CN116286849B - 调控花生含油率的基因AhWRI1及其应用 - Google Patents
调控花生含油率的基因AhWRI1及其应用 Download PDFInfo
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Abstract
本发明公开了调控花生含油率的基因AhWRI 1及其应用，涉及植物生物技术和植物分子育种技术领域，通过全基因组关联分析定位并克隆位于花生基因组A08染色体上的调控花生含油率的基因AhWRI 1；转录组测序分析和qRT‑PCR实验对该基因进行表达验证；构建过表达载体转化拟南芥对该基因进行过表达功能验证；对该基因外显子SNP变异位点开发KASP分子标记，利用该标记对双亲杂交育种后代开展分子辅助育种，选育高油花生新品种。本发明的AhWR I 1为花生含油率遗传改良提供理论依据；对AhWR I 1的SNP变异位点开发分子标记，进行杂交育种后代筛选，快速选育出高油花生新品种，具广阔应用前景。
Description
技术领域
本发明涉及植物生物技术和植物分子育种技术领域，具体涉及调控花生含油率的基因AhWRI1及其应用。
背景技术
花生是世界范围内广泛栽培的油料与经济作物，也是人们重要的植物油脂和植物蛋白质来源之一。我国花生种植面积常年保持在7000万亩，年总产1700万吨，种植面积和总产分别占世界种植面积和产量的18.9％和41.7％，其中国内一半以上的花生用于榨油，年产油量230万吨，占国产植物油的23％。目前国内花生油市场需求量与实际生产量之间仍存在巨大缺口。2020年，我国花生油进口量为22.6万吨，相比2019年上涨了31.40％，相比2013年上涨了247.69％，进口量增速较快。在日趋复杂严峻的国际环境下，我国也及时制定出多个有力政策，部署扩种油料作物，解决我国植物油供给不足。花生含油率和亩产分别是是大豆的2.5和1.2倍、油菜的1.5和2.0倍。因此，花生也是解决我国植物油供给不足的重要油料作物。中国当前花生主要推广品种平均含油率在51.0％，有研究表明花生含油率每提高1％相当于产量提高2％，加工企业效益提高7％以上(Freeman et al.,1999)。可见，挖掘花生含油率关键基因，通过植物生物技术或分子遗传改良技术，提高花生品种含油率对增加单位面积产油量具有重要意义。
20世纪以来，国内外研究者对花生含油量性状的遗传基础开展了大量的研究工作。多数研究者认为花生含油量为多基因控制的数量性状，易受环境影响，遗传效应与环境之间存在显著互作。有研究表明基因的加性效应在花生含油量遗传中起主要作用。研究者们通过不同世代种子含油量进行遗传分析，认为花生含油量是由2对主基因和多个微效基因共同控制。还有研究表明花生含油率具有超亲优势，其含量呈正态分布，符合主基因加多基因遗传，受基因的加性效应和环境的影响。随着分子标记的迅速发展，国内外研究者利用分子标记对花生含油率进行了QTL定位。另外有研究者利用7对SSR标记进行含油率性状的连锁分析，发现标记2A5与花生含油量基因紧密连锁，其中2A5-240和2A5-250分别与高、低含油率基因相关联，可用于花生含油率性状的分子标记辅助选择。还有研究者利用重组自交系连锁作图对花生含油率进行了QTL定位，在两个环境中鉴定到4个含油率QTLs，其中位于标记区间IPAHM103-PM36的位点在两个环境中都被检测到，分别解释了7.9％和9.1％的表型变异。也有研究者利用重组自交系构建了18个连锁群，检测到2个含油率QTLs，qF-1-1和qF-17-2，分别解释了5.3％和8.2％的表型变异。Huang等(2015)利用F2：3家系群体，在B03连锁群定位了1个含油率QTL，qOCB3，解释了14.4％的表型变异。再有研究者利用高密度遗传标记，定位了8个含油率QTLs，其中包括qOc-A10和qOc-A02两个主效QTLs，解释22.1和10.4％的表型变异。也还有研究者利用重组自交系定位到18个含油率QTLs，其中qOCB06可解释22.6％的表型变异，锁定在了B06染色体的0.6Mb区间内。另外还有研究者们通过对318份花生重组自交系群体进行基因组重测序，开展了含油率QTL定位，总共找到110个含油率、蛋白质含量和脂肪酸组分QTLs，并开发了KASP分子标记用于后续辅助育种。
综上可见，对花生含油率的遗传解析多停留在经典遗传学的QTL定位层面，定位区间较大，通常只能解释整个性状小部分的遗传变异，而对关键调控基因的挖掘及其应用鲜有报道，致使花生含油率遗传改良存在很大的盲目性和不可预测性，造成育种效率低、周期长。因此，挖掘花生含油率关键调控基因，并利用生物技术和分子育种技术进行育种应用，快速、有效的提高种子含油率成为花生油份遗传改良的当务之急。WRINKLED1(WRI1)属于AP2/EREBP家族转录因子，是包含60至70个氨基酸组成的植物特有的一类AP2/ERF结构域转录因子，于1998年首次在拟南芥中发现，其拟南芥突变体和过表达植株中，种子含油率分别降低80％或提高20％，是植物种子中重要的油份含量调控关键基因。有研究表明WRI1通过调控油脂合成中关键酶的表达来影响油脂含量，已相继在拟南芥、油菜、玉米、水稻、大豆等多种植物中克隆了WRI1基因并进行了功能分析。在花生中有研究者对WRI1基因家族进行了全基因组鉴定及表达分析，但是尚未见花生WRI1功能基因研究及相关基因应用的报道。
因此，本发明旨在提供一种调控花生含油率的基因AhWRI1及其应用，以解决上述问题，并基于现代生物技术和分子遗传改良技术，有力推动作物育种进入智能设计育种时代。
发明内容
本发明的目的是为了解决上述问题，提供调控花生含油率的基因AhWRI1及其应用，通过全基因组关联作图，从花生中定位并克隆到含油率功能基因AhWRI1，并利用该基因SNP变异的侧翼序列信息设计KASP引物，对花生的含油率进行分子遗传改良。
为了达到上述目的，本发明的技术方案如下：调控花生含油率的基因AhWRI1，所述基因AhWRI1位于花生A08染色体，全长基因组序列为9368bp，全长核苷酸正链序列如SEQ IDNO.1所示；所述基因AhWRI1直接来源于花生、大豆、油菜、向日葵或芝麻。
为了达到上述目的，本发明还提供调控花生含油率的基因AhWRI1在作物含油率性状上的遗传改良应用。
为了达到上述目的，本发明还提供一种调控花生含油率的基因AhWRI1编码的cDNA在作物含油率性状上的遗传改良应用。
进一步地，所述基因AhWRI1编码的cDNA至少有两种转录本形式，第一种转录本为AhWRI1.1，序列全长1092bp，如SEQ ID NO.2所示；第二种转录本为AhWRI1.2，序列全长891bp，如SEQ ID NO.3所示。
进一步地，所述基因AhWRI1的第一种转录本AhWRI1.1的编码长度为363个氨基酸的蛋白质，其氨基酸序列如SEQ ID NO.4所示；所述基因AhWRI1的第二种转录本AhWRI1.2编码长度为296个氨基酸的蛋白质，其氨基酸序列如SEQ ID NO.5所示。
为了达到上述目的，本发明还提供一种调控花生含油率的基因AhWRI1编码的氨基酸或多肽或蛋白质在作物含油率性状上的遗传改良应用。
为了达到上述目的，本发明还提供一种调控花生含油率的基因AhWRI1的启动子及其在作物含油率性状上的遗传改良应用；所述基因AhWRI1的启动子的核苷酸序列如SEQ IDNO.6所示。
为了达到上述目的，本发明还提供一种克隆花生含油率基因AhWRI1的引物对，所述引物对为AhWRI1-F和AhWRI1-R，其序列分别如SEQ ID NO.7和SEQ ID NO.8所示，该引物可以在花生的cDNA中，通过PCR克隆扩增所述基因AhWRI1的完整编码框，将扩增产物通过BsaI-HF酶切后，再利用T4 DNA ligase连接入过表达载体pEGOEP35S-H，构建成pEGOEP35S-H-AhWRI1过表达载体。
为了达到上述目的，本发明还提供一种花生含油率基因AhWRI1的SNP变异位点检测KASP分子标记，所述SNP变异位点检测KASP分子标记分别命名为AhWRI1-KASP-F1、AhWRI1-KASP-F2和AhWRI1-KASP-R，其引物序列分别如SEQ ID NO.9、SEQ ID NO.10和SEQID NO.11所示。
为了达到上述目的，本发明还提供一种高油花生新品种培育的KASP分子标记辅助选择方法，利用KASP标记在杂交后代中筛选具有提高含油率的基因型株系，通过分子育种获得高油花生新品种。
与现有技术相比，本方案的有益效果：
1、本发明提供了一种可调控花生含油率的基因AhWRI1，可通过AhWRI1表达量的改变调控花生种子含油率。
2、本发明提供了调控花生含油率的基因AhWRI1的启动子序列，对该启动子序列进行编辑突变可实现对AhWRI1基因表达量的调控，进而实现对花生含油率的控制。
3、本发明提供了调控花生含油率的基因AhWRI1的KASP分子标记序列，利用该分子标记可进行杂交育种后代筛选，获得高油花生新品种。
4、本发明综合利用全基因组重测序技术，通过全基因组关联分析方法定位并克隆了位于花生基因组A08染色体上的调控花生含油率的基因AhWRI1；通过转录组测序分析和qRT-PCR实验对该基因进行了表达验证；通过构建过表达载体转化拟南芥对该基因进行了过表达功能验证；通过对该基因外显子SNP变异位点开发KASP分子标记，并利用该标记对双亲杂交育种后代开展分子辅助育种，选育高油花生新品种。本发明的AhWRI1在调控花生含油率性状的作用，为花生含油率遗传改良提供了理论依据；本发明通过对AhWRI1的SNP变异位点开发分子标记，进行杂交育种后代筛选，能够快速选育出高油花生新品种，具有广阔的应用前景。
附图说明
图1是本发明实施例中花生RIL-JK重组自交系群体的含油率表型分布(A、花生RIL-JK重组自交系群体在2016和2017年的含油率表型分布；B、花生RIL-JK重组自交系群体两年平均含油率表型分布)；
图2是本发明实施例中花生含油率基因AhWRI1的定位及功能分析(A、基于RIL-JK重组自交系群体连锁作图在A07和A08定位了两个花生含油率QTL，qOilA07和qOilA08；B、花生含油率全基因组关联分析Manhattan图，其中A08显著性关联位点与连锁作图定位到的含油率QTL(qOilA08)位置一致；C、A08染色体Manhattan图及LDblock精细定位；D、含油率候选基因AhWRI1基因结构和变异分析；E、AhWRI1两种基因型含油率表型差异；F、AhWRI1候选基因在两个载体材料的不同组织qRT-PCR表达；G、AhWRI1候选基因在高油载体材料CY1016种子不同发育时期的qRT-PCR表达；H、AhWRI1候选基因两个不同转录本的AP2结构域预测；I、AhWRI1候选基因过表达转拟南芥植株、果荚和种子表型鉴定；J、AhWRI1候选基因过表达转拟南芥果荚长、果荚宽、种子长、种子宽显著性分析；K、AhWRI1候选基因过表达转拟南种子含油率显著性分析；L、AhWRI1候选基因过表达转拟南种子脂肪酸鉴定；ns、*和***分别表示t检验P>0.05、≤0.05和≤0.001)；
图3是本发明实施例中高含油率和低含油率材料含油率表型(A)及AhWRI1-KASP基因型验证(B)；
图4是本发明实施例中基于AhWRI1-KASP分子标记辅助选择的花生高油定向育种技术流程。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明的实施例及附图，对本发明的技术方案进行进一步详细地描述，显然，所描述的实施例仅仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
需要说明的是，在不冲突的情况下，本发明中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。
实施例1：
基于重组自交系群体连锁作图定位花生含油率QTL：
本实施例1利用一个含油率差异显著的两个亲本，分别为J7(含油率47.5％)和K17-15(含油率54.1％)，构建了杂交组合并通过单粒传获得了一个包含218个个体的F9代重组自交系RIL-JK。对该重组自交系进行了两年的含油率表型调查，群体两年平均含油率表型基本呈正态分布(如图1所示)。基于illumina的Hiseq X10平台开展群体的全基因组重测序，测序深度～10X。下机数据通过数据过滤，利用比对软件BWA将数据比对到参考基因组；利用Picard对比对结果进行排序并标记重复序列；通过GATK进行群体SNP-InDel变异检测，去除缺失比例高于10％的位点、去除杂合比例大于20％的位点、去除严重偏分离位点，最终获得14.4万个位点用于后续分析；统计每个样本的基因型缺失比例和杂合比例，基因型缺失比例大于30％的样本视为异常样本并去除，最终保留了200个分析的样本；使用Lep-MAP3，构建了一张包含20条连锁群的高密度物理图谱，总图距3328.4cM，标记间平均距离为0.024cM；结合重组自交系群体两年平均的含油率表型性状，使用R/qtl软件按照指定的步长进行QTL扫描，以LOD＝3为阈值标准，分别在连锁群A07和A08定位到2个含油率QTLs，分别命名为qOilA07和qOilA08，其中qOilA07的定位区间为0.0-8.0cM，贡献率为21.1％；qOilA08的定位区间为191.41-191.91cM，贡献率为18.4％；(如图2A)。
实施例2：
基于自然群体全基因组关联分析挖掘花生含油率关键基因：
本实施例利用一个包含390份花生种质资源材料构成的自然群体，开展了测序深度为～10X的全基因组重测序。经测序数据质量控制后，获得数据12.95Tb的测序数据，平均测序深度为10.95X，平均基因组覆盖率为97.21％；通过GATK、SAMTOOLS、ANNOVAR等软件将测序reads比对到参考基因组，并进行群体SNP的检测和注释，共获得880万个SNPs，平均密度为3.45个SNP/Kb。结合自然群体的含油率表型，利用EMMAX软件，采用混合线性模型，进行全基因组关联分析，以关联位点的-log10 Pvalue＝6为显著性阈值筛选标准，总共在5条染色体上找到32个显著性SNPs关联位点(如图2B)。其中，A08染色体关联到的位点SNP_44514436位于实施例1定位到的qOilA08附近，进一步验证了该定位区间的准确性。通过对该区间进行LD连锁不平衡分析，可以看出该显著性关联位点位于74.2Kb的LD block区间内，包含6个候选基因(如图2C)。对这些候选基因进行变异注释，其中3个候选基因的外显子存在非同义SNP变异；通过对这3个候选基因进行同源注释，其中1个基因AhWRI1与拟南芥WRI1(座位号为AT3G54320)具有高度同源性。在拟南芥中，WRI1基因具有AP2/EREB特殊的DNA结合结构域，参与调控拟南芥种子的油份代谢(Kong等，2019)。此外，该基因同样在油菜、大豆、玉米等作物中也有参与调控种子油份含量的相关报道。
进一步，通过对AhWRI1基因变异分析发现该基因在外显子区存在一个G/T非同义突变，导致氨基酸从精氨酸(Arg)到甲硫氨酸(Met)变异(如图2D)。该非同义突变的两种基因型分别为GG和TT，其中GG基因型可显著增加群体含油率，属于优势等位基因型(如图2E)。
实施例3：
花生含油率基因AhWRI1的表达验证、基因克隆和功能分析：
在研究材料中，分别挑选携带GG和TT不同基因型并且含油率差异明显的两份研究材料，编号分别为CY1016(含油率53.4％)和GH4238(含油率46.5％)，进行后续的表达验证、基因克隆和功能分析。
AhWRI1基因的表达分析：利用Plant RNA Kit(Omega)试剂盒参照其说明书，提取不同组织的总RNA；利用PrimeScript RT reagent Kit试剂盒，参照其说明书，将RNA反转录成cDNA模板；设计两条qRT-PCR引物，分别为AhWRI1-RT-F：GCAGATCCAGCAAATTTAGGTAAC和AhWRI1-RT-R：GTCATCCTCCAACCCACTACC，利用SYBR Green Master Mix试剂盒，参照其说明书在ABI VIIA7荧光定量PCR仪中进行扩增，反应条件为94℃2min，94℃15s，55℃15s，68℃30s，共45个循环；以2-ΔΔCt法进行数据分析。根、茎、叶、花、果壳和种子6个不同组织的qRT-PCR表达结果显示该基因在两个品种的种子中高度表达(如图2F)。而种子的5个不同发育时期的qRT-PCR表达结果显示该基因在种子发育快速膨大期表达最高，并且基因表达量与种子增大趋势保持一致(如图2G)。
AhWRI1基因的克隆：利用Plant RNA Kit(Omega)试剂盒参照其说明书，提取GG优势等位基因载体材料CY1016叶片总RNA，利用PrimeScript RT reagent Kit试剂盒，参照其说明书，将RNA反转录成cDNA模板；利用克隆引物对AhWRI1-F/R(SEQ ID NO.7/8)，以该cDNA为模板进行PCR扩增，将扩增产物回收纯化通过BsaI-HF酶切后，再利用T4 DNA ligase连接入过表达载体pEGOEP35S-H，构建成pEGOEP35S-H-AhWRI1过表达载体。PCR体系为25μL，其中包括12.5μL 2X PCR Buffer、1.0μL cDNA模板、PCR引物各1.0μL、1.0μL DNA Polymerase、8.5μL H2O。PCR反应程序为，94℃2min，94℃15s，55℃15s，68℃30s，共45个循环。克隆的基因AhWRI1共发现两种转录方式，第一种转录本(AhWRI1.1)全长1092bp，序列如SEQ ID NO.2所示，编码363个氨基酸，序列如SEQ ID NO.4所示；第二种转录本(AhWRI1.2)全长891bp，序列如SEQ ID NO.3所示，编码296个氨基酸，序列如SEQ ID NO.5所示。
AhWRI1基因功能分析：根据同源基因注释显示花生AhWRI1与拟南芥基因AT3G54320具有高度同源性，属于乙烯响应转录因子WRI1，其蛋白具有AP2特殊的DNA结合结构域。通过SMART数据库(http://smart.embl-heidelberg.de/)对蛋白结构域分析表明AhWRI1的两种转录本编码的蛋白均具有AP2结构域(如图2H)。在拟南芥中，WRI1参与调控种子的油份代谢；在油菜、大豆、玉米等作物中该同源基因也同样参与调控种子油份含量；并且通过在大豆中表达拟南芥的WRI1基因可提高大豆棕榈酸的积累，而通过在拟南芥中过表达油菜的wri1-like基因可提高拟南芥种子含油率和生物量。
进一步，本实施例利用35S为启动子构建过表达载体，通过拟南芥浸花法进行转化，利用潮霉素筛选、目的基因qRT-PCR和GFP荧光观察，筛选获得拟南芥阳性转基因植株。具体步骤包括，将AhWRI1基因克隆扩增产物通过BsaI-HF酶切后，再利用T4 DNA ligase连接进入过表达载体pEGOEP35S-H；将连接产物热激转化感受态大肠杆菌DH5a，涂布卡那霉素抗性LB琼脂平板，挑取单克隆进行PCR检测并将阳性克隆进行测序检测；选择测序正确的菌株摇菌，提取带有目的片段的质粒，即是AhWRI1过表达质粒pEGOEP35S-H-AhWRI1；将其转化农杆菌感受态细胞，涂布卡那霉素和利福平的YEB平板，挑取单克隆后通过PCR检测阳性后接种于4ml液体LB培养基中，28℃暗培养条件下，摇菌过夜18h；取过夜菌1ml，接种于100ml的LB培养液中，28℃暗培养条件下，培养过夜；用紫外分光光度计检测上述菌OD值(OD600在1.2～2.0之间)，倒入灭菌的50ml灭菌EP中，5000rpm，28℃离心8min，收集菌体；将拟南芥的花蕾部位置于含有农杆菌渗透液的EP管中5min，侵染结束放于真空干燥器中，之后在黑暗条件下培养24h后正常培养；将T0-T3的阳性株种子种植在潮霉素抗性MS平板，每一世代结合目的基因qRT-PCR和GFP荧光筛选，直到T4代阳性株；将阳性株种子点样在营养土，置于4℃冰箱进行春化3d，之后放置在22℃光照培养箱，设置5000lux光照强度，80％湿度，16h光照，8h黑暗，待结荚后收取种子进行表型鉴定。过表达AhWRI1转拟南芥结果显示转基因阳性株比野生型对照具有更大的植株生长量，并且提早开花(如图2I)；转基因阳性株的果荚和种子长度显著大于野生型对照，但果荚和种子宽度差异不显著(如图2I和J)；在种子油份含量上，过表达AhWRI1转基因植株种子的含油率比野生型对照显著提高了～4％左右(如图2K)，并且具有更高比例的不饱和脂肪酸含量(如图2L)。由此可见，AhWRI1过表达验证了该基因通过参与植物油份代谢具调控植物含油率，是控制花生含油率的关键候选基因。
实施例4：
花生含油率基因AhWRI1的KASP分子标记开发及验证：
根据实施例2分析结果表明AhWRI1基因外显子区存在一个G/T非同义突变，导致氨基酸从精氨酸(Arg)到甲硫氨酸(Met)变异(如图2D所示)，其中GG基因型可显著增加群体含油率，属于优势等位基因型。在该变异位点两端150bp侧翼序列设计引物，开发KASP标记，其中正向引物分别为AhWRI1-KASP-F1和AhWRI1-KASP-F2，引物序列分别为SEQ ID NO.9和SEQID NO.10所示，通用反向引物为AhWRI1-KASP-R，序列为SEQ ID NO.11所示(如下表1所示)。选取实施例1和实施例3中的4个研究材料，其中高含油率材料为K17-15(54.1％)和CY1016(53.4％)，低含油率材料为J7(47.5％)和GH4238(46.5％)。分别提取苗期叶片基因组DNA，利用AhWRI1-KASP标记引物对提取的基因组DNA进行PCR扩增。扩增产物利用KASP荧光分析仪Pherastar(LGC公司)，参照其操作指南，扫描并分析PCR结果。PCR反应总体系为5μl，包括基因组DNA 2.43μl，2×KASP Master Mix 2.5μl，KASP Assay Mix 0.07μl。其中，KASPAssay Mix包括，FAM荧光结合的特异序列正向引物(F1)、HEX荧光结合的特异序列正向引物(F2)、通用反向引物(R)和纯化水。PCR反应程序：第一步，94℃，15min；第二步，94℃，20s，61～55℃梯度PCR，1min，每个循环降低0.6℃，进行10个循环；第三步，94℃，20s，55℃，1min，进行26个循环；第四步，10℃保存。AhWRI1-KASP基因分型结果显示高含油率材料基因型均为GG型，低含油率材料基因型均为TT型(如图3所示)。
表1花生含油率基因AhWRI1的KASP引物序列
实施例5：
高油花生新品种培育的KASP分子标记辅助定向选择方法：
分别以实施例3中的高油材料CY1016(含油率53.4％)和低油材料GH4238(含油率46.5％)为父母本，开展高油杂交分子标记辅助定向育种(如图4所示)。利用单粒传的方法进行F2、F3、F4各分离世代的种植，直到F5代共获得311个单株；利用实施例4开发的AhWRI1-KASP标记及其基因分型方法，对这311个单株进行基因分型，保留具有GG优势基因型的单株，淘汰GT杂合基因型和TT弱势基因型单株，总共保留98个GG优势纯和基因型单株；这些保留单株在F6代种植成98个株系，收获后进行含油率测量，淘汰含油率低于50％的株系，总共保留21个株系；这些保留的株系在F7代种植成21个无重复小区，每个小区为一个品系，收获后进行含油率测量，淘汰含油率低于50％的品系，总共保留4个品系；这些保留的品系在F8种植成4个小区品系，每个品系3次重复，评价其含油率性状；最终获得的4个高油花生新品系含油率分别为53.6％、53.8％、55.4％和55.8％(如图4所示)，平均含油率达54.7％，比高油和低油亲本的含油率分别提高了1.3％和8.2％。这表明本发明开发的AhWRI1-KASP含油率标记可用于花生含油率性状的定向辅助育种。
根据本发明的上述实施例，本发明中提供的一种调控花生含油率的基因AhWRI1准确可靠；基于该含油率基因SNP变异位点开发的KASP分子标记方法可用于花生含油率分子标记辅助定向选择育种，技术可靠、经济、简便，有效的提高了花生含油率性状的育种效率。
以下为上述实施例中涉及的序列：
SEQ ID NO.1
ATTCACTGTTCTATTTTCCTTTTCTACTTCCTTTCCCTAGTTGTTTTCTACCTTGACTTCACTTCTTCTTAAGTTCCCACCACTCTAACAATAATCTTGACATTTTAATTtcagaaaaagataaatttatttatttatttatgttctcCCATTTGCCTAGTTATCAACTTGTCCAACTTTTTTATtattccttcttcatttcttaGTCTATCTCTCTGTTTTGGTAACCAACCTTAGGTTACCAGAAAAAGCTGTTAAAGCTGTTtcgatattaaaaatataataaacaatTACAATATTTACATTACATTAGTATTGTTTCTGTTTTTTATCCTAATGGGTCGCCTTTCATAGTTTCTACTGCTACAATTGAAGAATTGCTCTCTCAGATCTGTTAGATTTTGGATTTCATACTAGTAAGATCAGTTTAGTGTTCATCTTCATTTTTATTTACAttgtttcatcttcttcaatttattatgggttcttatttaattttttggcaATAGAGAAACTAGCAAGGTCTTATCAACAGTGATGATTGTGCACAGGACAGCTGCAAGGACAGAACCTCACACTGTTAGAGCCATCTAACCATTTATACATTTTTATCATCAATTACTTTTACTTGTTATATGTGGTTTAAAGAGAGGTCCACATGCACCAGCAGTGCATTACTCTCGGTCATTTTTAAGGTGCATGACAGAAAGCCCTCATATATATAGAATCTACTAAAACAGtcataaaagagagagagagggagttgaCACACATTCAAAGGCATTTTGTCTGTTCCgataaagaagaatagaaaacagaaaatacaCATTACATAGTGCaatattttcttcttattggacgaagagggtggtggtggtggtggtgagatGAAGAGGTCTCCTAAttcctcttcttcatcttcatcatcttctgtTGGTGTTGGTTTTGAattaaagattaataataatgataagtATCCAAGAAGcaaaaatagcagtaataatgtGAAGTTATCACAGAATAAAGTATTGAAGAAAACAAATAGCTATGACAATAGCAGTGTCAACAAAAGAAGCTCCATTTACAGAGGAGTAACCAGGtttgatttcatttttattttcatttgcatGTCTCTATTGACAGTTTTAATAATCAATAATATGTTTAATGATCCATAGGCATAGGTGGACAGGGAGGTTTGAAGCTCATCTGTGGGACAAGAATTCTTGGAATAATATTCAGAGTAAGAAGGGACGACAGggtaattaaattttttcattataCTATTTTCACTCTTATTTGTCCATCTTCATTTACTGTTTCAtaggtttattatttatttataaattttattaaactaaACTTACTCATAGCGCTTACAAACATAATGTATTATgatgaaaattgtttttgaatacAACTATAGAATTGTATTTTTTGAaatgataaatattttaatagatgtgtataaaaaaagtttaatttaattattcgaCGTACTTAAGTTTTATAAATTacatttgttatatatattttttttgcataATTTCTTCTAAACTGTACTCAATTTTGTTATATGATTTATGTTATcggttgatttttctttttttataattttctgtcCATTTCATACGGTCACAAAAATAGACAATAACAATGTTTTTGTGATCTTTACTAGCCAATTTAGAATCTTTACTATATTTGAATAACATTGAGTACCTGAAGAAagttttgaaacaaaaataagatttgaaacaaaaataattgcttgTCAGATCATTAATAATTGGATTTTACTAAGGTATTTGTTTATATATTGACATTCTTAAAAATTAGGATAATTCGATTATTTAAAAATTGAGAATATTCTTTAAAAACAATTACAATTCCTTTTAACAGCATTTACATGCACAAtatattttccattattatttgttatttttaaaattaatttactattaagctattaataaaaatgaattagattaaataaattaGTTAACAACTCAGCTAAGATTTAATACATATGACTTCTGCAAACATATATTAAAGTTAGAAATAAAATATAGatagtaaatatttttaaattattattcaaaTAGAGAATCCTACCTAAAGATGAATAGAATGACCATAAACATTTAAAGAGTTGCATTAGTCATTCTCTTAAATTATAGTGtattcaaaacatgaatgaaCGAATAAAAAGCAGTGGAAAATCAAGGAGATCTGTaatgttttgttttgttatttaTGTTAAGTGTTTTGACTTTATCCTGTGTTTTCACTTGCCATGTTTTAATCTGTTTATGACAATCTCTGCCATGACAACGTTCACATACACTTCTGTCGCTGCTTTAGTTTATTTAGGTAAGTCTCCCTAATAAATCTTGGATCTCACACAAGCTTtcggttttatgaatttttaattctaatgatatacttttaattatattgagtttttttacaaatttgggaaaatatatgttatattttttttaaaacaaaaaaatattaacttaCTTATTGTGACTTTACATGTGAATTTAGGGGCATACAATACAGAAGAAGCTGCTGCTCGCTCCTATGATCTTGCTGCTCTAAAGTATTGGGGACCTAATACAATCTTAAATTTTCCGGTATACTAATACATACATGCATATATGTATAAGAGGTCGCGAgttcgaatttttttatattttataaaaaaaattatttgtgaatAATTATGCTGCTTAGGAAATACTTAACAATTTGAATGATATCTTCATTAAaaattatagtaattttttttttaaaaactaacgagttataactcaaatgacatagtcttttttggtatttgatatttttattttcgatGTTTTATCTAAACGGGATTAGATTATTGGTAtttggtatttttatttttaatatttttgtttttaaaatgttgtaattttttttttacaaaatcctaaaaaattaaaatacaatccaAGCATGCAATTTTTGTTACATTGAAAGccaatttttcttatattttgggaagattatatataacttataattaaTTAACTTTGGATGAACAATGCAGATGGAAACATATGCCAAGGAAATTGAGGAAATGAAGGATGTTTCACAAACTGAATACTTGGCTTCTCTGCGAAGAAGGAGCAGTGGATTTTCAAGAGGTGTTTCCAAGTATCGTGGAGTGGCTAGGTTAGTTTATTTATACATCaccttttttatataaataatctttttttattatactaTTCATTCAAAAAATTGTGCAAGCATGAAGCTAAGATCATTTACATTCCTAATACACAATGCACGCGATACAATATTCTAaacgtattttaattttaaagtatttttatttctatcggtacactttatttatttatttttagcaaaaattacatttttttaaaatgttaaaatgacatttttttctcttataacttttaaaattttttttttaatccattttaaattttttgtaactctatccatttttttaaattattttttataaaaatatctttttacaaatttttttttgtgattaaattttgtttatcaaaataccttttaacaaattatttttttactgattaaattgtatttttactaaaatttctttaaaaaattttaatgactAAATTATGTTTTCATAAGATAACAAATGATCTTTTTAATgttcaaattattttttcaaaaacacaAATGGCAATCAACCTTGCTTCTGAAGGTTGTCTATGGAAGATCCATGGTTTTTCTTctccaaatttttaaaagatattttggtataaatacaatttaatcaataaaaaataatttgttaaaagatatttcgaaaacaaaatttaatcaaaaaataaaatttttgctaaaaataaagaaaaaaatatcatgttaaaatctctcaaaaaaaaaaagtgaaattttttcttctttttattgaaatatttcTGGATTTTTGAGTAGGCATCACCACAATGGAAGGTGGGAAGCACGAATTGGAAGAGTAAACGGCAACAAGTACCTTTATCTGGGGACCTATagtaagtaaaaaaaattttcactatTCCCTAAATATGCATGTTCCTAAACTCAACATTGTATCATAGGAAAGTGCTaagttaaaaaaaagaattacATCATAACTTACAAAATTTTAGAGTATTGTTATGtggctaataattttttttgaccaaaatatatgtatttgtttgaaaaataaatgaaaaagataataaagaagaaatagactacatattaattaaattaagtttGTTTAAATGTTAGTTAAAAAATAGAGGATTCCAATCATTTGTGTAAAATTTAAGCAACTAAGGATAACAGTTTAGCAGTGAAAACTGAAAATATACCATACACAAAATTTtaggtttaaattttttatgatggttgttaaaaaaattgaatttgaaagaaTGCAAAGCAACCTTTGTATTTGTCAAATATGCCGAATGATTCTGCAAGTGCAGTTGGCAGACTACTAAGTACCAAACTGCCCGTTTGTCTGTGACTCTGTCCCCACACCAACAATGCTCAAATACTTTATATAGTACTTTATAATGTTTAGACTTTAATTAAGTTAAGATTACCTCATTAACGCTATTTGACTATTTCAACAATTCCATTTAATGTTATCATTAAATTGTACCATGGTCCACCTGTGCTACCAGAATACATTTATACTTGATcggattaataattaaattaatctctaaaaaaatgagatgtttttaaatttattttaaaaagtttttttaattaaattaattattcaaagattataaatcaattatatttattttttagtgattttattaacaatttttatcaacaattaataataaaaaaacgttAATTAACAACATACATATACTTGATATGTCTAATTGAacgttaattaatatatttatcaaaatttattaatttaatctctttttttaattatataatctaTATTCGAAATATTTTCAATATAACTTAatgaataaattgataaattttcataaacatatatGATTTACGTTTAATTGAACATGTTAGGTATTATGTATGCTATCAATTAATATTTTACGTcattaattattgataaaaattgTTAAGGAGTATCTAAATGATAAGtatgattaatttgtaattttaaacgaccaattaattaaaaaaatagaaaataatttaaaaaatattttattttttaaagcaaaataaaataataaataataagaatttgCATGTTGTTGTGTTAGAggcacatttaagtaaattaatGTTATAAaaggttttctttttcatttgtttaAAAAATGTTCAGTGTGGCAAATCTGAGAGTTTTTAAAGTTAGTTGATAATACCTAAAATAAAGAGAGGGAAATTTAAATGgtataaaaagatttttatttaCCTAAAACAAAATAGAAATTAGCATGCAACAGGTGGAAACGGTACAGGGACACGTGCATTCCCACTTTCACATGTTGAATGTTGATGGATTCAACGTGTTCAAAAGTTGATAAGCACTTTATCATGAATCATAATTTCCTATTGTTCTATTACAATTACAAATCAAGCATTTTAATCTTAGTAAATGCTACAGTATTTAAAAGGTGGtgtttatttattacaaaaattaaaaaataatatttaatttaaaaatataacaataaataatttaaaaaaaattaaaatttattataaaaaataaattaaacaaaatttaaacaaTAATTCATAGACACTATAAAATTTGCCTTTAATCTTTTGCAACCTTACACCATCTTTCCCATGAAAATATTGTGGTATCTCTCGAGGGAAAAAAAGTTGTCTGATTTATtgccaataatatataattttaaaaaaatattgctaAAGCTAAGGTTAATATAAGCATTAAGAAAATTATttagtaaattatttttcaactaacattcggtcaattttttttctctctcctttttctttcatatctctttcaagttgaaagattttttttttttttagattgaaGTAGTAAGAGTAACTCCAATTCATACATAAATATAGACTACacacaatataaatatataaaaaagagagaagggAGATTGAGAGAGTCCCTTTCTTTATCGATAATATATATGGACTAATATTGGTCCCATATTGAGATTTTAcatgtattaattaaaataacaataattaatatttttaaataatatgttattattttattataataaataattaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaatatttattaaatttttaataagtttataattaataaaaatttgaaaataaacataaaacaatGTATACACATTTACTCCAATAATTCTCTATTTCTATCCCTATTACTCTTCTCTTTGTCCCTAATTATCTGCAGCAGATAATGTTTTTAtcctttatatattattaaaaaaaggaatgcactaattaatttagtgttAGACTCAACCAAAACATTTAGAATGTATATTAGATAAatgtgtttattattttttttaattgtaataataatttttttcactaattaattaattcatgGCTAATGATCTAAACCCAATAAAACTTAGAATATCAAAgattttttctttattaaataGAAATTGACTACTACTAAATAGTATTATTTTATGATTAGTGGTCAGGATCTATCCAACATTTGACTAGATTTTATAGATCTTATTATATCATCTTAATATTATATATTGAGTAAActttaaagtatttaaattttgtattaaaataatttttaaaattgtaattatatcaattatatttttttttttttttttattttatattattaaaaatagaataaaatattttttgtttttaaaatttgtaaagttaaaaaatattttaagttttttttaattttgtccaaaaaaataaaaatttttaaaaaaatttataactatagcaataattttataataacaaacaaacaagaaatcaagaaagatttgatcttctaaattttgattttcactttaaaggacaaagtgtgatctctcaccatttattttataggtaggaataagagaaaatataagaaaaaaaaaactattaagaGTAAAAGACACACTTTATCctataagtaaaattttaaatttagaggatccaaattcaaatcaaatagttataaaatatattattgttggattagtcCTAAATTTTTTCAAAGTTTAATTGTCAGAACGATATTTagtacaaataaaatttttttgggatataattaaaataaaataaaattagaagtatttttaaaattgttgacaaactaaaaaaatatactttatcttaaaaatataatctgtataattaaaatttgacagactattttaatatatatggtcaatttaaagaattattttagaaaataactCTAATATTATTAGTATTAGTATGTGAGATTATATACTATGATCTAGAGTTCAGTCAAAAAAATAGTTTATGAAATTGTTAATGTTTAAAGATGAATTATTAAAATACAATATttgatttagtttaataatttaatgttAGACACAAAAATATCTAACTTTgcataaactaaaataattaatgaacATCTTTGGAGGGCACGTGACTTCTACGCCTCCTAGTCGTAGTACTGCAAGTAATAACAAACTTTATGTGATTCTCTgtaataaacaattaaaaaattagatattattatgaagattttataattatattcatatgaaaatatttttcttttactattaaacaataaattataaaatttaattttgatatgttataaaagtgtgTTTTTTTAAAGTGTGACGAAATAAACAAAGCCACACtcttaacacaaaaatttttatgaaaaaatatttttagtattttcatttGAGTAAAtaccttaaaaaatatatttttttaactttattcTTTTATCAATAAATTAGCTACTAACCCTCTCTTTAagctattatattttttaatttgatttttgatatTGAAGACACTCAAGAAGAGGCGGCAGCAGCATATGATAGAGCAGCTATAGAGTACAGAGGAATAAATGCAGTAACAAATTTTGATATAAGCAATTATATTgacaaaataaagaagaatgagccGTCTATGAAAGAAGCACAAACACATATAACTCCAATCTCTACTGAATCTAAAGAAGCAGAAGTAGTGCAGCAGGAGGCTCCTTCCACAGTAGTGGCAGATCCAGCAAATTTAGGTAACCAGAAAAAAGGTAATataatacatatattatatattatatgtttaattaatatgttaatttctataattttattaaatttttaatttagatttttatatatattttttaattgaatctttacattattttttattttgtaattaggtcattttagtaaaaaaatattaaagctaattaaatatttttttataaaatatatgcgGTCGAGATTTAGTTATGTCAAATGTATTTTCATGAACTTTAGTTCATGTGCAATTTGTCTCCAAAATTTTTTAACAGTGCAATTTGACGCAAATTTGTTAAATGGTTGGATTTTTATTTACACTATTTGAAAACTTCAACAATTAATTTCACATTTTCTATGATAAAAATTTACATTTGACAatagatttggatcctctaaattttgaatttttattttagaggataaaATGTTATTTTCTATCCTTAAATGGTTTTTATCATATTTACTCTTGACcccacttataaaataaatgatgagaGATCATAtttattctctaaagtgaaattcaaactttagaggatctaaataCTTTGACAATATcaagtgaaaaaaattaaaaaagatctaTATCTATGCAGAAAATATGCAGAAGAAAAAAGAGCAGCCCAATACAGATATCAACGGCATACTTTCTCGGGATCTGAGATGGAGCTTCATGGATGCCGAATTATCCGAGTTCAACATGAATGATATTTTTCAAGGTGAAGGTAGTGGGTTGGAGGATGACATCAATCTTATGTGTATGGCTGATCCTGATGAGAGTAGTTTCAATCTGAACACTATCTTTGATGATCCTGGAAAATGTGGTGGAGATATGCATGTCTATGCCAATTATTAAGAAGCAAAAGTTCTCCTTCTTCTAATATGTGATCTCTTCAAGAAGCAAAAGTTCTTGTCCTGTTCTAGTATGGTTCTGTTTGAAAAATTGTTGGAGTTTGTTTTTCAGTTGTTGGTATCTGTTGTGATAGgtgaagaaaaaacaaaaaaataaaaaaataaaataaaaaaatgttaaagcaGTGTTGTTATTAAGGTAAGGATCGAGGTGAATGGATATCTATCTTTAGATCAATAATGTATGAAaaacttgaataaaaataatt
SEQ ID NO.2
ATGAAGAGGTCTCCTAATTCCTCTTCTTCATCTTCATCATCTTCTGTTGGTGTTGGTTTTGAATTAAAGATTAATAATAATGATAAGTATCCAAGAAGCAAAAATAGCAGTAATAATGTGAAGTTATCACAGAATAAAGTATTGAAGAAAACAAATAGCTATGACAATAGCAGTGTCAACAAAAGAAGCTCCATTTACAGAGGAGTAACCAGGCATAGGTGGACAGGGAGGTTTGAAGCTCATCTGTGGGACAAGAATTCTTGGAATAATATTCAGAGTAAGAAGGGACGACAGGGGGCATACAATACAGAAGAAGCTGCTGCTCGCTCCTATGATCTTGCTGCTCTAAAGTATTGGGGACCTAATACAATCTTAAATTTTCCGATGGAAACATATGCCAAGGAAATTGAGGAAATGAAGGATGTTTCACAAACTGAATACTTGGCTTCTCTGCGAAGAAGGAGCAGTGGATTTTCAAGAGGTGTTTCCAAGTATCGTGGAGTGGCTAGGCATCACCACAATGGAAGGTGGGAAGCACGAATTGGAAGAGTAAACGGCAACAAGTACCTTTATCTGGGGACCTATAACACTCAAGAAGAGGCGGCAGCAGCATATGATAGAGCAGCTATAGAGTACAGAGGAATAAATGCAGTAACAAATTTTGATATAAGCAATTATATTGACAAAATAAAGAAGAATGAGCCGTCTATGAAAGAAGCACAAACACATATAACTCCAATCTCTACTGAATCTAAAGAAGCAGAAGTAGTGCAGCAGGAGGCTCCTTCCACAGTAGTGGCAGATCCAGCAAATTTAGGTAACCAGAAAAAAGAAAATATGCAGAAGAAAAAAGAGCAGCCCAATACAGATATCAACGGCATACTTTCTCGGGATCTGAGATGGAGCTTCATGGATGCCGAATTATCCGAGTTCAACATGAATGATATTTTTCAAGGTGAAGGTAGTGGGTTGGAGGATGACATCAATCTTATGTGTATGGCTGATCCTGATGAGAGTAGTTTCAATCTGAACACTATCTTTGATGATCCTGGAAAATGTGGTGGAGATATGCATGTCTATGCCAATTATTAA
SEQ ID NO.3
ATGATCCATAGGCATAGGTGGACAGGGAGGTTTGAAGCTCATCTGTGGGACAAGAATTCTTGGAATAATATTCAGAGTAAGAAGGGACGACAGGGGGCATACAATACAGAAGAAGCTGCTGCTCGCTCCTATGATCTTGCTGCTCTAAAGTATTGGGGACCTAATACAATCTTAAATTTTCCGATGGAAACATATGCCAAGGAAATTGAGGAAATGAAGGATGTTTCACAAACTGAATACTTGGCTTCTCTGCGAAGAAGGAGCAGTGGATTTTCAAGAGGTGTTTCCAAGTATCGTGGAGTGGCTAGGCATCACCACAATGGAAGGTGGGAAGCACGAATTGGAAGAGTAAACGGCAACAAGTACCTTTATCTGGGGACCTATAACACTCAAGAAGAGGCGGCAGCAGCATATGATAGAGCAGCTATAGAGTACAGAGGAATAAATGCAGTAACAAATTTTGATATAAGCAATTATATTGACAAAATAAAGAAGAATGAGCCGTCTATGAAAGAAGCACAAACACATATAACTCCAATCTCTACTGAATCTAAAGAAGCAGAAGTAGTGCAGCAGGAGGCTCCTTCCACAGTAGTGGCAGATCCAGCAAATTTAGGTAACCAGAAAAAAGAAAATATGCAGAAGAAAAAAGAGCAGCCCAATACAGATATCAACGGCATACTTTCTCGGGATCTGAGATGGAGCTTCATGGATGCCGAATTATCCGAGTTCAACATGAATGATATTTTTCAAGGTGAAGGTAGTGGGTTGGAGGATGACATCAATCTTATGTGTATGGCTGATCCTGATGAGAGTAGTTTCAATCTGAACACTATCTTTGATGATCCTGGAAAATGTGGTGGAGATATGCATGTCTATGCCAATTATTAA
SEQ ID NO.4
MKRSPNSSSSSSSSSVGVGFELKINNNDKYPRSKNSSNNVKLSQNKVLKKTNSYDNSSVNKRSSIYRGVTRHRWTGRFEAHLWDKNSWNNIQSKKGRQGAYNTEEAAARSYDLAALKYWG PNTILNFPMETYAKEIEEMKDVSQTEYLASLRRRSSGFSRGVSKYRGVARHHHNGRWEARIGRVNGNKYLYLGTYNTQEEAAAAYDRAAIEYRGINAVTNFDISNYIDKIKKNEPSMKEAQTHITPISTESKEAEVVQQEAPSTVVADPANLGNQKKENMQKKKEQPNTDINGILSRDLRWSFMDAELSEFNMNDIFQGEGSGLEDDINLMCMADPDESSFNLNTIFDDPGKCGGDMHVYANY
SEQ ID NO.5
MIHRHRWTGRFEAHLWDKNSWNNIQSKKGRQGAYNTEEAAARSYDLAALKYWGPNTILNFPMETYAKEIEEMKDVSQTEYLASLRRRSSGFSRGVSKYRGVARHHHNGRWEARIGRVNGNKYLYLGTYNTQEEAAAAYDRAAIEYRGINAVTNFDISNYIDKIKKNEPSMKEAQTHITPISTESKEAEVVQQEAPSTVVADPANLGNQKKENMQKKKEQPNTDINGILSRDLRWSFMDAELSEFNMNDIFQGEGSGLEDDINLMCMADPDESSFNLNTIFDDPGKCGGDMHVYANY
SEQ ID NO.6
gtttttctctctaaatgacttgatgaaaaacaaaaaagaaacttAATTTATAGATAAACTTCTTGTATGTAAACTACTGGTAGCATAGCAACGATCTTGTTTCTCTATTCTATACCAAACAATTAATAATTACTATTAATTATGAGCTTCTCTCCATCCATTTGTTTACCATAAAAGTAGTATTAGCTTTCTaactacaaaattaattttattatttttaccctCTAAATCACCATCTAAGTTATAATTTTCTTCACCCGTAttataaaatagatttttttcttttatcatgtCCATGTCTAATGTATGATAGTAATTGGATACTGACAACAAGACTGGAATTGGATGTGGGACAAGTAGAAGATCAATGCCGGAGCTTGAAAGGAATTTTTGGAGggactaaaaattttattaaaaaattatataataatatttatattaaaataaaagctataaatttaattattttatttttaaatttattaattaaaatataggaTCACATGTGGTTAAGATTAACTAAAAAAGTAAATCTGATTTTCGTAAAAAAAAGTTGTTTAGATTAATaaaccaatttgattttaaataaaaaagccaatttaaaaaaaattactttttatatgGAAAAATTTGTTTTTCCCCGTAAAAacctatttttatttcaaaaaatcatttttaatctaaaaattgatttttttttcaaaaaattgattttttatttaaattatacaaaaacgCTTAcgacttataaattattttgtaatatttaaaaaaaataattttatttttgataatatttatctcTCACAAGTCTCAAGAGTAATTATCTTTGATATTATTTTCATtacaatcaaataatataatacaataaaataaaatgttaaatttttaataaaataaaaatatcaaaatttattaatataaacatTGTTGATACTACAATAACATTTAACTTATAatattgatttaaaataaaatattcactTTTTATTAATAACTGATATTATTACTAGTTATACTCATATACTGTAATTTTCATTATTCAAACTTGTTTTTCTtagaagtcaccaaaaaaaaacttGTTTTTCTTAGAGATAGAACTACTTACAAAAGTTTTGGGGGCTGTGACCCCATTGTCCTAACAAAACTCCGCCACTGCAGAAGATACCGACGTGATAGCTCGACCAGGGTCTCCGGCATAAACTCCTCCTCGTCATCATCTTCTGAAGAACCGTTATCGTTGCTATCCGCAATATATTCCTCGTCTGACCGTCAACGTCCATGTCATTCACTGAACTAGCAACGTGTATTGATGGTGGTGCTAAAGGTGGATTATCTTGCACAAAATTTGACTGCTTAGATCCACCGCCAGCGACATCACCAACTTCTGCAGAAAACTCCATCATTTGTTTGGCCATGATTCTTTCATGGAAGTCAAATATCAGTTGCACATGCTCGTCGTGCAGTCAAAACAAATCAAACGGAAAGACGCCAAAACAAACTAAACTGAAAGACTCCATTTTCTATTGGTGCCAGCAACCTATATCCTAACTATCCGATCACTTTTCTCCCACTACCAGCAACGCTACTCAATATCAGACTCTTCTGCTCCGACAATGAAGTAACTCGTCGGGTACGCAACAAAacggattttcacactaaaaaatGACCCATTATCACCATTACACACATAAAATTAAATATCTACTACTACTAGACATTATGGCtaatttggaaaagaaaataaacgtCATAATTTTCTACGTATTTTGTTTACCGTATAAATAAAACAATATTACATGTATTTGGTTTATACATAAACGAAATATATACGAAATACATAAACAAAATATAGACATCTGTGCATGTTAGTTTTATTTCAGTATAAACGAAATACATACGAAATAcatgtaatattattttatttatactgtaaacgaaataatacataaaatacaTTTTCCTAAAAacgtttaaaaatatttattttgataaataaaatatttatttaatttaaaaaaaaagcctATAAGCATGTGCCctctttattaatatttttatctgcataaacttaatatatatatatatatatatatatatataataatatatatatatatataataatatttttctacTTAATATGGATGTACAAATAGCTAGCATGAGGAactacatttttttttctatagcTTATTTTTTCTGTAGTTATttcctatatttttttaaatcatttacTTTTCAAAATTATTGTCATATAAATTGACTCCGTCATTATTATTTTGAGAAATTGAAATATTTATTGTAAAAAAGTCAGAGAAATTTCAAAAGTACAATTAcaaatttatatgattgagaacTATGAAATATTATCCAGTAACAAAATTATTGTAAGTTTAAAATCACAACCACTGCCATATTATACACATTAATAATATGTTTCTCTAATTGCAATACTTGATACATCAAATATACAACTAGTAAGTACGCATGGAGTACATATTTTTTATGCActctttattattaaaatatgttatttttgtattttgtgtgTTTTATAGTATATAcatgaataattttattattttaatttaataaattatattaatatttaaaatataacaaatagTCCGAGAATCTGATAATATGgttgaatttttataatttattaattttttggtaacttaacaaaaataaatttatatatgttagttataattatttataatttattatacaaatacaatatcttttaacaaagaaaatcaatcatttcatacaaaatttttaaataatttattcacATATCATGGTGGCAACGAACTTGGACAGATTCCCAAACAAGGAAAGCCAGCGTAGAACCCAAATCCTCATTGGTAGCATGTCTGCAGAGTGCCAAGTGTTAGAAACTATACACCAAGAGTAAGCGTAAAATTTCATCACCTATAGTCAATTAACTAACTCATCTTCAAATCTTTAACCCAAGTTCAAAACAAAACC
SEQ ID NO.7
CACCATGAAGAGGTCTCCTAATTCCTCTTCT
SEQ ID NO.8
CGCCATAATTGGCATAGACATGCATATCTCCA
SEQ ID NO.9
GAAGGTGACCAAGTTCATGCTGTGGTGGTGGTGAGATGAAGAG
SEQ ID NO.10
GAAGGTCGGAGTCAACGGATTGTGGTGGTGGTGAGATGAAGAT
SEQ ID NO.11
AATCTTTAATTCAAAACCAACACCA
以上具体实施例仅仅是对本发明的解释，其并不是对本发明的限制，本领域技术人员在阅读完本说明书后可以根据需要对本实施例做出没有创造性贡献的修改，但只要在本发明的权利要求范围内都受到专利法的保护。
Claims (2)
1.一种检测花生含油率基因AhWRI1的特异SNP的KASP分子标记引物组，其特征在于：所述KASP分子标记引物组中的引物分别命名为AhWRI1-KASP-F1、AhWRI1-KASP-F2和AhWRI1-KASP-R，所述引物序列分别如SEQ ID NO.9、SEQ ID NO.10和SEQ ID NO.11所示；
所述特异SNP位于花生基因组8号染色体上第44514436位核苷酸，所述特异SNP为G/T多态。
2.如权利要求1所述的KASP分子标记引物组在花生的高油性状的分子标记辅助选择育种中的应用。
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| CN117418036A (zh) * | 2023-11-28 | 2024-01-19 | 华智生物技术有限公司 | 一种与甘蓝型油菜含油量基因BnA05.OC紧密连锁的SNP分子标记及其应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000018963A1 (en) * | 1998-10-01 | 2000-04-06 | Monsanto Company | Methods for breeding for and screening of soybean plants with enhanced yields, and soybean plants with enhanced yields |
| CN111675756A (zh) * | 2020-07-03 | 2020-09-18 | 青岛农业大学 | 花生开花习性基因AhFH1及其等位变异的克隆与应用 |
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