CN116622766B - 杨树spl16和spl23基因在调控杨树休眠时期转换上的应用 - Google Patents
杨树spl16和spl23基因在调控杨树休眠时期转换上的应用 Download PDFInfo
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Abstract
本发明公开了杨树SPL16和SPL23基因在调控杨树顶芽休眠时期转换上的应用,所述杨树SPL16基因编码SEQ ID No.1所示的氨基酸序列的蛋白,所述SPL23基因编码SEQ IDNo.2所示的氨基酸序列的蛋白,通过CRISPR/Cas9编辑***敲除SPL16基因或同时敲除SPL16/SPL23基因,从而推迟杨树在感知秋季光周期变化(短日照条件)后顶芽生长停止和进入休眠期的时间。通过推迟林木在秋季短日照条件下的休眠时间能够延长树木的生长期,因此本发明在提高树木光合固碳和生物量积累方面具有较大的应用前景。
Description
技术领域
本发明属于分子生物学领域,具体涉及杨树SPL16和SPL23基因在调控杨树休眠时期转换上的应用。
背景技术
北温带的多年生林木生长具有明显的季节性特征。在秋季短日照条件下顶芽生长停止,休眠建立,随之叶片衰老并脱落,以增加植物对逆境的抵御能力,安全度过对树木生存不利的冬季环境。经过冬天一定时间的低温后,休眠解除,顶芽发生质变在来年春天开始萌芽。顶芽的休眠过程可以划分为3个阶段:生态休眠(ecodormancy)—内休眠(endodormancy)—生态休眠(Shim et al.,2014)。在生态休眠阶段顶端分生组织细胞保持一定对生长因子的反应能力,若环境条件适宜顶芽则可恢复生长。而处于内休眠阶段的干细胞对于外界环境的反应能力消失,只有经过一段时间的低温后才能够打破内休眠状态,恢复对环境的反应能力。
光周期(昼夜周期中光照期和暗期长短的交替变化)是一种决定树木季节性生长停止最佳时间的关键环境信号。当光周期短于生长的临界阈值(短日照,SD)时,茎尖分生组织生长停止,叶原基也不再形成嫩叶,而是形成包裹茎尖分生组织的芽鳞(Olsen,2010)。生长停止后,短日照进而导致芽过渡到内休眠,其特点是芽分生组织无法对生长促进信号做出反应,这对植物越冬至关重要(Tylewicz et al.,2008)。研究表明,高温和长日照能推迟生理休眠期的到来(Begum et al.,2007,2008;Tanino et al.,2010),低温可以诱导休眠解除,低温积累对于解除内休眠以及植物在来年春天的萌芽至关重要(Ding et al.,2014)。综上所述,树木的季节性生长主要受光周期和温度信号的调控。
光是一种重要的环境因子,既可以作为能量参与光合作用,又可以作为信号调节植物生长发育。研究表明,参与调控杨树生长停止及休眠的遗传途径与拟南芥中光周期介导的开花调控通路之间存在显著的保守性。高等植物对光的感知依赖于红光、远红光受体光敏色素和蓝光受体隐花色素。杨树的基因组有3个光敏色素(phytochrome)基因:PHYA、PHYB1和PHYB2(Howe et al.,1998)。最新研究表明,杨树phyB1和phyB2是顶芽季节性生长的促进因子(Ding et al.,2021)。属于bHLH转录因子家族的光敏色素互作因子(Phytochrome-Interacting Factor 8,PIF8)是杨树季节性生长的抑制因子。通过RNAi干扰的方式降低PIF8表达导致顶芽对短日照条件的敏感程度降低。转录测序分析显示PIF8通过间接调控FLOWERING LOCUS T(FT)、和BRANCHED 1(BRC1)的表达来控制顶芽对短日照的感应(Ding et al.,2021)。然而,作用于phyB-PIF8模块下游、介导调控FT2/BRC1基因的分子元件仍不清楚。
microRNA156(miR156)及其靶标基因SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE(SPL)是年龄途径的重要组分。miR156抑制SPL的表达,参与调控植物幼年期向成年期转变、成花转变和株型(分枝)等多个生长和发育过程。miR156在种胚和幼苗中表达水平较高,随着年龄的增加miR156表达减少,而SPL表达上调,从而促进植株的成熟和开花(Xu et al.,2016)。杨树基因组中具有保守的MIR156和SPL基因,但关于杨树miR156-SPL模块的研究仍处于起步阶段,其生物学功能仍鲜有报道。鉴于拟南芥SPL3、SPL4和SPL5转录因子在调控开花和分枝方面发挥重要作用。杨树中与拟南芥SPL3/4/5同源的SPL16和SPL23转录因子是否参与调控杨树顶芽的季节性生长值得深入探索。
发明内容
本发明所要解决的技术问题为:如何提供一种减弱杨树响应短日照条件,延缓顶芽生长停止和休眠时间的方法。
本发明的技术方案为:杨树SPL16或/和SPL23基因在调控杨树季节性生长过程中的休眠时期转换上的应用,所述杨树SPL16基因编码SEQ ID No.1所示的氨基酸序列的蛋白,所述SPL23基因编码SEQ ID No.2所示的氨基酸序列的蛋白。
进一步地,所述应用为通过CRISPR/Cas9***敲除SPL16基因或同时敲除SPL16/SPL23基因,从而推迟杨树在感知秋季光周期变化(短日照条件)后生长停止和建立休眠的时间。
进一步地,所述杨树SPL16基因的核苷酸序列如SEQ ID No.3所示,所述SPL23基因的核苷酸序列如SEQ ID No.4所示。
一种构建推迟进入秋季休眠期的杨树株系的方法,包括如下步骤:
(1)利用CRISPR/Cas9编辑技术,构建针对杨树SPL16基因或同时针对SPL16基因和SPL23基因的编辑载体;
(2)采用农杆菌介导的叶盘法将步骤(1)构建的编辑载体转入杨树叶片中;
(3)通过杨树组织培养、筛选阳性转基因植株,将长日照条件(16h光照/8h黑暗)培养的毛白杨野生型和转基因植株转移至短日照条件(10h光照/14h黑暗)进行处理,观察和统计顶芽表型变化,获得在短日照条件下顶芽休眠期延缓的的杨树株系。
进一步地,针对SPL16基因的编辑载体的sgRNA核苷酸序列如SEQ ID No.5和6所示,针对SPL23基因的编辑载体的sgRNA核苷酸序列如SEQ ID No.7和8所示。
一种靶向杨树SPL16/SPL23基因的sgRNA,所述sgRNA核苷酸序列如SEQ IDNo.5~SEQ ID No.8中的任一种。
一种基因编辑载体,含有SEQ ID No.5~SEQ ID No.8中的任一sgRNA。
本发明提供了SPL16和SPL23基因在调控杨树季节性生长时期转换中的应用。利用CRISPR/Cas9基因编辑技术对毛白杨中的SPL16和SPL23两个基因进行了基因编辑,使SPL16和SPL23基因的靶点产生碱基缺失、***或大片段删除。结果显示,spl16 L1单突中SPL16基因T2靶点有一个碱基缺失,spl16 L2单突中SPL16基因T2靶点有31个碱基删除。spl16/23L1双突中SPL16基因T1靶点有6个碱基缺失,SPL23基因T1靶点有1个碱基***。spl16/23L2双突中SPL16基因T1靶点有3个碱基缺失,SPL23基因T1靶点有5个碱基缺失。在正常培养的长日照条件下,突变体植株与野生型植株相比,侧枝数目显著增加,并且双突植株侧枝数>单突植株侧枝数>野生型植株侧枝数。将毛白杨野生型WT和spl16单突、spl16/23双突材料在长日照条件(16h光照/8h黑暗)培养2个月后,一部分材料转移到短日照条件(10h光照/14h黑暗)做处理,另一部分材料(对照组)继续在长日照条件下培养。在短日照处理大约15天之后,发现spl16单突、spl16/23双突材料相较于WT型杨树顶芽的生长停止和休眠时间推迟,表明突变体植株的顶芽在短日照条件下仍维持较高的生长活性。
与现有技术相比,本发明具有以下有益效果:
本发明表明单独敲除SPL16,或同时敲除SPL16/23基因后,可改变杨树季节性生长过程中的休眠时期转换。通过推迟林木在秋季短日照条件下的休眠时间能够延长树木的生长期,因此在提高树木光合固碳和生物量积累方面具有较大的应用前景。
附图说明
图1.毛白杨spl16单突变体和spl26/23双突变体材料的创制。(a)杨树SPL16和SPL23的基因结构以及第一个外显子中靶位点(T1,T2)的设计。(b)毛白杨spl16单突变体(两个株系,L1和L2)的基因型测序结果。
图2.毛白杨spl26/23双突变体材料的创制。
图3.杨树spl16单突变体和spl26/23双突变体材料的表型观察。(a)毛白杨野生型、spl16单突和spl26/23双突材料在长日照条件下的侧枝表型。(b)毛白杨野生型、spl16单突和spl26/23双突材料在长日照(LD)和短日照(SD)条件下的顶芽表型。(c)侧枝数目的统计分析。不同字母代表显著性差异(p<0.05,ANOVA分析)。(d)不同遗传背景杨树材料在短日照条件下顶芽的活性比较分析,展示突变体株系与野生型相比生长停止和休眠时间推迟。
具体实施方式
下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为从商业渠道购买得到的。
(1)SPL16和SPL23基因的CRISPR/Cas9敲除载体的构建
首先,从Phytozome数据库下载杨树SPL16基因(基因序列如SEQ ID No.3,编码蛋白如SEQ ID No.1)和SPL23基因(基因序列如SEQ ID No.4,编码蛋白如SEQ ID No.2)的基因组序列,利用SnapGene Viewer软件设计SPL16和SPL23基因特异的靶标序列(设计sgRNA)。为了保证基因编辑效率,对每个基因设计了2个最优的sgRNA靶标序列。
表1设计的sgRNA序列
靶向基因 | T1 | T2 |
SPL16 | ATGGAAACAAGCAAAGCAGAAGG | ATGGCGTCTATGGTATCGCTTGG |
SPL23 | ATGGGAACAAGCAAAGCTGAAGG | ATGTCGTTTATGGTATCGCTTGG |
之后将这些靶标序列通过同源重组的方式连入pYLCRISPR_Cas9P35S-H载体中,经过PCR测序验证载体无误后转化农杆菌。
(2)杨树的遗传转化和基因型鉴定
以毛白杨(Populus tomentosa Carrière)为受体材料,通过农杆菌介导的叶盘法将构建成功的CRISPR/Cas9载体导入杨树叶片中,通过杨树组织培养、筛选具有相应抗性的阳性转基因植株。
首先,将构建成功的CRISPR/Cas9载体转入农杆菌,在28℃培养箱培养2天,然后挑单克隆在添加卡那霉素的YEP培养基中,28℃摇床过夜培养。将过夜培养的农杆菌按1∶100比例加入添加卡那霉素的YEP培养基中,在28℃摇床振荡培养OD至0.4-0.6。随后在4℃低温离心机中进行离心,4000rpm,10min,收集菌体,用重悬液将菌体重悬后加入重悬液至30mL,在28℃摇床振荡培养45min。随后杨树叶片切为4-6mm叶盘,在农杆菌液中侵染10min,每3-5分钟振荡一次。10min后用无菌纸吸干叶盘表面菌液,然后放置共培养培养基上,暗培养2天,随后转移至有卡那霉素的培养基进行培养。
获得整株植株后,取植株叶片,用CTAB法提取植物DNA。为了鉴定其基因型,利用SPL16和SPL23基因的特异引物,扩增包含靶点序列的基因片段,通过PCR测序获得准确的基因编辑信息。具体结果为:spl16 L1单突中SPL16基因T2靶点有一个碱基缺失,spl16L2单突中SPL16基因T2靶点有31个碱基删除。spl16/23L1双突中SPL16基因T1靶点有6个碱基缺失,SPL23基因T1靶点有1个碱基***。spl16/23L2双突中SPL16基因T1靶点有3个碱基缺失,SPL23基因T1靶点有5个碱基缺失,如图1和图2所示。
(3)转基因植株的表型观察
将毛白杨野生型WT和spl16单突、spl16/23双突材料在长日照条件(16h光照/8h黑暗)培养2个月后,一部分材料转移到短日照条件(10h光照/14h黑暗)做处理,另一部分材料(对照组)继续在长日照条件下培养。根据前人研究对顶芽生长状态的评分标准(Rohde etal.,2011;Johansson et al.,2022),观察、记录和评价不同遗传背景的杨树在短日照处理后顶芽生长状态的变化。
结果如图3所示,在正常培养的长日照条件下,突变体植株与野生型植株相比,侧枝数目显著增加,并且双突植株>单突植株>野生型植株。将毛白杨野生型WT和spl16单突、spl16/23双突材料在长日照条件(16h光照/8h黑暗)培养2个月后,一部分材料转移到短日照条件(10h光照/14h黑暗)做处理,另一部分材料(对照组)继续在长日照条件下培养。在短日照处理大约15天之后,发现spl16单突、spl16/23双突材料相较于WT型杨树顶芽的生长停止和休眠时间推迟,表明突变体植株的顶芽在短日照条件下仍维持较高的生长活性。本发明表明单独敲除SPL16,或同时敲除SPL16/23基因后,可改变杨树季节性生长过程中的休眠时期转换。在秋季短日照条件下,延缓树木顶芽的生长停止和休眠时间能促进树木的生长期,因此在提高树木光合固碳和生物量积累方面具有较大的应用前景。
Claims (4)
1.杨树SPL16或/和SPL23基因在调控杨树季节性生长过程中顶芽的休眠时期转换上的应用,所述SPL16基因编码SEQ ID No.1所示的氨基酸序列的蛋白,所述SPL23基因编码SEQID No.2所示的氨基酸序列的蛋白,所述应用为通过CRISPR/Cas9编辑***敲除SPL16基因或同时敲除SPL16/SPL23基因,从而推迟杨树感知秋季光周期变化后顶芽生长停止和进入休眠期的时间。
2. 根据权利要求1所述的应用,其特征在于,所述SPL16基因的核苷酸序列如SEQ IDNo.3所示,所述SPL23基因的核苷酸序列如SEQ ID No.4所示。
3.一种构建减弱杨树响应短日照条件、延缓顶芽生长停止和休眠时间的杨树株系的方法,其特征在于,包括如下步骤:
(1)利用CRISPR/Cas9基因编辑技术,构建针对杨树SPL16基因或同时针对SPL16和SPL23基因的编辑载体;所述SPL16基因的核苷酸序列如SEQ ID No.3所示,所述SPL23基因的核苷酸序列如SEQ ID No.4所示;
(2)采用农杆菌介导的叶盘法将步骤(1)构建的编辑载体转入杨树叶片中;
(3)通过杨树组织培养、筛选阳性转基因植株,将长日照条件培养的毛白杨野生型和转基因植株转移至短日照条件进行处理,观察和统计顶芽表型变化,获得在短日照条件下顶芽休眠期延缓的的杨树株系,所述长日照条件是指16h 光照/8h 黑暗,短日照条件是指10h光照/14h 黑暗。
4. 根据权利要求3所述的方法,其特征在于,针对SPL16基因的编辑载体的sgRNA核苷酸序列如SEQ ID No.5和6所示,针对SPL23基因的编辑载体的sgRNA核苷酸序列如SEQ IDNo.7和8所示。
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102399270A (zh) * | 2010-09-15 | 2012-04-04 | 西南大学 | 毛白杨中的MYB类转录因子PtrMYB01及其cDNA的克隆方法及应用 |
CN106591320A (zh) * | 2015-10-15 | 2017-04-26 | 东北林业大学 | 促进提前开花的白桦BplSPL1基因及其编码蛋白 |
CN107058337A (zh) * | 2017-03-20 | 2017-08-18 | 中国农业科学院茶叶研究所 | 茶树开花基因CsFT及其编码蛋白 |
CN108409858A (zh) * | 2018-05-29 | 2018-08-17 | 天津农学院 | 一种番茄果实转录因子cnr多克隆抗体及其制备方法 |
CN110527687A (zh) * | 2019-07-25 | 2019-12-03 | 南京大学 | 一种水稻转录因子基因Osspl10及其应用 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
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US8030546B2 (en) * | 1998-09-22 | 2011-10-04 | Mendel Biotechnology, Inc. | Biotic and abiotic stress tolerance in plants |
US7989676B2 (en) * | 2006-08-31 | 2011-08-02 | Ceres, Inc. | Nucleotide sequences and corresponding polypeptides conferring modulated plant characteristics |
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Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102399270A (zh) * | 2010-09-15 | 2012-04-04 | 西南大学 | 毛白杨中的MYB类转录因子PtrMYB01及其cDNA的克隆方法及应用 |
CN106591320A (zh) * | 2015-10-15 | 2017-04-26 | 东北林业大学 | 促进提前开花的白桦BplSPL1基因及其编码蛋白 |
CN107058337A (zh) * | 2017-03-20 | 2017-08-18 | 中国农业科学院茶叶研究所 | 茶树开花基因CsFT及其编码蛋白 |
CN108409858A (zh) * | 2018-05-29 | 2018-08-17 | 天津农学院 | 一种番茄果实转录因子cnr多克隆抗体及其制备方法 |
CN110527687A (zh) * | 2019-07-25 | 2019-12-03 | 南京大学 | 一种水稻转录因子基因Osspl10及其应用 |
Non-Patent Citations (12)
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