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Volume 32 Issue 2
Jul.  2019
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Construction of the Expression Vector and RNAi Mediated by Bacteria Expressed dsRNA of Chitinase Gene from Hyphantria cunea

  • Corresponding author: ZHANG Zhen, zhangzhen@caf.ac.cn
  • Received Date: 2017-11-20
    Accepted Date: 2018-02-05
  • Objective To explore the feasibility of dsRNA expressed by HT115-mediated RNAi in Hyphantria cunea. Method Hyphantria cunea chitinase gene (HcChi) was chosen as the target gene. An interference fragment was designed and inserted into the expression vector L4440, and then transformed into the Escherichia coli strain HT115. The H. cunea larvae were fed on bacteria solution which was induced by IPTG. The growth of larvae were observed, and the mRNA level of HcChi was detected using qPCR. Result The HT115 strain containing HcChi-L4440 expression vector can express HcChi-dsRNA by IPTG induction. After fed on bacteria solution, the expression level of HcChi in H. cunea decreased significantly by 76.7%-90.3%. The body weight growth decreased about 40.7% compared with the control. Conclusion The RNA interference vector was constructed successfully. The effect of RNAi was observed in H. cunea by feeding method. This is the first time that developing a method of RNAi based on bacteria expressed dsRNA in H. cunea, which provides a new idea on the study of gene function and biological control of H. cunea.
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Construction of the Expression Vector and RNAi Mediated by Bacteria Expressed dsRNA of Chitinase Gene from Hyphantria cunea

    Corresponding author: ZHANG Zhen, zhangzhen@caf.ac.cn
  • Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing 100091, China

Abstract:  Objective To explore the feasibility of dsRNA expressed by HT115-mediated RNAi in Hyphantria cunea. Method Hyphantria cunea chitinase gene (HcChi) was chosen as the target gene. An interference fragment was designed and inserted into the expression vector L4440, and then transformed into the Escherichia coli strain HT115. The H. cunea larvae were fed on bacteria solution which was induced by IPTG. The growth of larvae were observed, and the mRNA level of HcChi was detected using qPCR. Result The HT115 strain containing HcChi-L4440 expression vector can express HcChi-dsRNA by IPTG induction. After fed on bacteria solution, the expression level of HcChi in H. cunea decreased significantly by 76.7%-90.3%. The body weight growth decreased about 40.7% compared with the control. Conclusion The RNA interference vector was constructed successfully. The effect of RNAi was observed in H. cunea by feeding method. This is the first time that developing a method of RNAi based on bacteria expressed dsRNA in H. cunea, which provides a new idea on the study of gene function and biological control of H. cunea.

  • RNA干扰(RNAi)是由双链RNA(dsRNA)引发的内源特异性基因转录后沉默机制[1]。dsRNA导入生物体内后,被细胞中称为Dicer的RNase Ⅲ分解成21~23 bp的小干扰RNA (siRNA),siRNA在沉默复合体(RISC)的作用下与目标mRNA结合,序列特异性地降解靶mRNA,阻止相应蛋白产物的合成,造成靶标基因的功能丧失[2]。自从这种快速且直接沉默特定基因的机制被发现以来,RNAi技术迅速在非模式动物的基因功能研究领域得到广泛应用[3]。在多种农林害虫中的成功试验[4-6],也证实了利用RNAi进行害虫防治的可能性和广泛性。

    RNAi技术在害虫防治领域的应用需要大剂量dsRNA,目前最常用的试剂盒合成方法价格昂贵、不能持续生产。利用转基因寄主植物表达昆虫源dsRNA对害虫有一定防效[4, 7],但植物转基因难度高、研究周期长,具有很大的局限性。用细菌表达dsRNA的方法,最早由Timmons和Fire开创使用,他们将带有靶基因片段的L4440重组载体转入BL21(DE3)菌株合成dsRNA,饲喂线虫引发RNAi[8]。随后他们从多个菌株中筛选出具有高dsRNA表达效率的HT115菌株,并在线虫中得到应用[9]。RNaseIII是细菌中普遍存在的dsRNA特异性核酸内切酶,由rnc基因编码,HT115菌株为rnc-突变不能降解自身合成的dsRNA[10]。HT115经过修饰,插入一个λDE3噬菌体衍生物,可通过IPTG诱导表达T7 RNA聚合酶[11]。HT115的四环素和Amp抗性,可在培养过程中分别排除rnc+反突变体和空载质粒。L4440载体序列的多克隆位点两侧各有一个相反方向的T7聚合酶启动子,转入HT115菌株后,可经IPTG诱导表达dsRNA[8]。在玉米上喷施表达菌液可抑制花叶病毒的感染[12],饲喂或注射细菌表达的dsRNA也能显著干扰甜菜夜蛾[13]、非洲甘薯象鼻虫[14]等害虫的生长,证明该技术在植物保护方面的应用潜力。

    美国白蛾(Hyphantria cunea(Drury))是我国重要检疫性害虫,其幼虫取食范围超过175种植物,包括几乎所有的园林树木、花卉及农作物[15]。美国白蛾繁殖力和传播能力强[16],适生范围广,严重威胁我国生态安全。目前对该害虫的防治手段主要有人工防治、化学防治和利用寄生天敌及性诱剂的生物防治等,这些手段具有一定的防治效果,但总体存在效率低、成本高、对生态环境不友好的问题[17],因此RNA干扰这种靶标性强、环境友好型的技术在害虫防控领域展现较大的应用潜力。几丁质主要存在于昆虫的表皮、中肠围食膜(占4%~20%)中,形成昆虫的外骨骼,起支撑和保护作用[18]。几丁质酶是几丁质水解的关键酶,在昆虫的变态发育中参与新旧表皮的更替和围食膜的降解,选择几丁质酶基因作为RNA干扰靶标,破坏昆虫几丁质表达通路,阻止昆虫的蜕皮、围食膜再生等生理过程,可达到害虫防治的目的[19]。本文以L4440质粒为载体,构建了利用大肠杆菌HT115表达美国白蛾几丁质酶dsRNA的体系,通过对美国白蛾幼虫饲喂细菌表达的dsRNA,检测该方法介导的RNAi在美国白蛾中的可行性。

1.   材料与方法
  • 菌株与载体:大肠杆菌HT115(DE3)由中国林科院资源昆虫研究所杨璞副研究员提供;L4440质粒购自美国Addgene机构。DH5α感受态细胞和T4 DNA连接酶购自北京天根生化科技公司,pMD 19-T质粒购自宝生物(大连)公司。

    试虫:供试美国白蛾幼虫由中国林科院昆虫病毒研究中心提供,饲喂人工饲料,全气候温室培养:温度25±1℃,光周期14L:10D,相对湿度60%80%。

  • 美国白蛾四龄幼虫个体总RNA提取参照RNeasy Mini Kit(QIAGEN)说明书进行,GoScript Reverse Transcription System(Promega)试剂盒反转录得到cDNA第一条链,该产物可作为HcChi基因扩增的模板用于后续实验。根据NCBI公布的美国白蛾HcChi基因序列全长(GenBank: U86877.1)[20],用Primer Premier 5.0软件设计扩增RNA干扰片段的引物序列,并在正反引物两端各加入一种酶切位点用于后续双酶切步骤。dsRNA目的片段大小为388 bp,位于几丁质酶基因编码区的前部,干扰该片段的转录可影响HcChi的转录及表达,在本实验室的前期试验中,已证明该dsRNA片段能显著抑制HcChi的表达。图 1展示了dsRNA及qPCR试验基因片段在几丁质酶基因中的位置示意。

    Figure 1.  Schematic diagram of dsRNA and qPCR site in HcChi gene

    F1: 5’-ATTTGCGGCCGCCACGCATCTCATCTACTCA-3’(下划线为Not Ⅰ酶切位点)

    R1: 5’-CGGCTAGCCGAACCTTTACCGACCCT-3’(下划线为Nhe Ⅰ酶切位点)

  • 以上述cDNA为模板,F1/R1引物进行PCR扩增,反应条件:95℃ 3 min预变性,95℃ 30 s,58℃ 30 s,72℃ 60 s,40个循环,72℃ 5 min终延伸。PCR产物按Omega公司试剂盒说明切胶回收,回收片段与pMD 19-T载体(TaKaRa)连接,连接产物转化至DH5α感受态细胞后,涂布于加有Amp、IPTG、Xgal(均购自天根公司)的固体LB培养基平板上,37℃过夜培养,挑取白色单菌落于含Amp的LB液体培养基中培养,菌液PCR筛选阳性克隆,确认阳性克隆取菌液交由上海生工公司测序。

    构建好的重组质粒HcChi-pMD 19T与L4440载体分别同时用Nhe Ⅰ和Not Ⅰ(购自NEB)进行双酶切,分别回收酶切产物。用T4连接酶将回收的HcChi基因片段和L4440质粒16℃过夜连接(反应体系:HcChL片段1 μL、L4440质粒7 μL、10× Buffer 1 μL、T4 DNA连接酶1 μL),连接产物命名为HcChi-L4440。将该重组干扰载体转入DH5α感受态细胞,涂布于含Amp的固体LB培养基平板上,37℃过夜培养,挑取白色单菌落于LB液体培养基中培养,菌液PCR及测序筛选阳性克隆。干扰表达载体构建过程如图 2所示。

    Figure 2.  Construction of RNA interference vector of HcChi gene

  • 将-80℃保存的HT115菌液在LB固体培养基中进行划线过夜培养,挑取单菌落转入LB液体培养基,37℃振荡培养过夜。以1: 10比例将菌液转入新的LB培养基中,继续培养至菌液OD600=0.3~0.5后置于冰上10 min,待菌液冷却后4℃ 5 000 r·min-1离心收集细胞沉淀。加入预冷的0.1 mol·L-1 CaCl2-MgCl2溶液重悬,冰浴30 min,4℃ 5 000 r·min-1离心收集细胞沉淀,加入预冷的0.1 mol·L-1 CaCl2溶液重悬,得到HT115感受态细胞,用于后续转化。

    用热激法将HcChi-L4440表达载体转入制备好的HT115感受态细胞中,涂布于LB固体培养基上,过夜培养后挑取单菌落接种到含50 mg·mL-1 Amp和12.5 mg·mL-1四环素的LB液体培养基中,菌液PCR筛选含有重组质粒的表达。

  • 将带有HcChi-L4440的HT115菌液接种于Amp和四环素抗性的LB液体培养基中,37℃过夜振荡培养,1:100比例将菌液转入新的LB培养基中,37℃,200 r·min-1培养至菌液OD600=0.6左右,加入IPTG终浓度至1.0 mmol·L-1进行诱导表达,相同条件继续培养4 h后收集菌液。参照细菌总RNA提取试剂盒(天根)说明书提取细菌总RNA,1%琼脂糖凝胶电泳检测RNA完整性。RNase A(天根)消化总RNA,去除单链RNA,获得HcChi-dsRNA。

  • 取180头正常饲养至2龄的美国白蛾幼虫,均分为两组。处理组饲喂含有经IPTG诱导后的HT115菌液浓缩液的人工饲料,对照组的人工饲料中添加未经过IPTG诱导的菌液,每天更换新鲜饲料和菌液,连续饲喂直到化蛹。饲喂后第5天和第10天,每组随机取4头幼虫分别提取总RNA,qPCR检测HcChi基因的表达变化,并持续监测幼虫的生长状态。HcChi基因和内参基因Actin的qPCR引物如下:

    F2: 5’-TCGGTCGTTCACTTTAGCAG-3’

    R2: 5’-TTTGTAAGCGTAGGGGCAT-3’

    Actin-F: 5’-GGTTACTCTTTCACCACCACAG-3’

    Actin-R: 5’-GGACTTCTCAAGGGAACTGC-3’

    qPCR试验在定量PCR仪(Roche)上进行,采用2-ΔΔCt方法分析结果。

  • 运用SPSS统计分析软件进行数据处理。统计美国白蛾取食细菌表达的dsRNA后不同时间HcChi基因表达水平的差异性(平均值+标准误),并进行单因素方差分析(n=5),显著性检验水平P<0.05。

2.   结果与分析
  • 提取4龄美国白蛾幼虫总RNA,反转录为cDNA,以F1/R1引物进行PCR扩增,扩增片段电泳结果显示介于250~500 bp之间(图 3 A),与预期大小388 bp相符。回收目的条带与pMD 19-T载体连接,命名为HcChi-pMD 19-T重组质粒,转入DH5α菌株并测序验证。

    Figure 3.  Clonging of RNAi fragment and construction of RNAi vector of HcChi

  • 用Nhe Ⅰ和Not Ⅰ分别双酶切HcChi-pMD 19-T和L4440质粒,凝胶电泳回收目的DNA片段和目标载体片段(图 3 B)。T4 DNA连接酶将HcChi基因片段与L4440连接,并转入DH5α感受态细胞,筛选阳性克隆,进行PCR和测序验证(图 3 C)。测序正确的菌株提取质粒HcChi-L4440,转入大肠杆菌HT115感受态细胞中,平板筛选,菌液PCR(图 4)和测序双重验证获得表达菌株。

    Figure 4.  PCR identification of HT115 expression strain

    HT115菌株中提取的重组质粒HcChi-L4440测序结果与原始序列用MEGA7.0软件进行比对,相似度99.23%(图 5),表明美国白蛾几丁质酶基因dsRNA片段成功转入L4440质粒中,与预期设计一致。

    Figure 5.  Sequences comparation of HcChi-L4440 recombinant vector

  • 为检测持续饲喂细菌表达的dsRNA后是否会引发美国白蛾幼虫HcChi基因的沉默,我们分别在饲喂第5天和第10天取样进行荧光定量PCR检测。结果显示(图 6),饲喂5天和10天后HcChi基因mRNA水平相对表达量分别下降了90.3 %和76.7 %,表明饲喂表达dsRNA的细菌可显著抑制美国白蛾幼虫HcChi基因的表达。

    Figure 6.  Expression levels of HcChi after feeding dsRNA expressed by HT115

    美国白蛾幼虫取食表达dsRNA的细菌后,生长发育显著滞后于对照。饲喂10天、15天、20天后,幼虫平均体重分别为63.5±5.30 mg、76.2±12.92 mg和45.9±7.69 mg,与对照相比分别减小了19.0%、36.2%和40.7%,且均有显著差异(P<0.05)(图 7)。图 7显示,饲喂试验20天后,对照组和处理组美国白蛾幼虫体重均显著下降,这可能由于试验用美国白蛾种群长期在室内无菌环境下人工饲养,对添加进人工饲料的HT115大肠杆菌敏感性较高,加之连续高浓度的菌液饲喂,影响了幼虫的取食消化,造成试验后期体重显著下降的现象。但与对照相比,取食经IPTG诱导生产HcChi-dsRNA的幼虫,在整个饲喂试验中都表现出较差的生长状态,该结果表明,随着饲喂天数的增加,美国白蛾幼虫受细菌表达的dsRNA干扰效应累加,导致取食困难、生长减缓、体重下降。

    Figure 7.  The effect of feeding dsRNA expressed by HT115 on the growth of H.cunea larvae

3.   讨论
  • 鳞翅目是昆虫纲中的第三大目,多数种类的幼虫为害农林植物。鳞翅目昆虫属全变态型,体内几丁质的正常合成和降解是昆虫生长发育的关键,因此与之相关的酶类成为利用生物技术防治害虫的重要研究方向[21]。RNAi技术的快速发展,使研究者更多地利用该方法研究昆虫几丁质酶系的功能,并试图得到新的害虫防治策略。通过RNAi对赤拟谷盗10个几丁质酶基因功能进行分析,发现TcCht5、TcCht10和TcCht7分别在虫态转化和翅原基的发育过程中起到关键作用,说明不同几丁质酶基因具有不同的生物学功能[22]。云杉卷叶蛾中有两种几丁质脱乙酰基酶CfCDA2的剪切体,幼虫期及预蛹期分别注射相应dsRNA均使其丧失蜕皮功能,导致畸形和高死亡率[23]。分别用注射和细菌表达几丁质合成酶A dsRNA处理甜菜夜蛾,可干扰幼虫蜕皮、破坏气管及中肠结构,导致其生长异常、死亡率显著提高[24-25]。上述研究表明与几丁质代谢相关的酶类为害虫防治研究提供了合适的靶标基因,具有很大的研究潜力。

    dsRNA导入昆虫体内的主要方式包括注射法[26]和饲喂法[27]等。微注射法可将精确定量的dsRNA注入昆虫血腔中,但会对昆虫造成不可逆的损伤,多用于实验室内的基因功能分析。饲喂法操作简单、对昆虫无机械损伤,昆虫可直接取食dsRNA溶液[27]、转基因植物[4]、纳米材料结合的dsRNA[6]和可表达dsRNA的细菌[14]等。其中,利用大肠杆菌表达dsRNA成本低廉、操作相对简便,可用于大规模的靶标基因筛选和干扰试验[28]。该方法中的表达载体一旦构建成功,可长期保存或利用注射和饲喂法研究昆虫的基因功能。王根洪等对家蚕注射细菌表达的FTZ-F1基因dsRNA,获得预期干扰表型,成功干扰了靶基因的表达[29]。石萌等构建了表达黄粉虫抗冻蛋白基因dsRNA的大肠杆菌HT115菌株,提纯后注射黄粉虫,对其抗冻蛋白基因的表达具有明显的抑制作用[30]。饲喂细菌表达的dsRNA对甘薯象鼻虫的毒性略低于注射,可能是由于dsRNA在其消化系统中被部分降解[14]。在小菜蛾、棉铃虫、大草蛉等鳞翅目害虫中,该方法也用于基因功能的研究[31-33]

    本研究利用大肠杆菌表达的几丁质酶dsRNA持续饲喂美国白蛾幼虫,没有造成幼虫蜕皮过程中的畸形表型,以及发育后期化蛹率和死亡率的显著降低,但与对照相比,幼虫的取食量显著减小,影响了幼虫的正常发育。这可能由于幼虫摄取混有dsRNA的人工饲料后,经消化系统转运到中肠,由于昆虫围食膜具有选择透过性,允许酶和小分子的通过[34],因此dsRNA可通过围食膜进入中肠细胞,导致中肠细胞中几丁质合成通路紊乱,影响围食膜的再生,从而降低围食膜的保护和吸收功能,使幼虫不能正常消化食物或更容易被食物中的病毒感染,最终导致幼虫的生长减缓。而在多种鳞翅目昆虫肠道中都发现一种专性降解dsRNA的核酸酶dsRNase[35-37],这种酶的存在能快速降解进入肠道的大部分dsRNA,因此除了作用在中肠及围食膜细胞上,可能仅有极少量的HcChi-dsRNA能经过组织间的转运作用于幼虫表皮,不能反应在个体水平的蜕皮过程中,这或可解释没有造成幼虫蜕皮过程中的畸形表型。干扰褐飞虱几丁质酶家族中10个基因的表达,其中有5个基因未出现明显的形态、存活率异常,可能由于这几个基因的功能与其它几丁质酶相比起辅助作用,当表达受到抑制时,其功能可被其它家族基因替代或弥补[38]。本研究中干扰HcChi基因表达后,可能刺激了其他相关基因的表达,弥补了该基因沉默对美国白蛾幼虫形态发育、存活能力的影响,因此未造成化蛹率和死亡率的显著降低。欧洲玉米螟中发现一种肠道特异性几丁质酶,饲喂幼虫dsRNA后,与对照相比幼虫围食膜上的几丁质含量增加26 %,体重减少54 %,说明几丁质含量的变化可能改变了围食膜的通透性,影响消化酶的转运和营养吸收[39]。用大肠杆菌表达的两种几丁质酶剪切体dsRNA饲喂粘虫(Mythimna separate(Walker))四龄幼虫,可导致其生长减缓、死亡率上升,但同样没有得到畸形表型[40]

4.   结论
  • 本研究构建了美国白蛾几丁质酶基因dsRNA的表达载体,在大肠杆菌HT115菌株中通过IPTG诱导表达dsRNA。对美国白蛾幼虫饲喂浓缩菌液,成功抑制几丁质酶基因在白蛾幼虫体内的表达,并导致幼虫生长发育迟缓。该体系在美国白蛾中首次建立,证实基于细菌表达dsRNA的策略能在美国白蛾中得到应用,也为美国白蛾的功能基因研究和生物防治提供了新的思路。同时,美国白蛾及其他昆虫几丁质酶基因的RNAi干扰机制尚不明确,具体机理还有待深入研究。

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