CN114990152B - 叶绿体表达gfp的转基因烟草在诱集鳞翅目害虫中的应用 - Google Patents

叶绿体表达gfp的转基因烟草在诱集鳞翅目害虫中的应用 Download PDF

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CN114990152B
CN114990152B CN202210424601.XA CN202210424601A CN114990152B CN 114990152 B CN114990152 B CN 114990152B CN 202210424601 A CN202210424601 A CN 202210424601A CN 114990152 B CN114990152 B CN 114990152B
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宁国贵
彭庆琳
项思雅
赵景
何超超
马梦妮
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Abstract

本发明公开了一种质体转化GFP的转基因烟草在诱集鳞翅目害虫中的应用；所述质体转化GFP的转基因烟草是将GFP基因序列连接到pLD载体上，再利用基因枪法得到。将质体转化GFP的转基因烟草植株在常规烟草田的四周条状种植，以集中引诱鳞翅目害虫成虫产卵和幼虫为害并集中诱杀。本发明将质体转化GFP的转基因烟草运用在田间引诱斜纹夜蛾和甜菜夜蛾等害虫，从而加强对害虫的引诱作用，附以灭杀策略可控制靶标保护田害虫虫口密度，达到绿色防控的目的。

Description

叶绿体表达GFP的转基因烟草在诱集鳞翅目害虫中的应用
技术领域
本发明涉及植物基因工程技术领域，具体涉及一种质体转化GFP的转基因烟草在诱集鳞翅目害虫中的应用。
背景技术
斜纹夜蛾(Spodoptera litura Fabricius)和甜菜夜蛾(Spodoptera exigua Hübner)等鳞翅目害虫是以幼虫取食叶片的多食性害虫，在多个国家和地区许多重要蔬菜和农田作物发生及为害严重。以斜纹夜蛾为例，其幼虫取食100多种植物的叶片(Cui et al.，2022)，包括茄果类、瓜果类、豆类、草莓和十字花科蔬菜(秦厚国等，2006)。斜纹夜蛾幼虫在叶片背面群集啃食叶肉，残留上表皮及叶脉，在叶片上形成不规则的透明斑，呈网纹状。幼虫有假死性，遇到惊扰后，四散爬离，或吐丝下坠落地。3龄后分散蚕食植物叶片、嫩茎，造成叶片缺刻、孔洞，残缺不堪，甚至将植株吃成光秆，也可取食花蕾、花等。由于产卵量大且集中，农作物常常受害严重，造成巨大的经济损失(姚文辉，2005)。对此类夜蛾类的防治主要依靠化学杀虫剂，但随着化学农药的长期大量使用，产生了此类害虫对多种杀虫剂产生了不同程度的抗药性、环境污染及农药残留等负面问题。
害虫防治的最佳策略是综合防治，除了化学农药防治，还包括农业防治、物理防治、生物防治等策略；它们的特点如下：
1.农业防治包括：作物种植要合理布局，抑制虫源，尽量避免与害虫嗜好作物连作；结合田间农事操作，人工摘除卵块及群集的幼虫等；
2.物理防治包括：利用成虫的趋性，在成虫发生期，用灯光或者性引诱剂诱杀等措施；
3.生物防治：保护田间的害虫自然天敌，或释放天敌等(孟宪佐，2000；严子华，2014；杨华等，2015；虞国跃等，2021)。
在现代绿色防控技术中，利用特定植物或化合物释放到田间集中诱集灭杀害虫，或者驱避害虫，均可以达到绿色防控害虫目的。这种使被保护的作物失去对害虫的引诱力(推)并将昆虫引向吸引源(拉)并将害虫杀死的防治策略即为“推-拉”策略。开发合适的“推-拉”策略应用植物，对于绿色防治夜蛾类等鳞翅目害虫具有重要意义。
发明内容
本发明的目的在于克服现有技术的不足，提供了一种质体转化GFP的转基因烟草在诱集鳞翅目害虫中的应用，本发明将GFP基因序列连接到pLD载体上，再利用基因枪法得到转基因烟草(Kwon et al 2013)。转基因烟草叶片在475nm激发光下呈绿色，而对照烟草呈红色，为叶绿体自发红光。利用该特点在在田间应用，加强对害虫的诱集作用，从而控制靶标田害虫虫口密度的质体转化GFP的转基因烟草开发与应用。
为实现上述目的，本发明所设计一种质体转化GFP的转基因烟草及在诱集鳞翅目害虫中的应用。
进一步地，所述鳞翅目害虫为斜纹夜蛾或甜菜夜蛾。
再进一步地，所述质体转化GFP的转基因烟草是将GFP基因序列连接到pLD载体上，再利用基因枪法得到。
再进一步地，所述应用的方法如下：
将质体转化GFP的转基因烟草植株在常规烟草田的四周条状种植，以集中引诱鳞翅目害虫成虫产卵和幼虫为害并集中诱杀。
本发明原理：
昆虫可以感知波长在650nm到300nm之间的光，包括光谱中的紫外线区域，以及生物自发荧光和荧光动植物发出的波长。特定波长的诱虫灯已被用作吸引害虫的有效工具，不仅可用来监测害虫的发生还可用于害虫诱杀，是害虫绿色防控技术的重要一环(Cook etal.，2007)。鉴于灯光诱杀的耗能性和专一性差等特点，本发明利用绿色荧光蛋白(GFP)是在水母等动物中存在的一种发光蛋白(首先从水母中分离出来的GFP在追踪标记生物体内蛋白质等方面得到广泛应用(Heim et al.，1995；Verkhusha et al.，2004年))，在475nm激发光光下激发下的最大发射波长为503nm左右(Keutgen et al.，2020)特点，将GFP蛋白表达到植物体内，得到叶绿体表达GFP的转基因烟草，该转基因烟草可以发出特定波长荧光而诱杀害虫。
本发明的有益效果：
1.本发明将质体转化GFP的转基因烟草运用在田间引诱斜纹夜蛾和甜菜夜蛾等害虫，从而加强对害虫的引诱作用，附以灭杀策略可控制靶标保护田害虫虫口密度，达到绿色防控的目的。
2.本发明通过测定斜纹夜蛾幼虫对离体的质体转化GFP的转基因烟草叶片的取食偏好、斜纹夜蛾幼虫对质体转化GFP的转基因烟草植株的取食偏好、斜纹夜蛾成虫对质体转化GFP的转基因烟草植株的产卵偏好，发现斜纹夜蛾对质体转化GFP的转基因烟草有产卵和取食偏好。
3.本发明在温室中测定了质体转化GFP的转基因烟草在田间的应用效果，发现与常规烟草临近种植时，质体转化GFP的转基因烟草显著引诱斜纹夜蛾、甜菜夜蛾等鳞翅目害虫在其上集中产卵并危害，而常规烟草几乎无虫害发生，显示出该烟草在诱集鳞翅目害虫方面的巨大应用潜力。
附图说明
图1为质体转化GFP的转基因烟草和对照烟草在475nm激发光下的表型对比图；
图2为质体转化GFP的转基因烟草和对照烟草叶绿体在475nm激发光下的对比图；
图3为质体转化GFP的转基因烟草和对照烟草植株在475nm激发光下的激发光谱对比图；
图4为斜纹夜蛾幼虫对离体的质体转化GFP的转基因烟草叶片选择性取食试验示意图；
图5为斜纹夜蛾幼虫对离体的质体转化GFP的转基因烟草叶片选择性取食试验操作图；
图6为斜纹夜蛾幼虫对质体转化GFP的转基因烟草选择性取食试验示意图；
图7为斜纹夜蛾成虫对质体转化GFP的转基因烟草选择性产卵操作图；
图8为斜纹夜蛾幼虫对离体的质体转化GFP的转基因烟草叶片选择性取食测试结果图；
图9为斜纹夜蛾幼虫对质体转化GFP的转基因烟草选择性取食测试结果图；
图10为斜纹夜蛾对质体转化GFP的转基因烟草选择性产卵测试结果图；
图11为温室内斜纹夜蛾幼虫对质体转化GFP的转基因烟草的危害测试结果图。
具体实施方式
下面结合具体实施例对本发明作进一步的详细描述，以便本领域技术人员理解。
实施例1:质体转化GFP的转基因烟草材料的获得
本发明使用的质体转化GFP的转基因烟草材料是从华中农业大学植物科学技术学院的金双侠教授课题组得来的。具体转化方法参考该课题组前期已发表文章：为了构建用于叶绿体转化的载体，使用pLD-GFP-His6-Factor Xa-retrocyclin-101(RC101)载体，对绿色荧光蛋白(GFP)编码序列进行PCR扩增模板并连接到BluntII Topo载体。再利用SalI和XbaI限制性内切酶将GFP编码序列剪切后连接到pLD载体中，得到用于烟草叶绿体转化的pLD-PTD-GFP载体质粒。在利用基因枪法将融合GFP荧光蛋白的载体质粒转入WT野生型烟草叶片中，通过对转基因烟草的DNA提取和PCR阳性鉴定，获得质体转化GFP的转基因烟草植株(Kwon et al 2013)。
实施例2:质体转化GFP的转基因烟草与对照烟草荧光检测
黑暗条件下，取质体转化GFP的转基因烟草和对照烟草各一盆，将紫外光发射器对准烟草，紫外光均匀照射在烟草上。质体转化GFP的转基因烟草的未抽出嫩叶呈红色，完全展开叶呈绿色；对照烟草的叶片均为红色。红色是烟草叶片中叶绿体自发荧光，绿色为GFP荧光蛋白发光(图1)。
通过梯度浓度蔗糖溶液粗提烟草叶片的叶绿体，在激光共聚焦显微镜下观测叶绿体的荧光。具体操作如下：取烟草叶片用双面刀片切成0.2cm宽的碎片；加入10ml提取buffer用均质仪进行样品充分均质化；用0.4um直径的细胞筛进行过滤；重复均质、过滤2-3次；4℃3000rpm离心20min，小心吸掉上清；用2-3ml提取buffer重悬沉淀，备用；提前1d用HSbuffer配置不同浓度的蔗糖梯度溶液(5％，15％，50％和75％)；按从高到低依次向离心管中缓慢加入3ml 75％,2ml 50％,3ml 15％和1ml 5％的蔗糖溶液，配置不连续蔗糖梯度；在梯度上层缓慢加入质体样品，4℃18000rpm水平离心90min；缓慢吸取15％和50％，以及50％和75％两个梯度之间的样品，即为完整质体；在样品中加入5ml的HS buffer，悬浮后4℃3000rpm离心30min，用1ml HS buffer重悬备用。取10μl样品，制作成玻片，在激光共聚焦显微镜下观测475nm激发光下叶绿体的荧光表现。对照烟草的叶绿体为红色荧光，质体转化GFP的转基因烟草的叶绿体为绿色荧光(图2)。
黑暗条件下，用虹谱光色红外光谱辐射计(OHSP-350S，杭州虹谱光色科技有限公司)测定475nm激发光下的质体转化GFP的转基因烟草和对照烟草的发射光谱。对照烟草检测到叶绿体自发荧光630-663nm波长的红光，质体转化GFP的转基因烟草除了检测到叶绿体自发荧光，还有488-620nm波长的绿光(图3)。
以上实验结果均表明，质体转化GFP的转基因烟草能发射出绿色荧光，而对照烟草只有叶片叶绿体自发红光。
实施例3:质体转化GFP的转基因烟草离体叶片的诱虫实验
采用图4、5所示的方法测定斜纹夜蛾幼虫对离体质体转化GFP的转基因烟草的取食偏好。取6周龄质体转化GFP的转基因烟草和对照烟草的第三片叶片，切成直径为1cm的叶盘，将质体转化GFP的转基因烟草和对照烟草叶片交叉放置在直径15厘米培养皿底部的湿滤纸上。将10只斜纹夜蛾3龄幼虫饥饿4h后，置于培养皿中央。分别于6h、12h和24h记录质体转化GFP的转基因烟草和对照烟草叶片上幼虫的数量。选择试验分别在光照和黑暗条件下进行，均设置8个重复。数据使用spss16.0进行卡方检验。
实验结果如图8所示。在连续光照条件下，斜纹夜蛾幼虫对质体转化GFP的转基因烟草有明显的偏好，6h：χ2＝8.53，P＝0.003；12h：χ2＝11.84，P＝0.001；24h：χ2＝11.84，P＝0.001。而在黑暗环境下，斜纹夜蛾幼虫在12h和24h对质体转化GFP的转基因烟草有显著的偏好，12h：χ2＝6.36，P＝0.012；24h：χ2＝10.67，P＝0.001。这些结果表明，质体转化GFP的转基因烟草对斜纹夜蛾幼虫有明显的引诱性。
实施例4:质体转化GFP的转基因烟草植株的诱虫实验
采用图6所示方法在整株烟草上进行选择试验，分别将2株6周龄的质体转化GFP的转基因烟草和对照烟草沿对角线种植在50×30×15cm的塑料盆边缘，植株根部铺设一层100目纱网，便于观察记录。植株在正常的养虫室(温度为26±1℃，RH:75％±5％，光照16L∶8D)中种植3d后放入纱网养虫笼内(50cm×50cm×50cm)，再将20头饥饿4h的斜纹夜蛾3龄幼虫接入塑料盆中。在接下来的3d中，每天记录转基因植株和对照植株上的幼虫数量，并拍摄烟草叶片被取食情况。共设置6次重复。数据使用spss16.0进行卡方检验。
测试结果如图9所示。斜纹夜蛾幼虫在接入48h时，对质体转化GFP的转基因烟草植株取食严重；72h时基本将质体转化GFP的转基因烟草植株完全取食，对对照烟草植株取食较少。这些结果表明，质体转化GFP的转基因烟草对斜纹夜蛾幼虫有显著的吸引性。
实施例5:质体转化GFP的转基因烟草植株的成虫产卵实验
使用如图7所示方法，在养虫室中，将4株6周龄质体转化烟草和对照烟草分别沿对角放置在50×50×50cm的养虫笼中，每个角落放置两株烟草。再放入8对初羽化成虫，并将含10％蔗糖溶液的棉球放入笼内为成虫提供营养。5d后，记录质体转化GFP的转基因烟草和对照烟草植株上的卵块数和产卵总数。每个实验重复6次。数据使用spss16.0进行卡方检验。
测试结果如图10所示。与对照烟草植株相比，斜纹夜蛾在质体转化GFP的转基因烟草植株上产卵块数显著较多、(χ2＝40.50，P＝0.006)。质体转化GFP的转基因烟草植株上的虫卵数量也比对照植株上的多(t＝5.97，P＝0.002)。结果表明，质体转化GFP的转基因烟草对斜纹夜蛾具有显著的产卵吸引性。
实施例6:质体转化GFP的转基因烟草植株的大田诱虫实验
在温室内测试质体转化GFP的转基因烟草对斜纹夜蛾幼虫的吸引作用。1)将5株3周龄的质体转化GFP的转基因烟草和5株3周龄的对照烟草分两行种植在一个塑料花盆(50×30×15cm)的两侧，共设置6个重复。2)将质体转化GFP的转基因烟草和对照烟草相邻种植在温室种植床上。两个测试均在30d后记录害虫的发生及危害情况。
测试结果如图11所示。在温室内质体转化GFP的转基因烟草被斜纹夜蛾幼虫为害严重。90％以上的质体转化GFP的转基因烟草植株受到严重破坏。而相邻的对照烟草受损率小于2％。除斜纹夜蛾外，在温室内受损的质体转化GFP的转基因烟草植株上还捕捉到甜菜夜蛾。以上两种害虫的排泄物和中肠中存在GFP荧光，表明这些害虫主要以质体转化GFP的转基因烟草为食。
其它未详细说明的部分均为现有技术。尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。

Claims (3)

1.一种叶绿体表达GFP的转基因烟草在诱集鳞翅目害虫中的应用，其特征在于：所述鳞翅目害虫为斜纹夜蛾。
2.根据权利要求1所述的应用，其特征在于：所述叶绿体表达GFP的转基因烟草是将GFP基因序列连接到pLD载体上，再利用基因枪法得到。
3.根据权利要求1所述的应用，其特征在于：所述应用的方法如下：
将叶绿体表达GFP的转基因烟草植株在常规烟草田的四周条状种植，以集中引诱鳞翅目害虫成虫产卵和幼虫为害并集中诱杀。
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