CN117247955A - 一种通过改造茶树CsHDH1基因来提高噻虫嗪降解能力的方法及应用 - Google Patents
一种通过改造茶树CsHDH1基因来提高噻虫嗪降解能力的方法及应用 Download PDFInfo
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Abstract
本发明涉及基因工程技术领域,提供了一种通过改造茶树CsHDH1基因来提高噻虫嗪降解能力的方法,通过基因工程途径使茶树3‑羟基异丁酸脱氢酶CsHDH1基因过表达;其中,CsHDH1基因的核苷酸序列如SEQ ID NO.1所示。本发明还提供了一种上述通过改造茶树CsHDH1基因来提高噻虫嗪降解能力的方法在培育增强农药代谢能力的茶树新品种方面的应用。本发明研究筛选到了对茶树常施杀虫剂噻虫嗪降解能力具有正向调控作用的CsHDH1基因,通过基因工程途径使茶树3‑羟基异丁酸脱氢酶CsHDH1基因过表达,能够获得具有更强噻虫嗪降解能力的茶树植株,为培育增强农药代谢能力的茶树新品种提供理论基础和基因资源。
Description
技术领域
本发明涉及基因工程技术领域,尤其涉及一种通过改造茶树CsHDH1基因来提高噻虫嗪降解能力的方法及应用。
背景技术
茶树(Camellia sinensis(L.)O.Kuntze)为山茶科山茶属植物,是我国重要的经济作物之一,具有巨大的经济价值。
目前,茶树的防虫措施仍以化学防治为主,其中,噻虫嗪作为化学杀虫剂,在茶园中被广泛应用。然而,在实际应用中发现,杀虫剂的不当或过量使用,可能导致茶叶中存在药物残留,从而对消费者的健康构成潜在威胁。
噻虫嗪是第二代新烟碱类杀虫剂,具有胃毒、触杀以及内吸性等特点,可选择性抑制害虫中枢神经系统中的烟碱乙酰胆碱酯酶受体(nAChR),从而阻断正常传导并使害虫神经突触膜电势改变,最终导致其麻痹死亡。
据此,急需获得一种可有效提高茶树降解噻虫嗪能力的方法,进一步为茶树应对外源胁迫的调节机制提供理论依据,同时还为培育增强农药代谢能力的茶树新品种提供理论基础。
发明内容
本发明所要解决的技术问题在于提供一种通过改造茶树3-羟基异丁酸脱氢酶的CsHDH1基因来提高噻虫嗪降解能力的方法及应用。
本发明采用以下技术方案解决上述技术问题:
一种通过改造茶树CsHDH1基因来提高噻虫嗪降解能力的方法,通过基因工程途径使茶树3-羟基异丁酸脱氢酶CsHDH1基因过表达;其中,CsHDH1基因的核苷酸序列如SEQ IDNO.1所示。
作为本发明的优选方式之一,所述CsHDH1基因编码的蛋白质序列如SEQ ID NO.2所示。
作为本发明的优选方式之一,所述基因工程途径指:
将SEQ ID NO.1所示CsHDH1基因片段连接至载体pCAMBIA1305.1,获得茶树表达载体pCAMBIA1305.1-CsHDH1;再将所述茶树表达载体pCAMBIA1305.1-CsHDH1转入EHA105农杆菌,利用农杆菌介导转入目标植株。
作为本发明的优选方式之一,通过烟草瞬时转化技术对CsHDH1基因的功能进行初步验证:将茶树表达载体pCAMBIA1305.1-CsHDH1,通过农杆菌介导将其在烟草中瞬时表达。与对照相比,在烟草中瞬时转化CsHDH1基因,显著降低了烟草中噻虫嗪的含量,同时随着时间的推移,其代谢产物噻虫胺的残留量也在逐渐减少。表明:瞬时表达CsHDH1基因能够增强目标植株对噻虫嗪及其代谢物噻虫胺的降解能力。
作为本发明的优选方式之一,所述CsHDH1基因表达产物用于正向调控目标植株的噻虫嗪降解能力。
一种上述通过改造茶树CsHDH1基因来提高噻虫嗪降解能力的方法在培育增强农药代谢能力的茶树新品种方面的应用。
原理:
本发明通过对噻虫嗪处理下的茶树代谢组数据和转录组数据进行分析,发现3-羟基异丁酸脱氢酶表达量随着处理时间的增加呈现显著正相关,同时代谢组的氨基酸类相关代谢途径也受到显著影响。随后,具体分析了茶树3-羟基异丁酸脱氢酶CsHDHs的多条基因的表达量随处理时间的变化情况,最终确定CsHDH1基因是在噻虫嗪解毒过程中可能起到重要作用的基因。接着,从茶树中首次克隆并验证了CsHDH1基因,通过对CsHDH1基因进行茶树原生质体亚细胞定位分析发现定位于细胞质,通过农杆菌介导的烟草瞬时转化技术,进一步证明了CsHDH1基因可以提高/增强植物对新烟碱类杀虫剂噻虫嗪的代谢降解能力。
本发明相比现有技术的优点在于:本发明研究中筛选到了对茶树“噻虫嗪降解能力”具有正向调控作用的CsHDH1基因,通过基因工程途径使茶树3-羟基异丁酸脱氢酶CsHDH1基因过表达,能够获得具有更强“噻虫嗪降解能力”的茶树植株,为培育增强农药代谢能力的茶树新品种提供理论基础和基因资源。
附图说明
图1是实施例2中茶树3-羟基异丁酸脱氢酶基因CsHDH1组织特异性表达模式分析图;
图2是实施例2中茶树3-羟基异丁酸脱氢酶基因CsHDH1表达谱;
图3是实施例3中PCR产物与载体pCAMBIA1305.1的酶切图(图中,A图为CsHDH1 PCR扩增产物;B图为CsHDH1构建连接pEASY-Blunt载体后质粒双酶切验证产物;C图为CsHDH1构建pCAMBIA1305.1载体后质粒双酶切验证产物;DNA分子量标准DL2000 bp/DL10000 bp);
图4是实施例4中激光共聚焦显微镜扫描结果图(图中,a、e图显示GFP绿色荧光单通道;b、f图显示叶绿体自发红色荧光单通道;c、g图显示明场;d、h图显示三通道共同显示图像;pCAMBIA1305.1空载体为对照);
图5是实施例5中烟草瞬时表达CsHDH1对噻虫嗪降解功能验证图(图中,A图为烟草瞬时表达CsHDH1对X剂量噻虫嗪的降解能力,推荐剂量:0.015kg a.i./ha;B图为烟草瞬时表达CsHDH1对2X剂量噻虫嗪的降解能力,2倍剂量:0.03kg a.i./ha;C图为烟草瞬时表达CsHDH1对3X剂量噻虫嗪的降解能力,3倍剂量:0.045kg a.i./ha)。
具体实施方式
下面对本发明的实施例作详细说明,本实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。同时,以下实施例中的所使用的试剂产品及实验方法,未经特别说明的,均为本领域常规试剂或方法,不再赘述。
实施例1、CsHDH1基因的筛选:
通过课题组前期对噻虫嗪处理下的茶树代谢组数据和转录组数据进行分析,发现3-羟基异丁酸脱氢酶表达量随着处理时间的增加呈现显著正相关,同时代谢组的氨基酸类相关代谢途径也受到显著影响。随后,具体分析了茶树3-羟基异丁酸脱氢酶CsHDHs各基因的表达量随处理时间的变化情况,最终确定CsHDH1基因是在噻虫嗪解毒过程中可能起到重要作用的基因。
其中,CsHDH1基因的具体核苷酸序列如SEQ ID NO.1所示,编码的蛋白质序列如SEQ ID NO.2所示。
实施例2、茶树CsHDH1组织特异性表达模式分析:
CsHDH1基因在茶树芽、花、果实、嫩叶、成熟叶、老叶、根、茎八个组织器官中的表达模式如图1所示。图1结果表明:CsHDH1在茶树根部特异高表达。
另外,图2通过分析茶树的转录组数据表明,与对照组(茶树未喷施噻虫嗪的空白组)相比,在噻虫嗪处理下,随着处理时间的增加,CsHDH1的表达量呈现出显著的正相关。
实施例3、CsHDH1基因的克隆与序列结构分析:
选择种植于安徽农业大学农萃园国家级良种“舒茶早”,取幼嫩根用于RNA的提取。采用RNAprep Pure Plant Kit(Tiangen,Beijing,China)试剂盒提取总RNA,具体步骤按照说明操作,用紫外分光度计测定其RNA含量和质量。
采用One-Step gDNA Removal and cDNA Synthesis SuperMix(TransGen Biotech,Beijing,China)试剂盒,将RNA反转录成cDNA。
根据3-羟基异丁酸脱氢酶基因CsHDH1的编码序列设计引物,并在引物上分别加上限制性酶切位点SpeI和BamHI。其中,上游引物为:5’-GGACTAGTATGTCTTCTCTCATCCCACAACTC-3’,下游引物为:5’-CGCGGATCC GACCAAAGTGGAACAAGACGAG-3’。
以cDNA第一链作为RT-PCR模板,常规方法做PCR,对茶树3-羟基异丁酸脱氢酶基因CsHDH1进行扩增。其中,50μL PCR反应体系为“Autoclaved,distilled water:(33-X)μL,10×PCR Buffer for KOD-Plus-Neo:5μL,2mM dNTPs:5μL 25mM MgSO4:3.0μL,上、下游引物各1.5μL,KOD-Plus-Neo(1U/μL):1μL,模板1μL”。反应程序如下为:98℃10sec,98℃10sec,57℃30sec,72℃2min,72℃10min,35个循环。
PCR产物CsHDH1基因经纯化回收后,连接到pEASY-Blunt Simple Cloning Vector载体(TransGen Biotech,Beijing,China)上得到pEASY-Blunt-CsHDH1质粒,转化大肠杆菌感受态细胞Trans1-T1,送生工公司测序,核苷酸序列如序列表SEQ ID NO.1所示。
对测序结果正确的基因序列进行限制性内切酶酶切后,将CsHDH1基因同载体pCAMBIA1305.1进行连接,构建表达重组质粒pCAMBIA1305.1-CsHDH1。
将构建的表达载体转化至将重组质粒化转至大肠杆菌感受态细胞Trans1-T1中,构建重组菌。
挑取若干阳性重组菌,提取质粒利用限制性内切酶进行酶切鉴定(结果如图3所示)。
实施例4、茶树CsHDH1的亚细胞定位:
为了进一步明确CsHDH1的亚细胞分布,本实施例对茶树CsHDH1进行了茶树原生质体亚细胞定位试验。首先将上述构建的CsHDH1亚细胞定位载体pCAMBIA1305-CsHDH1提取质粒,同时提取茶籽苗幼嫩叶片质体细胞,利用PEG转化方法将空载体pCAMBIA1305.1和pCAMBIA1305.1-CsHDH1表达载体质粒转入舒茶早原生质体中,转化完成后,暗培养24h,利用激光共聚焦显微镜观察GFP荧光记录并拍照。
图4是激光共聚焦显微镜观察结果图,图中,(a)(e)显示GFP绿色荧光单通道;(b)(f)显示叶绿体自发红色荧光单通道;(c)(g)显示明场;(d)(h)显示三通道共同显示图像。
由图4可知:空载体pCAMBIA1305.1的绿色荧光信号充满整个原生质体细胞,但没有特异的亚细胞定位区域,而pCAMBIA1305.1-CsHDH1的绿色荧光信号仅在茶树原生质体的细胞质中富集,未检测到其他部位发出荧光信号,研究结果初步明确CsHDH1定位于细胞质。
实施例5、瞬时表达CsHDH1基因在植株中降解噻虫嗪能力:
由于烟草瞬时转化技术为本领域较为成熟的技术,下面以烟草为例,进一步验证本发明瞬时表达CsHDH1基因在植株降解噻虫嗪能力的影响。
把pCAMBIA1305.1-CsHDH1载体转化到EHA105农杆菌当中,并通过常规PCR方法鉴定阳性克隆。挑取菌落PCR验证正确的单克隆,接种于5mL液体LB培养基(含50μg/mL rif和100μg/mL Spec),培养至OD600=0.8~1.2。取1mL过夜培养的农杆菌菌液,接入50mL液体LB培养基含(50μg/mL rif和100μg/mL Spec),28℃200r/min培养1~2d。5000r/min离心5min收集菌体,用10mM MgCl2和10mM 2-(N-吗啉代)乙磺酸调节pH为5.6的重悬液重悬菌体;再用MMA重悬菌体至菌液OD值为0.6~0.8,在菌液中加入乙酰丁香酮(每1mL菌液加入1μL AS溶液),室温孵育2h。选取长势良好的烟草,用一次性注射器将菌液注入烟草叶片背面,做好标记;注射后的烟草暗处理后,使用激光共聚焦显微镜观察基因是否在烟草中瞬时表达。
确定CsHDH1在烟草中瞬时表达后,对烟草进行不同浓度X(推荐剂量:0.015kga.i./ha)、2X(2倍剂量:0.03kg a.i./ha)和3X(3倍剂量:0.045kg a.i./ha)的噻虫嗪标准品(Dr.Ehrenstorfer,99.8%)喷施处理,取喷药后36h、48h和72h的烟草叶片,仅选择注射叶片制备样品。
将烟草叶样品在研钵中研磨、粉碎。称取粉碎后的茶鲜叶样品0.5g,置于50mL离心管中。加入5mL乙腈,超声提取10min。称取氯化钠(0.5g)和无水硫酸镁(0.5g)于试管中,涡旋2min。然后将离心管在室温下以5000r/min离心5min。取2mL上清液于事先盛有50mgPVPP,5mg PSA,20mg GCB,10mg C18和30mg无水硫酸镁的试管中。将提取液涡旋混合2min,然后以10000r/min离心10min。取上清液1mL氮吹至近干。残渣用1mL乙腈:水(15:85,v/v)复溶,过0.22μm的滤膜,待超高效液相色谱-串联质谱分析(UPLC-MS/MS)。
图5是烟草瞬时表达CsHDH1对噻虫嗪降解功能的验证;其中,TMX-CK-X指“烟草未瞬时表达CsHDH1基因、但喷施了X剂量噻虫嗪(TMX)农药的情况下,TMX对应残留量”;TMX-CsHDH1-X指“烟草瞬时表达CsHDH1基因后喷施X剂量噻虫嗪(TMX)农药的情况下,TMX对应残留量”;ClO-CK-X指“烟草未瞬时表达CsHDH1基因、但喷施了X剂量TMX的情况下,TMX代谢生成的噻虫胺(CLO)含量(CLO是TMX的代谢产物);CLO-CsHDH1-X指“烟草瞬时表达CsHDH1基因后喷施X剂量TMX农药的情况下,TMX代谢生成的CLO含量”;其他以此类推。
如图5所示,当处理浓度为X(推荐浓度:0.015kg a.i./ha)时,瞬时转化CsHDH1的烟草对噻虫嗪的降解最明显。在该浓度处理下,瞬时转化CsHDH1的烟草植株在36h、48h和72h时对噻虫嗪的降解率分别为69.1%、64.4%和59.6%。同时,噻虫胺残留量也在逐渐降低,推测可能是CsHDH1在烟草中瞬时转化后对噻虫胺也有一定的降解能力。在3X(3倍剂量:0.045kg a.i./ha)浓度处理下,发现在36h时瞬时转化烟草CsHDH1中的噻虫嗪降解了55.2%。
以上结果说明:瞬时表达CsHDH1的烟草对噻虫嗪具有较强的降解能力,并且在36h时对噻虫嗪的降解效率最高。根据以上结果我们初步推测“瞬时表达CsHDH1”可以增强植株对噻虫嗪的降解能力。当具体用于茶树时,只需将表达载体pCAMBIA1305.1-CsHDH1转入EHA105农杆菌,利用农杆菌介导转入茶树植株即可。本发明为利用分子辅助育种增强农药代谢能力的茶树新品种提供理论基础和功能基因资源。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (6)
1.一种通过改造茶树CsHDH1基因来提高噻虫嗪降解能力的方法,其特征在于,通过基因工程途径使茶树3-羟基异丁酸脱氢酶CsHDH1基因过表达;其中,CsHDH1基因的核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的通过改造茶树CsHDH1基因来提高噻虫嗪降解能力的方法,其特征在于,所述CsHDH1基因编码的蛋白质序列如SEQ ID NO.2所示。
3.根据权利要求1所述的通过改造茶树CsHDH1基因来提高噻虫嗪降解能力的方法,其特征在于,所述基因工程途径指:
将SEQ ID NO.1所示CsHDH1基因片段连接至载体pCAMBIA1305.1,获得茶树表达载体pCAMBIA1305.1-CsHDH1;再将所述茶树表达载体pCAMBIA1305.1-CsHDH1转入EHA105农杆菌,利用农杆菌介导转入目标植株。
4.根据权利要求3所述的通过改造茶树CsHDH1基因来提高噻虫嗪降解能力的方法,其特征在于,通过烟草瞬时转化技术对CsHDH1基因的功能进行初步验证:将茶树表达载体pCAMBIA1305.1-CsHDH1,通过农杆菌介导将其在烟草中瞬时表达。
5.根据权利要求1~4任一所述的通过改造茶树CsHDH1基因来提高噻虫嗪降解能力的方法,其特征在于,所述CsHDH1基因表达产物用于正向调控目标植株的噻虫嗪降解能力。
6.一种如权利要求1~5任一所述的通过改造茶树CsHDH1基因来提高噻虫嗪降解能力的方法在培育增强农药代谢能力的茶树新品种方面的应用。
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