CN114316015B - 一种抗虫蛋白hRI及其编码基因和应用 - Google Patents
一种抗虫蛋白hRI及其编码基因和应用 Download PDFInfo
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Abstract
本发明提供了一种抗虫蛋白hRI及其编码基因和应用，所述抗虫蛋白hRI的氨基酸序列如序列表中的序列1所示。所述基因序列如序列表中的序列2所示。上述的抗虫蛋白hRI和基因在提高植物抗虫性中的应用。本发明首次将hRI应用于植物抗虫研究，并取得很好的抗虫效果，转基因抗虫植物对害虫生长的抑制率（依据害虫体重计算）大于60%；与对照相比，转基因抗虫植物所受虫害非常轻微。
Description
技术领域
本发明涉及基因工程技术领域，具体涉及一种抗虫蛋白hRI及其编码基因和应用。
背景技术
虫害造成农作物的产量和品质损失，严重影响农业生产的稳定与可持续发展。采用化学杀虫剂来控制虫害，在保护了农作物的同时，也带来了如农药残留、害虫抗性进化、杀灭天敌、污染环境等严重问题。利用转基因技术开发转基因抗虫作物，具有高效、低成本、环保等特点。
目前农业生产上的转基因抗虫植物上表达的抗虫基因主要为来自苏云金芽孢杆菌的δ-内毒素(Bt毒蛋白)、蛋白酶/淀粉酶抑制(例如豇豆蛋白酶抑制剂CpTI、α-淀粉酶抑制剂)、植物介导RNAi等。
在植物抗虫基因工程领域，目前生产上可用的抗虫基因种类非常有限。长期使用这些抗虫基因，必然会造成害虫的抗性进化，使转基因植物抗虫性降低或丧失，从而使用农业生产重新面临虫害威胁。随着上述转基因抗虫植物的推广和种植年限的延长，害虫的抗性进化问题越来越突出，亟需寻求新的抗虫基因或手段。
发明内容
本发明的目的就是提供一种抗虫蛋白hRI及其编码基因和应用，以解决现有转基因植物优良抗虫基因较单一、昆虫抗性进化的问题，从而为植物抗虫基因工程提供新的抗虫基因资源。
本发明的目的是通过以下技术方案实现的：一种抗虫蛋白hRI，所述抗虫蛋白hRI的氨基酸序列如序列表中的序列1所示。
编码上述抗虫蛋白hRI的基因，所述基因序列如序列表中的序列2所示。
含有上述基因片段的重组载体。所述重组载体通过如下方法构建：将序列表中序列2所示的基因片段连接到pBIN438质粒上，从而构成植物表达载体pBIN438-hRI。
含有上述重组载体的重组菌株，所述重组菌株为根癌农杆菌。
上述的抗虫蛋白hRI和基因在提高植物抗虫性中的应用。
将上述的基因片段导入目的植物中得到转基因植物，抗虫蛋白hRI在转基因植物中表达，从而使所述转基因植物的抗虫性高于所述目的植物。
上述的基因片段是通过上述的重组载体导入目的植物中的。
所述抗虫是指抗棉铃虫。
所述目的植物为陆地棉或烟草。
本发明所用的抗虫蛋白hRI为人核糖核酸抑制因子(Human ribonucleaseinhibitor，hRI)，与RNA的降解抑制有关。hRI与RNase1、牛RNase A等核糖核酸酶结合极为牢固(解离常数3.5×10-14M-4.5×10-14M)。本发明首次将hRI应用于植物抗虫研究，并取得很好的抗虫效果，转基因抗虫植物对害虫生长的抑制率(依据害虫体重计算)大于60％；与对照相比，转基因抗虫植物所受害虫危害非常轻微。
目前已经报道用于植物抗虫研究的降解酶类抑制剂基因都是针对淀粉、蛋白质；本发明的技术基于长期的实验室验证、分析，具有坚实的实验证据和较好的应用价值，能够作为现有抗虫基因的储备资源，用于植物抗虫基因工程。
附图说明
图1重组hRI对棉铃虫中肠RNA酶活性的抑制活性。(a)hRI对dsRNA1的保护效果(b)hRI对dsRNA2的保护效果；M：DNA Marker；1：dsRNA未处理组；2：dsRNA+hRI未处理组；3-6：不同用量hRI实验组；7：RRI(商品化重组RNA抑制剂，Takara公司，2313Q)对照组；8：EGFP对照组。
图2pBIN438-hRI载体构建示意图。
图3转hRI基因烟草植株的基因组DNA的PCR鉴定。数字编号是转基因植株，WT是野生型植株。
图4转hRI基因烟草植株的RT-PCR鉴定。数字编号是转基因植株，WT是野生型植株。
图5 24h烟草叶片的被咬噬情况。hRI-2、hRI-11、hRI-12是三个表达hRI的烟草植株，WT是未转基因野生型对照。
图6取食转hRI烟草叶片棉铃虫体重变化。
具体实施方式
下面结合具体实施例对本发明的技术方案进行详细说明。本发明实施例中未提到的试验条件和操作均按本领域常规方法进行，本领域技术人员可参照相关技术书籍实施，如：格林、萨姆布鲁克等著《分子克隆实验指南》第4版(2017)，等。
实施例1
核糖核酸酶抑制因子hRI蛋白的活性分析
采用RT-PCR技术从人肺组织cDNA扩增得到hRI编码基因序列，所用引物序列为：
Hrif：5'-CACTCTTCACCTCCACCA-3'
hrir：5'-CTGAGCGTTTCTCTTCAAACC-3'
PCR反应性程序：预变性(95℃，3min)，35个扩增(95℃10s；52℃退火30s，72℃；72℃延伸90s)，以及再延伸(72℃，10min)。凝胶电泳，目的条带凝胶回收后，通过酶连克隆到pUCm-T载体，经过转化、筛选及测序验验证。
使用TC克隆方法(参考中国发明专利：一种基因定向克隆用TC载体及其制备和使用方法，专利号CN201210436709.7)制备可以进行TC定向克隆的原核表达载体pET17b-TC；经XcmI酶切回收酶切质粒载体片段。使用引物：
HRPepf:5'-ATGAGCCTGGACATCCAGAGCCTGGACATCCAGTGTG-3'
HRPepr:5'-TCAGGAGATGACCCTCAGGGATGGCTTGTCCTTCTCCA-3'
扩增pUCm-T上的hRI基因序列，凝胶电泳回收后与pET-17b-TC大片段连接。经转化、筛选、鉴定后得到hRI基因原核表达质粒，随后导入BL21(DE3)pLysS大肠杆菌感受态。
使用His-Tag技术进行了hRI蛋白分离与纯化：用IPTG诱导hRI蛋白表达，hRI蛋白表达产物经过SDS-PAGE检查；将诱导菌体用10mL Tris-HCl裂解液(含溶菌酶)重悬，4℃静置30min以裂解菌体，随后超声破碎；按High Affinity Ni-NTA Resin说明书对裂解样品进行咪唑洗涤和Ni柱洗脱，最后通过SDS-PAGE检查所得纯化重组hRI蛋白。
重组hRI蛋白对棉铃虫中肠液核糖核酸酶活性的抑制分析：以商品化的RNA抑制剂为对照，将绿色荧光蛋白(EGFP)或不同量的His-hRI分别与0.5μL棉铃虫中肠消化液、缓冲液混合，用蛋白洗脱液补至7μL，37℃金属浴30min后，分别加入60ng dsRNA并快速混匀，37℃金属浴1h，85℃水浴5min。用DEPC水配置的1％琼脂糖凝胶进行RNA电泳，以检查dsRNA的降解情况。结果如图1所示。
如图1所示，在同等实验条件下，阴性对照组(泳道1)的结果显示实验所用的缓冲液对dsRNA无降解作用；未加hRI的实验组(泳道8)dsRNA降解较为彻底，这反映了自然条件下棉铃虫中肠酶对取食dsRNA有较强的破坏作用；加入商品化的抑制剂RRI(泳道7)，dsRNA发生部分降解；不同hRI用量的实验组(泳道3、4、5、6)中，随着hRI的用量(0、0.25、0.5、1μL)的增加，dsRNA降解程度递减，棉铃虫中肠酶活性明显受到重组hRI蛋白的抑制作用，表明hRI对dsRNA具有明显的降解抑制效果。上述结果显示原核表达的hRI在抑制中肠液酶类降解dsRNA方面具有明显的作用。
实施例2
核糖核酸酶基因转基因烟草的获得
植物表达载体构建：设计特异性引物hripBf和hripSr，其中hripBf引入Kozak序列和BamH I酶切位点，hripSr引入Sal I酶切位点：
hripBf:5'-CGCGGATCCAACAATGGCTAGCCTGGACATCCAGA-3'
hripSr:5'-GCAGGTCGACCCTCAGGAGATGAC-3'
用该引物PCR扩增hRI基因并进行凝胶电泳回收。使用限制性内切酶BamH I和SalI对pBIN438-X质粒和回收的片段分别进行双酶切。酶切体系(50μL)为：限制性内切酶各0.8μL，相应内切酶Buffer 4μL，待酶切质粒34.3μL，37℃过夜酶切，电泳回收载体骨架和目的片段，并估测回收产物浓度。按照载体片段与目的片段按摩尔比1:3-1:10的比例进行酶连，转化及筛选鉴定。PCR鉴定引物为hripBf/Sr，酶切鉴定所用限制性内切酶为EcoR I和HindIII。构成的植物表达载体pBIN438X-hRI结构如图2。
无菌苗的获得及遗传转化：选取饱满的野生型烟草种子去除杂质，依次用酒精和NaClO溶液消毒，均匀种植于MS固体培养基(Murashige和Skoog固体培养基)中；置于植物生长箱中培养，条件为：温度28±1℃，相对湿度65±5％，光周期为16L:8D。在无菌条件下，将合适大小烟草幼苗转移到含有MS固体培养基的组培瓶中。待无菌苗株高达到约10cm时，取叶片进行农杆菌介导的叶盘转化。步骤如下：(1)叶片预培养：将长势良好的无菌野生烟草叶片剪切成方块(大小约为1cm×1cm)，用镊子将叶片按照背面朝向培养基均匀的铺在含有无抗生素的MS固体培养基上，人工气候箱中培养2d；(2)农杆菌扩培：从-80℃超低温冰箱中取含重组质粒pBIN438X-hRI的农杆菌，220rpm/min，28℃活化24h后转接到150mL LB液体培养基中进行扩大培养，培养至对数生长期；(3)菌液浓缩：4℃，4000rpm离心手机生长期菌体，经MS液体培养基洗涤2次后重悬于100mL MS液体培养基中；(4)侵染：将预培养叶片轻轻夹入MS液体重悬液中，震荡侵染15min，将叶片置于灭菌过得滤纸上，吸干多余液体，夹取叶片背面朝下放入含有共培养基的平板中，用报纸将平板包裹起来暗培养2d；(5)叶片清洗及选择培养；先用无菌ddH2O将叶片清洗3次，最后用MS液体培养基清洗1次，将叶片背面朝下置于选择分化培养基上进行选择培养；(6)继续培养：将分化出的芽切下，插到组培瓶中进行生根培养，最后当转基因烟草长至10cm左右时，用长镊子从组培瓶中轻轻取出再生植株，用流动的冷水冲洗根部，去除剩余组织培养基后移入土壤中。
转基因烟草的分子鉴定：用CTAB法提取烟草叶片的DNA，用特异性鉴定引物扩增DNA，检测hRI是否整合进烟草基因组中，引物序列：
RNH1igF:5'-CAGCAGTGCCAAGTGGTCAG-3'
RNH1igR:5-'CAATGCCGCACAGGTCCC-3'
转录水平的鉴定：用TRIzol试剂提取烟草新鲜叶片的总RNA并进行反转录，用野生型烟草总RNA作对照。用上述引物进行RT-PCR扩增，鉴定目的基因是否转录。鉴定过程中至少要独立提取两次，每次做两次PCR或RT-PCR鉴定以确保结果准确性。对转基因植株进行了基因组水平的鉴定(图3)和转录水平的鉴定(图4)。
实施例3
转基因烟草的抗虫性分析
将人工饲料切成0.5cm×0.5cm左右的小块，放置于无菌培养皿中，将棉铃虫卵初孵幼虫接到饲料上，长到2龄(体长0.42cm-0.62cm)用于虫试分析。取T1代单拷贝转基因株系挑选3株，每株挑选3片长势一致的烟草叶片饲喂棉铃虫幼虫，同时，选取同期种植的野生型烟草中长势一致的叶片饲喂棉铃虫作为对照。将相同基因的3株单拷贝烟草设置为组间重复以减少因基因插入位点不同而引起的误差；将同株烟草的3片叶片设置为组内重复试验，减少选取叶片造成的人为误差；每组棉铃虫体重称取3次，减少体重测量的偶然误差，后续数据处理时取其平均值。从2龄期的棉铃虫中挑选长势相同，体重相同的棉铃虫幼虫16条，放入装有叶片的锁扣盒中。每隔24h称量棉铃虫体重，计数棉铃虫的剩余数量，数据统计至120h。观察24h时的叶片咬噬状况并进行拍照记录。虫试期间，每24h清理一次锁扣盒并更换新鲜烟草叶片。最后根据统计的数据计算棉铃虫平均体重，利用IBM SPSS Statistics20软件通过单因素方差分析比较不同组别之间棉铃虫平均体重之间的差异性。用Excel2016软件根据统计分析结果绘制棉铃虫平均体重折线图。
对24h烟草叶片的咬噬情况进行拍照记录，结果图所示(图5)。结果显示转hRI实验组与对照组(野生型WT)在饲喂棉铃虫二龄幼虫24h后均出现了程度不同的咬噬损伤，其中野生型烟草叶片的咬噬情况较为严重，转hRI烟草叶片被咬噬取食的情况较轻。可见转hRI基因烟草抗虫性有明显提高。
统计取食野生型烟草和转hRI烟草24h、48h、72h、96h、120h五个时间段的棉铃虫平均体重增长情况(表1)并绘制棉铃虫平均体重增长折线图(图6)。
表1：取食转hRI叶片不同时间段棉铃虫平均体重(g)
注：同列中不同小写字母表示在P﹤0.05水平有显著性差异；同列中不同大写字母表示差异在P﹤0.01水平差异极显著。
体重增长趋势显示：同一时间段，取食转hRI烟草组显著低于取食野生型烟草组，48h时取食转hRI烟草组与取食野生型烟草组相比有显著性差异，72h开始差异达到极显著水平，结果说明转hRI烟草棉铃虫幼虫的生长有显著的抑制作用。取食转hRI烟草的3组棉铃虫平均体重之间没有显著性差异，表明在虫试分析中转hRI烟草的抑虫能力不受基因插入位点的影响。
序列表
<110> 河北大学
<120> 一种抗虫蛋白hRI及其编码基因和应用
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Val Arg Leu Asp Asp Cys Gly Leu Thr Glu Ala Arg Cys Lys Asp Ile
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Ser Ser Ala Leu Arg Val Asn Pro Ala Leu Ala Glu Leu Asn Leu Arg
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Ser Asn Glu Leu Gly Asp Val Gly Val His Cys Val Leu Gln Gly Leu
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Pro Thr Leu Gln Glu Leu His Leu Ser Asp Asn Leu Leu Gly Asp Ala
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Glu Lys Leu Gln Leu Glu Tyr Cys Ser Leu Ser Ala Ala Ser Cys Glu
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Pro Leu Ala Ser Val Leu Arg Ala Lys Pro Asp Phe Lys Glu Leu Thr
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Val Ser Asn Asn Asp Ile Asn Glu Ala Gly Val Arg Val Leu Cys Gln
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Gly Leu Lys Asp Ser Pro Cys Gln Leu Glu Ala Leu Lys Leu Glu Ser
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Cys Gly Val Thr Ser Asp Asn Cys Arg Asp Leu Cys Gly Ile Val Ala
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Ser Lys Ala Ser Leu Arg Glu Leu Ala Leu Gly Ser Asn Lys Leu Gly
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Asp Val Gly Met Ala Glu Leu Cys Pro Gly Leu Leu His Pro Ser Ser
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Arg Leu Arg Thr Leu Trp Ile Trp Glu Cys Gly Ile Thr Ala Lys Gly
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Cys Gly Asp Leu Cys Arg Val Leu Arg Ala Lys Glu Ser Leu Lys Glu
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Leu Ser Leu Ala Gly Asn Glu Leu Gly Asp Glu Gly Ala Arg Leu Leu
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atgagcctgg acatccagag cctggacatc cagtgtgagg agctgagcga cgctagatgg 60
gccgagctcc tccctctgct ccagcagtgc caagtggtca ggctggacga ctgtggcctc 120
acggaagcac ggtgcaagga catcagctct gcacttcgag tcaaccctgc actggcagag 180
ctcaacctgc gcagcaacga gctgggcgat gtcggcgtgc attgcgtgct ccagggcctg 240
cagaccccct cctgcaagat ccagaagctg agcctccaga actgctgcct gacgggggcc 300
ggctgcgggg tcctgtccag cacactacgc accctgccca ccctgcagga gctgcacctc 360
agcgacaacc tcttggggga tgcgggcctg cagctgctct gcgaaggact cctggacccc 420
cagtgccgcc tggaaaagct gcagctggag tattgcagcc tctcggctgc cagctgcgag 480
cccctggcct ccgtgctcag ggccaagccg gacttcaagg agctcacggt tagcaacaac 540
gacatcaatg aggctggcgt ccgtgtgctg tgccagggcc tgaaggactc cccctgccag 600
ctggaggcgc tcaagctgga gagctgcggt gtgacatcag acaactgccg ggacctgtgc 660
ggcattgtgg cctccaaggc ctcgctgcgg gagctggccc tgggcagcaa caagctgggt 720
gatgtgggca tggcggagct gtgcccaggg ctgctccacc ccagctccag gctcaggacc 780
ctgtggatct gggagtgtgg catcactgcc aagggctgcg gggatctgtg ccgtgtcctc 840
agggccaagg agagcctgaa ggagctcagc ctggccggca acgagctggg ggatgagggt 900
gcccgactgc tgtgtgagac cctgctggaa cctggctgcc agctggagtc gctgtgggtg 960
aagtcctgca gcttcacagc cgcctgctgc tcccacttca gctcagtgct ggcccagaac 1020
aggtttctcc tggagctaca gataagcaac aacaggctgg aggatgcggg cgtgcgggag 1080
ctgtgccagg gcctgggcca gcctggctct gtgctgcggg tgctctggtt ggccgactgc 1140
gatgtgagtg acagcagctg cagcagcctc gccgcaaccc tgttggccaa ccacagcctg 1200
cgtgagctgg acctcagcaa caactgcctg ggggacgccg gcatcctgca gctggtggag 1260
agcgtccggc agccgggctg cctcctggag cagctggtcc tgtacgacat ttactggtct 1320
gaggagatgg aggaccggct gcaggccctg gagaaggaca agccatccct gagggtcatc 1380
tcctga 1386
Claims (4)
1.抗虫蛋白hRI或其基因在提高植物抗虫性中的应用；所述抗虫蛋白hRI的氨基酸序列如序列表中的序列1所示，所述基因序列如序列表中的序列2所示，所述抗虫是指抗棉铃虫。
2.根据权利要求1所述的应用，其特征在于，将抗虫蛋白hRI的基因片段导入目的植物中得到转基因植物，抗虫蛋白hRI在转基因植物中表达，从而使所述转基因植物的抗虫性高于所述目的植物。
3.根据权利要求1所述的应用，其特征在于，抗虫蛋白hRI的基因片段是通过抗虫蛋白hRI的基因片段的重组载体导入目的植物中的；所述重组载体通过如下方法构建：将序列表中序列2所示的基因片段连接到pBIN438质粒上，从而构成植物表达载体pBIN438-hRI。
4.根据权利要求2或3所述的应用，其特征在于，所述目的植物为陆地棉或烟草。
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