CN103497950B - 一种小分子rna及其在害虫防治中的应用 - Google Patents
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Abstract
本发明公开了一种小分子RNA及其在害虫防治中的应用。本发明提供了一种小分子RNASli-miR-2771,通过体内、体外试验证实Sli-miR-2771可通过结合解毒酶谷胱甘肽转移酶 slgste1 的3’非编码区,抑制 slgste1 的表达,使斜纹夜蛾减少对有毒的植物次生物质的解毒能力,进而影响斜纹夜蛾进食植物与生长状况,达到害虫防治的目的。该发明为害虫防治提供了新思路和新途径,在害虫防治领域中将具有广阔的应用前景。
Description
技术领域
本发明属于基因工程技术领域,特别涉及一种小分子RNA及其在害虫防治中的应用。
背景技术
小分子RNA(miRNA)是一类长度约21~24核苷酸非编码的小分子RNA,广泛存在于动、植物体中。一系列实验表明miRNA可能是一类在进化上保守的、在生物体中具有重要调控作用的分子。
miRNA源自细胞核内编码miRNA的基因转录成pri-microRNA,接着被Drosha酶剪切为长度约70 bp呈发夹状的pre-miRNA,随后被核质/细胞质转运蛋白从细胞核内转运到胞质中,之后被Dicer酶剪切成长为18-26bp的miRNA双螺旋复合体。其中一条解螺旋,并结合到RNA诱导的基因沉默的复合物内,形成非对称的RISC复合物。这个复合物通过其中的miRNA与靶mRNA的3’ UTR互补配对结合,从而降解靶目标mRNA或阻断其翻译,实现对靶标基因的负调控。近年来尽管有大量的miRNA被鉴定,但仅少数miRNA的功能被阐明。有文献报道,miRNA在基因表达、信号转导、植物发育、抵抗胁迫、预防疾病等方面起着至关重要的作用。因此,研究对昆虫抗药性相关基因具有沉默功能的miRNA,并将其应用于农业害虫的综合治理成为近年研究的热点。
谷胱甘肽转移酶是一种多功能的二相解毒酶,它广泛存在于动物与植物中,它通过催化还原谷胱甘肽与亲电性有毒物质的结合作用而转化为亲水性的无毒物质; 同时谷胱甘肽转移酶还具有过氧化物酶活性,通过催化还原谷胱甘肽与脂质过氧化中间或终端产物作用,而参与细胞内因农药和重金属等引起的氧胁迫,而增加生物体的耐性。但是,生物体中的谷胱甘肽转移酶是一大家族基因,不同的同工酶可能有不同的作用底物与酶活性,而且虽然动物与植物体内都有谷胱甘肽转移酶,但对不同种类有毒物质的降解作用会不一样。
斜纹夜蛾为杂食性昆虫,可危害多达290多种植物,特别是十字花科的经济作物,对经济收入有很大的影响。目前害虫的防治仍以化学杀虫剂为主,但大量而长期的使用不仅造成了各地害虫的抗药性,使杀虫剂的剂量使用越来越多,进而造成环境的污染和影响食品的安全。申请人从农业害虫斜纹夜蛾中筛选出高活性的编码谷胱甘肽转移酶的解毒基因,以其为靶标,研制出防治害虫的抑制剂,为斜纹夜蛾的防治提供新思路。
发明内容
本发明的目的在于提供一种小分子RNA。
本发明的再一个目的在于提供该小分子RNA在害虫防治中的应用。
申请人从斜纹夜蛾中获得了一小分子RNA,命名为Sli-miR-2771,其序列为:UAACAUUAUGAGGAUGGGUUGAACUG(SED ID NO.1)。通过生物信息学分析,预测Sli-miR-2771可能调控slgste1,并通过荧光素酶报告载体实验、细胞转染实验、虫体注射实验以及实时荧光定量PCR等多种实验技术,证实了Sli-miR-2771可通过结合slgste1的3’非编码区,抑制slgste1的表达。虫体体内实验更证实了Sli-miR-2771可以通过抑制slgste1的表达,使斜纹夜蛾减少了对有毒的植物次生物质的解毒能力,进而影响斜纹夜蛾进食植物与生长状况,达到害虫防治的目的。该发明方法为害虫防治提供了新思路和新途径,在害虫防治领域中将具有广阔的应用前景。
附图说明
图1为注射miRNAs后斜纹夜蛾的体重统计结果;
图2为注射miRNAs72h后斜纹夜蛾的外型观察结果;
图3为注射miRNAs后进食芥菜的斜纹夜蛾中slgste1的表达量检测结果;
图4为荧光素酶报告系统验证miRNAs与slgste13’UTR结合的实验流程图;
图5为miRNAs与slgste13’UTR结合实验的荧光素酶活性测定结果;
图6为细胞株添加miRNAs后吲哚-3-甲醇(I3C)对slgste1的诱导结果;
图7为miR-2771与slgste1 3’ UTR结合的计算机模拟图。
具体实施方式
申请人用植物源杀虫剂、有机磷杀虫剂和拟除虫菊酯类杀虫剂、以及有机氯类的DDT对农业重要害虫斜纹夜蛾进行诱导实验,发现了一个谷胱甘肽转移酶对这些处理都有着高表达。经测序发现该酶为基因库中号码为AY506545的谷胱甘肽转移酶,按分类原则,将其重新命名为slgste1。把slgste1基因进行体外蛋白表达,并对它的酶催化作用进行分析,结果表明,它对芥菜的两种主要次生物质吲哚-3-甲醇(非挥发的)和烯丙基异硫氰酸酯(气味成分)具有解毒作用,对其他的次生物质如花椒毒素也有作用。
申请人从斜纹夜蛾中获得了一小分子RNA,命名为Sli-miR-2771,其序列为:UAACAUUAUGAGGAUGGGUUGAACUG(SED ID NO.1)。通过生物信息学分析,预测miR-2771可能调控slgste1,并通过荧光素酶报告载体实验、细胞转染实验、虫体注射实验以及实时荧光定量PCR等多种实验技术,证实了miR-2771可以通过结合slgste1的3’非编码区,抑制slgste1的表达。虫体体内实验更证实了miR-2771可以通过抑制slgste1的表达,使斜纹夜蛾减少了对有毒的植物次生物质的解毒能力,进而影响斜纹夜蛾进食植物与生长状况。
下面结合具体实施例,进一步阐述本发明内容。
以下实施例中所用Sli-miR-2771 mimics(后续简写为miR-2771 mimics)和NC mimics(阴性对照mimics)由上海吉玛制药技术有限公司合成,稀释浓度为2 μg/μl。当然,本领域技术人员也可选择通过构建载体,表达后纯化获得Sli-miR-2771。
实施例1
选取大小一致、健康状况一致的五龄第一天斜纹夜蛾幼虫,称量每头虫子的重量,随机分为miR-2771 mimics处理组和NC mimics对照 组,每组40头,重复3次。顺血液循环流动方向,用WPI微量注射器从斜纹夜蛾幼虫的侧腹部分别注射miR-2771 mimics和NC mimics,注射量为每头虫4 μg,注射后饲喂芥菜。注射后第1、2、3天观察虫子的形态变化并拍照取证,每天统计虫子体重的变化,并随机取3条虫子提取RNA,荧光定量PCR检测slgste1 mRNA的表达。
图1显示注射miRNAs后斜纹夜蛾幼虫的体重统计结果、图2显示注射miRNAs 72h后斜纹夜蛾幼虫的外型观察结果,可见,注射miR-2771 mimics后喂食芥菜,斜纹夜蛾生长速度变慢,无论从体重还是外型上,注射miR-2771 mimics的斜纹夜蛾进食及生长速度显著低于NC对照组。
将虫子样品按Trizol 法抽提总RNA,测定浓度后取2 μg 总RNA 进行反转录(参考TAKARA的M-MLV RTase cDNA Synthesis Kit说明书进行),得到的cDNA 产物稀释后用于荧光定量PCR的模板。所用PCR引物对SlGSTe1-realtime-F和SlGSTe1-realtime-R的序列如下:
SlGSTe1- realtime-F GCTGAACTCCGCCTATGAAA (SED ID NO.2);
SlGSTe1- realtime-R CCAGAAGGGATGCTGATGATAC (SED ID NO.3)。
荧光定量PCR反应体系如表1所示:
表1 荧光定量PCR反应体系
cDNA模板 | 1 μL |
SYBR? Premix Ex Taq? (2×) | 10 μL |
ROX Reference Dye(50×) | 0.4 μL |
SlGSTe1- realtime-F(10 μM) | 0.4 μL |
SlGSTe1- realtime-R(10 μM) | 0.4 μL |
ddH2O | 7.8 μL |
Total | 20 μL |
荧光定量PCR反应程序如下:95℃预变性10 s; 95℃ 5 s,60℃ 31 s,40个循环。
结果分析:采用相对定量的方法(2?ΔΔCt,Livak & Schmittgen, 2001)来确定目标基因相对内标基因(GAPDH,HQ012003)的相对表达倍数。应用SPSS16.0统计分析软件,采用ANOVA(多个处理间的两两比较)或独立样本T检验(两个样品间比较)来进行处理间的差异比较分析。结果如图3所示,可见,注射miR-2771 mimics 后进食芥菜的斜纹夜蛾中肠slgste1的表达受到抑制。
实施例2
1) 构建改良的检测载体IE1-pGL3-UTR和IE1-pGL3:
a) 在pGL3载体的报告荧光素酶基因上游插入启动子IE1,并在其下游插入slgste1 3’非编码区序列,重组质粒转化到DH5α菌,构建得到检测载体IE1-pGL3-UTR(如图4中左图所示);
b) 在pGL3载体的报告荧光素酶基因上游插入启动子IE1,其下游未插入slgste1 3’非编码区序列,重组质粒转化到DH5α菌,构建得到检测载体IE1-pGL3;
2) 转染前一天,将生长状况良好的Spli-221细胞转入十二孔板,加入1 mL的10%FBS Grace’s Insect培养基26℃恒温培养箱中培养过夜,当细胞密度长至80%~95%时,进行转染操作;
3) 转染实验:
按表2进行分组转染实验,荧光素酶报告系统验证miR-2771与slgste1 3’UTR结合的实验流程图如图4所示。
表2 转染实验设计
处理组1 | 处理组2 | 处理组3 | 处理组4 | |
miR-2771 mimics | + | + | —— | —— |
NC mimics | —— | —— | + | + |
IE1-pGL3 | + | —— | + | —— |
IE1-pGL3-UTR | —— | + | —— | + |
注:+ 表示添加有对应组份,——表示未添加对应组份。
a) 取1.6 μg miR-2771 mimics/ NC mimics,分别用Opti-MEM稀释到100 μL,混匀;取6 μL Lipofectamine? 2000稀释于100 μL Opti-MEM,混匀,室温放置30min;
b) 将稀释的mimics分别与稀释的Lipofectamine(共200 μL)混匀,制得miR-2771 mimics/ NC mimics转染混合物,室温静置15min;
c) 去除步骤2)细胞培养皿中的完全培养基,用无血清(FBS)的Grace’s Insect培养基清洗细胞两次,再加入300 μL无FBS的Grace’s Insect培养基,然后按表1加入200 μL转染混合物,并加入对应的检测载体,将检测载体和miRNAs共转染至细胞中,轻轻混匀;
d) 26℃恒温培养8 h后,更换培养基为含10%FBS的Grace’s Insect 培养基,继续培养,同时用10-4 mol/L的芥菜主要次生物质吲哚-3-甲醇诱导细胞;
e) 按照Promega荧光素酶检测试剂盒说明书操作,检测不同处理组的荧光强度;并分别在转染24h、36h后提取RNA,定量PCR检测slgste1 mRNA水平的表达。
miRNAs与slgste1 3’UTR结合实验的荧光素酶活性测定结果如图5所示,NC表示NC mimics阴性对照组,细胞共转染无义miRNA;IE1-pGL3表示未加slgste1 3’UTR片段的检测载体;IE1-pGL3-UTR表示加slgste1 3’UTR的检测载体。可见,共转染miR-2771和IE1-pGL3-UTR的细胞,其荧光素酶的表达率较共转染NC mimics和IE1-pGL3-UTR的阴性对照下降约100%,差异显著;而共转染miR-2771和IE1-pGL3的细胞,其荧光素酶的表达率与共转染NC mimics和IE1-pGL3的阴性对照相比无显著影响。因此,实验表明添加miR-2771进入斜纹夜蛾细胞株后,miR-2771可能通过与slgste1 3’UTR靶向结合,抑制了报告荧光素酶(Luciferase)的表达。图6为slgste1 表达量的检测结果,可见,细胞株添加miR-2771后抑制了10-4 mol/L芥菜主要次生物质I3C对slgste1的诱导。以上结果说明,miR-2771是通过与解毒酶基因slgste1 3’UTR结合而起作用的,其计算机模型图见附图7。
实施例1、2体内及体外实验都表明,芥菜及其次生物质I3C (吲哚-3-甲醇)都可以显著的促进slgste1 基因的表达(如图3、6所示),但是加入miR-2771后,slgste1 基因的表达显著受到了抑制,与没加miR-2771的对照组相比,slgste1 基因表达量明显减少。说明miR-2771是通过降解slgste1 mRNA,使斜纹夜蛾减少了对有毒的植物次生物质的解毒能力,而影响斜纹夜蛾进食植物与生长的。
<110> 华南师范大学
<120> 一种小分子RNA及其在害虫防治中的应用
<130>
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 26
<212> RNA
<213> 人工序列
<400> 1
uaacauuaug aggauggguu gaacug 26
<210> 2
<211> 20
<212> DNA
<213> 人工序列
<400> 2
gctgaactcc gcctatgaaa 20
<210> 3
<211> 22
<212> DNA
<213> 人工序列
<400> 3
ccagaaggga tgctgatgat ac 22
Claims (1)
1.一种序列为UAACAUUAUGAGGAUGGGUUGAACUG的小分子RNA在害虫防治中的应用;所述害虫为斜纹夜蛾。
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