc tttctgcatg tggctagccc agagcttcag 2640
catgctcttg aggtccagaa aatgtctgaa caagctgcta caaacagctt taaagaatta 2700
acttacattc gtcaagaatt aaggaaccca ctcaatggca tgcaatttac ttataactta 2760
ttgaagcctt ccgaattgac agaggatcag cggcaacttg tttcatctaa tgttctgtgt 2820
caggaccagc tgaaaaagat tttacatgac actgatcttg aaagcattga acagtgctat 2880
atggagacga acacagtaga gttcaacctt gaggaagctc tgaatacggt cctgatgcaa 2940
ggcattcctt tgggcaagga aaaacgtatt tctattgaac gtgattggcc tgtggaagtg 3000
tcacacatgt acatttacgg ggacaatata aggcttcagc aggtcctagc agattatctg 3060
gcttgcgccc ttcaattcac acaaccagct gaaggacata tcgtgctcca ggtcattccc 3120
aagaaggaaa acattgggtc tggcatgcag attgctcatt tggaattcag gattgtccat 3180
ccagctccag gcgttccaga ggccctgata caggagatgt tccagcacaa cccaggggtg 3240
tccagggagg gtctcggcct gtacataagc cagaagctgg tgaaaacgat gagcggcacg 3300
ttgcagtacc tacgagaagc cgacacctct tcgttcatca tcctgataga gttcccggtc 3360
gcccagctca gcagcaagcg gtccaagcct tcgccaagta aattctga 3408
<210> 4
<211> 1135
<212> PRT
<213> Zea mays L
<400> 4
Met Ser Ser Pro Ser Asn Asn Arg Gly Thr Cys Ser Arg Ser Ser Ser
1 5 10 15
Ala Arg Ser Lys His Ser Ala Arg Val Val Ala Gln Thr Pro Val Asp
20 25 30
Ala Gln Leu His Ala Asp Phe Glu Gly Ser Gln Arg His Phe Asp Tyr
35 40 45
Ser Ser Ser Val Gly Ala Ala Asn Arg Pro Ser Ala Ser Thr Ser Thr
50 55 60
Val Ser Thr Tyr Leu Gln Asn Met Gln Arg Gly Arg Tyr Ile Gln Pro
65 70 75 80
Phe Gly Cys Leu Leu Ala Val His Pro Asp Thr Phe Ala Leu Leu Ala
85 90 95
Tyr Ser Glu Asn Ala Pro Glu Met Leu Asp Leu Thr Pro His Ala Val
100 105 110
Pro Thr Ile Asp Gln Arg Asp Ala Leu Thr Ile Gly Ala Asp Val Arg
115 120 125
Thr Leu Phe Arg Ser Gln Ser Ser Val Ala Leu His Lys Ala Ala Thr
130 135 140
Phe Gly Glu Val Asn Leu Leu Asn Pro Ile Leu Val His Ala Arg Thr
145 150 155 160
Ser Gly Lys Pro Phe Tyr Ala Ile Leu His Arg Ile Asp Val Gly Leu
165 170 175
Val Ile Asp Leu Glu Pro Phe Asn Pro Ala Asp Val Pro Val Thr Ala
180 185 190
Ala Gly Ala Leu Lys Ser Tyr Lys Leu Ala Ala Lys Ala Ile Ser Arg
195 200 205
Leu Gln Ser Leu Pro Ser Gly Asn Leu Ser Leu Leu Cys Asp Val Leu
210 215 220
Val Arg Glu Val Ser Glu Leu Thr Gly Tyr Asp Arg Val Met Ala Tyr
225 230 235 240
Lys Phe His Glu Asp Glu His Gly Glu Val Ile Ser Glu Cys Arg Arg
245 250 255
Ser Asp Leu Glu Pro Tyr Leu Gly Leu His Tyr Pro Ala Thr Asp Ile
260 265 270
Pro Gln Ala Ser Arg Phe Leu Phe Met Lys Asn Lys Met Arg Met Ile
275 280 285
Cys Asp Phe Ser Ala Thr Pro Val Leu Ile Ile Gln Asp Gly Ser Leu
290 295 300
Ala Gln Pro Val Ser Leu Cys Gly Ser Thr Leu Arg Ala Ser His Gly
305 310 315 320
Cys His Ala Gln Tyr Met Ala Asn Met Gly Ser Val Ala Ser Leu Val
325 330 335
Met Ser Val Thr Ile Asn Asp Asp Glu Glu Glu Asp Gly Asp Thr Asp
340 345 350
Ser Asp Gln Gln Pro Lys Gly Arg Lys Leu Trp Gly Leu Val Val Cys
355 360 365
His His Thr Ser Pro Arg Phe Val Pro Phe Pro Leu Arg Tyr Ala Cys
370 375 380
Glu Phe Leu Leu Gln Val Phe Gly Ile Gln Leu Ser Lys Glu Val Glu
385 390 395 400
Leu Ala Ala Gln Ala Lys Glu Arg His Ile Leu Arg Thr Gln Thr Leu
405 410 415
Leu Cys Asp Met Leu Leu Arg Asp Ala Leu Val Gly Ile Phe Thr Gln
420 425 430
Ser Pro Asn Val Met Asp Leu Val Lys Cys Asp Gly Ala Ala Leu Tyr
435 440 445
Tyr Gln Asn Gln Val Leu Val Leu Gly Ser Thr Pro Ser Glu Ser Glu
450 455 460
Ile Lys Ser Ile Ala Thr Trp Leu Gln Glu Asn His Asp Gly Ser Thr
465 470 475 480
Gly Leu Ser Thr Asp Ser Leu Val Glu Ala Gly Tyr Pro Gly Ala Ala
485 490 495
Ala Leu Arg Glu Val Val Cys Gly Met Val Ala Ile Lys Ile Ser Ser
500 505 510
Lys Asn Phe Ile Phe Trp Phe Arg Ser His Thr Thr Lys Glu Ile Lys
515 520 525
Trp Ser Gly Ala Lys His Glu Pro Phe Asp Ala Asp Asp Asn Gly Arg
530 535 540
Lys Met His Pro Arg Ser Ser Phe Lys Ala Phe Leu Glu Val Val Lys
545 550 555 560
Trp Arg Ser Val Pro Trp Glu Asp Val Glu Met Asp Ala Ile His Ser
565 570 575
Leu Gln Leu Ile Leu Arg Asp Ser Leu Gln Gly Glu Asp Ala Asn Arg
580 585 590
Asn Asn Ile Arg Ser Ile Val Lys Ala Pro Ser Asp Asp Met Lys Lys
595 600 605
Leu Gln Gly Leu Leu Glu Leu Arg Thr Val Thr Asn Glu Met Val Arg
610 615 620
Leu Ile Glu Thr Ala Thr Ala Pro Val Leu Ala Val Asp Ile Ala Gly
625 630 635 640
Asn Ile Asn Gly Trp Asn Lys Lys Ala Ala Glu Leu Thr Gly Leu Pro
645 650 655
Val Met Glu Ala Ile Gly Arg Pro Leu Ile Asp Leu Val Val Ala Asp
660 665 670
Ser Val Glu Val Val Lys Gln Ile Leu Asp Ser Ala Leu Gln Gly Ile
675 680 685
Glu Glu Gln Asn Leu Glu Ile Lys Leu Lys Thr Phe His Glu Gln Glu
690 695 700
Cys Cys Gly Pro Val Ile Leu Met Ile Asn Ser Cys Cys Ser Arg Asp
705 710 715 720
Leu Ser Glu Lys Val Ile Gly Val Cys Phe Val Ala Gln Asp Leu Thr
725 730 735
Arg Gln Lys Met Ile Met Asp Lys Tyr Thr Arg Ile Gln Gly Asp Tyr
740 745 750
Val Ala Ile Ile Lys Asn Pro Ser Glu Leu Ile Pro Pro Ile Phe Met
755 760 765
Ile Asn Asp Leu Gly Ser Cys Leu Glu Trp Asn Lys Ala Met Gln Lys
770 775 780
Ile Thr Gly Met Lys Arg Glu Asp Ala Ile Asn Lys Leu Leu Ile Gly
785 790 795 800
Glu Val Phe Thr Leu His Asp Tyr Gly Cys Arg Val Lys Asp His Ala
805 810 815
Thr Leu Thr Lys Leu Ser Ile Leu Met Asn Ala Val Ile Ser Gly Gln
820 825 830
Asp Pro Glu Lys Leu Leu Phe Gly Phe Phe Gly Thr Gly Gly Lys Tyr
835 840 845
Ile Glu Ser Leu Leu Thr Val Asn Lys Arg Thr Asn Ala Glu Gly Lys
850 855 860
Ile Thr Gly Ala Leu Cys Phe Leu His Val Ala Ser Pro Glu Leu Gln
865 870 875 880
His Ala Leu Glu Val Gln Lys Met Ser Glu Gln Ala Ala Thr Asn Ser
885 890 895
Phe Lys Glu Leu Thr Tyr Ile Arg Gln Glu Leu Arg Asn Pro Leu Asn
900 905 910
Gly Met Gln Phe Thr Tyr Asn Leu Leu Lys Pro Ser Glu Leu Thr Glu
915 920 925
Asp Gln Arg Gln Leu Val Ser Ser Asn Val Leu Cys Gln Asp Gln Leu
930 935 940
Lys Lys Ile Leu His Asp Thr Asp Leu Glu Ser Ile Glu Gln Cys Tyr
945 950 955 960
Met Glu Thr Asn Thr Val Glu Phe Asn Leu Glu Glu Ala Leu Asn Thr
965 970 975
Val Leu Met Gln Gly Ile Pro Leu Gly Lys Glu Lys Arg Ile Ser Ile
980 985 990
Glu Arg Asp Trp Pro Val Glu Val Ser His Met Tyr Ile Tyr Gly Asp
995 1000 1005
Asn Ile Arg Leu Gln Gln Val Leu Ala Asp Tyr Leu Ala Cys Ala
1010 1015 1020
Leu Gln Phe Thr Gln Pro Ala Glu Gly His Ile Val Leu Gln Val
1025 1030 1035
Ile Pro Lys Lys Glu Asn Ile Gly Ser Gly Met Gln Ile Ala His
1040 1045 1050
Leu Glu Phe Arg Ile Val His Pro Ala Pro Gly Val Pro Glu Ala
1055 1060 1065
Leu Ile Gln Glu Met Phe Gln His Asn Pro Gly Val Ser Arg Glu
1070 1075 1080
Gly Leu Gly Leu Tyr Ile Ser Gln Lys Leu Val Lys Thr Met Ser
1085 1090 1095
Gly Thr Leu Gln Tyr Leu Arg Glu Ala Asp Thr Ser Ser Phe Ile
1100 1105 1110
Ile Leu Ile Glu Phe Pro Val Ala Gln Leu Ser Ser Lys Arg Ser
1115 1120 1125
Lys Pro Ser Pro Ser Lys Phe
1130 1135
<210> 5
<211> 23
<212> DNA
<213> Artifical sequence
<400> 5
gttctggagg taggtggaga cgg 23
<210> 6
<211> 23
<212> DNA
<213> Artifical sequence
<400> 6
gcacatcgca cagcaacgac agg 23
<210> 7
<211> 20
<212> DNA
<213> Artifical sequence
<400> 7
gccgctgccc cgcgcgggcc 20
<210> 8
<211> 20
<212> DNA
<213> Artifical sequence
<400> 8
accttagtgg gaaagggacg 20
<210> 9
<211> 20
<212> DNA
<213> Artifical sequence
<400> 9
gcccaagagt atcgtcgctg 20
<210> 10
<211> 20
<212> DNA
<213> Artifical sequence
<400> 10
cttagcggga aagggacaaa 20
<210> 11
<211> 64
<212> DNA
<213> Artifical sequence
<400> 11
gagccgcaag caccgaattg ttctggaggt aggtggagag ttttagagct agaaatagca 60
agtt 64
<210> 12
<211> 64
<212> DNA
<213> Artifical sequence
<400> 12
gagccgcaag caccgaattg cacatcgcac agcaacgacg ttttagagct agaaatagca 60
agtt 64
<210> 13
<211> 37
<212> DNA
<213> Artifical sequence
<400> 13
tgcttttttt aagctgctgt ttttgttagc cccatcg 37
<210> 14
<211> 19
<212> DNA
<213> Artifical sequence
<400> 14
aattcggtgc ttgcggctc 19
<210> 15
<211> 22
<212> DNA
<213> Artifical sequence
<400> 15
ggatccctaa ttggccctta ca 22
<210> 16
<211> 21
<212> DNA
<213> Artifical sequence
<400> 16
ggagcggtgg tcgcagctga a 21
<210> 17
<211> 40
<212> DNA
<213> Artifical sequence
<400> 17
tgtaagggcc aattagggat ccaaaaaaag caccgactcg 40
<210> 18
<211> 83
<212> DNA
<213> Artifical sequence
<400> 18
gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60
ggcaccgagt cggtgctttt ttt 83
<210> 19
<211> 37
<212> DNA
<213> Artifical sequence
<400> 19
tgcactgcac aagctgctgt ttttgttagc cccatcg 37
<210> 20
<211> 34
<212> DNA
<213> Artifical sequence
<400> 20
ggccagtgcc aagcttaaaa aaagcaccga ctcg 34
<210> 21
<211> 26
<212> DNA
<213> Artifical sequence
<400> 21
cttagacatg caatgctcat tatctc 26
<210> 22
<211> 18
<212> DNA
<213> Artifical sequence
<400> 22
ctggcgaaag ggggatgt 18
<210> 23
<211> 22
<212> DNA
<213> Artifical sequence
<400> 23
gtgtattccc tcttctcccc cc 22
<210> 24
<211> 22
<212> DNA
<213> Artifical sequence
<400> 24
ggcttgtatg atggcagacg ac 22
<210> 25
<211> 22
<212> DNA
<213> Artifical sequence
<400> 25
ctcgctgaaa ttccctcttc tt 22
<210> 26
<211> 21
<212> DNA
<213> Artifical sequence
<400> 26
gttggtcact gtcggtatcc c 21
<210> 27
<211> 87
<212> PRT
<213> Artifical sequence
<400> 27
Met Ser Leu Pro Ser Asn Asn Arg Arg Thr Cys Ser Arg Ser Ser Ser
1 5 10 15
Ala Arg Ser Lys His Ser Ala Arg Val Val Ala Gln Thr Pro Val Asp
20 25 30
Ala Gln Leu His Ala Glu Phe Glu Gly Ser Gln Arg His Phe Asp Tyr
35 40 45
Ser Ser Ser Val Gly Ala Ala Asn Arg Pro Ser Ala Ser Thr Ser Thr
50 55 60
Val Ser Thr Gly Ser Arg Trp Ala Ala Thr Ala Ala Trp Arg Trp Pro
65 70 75 80
Trp Gln Arg Ala Cys Thr Thr
85
<210> 28
<211> 180
<212> PRT
<213> Artifical sequence
<400> 28
Met Ser Leu Pro Ser Asn Asn Arg Arg Thr Cys Ser Arg Ser Ser Ser
1 5 10 15
Ala Arg Ser Lys His Ser Ala Arg Val Val Ala Gln Thr Pro Val Asp
20 25 30
Ala Gln Leu His Ala Glu Phe Glu Gly Ser Gln Arg His Phe Asp Tyr
35 40 45
Ser Ser Ser Val Gly Ala Ala Asn Arg Pro Ser Ala Ser Thr Ser Thr
50 55 60
Val Phe His Leu Pro Pro Glu His Ala Ala Gly Pro Leu His Pro Ala
65 70 75 80
Leu Arg Leu Pro Ala Arg Arg Pro Pro Gly His Leu Arg Ala Ala Arg
85 90 95
Leu Gln Arg Glu Arg Ala Arg Asp Ala Arg Pro His Ala Thr Arg Arg
100 105 110
Pro His His Arg Pro Ala Gly Cys Ala Arg His Arg Arg Arg Cys Ala
115 120 125
His Ala Leu Pro Leu Ala Glu Leu Arg Arg Ala Ser Gln Gly Arg Arg
130 135 140
Leu Arg Gly Gly Gln Pro Thr Gln Pro His Pro Arg Ala Arg Gln Asp
145 150 155 160
Val Gly Glu Ala Leu Leu Arg His Ile Ala Pro Asp Arg Arg Arg Pro
165 170 175
Cys His Arg Pro
180
<210> 29
<211> 224
<212> PRT
<213> Artifical sequence
<400> 29
Met Ser Leu Pro Ser Asn Asn Arg Arg Thr Cys Ser Arg Ser Ser Ser
1 5 10 15
Ala Arg Ser Lys His Ser Ala Arg Val Val Ala Gln Thr Pro Val Asp
20 25 30
Ala Gln Leu His Ala Glu Phe Glu Gly Ser Gln Arg His Phe Asp Tyr
35 40 45
Ser Ser Ser Val Gly Ala Ala Asn Arg Pro Ser Ala Ser Thr Ser Thr
50 55 60
Val His Leu Gln Asn Met Gln Arg Gly Arg Tyr Ile Gln Pro Phe Gly
65 70 75 80
Cys Leu Leu Ala Val His Pro Asp Thr Phe Ala Leu Leu Ala Tyr Ser
85 90 95
Glu Asn Ala Pro Glu Met Leu Asp Leu Thr Pro His Ala Val Pro Thr
100 105 110
Ile Asp Gln Arg Asp Ala Leu Gly Ile Gly Val Asp Val Arg Thr Leu
115 120 125
Phe Arg Ser Gln Ser Ser Val Ala Leu His Lys Ala Ala Ala Phe Gly
130 135 140
Glu Val Asn Leu Leu Asn Pro Ile Leu Val His Ala Arg Thr Ser Gly
145 150 155 160
Lys Pro Phe Tyr Ala Ile Leu His Arg Ile Asp Val Gly Leu Val Ile
165 170 175
Asp Leu Glu Pro Val Asn Pro Ala Asp Val Pro Val Thr Ala Ala Gly
180 185 190
Ala Leu Lys Ser Tyr Lys Leu Ala Ala Lys Ala Ile Ser Arg Leu Gln
195 200 205
Ser Leu Pro Ser Gly Asn Leu Ser Val Ala Val Arg Cys Ala Cys Pro
210 215 220
<210> 30
<211> 264
<212> DNA
<213> Artifical sequence
<400> 30
atgtcgttgc cgtcgaacaa ccggaggacg tgctcccgga gcagctctgc gcggtccaag 60
cacagcgcgc gggtggtggc acagacgccc gtggacgcgc agctgcacgc cgagttcgag 120
ggctcccagc gccacttcga ctactcctcg tcggtgggcg ccgccaaccg cccgtcggca 180
agcaccagca ccgtctcgac aggttcccgc tgggcagcga ctgcagcctg gagatggcct 240
tggcagcgag cttgtacgac ttga 264
<210> 31
<211> 543
<212> DNA
<213> Artifical sequence
<400> 31
atgtcgttgc cgtcgaacaa ccggaggacg tgctcccgga gcagctctgc gcggtccaag 60
cacagcgcgc gggtggtggc acagacgccc gtggacgcgc agctgcacgc cgagttcgag 120
ggctcccagc gccacttcga ctactcctcg tcggtgggcg ccgccaaccg cccgtcggca 180
agcaccagca ccgtcttcca cctacctcca gaacatgcag cggggccgct acatccagcc 240
cttcggctgc ctgctcgccg tccacccgga caccttcgcg ctgctcgcct acagcgagaa 300
cgcgcccgag atgctcgacc tcacgccaca cgccgtcccc accatcgacc agcgggatgc 360
gctcggcatc ggcgtcgatg tgcgcacgct cttccgctcg cagagctccg tcgcgcttca 420
caaggccgcc gccttcgggg aggtcaacct actcaacccc atcctcgtgc acgccaggac 480
gtcggggaag cccttctacg ccatattgca ccggatcgac gtcggccttg tcatcgacct 540
tga 543
<210> 32
<211> 681
<212> DNA
<213> Artifical sequence
<400> 32
atgtcgttgc cgtcgaacaa ccggaggacg tgctcccgga gcagctctgc gcggtccaag 60
cacagcgcgc gggtggtggc acagacgccc gtggacgcgc agctgcacgc cgagttcgag 120
ggctcccagc gccacttcga ctactcctcg tcggtgggcg ccgccaaccg cccgtcggca 180
agcaccagca ccgtctccac ctacctccag aacatgcagc ggggccgcta catccagccc 240
ttcggctgcc tgctcgccgt ccacccggac accttcgcgc tgctcgccta cagcgagaac 300
gcgcccgaga tgctcgacct cacgccacac gccgtcccca ccatcgacca gcgggatgcg 360
ctcggcatcg gcgtcgatgt gcgcacgctc ttccgctcgc agagctccgt cgcgcttcac 420
aaggccgccg ccttcgggga ggtcaaccta ctcaacccca tcctcgtgca cgccaggacg 480
tcggggaagc ccttctacgc catattgcac cggatcgacg tcggccttgt catcgacctt 540
gagccggtca atccagccga cgtgccagtc accgccgcgg gcgcgctcaa gtcgtacaag 600
ctcgctgcca aggccatctc caggctgcag tcgctgccca gcgggaacct gtctgttgct 660
gtgcgatgtg cttgtccgtg a 681
<210> 33
<211> 179
<212> PRT
<213> Artifical sequence
<400> 33
Met Ser Ser Pro Ser Asn Asn Arg Gly Thr Cys Ser Arg Ser Ser Ser
1 5 10 15
Ala Arg Ser Lys His Ser Ala Arg Val Val Ala Gln Thr Pro Val Asp
20 25 30
Ala Gln Leu His Ala Asp Phe Glu Gly Ser Gln Arg His Phe Asp Tyr
35 40 45
Ser Ser Ser Val Gly Ala Ala Asn Arg Pro Ser Ala Ser Thr Ser Thr
50 55 60
Val Tyr Leu Pro Pro Glu His Ala Ala Gly Pro Leu His Pro Ala Leu
65 70 75 80
Arg Leu Pro Ala Arg Arg Pro Pro Gly His Leu Arg Ala Ala Arg Leu
85 90 95
Gln Arg Glu Arg Ala Gly Asp Ala Arg Pro His Ala Thr Arg Gly Pro
100 105 110
Asn His Arg Pro Ala Gly Arg Ala His His Arg Arg Arg Arg Ala His
115 120 125
Ala Leu Pro Leu Ala Glu Leu Arg Arg Ala Ser Gln Gly Arg His Leu
130 135 140
Arg Gly Gly Gln Pro Ala Gln Pro His Pro Arg Ala Arg Gln Asp Val
145 150 155 160
Gly Glu Ala Leu Leu Arg His Ile Ala Pro Asp Arg Arg Arg Pro Cys
165 170 175
His Arg Pro
<210> 34
<211> 180
<212> PRT
<213> Artifical sequence
<400> 34
Met Ser Ser Pro Ser Asn Asn Arg Gly Thr Cys Ser Arg Ser Ser Ser
1 5 10 15
Ala Arg Ser Lys His Ser Ala Arg Val Val Ala Gln Thr Pro Val Asp
20 25 30
Ala Gln Leu His Ala Asp Phe Glu Gly Ser Gln Arg His Phe Asp Tyr
35 40 45
Ser Ser Ser Val Gly Ala Ala Asn Arg Pro Ser Ala Ser Thr Ser Thr
50 55 60
Val Phe His Leu Pro Pro Glu His Ala Ala Gly Pro Leu His Pro Ala
65 70 75 80
Leu Arg Leu Pro Ala Arg Arg Pro Pro Gly His Leu Arg Ala Ala Arg
85 90 95
Leu Gln Arg Glu Arg Ala Gly Asp Ala Arg Pro His Ala Thr Arg Gly
100 105 110
Pro Asn His Arg Pro Ala Gly Arg Ala His His Arg Arg Arg Arg Ala
115 120 125
His Ala Leu Pro Leu Ala Glu Leu Arg Arg Ala Ser Gln Gly Arg His
130 135 140
Leu Arg Gly Gly Gln Pro Ala Gln Pro His Pro Arg Ala Arg Gln Asp
145 150 155 160
Val Gly Glu Ala Leu Leu Arg His Ile Ala Pro Asp Arg Arg Arg Pro
165 170 175
Cys His Arg Pro
180
<210> 35
<211> 81
<212> PRT
<213> Artifical sequence
<400> 35
Met Ser Ser Pro Ser Asn Asn Arg Gly Thr Cys Ser Arg Ser Ser Ser
1 5 10 15
Ala Arg Ser Lys His Ser Ala Arg Val Val Ala Gln Thr Pro Val Asp
20 25 30
Ala Gln Leu His Ala Asp Phe Glu Gly Ser Gln Arg His Phe Asp Tyr
35 40 45
Ser Ser Ser Val Gly Ala Ala Asn Arg Pro Ser Ala Ser Thr Ser Pro
50 55 60
Met Val Ala Met His Ser Thr Trp Gln Thr Trp Val Leu Leu His Arg
65 70 75 80
Leu
<210> 36
<211> 542
<212> PRT
<213> Artifical sequence
<400> 36
Ala Thr Gly Thr Cys Gly Thr Cys Gly Cys Cys Gly Thr Cys Gly Ala
1 5 10 15
Ala Cys Ala Ala Cys Cys Gly Thr Gly Gly Gly Ala Cys Gly Thr Gly
20 25 30
Cys Thr Cys Cys Cys Gly Gly Ala Gly Cys Ala Gly Cys Thr Cys Thr
35 40 45
Gly Cys Gly Cys Gly Gly Thr Cys Cys Ala Ala Gly Cys Ala Cys Ala
50 55 60
Gly Cys Gly Cys Gly Cys Gly Gly Gly Thr Gly Gly Thr Gly Gly Cys
65 70 75 80
Gly Cys Ala Gly Ala Cys Gly Cys Cys Cys Gly Thr Gly Gly Ala Cys
85 90 95
Gly Cys Gly Cys Ala Gly Cys Thr Gly Cys Ala Cys Gly Cys Cys Gly
100 105 110
Ala Thr Thr Thr Cys Gly Ala Gly Gly Gly Cys Thr Cys Cys Cys Ala
115 120 125
Gly Cys Gly Cys Cys Ala Cys Thr Thr Cys Gly Ala Cys Thr Ala Cys
130 135 140
Thr Cys Ala Thr Cys Cys Thr Cys Gly Gly Thr Gly Gly Gly Cys Gly
145 150 155 160
Cys Cys Gly Cys Cys Ala Ala Cys Cys Gly Cys Cys Cys Gly Thr Cys
165 170 175
Gly Gly Cys Cys Ala Gly Cys Ala Cys Gly Ala Gly Cys Ala Cys Cys
180 185 190
Gly Thr Cys Thr Cys Cys Ala Cys Cys Thr Ala Cys Cys Thr Cys Cys
195 200 205
Ala Gly Ala Ala Cys Ala Thr Gly Cys Ala Gly Cys Gly Gly Gly Gly
210 215 220
Cys Cys Gly Cys Thr Ala Cys Ala Thr Cys Cys Ala Gly Cys Cys Cys
225 230 235 240
Thr Thr Cys Gly Gly Cys Thr Gly Cys Cys Thr Gly Cys Thr Cys Gly
245 250 255
Cys Cys Gly Thr Cys Cys Ala Cys Cys Cys Gly Gly Ala Cys Ala Cys
260 265 270
Cys Thr Thr Cys Gly Cys Gly Cys Thr Gly Cys Thr Cys Gly Cys Cys
275 280 285
Thr Ala Cys Ala Gly Cys Gly Ala Gly Ala Ala Cys Gly Cys Gly Cys
290 295 300
Cys Gly Gly Ala Gly Ala Thr Gly Cys Thr Cys Gly Ala Cys Cys Thr
305 310 315 320
Cys Ala Cys Gly Cys Cys Ala Cys Ala Cys Gly Cys Gly Gly Thr Cys
325 330 335
Cys Cys Ala Ala Cys Cys Ala Thr Cys Gly Ala Cys Cys Ala Gly Cys
340 345 350
Gly Gly Gly Ala Cys Gly Cys Gly Cys Thr Cys Ala Cys Cys Ala Thr
355 360 365
Cys Gly Gly Cys Gly Cys Cys Gly Ala Cys Gly Thr Gly Cys Gly Cys
370 375 380
Ala Cys Gly Cys Thr Cys Thr Thr Cys Cys Gly Cys Thr Cys Gly Cys
385 390 395 400
Ala Gly Ala Gly Cys Thr Cys Cys Gly Thr Cys Gly Cys Gly Cys Thr
405 410 415
Thr Cys Ala Cys Ala Ala Gly Gly Cys Cys Gly Cys Cys Ala Cys Cys
420 425 430
Thr Thr Cys Gly Gly Gly Gly Ala Gly Gly Thr Cys Ala Ala Cys Cys
435 440 445
Thr Gly Cys Thr Cys Ala Ala Cys Cys Cys Cys Ala Thr Cys Cys Thr
450 455 460
Cys Gly Thr Gly Cys Ala Cys Gly Cys Cys Ala Gly Gly Ala Cys Gly
465 470 475 480
Thr Cys Gly Gly Gly Gly Ala Ala Gly Cys Cys Cys Thr Thr Cys Thr
485 490 495
Ala Cys Gly Cys Cys Ala Thr Ala Thr Thr Gly Cys Ala Cys Cys Gly
500 505 510
Gly Ala Thr Cys Gly Ala Cys Gly Thr Cys Gly Gly Cys Cys Thr Thr
515 520 525
Gly Thr Cys Ala Thr Cys Gly Ala Cys Cys Thr Thr Gly Ala
530 535 540
<210> 37
<211> 543
<212> DNA
<213> Artifical sequence
<400> 37
atgtcgtcgc cgtcgaacaa ccgtgggacg tgctcccgga gcagctctgc gcggtccaag 60
cacagcgcgc gggtggtggc gcagacgccc gtggacgcgc agctgcacgc cgatttcgag 120
ggctcccagc gccacttcga ctactcatcc tcggtgggcg ccgccaaccg cccgtcggcc 180
agcacgagca ccgtcttcca cctacctcca gaacatgcag cggggccgct acatccagcc 240
cttcggctgc ctgctcgccg tccacccgga caccttcgcg ctgctcgcct acagcgagaa 300
cgcgccggag atgctcgacc tcacgccaca cgcggtccca accatcgacc agcgggacgc 360
gctcaccatc ggcgccgacg tgcgcacgct cttccgctcg cagagctccg tcgcgcttca 420
caaggccgcc accttcgggg aggtcaacct gctcaacccc atcctcgtgc acgccaggac 480
gtcggggaag cccttctacg ccatattgca ccggatcgac gtcggccttg tcatcgacct 540
tga 543
<210> 38
<211> 1009
<212> DNA
<213> Artifical sequence
<400> 38
atgtcgtcgc cgtcgaacaa ccgtgggacg tgctcccgga gcagctctgc gcggtccaag 60
cacagcgcgc gggtggtggc gcagacgccc gtggacgcgc agctgcacgc cgatttcgag 120
ggctcccagc gccacttcga ctactcatcc tcggtgggcg ccgccaaccg cccgtcggcc 180
agcacgagca ccgtctccac ctacctccag aacatgcagc ggggccgcta catccagccc 240
ttcggctgcc tgctcgccgt ccacccggac accttcgcgc tgctcgccta cagcgagaac 300
gcgccggaga tgctcgacct cacgccacac gcggtcccaa ccatcgacca gcgggacgcg 360
ctcaccatcg gcgccgacgt gcgcacgctc ttccgctcgc agagctccgt cgcgcttcac 420
aaggccgcca ccttcgggga ggtcaacctg ctcaacccca tcctcgtgca cgccaggacg 480
tcggggaagc ccttctacgc catattgcac cggatcgacg tcggccttgt catcgacctt 540
gagccgttca acccagcaga cgtgccagtc acggccgcgg gcgcgcttaa gtcgtacaag 600
ctcgccgcca aggccatctc caggctgcag tcgctgccca gcgggaacct gtcgttgctg 660
tgcgatgtgc ttgtccgtga ggtgagcgag ctcacgggct atgaccgggt catggcgtac 720
aagttccatg aggatgagca cggtgaggtc atttctgagt gcaggaggtc cgatctggag 780
ccgtatcttg gcctgcacta cccagccacc gacatcccgc aggcgtccag gtttctgttt 840
atgaagaaca aaatgcggat gatatgtgat ttctctgcca ctccagtgct gatcattcag 900
gatggcagcc ttgcacagcc cgtcagcctc tgtggttcta ccctcagggc ttcccatggt 960
tgccatgcac agtacatggc aaacatgggt tctgttgcat cgcttgtga 1009

Claims (10)

1.玉米ZmPHYCs突变型蛋白，其特征在于，该突变型蛋白由ZmPHYC1突变型蛋白和ZmPHYC2突变型蛋白组成；其中，所述ZmPHYC1突变型蛋白的氨基酸序列为SEQ ID No.27-SEQ ID No.29示，其编码基因的核苷酸序列为SEQ ID No.30-SEQ ID No.32所示；所述ZmPHYC2突变型蛋白的氨基酸序列为SEQ ID No.33-SEQ ID No.35所示，其编码基因的核苷酸序列为SEQ ID No.36-SEQ ID No.38所示。
2.权利要求1所述的玉米ZmPHYCs突变型蛋白编码基因，其特征在于，所述ZmPHYC1突变型蛋白编码基因的核苷酸序列为SEQ ID No.30-SEQ ID No.32所示；所述ZmPHYC2突变型蛋白编码基因的核苷酸序列为SEQ ID No.36-SEQ ID No.38所示。
3.含有权利要求2所述编码基因的重组植物表达载体。
4.一种ZmPHYCs基因编辑载体，其特征在于，其构建方法包括：
(Ⅰ)获取玉米U6-1和U6-2启动子片段；
(Ⅱ)制备sgRNA表达盒：
(Ⅲ)将步骤(Ⅱ)获得的2个sgRNA表达盒依次连接CPB-Ubi-hspcas9载体，即得。
5.按照权利要求4所述的ZmPHYCs基因编辑载体，其特征在于，步骤(Ⅱ)中所述的制备sgRNA表达盒包括以下步骤：
(A)采用重叠PCR的方法将带有接头的靶点序列和sgR骨架序列融合在一起获得2个PCR产物；其中，PCR的上游引物分别为SEQ ID No.11-SEQ ID No.12所示；下游引物为SEQ IDNo.17所示，模板序列为SEQ ID No.18所示；
(B)再次用重叠PCR的方法将步骤(A)获得的2个融合PCR片段分别与U6-1启动子片段和U6-2启动子片段连接获得2个PCR产物，即为sgRNA连接产物；其中，该重叠PCR中所用到的上下游引物序列为SEQ ID No.19/SEQ ID No.17和SEQ ID No.19/SEQ ID No.20所示；所用到的模板是U6-1或U6-2启动子片段以及步骤(A)扩增得到的2个PCR产物。
6.权利要求1所述的玉米ZmPHYCs突变型蛋白在调控玉米开花期中的应用。
7.权利要求2所述的编码基因在调控玉米雄穗开花期中的应用。
8.权利要求3所述的ZmPHYCs基因编辑载体在调控玉米雄穗开花期中的应用。
9.按照权利要求6或7所述的应用，其特征在于，所述的调控玉米开花期是在长日照条件下使玉米雄穗开花期提前。
10.一种使植物雄穗开花期提前的方法，其特征在于，包括：(1)将权利要求2所述的编码基因可操作的与调控元件连接后构建得到重组植物表达载体；(2)将所构建的重组植物表达载体转化到植物中筛选得到开花提前的植物新品种；或者，将构建的ZmPHYCs基因编辑载体转化到植物中筛选得到开花提前的植物新品种；优选的，所述的植物是玉米。
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