CN113481211B - 果胶甲酯酶抑制因子基因GhPMEI39及其编码蛋白的应用 - Google Patents
果胶甲酯酶抑制因子基因GhPMEI39及其编码蛋白的应用 Download PDFInfo
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- CN113481211B CN113481211B CN202110879491.1A CN202110879491A CN113481211B CN 113481211 B CN113481211 B CN 113481211B CN 202110879491 A CN202110879491 A CN 202110879491A CN 113481211 B CN113481211 B CN 113481211B
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Abstract
本发明涉及植物性能基因调控领域,具体而言,涉及果胶甲酯酶抑制因子基因GhPMEI39及其编码蛋白的应用。果胶甲酯酶抑制因子基因GhPMEI39的基因序列如SEQIDNO:1所示。本发明利用公开的数据库筛选目的基因,设计引物克隆目的基因,构建其棉花转基因和拟南芥转基因材料,分析过量和组成性表达果胶甲酯酶抑制蛋白后植物发育表型,验证了果胶甲酯酶抑制因子基因GhPMEI39在植物开花和花序形态等相关功能中的作用。
Description
技术领域
本发明涉及植物性能基因调控领域,具体而言,涉及果胶甲酯酶抑制因子基因GhPMEI39及其编码蛋白的应用。
背景技术
维管束是植物重要的运输通道,其主要由韧皮部和木质部细胞组成,负责运输植物发育过程当中的水分、无机盐、有机物质、各种蛋白、氨基酸等,从而调控植物的营养生长和生殖生长。以前的研究表明CLE25、APL等基因在调控植物韧皮部起始及发育中发挥重要作用。PMEI是细胞壁果胶甲酯酶抑制因子,其在调节细胞壁的韧性、通透性和生化属性方面发挥重要作用,从而影响种子萌发、病菌侵入等多种植物发育以及与外界因素的互作等。
HG是最丰富的果胶聚合物,HG甲基酯化对细胞壁的流变特性和植物发育有显著影响。去甲基酯化的HG可以很容易地被果胶降解酶(例如内聚半乳糖醛酸酶)水解,或者通过促进分子间钙键的形成和所谓的蛋盒模型结构形成刚性凝胶,这意味着去甲基酯化的HG具有双重作用。同时,HG甲基酯化还可以阻碍钙桥的建立并增加细胞壁的柔韧性。HG的甲基酯化(DM)程度被认为是通过果胶甲基转移酶(PME)和PME/转化酶抑制剂(PMEI)的反作用通过甲基的转移和释放介导的,它们形成了一个大的植物序列家族,命名为PMEI相关蛋白(PMEI-RP)通过不可或缺的发夹基序直接相互作用。
开花是植物重要的性状,关系到农作物的产量和观赏植物的经济价值,但其具体的调控机制还不清楚。
有鉴于此,特提出本发明。
发明内容
现有的研究证明,果胶甲酯酶主要影响植物抗病性,如细菌或真菌病害。本发明应用基因克隆技术和植物转基因验证了一个棉花果胶甲酯酶抑制因子(Pectinmethylesterases inhibitor)基因GhPMEI39及其编码蛋白在植物中的作用。
基于上述内容,本发明提供了以下技术方案:
果胶甲酯酶抑制因子基因GhPMEI39,果胶甲酯酶抑制因子基因GhPMEI39的基因序列如SEQ ID NO:1所示。
本发明提供了果胶甲酯酶抑制因子基因GhPMEI39,利用生物学手段,从棉花中克隆得到。
本发明利用公开的数据库筛选目的基因,设计引物克隆目的基因,构建其棉花转基因和拟南芥转基因材料,分析过量和组成性表达果胶甲酯酶抑制蛋白后植物发育表型,验证了果胶甲酯酶抑制因子基因GhPMEI39在植物开花和花序形态等相关功能中的作用。
具体地,本发明还提供了果胶甲酯酶抑制因子基因GhPMEI39或其同源物或编码的蛋白在调控植物以下中的任一种或多种性能方面的应用:
(a)维管束组织分化;
(b)维管束组织发育;
(c)花序分化;
(d)花序发育;
(e)花蕾分化;
果胶甲酯酶抑制因子基因GhPMEI39的基因序列如SEQ ID NO:1所示。
进一步地,所述植物维管束组织分化包括维管束组织数量;
维管束组织发育包括维管束韧皮部和维管束木质部的细胞数量;
所述花序分化包括花序数量;
所述花序发育包括花序维管束组织的细胞数量;
所述花蕾分化包括花蕾数量。
进一步地,所述维管束组织包括茎和叶器官中的维管束组织。
本发明提供的果胶甲酯酶抑制因子基因GhPMEI39,经试验发现,在维管束组织方面,其能够影响维管束组织数量,并且改变维管束韧皮部和维管束木质部的细胞数量;在花序方面,其能够影响花序数量,并且改变花序维管束组织的细胞数量;在花蕾方面,其能够改变花蕾数量。
其中,所述茎包括初生茎、第一莲座叶。
具体地,所述植物为棉花,通过所述果胶甲酯酶抑制因子基因GhPMEI39的过表达促进所述棉花果枝茎的发育和增加花蕾的数量。
若植物为棉花等类似经济作物,通过过表达果胶甲酯酶抑制因子基因GhPMEI39,能够促进所述棉花果枝茎的发育和增加花蕾的数量,进而达到棉花增产的效果。
需要说明的是,本发明中的棉花可为各种种植棉,如陆地棉(Gossypiumhirsutum)、海岛棉(Gossypium barbadense)、亚洲棉(Gossypium arboreum)和草棉(Gossypium herbaceum)以及这些棉花品种的变种。也就是说,存在与本发明基因GhPMEI39相同或类似基因的棉花品种也均在本发明的保护范围内。类似基因如可以与本发明基因GhPMEI39序列相似度达80%以上,也可以是相似度达85%以上或90%以上或95%以上或96%以上或97%以上或98%以上或99%以上等等。
所述植物为拟南芥,通过所述果胶甲酯酶抑制因子基因GhPMEI39的过表达增加所述拟南芥花序的数量、增加主茎和莲座叶叶片的横向直径、增加花蕾数量。
进一步地,所述植物为拟南芥,所述果胶甲酯酶抑制因子基因GhPMEI39的过表达增加所述第一片莲座叶的韧皮部细胞数量、增加初生茎的维管组织数量、增加单个维管组织的韧皮部数量。
若植物为拟南芥等类似十字花科植物,可以通过过表达果胶甲酯酶抑制因子基因GhPMEI39提高拟南芥的茎和叶的厚实度,增加花序和花蕾的数量,增加该类植物的产量。
本发明提供的果胶甲酯酶抑制因子基因GhPMEI39的应用并不限于上述植物,还可以用于其他植物如观赏植物的花序性状调节和木本植物木材的硬度调控中。
进一步地,所述植物包括十字花科植物、观赏植物、经济作物、木本植物。
进一步地,所述十字花科植物包括拟南芥、萝卜、油菜、卷心菜;
所述观赏植物包括菊花、玫瑰、月季、紫罗兰;
所述经济作物包括棉花、油料、糖料、烟叶、麻类、苜蓿;
所述木本植物包括杨树、松树、柏树。
本发明中,果胶甲酯酶抑制因子基因GhPMEI39在应用于不同植物时,所用的基因序列可以是果胶甲酯酶抑制因子基因GhPMEI39或其同源物,同源物既包括不同植物中的果胶甲酯酶抑制因子基因,也包括自身植物中的果胶甲酯酶抑制因子基因。一般同源基因的序列相似度在80%以上,如在不同植物中,相似度可以达85%以上或90%以上或95%以上或96%以上或97%以上或98%以上或99%以上等等。
本发明还提供了一种棉花品种的检测方法,检测待测样品是否含有过表达的果胶甲酯酶抑制因子基因GhPMEI39或由过表达的果胶甲酯酶抑制因子基因GhPMEI39产生的产物;
若检测到,则判断待测样品具有更高棉花产量性能;
其中,所述果胶甲酯酶抑制因子基因GhPMEI39的基因序列如SEQ ID NO:1所示。
由于过表达的果胶甲酯酶抑制因子基因GhPMEI39具有上述所述的增加棉花产量的作用,在不同棉花品种或者在育种中,若想挑选出含有过表达的果胶甲酯酶抑制因子基因GhPMEI39的植株,则需要通过检测待测样品中是否含有过表达的果胶甲酯酶抑制因子基因GhPMEI39。检测并不限于仅对于果胶甲酯酶抑制因子基因GhPMEI39的检测,还可以检测其过表达的标志物、过表达的产物等。检测由果胶甲酯酶抑制因子基因GhPMEI39产生的产物可以通过多种手段进行,比如ELISA检测试剂盒等。
进一步地,通过过表达果胶甲酯酶抑制因子基因GhPMEI39的元件设计的引物对或探针或芯片对待测样品进行检测。
进一步地,所述引物对的核酸序列如SEQ ID NO.2和SEQ ID NO.3所示。
进一步地,所述待测样品包括适宜于有性繁殖、无性繁殖或可再生的细胞的组织培养的材料。
适宜于有性繁殖的材料,如选自花粉、子房、胚珠、胚囊等;
适宜于无性繁殖的材料如可以选自插枝、根、茎、原生质体等;
适宜于可再生的细胞的组织培养的材料如可以选自叶、花粉、胚、子叶、下胚轴、分生组织细胞、根、根端、花药、花、种子和茎等。
进一步地,所述待检测样本包括以下材料中的任一种:种子、叶、根、茎、胚根、胚芽。
本发明还提供了一种增加棉花产量的育种方法,将含有过表达的果胶甲酯酶抑制因子基因的片段重组到棉花基因组中;
筛选得到含有过表达果胶甲酯酶抑制因子基因元件的植株;
所述果胶甲酯酶抑制因子基因GhPMEI39的基因序列如SEQ ID NO:1所示。
本发明中,将目的片段重组到棉花基因组中,该过程会涉及一些转化的手段,如常见的根癌农杆菌介导法、基因枪法、PEG法、激光微束穿刺法、显微注射法和花粉介导法等。这些方法按照本领域的技术步骤可完成。
筛选得到含有过表达果胶甲酯酶抑制因子基因GhPMEI39元件的植株,该处的植株既可以表示植物的繁殖材料,也可以为植株非繁殖用材料,即为基因转化后的植株自身的一部分材料,这些材料包括但不限于块茎、枝、根、原生质体、叶、胚、子叶、下胚轴、分生组织细胞、根端、花药、花、种子、花粉、子房、胚珠、胚囊。
筛选得到含有过表达果胶甲酯酶抑制因子基因GhPMEI39的植株可通过多种手段实现,如分子生物学手段,包括但不限于通过果胶甲酯酶抑制因子基因GhPMEI39的引物对或探针或芯片对待测样品进行检测。
如检测由过表达果胶甲酯酶抑制因子基因GhPMEI39产生的产物,过表达载体上可设置一些表达可视的标记,如ELISA检测试剂盒可检测到的,或通过荧光在特定情况下显示来标记。
利用本发明的果胶甲酯酶抑制因子基因GhPMEI39辅助棉纤维优质性能育种,能够早期筛选出目标棉花,节省时间。
与现有技术相比,本发明的有益效果至少包括如下方面:
(1)本发明提供了果胶甲酯酶抑制因子基因GhPMEI39,利用公开的数据库筛选目的基因,设计引物克隆得到目的基因。
(2)通过构建其棉花转基因和拟南芥转基因材料,分析过量和组成性表达果胶甲酯酶抑制蛋白后植物发育表型,首次发现了果胶甲酯酶抑制因子基因GhPMEI39在植物开花和花序形态等相关功能中的作用。
(3)本发明提供的果胶甲酯酶抑制因子基因GhPMEI39可应用于多种植物,如经济作物棉花等类似作物的增产中,还可以用于其他植物如观赏植物的花序性状调节和木本植物木材的硬度调控中。
(4)本发明提供的果胶甲酯酶抑制因子基因GhPMEI39有望在其他植物发现与其类似作用的基因,为进一步的研究提供良好的技术支持。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,以下将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1为本发明实施例1中SDS-PAGE分析大肠杆菌BL21(DE3)表达的重组GhPMEI39蛋白电泳图;
图2为本发明实施例1中GhPMEI39蛋白对拟南芥茎PME粗提物的体外抑制活性柱形图;
图3为本发明实施例1中GhPMEI39蛋白亚细胞定位图;
图4为本发明实施例2中GhPMEI39超表达拟南芥植株基因鉴定以及表达情况图;
图5为本发明实施例2中GhPMEI39超表达拟南芥植株表型图;
图6为本发明实施例2中GhPMEI39超表达拟南芥第一片莲座叶以及主茎韧皮部结构相关图;
图7为本发明实施例2中拟南芥初生茎横断面维管组织中果胶免疫定位图;
图8为本发明实施例3中超表达转基因棉花鉴定相关图;
图9为本发明实施例3中超表达GhPMEI39转基因棉花生殖生长表型观察图。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限制本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
实施例1
1、棉花果胶甲酯酶抑制编码基因(GhPMEI39)的克隆
GhPMEI39 cDNA是以中棉所24(ZM24)棉花5DPA胚珠总cDNA为模板,以5′-CACCATGGGAATTCTCTTGCCTCCT-3′(sense)和5′-TACAAACCTATCAACTATTCT-3′(antisense)为引物PCR扩增获得553bp基因序列(如SEQ ID NO:1所示)。利用gateway构建pENTR-GhPMEI39入门克隆并进行测序验证。
2、GhPMEI39蛋白在大肠杆菌中的表达、纯化和酶活验证
(1)GhPMEI39蛋白原核诱导与纯化
利用Infusion技术和pColdTF表达载体,扩增GhPMEI39特异PCR产物使其5’和3’最末端分别带有和线性化载体两末端一致的序列(BamHI/ECORI双酶切线性化),通过参照Ultra One Step Cloning Kit(Vazyme,Cat.#115-02)无缝克隆方法获得pColdTF-GhPMEI39重组质粒并包含有6×His-Tag标签。pColdTF-GhPMEI39重组质粒转入大肠杆菌BL21(DE3)感受态细胞中。转化细胞在含有50μg/ml氨苄霉素的Luria-Bertani(LB)培养基中活化、培养。当细胞密度在600nm光密度(OD600)达到0.4-0.6时,向LB培养基中添加终浓度为0.5mM的异丙基-1-硫-丙二醇-D-半乳糖苷(IPTG)。16℃培养过夜后4℃5000g离心10mins,沉淀细胞重悬在binding buffer(50mM Tris-HCl,150mM NaCl,pH7.5)。超声波处理悬浮液,裂解物4℃12000g离心10mins。参考Ni SepharoseTM6 Fast Flow(GEHealthcare;Cat.#11-0008-87)纯化方法,将上清液加在带镍的his结合柱上,收集穿柱液后分别用包含30mM、50mM以及100mM咪唑的洗脱缓冲液洗脱目的蛋白,收集洗脱液。SDS-PAGE检测纯化效果如图1所示。
图1中,M为蛋白marker;未诱导表示IPTG诱导前pColdTF-GhPMEI39转化细菌的细胞裂解物;诱导表示IPTG诱导下pColdTF-GhPMEI39转化细菌的细胞裂解物;上清表示IPTG诱导下pColdTF-GhPMEI39转化细菌的细胞裂解物上清;沉淀表示IPTG诱导下pColdTF-GhPMEI39转化细菌的细胞裂解物沉淀;纯化显示了纯化的GhPMEI39蛋白;空载显示了纯化的pColdTF空载蛋白;WB显示了使用GhPMEI39自身抗体对纯化GhPMEI39蛋白的Westernblot分析;代表IPTG诱导下的GhPMEI39蛋白。
(2)酶活检测
在此基础上,进一步测定了GhPMEI39蛋白抑制果胶甲酯酶PME的活性,方法具体如下:为了获取PME粗提取物,将1g拟南芥茎(1个月)组织用液氮研磨后溶解在1ml提取缓冲液(PBSpH7.4,1M氯化钠)中形成匀浆,在冰上静置1h(中间上下颠倒混匀3次)后,将匀浆在11000g下离心10min,重复一次,收集上清液,冰封保存。蛋白质浓度用NanoDrop2000分光光度计(Thermo Science,美国)测量。PMEI纯化蛋白和PME粗提取物在酶活测定之前放置于25℃水浴30mins。每个反应管中加入反应液混合物包括1ml0.5%(w/v)果胶,0.01%溴麝香草酚蓝400μl,1.50ml蒸馏水(混合物pH=7.5)后再加入100μl不同比例的PME:GhPMEI39混合物(1:0,1:0.5,1:1以及1:2),30℃温浴16h,检测反应物在620nm波长下的OD值,计算方式为不同比例的PME:PMEI反应物在1s内ΔOD的值。本实验以pColdTF空载诱导蛋白为印象对照。结果如图2所示,随着PME:GhPMEI39比例从1:0到1:2,相应反应液中的PME酶活也从100%降至24%,而阴性对照pColdTF空载诱导蛋白加入后反应液中PME酶活并未改变,说明GhPMEI39蛋白具有抑制PME活性的能力。
图2中,pColdTF-GhPMEI39或空载体(pColdTF-,EV)粗蛋白是从表达pColdTF-GhPMEI39或空载体(达pColdTF,EV)的大肠杆菌BL21细胞中,经IPTG诱导后提取的。误差条表示S.E.M.(n≥3)。1:0、1:0.5、1:1、1:2代表拟南芥茎中PME粗提取与大肠杆菌BL21中pColdTF-GhPMEI39或空载体pColdTF粗提蛋白的比例,其中白色柱状代表PME:pColdTF-GhPMEI39的反应液,而黑色柱代表PME:pColdTF-空载的反应液。
3、GhPMEI39蛋白亚细胞定位分析
载体pCAMBIA2300:GhPMEI39:YFP的构建,GhPMEI39 CDS PCR产物克隆进入Kpn1/Asc1YFP(黄色荧光蛋白)基因上游,产生GhPMEI39:YFP融合蛋白转入GV3101农杆菌后通过Floral-dip方法得到35S:GhPMEI39:YFP超表达转基因拟南芥。以生长4 Days的拟南芥阳性幼苗以及WT的根为观察对象,用LSM 510激光共聚焦扫描显微观察,绿色(YFP)为GhPMEI39蛋白定位,红色(FM4-64,细胞膜染料)荧光标记为细胞膜(如图3)。为了确定GhPMEI39:YFP细胞壁或者细胞膜的具体定位,幼苗根用0.4M NaCl处理20mins后观察。研究结果如下图,GhPMEI39蛋白位于细胞壁与细胞膜之间即为外泌蛋白。
图3中,Before代表0.4M NaCl处理幼苗前定位,After代表0.4M NaCl处理幼苗根出现质壁分离后定位。
实施例2
1、GhPMEI39转基因拟南芥载体构建
利用pENTR-GhPMEI39和Gateway LR反应,GhPMEI39 cDNA片段克隆进入目的载体PGWB2(CaMV 35S启动子)。pGWB2-GhPMEI39 Ti质粒转入根癌农杆菌菌株GV3101后通过Floral-dip方法得到35S:GhPMEI39超表达转基因拟南芥。
2、DNA提取和GhPMEI39表达量检测
获取拟超表达转基因GhPMEI39拟南芥植株共12株,提取10株拟转基因以及WT植株DNA,以CaMV35S5′-AACACGGGGGACTCTAGA-3′(sense)和GhPMEI395′-TACAAACCTATCAACTATTCT-3′(antisense)为引物扩增检测DNA,12株超表达转基因GhPMEI39拟南芥植株均出现条带(图4A)。通过对9株拟南芥茎中GhPMEI39表达量检测后发现,与阴性对照WT相比,6株转基因植株表达量均上调(图4B、4C)。
图4中,A代表GhPMEI3912株转基因拟南芥DNA鉴定图,B、C分别为GhPMEI39转基因拟南芥植株RNA表达水平的半定量以及qRT-PCR鉴定结果图,Bar值表示三个重复PCR的标准差。
3、GhPMEI39转基因拟南芥表型观察
进一步选取GhPMEI39-5,8,93个株系获得单拷贝纯合子来进行表型观察,结果如图5所示。图5中,A代表GhPMEI39OE-5,8,9三个转基因系拟南芥以及WT植株表型图,B、C、D、E分别为GhPMEI39转基因拟南芥与野生型植株初生花序、花蕾、第一片莲座叶以及主茎图。F为GhPMEI39OE-5,8,9三个转基因系拟南芥及WT植株主茎果荚图。
通过对三个转基因株系以及WT植株的观察,发现超表达GhPMEI39转基因植株OE-5,8,9三个株系的主茎、莲座叶叶片横向直径均增加(图5A、5D、5E),更重要的是由于GhPMEI39基因的超表达,拟南芥的初生主茎花序由野生型的一个转变为两个或者三个不等,同时花蕾数量也显著增加(图5A-5C),从而导致主茎上一个果荚生长点可以同时产生两个甚至三个果荚的表型(图5F)。
4、GhPMEI39超表达引起拟南芥第一片莲座叶、初生茎维管组织细胞数量增加
为了探索造成GhPMEI39三个转基因株系以及WT植株叶片和主茎横向直径增加的原因,进一步对超表达GhPMEI39转基因植株OE-5,8,9三个株系的主茎、莲座叶叶片的维管组织结构通过石蜡切片的方式进行观察,结果如图6所示。图6中,A代表GhPMEI39OE-5,8,9三个转基因系拟南芥以及WT植株第一片莲座叶叶脉横切,B为A图中维管组织放大图,C为WT以及OE系主茎维管组织结构图D为C图单个维管组织方法图,E为GhPMEI39转基因拟南芥与野生型植株第一片莲座叶韧皮部细胞数量,初生花序总的维管组织数量以及总的韧皮部数量统计,其中n=6。
由于GhPMEI39超表达,第一片莲座叶的韧皮部细胞数量由WT的69增加为OE系的108、117、121等(图6A、6B、6E)同时,初生茎的维管组织数量也由WT的6-8增加到OE转基因材料中的12-15,并且单个维管组织的韧皮部数量也显著增加,从而导致整个初生茎韧皮部细胞数量从WT的330显著增加至1079-1181(图6C、6D、6E)。
5、GhPMEI39超表达拟南芥初生茎甲酯化程度(DM)增加
通过用代表不同甲酯化程度的两种果胶抗体LM19和LM20对三种OE系以及WT拟南芥植株初生茎维管组织横截面进行免疫观察发现,在OE系中,作为低甲酯化果胶指标的LM19荧光均较弱,高甲酯化果胶标签LM20较强,而WT呈现相反趋势(图7),图7中,LM19代表低甲酯果胶,而LM20代表高甲基酯果胶,所有图像Bar=300μm。
结果表明甲酯化果胶含量在OE系中较高,即GhPMEI39超表达拟南芥初生茎甲酯化程度(DM)增加。
实施例3
1、GhPMEI39转基因棉花构建
利用pENTR-GhPMEI39和GatewayLR反应,GhPMEI39cDNA片段克隆进入目的载体PGWB2(CaMV35S启动子)。pGWB2-GhPMEI39Ti质粒转入根癌农杆菌菌株LBA4404,以7天的ZM24无菌棉花幼苗(暗处理5天光照2天)下胚轴为外植体进行浸染培育,从而获取转基因超表达棉花。
2、DNA提取和GhPMEI39表达量检测
用植物基因组DNA提取试剂盒(百泰克,Cat#DP3112)提取所有组培获得的拟转基因以及ZM24棉花的嫩叶基因组DNA,以DNA为模板,CaMV35S-F:5′-AACACGGGGGACTCTAGA-3′(sense)和GhPMEI39-R:5′-TACAAACCTATCAACTATTCT-3′(antisense)为引物PCR扩增,凝胶检测有562bp片段的植株则进一步检测其表达量。
为确定GhPMEI39在转基因和野生型棉花植株中的表达水平,于上午8:00-9:00采集转基因GhPMEI39和野生型ZM24棉花5DPA胚珠,液氮浸泡后置于-80℃冰箱保存,或者立即研磨后按照多糖多酚植物总RNA提取试剂盒(Tiangen,Cat.#DP441)说明书从所有样本中分离出RNA。使用反转录酶(Invitrogen,Cat.#18080-093)对大约1μg总RNA进行了反转录得到cDNA。qRT-PCR以GhUBQ7(DQ116441)为内参基因,引物如下5′-CCCGAAACCTGCATAAAATG-3′(sense)和5′-GCGATCAAATTGGTTTTCGT-3′(antisense)。
将-80℃保存的转基因与野生型棉花的5DPA胚珠取出在液氮中研磨成粉后加入蛋白提取液(150mM NaCl;1M Tris-HCl,pH=7.5;0.1%NP40;0.1%TritonX-100)。充分混匀后冰上静置30mins,12000g,4℃高速离心10mins,取上清。GhPMEI39多克隆抗体的是由上海友科公司制作的。Westernblotting实验按照之前的报道进行。
获取拟超表达转基因GhPMEI39棉花植株共146株,提取所有拟转基因以及WT植株DNA,以CaMV35S5′-AACACGGGGGACTCTAGA-3′(sense)和GhPMEI39-R:5′-TACAAACCTATCAACTATTCT-3′(antisense)为引物扩增检测DNA,如图8的DNA所示,共10个超表达转基因GhPMEI39棉花系出现条带。
通过对其中7株植株的5DPA胚珠中GhPMEI39表达量检测后发现(图8的RNA),与阴性对照WT相比,所有转基因植株表达量均上调,随机选取表达量低中高GhPMEI39-8,9,13,15,17五个株系5DPA胚珠蛋白,以GhPMEI39自身蛋白抗体来进行westernblot检测后发现,与WT相比四个株系GhPMEI39-9,13,15,17蛋白水平也上调(图8的Protein),说明这四个株系属实为转基因GhPMEI39超表达植株。
3、转基因植株的表型分析
通过对三个转基因株系以及ZM24植株的营养生长以及生殖生长阶段观察发现,超表达GhPMEI39转基因植株OE-13,15,17营养生长与ZM24相比无差异,但是在生殖生长阶段,超表达GhPMEI39后,促使果枝出现花芽簇表型(图9A、9B),并且花芽簇可以进一步发育成花甚至成熟棉铃(图9C)。同时将三个OE系与ZM24果枝茎结构进行观察发现,超表达GhPMEI3可促使果枝茎中韧皮部的发育(图9D)。
图9中,A从左向右依次为ZM24及超表达GhPMEI39转基因棉花系OE-13,15,17现蕾阶段整株表型,图B为图A中不同株系花蕾分化放大图,图C为ZM24及OE系果枝分枝茎中维管组织结构观察,其中bar=600μm,图D为转基因株系OE-13形成的串铃以及成熟纤维图。
综述以上研究,发现GhPMEI39的过量表达促进了植物维管组织,尤其韧皮部的发育,从而改变了花序分化和花序特征。
其中,DPA为days post anthesis的缩写,含义为开花后天数。
尽管已用具体实施例来说明和描述了本发明,然而应意识到,在不背离本发明的精神和范围的情况下可以作出许多其它的更改和修改。因此,这意味着在所附权利要求中包括属于本发明范围内的所有这些变化和修改。
序列表
<110> 中国农业科学院棉花研究所
<120> 果胶甲酯酶抑制因子基因GhPMEI39及其编码蛋白的应用
<130> 2021
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<213> 中棉所24()
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cacaatgctg aagtccccga aacctgcata aaatgcgtaa aatctgattc tcgaagtcaa 180
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gctctccgag gttgcgagaa agggttttac tacacgaaaa ccaatttgat cgctgcgacg 360
aaccgattga aggggaaaga atatgatcaa acgaatcttc tggtgaaaca agcgctcgaa 420
gaagaatttg tttgtaagat gaaagtggag gttttacgat ttaactttcc gagtagtgtc 480
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Claims (5)
1.果胶甲酯酶抑制因子基因GhPMEI39或其编码的蛋白在调控植物以下性能方面的应用:
(b)维管束组织发育;
(d)花序发育;
果胶甲酯酶抑制因子基因GhPMEI39的基因序列如SEQ ID NO:1所示,通过果胶甲酯酶抑制因子基因GhPMEI39的过表达对植物的性能进行调控;
其中:
维管束组织发育包括增加维管束韧皮部的细胞数量;
所述花序发育包括增加花序维管束组织的细胞数量。
2.根据权利要求1所述的应用,其特征在于,所述维管束组织包括茎和叶器官中的维管束组织。
3.根据权利要求1所述的应用,其特征在于,所述植物包括十字花科植物和经济作物;
其中:
所述十字花科植物包括拟南芥;
所述经济作物包括棉花。
4.根据权利要求3所述的应用,其特征在于,所述植物为棉花,通过所述果胶甲酯酶抑制因子基因GhPMEI39的过表达促进棉花果枝茎的发育和增加花蕾的数量;
所述植物为拟南芥,通过所述果胶甲酯酶抑制因子基因GhPMEI39的过表达增加拟南芥花序的数量、增加主茎和莲座叶叶片的横向直径、增加花蕾数量。
5.根据权利要求4所述的应用,其特征在于,所述植物为拟南芥,通过所述果胶甲酯酶抑制因子基因GhPMEI39的过表达增加第一片莲座叶的韧皮部细胞数量、增加初生茎的维管组织数量、增加初生茎的单个维管组织的韧皮部数量。
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Pectinmethylesterases (PME) and Pectinmethylesterase Inhibitors (PMEI) Enriched during Phloem Fiber Development in Flax (Linum usitatissimum);David Pinzon-Latorre et al.;《PLoS ONE》;20140814;第9卷(第8期);第1-17页 * |
PREDICTED: Gossypium hirsutum uncharacterized LOC107962267 (LOC107962267), mRNA, XM_016898587.2;GenBank;《GenBank》;20210425;第1-2页 * |
桃果胶甲酯酶抑制因子PMEI基因家族的鉴定及表达分析;冀美玲等;《植物生理学报》;20171231;第53卷(第11期);第1988-1996页 * |
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CN113481211A (zh) | 2021-10-08 |
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