CN112359049A - 一种岷江百合几丁质酶基因LrCHI2及其应用 - Google Patents
一种岷江百合几丁质酶基因LrCHI2及其应用 Download PDFInfo
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- CN112359049A CN112359049A CN202011434026.9A CN202011434026A CN112359049A CN 112359049 A CN112359049 A CN 112359049A CN 202011434026 A CN202011434026 A CN 202011434026A CN 112359049 A CN112359049 A CN 112359049A
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Abstract
本发明公开了一种岷江百合几丁质酶基因LrCHI2,其核苷酸序列如SEQ ID NO:1所述,编码如SEQ ID NO:2所示氨基酸序列的蛋白质。本发明通过功能基因组学相关技术研究证实LrCHI2基因具有提高植物对病原真菌抗性的功能。将本发明抗真菌基因LrCHI2构建到植物表达载体上并转入烟草中过量表达,超表达LrCHI2的转基因烟草有着较强的抗稻黑孢霉、织球壳菌、尖孢镰刀菌侵染的能力。
Description
技术领域
本发明属于分子生物学以及基因工程技术领域,具体涉及一种具有抗真菌侵染能力的岷江百合几丁质酶基因LrCHI2及其应用。
背景技术
植物体拥有多种保护自身免受各种病原体侵害的机制。在植物体受到病原体攻击时,植物通过迅速改变基因表达来对病原菌和外部胁迫做出反应,诱导一些特殊蛋白的重新合成,其中大部分是病程相关蛋白(pathogenesis-related proteins, PRs),这类蛋白是植物防御体系的重要组成部分。PRs在植物中主要表现为几丁质酶(chitinase, CHI)、葡聚糖酶(dextranase)和类甜蛋白(thaumatin-like proteins, TLPs)等。根据氨基酸序列、血清学关系和生物活性,将PR蛋白分为17类(Kaur A, Pati PK, Pati AM, Nagpal AK.In-silico analysis of cis-acting regulatory elements of pathogenesis-relatedproteins of Arabidopsis thaliana and Oryza sativa. Plos One. 2017, 12(9):e0184523.)。PR3基因家族编码的几丁质酶在植物抗病防卫反应中备受关注。
几丁质,又称甲壳素或甲壳质,以N-乙酰-D-氨基葡萄糖为单体,以β-1, 4-糖苷键连结而成的生物多聚体,是自然界中除纤维素外储量最大的生物多聚糖。在自然界几丁质广泛存在于真菌、硅藻、节肢动物和原生动物等生物中,是绝大部分真菌细胞壁的结构物质,占细胞壁干重的40%~60%,但在高等植物中尚未发现有几丁质的存在。几丁质酶是广泛存在于微生物和植物体内的一类蛋白质,是植物体中与防御有关的一种次生水解酶。由于在大部分真菌的细胞壁中含有几丁质,而植物细胞中不含几丁质,却有几丁质酶,故认为它能催化真菌细胞壁的重要成分几丁质的水解,从而抑制真菌的生长增殖,提高植物的抗真菌能力。植物几丁质酶属于一种诱导性酶,在正常情况下表达量很低或者不表达(Dana M,Pintor J, Cubero B. Transgenic tobacco plants overexpressing chitinases offungal origin show enhanced resistance to biotic and abiotic stress agents.Plant physiology. 2006, 142(2): 722-730.)。植物几丁质酶存在于植物的根、茎、种子、花中,它们的表达存在组织特异性。大多植物几丁质酶相对分子质量都比昆虫几丁质酶相对分子质量小,其中最小的几丁质酶大小为22 kDa。植物几丁质酶根据氨基酸的数量、结构特征和酶的活性特征被划分为6类。其中,Class Ⅰ几丁质酶具有N-端富含半胱氨酸的几丁质结合区、C-端催化区以及结合区和催化区连接的可变区的结构特征。ClassⅡ几丁质酶主要有烟草(Nicotiana tabacum)酸性同功酶(PR-P和PR-Q)、大麦(Hordeum vulgare)几丁质酶和矮牵牛(Petunia hybrida)几丁质酶,定位于细胞间隙,含有催化区,但缺少几丁质结合区及交连区,有些成员还保留了部分交连区。Class Ⅲ几丁质酶既具有溶菌酶活性又具有几丁质酶活性,但与Class Ⅰ和Class Ⅱ无同源性,无几丁质结合区,一般位于细胞间隙。Class Ⅳ几丁质酶有甜菜(Beta vulgaris)碱性几丁质酶,菜豆(Phaseolus vulgaris)酸性PR-4几丁质酶,由几丁质结合区、交连区及催化区组成。近年来,随着对其作用机理、生化性质、表达调控的深入研究,利用几丁质酶基因培育抗真菌植物新品种成为植物防御真菌病害的一种有效途径。正是由于几丁质酶在植物抗真菌病害中起着重要的作用,因而成为近年来抗真菌病害研究的热点之一。
植物中的N-乙酰葡萄糖胺以糖酯键形式存在,而非线性同聚物,即植物中缺少几丁质酶的有效底物。所以植物的几丁质酶可以降解病原真菌细胞壁中的几丁质,破坏细胞新物质的沉积致使病原体死亡,而且产生的细胞壁碎片具有诱导作用,从而刺激寄主植物的抗病反应。大量研究表明,几丁质酶抑制菌丝生长,使其顶端细胞壁变薄进一步引发气球状突起,最终导致原生质膜破裂。几丁质酶通过降解菌丝末端新合成的几丁质,破坏菌丝端部正常生长,以达到抑制菌丝生长的目的。菌丝体顶端,相对裸露的新生几丁质易被几丁质酶水解,成熟细胞(远离菌丝顶端)细胞壁中形成交错的几丁质-葡聚糖纤丝,且外被其他多糖及蛋白,所以成熟细胞壁几丁质层不易接触到几丁质酶,难以被降解。因此,几丁质酶与β-1, 3-葡聚糖酶在植物防卫反应中常协同表达,从而增强了植物的抗病性(孟亮, 崔德才. 植物几丁质酶及其在抗真菌病害中的应用. 生物技术通讯. 2004, (04): 420-422.)。
百合是百合科(Liliaceae)百合属(Lilium)植物的总称,属多年生球根类花卉。在种球繁殖及鲜切花生产过程中,百合易受到真菌、病毒、细菌等多种病原菌的危害。目前发现的百合病害达几十种之多,其中由镰刀属(Fusarium spp.)真菌引起的枯萎病(又称为基腐病、茎腐病)是百合生产中危害最严重的病害。镰刀菌侵染百合种球后引起基盘坏死、鳞片腐烂脱落,造成种球质量下降;植株感染镰刀菌后叶片变黄、萎蔫下垂,植株提早枯萎死亡,严重影响百合切花的产量和品质。其中尖孢镰刀菌(F. oxysporum)致病性最强、分离频率最高,是百合枯萎病的主要致病菌。岷江百合(L. regale Wilson)为我国特有种,仅分布于岷江流域海拔800~2700m的河谷到山腰的岩石缝中,具有强的抗枯萎病性,是现代百合育种的重要种质资源。
发明内容
本发明的目的是提供一种岷江百合几丁质酶基因LrCHI2,并将该基因应用在提高烟草对稻黑孢霉(Nigrospora oryzae)、织球壳菌(Plectosphaerella cucumerina)、尖孢镰刀菌(F. oxysporum)的抗性中。
本发明从岷江百合中克隆获得的具有抗真菌活性的几丁质酶的全长基因,几丁质酶基因LrCHI2核苷酸序列如SEQ ID NO:1所示,该基因cDNA全长序列为1313 bp,包含一个933 bp的开放阅读框、19 bp的5’非翻译区、361 bp的3’非翻译区,编码如SEQ ID NO:2所示氨基酸序列的蛋白质。
本发明中LrCHI2基因的编码区是序列表SEQ ID NO:1中第20-952位所示的核苷酸序列;本发明分离克隆岷江百合的一个抗真菌相关基因的完整cDNA片段,利用根癌农杆菌(Agrobacterium tumefaciens)介导将目的基因转入受体植物中并过量表达,通过进一步实验验证该基因是否具有抗真菌的活性,为后期利用该基因改良烟草及其他植物抵御真菌病害的能力奠定基础,发明人将这个基因命名为LrCHI2。
上述LrCHI2基因可以提高烟草的抗真菌特性,具体操作如下:
(1)采用异硫氰酸胍法提取百合根部的总RNA,以提取的RNA为模板,以oligo(dT)18为反转录引物,通过逆转录-聚合酶链式反应(reverse transcription-polymerase chainreaction,RT-PCR)扩增出LrCHI2编码区,然后将其连接到pGEM-T载体上,经测序获得具有目的基因的克隆;
(2)用限制性内切酶BamHI和EcoRI酶切pGEM-T-LrCHI2,通过胶回收得到目的基因片段,用同样的内切酶酶切植物表达载体pCAMBIA2300s,胶回收获得所需载体大片段,再将所获得LrCHI2基因片段与pCAMBIA2300s片段连接,构建植物超表达载体,之后将所构建的重组载体通过根癌农杆菌介导转入烟草中表达;
(3)以重组载体pCAMBIA2300s-LrCHI2上具有的抗性标记筛选转化子,并通过PCR检测得到真正的转基因植株,分析转基因植物的LrCHI2表达水平和对真菌的抗性。
本发明为提高植物对真菌病害的抗性提供了一种新的方法,通过基因工程手段培育抗病植物可以克服传统育种的不足,不仅育种周期缩短,而且操作简单,容易获得高抗材料。本发明来自岷江百合的LrCHI2基因能增强植物对真菌的抗性,将该基因导入烟草中,可以产生抗真菌的新品种和新材料。利用基因工程技术培育抗性植物品种和材料具有明显的优势和不可取代的重要性;它不仅可以为大规模生产农作物、药材、园艺植物等提供方便,大量减少化学农药的使用,还可以为农业生产节约成本、减小环境污染,因此本发明具有广阔的市场应用前景。
附图说明
图1是本发明部分LrCHI2转基因烟草基因组DNA的PCR检测结果,其中Marker为DL15000 DNA Marker (大连宝生物),由15,000 bp、10,000 bp、5,000 bp、3,000 bp、2,000bp、1,000 bp、1,500 bp、750 bp、500 bp、250 bp以及100 bp DNA片段组成;阳性对照为质粒pGEM-T-LrCHI2为模板的PCR反应产物;WT为非转基因烟草(野生型,wild type)总DNA为模板的PCR反应产物;
图2是本发明部分阳性LrCHI2转基因烟草中LrCHI2转录水平的表达分析结果图;图中:Marker是DL15000 DNA Marker (大连宝生物);阳性对照为质粒pGEM-T-LrCHI2为模板的PCR反应产物;WT是非转基因烟草总RNA逆转录cDNA为模板的PCR产物。
图3是本发明部分阳性LrCHI2转基因烟草体外抗真菌活性的抑菌效果图;其中A、B、C图示中的真菌分别是稻黑孢霉、织球壳菌、尖孢镰刀菌;WT为野生型烟草的总蛋白;Buffer为空白对照,即无蛋白对照(用于提取蛋白的缓冲液)。
具体实施方式
下面通过附图和实施例对本发明作进一步详细说明,但本发明保护范围不局限于所述内容,实施例中方法如无特殊说明均为常规方法,使用的试剂如无特殊说明均为常规市售试剂或按常规方法配制的试剂。
实施例1:LrCHI2 cDNA克隆以及序列分析
取岷江百合根部提取总RNA,用液氮将岷江百合根部研磨成粉末,然后转入离心管中,采用异硫氰酸胍法提取总RNA,采用逆转录酶M-MLV (promega)以总RNA为模板合成cDNA第一链,反应体系和操作过程为:取5μg Total RNA,依次加入50ng oligo(dT)、2μL dNTP(2.5mmol/L each)、DEPC水至反应体积为14.5μL;混匀后,70℃加热变性5min后迅速在冰上冷却5min,然后依次加入4μL 5×First-stand buffer、0.5μL RNasin (200U)、1μL M-MLV(200U),混匀并短时离心,42℃温浴1.5h,取出后70℃加热10min,终止反应,cDNA第一链合成后置于-20℃保存备用。
以合成的第一链cDNA为模板,扩增目的基因LrCHI2。所用上下游引物序列分别为5’CGGGGACACAACTCATACAATGGCA3’及5’GAAAAATATAGTCATAGACTGTTAC3’。采用AdvantageTM 2PCR Enzyme(Clontech)扩增出目的基因;PCR反应条件:95℃ 1min;95℃ 30s,60℃ 30s,72℃ 50s,30个循环;72℃ 5min;反应体系(10μL)为1μL cDNA、1μL 10×Advantage 2 PCRBuffer、0.5μL 50×dNTP Mix (10 mM each)、0.2μL 正向引物(10 μM)、0.2μL 反向引物(10 μM)、0.2μL Advantage 2 PCR Polymerase Mix、6.9μL PCR-Grade water;PCR结束后,取5μL用于琼脂糖凝胶电泳,以检测扩增产物的特异性以及大小。
对PCR产物进行TA克隆,使用的试剂盒为pGEM-T easy Vector System I(Promega, USA),反应体系和操作过程为:取1.5μL PCR产物,依次加入1μL pGEM-T Vector(50 ng/μL)和2.5μL 2×Ligation solutionⅠ,混匀后置于16℃过夜反应。采用热激转化法将连接产物转入大肠杆菌DH5α中。使用含有氨苄青霉素(ampicillin,Amp)的LB固体培养基筛选阳性克隆,挑选若干个单菌落,摇菌后用扩增LrCHI2的特异引物鉴定出多克隆位点插入LrCHI2的克隆,将所鉴定的克隆进行测序,最终获得的LrCHI2全长cDNA为1313bp,通过NCBI ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html)分析发现其包含一个933bp的开放读码框(见序列表),LrCHI2编码一个含310个氨基酸的蛋白质,其分子量约为32.46 KDa,等电点约为5.65。LrCHI2编码的蛋白质序列具有GH19家族的保守结构域,与一些植物的几丁质酶具有较高的同源性,这表明其属于岷江百合中的几丁质酶基因。
实施例2:植物超表达载体构建
采用SanPrep柱式质粒DNA小量抽提试剂盒(上海生工)提取插入LrCHI2的大肠杆菌质粒pGEM-T-LrCHI2以及植物表达载体pCAMBIA2300s的质粒,取1μL用于琼脂糖凝胶电泳以检测所提取质粒的完整性及浓度高低;用限制性内切酶BamHI (TaKaRa)和EcoRI (TaKaRa)分别对质粒pGEM-T-LrCHI2和pCAMBIA2300s进行双酶切(100μL体系),反应体系和操作过程为:取20μL pGEM-T-LrCHI2和pCAMBIA2300s质粒、依次加入10μL 10×K buffer、4μL BamHI、6μL EcoRI、60μL ddH2O,混匀后短时离心,置于37℃过夜反应;将所有酶切产物点于琼脂糖凝胶中进行电泳,然后对LrCHI2片段和pCAMBIA2300s载体大片段分别进行胶回收,整个过程使用SanPrep柱式DNA胶回收试剂盒(上海生工);取1μL回收产物通过琼脂糖凝胶电泳检测回收片段的大小以及浓度,置于-20℃保存备用。
利用T4 DNA Ligase (TaKaRa),将回收的LrCHI2片段和pCAMBIA2300s载体片段连接起来,反应体系(20μL)和操作过程为:取10μL LrCHI2片段依次加入2μL pCAMBIA2300s载体DNA、2μL 10×T4 DNA Ligase Buffer、1μL T4 DNA Ligase、5μL ddH2O,混匀后短时离心,然后16℃水浴过夜反应。接着采用热激转化法将连接产物转入大肠杆菌DH5α中,用含有50mg/L卡那霉素(kanamycin,Km)的固体培养基筛选阳性克隆。挑选单菌落摇菌,以菌液为模板用扩增LrCHI2的特异引物进行PCR,挑选出LrCHI2与pCAMBIA2300s成功连接的克隆,所检测的菌株若为阳性,加入甘油并置于-80℃保存备用。
采用SanPrep柱式质粒抽提试剂盒(上海生工)提取并纯化上述大肠杆菌中的pCAMBIA2300s-LrCHI2质粒。随后用液氮冻融法将上述构建的植物表达载体pCAMBIA2300s-LrCHI2转入根癌农杆菌LBA4404感受态细胞中。操作步骤为:取2μg pCAMBIA2300s-LrCHI2质粒加入含有200μL感受态细胞的离心管中,轻轻混匀后冰浴5min,随后转入液氮中冷冻1min,然后迅速置于37℃水浴5min,之后立即冰浴2min,加入800μL LB液体培养基于28℃振荡培养4h。将活化后的农杆菌涂于含有50mg/L Km的LB固体培养基上,28℃静止培养。挑选单菌落摇菌,再用扩增LrCHI2的特异性引物进行PCR,检测pCAMBIA2300s-LrCHI2是否转入农杆菌中,对于阳性克隆,加入甘油后置于-80℃保存备用。
实施例3:农杆菌介导的植物遗传转化以及转基因植物筛选
本实验的转基因受体是烟草,将烟草种子用75%的酒精浸泡30s,用无菌水洗涤后用0.1%的HgCl2浸泡8min,然后再用无菌水洗涤若干次,播种于1/2 MS培养基上,28℃暗培养6d,发芽后转至光照培养箱(25℃,16 h/d光照),以后每月用1/2MS培养基继代一次。
从-80℃冰箱中取出保存的含有pCAMBIA2300s-LrCHI2质粒的农杆菌LBA4404菌种,接种于5mL含有50mg/L Km和20mg/L利福平的LB液体培养基中,28℃培养至培养基浑浊。吸取1mL浑浊的菌液至含有50mg/L Km的LB固体培养基上,28℃培养48h;随后将LB固体培养基上的农杆菌刮下适量接种于附加有20mg/L的乙酰丁香酮的MGL液体培养基中,28℃振荡培养2-3h以活化农杆菌。
取烟草无菌苗叶子切成1cm2左右的叶盘,完全浸泡于上述含有活化农杆菌的MGL液体培养基中,浸染时间为15min,用无菌滤纸吸干叶片表面的菌液,将叶盘置于共培养基上进行室温培养,烟草转化的共培养基为MS+0.02mg/L 6-BA+2.1mg/L NAA+30g/L sucrose+6g/L琼脂,22℃黑暗条件下共培养2天。
将共培养后的叶盘转到加有抗生素的MS筛选培养基中分化成苗,同时筛选转基因植株。烟草筛选培养基为MS+0.5mg/L 6-BA+0.1mg/L NAA+30g/L sucrose+6g/L琼脂+50mg/L Km+200mg/L头孢霉素(cefotaxime sodium salt,Cef);筛选培养时将培养瓶转移至光照培养箱培养(25℃,16h/d光照,8h/d黑暗),待烟草长出芽后用含有50mg/L Km和200mg/LCef的MS培养基继代培养,因烟草愈伤分化率较高,故需要对再生植株进行进一步筛选,将烟草再生苗移至含有50mg/L Km的MS培养基上使其生根,最后选用生根较好的再生苗做进一步的检测。
采用CTAB法提取转基因烟草植株叶片的基因组DNA,将提取的基因组DNA取1μL通过琼脂糖凝胶电泳检测其完整性和浓度,以转基因植株的基因组DNA为模板用扩增LrCHI2的特异引物进行PCR,PCR结束后,取8μL产物用于琼脂糖凝胶电泳以检测阳性转基因植株,部分烟草转基因植株的扩增结果如图1所示,LrCHI2转基因烟草共筛选到28株阳性转基因植株。
实施例4:转基因烟草中LrCHI2的表达分析以及转基因植株抗真菌活性分析
取阳性转基因单株以及非转基因烟草(野生型)的嫩叶提取总RNA,逆转录生成cDNA第一链,并以此为模板用扩增LrCHI2的特异引物进行PCR,根据PCR结果分析各转基因单株中LrCHI2转录水平的表达,总RNA提取以及RT-PCR的方法与实施例1中相同。PCR结束之后,取8μL用于琼脂糖凝胶电泳,部分单株的检测结果如图2所示,共检测到21个转基因单株中LCHI2在转录水平大量表达,这些单株的编号为1~21。
将实验室保存的几种真菌接种于PDA固体培养基(200g/L马铃薯、15g/L琼脂、20g/L葡萄糖)上,28℃暗培养,待菌落生长至直径约为2~3cm时添加蛋白,分析转基因植株体外抗真菌活性。供试真菌共有3种:稻黑孢霉、织球壳菌、尖孢镰刀菌。为了防止其它杂菌污染所提取的蛋白,整个植物蛋白提取过程均是无菌操作。首先取1g转基因烟草单株(编号分别为2、3、11)及野生型叶片放入研钵中,加入1mL蛋白提取液(1M NaCl、0.1M 乙酸钠、1%PVP,pH6.0),充分研磨,转入1.5mL离心管中,混匀后4℃静置过夜;4℃离心30min (12,000g/min),取上清于新的1.5mL离心管中,并取适量用紫外分光光度仪测定总蛋白浓度。将转基因和野生型植株的总蛋白浓度调整至0.2μg/μL,然后分别取20μL滴于各真菌培养基的无菌滤纸上。在每个真菌的平板上除了添加不同转基因烟草植株的总蛋白,同时平行添加野生型烟草的总蛋白和空白对照(蛋白提取液);28℃培养几天后观察真菌生长的情况,并据此来评价LrCHI2转基因烟草的体外抗真菌活性,结果如图3所示,LrCHI2转基因烟草蛋白对稻黑孢霉、织球壳菌、尖孢镰刀菌的生长具有明显的抑制作用。
序列表
<110> 昆明理工大学
<120> 一种岷江百合几丁质酶基因LrCHI2及其应用
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Claims (2)
1.一种岷江百合几丁质酶基因LrCHI2,其特征在于:其核苷酸序列如SEQ ID NO:1所示,编码如SEQ ID NO:2所示氨基酸序列的蛋白质。
2.权利要求1所述的岷江百合几丁质酶基因LrCHI2在提高烟草对稻黑孢霉(Nigrospora oryzae)、织球壳菌(Plectosphaerella cucumerina)、尖孢镰刀菌(Fusarium oxysporum)抗性中的应用。
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CN113604477A (zh) * | 2021-08-20 | 2021-11-05 | 昆明理工大学 | 一种岷江百合defensin抗菌肽基因LrDef1及应用 |
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