CN113337536B - Rs2z32基因作为植物免疫负调控因子在提高作物抗性中的应用 - Google Patents

Rs2z32基因作为植物免疫负调控因子在提高作物抗性中的应用 Download PDF

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CN113337536B
CN113337536B CN202110405120.XA CN202110405120A CN113337536B CN 113337536 B CN113337536 B CN 113337536B CN 202110405120 A CN202110405120 A CN 202110405120A CN 113337536 B CN113337536 B CN 113337536B
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董莎萌
黄杰
王源超
吴宏伟
闫东
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Nanjing Agricultural University
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Abstract

本发明属于植物分子生物学与植物遗传工程领域，具体公开了RS2Z32基因作为植物免疫负调控因子在提高作物抗性中的应用，沉默番茄中RS2Z32基因可以显著增强植物免疫，提高番茄对致病疫霉的抗性，是一种理想的可用于改良作物抗性的易感基因。利用CRISPR‑Cas9等基因编辑技术在番茄、马铃薯、烟草、水稻、小麦、玉米、大豆、棉花等植物中敲除该RS2Z32基因，可以增强植物对不同病虫害和逆境胁迫的抵抗能力，从而提高作物的田间抗性。本发明可用于作物抗性改良方面。

Description

RS2Z32基因作为植物免疫负调控因子在提高作物抗性中的 应用
技术领域
本发明属于植物分子生物学与植物遗传工程领域，具体地说，本发明涉及剪切因子基因 RS2Z32作为植物免疫负调控因子在提高作物抗性中的应用。
背景技术
可变剪切是一种重要的转录后修饰过程，同一个基因mRNA的可变剪切可以产生多个 不同的转录本，可变剪切不仅可以增加转录的丰富性和蛋白的多样性，而且可变剪切过程中 产生的一些副产物也能参与基因的表达调控。随着转录组测序技术的快速发展，人们对可变 剪切的认识和理解也逐渐加深，在人类基因组中大约有95％的基因会发生可变剪切，在植物 基因组中会有60％-70％的基因会发生可变剪切。可变剪切是由剪切复合体来完成的，一般 认为剪切复合体包括5个核心的小核核糖核蛋白(snRNPs:U1,U2,U4,U5,U6)以及数百个 剪切辅助蛋白。最新研究结果发现，在拟南芥和水稻中分别有436个和362个辅助蛋白参与 其可变剪切过程，其中富含丝氨酸和精氨酸的SR蛋白以及富含甘氨酸的GRP蛋白是目前 植物中研究最为深入的剪切因子蛋白。
据报道，拟南芥中SR蛋白AtSR45可以负调控植物免疫；番茄中的SlSR30和SlSR45蛋白也可以在番茄与致病疫霉的互作过程中发挥重要作用；细菌P.syringae在拟南芥AtGRP7的T-DNA插入突变体上发病更加严重，这些结果表明剪切因子蛋白在植物响应外 界的生物胁迫过程中发挥着重要作用。番茄中的RS2Z32蛋白作为SR蛋白家族中的一员， 目前未见其在生物与非生物胁迫过程中的研究报道，并且RS2Z32的同源基因在马铃薯、烟 草、大豆、水稻、玉米和小麦、棉花等作物中也高度保守，因此研究RS2Z32基因在植物中 的抗性作用机制具有非常重要的理论价值与实际应用价值。
我国是一个农业大国，每年因各类病虫害引起的粮食损失高达数千万吨，严重威胁我国粮食生产安全和农业经济的可持续发展。长期以来人们一直使用化学农药对农作物病虫害进行防治，这不仅破坏了生态系统的平衡还威胁人类的健康生活。因此，利用基因编辑技术可以对植物中一些“不良基因”进行敲除从而达到去劣存优的目标，还能够明显缩短育种年限，因此在作物抗性改良上更为安全和快捷。目前，CRISPR/Cas9基因编辑技术已广泛运用到植物基因编辑过程中，利用该技术敲除植物內源的免疫负调控因子RS2Z32可以提高植物对不同病原菌的抗性。因此，研究并利用抗性关键基因RS2Z32对于植物抗病品种的改良具有非常重要的意义。
发明内容
本发明的目的在于提供剪切因子基因RS2Z32作为负调控因子在农业生产中的应用；具 体是在提高作物抗性或提高抗性作物定向育种中的应用。
本发明的目的可以通过以下技术方案实现：
剪切因子基因RS2Z32作为负调控因子在提高作物抗性或提高抗性作物定向育种中的应 用。所述的作物抗性为作物的抗病性、抗虫性和抗逆性中的至少一种。所述的提高抗性作物 定向育种为提高作物抗病性、抗虫性或抗逆性的定向育种。
上述的应用，在作物基因组中沉默或敲除剪切因子基因RS2Z32能够提高作物抗性(即 提高作物对不同病虫害和逆境胁迫的抗性)。
上述的剪切因子基因RS2Z32为如下(1)或(2)：
(1)具有如SEQ ID NO.1所示的核苷酸序列；
(2)与SEQ ID NO.1相比具有至少60％以上同源性的核苷酸序列；优选为，与SEQID NO.1相比具有至少70％以上同源性的核苷酸序列；进一步优选为，与SEQ ID NO.1相比具有至少80％以上同源性的核苷酸序列；更进一步优选为，与SEQ ID NO.1相比具有至少90％以上同源性的核苷酸序列；最优选为，与SEQ ID NO.1相比具有至少95％以上同源性的核苷酸序列。
上述的应用，构建重组载体用于沉默、敲除或改造作物基因组中的剪切因子基因RS2Z32；构建沉默载体的方法是将如SEQ ID NO.2所示的沉默片段插入到植物转化质粒中，所述的植物转化质粒优选为病毒沉默载体TRV2，也可以为PbinGFP2、pCambia等植物 转化质粒；优选的，将RS2Z32的特异性沉默片段插入到沉默载体TRV2上得到重组载体 TRV2-RS2Z32。
构建敲除载体的方法是将如SEQ ID NO.3所示的sgRNA序列插入到含有CRISPR/Cas9 基因编辑功能的植物转化质粒，所述的含有CRISPR/Cas9基因编辑功能的植物转化质粒优 选为植物转化载体BGK01，也可以为其他类型的含有CRISPR/Cas9基因编辑功能的植物转 化质粒。优选的，将敲除RS2Z32的sgRNA序列插入到植物转化质粒BGK01中得到重组载体BGK01-RS2Z32。
用于特异性沉默剪切因子基因RS2Z32的沉默片段、重组沉默载体或者转化体，或者用 于特异性敲除剪切因子基因RS2Z32的sgRNA序列、重组敲除载体或者转化体在提高作物 抗性或提高抗性作物定向育种中的应用。
上述的用于特异性沉默剪切因子基因RS2Z32的沉默片段的核苷酸序列如SEQ IDNO.2 所示；所述的重组沉默载体是将所述的沉默片段插入到植物转化质粒中得到的；所述的植物 转化质粒优选为病毒沉默载体TRV2，也可以为PbinGFP2、pCambia等植物转化质粒；所述 的转化体是由所述的重组沉默载体导入宿主细胞得到的；所述的宿主细胞优选大肠杆菌或农 杆菌细胞等。
上述的用于特异性敲除剪切因子基因RS2Z32的sgRNA序列的核苷酸序列如SEQ IDNO.3所示；所述的重组敲除载体是将所述的sgRNA序列插入到含有CRISPR/Cas9基因编 辑功能的植物转化质粒中得到的；所述的植物转化质粒优选为植物转化载体BGK01，也可 以为其他类型的含有CRISPR/Cas9基因编辑功能的植物转化质粒；所述的转化体是由所述 的重组敲除载体导入宿主细胞得到的；所述的宿主细胞优选大肠杆菌或农杆菌细胞等。
剪切因子蛋白RS2Z32作为负调控因子在提高作物抗性或提高抗性作物定向育种中的应 用，其特征在于，该剪切因子蛋白RS2Z32是如下(a)～(c)中的至少一种：
(a)由剪切因子基因RS2Z32编码的蛋白；
(b)与所述剪切因子基因RS2Z32编码的蛋白相比，在氨基酸序列上具有不小于60％ 相似性且能够作为负调控因子提高作物抗性的同源蛋白；
(c)与所述剪切因子基因RS2Z32的编码蛋白相比，在氨基酸序列上具有不小于60％ 相似性且能够作为负调控因子提高作物抗性的由RS2Z32天然等位基因编码的蛋白。
上述的由剪切因子基因RS2Z32编码的蛋白具有如SEQ ID NO.4所示的氨基酸序列，或 将如SEQ ID NO.4所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或添加和/或缺失 且能够作为负调控因子提高作物抗性的由SEQ ID NO.4衍生出来的蛋白；
上述的与剪切因子基因RS2Z32编码的蛋白相比，在氨基酸序列上具有不小于60％相似 性且能够作为负调控因子提高作物抗性的同源蛋白或由RS2Z32天然等位基因编码的蛋白， 其氨基酸序列如SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ IDNO.9、SEQ ID NO.10或SEQ ID NO.11所示；或者将SEQ ID NO.5、SEQ ID NO.6、SEQ IDNO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10或SEQ ID NO.11所示的氨基酸序列经 过一个或几个氨基酸残基的取代和/或添加和/或缺失且能够作为负调控因子提高作物抗性的 由SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ ID NO.10或SEQ ID NO.11衍生出来的蛋白。
用于沉默、敲除或改造剪切因子基因RS2Z32的重组载体、转基因细胞系或重组工程细 菌在提高作物抗病性、抗虫性或抗逆性育种中的应用。
一种提高作物抗性的方法，该方法通过沉默、敲除或改造作物基因组中剪切因子基因 RS2Z32能够提高作物对不同病虫害和环境逆境胁迫的抗性。
上述的RS2Z32基因、上述的RS2Z32蛋白、上述的RS2Z32特异性沉默片段、上述的RS2Z32沉默载体、上述的特异性敲除RS2Z32的sgRNA序列、上述的RS2Z32敲除载体、 上述的RS2Z32转化体、针对RS2Z32基因的沉默材料或者基因编辑材料在提高作物抗性或 提高抗病性、抗虫性或抗逆性作物育种中的应用。所述的作物优选为马铃薯、烟草、大豆、 水稻、玉米、小麦、棉花以及蔬菜和果树等农作物。所述的作物抗病性为农作物对病原物引 起病害的抗性，所述的病原物为能够侵染经济作物和主要粮食作物的致病菌、病毒和线虫 等，可以为卵菌、真菌或细菌，如疫霉菌、霜霉菌、稻瘟菌、镰刀菌、青枯菌、丁香假单胞 菌、病毒和线虫等。所述的作物抗虫性为农作物对害虫引起虫害的抗性，所述的害虫为能够 侵染经济作物和主要粮食作物的害虫等，可以为叶蝉、飞虱、蝗虫、棉铃虫、草地贪夜蛾、 玉米螟、马铃薯瓢虫等。所述的作物抗逆性为农作物对不利的环境因素引起的抗性，所述的 不利环境因素可以为干旱胁迫、低温胁迫、高温胁迫、盐胁迫等。
上述的RS2Z32基因在提高作物抗性或提高抗病性、抗虫性或抗逆性作物育种中的应 用，沉默或敲除作物基因组中RS2Z32基因可以显著增强作物的抗性，培育高抗的作物，还 可以增加作物的产量。尤其在马铃薯、烟草、大豆、水稻、玉米、小麦、棉花等作物中沉默或敲除该基因可以获得对不同病虫害和逆境胁迫的抗性或增加产量的品种。
上述的RS2Z32基因、上述的RS2Z32蛋白、上述的RS2Z32特异性沉默片段、上述的RS2Z32沉默载体、上述的特异性敲除RS2Z32的sgRNA序列、上述的RS2Z32敲除载体、 上述的RS2Z32转化体、针对RS2Z32基因的沉默材料或者基因编辑材料在增加作物产量中 的应用。在作物中沉默或敲除RS2Z32基因后能增加作物产量，优选在马铃薯、烟草、大 豆、水稻、玉米、小麦、棉花等作物中沉默或敲除RS2Z32基因能够获得增强抗病性或抗虫 性或抗逆性和/或增加产量的品种。
本研究发现番茄剪切因子RS2Z32基因参与植物-疫霉菌的互作过程。RS2Z32基因在致 病疫霉侵染番茄过程中诱导表达，在番茄中沉默內源的RS2Z32基因可以显著提高植物对疫 霉菌的抗性，INF1蛋白能在沉默TRV2-RS2Z32的番茄叶片上触发更强的免疫反应，在番茄 中敲除RS2Z32基因可以显著提高植物对疫霉菌的抗性。RS2Z32基因对疫霉菌的抗病性起着 非常重要的作用，对番茄中RS2Z32基因的研究可带动许多其它植物(如马铃薯、烟草、大 豆、水稻、玉米、小麦、棉花等)中剪切因子基因的相关研究。这些研究能更好的阐明植物 剪切因子蛋白对不同病虫害和逆境胁迫的抗性，为农作物的抗病或抗虫或抗逆品种选育提供 优质的抗性资源。
本发明的有益效果：
本发明所述RS2Z32基因编码的蛋白质通过干扰寄主可变剪切过程从而增强植物抗病 性。在番茄中沉默或敲除RS2Z32基因可以显著增强番茄对致病疫霉的抗性。本发明可以运 用在作物抗性品种改良的方面，有望提高作物对不同病虫害和逆境胁迫的抗性，从而达到减 肥减药的目的。
附图说明
图1实时荧光定量PCR技术检测RS2Z32基因的沉默效率。RS2Z33为RS2Z32的同源基因，TRV2-RS2Z32载体能特异性沉默RS2Z32基因，并不能沉默其同源基因RS2Z33。其中TRV2-GFP载体为阴性对照。
图2 TRV2-GFP和TRV2-RS2Z32沉默的番茄叶片上接种致病疫霉后的发病症状。
图3 TRV2-GFP和TRV2-RS2Z32沉默的番茄叶片上接种致病疫霉后的病斑面积统计分析。
图4用INF1的纯蛋白分别处理沉默TRV2-GFP和TRV2-RS2Z32的番茄叶片，INF1能在沉 默TRV2-RS2Z32的番茄叶片上触发更强的活性氧反应。
图5 RS2Z32基因敲除突变体的制备。
图6野生型和rs2z32#9的番茄叶片上接种致病疫霉后的发病症状。
图7野生型和rs2z32#9的番茄叶片上接种致病疫霉后的病斑面积统计分析。
具体实施方式
以下的实施例是为了更好地理解本发明专利，但并不限定于本发明专利。以下实施例中 的实验方法，如无特殊的解释和说明，均为常规的实验操作方法。下述实施例中所用到的实 验材料，如无特殊的解释和说明，均从日常试剂商店购买获得。本发明实施例中所涉及的所 有引物均在上海生工生物工程公司合成。
实施例1.构建TRV2-RS2Z32的沉默载体
(1)番茄叶片的总RNA提取：以番茄的叶片为原材料，利用英俊公司的RNA提取试剂盒(Cat no.12183018A)提取番茄叶片的总RNA，然后利用NanoDrop分光光度计测定总RNA的浓度并记录。
(2)反转录产生cDNA：取1μg的总RNA作为模板，利用南京诺唯赞生物科技有限 公司的反转录试剂盒(R223-01)，按照试剂盒中的说明书进行操作获得cDNA，以此作为 模板用来扩增RS2Z32的沉默片段。
RS2Z32基因沉默片段的PCR引物序列为：
上游引物：SEQ ID NO.12
(5’-CGACAAGACCCTGCAAAAGAAGTCCAGTAGAGA-3’)
下游引物：SEQ ID NO.13
(5’-GAGAAGAGCCCTGCATTAGGGTGACTCACTGCC-3’),
(3)RS2Z32基因沉默片段的克隆：利用南京诺唯赞公司
Figure BDA0003021962870000051
Max MasterMix (P525)配置100μL的反应体系(2×Phanta Max Master Mix为50μL，上下游引物各为4μL，模板为2μL，加水补充总体积至100μL)，PCR反应的程序为95℃预变性3分钟， 95℃变性15秒，60℃退火15秒，72℃延伸15秒，30次循环，72℃延伸5分钟，最后10℃ 延伸。待PCR反应结束后向产物中加入核酸上样缓冲液loading buffer，并在琼脂糖凝胶上 进行电泳分离并切取300bp大小的胶条，利用Takara公司的DNA凝胶回收试剂盒(Code No.9762)按照试剂盒中的说明书操作回收目的产物，随后用NanoDrop分光光度计测定 PCR产物的浓度并记录。
(4)TRV2-RS2Z32载体的构建：利用限制性内切酶PstI酶切TRV2空载体，并在琼脂糖凝胶上进行电泳分离，回收后利用NanoDrop分光光度计来测定回收后的载体浓度。然后利用南京诺唯赞公司的单片段连接试剂盒(C112)，按照试剂盒中的说明书进行操作将RS2Z32的沉默片段连接到PstI酶切过的TRV2载体上。随后将连接的产物转入到大肠杆菌感受态细胞中，最后利用涂布棒均匀涂布在含卡纳(50μg/mL)抗性的LB平板上，37℃培 养12小时后利用TRV2的载体引物(SEQ ID NO.14和SEQ ID NO.15)进行菌落PCR扩增 验证，选取条带大小正确的菌落并摇菌提取质粒，送往上海生工生物工程公司进行DNA测 序，测序所得的序列与SEQ ID NO.2所示的序列进行比对。最后将测序正确的TRV2- RS2Z32质粒利用电激转化的方法转化到GV3101农杆菌中，用于后续的番茄中RS2Z32基 因的沉默。
TRV2的载体引物序列为：
上游引物：SEQ ID NO.14
(5’-GATGGACATTGTTACTCAAG-3’)
下游引物：SEQ ID NO.15
(5’-CGAGAATGTCAATCTCGTAG-3’)
实施例2.在沉默RS2Z32基因的番茄叶片上接种致病疫霉
(1)农杆菌培养：利用TRV2的载体引物(SEQ ID NO.14和SEQ ID NO.15)对转入TRV2-RS2Z32质粒的农杆菌进行菌落PCR验证，挑取成功转入TRV2-RS2Z32载体的农杆 菌，并接种到含有液体LB(卡纳浓度50μg/mL，利福平浓度50μg/mL)的试管中进行培 养。在28℃，220rpm的摇床上培养12小时左右，然后将培养的农杆菌菌液收集在2mL的 离心管中，5000rpm离心3-4分钟收集农杆菌的菌体。随后用烟草注射缓冲液(10mM MgCl2,10mM MES,pH＝5.6,200μM AS)重悬菌体1-2次，利用分光光度计测定出菌液的 OD600浓度。用缓冲液稀释菌液，将TRV1和TRV2-RS2Z32(TRV1和TRV2-GFP作为阴性 对照)菌液按照1:1的比例等体积混匀，使得最终的浓度各为OD600＝0.5。
(2)沉默番茄的RS2Z32基因：选取刚刚长出2片子叶的番茄幼嫩小苗，将配好的农杆菌菌液注射到番茄叶片上，注射后将番茄置于温室中(24℃/14h光照和22℃/10h黑暗)继续培养。
(3)检测番茄中RS2Z32基因的沉默效率：选取生长4-6周的番茄叶片，随后将叶片放 入研钵中，并加入液氮进行研磨，反复研磨3-4次，再利用英俊公司RNA提取试剂盒(Catno.12183018A)，按照说明书中的操作流程提取番茄叶片的总RNA。随后用NanoDrop分 光光度计测定提取的RNA浓度，利用1μg RNA作为原始的模板，利用南京诺唯赞公司的 反转录试剂盒(R223-01)，按照试剂盒中的说明书进行操作获得cDNA。取适量的cDNA 作为qRT-PCR的模板，用于后续的沉默效率检测(图1)。
RS2Z32基因的定量PCR扩增引物序列为：
上游引物：SEQ ID NO.16
(5’-GTTACCGCTGTGGAGACCGA-3’)
下游引物：SEQ ID NO.17
(5’-CACAGGAGACCTGGATCGGC-3’)
(4)沉默RS2Z32基因的致病性检测：收集沉默了RS2Z32基因的番茄叶片和对照番茄 的叶片，在叶片上接种10μl的致病疫霉(200个/μl)游动孢子，然后将接种的番茄叶片放置在18度的培养箱中进行培养，接种5-6天后观察致病疫霉的发病症状，并记录侵染的病斑大小。统计结果表明在番茄中沉默了RS2Z32基因后能显著提高番茄对致病疫霉的抗性(图2)。此外，病斑的大小统计结果也表明与对照相比致病疫霉在沉默RS2Z32的番茄叶 片上的侵染病斑明显减小(图3)。以上这些证据证实，沉默番茄内源的RS2Z32基因可以 显著提高番茄对致病疫霉的抗性。
实施例3.在沉默RS2Z32基因的番茄叶片上检测INF1触发的活性氧
选取8片沉默RS2Z32基因的番茄叶片(注射TRV2-GFP的番茄叶片作为阴性对照)，利用打孔器从每个叶片上选取6个叶碟，放入96孔酶标板中并加入100μl的灭菌水，室温 放置12小时左右(减少叶片损伤对活性氧测定造成的影响)。然后将灭菌水吸出，加入 200μl的反应液(35.4μg/mL的luminol；10μg/mL的peroxidase；1μM的INF1纯蛋白)，随 后立即放入Promega GloMax 96微孔板发光检测仪器中检测ROS的产生情况，初步发现沉 默RS2Z32基因的番茄叶片上INF1触发更强的活性氧，表明沉默了番茄的RS2Z32基因后增 强了植物免疫(图4)。
实施例4.在敲除RS2Z32基因的番茄叶片上接种致病疫霉
(1)RS2Z32基因敲除突变体的制备：利用稳定转基因的方法将重组载体BGK01-RS2Z32转化到Micro-Tom的番茄品种中并获得RS2Z32基因敲除突变体。利用RS2Z32基因 敲除突变体的叶片为原材料，然后利用天根DNA提取试剂盒(DP305)提取番茄叶片的总 DNA，取适量的DNA作为PCR的模板，用于扩增RS2Z32基因的序列，如图5所示，一共 获得了2种不同类型的RS2Z32基因敲除突变体，这些突变体中RS2Z32基因都会发生移码 突变，可以用于后续的实验。
RS2Z32基因敲除突变体的验证引物序列为：
上游引物：SEQ ID NO.18
(5’-AGGAGAGGAGCTAATTGGGTAG-3’)
下游引物：SEQ ID NO.19
(5’-ATTGGGCAAAAACATAGAAACCT-3’)
(2)RS2Z32基因敲除突变体的致病性检测：选取生长3-4周的RS2Z32基因敲除突变体番茄和对照野生型番茄的叶片，在叶片上接种500个左右的致病疫霉游动孢子，然后将接种的番茄叶片放置在18度的培养箱中进行培养，接种4天后观察致病疫霉的发病症状，并记录侵染的病斑大小。统计结果表明在番茄中敲除了RS2Z32基因后能显著提高番茄对致病疫霉的抗性(图6)。此外，病斑的大小统计结果也表明，与对照相比致病疫霉在敲除 RS2Z32的番茄叶片上的侵染病斑明显减小(图7)。以上这些证据证实，敲除番茄内源的 RS2Z32基因可以显著提高番茄对致病疫霉的抗性。
序列表
SEQ ID NO.1(RS2Z32全长核苷酸序列)
ATGCCGCGGTATGATGACAGGTATGGTGGCACACGCCTATATGTTGGACATTTGTCTTC CCGGACGCGATCTCGAGACTTGGAGGACGTCTTTAGCAGATATGGGAGAGTACGTGAT GTGGATATGAAGCGTGACTATGCTTTTGTGGAGTTTAGTGATCCTCGAGATGCCGATGA TGCAAGATACGGCCTAAATGGGCGAGATGTTGATGGAAGTCGTGTTATCGTGGAGTTC GCCAAAGGGGTGCCTCGTGGTCCAGGTGGATCTCGAGAGTTTGGTGGCAGAGGTCCTCCTCCAGGTACTGGTCGTTGCTTTAATTGTGGAATTGATGGACATTGGGCTCGAGATTGT AAAGCTGGGGACTGGAAGAACAAGTGTTACCGCTGTGGAGACCGAGGTCATATAGAA AGGAACTGTCAGAATAGCCCCAAGAAATTGAAACGTGACCGAAGTTATTCCCGCTCAC CATCTCCTCGGCGTGGAAGAAGCCGCAGCCGCAGCCGCAGTTACAGCAGAGGTCGTAG CTACAGCCGATCCAGGTCTCCTGTGAAGAGGGACCGTAGCATTGAGCGTGAAGAAAAG AGATCAAGGAGTCCTCGCCATCATAGGTCATCACCACCTCCATCAAAGGGAAGGAAGC ACAGCCTTTCACCTGATGAAAGAAGTCCAGTAGAGAGAGGTACACCATCACCAAGGGA TGATAGGGCAACCAATGGTTCTGACCGCAGCAGGAGCCCTAAAGATGATGTTAGAATG GATGAACGCGGAGATATTAGCCCTGTTGAAGAAAATGGCCGTAGTCGCAGCAATAGCC CCATCCATAGGGAGGACAGAAGCCCAGTGGAAGACGGTAGCCCAACAGGTGACTATG AAAATCATGGTTCTCCAAGGGGTTCTCCAAGGGGCAGTGAGTCACCCTAA
SEQ ID NO.2(RS2Z32特异性沉默片段的核苷酸序列)
AAAGAAGTCCAGTAGAGAGAGGTACACCATCACCAAGGGATGATAGGGCAACCAATG GTTCTGACCGCAGCAGGAGCCCTAAAGATGATGTTAGAATGGATGAACGCGGAGATAT TAGCCCTGTTGAAGAAAATGGCCGTAGTCGCAGCAATAGCCCCATCCATAGGGAGGAC AGAAGCCCAGTGGAAGACGGTAGCCCAACAGGTGACTATGAAAATCATGGTTCTCCAA GGGGTTCTCCAAGGGGCAGTGAGTCACCCTAA
SEQ ID NO.3(特异性敲除RS2Z32的sgRNA序列)
CCGCGGTATGATGACAGGTA
SEQ ID NO.4(番茄中RS2Z32的全长蛋白氨基酸序列)
MPRYDDRYGGTRLYVGHLSSRTRSRDLEDVFSRYGRVRDVDMKRDYAFVEFSDPRDADD ARYGLNGRDVDGSRVIVEFAKGVPRGPGGSREFGGRGPPPGTGRCFNCGIDGHWARDCKA GDWKNKCYRCGDRGHIERNCQNSPKKLKRDRSYSRSPSPRRGRSRSRSRSYSRGRSYSRSR SPVKRDRSIEREEKRSRSPRHHRSSPPPSKGRKHSLSPDERSPVERGTPSPRDDRATNGSDRS RSPKDDVRMDERGDISPVEENGRSRSNSPIHREDRSPVEDGSPTGDYENHGSPRGSPRGSES P
SEQ ID NO.5(马铃薯中RS2Z32的同源蛋白氨基酸序列)
MPRYDDRYGGTRLYVGHLSSRTRSRDLEDVFSRYGRVRDVDMKRDYAFVEFSDPRDADD ARYGLNGRDVDGSRVIVEFAKGVPRGPGGSREFGGRGPPPGTGRCFNCGIDGHWARDCKA GDWKNKCYRCGDRGHIERNCQNSPKKLKRDRSYSRSPSPRRGRSRSRSYSRGRSYSRSRSP VKRDRSIEREEKRSRSPRQHRPSPPPSKGRKHSPSPDERSPQERGTPSPRDDRATNGSDRSRS PKDDARMDERGDISPVEENGRSRSNSPIHREDRSPVEDGSPTGDYENHGSPRGSESP
SEQ ID NO.6(烟草中RS2Z32的同源蛋白氨基酸序列)
MPRYDDRYGGTRLYVGHLSSRTRSRDLEDVFSRYGRVRDVDMKRDYAFVEFSDPRDADDA RYGLNGRDVDGSRITVEFAKGVPRGPGGSREFGGRGPPPGTGRCFNCGLDGHWARDCKAG DWKNKCYRCGERGHIERNCQNSPKKLERGRSYSRSPSPRHGRSRSRSRSYSGGRSYSRSRS PVRRDSRSPVKRDRSIEHEERKSRSPRPRRSSPPPSKGRKYSPSPDKRSPQERGTPSPKGDRA ANGSEYSRSPTDDAGIDEHRNLSPIEENGRSRSNSPIHRDNGSPVENGSPMGADENHGSPRG SESP
SEQ ID NO.7(大豆中RS2Z32的同源蛋白氨基酸序列)
MPRYDDKYGNTRLYVGHLSSRTRSRDLERAFSRYGRVRGVDMKNDFAFVDFSDPRDADD ARYNLDGRDVDGSRIIVEFAKGAPRGSREYLGRGPPPGSGRCFNCGLDGHWARDCKAGD WKNKCYRCGERGHIERNCKNSPKKLSTRRGRSYSRSPVRSRSPHRGRSRDRSYSRDRSYSR SRSPVRREESPVREDRSQSPDHKNSPQPSKTRKHSPSPDRSPQKRGDTSPDNDRLSRSPARD PEDRGYDSPKVNGRSGSPSCSPRDDDRSPIDDDNNHRHSPRGSESP
SEQ ID NO.8(水稻中RS2Z32的同源蛋白氨基酸序列)
MPRYDDRYGSTRLYVGRLSSRTRSRDLEYHFSRYGRIREVELKRDYAFIEFSDPRDAEEAR YNLDGRDVDGSRILVEFAKGVPRGPGGSREYMGRGPPPGTGRCFNCGIDGHWARDCKAG DWKNKCYRCGERGHIERNCQNSPRNLRRERSYSCSPSPRRGRGRSRSYSRSRSRSRSYSRSR SRSLSGSPRARRELERSRSLSYSRSPRRSISPAANEKKRSPTPDGSRSPRSPQDQVSPPPKDNA ERNGSDHGDSPRGRENSRSPSDGYRSPAAANGRSPSPRNNGSPSPMDNGSRSPRDGNGDG GSRGGSRSPRPSESPEA
SEQ ID NO.9(玉米中RS2Z32的同源蛋白氨基酸序列)
MGQKAVQAHRRSSSAQPRTTSEFHLRRRRHHQFLSSRIEIPPTTSVPLPFPPLARRLHADPGD MPRYDDRYGGTRLYVGRLATRTRSRDLEHLFGRYGRIREVELKRDYAFIEFSDHRDADEA RYQLDGRDVDGSRIVVEFAKGVPRGSGGSREYMGRGPPPGTGRCFNCGVDGHWARDCKA GDWKNKCYRCGERGHIERNCQNSPRSLRRERSYSRSPSPRRGRGRSRSYGRSRSRSRSYSR SRSLSRSPRGGRRDRDDRRSRSPSYSRSPMRSASPPVKEKERSPAPDGSRSPRSRSPQDQVM SPPPKDNGEGNGSDRGGSPRGRENSRSCSRSRSPSGSNRSPAANGRSRSPGGERSPSPGGERSPSPRGERSPSPRADRSPSPNGNGNNDAAADVDADAAARSGSPTGSKSP
SEQ ID NO.10(小麦中RS2Z32的同源蛋白氨基酸序列)
MPRYDDRYGNARLYVGRLSSRTRSRDLEYLFSKYGRIREVELKRDYAFIEYSDPRDADEAR YNLDGRDVDGSRIIVEFAKGVPRGSGGSREREYVGRGPPPGTGRCFNCGIDGHWARDCKA GDWKNKCYRCGERGHIERNCQNSPRSLRRERSYSRSPSPRRGRARSRSYSRSRSYSRSRSRS YSESPRGRRTERDERRSRSISYSRSPRRSLSPGGKEMDRSPTPDRSRSPRRSISPVAKDNGDS PRGRETSRSPSDGYRSPVANGRSPRSPVNNGSPSPTRDNRASPSLRGNNGSPSPKGNGNGGS PSPRGNGDDDGRRGSGSPRGRSVSP
SEQ ID NO.11(棉花中RS2Z32的同源蛋白氨基酸序列)
MPRYDDRRGGTRLYVGHLSSRTRSRDLEDMFSRYGRVRDVDMKRDYAFVEFSDPRDADD ARYALNGRDMDGSRMIVEFAKGVPRGSGGSRDYPGRGPTPGSGRCFNCGIDGHWARDCK AGDWKNKCYRCGERGHIERNCQNSPKKLRPRSYSRSPSPYRGRSRSRSYSRGRSDSRSRSP VKRDRSYEREDRISRSPKRHKGSPSPSQGRKHSPAPDERRPQEGGGPSPKDRRHANGSDYS ASPRGRSRSPDADAGAEDGAYRSSRKENGHSRSLSPPPRDDRSPVYDDDDDDDNHASTRR GESN
SEQ ID NO.12(人工合成DNA序列)
(5’-CGACAAGACCCTGCAAAAGAAGTCCAGTAGAGA-3’)
SEQ ID NO.13(人工合成DNA序列)
(5’-GAGAAGAGCCCTGCATTAGGGTGACTCACTGCC-3’)
SEQ ID NO.14(人工合成DNA序列)
(5’-GATGGACATTGTTACTCAAG-3’)
SEQ ID NO.15(人工合成DNA序列)
(5’-CGAGAATGTCAATCTCGTAG-3’)
SEQ ID NO.16(人工合成DNA序列)
(5’-GTTACCGCTGTGGAGACCGA-3’),
SEQ ID NO.17(人工合成DNA序列)
(5’-CACAGGAGACCTGGATCGGC-3’)
SEQ ID NO.18(人工合成DNA序列)
(5’-AGGAGAGGAGCTAATTGGGTAG-3’)
SEQ ID NO.19(人工合成DNA序列)
(5’-ATTGGGCAAAAACATAGAAACCT-3’)
序列表
<110> 南京农业大学
<120> RS2Z32基因作为植物免疫负调控因子在提高作物抗性中的应用
<160> 19
<170> SIPOSequenceListing 1.0
<210> 1
<211> 921
<212> DNA
<213> 番茄(Lycopersicon esculentum Mill.)
<400> 1
atgccgcggt atgatgacag gtatggtggc acacgcctat atgttggaca tttgtcttcc 60
cggacgcgat ctcgagactt ggaggacgtc tttagcagat atgggagagt acgtgatgtg 120
gatatgaagc gtgactatgc ttttgtggag tttagtgatc ctcgagatgc cgatgatgca 180
agatacggcc taaatgggcg agatgttgat ggaagtcgtg ttatcgtgga gttcgccaaa 240
ggggtgcctc gtggtccagg tggatctcga gagtttggtg gcagaggtcc tcctccaggt 300
actggtcgtt gctttaattg tggaattgat ggacattggg ctcgagattg taaagctggg 360
gactggaaga acaagtgtta ccgctgtgga gaccgaggtc atatagaaag gaactgtcag 420
aatagcccca agaaattgaa acgtgaccga agttattccc gctcaccatc tcctcggcgt 480
ggaagaagcc gcagccgcag ccgcagttac agcagaggtc gtagctacag ccgatccagg 540
tctcctgtga agagggaccg tagcattgag cgtgaagaaa agagatcaag gagtcctcgc 600
catcataggt catcaccacc tccatcaaag ggaaggaagc acagcctttc acctgatgaa 660
agaagtccag tagagagagg tacaccatca ccaagggatg atagggcaac caatggttct 720
gaccgcagca ggagccctaa agatgatgtt agaatggatg aacgcggaga tattagccct 780
gttgaagaaa atggccgtag tcgcagcaat agccccatcc atagggagga cagaagccca 840
gtggaagacg gtagcccaac aggtgactat gaaaatcatg gttctccaag gggttctcca 900
aggggcagtg agtcacccta a 921
<210> 2
<211> 263
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
aaagaagtcc agtagagaga ggtacaccat caccaaggga tgatagggca accaatggtt 60
ctgaccgcag caggagccct aaagatgatg ttagaatgga tgaacgcgga gatattagcc 120
ctgttgaaga aaatggccgt agtcgcagca atagccccat ccatagggag gacagaagcc 180
cagtggaaga cggtagccca acaggtgact atgaaaatca tggttctcca aggggttctc 240
caaggggcag tgagtcaccc taa 263
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ccgcggtatg atgacaggta 20
<210> 4
<211> 306
<212> PRT
<213> 番茄(Lycopersicon esculentum Mill.)
<400> 4
Met Pro Arg Tyr Asp Asp Arg Tyr Gly Gly Thr Arg Leu Tyr Val Gly
1 5 10 15
His Leu Ser Ser Arg Thr Arg Ser Arg Asp Leu Glu Asp Val Phe Ser
20 25 30
Arg Tyr Gly Arg Val Arg Asp Val Asp Met Lys Arg Asp Tyr Ala Phe
35 40 45
Val Glu Phe Ser Asp Pro Arg Asp Ala Asp Asp Ala Arg Tyr Gly Leu
50 55 60
Asn Gly Arg Asp Val Asp Gly Ser Arg Val Ile Val Glu Phe Ala Lys
65 70 75 80
Gly Val Pro Arg Gly Pro Gly Gly Ser Arg Glu Phe Gly Gly Arg Gly
85 90 95
Pro Pro Pro Gly Thr Gly Arg Cys Phe Asn Cys Gly Ile Asp Gly His
100 105 110
Trp Ala Arg Asp Cys Lys Ala Gly Asp Trp Lys Asn Lys Cys Tyr Arg
115 120 125
Cys Gly Asp Arg Gly His Ile Glu Arg Asn Cys Gln Asn Ser Pro Lys
130 135 140
Lys Leu Lys Arg Asp Arg Ser Tyr Ser Arg Ser Pro Ser Pro Arg Arg
145 150 155 160
Gly Arg Ser Arg Ser Arg Ser Arg Ser Tyr Ser Arg Gly Arg Ser Tyr
165 170 175
Ser Arg Ser Arg Ser Pro Val Lys Arg Asp Arg Ser Ile Glu Arg Glu
180 185 190
Glu Lys Arg Ser Arg Ser Pro Arg His His Arg Ser Ser Pro Pro Pro
195 200 205
Ser Lys Gly Arg Lys His Ser Leu Ser Pro Asp Glu Arg Ser Pro Val
210 215 220
Glu Arg Gly Thr Pro Ser Pro Arg Asp Asp Arg Ala Thr Asn Gly Ser
225 230 235 240
Asp Arg Ser Arg Ser Pro Lys Asp Asp Val Arg Met Asp Glu Arg Gly
245 250 255
Asp Ile Ser Pro Val Glu Glu Asn Gly Arg Ser Arg Ser Asn Ser Pro
260 265 270
Ile His Arg Glu Asp Arg Ser Pro Val Glu Asp Gly Ser Pro Thr Gly
275 280 285
Asp Tyr Glu Asn His Gly Ser Pro Arg Gly Ser Pro Arg Gly Ser Glu
290 295 300
Ser Pro
305
<210> 5
<211> 300
<212> PRT
<213> 马铃薯(Solanum tuberosum)
<400> 5
Met Pro Arg Tyr Asp Asp Arg Tyr Gly Gly Thr Arg Leu Tyr Val Gly
1 5 10 15
His Leu Ser Ser Arg Thr Arg Ser Arg Asp Leu Glu Asp Val Phe Ser
20 25 30
Arg Tyr Gly Arg Val Arg Asp Val Asp Met Lys Arg Asp Tyr Ala Phe
35 40 45
Val Glu Phe Ser Asp Pro Arg Asp Ala Asp Asp Ala Arg Tyr Gly Leu
50 55 60
Asn Gly Arg Asp Val Asp Gly Ser Arg Val Ile Val Glu Phe Ala Lys
65 70 75 80
Gly Val Pro Arg Gly Pro Gly Gly Ser Arg Glu Phe Gly Gly Arg Gly
85 90 95
Pro Pro Pro Gly Thr Gly Arg Cys Phe Asn Cys Gly Ile Asp Gly His
100 105 110
Trp Ala Arg Asp Cys Lys Ala Gly Asp Trp Lys Asn Lys Cys Tyr Arg
115 120 125
Cys Gly Asp Arg Gly His Ile Glu Arg Asn Cys Gln Asn Ser Pro Lys
130 135 140
Lys Leu Lys Arg Asp Arg Ser Tyr Ser Arg Ser Pro Ser Pro Arg Arg
145 150 155 160
Gly Arg Ser Arg Ser Arg Ser Tyr Ser Arg Gly Arg Ser Tyr Ser Arg
165 170 175
Ser Arg Ser Pro Val Lys Arg Asp Arg Ser Ile Glu Arg Glu Glu Lys
180 185 190
Arg Ser Arg Ser Pro Arg Gln His Arg Pro Ser Pro Pro Pro Ser Lys
195 200 205
Gly Arg Lys His Ser Pro Ser Pro Asp Glu Arg Ser Pro Gln Glu Arg
210 215 220
Gly Thr Pro Ser Pro Arg Asp Asp Arg Ala Thr Asn Gly Ser Asp Arg
225 230 235 240
Ser Arg Ser Pro Lys Asp Asp Ala Arg Met Asp Glu Arg Gly Asp Ile
245 250 255
Ser Pro Val Glu Glu Asn Gly Arg Ser Arg Ser Asn Ser Pro Ile His
260 265 270
Arg Glu Asp Arg Ser Pro Val Glu Asp Gly Ser Pro Thr Gly Asp Tyr
275 280 285
Glu Asn His Gly Ser Pro Arg Gly Ser Glu Ser Pro
290 295 300
<210> 6
<211> 310
<212> PRT
<213> 烟草(Nicotiana tabacum L.)
<400> 6
Met Pro Arg Tyr Asp Asp Arg Tyr Gly Gly Thr Arg Leu Tyr Val Gly
1 5 10 15
His Leu Ser Ser Arg Thr Arg Ser Arg Asp Leu Glu Asp Val Phe Ser
20 25 30
Arg Tyr Gly Arg Val Arg Asp Val Asp Met Lys Arg Asp Tyr Ala Phe
35 40 45
Val Glu Phe Ser Asp Pro Arg Asp Ala Asp Asp Ala Arg Tyr Gly Leu
50 55 60
Asn Gly Arg Asp Val Asp Gly Ser Arg Ile Thr Val Glu Phe Ala Lys
65 70 75 80
Gly Val Pro Arg Gly Pro Gly Gly Ser Arg Glu Phe Gly Gly Arg Gly
85 90 95
Pro Pro Pro Gly Thr Gly Arg Cys Phe Asn Cys Gly Leu Asp Gly His
100 105 110
Trp Ala Arg Asp Cys Lys Ala Gly Asp Trp Lys Asn Lys Cys Tyr Arg
115 120 125
Cys Gly Glu Arg Gly His Ile Glu Arg Asn Cys Gln Asn Ser Pro Lys
130 135 140
Lys Leu Glu Arg Gly Arg Ser Tyr Ser Arg Ser Pro Ser Pro Arg His
145 150 155 160
Gly Arg Ser Arg Ser Arg Ser Arg Ser Tyr Ser Gly Gly Arg Ser Tyr
165 170 175
Ser Arg Ser Arg Ser Pro Val Arg Arg Asp Ser Arg Ser Pro Val Lys
180 185 190
Arg Asp Arg Ser Ile Glu His Glu Glu Arg Lys Ser Arg Ser Pro Arg
195 200 205
Pro Arg Arg Ser Ser Pro Pro Pro Ser Lys Gly Arg Lys Tyr Ser Pro
210 215 220
Ser Pro Asp Lys Arg Ser Pro Gln Glu Arg Gly Thr Pro Ser Pro Lys
225 230 235 240
Gly Asp Arg Ala Ala Asn Gly Ser Glu Tyr Ser Arg Ser Pro Thr Asp
245 250 255
Asp Ala Gly Ile Asp Glu His Arg Asn Leu Ser Pro Ile Glu Glu Asn
260 265 270
Gly Arg Ser Arg Ser Asn Ser Pro Ile His Arg Asp Asn Gly Ser Pro
275 280 285
Val Glu Asn Gly Ser Pro Met Gly Ala Asp Glu Asn His Gly Ser Pro
290 295 300
Arg Gly Ser Glu Ser Pro
305 310
<210> 7
<211> 287
<212> PRT
<213> 大豆(Glycine max (Linn.)
<400> 7
Met Pro Arg Tyr Asp Asp Lys Tyr Gly Asn Thr Arg Leu Tyr Val Gly
1 5 10 15
His Leu Ser Ser Arg Thr Arg Ser Arg Asp Leu Glu Arg Ala Phe Ser
20 25 30
Arg Tyr Gly Arg Val Arg Gly Val Asp Met Lys Asn Asp Phe Ala Phe
35 40 45
Val Asp Phe Ser Asp Pro Arg Asp Ala Asp Asp Ala Arg Tyr Asn Leu
50 55 60
Asp Gly Arg Asp Val Asp Gly Ser Arg Ile Ile Val Glu Phe Ala Lys
65 70 75 80
Gly Ala Pro Arg Gly Ser Arg Glu Tyr Leu Gly Arg Gly Pro Pro Pro
85 90 95
Gly Ser Gly Arg Cys Phe Asn Cys Gly Leu Asp Gly His Trp Ala Arg
100 105 110
Asp Cys Lys Ala Gly Asp Trp Lys Asn Lys Cys Tyr Arg Cys Gly Glu
115 120 125
Arg Gly His Ile Glu Arg Asn Cys Lys Asn Ser Pro Lys Lys Leu Ser
130 135 140
Thr Arg Arg Gly Arg Ser Tyr Ser Arg Ser Pro Val Arg Ser Arg Ser
145 150 155 160
Pro His Arg Gly Arg Ser Arg Asp Arg Ser Tyr Ser Arg Asp Arg Ser
165 170 175
Tyr Ser Arg Ser Arg Ser Pro Val Arg Arg Glu Glu Ser Pro Val Arg
180 185 190
Glu Asp Arg Ser Gln Ser Pro Asp His Lys Asn Ser Pro Gln Pro Ser
195 200 205
Lys Thr Arg Lys His Ser Pro Ser Pro Asp Arg Ser Pro Gln Lys Arg
210 215 220
Gly Asp Thr Ser Pro Asp Asn Asp Arg Leu Ser Arg Ser Pro Ala Arg
225 230 235 240
Asp Pro Glu Asp Arg Gly Tyr Asp Ser Pro Lys Val Asn Gly Arg Ser
245 250 255
Gly Ser Pro Ser Cys Ser Pro Arg Asp Asp Asp Arg Ser Pro Ile Asp
260 265 270
Asp Asp Asn Asn His Arg His Ser Pro Arg Gly Ser Glu Ser Pro
275 280 285
<210> 8
<211> 323
<212> PRT
<213> 水稻(Oryza sativa L.)
<400> 8
Met Pro Arg Tyr Asp Asp Arg Tyr Gly Ser Thr Arg Leu Tyr Val Gly
1 5 10 15
Arg Leu Ser Ser Arg Thr Arg Ser Arg Asp Leu Glu Tyr His Phe Ser
20 25 30
Arg Tyr Gly Arg Ile Arg Glu Val Glu Leu Lys Arg Asp Tyr Ala Phe
35 40 45
Ile Glu Phe Ser Asp Pro Arg Asp Ala Glu Glu Ala Arg Tyr Asn Leu
50 55 60
Asp Gly Arg Asp Val Asp Gly Ser Arg Ile Leu Val Glu Phe Ala Lys
65 70 75 80
Gly Val Pro Arg Gly Pro Gly Gly Ser Arg Glu Tyr Met Gly Arg Gly
85 90 95
Pro Pro Pro Gly Thr Gly Arg Cys Phe Asn Cys Gly Ile Asp Gly His
100 105 110
Trp Ala Arg Asp Cys Lys Ala Gly Asp Trp Lys Asn Lys Cys Tyr Arg
115 120 125
Cys Gly Glu Arg Gly His Ile Glu Arg Asn Cys Gln Asn Ser Pro Arg
130 135 140
Asn Leu Arg Arg Glu Arg Ser Tyr Ser Cys Ser Pro Ser Pro Arg Arg
145 150 155 160
Gly Arg Gly Arg Ser Arg Ser Tyr Ser Arg Ser Arg Ser Arg Ser Arg
165 170 175
Ser Tyr Ser Arg Ser Arg Ser Arg Ser Leu Ser Gly Ser Pro Arg Ala
180 185 190
Arg Arg Glu Leu Glu Arg Ser Arg Ser Leu Ser Tyr Ser Arg Ser Pro
195 200 205
Arg Arg Ser Ile Ser Pro Ala Ala Asn Glu Lys Lys Arg Ser Pro Thr
210 215 220
Pro Asp Gly Ser Arg Ser Pro Arg Ser Pro Gln Asp Gln Val Ser Pro
225 230 235 240
Pro Pro Lys Asp Asn Ala Glu Arg Asn Gly Ser Asp His Gly Asp Ser
245 250 255
Pro Arg Gly Arg Glu Asn Ser Arg Ser Pro Ser Asp Gly Tyr Arg Ser
260 265 270
Pro Ala Ala Ala Asn Gly Arg Ser Pro Ser Pro Arg Asn Asn Gly Ser
275 280 285
Pro Ser Pro Met Asp Asn Gly Ser Arg Ser Pro Arg Asp Gly Asn Gly
290 295 300
Asp Gly Gly Ser Arg Gly Gly Ser Arg Ser Pro Arg Pro Ser Glu Ser
305 310 315 320
Pro Glu Ala
<210> 9
<211> 416
<212> PRT
<213> 玉米(Zea mays L.)
<400> 9
Met Gly Gln Lys Ala Val Gln Ala His Arg Arg Ser Ser Ser Ala Gln
1 5 10 15
Pro Arg Thr Thr Ser Glu Phe His Leu Arg Arg Arg Arg His His Gln
20 25 30
Phe Leu Ser Ser Arg Ile Glu Ile Pro Pro Thr Thr Ser Val Pro Leu
35 40 45
Pro Phe Pro Pro Leu Ala Arg Arg Leu His Ala Asp Pro Gly Asp Met
50 55 60
Pro Arg Tyr Asp Asp Arg Tyr Gly Gly Thr Arg Leu Tyr Val Gly Arg
65 70 75 80
Leu Ala Thr Arg Thr Arg Ser Arg Asp Leu Glu His Leu Phe Gly Arg
85 90 95
Tyr Gly Arg Ile Arg Glu Val Glu Leu Lys Arg Asp Tyr Ala Phe Ile
100 105 110
Glu Phe Ser Asp His Arg Asp Ala Asp Glu Ala Arg Tyr Gln Leu Asp
115 120 125
Gly Arg Asp Val Asp Gly Ser Arg Ile Val Val Glu Phe Ala Lys Gly
130 135 140
Val Pro Arg Gly Ser Gly Gly Ser Arg Glu Tyr Met Gly Arg Gly Pro
145 150 155 160
Pro Pro Gly Thr Gly Arg Cys Phe Asn Cys Gly Val Asp Gly His Trp
165 170 175
Ala Arg Asp Cys Lys Ala Gly Asp Trp Lys Asn Lys Cys Tyr Arg Cys
180 185 190
Gly Glu Arg Gly His Ile Glu Arg Asn Cys Gln Asn Ser Pro Arg Ser
195 200 205
Leu Arg Arg Glu Arg Ser Tyr Ser Arg Ser Pro Ser Pro Arg Arg Gly
210 215 220
Arg Gly Arg Ser Arg Ser Tyr Gly Arg Ser Arg Ser Arg Ser Arg Ser
225 230 235 240
Tyr Ser Arg Ser Arg Ser Leu Ser Arg Ser Pro Arg Gly Gly Arg Arg
245 250 255
Asp Arg Asp Asp Arg Arg Ser Arg Ser Pro Ser Tyr Ser Arg Ser Pro
260 265 270
Met Arg Ser Ala Ser Pro Pro Val Lys Glu Lys Glu Arg Ser Pro Ala
275 280 285
Pro Asp Gly Ser Arg Ser Pro Arg Ser Arg Ser Pro Gln Asp Gln Val
290 295 300
Met Ser Pro Pro Pro Lys Asp Asn Gly Glu Gly Asn Gly Ser Asp Arg
305 310 315 320
Gly Gly Ser Pro Arg Gly Arg Glu Asn Ser Arg Ser Cys Ser Arg Ser
325 330 335
Arg Ser Pro Ser Gly Ser Asn Arg Ser Pro Ala Ala Asn Gly Arg Ser
340 345 350
Arg Ser Pro Gly Gly Glu Arg Ser Pro Ser Pro Gly Gly Glu Arg Ser
355 360 365
Pro Ser Pro Arg Gly Glu Arg Ser Pro Ser Pro Arg Ala Asp Arg Ser
370 375 380
Pro Ser Pro Asn Gly Asn Gly Asn Asn Asp Ala Ala Ala Asp Val Asp
385 390 395 400
Ala Asp Ala Ala Ala Arg Ser Gly Ser Pro Thr Gly Ser Lys Ser Pro
405 410 415
<210> 10
<211> 333
<212> PRT
<213> 小麦(Triticum aestivum L.)
<400> 10
Met Pro Arg Tyr Asp Asp Arg Tyr Gly Asn Ala Arg Leu Tyr Val Gly
1 5 10 15
Arg Leu Ser Ser Arg Thr Arg Ser Arg Asp Leu Glu Tyr Leu Phe Ser
20 25 30
Lys Tyr Gly Arg Ile Arg Glu Val Glu Leu Lys Arg Asp Tyr Ala Phe
35 40 45
Ile Glu Tyr Ser Asp Pro Arg Asp Ala Asp Glu Ala Arg Tyr Asn Leu
50 55 60
Asp Gly Arg Asp Val Asp Gly Ser Arg Ile Ile Val Glu Phe Ala Lys
65 70 75 80
Gly Val Pro Arg Gly Ser Gly Gly Ser Arg Glu Arg Glu Tyr Val Gly
85 90 95
Arg Gly Pro Pro Pro Gly Thr Gly Arg Cys Phe Asn Cys Gly Ile Asp
100 105 110
Gly His Trp Ala Arg Asp Cys Lys Ala Gly Asp Trp Lys Asn Lys Cys
115 120 125
Tyr Arg Cys Gly Glu Arg Gly His Ile Glu Arg Asn Cys Gln Asn Ser
130 135 140
Pro Arg Ser Leu Arg Arg Glu Arg Ser Tyr Ser Arg Ser Pro Ser Pro
145 150 155 160
Arg Arg Gly Arg Ala Arg Ser Arg Ser Tyr Ser Arg Ser Arg Ser Tyr
165 170 175
Ser Arg Ser Arg Ser Arg Ser Tyr Ser Glu Ser Pro Arg Gly Arg Arg
180 185 190
Thr Glu Arg Asp Glu Arg Arg Ser Arg Ser Ile Ser Tyr Ser Arg Ser
195 200 205
Pro Arg Arg Ser Leu Ser Pro Gly Gly Lys Glu Met Asp Arg Ser Pro
210 215 220
Thr Pro Asp Arg Ser Arg Ser Pro Arg Arg Ser Ile Ser Pro Val Ala
225 230 235 240
Lys Asp Asn Gly Asp Ser Pro Arg Gly Arg Glu Thr Ser Arg Ser Pro
245 250 255
Ser Asp Gly Tyr Arg Ser Pro Val Ala Asn Gly Arg Ser Pro Arg Ser
260 265 270
Pro Val Asn Asn Gly Ser Pro Ser Pro Thr Arg Asp Asn Arg Ala Ser
275 280 285
Pro Ser Leu Arg Gly Asn Asn Gly Ser Pro Ser Pro Lys Gly Asn Gly
290 295 300
Asn Gly Gly Ser Pro Ser Pro Arg Gly Asn Gly Asp Asp Asp Gly Arg
305 310 315 320
Arg Gly Ser Gly Ser Pro Arg Gly Arg Ser Val Ser Pro
325 330
<210> 11
<211> 304
<212> PRT
<213> 棉花(Gossypium spp)
<400> 11
Met Pro Arg Tyr Asp Asp Arg Arg Gly Gly Thr Arg Leu Tyr Val Gly
1 5 10 15
His Leu Ser Ser Arg Thr Arg Ser Arg Asp Leu Glu Asp Met Phe Ser
20 25 30
Arg Tyr Gly Arg Val Arg Asp Val Asp Met Lys Arg Asp Tyr Ala Phe
35 40 45
Val Glu Phe Ser Asp Pro Arg Asp Ala Asp Asp Ala Arg Tyr Ala Leu
50 55 60
Asn Gly Arg Asp Met Asp Gly Ser Arg Met Ile Val Glu Phe Ala Lys
65 70 75 80
Gly Val Pro Arg Gly Ser Gly Gly Ser Arg Asp Tyr Pro Gly Arg Gly
85 90 95
Pro Thr Pro Gly Ser Gly Arg Cys Phe Asn Cys Gly Ile Asp Gly His
100 105 110
Trp Ala Arg Asp Cys Lys Ala Gly Asp Trp Lys Asn Lys Cys Tyr Arg
115 120 125
Cys Gly Glu Arg Gly His Ile Glu Arg Asn Cys Gln Asn Ser Pro Lys
130 135 140
Lys Leu Arg Pro Arg Ser Tyr Ser Arg Ser Pro Ser Pro Tyr Arg Gly
145 150 155 160
Arg Ser Arg Ser Arg Ser Tyr Ser Arg Gly Arg Ser Asp Ser Arg Ser
165 170 175
Arg Ser Pro Val Lys Arg Asp Arg Ser Tyr Glu Arg Glu Asp Arg Ile
180 185 190
Ser Arg Ser Pro Lys Arg His Lys Gly Ser Pro Ser Pro Ser Gln Gly
195 200 205
Arg Lys His Ser Pro Ala Pro Asp Glu Arg Arg Pro Gln Glu Gly Gly
210 215 220
Gly Pro Ser Pro Lys Asp Arg Arg His Ala Asn Gly Ser Asp Tyr Ser
225 230 235 240
Ala Ser Pro Arg Gly Arg Ser Arg Ser Pro Asp Ala Asp Ala Gly Ala
245 250 255
Glu Asp Gly Ala Tyr Arg Ser Ser Arg Lys Glu Asn Gly His Ser Arg
260 265 270
Ser Leu Ser Pro Pro Pro Arg Asp Asp Arg Ser Pro Val Tyr Asp Asp
275 280 285
Asp Asp Asp Asp Asp Asn His Ala Ser Thr Arg Arg Gly Glu Ser Asn
290 295 300
<210> 12
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
cgacaagacc ctgcaaaaga agtccagtag aga 33
<210> 13
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gagaagagcc ctgcattagg gtgactcact gcc 33
<210> 14
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gatggacatt gttactcaag 20
<210> 15
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
cgagaatgtc aatctcgtag 20
<210> 16
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gttaccgctg tggagaccga 20
<210> 17
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
cacaggagac ctggatcggc 20
<210> 18
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
aggagaggag ctaattgggt ag 22
<210> 19
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
attgggcaaa aacatagaaa cct 23

Claims (5)

1.沉默或敲除剪切因子基因RS2Z32在提高番茄致病疫霉抗性或抗致病疫霉番茄定向育种中的应用，所述剪切因子基因RS2Z32的核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的应用，其特征在于，构建重组载体用于沉默或敲除剪切因子基因RS2Z32；构建重组沉默载体的方法是将如SEQ ID NO.2所示的核苷酸序列插入到植物转化质粒中；构建重组敲除载体的方法是将如SEQ ID NO.3所示的sgRNA序列插入到含有CRISPR/Cas9基因编辑功能的植物转化质粒中。
3.用于特异性沉默核苷酸序列如SEQ ID NO.1所示剪切因子基因RS2Z32的沉默片段、重组沉默载体或者转化体，或者用于特异性敲除核苷酸序列如SEQ ID NO.1所示剪切因子基因RS2Z32的sgRNA序列、重组敲除载体或者转化体在提高番茄致病疫霉抗性或抗致病疫霉番茄定向育种中的应用。
4.根据权利要求3所述的应用，其特征在于，所述的用于特异性沉默剪切因子基因RS2Z32的沉默片段的核苷酸序列如SEQ ID NO.2所示；所述的重组沉默载体是将所述的沉默片段插入到植物转化质粒中得到的；所述的转化体是由所述的重组沉默载体导入宿主细胞得到的；所述的用于特异性敲除剪切因子基因RS2Z32的sgRNA的核苷酸序列如SEQ IDNO.3所示；所述的重组敲除载体是将所述的sgRNA序列插入到含有CRISPR/Cas9基因编辑功能的植物转化质粒中得到的；所述的转化体是由所述的重组敲除载体导入宿主细胞得到的。
5.一种提高作物抗性的方法，其特征在于，该方法通过沉默或敲除核苷酸序列如SEQID NO.1所示的剪切因子基因RS2Z32能够提高番茄对致病疫霉的抗性。
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