CN105925591B - 一种毛竹木聚糖合成关键基因PeIRX10的克隆及其应用 - Google Patents
一种毛竹木聚糖合成关键基因PeIRX10的克隆及其应用 Download PDFInfo
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Abstract
本发明属于分子生物学和基因工程领域,具体涉及一个参与木聚糖合成的毛竹糖基转移酶(glycosyltransferases,GTs)关键基因PeIRX10,该基因具有SEQ ID No:1所示的核苷酸序列。本发明为了解次生细胞壁的形成,阐明毛竹组织快速生长的分子基础提供了一个有力工具;实现了利用PeIRX10能够修复拟南芥突变体中木聚糖合成缺陷和互补拟南芥突变体中次生细胞壁缺陷。
Description
技术领域
本发明属于分子生物学和基因工程领域,具体涉及一个参与木聚糖合成的毛竹糖基转移酶(glycosyltransferases,GTs)关键基因PeIRX10的克隆和功能初探。
背景技术
半纤维素木聚糖(Xylan)是植物中含量仅次于纤维素的多糖成分,在维持细胞壁的稳定性与完整性中发挥重要作用,是重要的可再生生物质资源。木聚糖由位于高尔基体内的GTs合成。GTs是一系列参与催化双糖、聚糖和糖复合物中糖链合成的一类酶,存在于所有生物中,不仅参与了细胞壁主要成分的合成,还催化各种分子的糖基并参与发育、信号转导、防御等生物学过程。
IRX10基因是参与半纤维素木聚糖主链延伸的关键基因,对植物次生壁正常的加厚有重要作用。如果IRX10基因缺失,则花序和茎中木糖含量下降,细胞壁表现为不规则的木质部而导致次生细胞壁缺陷。目前,已有植物的IRX10基因功能研究主要集中在拟南芥、水稻等模式植物上,然而毛竹中参与木聚糖合成的关键基因PeIRX10至今仍未见报道。
毛竹(Phyllostachys edulis)是我国种植面积最大的经济竹种,被广泛应用于食品、建材、纺织、生物质能源等领域。已经发现毛竹的快速生长过程中伴随着次生细胞壁生长。因此,鉴别出毛竹中合成半纤维素木聚糖的相关基因PeIRX10,对于了解次生细胞壁的形成,阐明毛竹组织快速生长的分子基础具有重要的科学意义。
发明内容
针对目前尚没有找到引起毛竹次生细胞生长的原因,本发明旨在提供一个新的来源于毛竹的参与木聚糖合成的关键基因PeIRX10,从而为了解次生细胞壁的形成,阐明毛竹组织快速生长的分子基础提供了一个有力工具。本发明的目的还在于提供基因PeIRX10在植物遗传转化中的应用,以实现利用基因PeIRX10能够修复拟南芥突变体中木聚糖合成缺陷和互补拟南芥突变体中次生细胞壁缺陷。
为实现本发明的发明目的,发明人提供如下技术方案:
本发明的目的首先是提供一个新的来源于毛竹的参与木聚糖合成的关键基因PeIRX10,该基因具有SEQ ID No:1所示的核苷酸序列。
如附图1所示:本发明提供的毛竹木聚糖合成关键基因PeIRX10在竹笋中的表达水平最高,茎、根和花序中表达量的表达量相对较低,在叶片中的表达水平最低。PeIRX10的这一表达模式与竹笋具有快速生长的特性有关。
本发明的另一个目的是提供PeIRX10在植物遗传转化中的应用,一方面包括毛竹木聚糖合成关键基因PeIRX10在制备转基因植物中的应用,所述的植物为拟南芥;另一方面包括毛竹木聚糖合成关键基因PeIRX10在修复拟南芥突变体中木聚糖合成缺陷和互补拟南芥突变体中次生细胞壁缺陷上的应用。本发明利用基因工程技术将PeIRX10基因重组转入具有irx10irx10l双突背景的拟南芥植株中,获得过表达PeIRX10基因的转基因拟南芥植株,PeIRX10基因过表达得到的互补的植株表型与野生型几乎一致(如表2和图3所示)。通过单克隆抗体LM10进行标记,对互补植株做免疫荧光检测,显示互补型拟南芥茎的横切面切片中木质部木聚糖具有强烈的免疫信号(如图5所示),说明PeIRX10基因能够修复拟南芥突变体中木聚糖合成缺陷,在毛竹木聚糖合成中起重要作用。
与现有技术相比,本发明具有如下优点:
1、本发明找到了一个新的来源于毛竹的参与木聚糖合成的关键基因PeIRX10,从而为了解次生细胞壁的形成,阐明毛竹组织快速生长的分子基础提供了具有重要的科学意义。
2、本发明利用基因PeIRX10能够修复拟南芥突变体中木聚糖合成缺陷和互补拟南芥突变体中次生细胞壁缺陷,在毛竹木聚糖合成中起着重要作用。
附图说明
附图1是毛竹PeIRX10基因在不同组织器官中的表达模式分析柱形图。
附图2是转PeIRX10基因拟南芥植株RT-PCR检测,
其中1代表野生型拟南芥;2代表irx10irx10l双突纯合植株;3-6则为转PeIRX10基因植株。
附图3是野生型、双突植株和互补型拟南芥植株的表型变化。
附图4是野生型、irx10irx10l双突和互补植株茎的次生细胞壁变化。
附图5是过表达PeIRX10基因的拟南芥茎部切片的LM10化学免疫观察,
其中a代表野生型拟南芥;b代表irx10irx10l双突纯合植株;c则为转PeIRX10基因植株。
具体实施方式
下面结合实施例和说明书附图,更具体地说明本发明的内容。应当理解,本发明的实施并不局限于下面的实施例,对本发明所做的任何形式上的变通和/或改变都将落入本发明保护范围。
在本发明中,若非特指,所有的份、百分比均为重量单位,所有的设备和原料等均可从市场购得或是本行业常用的。若无特别指明,实施例采用的方法为本领域通用技术。
实施例中未注明具体条件的实验方法,是按照常规条件,Sambrook等作者的分子克隆实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件进行。
实施例1毛竹PeIRX10基因表达
(一)实验方法
1.毛竹材料
不同组织的毛竹材料取自1年实生苗的根、茎、叶、花序和幼笋,液氮速冻后保存–80℃,用于总RNA的提取。
(1)RNA的提取和cDNA的合成
通过毛竹中已完成的测序的基因序列数据库和其相应的蛋白序列数据库进行BLAST检索及比对分析,得到PeIRX10基因(PH01004923G0080;SEQ ID No:1)的核苷酸序列,设计引物,如下:
PeIRX10-F1:5′-CCTGAACCACATGTTTGCCG-3′(SEQ ID No:2)
PeIRX10-R1:5′-AATCGCACTGCGCATCATTC-3′(SEQ ID No:3)
用RNA提取试剂盒(OMEGA)提取毛竹各样品的总RNA。采用PrimeScriptII1stStrand cDNA Synthesis Kit(Takara)试剂盒进行反转录,反转录的反应体系为:总RNA 2μg,Oligo(dT)1μL,酶0.5μL,RNA酶抑制剂0.5μL,dNTP 2μL,加ddH2O至总体系为20μL;反应条件为:37℃15min;85℃5s;引物合成在中美泰和生物技术(北京)有限公司进行。
(2)RT-PCR分析
内参选用β-actin基因,引物如下:
ACTIN-F:TGAGCTTCCTGATGGGCAAG(SEQ ID No:4);
ACTIN-R:CCTGATATCCACGTCGCACTT(SEQ ID No:5)。
以步骤(1)得到的cDNA为模板,用步骤(1)中的引物进行PCR扩增,测序显示,得到PeIRX10基因(核苷酸序列如SEQ ID No:1所示)。RT-PCR扩增的反应体系为:cDNA 3μL,10×buffer 5μL,LA Taq 0.5μL,dNTP 8μL,PeIRX10-F1 1μL,PeIRX10-R1 1μL,32.5μL ddH2O,共50μL;反应程序为:95℃30s;95℃5s,60℃30s,40个循环;熔点曲线检测程序为,从60℃按照0.6℃/s速率上升至95℃,连续读取荧光信号值。所用仪器为CFX96实时荧光定量PCR仪(Bio-Rad,美国),每次检测都包括以H2O作反应模板的阴性对照,检测数据采用相对定量△Ct方法,用内参基因β-actin的Ct值进行归一化。
(二)实验结果
在毛竹不同的组织器官中PeIRX10基因的表达量存在差异(参见附图1),其中在竹笋中的表达水平最高,茎、根和花序中表达量的表达量相对较低,在叶片中的表达水平最低。
实施例2拟南芥中毛竹PeIRX10基因的过表达
(一)实验方法
1.表达载体的构建
基于Gateway系统用于PCR扩增毛竹PeIRX10全长cDNA序列,扩增引物为:
PeIRX10-F:
5′-
GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGAGGAGGTGGGTCTTGGCC-3′(SEQ ID No:6);
PeIRX10-R:
5′-
GGGGACCACTTTGTACAAGAAAGCTGGGTCCCAAGGCTTCAGGTCGCCCACCG-3′(SEQ ID No:7)。
PCR反应体系为20μL:2×PrimeSTAR Max Premix 10μL,上下游引物(10μmol L-1)各0.5μL,模板2μL,dd H2O 7μL。反应程序:94℃预变性5min;94℃30s,55℃1min 30s,72℃30s,35个循环;72℃延伸10min。将扩增产物与pDONR207连接,转化DH5α,得到阳性克隆,提取质粒后进行质粒PCR鉴定,反应体系:回收的PCR目的片段3μL,pDONR207载体1μL,BPClonase Enzyme Mix1μL,然后测序。将测序正确的重组质粒与pEarlyGate101进行LR重组反应,LR反应体系:pDONR207-PeIRX10 3μL,pEarlyGate101 1μL,LR Clonase Enzyme Mix1μL,然后转化大肠杆菌DH5α,培养后提取质粒并测序。将测序正确的重组质粒转化农杆菌,获得的阳性菌落用蘸花法转化到拟南芥irx10l(-/-)irx10(+/-)杂合植株中。
2.转基因植株鉴定
经基因工程技术转化irx10l(-/-)irx10(-/-)双突拟南芥,获得转基因植株后,通过PCR和RT-PCR手段进行验证,引物为:
IRX10-F:5′-CCACTCGGAGGACTTGGA-3′(SEQ ID No:8),
IRX10-R:5′-GGAAAAAGCCATTGAAAG GG-3′(SEQ ID No:9),
T-DNA插入引物:
LBa1:5′-TGGTTCACGTAGTGGGCCATCG-3′(SEQ ID No:10),
提取四周大的三种基因型的拟南芥植株的RNA,反转录成cDNA,方法如实施例1所述,内参基因同实施例1,RT-PCR反应体系和反应程序同实施例1。
(二)实验结果
通过RT-PCR检测,说明PeIRX10基因已经转入拟南芥并表达(参见附图2),在附图2中,其中1代表野生型拟南芥;2代表irx10irx10l双突纯合植株;3-6则为转PeIRX10基因植株。在irx10irx10l双突植株背景下,在PeIRX10基因过表达得到互补的拟南芥植株的表型大小与野生型几乎一致(参见附图3),同时具有正常的株高、茎粗以及叶片大小和数量(参见表1)。
表1野生型,双突植株和互补型拟南芥植株的表型分析
实施例3细胞壁木聚糖免疫定位
(一)实验方法
分别取生长八周的野生型、双突植株和互补型拟南芥植株基部的茎,切成1mm厚的切片,用0.1M磷酸盐缓冲液(pH 7.2)洗涤切片5~10min;用新鲜的3%脱脂牛乳浸泡切片1h并不断吹打脱脂牛乳;去除脱脂牛乳,并用PBS缓冲液洗涤切片5min;用大鼠抗木聚糖抗体LM10(Plantprobes)孵育切片2h,然后用PBS缓冲液洗涤切片,以洗净未结合的一抗;用稀释50倍的FITC-羊抗大鼠抗体(Zomanbio,Cat.Z1319)孵育2h,然后用PBS缓冲液洗涤切片10次,以洗净未结合的二抗;最后将切片固定于载玻片上,置于激光共聚焦电子显微镜(ZeissLSM710,495nm)下观察拍照,结果如附图4和附图5所示。
(二)实验结果
1.PeIRX10基因能互补拟南芥irx10irx10l双突植株次生细胞壁缺陷
irx10irx10l双突植株纯合植株次生细胞壁生长基本缺失,在维管束间纤维和木质部导管细胞的细胞壁明显变薄(参见附图4的B和E)。PeIRX10基因的互补植株中维管束间纤维和木质部导管细胞的细胞壁厚度已经与野生型拟南芥近乎相近(参见附图4的A和D)。表明PeIRX10基因能够互补拟南芥irx10irx10l植株细胞壁的次生加厚缺陷。
2.PeIRX10基因可以修复拟南芥irx10irx10l双突植株细胞壁木聚糖合成缺失
表2野生型、双突和互补型拟南芥植株茎部细胞壁单糖组分含量比较
样品 | 野生型 | irx10irx10l | irx10irx10l±PeIRX10 |
鼠李糖 | 9.4±0.04Bc | 11.3±0.03BCa | 10.3±0.07Bb |
海藻糖 | 2.2±0.02Cb | 4.5±0.05Ca | 2.6±0.05Cb |
阿拉伯糖 | 11.1±0.03ABCc | 38.6±2.12ABa | 16.8±0.49ABCb |
木糖 | 102.0±5.63Aa | 8.3±0.11BC c | 94.1±4.96Ab |
甘露糖 | 17.2±0.09ABc | 20.4±3.17Ba | 18.5±1.06BCb |
半乳糖 | 17.6±1.08BCb | 41.2±3.17ABa | 19.4±3.34Aab |
葡萄糖 | 19.84±1.98Cb | 75.32±4.26Aa | 26.32±2.56ABa |
从表2可以看出,与野生型相比,irx10irx10l双突植株中木糖含量减少至8%左右,而细胞壁中其他单糖(例如阿拉伯糖、半乳糖和葡萄糖等)的含量与野生型相比则有明显增加。而在互补植株中,木糖含量以及其他单糖的含量与野生型相近,这说明在irx10irx10l突变体中,PeIRX10基因的过表达可以使其细胞壁中的单糖含量恢复到与野生型相似。
在野生型和互补型拟南芥茎的横切切片中木质部具有强烈的免疫信号,而在irx10irx10l双突植株中没有检测到免疫信号(参见附图5),说明具有双突背景的互补植株中PeIRX10基因的过表达使irx10irx10l双突植株中木聚糖缺乏得到恢复。
尽管发明人已经对本发明的技术方案做了较为详细的阐述和列举,应当理解,对于本领域一个熟练的技术人员来说,对上述实施例作出修改和/或变通或者采用等同的替代方案是显然的,都不能脱离本发明精神的实质,本发明中出现的术语用于对本发明技术方案的阐述和理解,并不能构成对本发明的限制。
Claims (3)
1.一种毛竹木聚糖合成关键基因PeIRX10,其特征在于其核苷酸序列如SEQ ID NO:1所示。
2.权利要求1 所述的毛竹木聚糖合成关键基因PeIRX10 在制备转基因植物中的应用,其特征在于,所述的植物为拟南芥。
3.权利要求1 所述的毛竹木聚糖合成关键基因PeIRX10在修复拟南芥突变体中木聚糖合成缺陷和互补拟南芥突变体中次生细胞壁缺陷上的应用。
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