CN112175946B - 一种靶向Trypsin-9基因的miR-283-3p及其应用 - Google Patents

一种靶向Trypsin-9基因的miR-283-3p及其应用 Download PDF

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CN112175946B
CN112175946B CN202010614473.6A CN202010614473A CN112175946B CN 112175946 B CN112175946 B CN 112175946B CN 202010614473 A CN202010614473 A CN 202010614473A CN 112175946 B CN112175946 B CN 112175946B
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金丰良
许小霞
花艳艳
李树忠
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Abstract

本发明公开了一种靶向Trypsin‑9基因的miR‑283‑3p及其应用，所述miR‑283‑3p序列如SEQ ID NO:1所示，Trypsin‑9基因序列如SEQ ID NO:2所示。并验证了miR‑283‑3p负调控Trypsin‑9的表达，进一步利用SDS‑PAGE电泳检测了高表达和抑制miR‑283‑3p的表达后，中肠液对原毒素Cry1Ac的活化作用，明确了miR‑283‑3p靶向Trypsin‑9可介导原毒素Cry1Ac的活化作用，可应用miR‑283‑3p作为分子靶标来防控小菜蛾等十字花科蔬菜害虫。

Description

一种靶向Trypsin-9基因的miR-283-3p及其应用
技术领域
本发明涉及农业生物技术领域，更具体地，涉及一种靶向Trypsin-9基因的 miR-283-3p及其应用。
背景技术
微小RNA(miRNA，microRNA)是一类由基因组编码产生的21～24nt的非编码单链小分子RNA。在生物体中广泛存在，参与细胞增殖、分化、发育、代谢、凋亡等多种生理活动。它们由RNA聚合酶转录出初始转录本(pri-miRNA)，pri-miRNA在内切酶(Drosha)的剪切下形成70-100nt茎环结构的前体miRNA (pre-miRNA)，在Ran-GTP的介导下，转运蛋白Exportin5将前体miRNA转运到细胞质中，后进一步被内切酶Dicer剪切形成22nt的未成熟的双链RNA(imperfect miRNA)，再与具有Dicer酶和解螺旋酶活性的AgoⅠ蛋白结合，双链被裂开，一条被降解，而另一条形成成熟的miRNA后参与沉默复合体 (RNA-induced silencingcomplex，miRISC)的形成。通过碱基互补配对的方式和mRNA结合，在转录后水平对靶基因进行负调控从而降解mRNA的转录，或者抑制mRNA翻译成蛋白。
随着克隆和高通量测序技术和以及生物信息学的飞速发展，掀起了生命科学领域miRNA研究的研究热潮。目前鉴定miRNA靶基因的常用策略是利用生物信息学软件预测，如miRanda，TargetScan和TargetScanS，RNAhybrid， DIANA-microT，PicTar等。由于计算机模拟在预测miRNA靶基因时存在一定的局限性，利用生物学实验方法可以进一步证明miRNA与靶基因的调控关系。
目前鉴定miRNA与靶基因的关系。主要包括三最直接的方法是，利用荧光定量PCR的方法在mRNA水平上检测miRNA及mRNA的表达水平从而确定 miRNA与靶基因的对应关系。而miRNA的靶位点的鉴定最常用的方法是荧光素酶报告基因法。其基本原理是首先构建荧光素酶表达载体，将筛选的miRNA靶基因的片段构建到荧光素酶基因的3’UTR中，然后将荧光素酶基因表达质粒转染细胞，通过咋细胞水平上添加miRNA的抑制剂或模拟物。检测荧光素酶的活性水平反应转染的片段中是否含有miRNA的靶位点。而体内进一步鉴定靶基因的方法是，直接向生物体注射或者饲喂miRNA模拟物mimic或者抑制物inhibitor，后利用荧光定量PCR的方法检测靶基因的mRNA表达水平，来确定miRNA与靶基因的靶标调控关系。对棉铃虫幼虫饲喂ha-miR-2002b mimics使其过表达处理后发现其靶标基因Ha-TLP转录水平急剧下，同时幼虫的生长发育收到影响；相反饲喂miRNA抑制剂inhibitor后转录水平显著上调，说明miRNA通过靶向 Ha-TLP来调控棉铃虫的生长发育。
昆虫胰蛋白酶由中肠肠壁细胞产生分泌进人中肠，主要在取食期的幼虫中肠中被检测到，是昆虫碱性中肠环境中主要的一种丝氨酸蛋白酶(董育新等，1998)，在蛋白的消化和Bt毒素的毒性调节中起重要作用。通过寻找靶向昆虫胰蛋白酶的miRNA可以为农业昆虫的防治提供更多选择。
发明内容
本发明的目的在于克服现有技术中存在的上述缺陷和不足，提供一种靶向Trypsin-9基因的miR-283-3p。
本发明的第二个目的在于提供一种提高miR-283-3p表达的miRNA模拟物。
本发明的第三个目的在于抑制所述miR-283-3p表达的miRNA抑制剂。
本发明的第四个目的在于提供所述miR-283-3p和miR-283-3p抑制剂的应用。
本发明的上述目的是通过以下技术方案给予实现的：
一种靶向Trypsin-9基因的miR-283-3p，所述miR-283-3p序列如SEQ ID NO:1所示，Trypsin-9基因序列如SEQ ID NO:2所示。
本发明筛选了到了靶向昆虫胰蛋白酶Trypsin-9基因的miR-283-3p， miR-283-3p中22个核苷酸识别Trypsin序列中18个核苷酸，具体是靶向基因 Trypsin9的CDS序列，该序列如SEQ ID NO：6所示。当miR-283-3p的抑制剂 (inhibitor)添食小菜蛾后，该抑制剂能够显著抑制miR-283-3p的表达，显著提高靶标基因Trypsin-9在小菜蛾中肠中的表达，可显著提高小菜蛾中肠液对Bt前毒素的活化，显著提高苏云金芽孢杆菌Cry1Ac的活力及对小菜蛾的致死率。而 miR-283-3p的模拟物(mimic)添食小菜蛾后，该模拟物能显著抑制靶标基因Trypsin-9的表达，将添加miR-283-3p模拟物的中肠液与原毒素温育后，发现降低了苏云金芽孢杆菌Cry1Ac的活力。而且添食miR-283-3p模拟物可降低 Trypsin-9的表达，减弱了小菜蛾对营养蛋白的消化和吸收，进一步饲喂Cry1Ac 后发现小菜蛾死亡率显著降低。表明所述miR-283-3p负调控Trypsin-9基因的表达进而调控Bt毒蛋白活力及昆虫中肠对营养蛋白的消化和吸收，可进一步利用 miR-283-3p作为分子靶标防控十字花科蔬菜害虫。
本发明还提供一种提高所述miR-283-3p表达的miRNA模拟物，包括正义链和反义链，其序列依次如SEQ ID NO:3～4所示；
一种抑制所述miR-283-3p表达的miRNA抑制剂。
优选地，所述miRNA抑制剂其序列如SEQ ID NO:5所示。
本发明提供的miR-283-3p的模拟物和抑制剂能任意调控Trypsin-9的表达，进一步调控Cry1Ac的活力及其它营养蛋白的消化和吸收，为后期运用 miR-283-3p控制小菜蛾危害十字花科蔬菜提供分子靶标。
本发明还提供所述miR-283-3p在靶向负调控Trypsin-9基因表达进而调控 Bt毒蛋白活力及营养蛋白的消化和吸收中的应用。
因此本发明还提供所述miR-283-3p作为分子靶标在防治十字花科蔬菜害虫或在制备防治十字花科蔬菜害虫药剂中的应用。
或miR-283-3p抑制剂在防治十字花科蔬菜害虫或在制备防治十字花科蔬菜害虫药剂中的应用。
一种防治十字花科蔬菜害虫的方法，通过抑制害虫体内miR-283-3p的表达并施用Bt毒蛋白；抑制miR-283-3p可提高Trypsin-9基因的表达，提高Bt毒蛋白活力及害虫对营养蛋白的消化和吸收。
优选地，是通过施用上述的miR-283-3p抑制剂以抑制害虫体内miR-283-3p 的表达。
一种防治十字花科蔬菜害虫的药剂，包含用于抑制害虫体内miR-283-3p表达的制剂及Bt毒蛋白。
优选地，所述为上述的miR-283-3p抑制剂。
优选地，所述Bt毒蛋白为BtCry1Ac。
本发明还提供miR-283-3p在制备抗十字花科蔬菜害虫的转基因植物中的应用。通过将miR-283-3p的前体的抑制剂转进十字花科作物中，当鳞翅目害虫小菜蛾等危害十字花科蔬菜时，由于转进了miR-283-3p抑制剂，Trypisn-9的高表达，进一步活化Bt毒蛋白，提高了Bt的杀虫效果，达到预防小菜蛾目的。对于 Trypsin基因高度保守的对于其他害虫同样适用。
优选地，所述十字花科蔬菜害虫具有与SEQ ID NO:2所示Trypsin基因高度保守的鳞翅目害虫。
进一步优选地，所述害虫为小菜蛾。
与现有技术相比，本发明具有以下有益效果：
本发明提供了一种靶向Trypsin-9基因的miR-283-3p，所述miR-283-3p序列如SEQID NO:1所示，Trypsin-9基因序列如SEQ ID NO:2所示。所述miR-283-3p 负调控Trypsin-9的表达，进而调控Bt毒蛋白活力及昆虫中肠对营养蛋白的消化和吸收，提高害虫的死亡率，可应用所述miR-283-3p作为分子靶标来防控小菜蛾等十字花科蔬菜害虫。
附图说明
图1为本发明实施例中miR-283-3p靶向Trypsin-9基因的表达趋势图。
图2为本发明实施例中重组质粒psiCHECK-2-Trypsin-9构建图和酶切图。注：M1：分子量标记DL2000；1：PxTrypsin-9PCR扩增产物；2：psi-checck-2 空载体双酶切；3：psi-CHECK-2-PxTrypsin-9重组质粒双酶切；M2：M10000。
图3为发明实施例中荧光素酶活性图。注：miRNA模拟物与 psi-CHECK2-Trypsin9共转染48h后，使用HEK293T细胞进行双荧光素酶测定，验证miR-283-3p与靶基因Trypsin9的相互作用。作图数值为平均值±SEM(n＝3)，通过SPSS 25对数值进行独立样本T检验差异显著性分析。(**代表显著水平 p≤0.01)。
图4为本发明实施例中体内miR-283-3p抑制物与靶基因的对应关系验证。注：A：添食inibitor24、48、60h后miR-283-3p的表达分析，B：添食inibitor24、 48、60h后Trypsin9的表达分析。作图数值为平均值±SEM(n＝3)，通过SPSS 25对数值进行独立样本T检验差异显著性分析。(*代表显著水平p≤0.05，*** 代表显著水平p≤0.001)。
图5为本发明实施例中体内miR-283-3p模拟物与靶基因的对应关系验证。注：A：添食mimic 24、48、60h后miR-283-3p的表达分析，B：添食mimic 24、 48、60h后Trypsin9的表达分析。作图数值为平均值±SEM(n＝3)，通过SPSS 25对数值进行独立样本T检验差异显著性分析。(**代表显著水平p≤0.01， ***代表显著水平p≤0.001)。
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小菜蛾Trypsin-9基因的CDS片段的克隆
利用Trizol法提取小菜蛾中肠的总RNA，反转录合成cDNA的一条链，并以此为模板进行PCR克隆，包含全部CDS区域的Trypsin-9目的基因的引物序列为F： 5’-GCAGAGGATTGTGGGTGGAT-3’；R：5’-TTGACTCCAGGGTACTCGGC-3’ PCR产物用1％的琼脂糖凝胶电泳分离并回收纯化，经分析其序列大小为820bp。回收得到的PCR产物连接到pMD18-TVector，并将连接产物转化到感受态细胞 DH5α中，将阳性克隆菌株送去测序鉴定，其序列如SEQ ID NO:2所示。将测序鉴定正确的菌株以甘油菌保存于-80℃。
实施例2 miR-283-3p靶向Trypsin-9的预测
基于实验室测定的Cry1Ac处理后的小菜蛾中肠转录组RNA-Seq数据，初步筛选到了miR-283-3p，其核酸序列信息如SEQ ID NO:1所示，经miRanda， RNAhybrid和TargetScan三种软件预测了其结合位点，并以Trypsin-9特异性区域为引物F：5’-GTCTGCACCGCAGGAGAAAC-3’；R： 5’-TCCTCATGATGGCGAGGTCA-3’，qRT-PCR检测miR-283-3p靶向PxTrypsin-9 的预测，结果如图1所示，Cry1Ac原毒素诱导后miR-283-3p在24h和36h强烈诱导表达，而靶基因Trypsin-9则强烈下调。
实施例3细胞表达质粒的构建
上述获得cDNA为模板进行PCR克隆，包含预测靶位点的引物序列为F： 5’-CCCTCGAGACTTCCTCCCTGGATTGG-3’(下划线表示设计的XhoI酶切位点)；R：5’-GGGTTTAAACGGGGTCGGACGATTTTGA-3’(下划线表示设计的 PmeI酶切位点)，构建策略如图2左图所示。PCR产物用1％的琼脂糖凝胶电泳分离并回收纯化，回收得到的PCR产物与psiCHECK-2空载体用XhoI和PmeI同时进行双酶切，酶切产物连接到psiCHECK-2Vector并将连接产物转化到感受态细胞 DH5α中，抽提质粒XhoI/PmeI双酶切鉴定，酶切鉴定结果如图2右图所示，将测序正确的菌株以甘油菌的形式保存。
实施例4 293T荧光素酶活性测定
miR-283-3p模拟物mimic的合成：
模拟物正义链Mimic Sense：5’-GUCUCACGAUCAACCAUAGGU-3’；
模拟物反义链Mimic Anti-sense：5’-ACCUAUGGUUGAUCGUGAGACUU-3’；
将实施例3所制备的报告重组质粒和合成的miR-283-3p模拟物mimic与psiCHECK-2质粒共转染HEK293T细胞，转染前1天将HEK293T细胞按5×105的密度接种于48孔板，含10％胎牛血清培养基，置于5％CO2培养箱37℃过夜培养；将报告重组质粒和合成miR-283-3p mimic，以及piCHECK-2质粒分别转染HEK293T 细胞；转染24h后，加入PBS洗涤细胞，尽可能的弃净各孔内的残留液体。用双荧光素酶报告系统试剂盒检测各组荧光素酶活性。通过Dual-Luciferase报告基因检测系统测定各实验组中萤火虫荧光素酶的荧光值(FLuc)和海肾荧光素酶的荧光值(RLuc)，取F/R为相对活性值进行数据的统计。结果如图3所示， miR-283-3p能够识别Trypsin-9基因，并负调控Trypsin-9基因的表达。
实施例5活体内添食miR-283-3p抑制剂和模拟物
挑取长势一致的3龄的小菜蛾幼虫，将人工饲料/g与inhibitor和人工饲料/g与mimic比列为1:500μL(1μM)饲喂小菜蛾，12h更换一次饲料，用DEPC为对照。饲喂24，48h，60h后分别对小菜蛾对照组与处理组miR-283-3p inhibitor和 miR-283-3p mimic的中肠组织解剖，利用qRT-PCR检测miR-283-3p及其靶基因 Trypsin-9的转录水平。结果如图4和图5所示。由图4可知小菜蛾添食miR-283-3p 的inhibitor后，qRT-PCR检测发现添食miR-283-3p24h、48h、60h后，miR-283-3p 表达量显著降低(图4A)；同时Trypsin-9的表达在60h显著高于对照(图4B)。由图5可知小菜蛾添食miR-283-3p的mimic后，qRT-PCR检测miR-283-3p表达量显著高于对照(图5A)；同时，小菜蛾添食miR-283-3p mimic24h、48h、60h后 Trypsin-9，表达量显著降低，尤其在60h，Trypsin-9的表达量显著低于对照(图 5B)。图4和图5可知，说明miR-283-3p负调控Trypsin-9基因的表达。
为了进一步直观的观察Trypsin-9对于原毒素的活化过程中的作用。我们将 miR-283-3p mimic和miR-283-3p inhibitor分别添食小菜蛾，24h进一步添食Cry1Ac，每隔12h观察死亡率。结果表明，发现添加了miR-283-3p mimic，进一步添加 Cry1Ac，小菜蛾死亡率大大降低。当添加了miR-283-3p inhibitor后，进一步添加 Cry1Ac，小菜蛾死亡率却显著提高。说明Trypsin-9在Cry1Ac原毒素的活化起重要作用，可能参与小菜蛾中肠免疫防御BtCry1Ac，而miR-283-3p可通过调控 Trypsin-9的表达，影响上述功能，表明可将miR-283-3p作为分子靶标来防控小菜蛾等十字花科蔬菜害虫。
序列表
<110> 华南农业大学
<120> 一种靶向Trypsin-9基因的miR-283-3p及其应用
<141> 2020-06-30
<160> 5
<170> SIPOSequenceListing 1.0
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<213> 小菜蛾(Plutella xylostella)
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gucucacgau caaccauagg u 21
<210> 2
<211> 820
<212> DNA
<213> 小菜蛾(Plutella xylostella)
<400> 2
agcataattc aaattcagtt accagtagtc acaatgcgtt ctcttttcct gttagttttg 60
ggagcaattg cagtgtcggg ggcggctgtc cctcgcactt cgcagaggat tgtgggtgga 120
tccgccacca ccatcgaccg gtatccgttc ggtgcagtcg ttctgttcct ctcgaatgga 180
ggattcttcc gccagcattg cggagggagc atcatcaatg acaatgcagt cttgactgct 240
gcgcactgtc tgcaccgcag gagaaacgac cagttccgca tccgtgtcgg ttcaacccaa 300
gccagcagcg gcggcagcgt gcacgccgtc aaccggctaa tctcacatgc gagcttcaac 360
cacaacaccc aggacaatga cctcgccatc atgaggacca ccacccagat caacttcctc 420
cctggattgg tcgcggctgg caccttcgct gggtctaact acaacgtgcc cgacggcgcc 480
tccgtctggg ctatcggctg gggagctgga cgacccaacg gccccggatc cgagcagctg 540
cgtcacgtgg agatctggac cgtgaaccag gcggtctgca gggctcgcta ccagaacatc 600
agaatgactg ttactgacaa catgctgtgc tcgggctggc tggacgtggg cggccgcgac 660
cagtgcacgg gagactctgg tggccccctc cttcatgata atgtggtcat tggagtctca 720
tcgtggggcc agggctgtgc gtcggccgag taccctggag tcaacgtgcg cgtgtctcgc 780
tacattgact ggattcgcac aaacgcgtag gagaaatgtt 820
<210> 3
<211> 21
<212> RNA
<213> 小菜蛾(Plutella xylostella)
<400> 3
gucucacgau caaccauagg u 21
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<213> 小菜蛾(Plutella xylostella)
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accuaugguu gaucgugaga cuu 23
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<212> RNA
<213> 小菜蛾(Plutella xylostella)
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accuaugguu gaucgugaga c 21

Claims (3)

1.小菜蛾miR-283-3p的miRNA inhibitor在防治小菜蛾或在制备防治小菜蛾药剂中的应用。
2.一种防治小菜蛾的方法，其特征在于，通过小菜蛾miR-283-3p的miRNA inhibitor抑制小菜蛾体内miR-283-3p的表达并施用Bt毒蛋白BtCry1Ac。
3.一种防治小菜蛾的药剂，其特征在于，包含用于抑制小菜蛾体内miR-283-3p表达的miRNA inhibitor及Bt毒蛋白BtCry1Ac。
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