CN114606259A - 一类cle小肽在控制芽再生和作为植物组培增殖调节剂中的应用 - Google Patents
一类cle小肽在控制芽再生和作为植物组培增殖调节剂中的应用 Download PDFInfo
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Abstract
本发明公开了一类CLE小肽在控制芽再生和作为植物组培增殖调节剂的应用,属于植物生物技术领域。本发明发现了CLE1‑7小肽具有控制植物芽再生或作为植物组织培养增殖调节剂的应用,可通过基因编辑使CLE1‑7基因中的一种或多种的功能缺失提高植物组织培养中的芽再生能力,或过提高CLE1‑7基因中的一种或多种的表达抑制植物组织培养中的芽再生能力,或通过向组织培养基中添加CLE1‑7小肽中的一种或多种抑制植物组织培养中的芽再生能力。
Description
技术领域
本发明属于植物生物技术领域,具体涉及一类可以调节植物重编程、芽再生、抑制植物外植体芽增殖能力的小肽和它们充当植物组培调节剂的应用。
背景技术
组织培养是实现植物无性繁殖和大规模扩繁的常用植物生物技术手段之一。具体是,在无菌培养的条件下,通过调整培养基中的植物激素的种类、含量和配比,主要包括生长素类与细胞分裂素类,从而诱导合适的外植体形成胚性愈伤,并进一步经历愈伤增殖和分化等过程获取高质量的再生芽,再生芽在合适的生长调节剂,一般是高浓度细胞分类素的作用下实现生根、并最后通过练苗等步骤获取大量再生苗。获取高质量的再生芽是影响植物组培成败的关键环节。
组培常用的芽再生过程需要经历两个阶段:第一步在高浓度生长素的诱导下形成具有多潜能性的胚性愈伤,第二步愈伤在高浓度细胞分裂素的诱导启动芽再生。芽再生芽的过程与植物干细胞活性有关,但不同于茎尖分生组织发育和根尖分生组织发育,与植物种类、基因型、外植体类型、培养条件和生长调节剂等因素有关,但是具体的调控机制尚不清楚。对组培生产而言,研究影响芽再生的新型植物调节剂具有广阔的应用价值。
生长调节剂一般指的是植物激素。高等植物普遍存在一类序列和功能保守的CLAVATA3/EMBRYO SURROUNDING REGION-RELATED(CLE)小肽,简称CLE小肽,被认为是一种新型多肽植物激素。CLE小肽的已知功能是维持植物分生组织区的干细胞活性,目前研究较为清楚的是CLV3。CLE1、CLE2、CLE3、CLE4、CLE5、CLE6和CLE7(CLE1-7)是一族亲缘关系紧密的CLE小肽,它们的氨基酸序列在不同物种高度保守。拟南芥CLE1-7基因合成4种小肽,分别是:CLE1/3/4p(CLE1、CLE3和CLE4的氨基酸序列一致):RLSPGGPDPRHH;CLE2p:RLSPGGPDPQHH;CLE5/6p(CLE5和CLE6的氨基酸序列一致):RVSPGGPDPQHH;CLE7p:RFSPGGPDPQHH。超表达这些小肽基因可以互补CLV3缺失的表型。此外,它们还有哪些生理学功能,仍未见公开报道。
获取高质量的再生芽是影响植物组培成败的关键环节。植物激素充当生长调节剂,在芽再生过程中发挥至关重要的作用。即便尝试调整培养基中的植物激素的种类、含量和配比等因子,某些植物仍很难再生芽。对组培生产而言,研究影响芽再生的新型植物调节剂具有广阔的应用价值。
发明内容
本发明的目的在于提供CLE1-7小肽在控制芽再生和作为植物组培增殖调节剂中的应用。
本发明发现CLE1、CLE2、CLE3、CLE4、CLE5、CLE6和CLE7(CLE1-7)编码基因的缺失促进拟南芥下胚轴外植体在芽诱导培养基(shoot-induction medium,SIM)上形成再生芽。
本发明发现与野生型相比,CLE4和CLE7超表达植株芽再生受到显著抑制。与之一致的是,外源施加人工合成CLE1-7小肽,即CLE1/3/4p、CLE2p、CLE5/6p和CLE7p,同样抑制拟南芥芽再生。
本发明发现CLE1-7通过影响植物保守的干性因子WUSCHEL(WUS)活性调控拟南芥芽再生过程,表明这类保守的小肽激素调控植物芽再生的新功能在高等植物中具有普遍意义。
基于以上发现,本发明提供一类小肽CLE1、CLE2、CLE3、CLE4、CLE5、CLE6和CLE7(CLE1-7)在控制植物芽再生中的应用。
在一些实施方案中,可通过基因编辑使CLE1-7基因中的一种或多种的功能缺失提高植物在组织培养中的芽再生能力。
在一些实施方案中,可通过提高CLE1-7基因中的一种或多种的表达抑制植物在组织培养中的芽再生能力。
在一些实施方案中,可通过向组织培养基中添加CLE1-7小肽中的一种或多种抑制植物在组织培养中的芽再生能力。
本发明还提供了一类小肽CLE1、CLE2、CLE3、CLE4、CLE5、CLE6和CLE7(CLE1-7)作为植物组织培养增殖调节剂的应用,其具体为通过加入CLE1-7小肽中的一种或多种抑制植物在组织培养中的芽再生能力,以获取高质量再生芽。
上述植物为含干性因子WUSCHEL(WUS)的植物。在一些实施方案中,所述的植物为拟南芥。
在一些实施方案中,上述CLE1-7小肽的氨基酸序列分别如下:
CLE1/3/4p(CLE1、CLE3和CLE4的氨基酸序列一致):RLSPGGPDPRHH(SEQ ID NO.1);
CLE2p:RLSPGGPDPQHH(SEQ ID NO.2);
CLE5/6p(CLE5和CLE6的氨基酸序列一致):RVSPGGPDPQHH(SEQ ID NO.3);
CLE7p:RFSPGGPDPQHH(SEQ ID NO.4)。
在一些实施方案中,上述CLE1-7小肽的编码基因的核苷酸序列分别如下:
CLE1基因:ATGGCTAACTTGAAATTCTTGCTGTGCTTGTTCTTGATCTGCGTTTCCTTATCGCGTTCATCAGCGTCTCGACCGATGTTCCCAAACGCAGACGGGATTAAACGAGGGCGTATGATGATAGAAGCAGAGGAAGTGTTGAAAGCGAGTATGGAGAAGCTAATGGAGAGAGGTTTTAATGAGTCCATGAGACTCAGTCCTGGAGGTCCCGATCCTCGCCATCACTAA(SEQ ID NO.5);
CLE2基因:ATGGCTAAGTTAAGCTTCACTTTCTGCTTCTTGTTGTTTCTTCTGTTATCCTCAATCGCCGCTGGAAGCCGCCCTCTTGAGGGGGCTCGGGTCGGGGTGAAGGTGAGAGGCCTAAGCCCTTCTATCGAGGCTACGAGTCCGACTGTAGAGGATGATCAAGCTGCGGGTAGCCATGGGAAATCTCCAGAGCGGTTAAGCCCAGGAGGACCCGACCCACAACATCACTAG(SEQ ID NO.6);
CLE3基因:ATGGCAAGTCTCAAGTTATGGGTTTGTTTGGTCCTGCTTCTAGTACTCGAATTGACATCGGTGCACGAATGTCGACCATTGGTTGCCGAAGAGAGATTTTCTGGTTCAAGTCGTTTGAAAAAGATAAGACGTGAACTTTTTGAGAGGTTAAAAGAGATGAAGGGGAGATCAGAAGGCGAAGAGACGATCCTTGGAAATACTCTTGACTCAAAGCGGCTTAGTCCCGGTGGTCCTGACCCGAGGCATCACTGA(SEQ ID NO.7);
CLE4基因:ATGGCAAGTTTCAAGTTATGGGTTTGCCTTATATTGCTTCTACTCGAGTTCTCGGTGCATCAATGCCGACCACTGGTTGCGGAAGAGAGCCCTTCAGATTCAGGTAACATAAGAAAGATTATGAGGGAACTTCTCAAAAGATCAGAAGAGCTGAAGGTAAGATCGAAAGACGGCCAAACGGTTCTAGGCACCCTTGATTCAAAGCGGCTCAGCCCTGGTGGGCCGGACCCTAGACATCACTAA(SEQ ID NO.8);
CLE5基因:ATGGCGACTTTGATCCTCAAGCAAACTCTAATCATACTCCTAATCATATTTTCATTACAAACCTTAAGTTCTCAAGCTCGAATCCTCCGTTCATATCGTGCCGTGTCCATGGGCAATATGGATAGTCAGGTTCTCCTACATGAACTCGGGTTTGATCTCTCTAAGTTCAAAGGTCATAACGAGAGGCGATTTTTAGTGAGTTCCGACAGGGTTTCACCCGGAGGTCCCGATCCACAACACCATTGA(SEQ ID NO.9);
CLE6基因:ATGGCGAATTTGATCCTTAAGCAATCTCTAATCATACTCCTAATCATATATTCAACACCAATCTTGAGTTCTCAAGCTCGAATCCTCCGTACATATCGCCCCACAACCATGGGCGATATGGATAGTCAGGTTCTCCTACGTGAACTCGGGATTGATCTCTCTAAGTTCAAAGGTCAAGACGAGAGACGGTTTTTAGTGGATTCCGAAAGGGTTTCTCCGGGGGGTCCTGATCCACAACACCATTGA(SEQ ID NO.10);
CLE7基因:ATGGCTTCTAAAGCGTTATTGTTATTTGTTATGCTCACCTTTCTATTGGTAATTGAAATGGAAGGGAGGATACTTCGGGTGAATTCAAAGACTAAAGATGGTGAGAGCAACGATCTTTTGAAACGGTTAGGTTACAATGTTTCTGAACTAAAGCGTATTGGCCGAGAGCTTTCCGTCCAAAACGAAGTAGATAGGTTTTCTCCAGGAGGGCCTGACCCTCAACATCACTCTTATCCTCTGTCTTCAAAACCTAGAATTTGA(SEQ ID NO.11)。
本发明的优点在于:
(1)本发明发现了CLE1、CLE2、CLE3、CLE4、CLE5、CLE6和CLE7(CLE1-7)控制芽再生的新功能。
(2)当前植物基因编辑技术的日趋成熟,越来越多的植物基因组被测序,使得在不同植物中敲除或者超表达基因变得可行。敲除一个或者全部的CLE1、CLE2、CLE3、CLE4、CLE5、CLE6和CLE7(CLE1-7)小肽,将显著提高植物组培过程中芽再生的能力,这个应用对那些愈伤难以再生芽的植物而言尤为重要。
(3)人工合成CLE1、CLE2、CLE3、CLE4、CLE5、CLE6和CLE7(CLE1-7)小肽的优点是:便于获取、便于保存、容易配制、价格适合。将上述人工合成小肽作为一种抑制性生长调节剂,添加至SIM培养基,可以拮抗高浓度细胞分裂素,从而获取适量的高质量再生芽。
附图说明
图1是不同CLE基因型(突变体)和野生型的再生芽数目统计。星号代表显著性差异。统计方法:Dunnett多重比较检验,单向方差分析(**P<0.01,****P<0.0001)。cle4-cr2和cle4c是CLE4的两个等位突变体,cle4c cle5c cle6c是CLE4/5/6三突变体,cle7-cr1是CLE7突变体,cle1-7sept是CLE1-7七突变体。这些单突变体和多突变体再生芽数目显著多于野生型对照。
图2是CLE4和CLE7超表达材料和野生型的再生芽数目统计。#1和#2代表独立的转基因家系。星号代表显著性差异。统计方法:Dunnett多重比较检验,单向方差分析(****P<0.0001)。
图3是外源施加人工合成CLE小肽处理后再生芽数目统计。星号代表显著性差异。统计方法:Dunnett多重比较检验,单向方差分析(*P<0.05,**P<0.01,****P<0.0001)。
图4是CLE1-7通过调控WUS基因活性影响芽再生的能力。(A)在SIM培养基外源施加CLE1/3/4p和CLE7p,与不外施(对照)相比,在2天、5天和6天,分别观察pWUS::GUS染色强度,发现外施CLE1/3/4p和CLE7p导致pWUS::GUS强度减弱。(B)利用RT-qPCR检测cle1-7sept(CLE1-7七突变体)WUS基因的表达活性,发现在SIM培养基培养6天和8天,与野生型对照相比,WUS在cle1-7sept的表达增强。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
若未特别指明,下述实施例所用的技术手段为本领域技术人员所熟知的常规手段;所用实验方法均为常规方法,并可按照已描述的重组技术(参见分子克隆实验指南,第4版,冷泉港实验室出版社,冷泉港,纽约)完成;所用的材料、试剂等,均可从商业途径得到。
下述实施例中的组织培养所用培养基及培养条件如下,CIM培养基配方是:4.4gMS基本盐(含维他命;basal medium with vitamins),0.5g/L磺酸甲酯(methylestersulfonate),20g/L蔗糖,2.2mM 2,4-D,0.2mM激动素(kinetin)和8g/L琼脂,pH 5.7;SIM培养基配方是:4.4gMS基本盐(含维他命),0.5g/L磺酸甲酯,20g/L蔗糖,5mM激动素,0.9mM吲哚-3-乙酸(indole-3-acetic acid)和8g/L琼脂,pH 5.7;培养温度是22℃,光周期是16h光/8h暗。
实施例1CLE1、CLE2、CLE3、CLE4、CLE5、CLE6和CLE7(CLE1-7)编码基因的缺失促进拟南芥下胚轴外植体在芽诱导培养基(shoot-induction medium,SIM)上形成再生芽。
实施例1中涉及到的拟南芥材料共有16份。其中1份是野生型(col-0),15份分别是CLE1-7编码基因的缺失突变体(包括单突变体和多突变体)。如下11份单突变体是利用CRISPR/Cas9基因编辑技术创制:cle1-cr2,cle2-cr1,cle3-cr1,cle3-cr2,cle4-cr2,cle5-cr1,cle5c,cle6-cr1,cle6-cr2,cle6c和cle7-cr1(详见文献Yamaguchi Y L,IshidaT,Yoshimura M,et al.A collection of mutants for CLE-peptide-encoding genes inArabidopsis generated by CRISPR/Cas9-mediated gene targeting[J].Plant andCell Physiology,2017,58(11):1848-1856.)。
利用CRISPR/Cas9基因编辑技术创制cle4c单突变体,cle4c cle5c双突变体和cle4c cle5c cle6c三突变体。具体如下:CLE4-sgRNA(含PAM)序列是:GGTGCATCAATGCCGACCACTGG。CLE5-sgRNA(含PAM)序列是:CCGTGTCCATGGGCAATATGGAT。CLE6-sgRNA(含PAM)序列是:CCACAACCATGGGCGATATGGAT(Yamaguchi Y L,Ishida T,Yos himuraM,et al.A collection of mutants for CLE-peptide-encoding genes in Arabidopsisgenerated by CRISPR/Cas9-mediated gene targeting[J].Plant and CellPhysiology,2017,58(11):1848-1856.)。利用Gateway克隆将含有CLE4-sgRNA或同时含有CLE4/5/-sgRNA或CLE4/5/6/-sgRNA的At-psgR-Cas9质粒整合到表达载体pEarleyGate301。通过热激法将含有sgRN A的终载体转化至农杆菌GV3101。利用花序浸染法转化拟南芥。经测序,cle4c纯合子在位于起始密码子ATG下游+151发生了“C”的插入突变。经测序,cle4c cle5c cle6c纯合子分别在CLE4基因起始密码子ATG下游+151发生了“C”的插入突变,在CLE5基因起始密码子ATG下游+214发生了“A”的缺失突变,在CLE5基因起始密码子ATG下游+109发生了“ACCA”的缺失突变。
利用CRISPR/Cas9基因编辑技术创制cle1-7sept七突变体。具体如下:CLE1、CLE2、CLE3、CLE4、CLE5和CLE6的sgRNA序列参考已发表文献(Yamaguchi Y L,Ishida T,Yoshimura M,et al.A collection of mutants for CLE-peptide-encoding genes inArabidopsis generated by CRISPR/Cas9-mediated gene targeting[J].Plant andCell Physiology,2017,58(11):1848-1856.)。CLE1/3/4和CLE2/5/6的sgRNA分别串联融合成两个片段,分别整合到pDe-Cas9载体后,经Gateway克隆进一步整合到表达载体pGWB501。终载体经农杆菌转化后,利用花序浸染法转化到上述的cle7-cr1突变体。通过筛选和测序,获得cle1-7sept七突变体。
所有遗传材料经过暗培养7天,取1cm的下胚轴作为外植体。将外植体转移到CIM培养基后,继续培养7天至形成愈伤。将愈伤转移至SIM培养基,继续培养21天。利用NikonSMZ745T体式显微镜观察、拍照和计数每个外植体愈伤的再生芽数目。再生芽的定义是一个被3片叶子包围的分生组织样结构。所有实验数据收集3次生物学重复。结果表明,CLE1-7编码基因的缺失促进拟南芥下胚轴外植体在SIM培养基上形成再生芽。以cle4-cr2和cle4c(CLE4的两个等位突变体)、cle4c cle5c cle6c(CLE4/5/6三突变体)、cle7-cr1(CLE7突变体)、cle1-7sept(CLE1-7七突变体)的表型最为显著(图1)。
实施例2与野生型相比,CLE4和CLE7超表达植株芽再生受到显著抑制。
由于拟南芥CLE1-7基因仅合成4种小肽,而且从上述突变体表型分析来看,CLE4和CLE7对芽再生的贡献作用尤为突出,因此仅利用自身启动子驱动CLE4和CLE7获取转基因超表达材料。每个基因的超表达材料保留2个独立家系。对超表达材料开展再生芽表型考察。
实施例2中涉及到的CLE4和CLE7超表达植株创制方法如下:包含CLE4和CLE7 5’-端和3’-端调控元件的基因组片段经PCR扩增后克隆至pDONR201载体,经Gateway克隆进一步整合到表达载体pBGWFS7,获取pCLE4:CLE4:CLE4ter和pCLE7:CLE7:CLE7ter。终载体经农杆菌转化后,利用花序浸染法转化到拟南芥,获取的转基因家系经PCR表达量鉴定,各自保留两个家系。
克隆引物序列分别是:
CLE4-OE-F:5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTGGTCACCAATCAAA-3',
CLE4-OE-R:5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTAAATCCGAATGT-3';
CLE7-OE-F:5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTGGTTTAGCTTCACA-3',
CLE7-OE-R:5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTAAGCTACGTTTG-3'。
所有遗传材料经过暗培养7天,取1cm的下胚轴作为外植体。将外植体转移到CIM培养基后,继续培养7天至形成愈伤。将愈伤转移SIM培养基,继续培养21天。利用NikonSMZ745T体式显微镜观察、拍照和计数每个外植体愈伤的再生芽数目。再生芽的定义是一个被3片叶子包围的分生组织样结构。所有实验数据收集3次生物学重复。结果表明,CLE4和CLE7基因超表达显著抑制拟南芥下胚轴外植体在SIM培养基上形成再生芽(图2)。
实施例3外源施加人工合成CLE1-7小肽同样抑制拟南芥芽再生。
CLE1-7是一族亲缘关系紧密的CLE小肽,它们的氨基酸序列在不同物种高度保守。拟南芥CLE1-7基因合成4种小肽,分别是:CLE1/3/4p(CLE1、CLE3和CLE4的氨基酸序列一致):RLSPGGPDPRHH;CLE2p:RLSPGGPDPQHH;CLE5/6p(CLE5和CLE6的氨基酸序列一致):RVSPGGPDPQHH;CLE7p:RFSPGGPDPQHH。
上述的遗传证据表明,拟南芥(植物)体内CLE1-7的累积水平与芽再生能力呈负相关。因此,在SIM培养基里外施人工合成小肽将抑制野生型拟南芥芽再生能力。
基于此,野生型拟南芥幼苗经过暗培养7天,取1cm的下胚轴作为外植体。将外植体转移到CIM培养基后,继续培养7天至形成愈伤。将愈伤转移至含有不同浓度人工合成小肽(即0μM,5μM和10μM)的SIM培养基,继续培养21天。利用Nikon SMZ745T体式显微镜观察、拍照和计数每个外植体愈伤的再生芽数目。再生芽的定义是一个被3片叶子包围的分生组织样结构。所有实验数据收集3次生物学重复。结果表明,外源施加人工合成CLE1-7小肽显著抑制拟南芥芽再生(图3)。
实施例4CLE1-7通过影响植物保守的干性因子WUSCHEL(WUS)活性调控拟南芥芽再生过程。
实施例4中涉及的pWUS::GUS是WUS启动子驱动的转基因启动子融合报告子,是转基因植物(Cui Y,Rao S,Chang B,et al.AtLa1 protein initiates IRES-dependenttranslation of WUSCHEL mRNA and regulates the stem cell homeostasis ofArabidopsis in response to environmental hazards[J].Plant,Cell&Environment,2015,38(10):2098-2114.)。
利用GUS染色法,通过观察pWUS::GUS强度,分析外源施加CLE小肽对再生芽过程中GUS活性的影响。pWUS::GUS经过暗培养7天,取1cm的下胚轴作为外植体。将外植体转移到CIM培养基后,继续培养7天至形成愈伤。将愈伤转移至含有不同浓度人工合成小肽(即0μM和10μM)的SIM培养基,继续培养2天、5天和6天,分别观察pWUS::GUS染色强度,发现外施CLE1/3/4p和CLE7p导致pWUS::GUS强度减弱(图4A)。
利用RT-qPCR检测cle1-7sept(CLE1-7七突变体)WUS基因的表达活性,发现该突变体在SIM培养基培养6天和8天,与野生型对照相比,WUS在cle1-7sept的表达增强(图4B)。
综上所述,这些体内的遗传证据,即突变体和超表达材料的再生芽表型分析和外施CLE小肽的结果,支持CLE1-7小肽是控制芽再生的生长调节因子。他们通过影响保守因子WUS发挥这个新功能。从植物系统发育分析的角度,本发明提供一种通过基因遗传操作和外施人工合成CLE小肽控制不同植物组培再生芽效率的实施方法。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110> 陕西师范大学
湖北医药学院
<120> 一类CLE小肽在控制芽再生和作为植物组培增殖调节剂中的应用
<141> 2022-01-27
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213> 拟南芥(Arabidopsis thaliana)
<400> 1
Arg Leu Ser Pro Gly Gly Pro Asp Pro Arg His His
1 5 10
<210> 2
<211> 12
<212> PRT
<213> 拟南芥(Arabidopsis thaliana)
<400> 2
Arg Leu Ser Pro Gly Gly Pro Asp Pro Gln His His
1 5 10
<210> 3
<211> 12
<212> PRT
<213> 拟南芥(Arabidopsis thaliana)
<400> 3
Arg Val Ser Pro Gly Gly Pro Asp Pro Gln His His
1 5 10
<210> 4
<211> 12
<212> PRT
<213> 拟南芥(Arabidopsis thaliana)
<400> 4
Arg Phe Ser Pro Gly Gly Pro Asp Pro Gln His His
1 5 10
<210> 5
<211> 225
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 5
atggctaact tgaaattctt gctgtgcttg ttcttgatct gcgtttcctt atcgcgttca 60
tcagcgtctc gaccgatgtt cccaaacgca gacgggatta aacgagggcg tatgatgata 120
gaagcagagg aagtgttgaa agcgagtatg gagaagctaa tggagagagg ttttaatgag 180
tccatgagac tcagtcctgg aggtcccgat cctcgccatc actaa 225
<210> 6
<211> 228
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 6
atggctaagt taagcttcac tttctgcttc ttgttgtttc ttctgttatc ctcaatcgcc 60
gctggaagcc gccctcttga gggggctcgg gtcggggtga aggtgagagg cctaagccct 120
tctatcgagg ctacgagtcc gactgtagag gatgatcaag ctgcgggtag ccatgggaaa 180
tctccagagc ggttaagccc aggaggaccc gacccacaac atcactag 228
<210> 7
<211> 252
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 7
atggcaagtc tcaagttatg ggtttgtttg gtcctgcttc tagtactcga attgacatcg 60
gtgcacgaat gtcgaccatt ggttgccgaa gagagatttt ctggttcaag tcgtttgaaa 120
aagataagac gtgaactttt tgagaggtta aaagagatga aggggagatc agaaggcgaa 180
gagacgatcc ttggaaatac tcttgactca aagcggctta gtcccggtgg tcctgacccg 240
aggcatcact ga 252
<210> 8
<211> 243
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 8
atggcaagtt tcaagttatg ggtttgcctt atattgcttc tactcgagtt ctcggtgcat 60
caatgccgac cactggttgc ggaagagagc ccttcagatt caggtaacat aagaaagatt 120
atgagggaac ttctcaaaag atcagaagag ctgaaggtaa gatcgaaaga cggccaaacg 180
gttctaggca cccttgattc aaagcggctc agccctggtg ggccggaccc tagacatcac 240
taa 243
<210> 9
<211> 246
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 9
atggcgactt tgatcctcaa gcaaactcta atcatactcc taatcatatt ttcattacaa 60
accttaagtt ctcaagctcg aatcctccgt tcatatcgtg ccgtgtccat gggcaatatg 120
gatagtcagg ttctcctaca tgaactcggg tttgatctct ctaagttcaa aggtcataac 180
gagaggcgat ttttagtgag ttccgacagg gtttcacccg gaggtcccga tccacaacac 240
cattga 246
<210> 10
<211> 246
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 10
atggcgaatt tgatccttaa gcaatctcta atcatactcc taatcatata ttcaacacca 60
atcttgagtt ctcaagctcg aatcctccgt acatatcgcc ccacaaccat gggcgatatg 120
gatagtcagg ttctcctacg tgaactcggg attgatctct ctaagttcaa aggtcaagac 180
gagagacggt ttttagtgga ttccgaaagg gtttctccgg ggggtcctga tccacaacac 240
cattga 246
<210> 11
<211> 261
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 11
atggcttcta aagcgttatt gttatttgtt atgctcacct ttctattggt aattgaaatg 60
gaagggagga tacttcgggt gaattcaaag actaaagatg gtgagagcaa cgatcttttg 120
aaacggttag gttacaatgt ttctgaacta aagcgtattg gccgagagct ttccgtccaa 180
aacgaagtag ataggttttc tccaggaggg cctgaccctc aacatcactc ttatcctctg 240
tcttcaaaac ctagaatttg a 261
Claims (9)
1.一类CLE小肽在控制植物芽再生中的应用,其特征在于:所述的CLE小肽包括CLE1、CLE2、CLE3、CLE4、CLE5、CLE6和CLE7;
其中,CLE1、CLE3和CLE4的氨基酸序列为:RLSPGGPDPRHH;
CLE2的氨基酸序列为:RLSPGGPDPQHH;
CLE5和CLE6的氨基酸序为:RVSPGGPDPQHH;
CLE7的氨基酸序为:RFSPGGPDPQHH。
2.根据权利要求1所述的应用,其特征在于:通过使CLE1-7基因中的一种或多种的功能缺失提高植物在组织培养中的芽再生能力。
3.根据权利要求1所述的应用,其特征在于:通过提高CLE1-7基因中的一种或多种的表达抑制植物在组织培养中的芽再生能力。
4.根据权利要求1所述的应用,其特征在于:通过向组织培养基中添加CLE1-7小肽中的一种或多种抑制植物在组织培养中的芽再生能力。
5.一类CLE小肽作为植物组织培养增殖调节剂的应用,其特征在于:所述的CLE小肽包括CLE1、CLE2、CLE3、CLE4、CLE5、CLE6和CLE7;
其中,CLE1、CLE3和CLE4的氨基酸序列为:RLSPGGPDPRHH;
CLE2的氨基酸序列为:RLSPGGPDPQHH;
CLE5和CLE6的氨基酸序为:RVSPGGPDPQHH;
CLE7的氨基酸序为:RFSPGGPDPQHH。
6.根据权利要求5所述的应用,其特征在于:通过加入CLE1-7小肽中的一种或多种抑制植物在组织培养中的芽再生能力。
7.根据权利要求1-6任一项所述的应用,其特征在于:所述的植物为含干性因子WUSCHEL的植物。
8.根据权利要求7所述的应用,其特征在于:所述的植物为拟南芥。
9.根据权利要求1-6任一项所述的应用,其特征在于:所述的CLE小肽的编码基因的核苷酸序列分别如下:
CLE1基因:核苷酸序列如SEQ ID NO.5所示;
CLE2基因:核苷酸序列如SEQ ID NO.6所示;
CLE3基因:核苷酸序列如SEQ ID NO.7所示;
CLE4基因:核苷酸序列如SEQ ID NO.8所示;
CLE5基因:核苷酸序列如SEQ ID NO.9所示;
CLE6基因:核苷酸序列如SEQ ID NO.10所示;
CLE7基因:核苷酸序列如SEQ ID NO.11所示。
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