CN116098057A - 一种提高玉米抗草地贪夜蛾幼虫能力的方法 - Google Patents
一种提高玉米抗草地贪夜蛾幼虫能力的方法 Download PDFInfo
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Abstract
本发明属于病虫害防治技术领域，具体公开一种提高玉米抗草地贪夜蛾幼虫能力的方法，本发明具体公开了一系列实验，揭示了玉米Zm00001d017111基因突变为突变体gl8会引起玉米脂质代谢的改变以及虫害诱导茉莉酸等一系列抗虫防御反应的上调，进而抑制草地贪夜蛾幼虫的生长。本发明还公开了上述方法在作物育种尤其在玉米种质资源改良等方面的应用，具有重要的经济价值和广阔的应用前景。
Description
技术领域
本发明属于病虫害防治领域，具体涉及一种提高玉米抗草地贪夜蛾幼虫能力的方法。
背景技术
草地贪夜蛾（Spodoptera frugiperda）是一种具有很强入侵性、迁飞性和暴发性的鳞翅目夜蛾科害虫，被列为世界十大植物害虫之一。草地贪夜蛾源于美洲，现已扩散至非洲、亚洲、大洋洲等世界主要大陆，于2019年入侵我国。据统计，草地贪夜蛾可以危害76科353种寄主植物，包括玉米、小麦、水稻、大豆、花生、油菜、蔬菜等多种重要农作物；另外，草地贪夜蛾属于暴食性害虫，尤其嗜好取食农作物的幼嫩生长点和繁殖器官，严重影响农作物的产量和品质。在自然环境中，植物面临着多种病虫害的威胁，严重影响了植物的生长繁殖和生产力。据估计，在小麦、水稻、玉米、油菜和大豆等重要作物中，这些胁迫因素至少造成了约20%的产量损失，严重影响了世界农业的发展。植物表皮蜡质作为抵挡病原微生物和植食性昆虫的第一道屏障，在调控植物与病虫害互作方面起着重要作用。目前，表皮蜡质的相关研究主要集中在蜡质的生物合成、转运和调控等方面，关于表皮蜡质对植物抗虫性的影响主要集中于其在蜡质的物理防御作用。首先，表皮蜡质可以使植物表面光滑进而影响大多数昆虫的附着；其次，一些害虫可以通过识别叶片上表皮和下表皮的蜡质覆盖率、结构或化学成分的差异来辨别潜在的产卵地点；第三，昆虫的卵子积累会显著影响植物表皮蜡质的组成进而调控天敌行为。目前，表皮蜡质对植物激素合成及其调控次生代谢的影响机制研究较为缺乏。因此，解析表皮蜡质对于玉米抗虫性的影响及机制，可以为发掘玉米新型草地贪夜蛾防控机制提供技术支持。
本发明关注的玉米Zm00001d017111基因编码3-酮酰基还原酶，是玉米苗期叶片表皮蜡质合成所必须的关键酶（Dietrich, C.R., Perera, M.A., M, D.Y.-N., Meeley,R.B., Nikolau, B.J., and Schnable, P.S. (2005). Characterization of two gl8paralogs reveals that the 3-ketoacyl reductase component of fatty acidelongase is essential for maize (Zea mays L.) development. Plant J. 42: 844-861.）。尽管已知该基因编码超长链脂肪酸合成的重要酶类（Xu, X., Dietrich, C.R.,Lessire, R., Nikolau, B.J., and Schnable, P.S. (2002). The endoplasmicreticulum-associated maize gl8 protein is a component of the acyl-coenzyme Aelongase involved in the production of cuticular waxes. Plant Physiol. 128:924-934.），但是其对玉米脂质的合成、次生物质代谢等抗虫化学防御指标的影响尚不清楚。
发明内容
为解决上述技术问题，本发明提出一种本发明提出一种提高玉米抗草地贪夜蛾幼虫能力的方法。
本发明的技术方案是这样实现的：本发明提出一种提高玉米抗草地贪夜蛾幼虫能力的方法,所述方法包括:采用EMS的方法诱导玉米Zm00001d017111基因突变为突变体
gl8的步骤。
本发明还在提供所述的提高玉米抗草地贪夜蛾幼虫能力的方法在玉米种质资源改良中的应用，以及在制备抗草地贪夜蛾幼虫转基因玉米中的应用。
目前虽然已知表皮蜡质能够从物理层面影响害虫的行为和取食，但是玉米苗期表皮蜡质如何影响草地贪夜蛾幼虫的取食和生长尚不清楚。玉米Zm00001d017111基因在玉米苗期EMS诱变为突变体
gl8后（本发明将玉米Zm00001d017111基因在玉米苗期EMS诱变突变体命名为突变体
gl8），草地贪夜蛾幼虫的取食量显著增加，但草地贪夜蛾的生长受到显著抑制，说明该基因突变为突变体
gl8后能够影响玉米苗期的抗虫性。
目前关于玉米Zm00001d017111基因在超长链脂肪酸合成中的功能已经明晰，但是该基因如何影响玉米的脂质代谢以及与抗虫相关的植物激素、次生物质含量以及重要抗虫基因的表达情况尚不明晰。玉米Zm00001d017111基因突变为突变体
gl8后，玉米苗期叶片脂质组发生显著变化，同时使用草地贪夜蛾幼虫唾液模拟虫害处理，在虫害诱导下，OPDA、JA和JA-Ile均在突变体中含量更高。对植物抗虫起重要作用的ABA以及乙烯合成前体ACC的含量在虫害诱导下也在突变体中显著增高，但是SA的含量在突变体和野生型中并无显著差异。同时，在虫害诱导处理下，突变体叶片中总黄酮和HDMBOA-Glc的含量明显高于野生型，蛋白酶抑制剂相关基因ZmCyst、ZmMPI、ZmSerPIN和杀虫核糖体失活蛋白基因ZmRIP2的表达量也明显高于野生型，且差异极显著。
本发明具有以下有益效果：
1、本发明以世界十大植物害虫之一的草地贪夜蛾为研究对象，提出一种提高玉米抗草地贪夜蛾幼虫能力的方法，具有重要的经济价值。
2、本发明公开了一系列实验，揭示了玉米Zm00001d017111基因突变为突变体gl8会引起玉米脂质代谢的改变以及虫害诱导茉莉酸等一系列抗虫防御反应的上调，进而抑制草地贪夜蛾幼虫的生长。上述发现能够为综合调控玉米苗期叶片的物理及化学防御抗虫性提供理论和技术支持。
3、本发明的提供了提高玉米抗草地贪夜蛾幼虫能力的方法在玉米种质资源改良中的应用、在制备抗草地贪夜蛾幼虫转基因玉米中的应用以及在制备抗草地贪夜蛾幼虫转基因玉米的生物材料中的应用，而具有广阔的应用前景。
总之，Zm00001d017111基因突变为突变体gl8后，不仅影响玉米苗期表皮蜡质的合成和积累，还会影响玉米苗期对草地贪夜蛾幼虫的化学防御作用，抑制了草地贪夜蛾幼虫的生长。
附图说明
图1野生型和
gl8突变体中草地贪夜蛾幼虫的取食和生长指标测定
图1A. 在野生型和
gl8突变体中草地贪夜蛾幼虫的取食面积，误差线为标准方差，n = 15，T检验，* p < 0.05。图1B. 在野生型和gl8突变体中草地贪夜蛾幼虫的取食称重，误差线为标准方差，n = 45，T检验，* p < 0.05。
图2玉米叶片在对照和虫害诱导处理下野生型和
gl8突变体玉米叶片中激素含量测定
图2A. 12-氧代植物二烯酸（OPDA）的含量; 图2B. 茉莉酸（JA）的含量; 图2C. 茉莉酰基-L-异亮氨酸（JA-Ile）的含量; 图2 D. 水杨酸（SA）的含量; 图2E.1-氨基环丙烷-1-羧酸（ACC）的含量; 图2F. 脱落酸（ABA）的含量。误差线为标准误差，n = 4，T检验，* p <0.05，** p < 0.01，*** p < 0.001
图3.玉米叶片在对照和虫害诱导处理下，野生型和
gl8突变体中玉米次生代谢物测定和抗虫相关基因相对表达量分析
图3A. 玉米叶片中次生代谢物总黄酮的含量;图3B. 玉米叶片中次生代谢物 2-羟基-4，7-二甲氧基-1，4-苯并恶嗪-3-β-葡萄糖苷（HDMBOA-Glc）的含量;图3C. 蛋白酶抑制剂相关基因
ZmCyst的表达量;图3D.蛋白酶抑制剂相关基因
ZmMPI的表达量;图3E. 蛋白酶抑制剂相关基因
ZmSerPIN的表达量;图3F. 杀虫核糖体失活蛋白基因
ZmRIP2的表达量。误差线为标准误差，n = 3-6，T检验，* p < 0.05，** p < 0.01，*** p < 0.001。
图4. 玉米叶片在对照和虫害诱导处理下，野生型和
gl8突变体的玉米叶片中所有脂质物质含量分析
图4A. 玉米叶片中所有脂质物质含量的主成分（PCA）的含量;图4 B. 野生型和
gl8突变体之间玉米叶片中差异脂质的KEGG富集的含量；图4C.玉米叶片中总游离脂肪酸的含量;图4D.玉米叶片中 C18游离脂肪酸中硬脂酸、油酸、亚油酸和亚麻酸的含量。误差线为标准误差，n = 5，T检验，* p < 0.05，** p < 0.01，*** p < 0.001。
具体实施方式
本发明材料中所运用的术语简介如下：
| 英文缩写 | 英文名称 | 中文名称 |
| 无 | stearic acid | 硬脂酸 |
| 无 | oleic acid | 油酸 |
| 无 | linoleic acid | 亚油酸 |
| 无 | linolenic acid | 亚麻酸 |
| 无 | flavonoid | 黄酮 |
| EMS | Ethyl methane sulfonate | 甲基磺酸乙酯 |
| JA | Jasmonic acid | 茉莉酸 |
| LOX | Lipoxygenases | 脂氧合酶 |
| OS | Oral secretions | 昆虫口腔分泌物 |
| ABA | Abscisic Acid | 脱落酸 |
| ACC | 1-Aminocyclopropane-1-Carboxylic Acid | 1-氨基环丙烷-1-羧酸 |
| PCA | Principal component analysis | 主成分分析 |
| SA | Salicylic acid | 水杨酸 |
| JA-Ile | Jasmonoyl-L-isoleucine | 茉莉酰基-L-异亮氨酸 |
| HDMBOA-Glc | 2-(2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one)-β-D-glucopyranose | 2-羟基-4，7-二甲氧基-1，4-苯并恶嗪-3-β-葡萄糖苷 |
| OPDA | 12–oxo-phytodienoic acid | 12-氧代植物二烯酸 |
| KEGG | Kyoto encyclopedia of genes and genomes | 京都基因与基因组百科全书 |
| VLCFA | Very-long-chain fatty acids | 超长链脂肪酸 |
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。下面结合实施例对本发明作进一步的解释说明，在介绍具体实施例前，就下述实施例中涉及部分生物材料等背景情况简要介绍说明如下。
生物材料：
玉米Zm00001d017111基因的CDS长度为981 bp，编码327个氨基酸，编码3-酮酰基还原酶，GC含量较高。玉米Zm00001d017111基因突变体即gl8突变体是从EMS诱变玉米自交系Ye478创建的突变体库中筛选获得，Ye478自交系玉米植株花粉成熟后，于诱变前1天下午抖去雄穗上散出的花粉并套袋，诱变当日上午在散粉高峰期取回袋中的花粉；将取回的花粉经过100目筛子过滤后，加入到浓度为1×10-3的EMS诱变剂（EMS:石蜡体积比=1:1000）中处理45分钟，期间每隔4-5分钟将诱变剂中的花粉震荡摇匀；处理后的花粉用毛笔蘸取涂抹在Ye478自交系雌穗的花丝上，并将雌穗套袋；5-6周玉米成熟后收取M1代的突变体种子，严格自交后获得M2代Ye478的EMS突变体库（方法来自库来宝，（2007），EMS玉米花粉诱变及根系突变体筛选，硕士学位论文，中国农业大学）。经过表型观察，筛选出表皮蜡质缺失的突变体，该突变体经鉴定，Zm00001d017111的第一个外显子上第200个碱基G突变为A，导致突变位点处的甘氨酸突变为天冬氨酸，鉴定引物为gl8-F（其核苷酸序列：TGTCCGTCCATACGAACTCTACGAC）和gl8-R（其核苷酸序列：CACGAAAGTCCGCACCTCGG）。上述材料均可从商业途径得到或利用公开的技术合成。
实验所用草地贪夜蛾幼虫使用人工饲料（玉米粉和黄豆粉为原料）进行单头培养，10%蜂蜜水喂食成虫。草地贪夜蛾世代培养在温度26 ± 2℃，湿度60 ± 10%，光周期16 h :8 h（L : D）的人工培养箱中完成。
实施例1 野生型和gl8突变体中草地贪夜蛾幼虫的取食和生长指标测定
取食面积实验：首先在直径为9 cm的培养皿底部覆盖滤纸，无菌水将滤纸润湿备用；取野生型和gl8突变体4叶期幼苗的第3片叶片中间部分（约为叶长1/2）放入培养皿中；将饥饿3 - 5 h的3龄初草地贪夜蛾幼虫接入叶片上；于0.5 h、1.5 h、3 h、6 h、8 h观察草地贪夜蛾的取食情况，8 h时将草地贪夜蛾幼虫去除；叶片的取食面积用ImageJ软件进行统计，结果发现在离体的情况下，草地贪夜蛾幼虫取食gl8突变体叶片的面积显著高于取食野生型叶片，说明玉米表皮蜡质可以抑制害虫取食（图1 A）。
取食称重实验：在温室大棚中，环境参数为：环境温度26 ± 4°C，湿度60 ± 5%，光周期为16 h : 8 h（L : D）。将野生型和gl8突变体在营养钵中培养至4叶期幼苗，毎盒营养钵中种植4颗，当玉米第3片完全展开时，放置3头3龄末的草地贪夜蛾幼虫（放置前先对幼虫称重记录初始重量），玉米苗被透气且透明的塑料袋包裹，以防止幼虫的逃逸。4天后对草地贪夜蛾的数量及体重增长量进行统计。结果显示，当草地贪夜蛾取食活体gl8突变体和野生型植株时，取食
gl8突变体的草地贪夜蛾幼虫增长速率显著低于取食野生型的幼虫（图1B）。
实施例2 虫害诱导处理下野生型和gl8突变体激素含量测定
植物激素对植物抗虫防御反应起着重要的调控作用，由于植物对咀嚼式口器昆虫的抗性主要受到茉莉酸通路的调控，发明人通过对野生型和gl8突变体本底以及模拟虫害诱导下多种激素的含量进行检测，着重分析了茉莉酸通路中OPDA、JA和JA-Ile的含量。结果表明，在虫害诱导下，OPDA、JA和JA-Ile均在gl8突变体中含量更高。此外，对植物抗虫起重要作用的ABA和ACC含量，在虫害诱导下均在gl8突变体中显著增高，但是SA的含量在野生型和gl8突变体中并无显著差异（图2）。结果表明在gl8突变体中影响了玉米的激素合成，进而影响植物的抗虫防御反应。
草地贪夜蛾唾液收集的方法：草地贪夜蛾卵孵化后喂食玉米叶片至6龄期。唾液收集时将草地贪夜蛾固定于拇指间，移液器轻触草地贪夜蛾口器加以刺激，幼虫会分泌唾液，此时进行收集并将唾液置于冰上的2 mL离心管中。收集的唾液在4°C，12,000 rpm，离心10min，去除食物残渣。取上清液并使用无菌水1 : 1稀释，保存于-80°C备用。
模拟草地贪夜蛾虫害胁迫处理方法：为了模拟草地贪夜蛾对玉米叶片的损伤，当玉米幼苗第3片完全展开时，用手术刀片对第3片叶下表皮（处理位置距叶尖4 cm处）刮40次，直至刮伤处叶肉组织完全破坏，操作过程中不伤及主叶脉，刮伤面积约为3 - 4 cm2，刮伤后立即在损伤部位加10 μL草地贪夜蛾唾液（Wounding + OS）。
植物激素含量测定：利用模拟草地贪夜蛾虫害诱导处理的方法，对野生型和gl8突变体幼苗模拟虫害诱导0 h、1.5 h时取样，用锡箔纸包裹后迅速置于液氮中。在研钵中用液氮迅速研磨成粉末。取100 mg样品，加入1 mL含内标物（D-CKs、D3-BRs、D5-JA、D6-ABA、D2-IAA、D4-SA、D5-OPDA、D4-ACC，各100 ng）的提取液进行提取，并采用高效液相色谱-串联质谱（HPLC-MS2）系统进行分析。具体操作如下：在4°C过夜孵育提取后，4°C，12,000 rpm，离心15 min，收集上清液并将其转移到MCX-WAX柱，再向柱子中加入1.2 mL 90%甲醇，收集并混合流下来的液体。将MCX柱与WAX柱分开，MCX柱依次用1.5 mL的2%甲酸+5%甲醇、1.5 mL的5%甲醇和1.5 mL的甲醇洗涤，再用1.5 mL的5%氨水+80%甲醇洗脱，最后用100 μL 20%甲醇重新溶解洗脱液，并用UPLC-MS2对样品中BRs、CKs和ACC进行分析；WAX柱子依次用2 mL的5%甲酸、1.5 mL的甲醇和1.5 mL的5%氨水+80%甲醇依次洗脱，真空干燥后用40%甲醇重新溶解，用于GAs、IAA、SA、JA及ABA等分析。
实施例3 野生型和gl8突变体中玉米次生代谢物测定和蛋白酶抑制剂表达分析
为进一步明确gl8突变体是否对草地贪夜蛾具有更强的化学防御，导致草地贪夜蛾幼虫体重增长速率低于野生型，发明人检测了总黄酮、苯并噁嗪类化合物以及蛋白酶抑制剂等玉米中典型的抗虫类次生代谢物质在野生型和gl8突变体中的差异。如图3所示，在虫害诱导处理下，gl8突变体叶片中总黄酮和HDMBOA-Glc的含量明显高于野生型（图3 A-B），蛋白酶抑制剂相关基因ZmCyst、ZmMPI、ZmSerPIN和杀虫核糖体失活蛋白基因ZmRIP2的表达量也明显高于野生型，且差异极显著（图3 C-F）。说明在gl8突变体中能够一定程度激活植物的化学防御，有助于抵御虫害。
总黄酮含量测定方法：第一，标准曲线的建立；取精密芦丁标准品5 mg放于烧杯中，加70%乙醇溶液适量，转移至5 mL容量瓶中定容，摇匀备用，分别移取芦丁标准品溶液0mL、0.5 mL、1 mL、2 mL、3 mL、4 mL、5 mL于5 mL容量瓶中，加70%乙醇补至5 mL，之后加5%亚硝酸钠0.4 mL，摇匀静置5 min，加10%硝酸铝0.4 mL，摇匀静置5 min，加入1 M氢氧化钠4mL。用70%乙醇溶液定容至10 mL，在λ = 495 nm测吸光值A。芦丁标准品溶液浓度为0 mg/mL时仪器调零。回归方程：Y = 304.85 × X + 0.7381（X为吸光值，Y为芦丁标准品浓度），R²= 0.9998。第二，样品处理及测定；取玉米叶片组织立即用液氮冷冻后真空冷冻干燥，研磨成粉状，精密称取各样品0.1 g置于1.5 mL离心管中，加70%乙醇溶液，称重；40°C 30 Hz超声1 h后再次称重，并用70%乙醇补充损失的重量，摇匀后用滤纸过滤待用。样品中总黄酮含量测定，同第一步操作。
苯并噁嗪类化合物含量测定方法：利用模拟草地贪夜蛾虫害诱导处理的方法，对野生型和gl8突变体4叶期幼苗完全展开的第三片叶模拟虫害诱导，0 h、1.5 h时取样，用锡箔纸包裹后迅速置于液氮中，在研钵中用液氮迅速研磨成粉末，然后取研磨好的粉末50 mg于2 mL离心管中，加入500 μL提取液（50%甲醇 + 50% H2O + 0.5%甲酸），涡旋1 min，4°C，12,000 rpm，离心15 min，吸取上清液，然后4°C，12,000 rpm，离心5 min，吸取100 μL上清液至进样小瓶，并对其进行超高速液相色谱-串联质谱UHPLC-MS分析。
抗虫相关基因表达量的分析方法：第一，Trizol法提取总RNA；取约100 mg玉米叶片材料，在预冷的研钵中使用液氮充分研磨3 - 4次，将粉末转入装有1 mL Trizol提取液的1.5 mL离心管中，做好标记，使用旋涡震荡仪混匀，室温静置5 - 8 min，向离心管中加入200 μL氯仿，剧烈摇匀5 - 10 min，然后室温静置3 - 5 min，随后4°C、12,000 rpm，离心15min，吸取500 μL上清液转入新的1.5 mL离心管中，然后向离心管中加入500 μL异丙醇，轻柔摇匀，室温静置10 - 30 min，随后4°C、12,000 rpm，离心15 min，弃上清，再用70%乙醇清洗沉淀，4°C、12,000 rpm，离心5 min，重复沉淀清洗操作，然后放置于冰上晾干，加入30 μLDEPC水溶解RNA，使用Nanodrop One测定各样品RNA浓度，经A260/A280值评估提取RNA质量，等待进行下一步实验，-80℃保存。第二，实时荧光定量qRT-PCR；首先将上述提取的RNA进行反转录，取上述RNA 2 μg， Oligo (dT) primer 1 μL，用RNase-free ddH2O补至12 μL，混匀，72°C，5 min后置于冰上5 min，然后在反应液中加入M-MLV bufer 5 μL，dNTP 2 μL，RNase-free ddH2O 4.5 μL，RRI 0.5 μL，M-MLV 1 μL，混匀，在PCR仪中完成37°C，90 min；75°C，10 min反应，反应结束后利用实时荧光定量qRT-PCR法检测玉米抗虫相关基因的相对表达量；实时荧光定量qRT-PCR检测的反应体系为2 × SYBR Green PCR Master Mix 10 μL，正反向引物均为0.4 μL，模板cDNA 5 μL，RNase-Free Water 4.2 μL，反应程序为 95°C5min预变性；95°C 10 s，60°C 30s共40个循环反应；溶解曲线，95°C 15 s，60°C 60 s，95°C15 s。ZmUbiquitin2基因作为玉米内参基因，每个样本基因设3个重复，按相对定量法（2-ΔΔt法）计算相关基因表达量。玉米抗虫相关基因的定量引物如下所示：
ZmUBIQUITIN2-F: TGGTTGTGGCTTCGTTGGTT
ZmUBIQUITIN2-R: GCTGCAGAAGAGTTTTGGGTACA
ZmCyst-F: GGACATGAGCTGGCGATTTT
ZmCyst-R: CAAGGAGCACAACAGGCAGA
ZmMPI-F: GAAGGTGATCCTCAAGGACAAG
ZmMPI-R: GAAGATGCGGACACGGTTAG
ZmSerPIN-F: ACCTGATGCACTGCTTGCAC
ZmSerPIN-R: GACGGAGGAGGAAGGAGGAG
ZmRIP2-F: GAGATCCCCGACATGAAGGA
ZmRIP2-R: CTGCGCTGCTGCGTTTT
实施例4 野生型和gl8突变体的脂质组分析
在温室大棚中，选取生长状态一致的野生型和gl8突变体4叶期幼苗，在第三片成熟叶片上模拟草地贪夜蛾虫害胁迫处理。分别取胁迫处理0 h、0.5 h的野生型和gl8突变体玉米叶片，每个处理取6株材料叶片，装在50 mL离心管液氮速冻，取5个重复，将上述样品干冰冷冻送至武汉迈特维尔生物科技有限公司进行脂质组分析。通过分析野生型和gl8突变体分别在对照和虫害诱导处理下的脂质组，从PCA结果可以看出，野生型和gl8突变体的脂质组存在显著差异（PC1, 41.54%），而虫害诱导对二者脂质组的影响差异较小（图4 A）。通过对差异脂质进行了KEGG富集分析，发现脂质组中受到显著影响的通路包括代谢和次生代谢通路、亚油酸和亚麻酸通路，此外，甘油酯和甘油磷脂类代谢也受到显著影响（图4 B）。脂质代谢分析表明，总游离脂肪酸以及C18结构游离脂肪酸的含量在gl8突变体中显著上升（图4 C-D）。
上述发明人的研究证实，玉米Zm00001d017111基因突变为突变体gl8会引起玉米脂质代谢的改变以及虫害诱导茉莉酸等一系列抗虫防御反应的上调，进而抑制草地贪夜蛾幼虫的生长。体现出本发明的创新之处。上述成果丰富了表皮蜡质对于植物抗虫的多重生物学功能。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (3)
1.一种提高玉米抗草地贪夜蛾幼虫能力的方法,其特征在于,包括采用EMS的方法诱导玉米Zm00001d017111基因突变为突变体gl8的步骤。
2.权利要求1所述的提高玉米抗草地贪夜蛾幼虫能力的方法在玉米种质资源改良中的应用。
3.权利要求1所述的提高玉米抗草地贪夜蛾幼虫能力的方法在制备抗草地贪夜蛾幼虫转基因玉米中的应用。
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