CN114774440B - 一种柱花草多聚半乳糖醛酸酶基因SgPG1及其应用 - Google Patents
一种柱花草多聚半乳糖醛酸酶基因SgPG1及其应用 Download PDFInfo
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Abstract
本发明公开了一种柱花草多聚半乳糖醛酸酶基因SgPG1及其应用。本发明提供的多聚半乳糖醛酸酶基因SgPG1的核苷酸序列如SEQ ID NO:1所示,多聚半乳糖醛酸酶SgPG1的氨基酸序列如SEQ ID NO:2所示。本发明表明,通过转基因柱花草毛根表达体系,证明SgPG1基因的表达能够促进柱花草根尖类边缘细胞的形成,同时证明了该基因的作用机制是通过降解低甲酯化的同型半乳糖醛酸,从而提高了细胞壁中高甲酯化的同型半乳糖醛酸比例,表明SgPG1基因具有选择性降解细胞壁低甲酯果胶从而改变细胞壁组分的功能,从而提高了植物根系对铝毒的耐受。
Description
技术领域
本发明属于基因工程技术领域。更具体地,涉及一种柱花草多聚半乳糖醛酸酶基因SgPG1及其应用。
背景技术
柱花草(Stylosanthes guianensis)是豆科多年丛生性草本植物,约50个品种,原产于拉丁美洲。因其产量高、草质好、易于种植等特点,成为了热带和亚热带地区广泛种植的优良牧草,是重要的豆科牧草,可作为饲草喂养牲畜、作为绿肥覆盖果园和改良土壤等。而热带以及亚热带地区主要是以酸性土壤为主,在长期的自然进化和人工选育过程中,柱花草具有较强的耐铝毒能力。铝毒和低磷是酸性土壤中限制作物生长的两个主要障碍因素,酸性土壤中活性态铝(A13+)含量较高,会抑制根系生长而影响作物产量。柱花草是优质的热带豆科牧草,对酸性土壤具有良好的适应性,研究柱花草适应铝毒的生理与分子机制对培育耐铝毒作物新品种具有重要意义。
前期的研究结果显示,剥落细胞(BC)能够帮助植物适应铝毒胁迫,也有研究表明,在铝处理条件下,移除BCs会显著抑制根的伸长,增加根尖铝的积累,这表明BCs是保护根尖免受铝毒的一道屏障(Brigham et al.,2001)。虽然根尖缘细胞(BCs)或根尖类边缘细胞(BLCs),是指从植株根冠产生的一类活细胞。但是,BLCs细胞与BCs细胞不同,其中BCs细胞是单个细胞,分散在根尖的粘液中形成松散的粘连结构;而BLCs细胞则以细胞连着细胞,形成致密的细胞鞘结构,粘连在根尖部位。因此,BLCs与BCs在某些生理生化功能上可能存在差异。
在BLCs细胞/BCs细胞从根尖分离的时候,与表皮细胞连接的细胞壁中的果胶成分会降解。而多聚半乳糖醛酸酶(Polygalacturonase,简称PG)是一类可催化果胶中多聚半乳糖醛酸裂解的果胶降解酶(McCarthy et al.,2014)。而目前对于PG基因和蛋白的研究中,多数PG是分离自茄属植、花生、烟草和拟南芥。虽然在拟南芥中的研究已揭示多聚半乳糖醛酸酶基因RCPG(At1g65570)参与了拟南芥根尖剥落细胞的形成,即该基因的突变会导致拟南芥根尖单个剥落细胞形成粘连状,而该基因的超量表达会导致剥落细胞单个细胞剥落(Kamiya et al.,2016)。但是关于多聚半乳糖醛酸酶基因是否也参与了类边缘细胞从根尖的脱落尚无明确的报道。而且,多聚半乳糖醛酸酶介导的细胞壁果胶成分的变化是否也参与了植物对土壤中铝毒害的忍耐机制尚未清楚。而在柱花草中对多聚半乳糖醛酸酶基因的研究分析也鲜有报道。
发明内容
本发明通过同源克隆,首次在柱花草中克隆到了一个表达水平与柱花草根尖类边缘细胞发育过程高度吻合的多聚半乳糖醛酸酶基因SgPG1,并对柱花草多聚半乳糖醛酸酶基因进行研究分析,为柱花草类边缘细胞的形成以及作用机制提供更多的理论基础。
本发明的目的是提供一种柱花草多聚半乳糖醛酸酶基因SgPG1。
本发明第二个目的是提供一种柱花草多聚半乳糖醛酸酶SgPG1。
本发明第三个目的是提供所述柱花草多聚半乳糖醛酸酶基因SgPG1和柱花草多聚半乳糖醛酸酶SgPG1的应用。
本发明第四个目的是提供一种重组表达载体。
本发明第五个目的是提供一种基因工程菌。
本发明第六个目的是提供一种提高植物根尖铝毒耐受能力的方法。
本发明上述目的通过以下技术方案实现:
申请人通过同源克隆,首次在柱花草中克隆到了一个表达水平与柱花草根尖类边缘细胞发育过程高度吻合的多聚半乳糖醛酸酶基因SgPG1。经测序后进行序列对比,将该基因归于多聚半乳糖醛酸酶基因基因家族,为柱花草多聚半乳糖醛酸酶基因SgPG1,其核苷酸序列如SEQ ID NO:1所示,序列长度为1425bp,其多聚半乳糖醛酸酶的氨基酸序列如SEQ IDNO:2所示。
通过转基因柱花草毛根表达体系,证明SgPG1基因的表达能够促进柱花草根尖类边缘细胞的形成。同时证明了该基因的作用机制是通过降解低甲酯化的同型半乳糖醛酸,从而提高了细胞壁中高甲酯化的同型半乳糖醛酸比例,从而提高了植物对铝毒的耐受能力。
因此,本发明提供的多聚半乳糖醛酸酶基因SgPG1或所述多聚半乳糖醛酸酶SgPG1在促进植物根尖类边缘细胞形成,或在制备促进植物根尖类边缘细胞形成的制剂,在提高植物铝毒耐受能力,在制备提高植物铝毒耐受能力的制剂以及在构建高铝毒耐受能力的转基因植物中的应用都在本发明保护范围内。
本发明提供一种重组表达载体,含有所述柱花草多聚半乳糖醛酸酶基因SgPG1。
本发明提供一种基因工程菌,含有上述重组表达载体。
本发明提供所述的表达载体或所述的基因工程菌在构建转基因植物或制备促进植物根尖类边缘细胞形成的制剂中的应用。
本发明还提供一种提高植物根尖铝毒耐受能力的方法,在植物中超量表达多聚半乳糖醛酸酶编码基因SgPG1。
优选地,所述植物为双子叶植物。
进一步优选地,所述所述双子叶植物为柱花草。
本发明具有以下有益效果:
本发明首次在柱花草中克隆到了一个表达水平与柱花草根尖类边缘细胞发育过程高度吻合的多聚半乳糖醛酸酶基因SgPG1。通过转基因柱花草毛根表达体系,证明SgPG1基因的表达能够促进柱花草根尖类边缘细胞的形成。因此,本发明提供的柱花草多聚半乳糖醛酸酶基因SgPG1制备促进植物根尖类边缘细胞形成的制剂以及构建转基因植物,均具有广泛的应用前景。同时证明了该基因的作用机制是通过降解低甲酯化的同型半乳糖醛酸,从而提高了细胞壁中高甲酯化的同型半乳糖醛酸比例,从而提高了植物对铝毒的耐受能力,表明SgPG1基因是一个重要的耐铝基因,该基因也是第一个报道功能与植物耐铝相关的多聚半乳糖醛酸酶基因,该基因的获得丰富了植物耐铝毒的基因库。
附图说明
图1为SgPG1在柱花草根尖的组织定位与SgPG1的亚细胞定位分析;
图2为SgPG1蛋白化学性质分析;
图3为外源添加SgPG1酶液对根尖果胶成分的影响;
图4为超量表达SgPG1基因对柱花草毛状根根尖BLCs形成的影响;
图5为超量表达SgPG1基因对柱花草毛状根和拟南芥耐受铝毒的影响。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
实施例1 SgPG1基因的克隆表达分析
1、SgPG1基因的克隆
根据柱花草根尖发育的转录组数据,在高度保守区域设计上游引物5’-ATGGAAACCATTCAATTGTTGTTC-3’(SEQ ID NO:3)和下游引物5’-TTAAGTGCATTCGAAAGTAGAAGGGT-3’(SEQ ID NO:4),并按照TRIzol一步法提取热研2号柱花草(来自中国热带农业科学院热带作物品种资源研究所热带牧草研究室)根尖总RNA,然后反转录得到的cDNA作为模板进行PCR扩增。
PCR反应体系为:总共50μL体系,包含10μM正向和反向引物各1μL,10×PCR buffer5μL,2.5mM dNTP 4μL,Taq酶0.5μL,cDNA模板量2.5μL,然后用灭菌ddH2O补足50μL;
PCR反应程序为:98℃1分钟;98℃30秒,58℃30秒,72℃2分钟,扩增35个循环;72℃延伸10分钟。
扩增的PCR产物通过1%琼脂糖胶电泳,再通过Golden view核酸染料染色后,在凝胶成像系统成像。采用DNA凝胶回收试剂盒(擎科生物,广州)回收长度为1425bp的PCR产物。回收得到PCR产物克隆到Trans-T1(全式金公司,中国)载体上进行测序鉴定,获得柱花草SgPG1基因全长cDNA序列SEQ ID NO:1,其氨基酸序列如SEQ ID NO:2所示。
2、载体的构建
(1)超量表达载体的构建:
以上述获得的柱花草根尖cDNA为模板,设计SgPG1特异引物,并采用上游特异引物5’-CCGGGGATCCTCTAGAATGGAAACCATTCAATTGTTGTTC-3’(SEQ ID NO:5)和下游特异引物5’-GCAGGTCGACTCTAGATTAAGTGCATTCGAAAGTAGAAGGGT-3’(SEQ ID NO:6)扩增SgPG1基因ORF的1425bp片段,PCR片段回收测序无误后,通过酶XbalⅠ对目的载体进行单酶切后,将SgPG1基因连接到目的载体ptf101s。采用转化大肠杆菌Trelief TM5α(擎科生物,广州),进行测序分析,测序无误后转化发根农杆菌MSU440(唯地生物,上海),用于农杆菌介导的柱花草下胚轴转化法进行柱花草和菜豆毛根转化。
(2)亚细胞定位分析表达载体的构建:
提取柱花草根部RNA,并将其反转录成cDNA,以该cDNA为模板,设计SgPG1特异引物,并用上游特异引物5’-CTCTAGCGCTACCGGTATGGAAACCATTCAATTGTTGTTC-3’(SEQ ID NO:7)和下游特异引物5’-CATGGTGGCGACCGGTGCAGTGCATTCGAAAGTAGAAGGGT-3’(SEQ ID NO:8)扩增SgPG1开放阅读框片段,PCR片段回收测序无误后,将SgPG1基因PCR片段连接到AgeⅠ单酶切线性化目的载体pEGAD。
(3)组织定位分析载体的构建:
采用CTAB方法提取柱花草根尖DNA,以其为模板,设计SgPG1特异引物,并用上游特异引物5’-CTATGACATGATTACGAATTCTTCTCTATCACACCCGTGAGGCT-3’(SEQ ID NO:9)和下游特异引物5’-GACTGACCTACCCGGGGATCCGAAGGAGGAAGAATGGCTAAGGTC-3’(SEQ ID NO:10)扩增SgPG1起始密码子上有2000bp的序列,并将其克隆入融合GUS的表达载体ptf102,使GUS标签蛋白融合在SgPG1的C-末端。构建成功的proSgPG1-GUS载体用于后续实验。
(4)重组蛋白载体的构建:
提取柱花草根部RNA,并将其反转录成cDNA,以该cDNA为模板,设计SgPG1特异引物,并用上游特异引物5’-GAATTCCCGGGTCGACCATGACATCAACATTGGAAATG-3’(SEQ ID NO:11)和下游特异引物5’-GGCCGCTCGAGTCGACTTAAGTGCATTCGAAAGTAGAAGGGT-3’(SEQ ID NO:12)扩增SgPG1开放阅读框片段,并将其克隆入大肠杆菌表达载体pGEX6P-3,使融合蛋白表达时GST-Tag融合在SgPG1的C-末端。构建成功的pGEX6P-3-SgPG1载体用于后续实验。
3、SgPG1的亚细胞定位及其基因的表达模式分析
(1)SgPG1的亚细胞定位分析:
通过菜豆毛状根转化法,将装载了SgPG1-GFP载体和pEGAD空载体导入菜豆毛状根进行稳定表达。然后用激光共聚焦显微镜(Zeiss,德国)观察柱花草毛状根的GFP和PI荧光信号。
结果如图1A所所示为SgPG1的亚细胞定位分析,其中图1A为菜豆毛状根表皮亚细胞定位结果,左列为GFP荧光信号,中间为PI信号,右列为GFP和PI信号的融合结果,图中标尺为20μm,结果表明柱花草SgPG1定位于细胞壁。
(2)SgPG1基因的表达模式分析:
SgPG1基因表达的组织特异性分析,取约0.5g柱花草热研2号种子,搓去种皮,常规消毒后置于MS培养基(0.4125g·L-1NH4NO3、0.48g·L-1KNO3、0.11g·L-1CaCl2·2H2O、0.0925g·L-1MgSO4·7H2O、0.0425g·L-1KH2PO4、0.2075mg·L-1KI、1.54mg L-1H3BO3、5.56mg·L-1MnSO4·4H2O、2.65mg·L-1ZnSO4.7H2O、0.0625mg·L-1Na2MoO4·2H2O、0.00625mg·L-1CuSO4·5H2O、0.00625mg·L-1CoCl2·6H2O、9.325mg·L-1Fe-EDTA;1%蔗糖)上萌发,分别提取萌发1~5天的柱花草幼苗根尖的RNA。
将上述RNA反转录成cDNA,进一步用定量PCR检测SgPG1的表达模式。柱花草的看家基因SgEFα作为内参。用于定量PCR检测基因表达量的引物分别为:
柱花草SgEFα基因的引物为:
SgEFαF:5’-CACTTCAGGACGTGTACAAGATC-3’(SEQ ID NO:13);
SgEFαR:5’-CTTGGAGAGCTTCATGGTGCA-3’(SEQ ID NO:14);
SgPG1基因的引物为:
SgPG1F:5’-TTCTTCACCTAAACTCAGCCCC-3’(SEQ ID NO:15);
SgPG1 R:5’-TCTCAAACCTGTCACTGATCCC-3’(SEQ ID NO:16);
结果如图1B~C所示,SgPG1在柱花草根尖的组织定位分析,其中图1B为SgPG1基因启动子融合GUS在根部的染色结果;GUS染色结果表明,在整个根横截面上都可以观察到用空载(35S::GUS)转化的对照毛状根的GUS染色。然而,pSgPG1::GUS毛状根的GUS染色主要在表皮细胞和周周期细胞中观察到。
图1C为SgPG1基因在柱花草幼苗根尖和根尖类边缘细胞的表达模式分析,图中数据为4个重复的平均值和标准误差。在萌发后120小时内,柱花草根尖中SgPG1的表达水平在48小时达到最高水平。在48、72、96和120小时,根尖中SgPG1的表达分别是24小时的14.9、6.9、9.9和9.1倍。同样,在萌发后48小时,观察到SgPG1在BLCs中表达水平最高,随后显著降低。
4、SgPG1蛋白的酶理化性质分析
(1)蛋白纯化:
将构建好的表达载体(pGEX6P-3-SgPG1载体)转化至大肠杆菌BL21(唯地生物,上海)中,将菌体加入50mL含有氨苄抗性的YEP培养液中,培养至波长600的分光光度计测定OD为0.5~0.6时,加入IPTG至最终浓度为1mM,于28℃摇床诱导培养4h后,各加入3mL的100mM浓度PMSF和DTT,摇匀后分装至50mL离心管中,在4℃的离心机5000rpm离心10min,去掉上清并加入50mM的PBS缓冲液重悬,于35pis高压破碎,得到澄清透亮菌液后5000rpm离心10分钟,取上清于新的50mL离心管中,加入3mL GSH磁珠,封口后置于冰上,放到冷库摇床中结合5h。
蛋白上清与磁珠结合完成后,用预冷的PBS缓冲液洗去杂蛋白,当测定洗涤的缓冲液蛋白含量为0时,加入3mL预冷的蛋白洗脱液(pH=5.0)洗脱磁珠上的重组蛋白,并用考马斯亮蓝的方法测定蛋白浓度,取适量进行SDS聚丙烯酰胺凝胶电泳和Western Blot分析,验证结果表明SgPG1酶液中无杂蛋白,即可进行后续试验。
(2)SgPG1重组蛋白的酶活力测定:
将纯化的0.004mL(0.1mg/mL)SgPG1蛋白与0.06mL 0.5%(w/v)聚半乳糖醛酸和0.136mL醋酸缓冲液(pH 5.0)混合,然后在37℃下培养30min。随后,添加0.2mL DNS试剂,将混合物在100℃下煮沸5min以停止反应。冷却至室温后,将1.6mL蒸馏水与所得样品混合,并使用分光光度计在540nm处测量吸光度。同时采用美国Sigma公司的D-半乳糖醛酸作为标准物质,SgPG1酶活性定义为每毫克SgPG1蛋白每分钟释放的D-半乳糖醛酸(毫克蛋白质-1.min-1)。
将纯化的SgPG1蛋白分别与0.06ml0.5%(w/v)聚半乳糖醛酸在一系列不同pH值的缓冲液中孵育,包括甘氨酸-盐酸缓冲液(pH 3~5)、乙酸-醋酸钠缓冲液(pH 5~6)、Tris-HCl-MES缓冲液(pH 6~7)或Tris-HCl缓冲液(ph7~9)。培养30分钟后,参照上述活力测定方法(在每个实验,将最高活性设置为100%,以便与其他条件下的活性进行比较)。
结果如图2所示,其中图2A为纯化后的SgPG1蛋白在pH 3~8范围内的活性,图2B为纯化后的SgPG1蛋白在10~90℃范围内的活性,在pH为3~6时,SgPG1对多聚半乳糖醛酸的催化活性随pH的升高而增加;当pH为6时,其酶活性达到最大值,但随着pH继续升高,SgPG1的酶活性逐渐降低;在pH为8时,其酶活几乎完全丧失。另一方面,在温度为10℃时,SgPG1的酶活性接近为零,在温度10~50℃时,随温度升高,SgPG1的活性增加,当温度为50℃时,其酶活性达到最大值,此后随温度升高酶活性逐渐降低,而温度达到90℃时,SgPG完全失活。所以,SgPG1的多聚半乳糖醛酸酶活性最适pH为6,最适温度为50℃。
(3)SgPG1蛋白的体内活性测定:
将柱花草根尖的横截面放在载玻片上,用滤纸除去多余的PBS缓冲液。随后,采用100ml(0.1mg/mL)的活性SgPG1蛋白和失性的SgPG1蛋白在40℃孵育30min后,用PBS洗涤3次,每次5min,进行免疫组织学分析,在激光共聚焦荧光显微镜(Zeiss,德国)下观察荧光信号。结果如图3所示,其中采用了JIM 5和JIM 7两种果胶抗体,图中标尺为20μm。从图中可看出添加SgPG1酶液处理后,JIM 5抗体识别的跟尖细胞细胞壁荧光强度变化与JIM 7抗体识别的荧光信号正好相反,添加SgPG1酶液后JIM 5抗体识别的荧光信号明显减弱,而JIM 7抗体在根尖细胞细胞壁上的信号增强。说明外源添加SgPG1酶液会降低根尖细胞细胞壁上低甲基化HG的含量,同时增加高甲基化HG的含量。
实施例2转基因实验
1、转基因柱花草毛状根的获得
采用实施例1中构建好的表达载体(pTF101s-SgPG1载体)转化至发根农杆菌MSU440(唯地生物,上海)中,采用农杆菌介导的柱花草下胚轴转化法获得柱花草毛状根。同时设置空载体对照为:同实施例1中的构建方法,将表达载体ptf101s转化到柱花草,获得转pTF101s空载对照株系(CK)。
2、转基因材料的检测
(1)转基因柱花草毛状根的检测:
在转基因后的柱花草毛状根形成后14天,取根部样品提取DNA和RNA,然后用PCR扩增后进行定量PCR检测超量表达的效果,PCR扩增和定量PCR采用的反应体系如下表1和表2所示。
表1 PCR反应体系
表2定量PCR反应体系
检测体系 | 体积(μL) |
2×Go Tap qPCR Master Mix | 10μL |
上游引物(10mol/L) | 0.6μL |
下游引物(10mol/L) | 0.6μL |
Mili-Q水 | 补至20μL |
DNA | 2μL |
PCR扩增反应条件:95℃预变性1min,然后按95℃变性15s,55-60℃退火15s,进行35-40个循环,最后72℃延伸30s。
qPCR反应条件:95℃1分钟,95℃15秒,60℃60秒,72℃30秒,进行40个循环。
定量PCR确认得到SgPG1高表达量的不同转基因拟南芥株系。
3、转基因柱花草毛状根表型分析
(1)毛状根有无BLCs的分析:
将上述定量PCR检测后得到的阳性转基因柱花草毛状根以及转化空载的毛状根在23℃光照条件下(16h:8h光照:黑暗)中培养14d后,收集并统计具有BLCs的毛状根数量。
(2)转基因毛状根根尖细胞壁果胶成分的分析:
将阳性转基因柱花草毛状根以及对照毛状根培养14天后,取毛状根根尖琼脂包埋横切后,进行上述免疫组织学分析,在共聚焦荧光显微镜下观察并拍摄。
结果如图4所示,其中图4A为有无BLCs的柱花草毛状根的表型分析,观察到超量表达SgPG1的毛状根约62%显示出明显的BLCs形成,而对照毛状根只有29%的显示出明显的BLCs形成。
图4B为超量表达SgPG1对柱花草毛状根BLCs形成的影响,CK是转化空载的对照株系,SgPG1-OX是的超表达株系。对转基因毛状根中的果胶表位也进行了研究,结果表明,与CK毛状根相比,超量表达SgPG1毛状根的JIM5抗体标记较低,说明超量表达SgPG1毛状根的细胞壁低甲酯化多聚半乳糖醛酸含量降低,但JIM7抗体标记较高。
实施例3超量表达SgPG1对植物根系耐受铝毒的影响
1、柱花草毛状根的铝溶液处理
采用实施例2中已获得的柱花草毛状根。将超量表达SgPG1的柱花草毛状根与空载对照株系(CK)在含或不含10mM AlCl3的0.5mM CaCl2溶液中培养两天,拍摄处理前后的毛状根照片。使用ImageJ软件测量处理前后的根系长度,计算获得根系相对生长速率。
2、转基因拟南芥的铝处理
(1)转基因拟南芥的获得:
采用实施例1中构建好的表达载体(pTF101s-SgPG1载体)转化至根癌农杆菌Gv3101(唯地生物,上海)中,采用农杆菌介导的拟南芥花粉转化法获得转基因拟南芥。
(2)转基因拟南芥的铝处理:
为了进行耐铝性分析,将野生型和转基因拟南芥的表面灭菌种子播种在含有0.5%琼脂(含或不含10mM AlCl3)的1/30Hoagland培养基(pH=4.5)上。处理5天后,拍摄幼苗照片,并使用图像J测量根长。
结果如图5所示为超量表达SgPG1对柱花草毛状根耐受铝毒的影响,其中图5A为柱花草毛状根铝处理后的苏木精染色情况,CK为转化空载的对照毛状根,OX为超量表达株系,*号表示同一指标在超量表达毛状根与对照毛状根之间的显著性比较:***表示显著水平P<0.001时,差异极显著,图中标尺为20μm;从图中可看出,与转化了空载的毛状根相比,超量表达SgPG1的柱花草毛状根在处理后的苏木精染色程度显著降低,说明超量表达SgPG1减少柱花草根尖铝的富集。
图5B为柱花草毛状根铝处理后的相对生长速率;从图中可以看出,与对照的毛状根相比,超量表达SgPG1提高了柱花草根系在铝处理下的相对生长速率,说明超量表达SgPG1可以提高柱花草对铝毒的耐受。
图5C为拟南芥铝处理后的生长情况,WT为野生型拟南芥,OX为超量表达株系;从图中可以看出,与野生型拟南芥相比,超量表达SgPG1促进了拟南芥在铝处理条件下的根长。
图5D为不同基因型拟南芥铝处理后的根系相对生长速率,*号表示同一指标在超量表达拟南芥与对照拟南芥之间的显著性比较:***表示显著水平P<0.001时,差异极显著。从图中可以看出,与野生型拟南芥相比,超量表达SgPG1提高了拟南芥在铝处理下的相对生长速率,说明超量表达SgPG1可以提高拟南芥对铝毒的耐受。
综上结果表明,超量表达SgPG1基因可以降解低甲酯化的同型半乳糖醛酸,从而提高了细胞壁中高甲酯化的同型半乳糖醛酸比例,促进柱花草根尖类边缘细胞的形成,从而提高了植物对铝毒的耐受能力。该基因在其他物种如拟南芥中异源表达后,也能够促进拟南芥对铝毒害的耐受性。
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> 华南农业大学
<120> 一种柱花草多聚半乳糖醛酸酶基因SgPG1及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1425
<212> DNA
<213> 柱花草多聚半乳糖醛酸酶基因SgPG1( SIPOSequenceListing 1.0)
<400> 1
atggaaacca ttcaattgtt gttcatgatt ttgatttctg tgattctctt caaccatgac 60
atcaacattg gaaatgtgga aggaagatac cattatcata agaaaaattc ccctgttcct 120
actaatccta atccccctgc ttctgattac tctcctgagc cacaagttcc ttcaacacct 180
tccacacctt accccaatga ccctcctcct caagattctt ctccagacgg cgtcttcgat 240
ctgaggtcgt tcggagccgt tggagatggc ttagcagatg acacagcagc atttagggca 300
gcatggaaag ctgcttgtgc tgttgagtca ggtgttgttc ttgctccaga gaactactct 360
tttaaggtca cttcaagtat tctctcaggt ccatgcaagc ctggattagt actccaagtg 420
gatggtacat tgatggcacc agatggaccg gattcatggc cggaagccga tagccggaat 480
caatggctag tgttttatag acttgatcaa atgtcactta atggcacagg aaccattgaa 540
ggcaatggag ataagtggtg ggatctcccc tgcaaacctc acaggagtga agatggaaaa 600
acagtttcag gaccatgtgg cagccctgct atgatgaggt tcttcatgag cagcaacttg 660
aaggtgagtg gtctgagaat ccagaacagt cctcagttcc acatgatttt caatggctgc 720
caaggagtgc agatagatag gctgtccatt tcttcaccta aactcagccc caacactgat 780
ggaatccatg ttgaaaactc taagtctgtt ggaatataca ataccatgat aagcaatggt 840
gatgactgca tttcaattgg acctggcact gcaaacgtgg acatagatgg tgttacttgt 900
ggtcctagcc atgggattag cattggaagc cttggagtgc ataattctca agcatgtgtg 960
tccaacttaa cagttaagaa caccatcata aaagaatcag acaatgggct aagaatcaag 1020
acatggcaag gtgggatggg atcagtgaca ggtttgagat ttgagaatat ccaaatggaa 1080
aatgttagga actgcataat catagaccag tactactgct tgtcaaagga atgccataac 1140
caaacttcag ctgttcatgt gaatgatgtg tcctacaaga acattaaggg tacctatgat 1200
gttaggaccc ctccaattca ctttgcatgc agtgacactg ttgcttgcac aaacataaca 1260
ctctctgagg ttgagctttt tccatatgaa ggagagttgc ttgatgaccc tttctgttgg 1320
aatgcttatg ggacacagga gacttggact atacctccaa tcaattgctt aagggaaggt 1380
gaccctgaga ctgtggcaga cccttctact ttcgaatgca cttaa 1425
<210> 2
<211> 474
<212> PRT
<213> 柱花草多聚半乳糖醛酸酶SgPG1( SIPOSequenceListing 1.0)
<400> 2
Met Glu Thr Ile Gln Leu Leu Phe Met Ile Leu Ile Ser Val Ile Leu
1 5 10 15
Phe Asn His Asp Ile Asn Ile Gly Asn Val Glu Gly Arg Tyr His Tyr
20 25 30
His Lys Lys Asn Ser Pro Val Pro Thr Asn Pro Asn Pro Pro Ala Ser
35 40 45
Asp Tyr Ser Pro Glu Pro Gln Val Pro Ser Thr Pro Ser Thr Pro Tyr
50 55 60
Pro Asn Asp Pro Pro Pro Gln Asp Ser Ser Pro Asp Gly Val Phe Asp
65 70 75 80
Leu Arg Ser Phe Gly Ala Val Gly Asp Gly Leu Ala Asp Asp Thr Ala
85 90 95
Ala Phe Arg Ala Ala Trp Lys Ala Ala Cys Ala Val Glu Ser Gly Val
100 105 110
Val Leu Ala Pro Glu Asn Tyr Ser Phe Lys Val Thr Ser Ser Ile Leu
115 120 125
Ser Gly Pro Cys Lys Pro Gly Leu Val Leu Gln Val Asp Gly Thr Leu
130 135 140
Met Ala Pro Asp Gly Pro Asp Ser Trp Pro Glu Ala Asp Ser Arg Asn
145 150 155 160
Gln Trp Leu Val Phe Tyr Arg Leu Asp Gln Met Ser Leu Asn Gly Thr
165 170 175
Gly Thr Ile Glu Gly Asn Gly Asp Lys Trp Trp Asp Leu Pro Cys Lys
180 185 190
Pro His Arg Ser Glu Asp Gly Lys Thr Val Ser Gly Pro Cys Gly Ser
195 200 205
Pro Ala Met Met Arg Phe Phe Met Ser Ser Asn Leu Lys Val Ser Gly
210 215 220
Leu Arg Ile Gln Asn Ser Pro Gln Phe His Met Ile Phe Asn Gly Cys
225 230 235 240
Gln Gly Val Gln Ile Asp Arg Leu Ser Ile Ser Ser Pro Lys Leu Ser
245 250 255
Pro Asn Thr Asp Gly Ile His Val Glu Asn Ser Lys Ser Val Gly Ile
260 265 270
Tyr Asn Thr Met Ile Ser Asn Gly Asp Asp Cys Ile Ser Ile Gly Pro
275 280 285
Gly Thr Ala Asn Val Asp Ile Asp Gly Val Thr Cys Gly Pro Ser His
290 295 300
Gly Ile Ser Ile Gly Ser Leu Gly Val His Asn Ser Gln Ala Cys Val
305 310 315 320
Ser Asn Leu Thr Val Lys Asn Thr Ile Ile Lys Glu Ser Asp Asn Gly
325 330 335
Leu Arg Ile Lys Thr Trp Gln Gly Gly Met Gly Ser Val Thr Gly Leu
340 345 350
Arg Phe Glu Asn Ile Gln Met Glu Asn Val Arg Asn Cys Ile Ile Ile
355 360 365
Asp Gln Tyr Tyr Cys Leu Ser Lys Glu Cys His Asn Gln Thr Ser Ala
370 375 380
Val His Val Asn Asp Val Ser Tyr Lys Asn Ile Lys Gly Thr Tyr Asp
385 390 395 400
Val Arg Thr Pro Pro Ile His Phe Ala Cys Ser Asp Thr Val Ala Cys
405 410 415
Thr Asn Ile Thr Leu Ser Glu Val Glu Leu Phe Pro Tyr Glu Gly Glu
420 425 430
Leu Leu Asp Asp Pro Phe Cys Trp Asn Ala Tyr Gly Thr Gln Glu Thr
435 440 445
Trp Thr Ile Pro Pro Ile Asn Cys Leu Arg Glu Gly Asp Pro Glu Thr
450 455 460
Val Ala Asp Pro Ser Thr Phe Glu Cys Thr
465 470
Claims (9)
1.一种柱花草多聚半乳糖醛酸酶基因SgPG1,其特征在于,其核苷酸序列如SEQ ID NO:1所示。
2.一种柱花草多聚半乳糖醛酸酶SgPG1,其特征在于,其氨基酸序列如SEQ ID NO:2所示。
3.权利要求1所述多聚半乳糖醛酸酶基因SgPG1或权利要求2所述多聚半乳糖醛酸酶SgPG1在促进植物根尖类边缘细胞形成或在制备促进植物根尖类边缘细胞形成的制剂中的应用,其特征在于,所述植物为拟南芥或柱花草。
4.权利要求1所述多聚半乳糖醛酸酶基因SgPG1或权利要求2所述多聚半乳糖醛酸酶SgPG1在提高植物铝毒耐受能力或在制备提高植物铝毒耐受能力的制剂中的应用,其特征在于,所述植物为拟南芥或柱花草。
5.权利要求1所述多聚半乳糖醛酸酶基因SgPG1在构建高铝毒耐受能力的转基因植物中的应用,其特征在于,所述植物为拟南芥或柱花草。
6.一种重组表达载体,其特征在于,含有权利要求1所述柱花草多聚半乳糖醛酸酶基因SgPG1。
7.一种基因工程菌,其特征在于,含有权利要求6所述重组表达载体。
8.权利要求6所述重组表达载体或权利要求7所述基因工程菌在构建转基因植物或制备促进植物根尖类边缘细胞形成的制剂中的应用,其特征在于,所述植物为拟南芥或柱花草。
9.一种提高植物根尖铝毒耐受能力的方法,其特征在于,在植物中超量表达多聚半乳糖醛酸酶编码基因SgPG1;所述植物为拟南芥或柱花草。
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