CN113801886B - Application of BZR1 gene in regulating plant resistance to pest stress - Google Patents

Abstract

Translated fromChinese

本发明公开了BZR1基因在调控植物对虫害胁迫抗性中的应用，属于生物技术领域。所述应用为：利用生物技术手段使得所述BZR1基因功能缺失，进而提高植物对虫害胁迫的抗性；所述BZR1基因的编码序列如SEQ ID NO.1所示或与SEQ ID NO.1所示序列具有至少70％同源性且编码的蛋白在功能上等价。本发明首次公开BZR1基因在调控植物对虫害胁迫尤其是棉铃虫抗性方面的用途，通过基因编辑技术缺失BZR1基因功能，可显著增强植物对对棉铃虫的抗性，可将其应用到抗虫害胁迫植株品种的培育中。

The invention discloses the application of BZR1 gene in regulating the resistance of plants to pest stress, and belongs to the field of biotechnology. The application is as follows: using biotechnological means to make the BZR1 gene function deficient, thereby improving the resistance of plants to pest stress; the coding sequence of the BZR1 gene is as shown in SEQ ID NO.1 or as shown in SEQ ID NO.1. The sequences shown have at least 70% homology and the encoded proteins are functionally equivalent. The present invention discloses for the first time the use of BZR1 gene in regulating plant resistance to pest stress, especially cotton bollworm. Deleting the function of BZR1 gene through gene editing technology can significantly enhance the plant's resistance to cotton bollworm, and can be applied to insect pest resistance Stressed plant varieties are being developed.

Description

Translated fromChinese

BZR1基因在调控植物对虫害胁迫抗性中的应用Application of BZR1 gene in regulating plant resistance to pest stress

技术领域technical field

本发明涉及生物技术领域，具体涉及一种通过缺失BZR1基因功能来提高植物对虫害尤其是棉铃虫抗性的方法。The invention relates to the field of biotechnology, in particular to a method for improving plant resistance to insect pests, especially cotton bollworm by deleting the function of BZR1 gene.

背景技术Background technique

番茄(Solanum lycopersicum L.)是世界上消费和种植最广泛的蔬菜之一。然而，在整个生长周期中，番茄面临各种病虫的侵袭，导致了巨大的经济损失。若大量施用杀虫剂防治，则会带来农药残留、环境污染和耐药性等问题。鉴于番茄具备生命周期相对较短、基因组较小、遗传多样性高、转基因和栽培容易等优点，加之全基因组测序完成和分子遗传学研究的发展，番茄已经成为研究植物与病虫害互作的经典模式植物。因此，以番茄为对象，探究植物抵御虫害的分子机制对于虫害控制和农产品安全生产具有积极的生态、经济意义。Tomato (Solanum lycopersicum L.) is one of the most widely consumed and grown vegetables in the world. However, throughout the growth cycle, tomatoes face various pests and diseases, resulting in huge economic losses. If a large number of pesticides are used for control, it will bring problems such as pesticide residues, environmental pollution and drug resistance. In view of the advantages of tomato's relatively short life cycle, small genome, high genetic diversity, easy transgenic and cultivation, coupled with the completion of whole genome sequencing and the development of molecular genetics research, tomato has become a classic model for studying the interaction between plants and pests and diseases. plant. Therefore, taking tomato as an object, exploring the molecular mechanism of plants resisting pests has positive ecological and economic significance for pest control and safe production of agricultural products.

棉铃虫(Helicoverpa armigera)是在中国棉区和蔬菜种植区均有发生的重要作物虫害。棉铃虫取食范围广，寄主植物多达30多科200余种，其中包括重要的园艺作物——番茄。棉铃虫在番茄种植的整个周期均可发生，其最大的危害是专蛀果实，引起果实腐变，产量损失达30％以上，已经成为发展晚茬番茄生产的一个主要障碍因素。因此，探究番茄内在防御棉铃虫的分子机制对培育抗虫新品种具有一定的指导意义。Cotton bollworm (Helicoverpa armigera) is an important crop pest that occurs in both cotton and vegetable growing areas in China. The cotton bollworm feeds on a wide range and hosts more than 200 species of more than 30 families, including an important horticultural crop-tomato. Cotton bollworm can occur in the whole cycle of tomato planting, and its biggest damage is fruit borer, causing fruit decay and yield loss of more than 30%, which has become a major obstacle to the development of late crop tomato production. Therefore, exploring the molecular mechanism of tomato's intrinsic defense against cotton bollworm has certain guiding significance for breeding new insect-resistant varieties.

作为固着生物，植物已经进化出大量的防御策略来对抗草食动物，以成功生存和繁殖。植物对草食动物攻击的防御反应是由植物激素(Pieterse,C.M.J.等，“SignallingNetworks Involved in Induced Resistance.”In Induced Resistance for PlantDefense，2014，58–80)介导的，其中，茉莉酸(JA)信号通路已被公认为是调控植物对草食动物防御反应的核心通路(Howe GA等，“Modularity in jasmonate signaling formultistress resilience.”Annu.Rev.Plant Biol.，2018，69，387–415)。当草食动物攻击时，JA信号激活，修饰植物的物理和化学表型，从而增加植物的抵御能力。过去20年的深入研究揭示了JA通路的重要分子组成(Howe GA等，“Modularity in jasmonate signalingfor multistress resilience.”Annu.Rev.Plant Biol.，2018，69，387–415)。在植物受到损伤时，脂酰水解酶从质体膜中的半乳糖脂中释放α-亚麻酸(18:3)，由13-脂肪加氧酶氧化，然后环氧化和环化反应生成12-氧代-植二烯酸(OPDA)。OPDA被运输到过氧化物酶体中，经还原和乙酰辅酶A氧化酶(Acx1)等介导的三个β氧化循环形成(+)-7-iso-JA，通过差向异构转变为较为稳定的反式构型，即JA。进一步，JA与异亮氨酸结合形成具有活性的衍生物茉莉酸-异亮氨酸(JA-Ile)。JA-Ile结合由COI1、JAZ及肌醇聚磷酸盐辅因子组成的受体，致使抑制因子JAZ被26S蛋白酶体降解，从而释放MYC转录因子，激活下游防御基因的表达。虽然JA信号转导途径已经得到了广泛的研究，但对于JA合成途径的调控机制亟待进一步的探究。As sessile organisms, plants have evolved numerous defense strategies against herbivores for successful survival and reproduction. Plant defense responses to herbivore attack are mediated by phytohormones (Pieterse, C.M.J. et al., "Signalling Networks Involved in Induced Resistance." In Induced Resistance for Plant Defense, 2014, 58–80), in which jasmonic acid (JA) signaling The pathway has been recognized as a core pathway regulating plant defense responses to herbivores (Howe GA et al., "Modularity in jasmonate signaling for multistress resilience." Annu. Rev. Plant Biol., 2018, 69, 387–415). When attacked by herbivores, JA signaling activates and modifies the plant's physical and chemical phenotype, thereby increasing plant resilience. Intensive research over the past 20 years has revealed an important molecular composition of the JA pathway (Howe GA et al., "Modularity in jasmonate signaling for multistress resilience." Annu. Rev. Plant Biol., 2018, 69, 387–415). Upon plant injury, fatty acyl hydrolases release α-linolenic acid (18:3) from galactolipids in the plastid membrane, oxidized by 13-lipoxygenase, followed by epoxidation and cyclization to 12 -Oxo-phytadienoic acid (OPDA). OPDA is transported into peroxisomes, and forms (+)-7-iso-JA through three β-oxidation cycles mediated by reduction and acetyl-CoA oxidase (Acx1), etc. The stable trans configuration, or JA. Further, JA combines with isoleucine to form an active derivative, jasmonic acid-isoleucine (JA-Ile). JA-Ile binds to a receptor composed of COI1, JAZ, and inositol polyphosphate cofactors, causing the inhibitory factor JAZ to be degraded by the 26S proteasome, thereby releasing the MYC transcription factor and activating the expression of downstream defense genes. Although the JA signal transduction pathway has been extensively studied, the regulatory mechanism of the JA synthesis pathway needs to be further explored.

转录调控在植物抗性形成调控网络中起到关键的作用，是研究植物产生防御反应的关键所在。转录调控是由转录因子通过结合启动子区域的顺式作用元件，调控一系列基因的表达，以抵御病虫害的侵袭。BZR1(BRASSINAZOLE RESISTANT 1)为油菜素内酯信号通路中的关键转录因子，参与植物细胞伸长、细胞分裂、开花及种子发育等多个生长发育过程，还发现其在抵御高低温、盐胁迫等非生物胁迫中发挥重要作用(Qianfeng Li等，“Thebrassinosteroid-regulated transcription factors BZR1/BES1 function as acoordinator in multisignal-regulated plant growth.”BBA-Gene RegulatoryMechanisms，2018，861，561–571；Hui Li等，“BZR1 Positively Regulates FreezingTolerance viaCBF-Dependent and CBF-Independent Pathwaysin Arabidopsis.”Molecular Plant，2017，10，545-559)。但是对于BZR1在虫害胁迫中的作用及机制仍不清楚。因此，研究番茄SlBZR1基因应对棉铃虫的抗性机制对于抗棉铃虫番茄品种的选育具有一定的指导意义。Transcriptional regulation plays a key role in the regulation network of plant resistance formation, and is the key to study the defense response of plants. Transcriptional regulation is that transcription factors regulate the expression of a series of genes by binding cis-acting elements in the promoter region to resist the invasion of pests and diseases. BZR1 (BRASSINAZOLE RESISTANT 1) is a key transcription factor in the brassinolide signaling pathway, which is involved in many growth and development processes such as plant cell elongation, cell division, flowering and seed development. It has also been found to resist high and low temperature, salt stress, etc. plays an important role in abiotic stress (Qianfeng Li et al., "Thebrassinosteroid-regulated transcription factors BZR1/BES1 function as acoordinator in multisignal-regulated plant growth." BBA-Gene Regulatory Mechanisms, 2018, 861, 561–571; Hui Li et al., " BZR1 Positively Regulates Freezing Tolerance via CBF-Dependent and CBF-Independent Pathways in Arabidopsis." Molecular Plant, 2017, 10, 545-559). However, the role and mechanism of BZR1 in insect stress is still unclear. Therefore, studying the resistance mechanism of tomato SlBZR1 gene to H. armigera has certain guiding significance for the breeding of tomato varieties resistant to H. armigera.

发明内容SUMMARY OF THE INVENTION

本发明的目的在于提供一种可调控植物对虫害胁迫尤其是棉铃虫抗性的基因，通过基因改造达到提高植物对棉铃虫抗性的目的，为培育抗虫害的植物品种提供依据。The purpose of the present invention is to provide a gene that can regulate the resistance of plants to pest stress, especially cotton bollworm, achieve the purpose of improving plant resistance to cotton bollworm through genetic modification, and provide a basis for cultivating pest-resistant plant varieties.

为实现上述目的，本发明采用如下技术方案：To achieve the above object, the present invention adopts the following technical solutions:

本发明提供了BZR1基因在调控植物对虫害胁迫抗性中的应用，所述应用为：利用生物技术手段使得所述BZR1基因功能缺失，进而提高植物对虫害胁迫的抗性；The present invention provides the application of BZR1 gene in regulating plant's resistance to pest stress.

所述BZR1基因的编码序列如SEQ ID NO.1所示或与SEQ ID NO.1所示序列具有至少70％同源性且编码的蛋白在功能上等价。The coding sequence of the BZR1 gene is shown in SEQ ID NO. 1 or has at least 70% homology with the sequence shown in SEQ ID NO. 1 and the encoded protein is functionally equivalent.

进一步的，所述BZR1基因编码的蛋白质负调控茉莉酸的合成。Further, the protein encoded by the BZR1 gene negatively regulates the synthesis of jasmonic acid.

进一步的，所述BZR1基因编码的蛋白质结合到茉莉酸合成相关基因Acx1的启动子上，负调控Acx1基因的转录，降低茉莉酸的含量。Further, the protein encoded by the BZR1 gene binds to the promoter of the jasmonic acid synthesis-related gene Acx1, negatively regulates the transcription of the Acx1 gene, and reduces the content of jasmonic acid.

本发明研究表明，BZR1基因编码的蛋白负调控茉莉酸(JA)合成相关基因Acx1(Acyl-CoA oxidase 1)的转录，Acx1基因编码的乙酰辅酶A氧化酶为JA合成的关键酶，茉莉酸在植物抵御生物胁迫中具有重要作用。通过缺失BZR1基因功能，可显著提高植物对虫害胁迫的抗性。The research of the present invention shows that the protein encoded by the BZR1 gene negatively regulates the transcription of Acx1 (Acyl-CoA oxidase 1), a gene related to jasmonic acid (JA) synthesis, and the acetyl-CoA oxidase encoded by the Acx1 gene is the key enzyme in the synthesis of JA. It plays an important role in plant resistance to biotic stress. Deletion of BZR1 gene function can significantly improve plant resistance to pest stress.

进一步的，所述应用包括：利用基因突变、基因敲除、基因干扰或基因沉默技术导致所述BZR1基因的表达降低或缺失，从而获得对虫害胁迫抗性增强的突变体植株。Further, the application includes: using gene mutation, gene knockout, gene interference or gene silencing technology to reduce or delete the expression of the BZR1 gene, thereby obtaining mutant plants with enhanced resistance to pest stress.

进一步的，所述植物为番茄。Further, the plant is tomato.

进一步的，所述虫害胁迫为棉铃虫。Further, the pest stress is cotton bollworm.

本发明对番茄SlBZR1(基因编号：Solyc04g079980)进行序列分析，查找PAM序列，将NGG前的20bp的序列定义为sgRNA，选定位于基因蛋白编码区上且具有高度特异性的sgRNA序列。通过基因编辑技术和组培技术，构建了SlBZR1基因的CRISPR/Cas9载体，并筛选获得了遗传稳定并且不含外源Cas9蛋白的纯合突变体株系。通过接种棉铃虫发现突变体植株能够显著增强番茄对棉铃虫的抗性。The present invention carries out sequence analysis on tomato SlBZR1 (gene number: Solyc04g079980), searches for the PAM sequence, defines the 20bp sequence before NGG as sgRNA, and selects the highly specific sgRNA sequence located on the protein coding region of the gene. Through gene editing technology and tissue culture technology, the CRISPR/Cas9 vector of SlBZR1 gene was constructed, and a homozygous mutant line with stable genetics and no exogenous Cas9 protein was obtained by screening. It was found that the mutant plants could significantly enhance the resistance of tomato to Helicoverpa armigera by inoculation with Helicoverpa armigera.

所述SlBZR1基因的全基因DNA序列如SEQ ID NO.4所示，蛋白编码区的核苷酸序列如SEQ ID NO.1所示，其蛋白编码区长度为987bp；该SlBZR1基因编码的蛋白为油菜素内酯信号通路中的关键转录因子，由328个氨基酸组成，其序列如SEQ ID NO.2所示。The complete gene DNA sequence of the SlBZR1 gene is shown in SEQ ID NO.4, the nucleotide sequence of the protein coding region is shown in SEQ ID NO.1, and the protein coding region length is 987bp; the protein encoded by the SlBZR1 gene is The key transcription factor in the brassinolide signaling pathway is composed of 328 amino acids, and its sequence is shown in SEQ ID NO.2.

在棉铃虫胁迫条件下，与野生型番茄(WT)相比，bzr1缺失突变体中茉莉酸含量显著上升。因此，缺失番茄Slbzr1基因有利于提高番茄对棉铃虫的抗性。Under H. armigera stress, the content of jasmonic acid was significantly increased in bzr1 deletion mutants compared with wild-type tomato (WT). Therefore, deletion of the tomato Slbzr1 gene is beneficial to improve the resistance of tomato to H. armigera.

SlBZR1基因编码蛋白负调控JA合成相关基因Acx1(基因编号：Solyc08g078390)的转录。进一步的，所述Acx1基因启动子的核苷酸序列如SEQ ID NO.3所示。The protein encoded by the SlBZR1 gene negatively regulates the transcription of the JA synthesis-related gene Acx1 (gene number: Solyc08g078390). Further, the nucleotide sequence of the Acx1 gene promoter is shown in SEQ ID NO.3.

本发明还提供了一种抗棉铃虫番茄的培育方法，包括以下步骤：The present invention also provides a method for cultivating tomato resistant to bollworm, comprising the following steps:

(1)在SlBZR1基因的蛋白编码区选取含有PAM结构的靶标片段，以靶标片段PAM结构前20个碱基为依据，进行引物设计，构建CRISPR/Cas9载体；所述SlBZR1基因的核苷酸序列如SEQ ID NO.4所示；(1) Select a target fragment containing a PAM structure in the protein coding region of the SlBZR1 gene, design primers based on the first 20 bases of the PAM structure of the target fragment, and construct a CRISPR/Cas9 vector; the nucleotide sequence of the SlBZR1 gene As shown in SEQ ID NO.4;

(2)构建含步骤(1)所述CRISPR/Cas9载体的农杆菌基因工程菌；(2) constructing an Agrobacterium genetically engineered bacterium containing the CRISPR/Cas9 vector described in step (1);

(3)将步骤(2)所述基因工程菌转化番茄子叶，获得不含外源Cas9蛋白、且靶标序列发生变异的稳定遗传的纯合突变体株系。(3) Transforming the tomato cotyledons with the genetically engineered bacteria described in step (2) to obtain a stable genetic homozygous mutant line that does not contain exogenous Cas9 protein and has a mutated target sequence.

进一步的，所述靶标片段PAM结构前20bp的核苷酸序列如SEQ ID NO.5所示。Further, the nucleotide sequence of the first 20 bp of the PAM structure of the target fragment is shown in SEQ ID NO.5.

本发明具备的有益效果：The beneficial effects that the present invention has:

本发明首次公开BZR1基因在调控植物对虫害胁迫尤其是棉铃虫抗性方面的用途，通过基因编辑技术缺失BZR1基因功能，可显著增强植物对对棉铃虫的抗性，可将其应用到抗虫害胁迫植株品种的培育中。The present invention discloses for the first time the application of BZR1 gene in regulating plant resistance to pest stress, especially cotton bollworm. Deleting the function of BZR1 gene through gene editing technology can significantly enhance the plant's resistance to cotton bollworm, and can be applied to insect pest resistance Stressed plant varieties are being developed.

附图说明Description of drawings

图1为CRISPR/Cas9介导的敲除SlBZR1的纯合突变体植株的基因编辑位点及编码氨基酸的变化；Fig. 1 shows the changes of gene editing site and encoded amino acid of CRISPR/Cas9-mediated knockout of SlBZR1 homozygous mutant plants;

其中，WT为未转基因的野生型番茄，bzr1为SlBZR1基因敲除的突变体番茄；与WT相比，突变体在sgRNA的位置发生10bp的碱基缺失，导致蛋白翻译提前终止。Among them, WT is a non-transgenic wild-type tomato, and bzr1 is a mutant tomato with SlBZR1 gene knockout. Compared with WT, the mutant has a 10 bp base deletion at the position of sgRNA, resulting in premature termination of protein translation.

图2为番茄SlBZR1基因敲除的突变体植株接种棉铃虫3天后的棉铃虫表型(A)及重量(B)；Fig. 2 is the phenotype (A) and weight (B) of cotton bollworm 3 days after tomato SlBZR1 knockout mutant plants are inoculated with cotton bollworm;

其中，棉铃虫体型、重量越大，表示棉铃虫取食越多，番茄植株受虫害越严重；小写字母a、b代表不同植株之间在T检验中P<0.05水平上的差异显著。Among them, the larger the size and weight of the bollworm, the more the bollworm feeds, and the more serious the tomato plant is affected by the insect; the lowercase letters a and b represent significant differences between different plants at the level of P<0.05 in the T test.

图3为番茄SlBZR1基因敲除的突变体植株接种棉铃虫3天后的叶片表型(A)及重量(B)；Fig. 3 is the leaf phenotype (A) and weight (B) of tomato SlBZR1 knockout mutant plants 3 days after inoculation with Helicoverpa armigera;

其中，番茄叶片受损越多，重量越小，表示棉铃虫取食越多，番茄植株受虫害越严重；小写字母a、b代表不同植株之间在T检验中P<0.05水平上的差异显著。Among them, the more damaged tomato leaves and the smaller the weight, the more feeding by the cotton bollworm, the more serious the tomato plant is infested; the lowercase letters a and b represent significant differences between different plants at the level of P<0.05 in the T test. .

图4为番茄SlBZR1基因敲除的突变体植株进行机械伤加棉铃虫口器分泌物处理(W+OS)模拟棉铃虫取食后的JA含量；Figure 4 shows the JA content of tomato SlBZR1 knockout mutant plants after mechanical injury and cotton bollworm mouthpart secretion treatment (W+OS) to simulate the feeding of cotton bollworm;

其中，JA含量越多，代表植株抗虫性越强；小写字母a、b代表不同植株之间在单因素ANOVA检验中P<0.05水平上的差异显著。Among them, the more JA content, the stronger the insect resistance of the plant; the lowercase letters a and b represent significant differences between different plants at the P<0.05 level in the single-factor ANOVA test.

图5为SlBZR1蛋白调控Acx1基因转录的荧光素酶报告基因检测比值；Fig. 5 is the luciferase reporter gene detection ratio of SlBZR1 protein regulating Acx1 gene transcription;

其中，小写字母a、b代表不同处理之间在T检验中P<0.05水平上的差异显著。Among them, lowercase letters a and b represent significant differences between different treatments at the P<0.05 level in the T test.

具体实施方式Detailed ways

下面结合具体实施例对本发明作进一步描述，以下列举的仅是本发明的具体实施例，但本发明的保护范围不仅限于此。The present invention will be further described below in conjunction with specific embodiments, the following are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto.

下述实施例中所用实验方法如无特殊说明，均为常规方法。The experimental methods used in the following examples are conventional methods unless otherwise specified.

下述实施例中所用实验材料、试剂等，如无特殊说明，均可从商业途径得到。The experimental materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

下述实施例中采用的番茄品种为番茄常规品种Condine Red。The tomato variety used in the following examples is the conventional tomato variety Condine Red.

实施例1番茄SlBZR1缺失突变体植株的制备与鉴定Example 1 Preparation and identification of tomato SlBZR1 deletion mutant plants

1、含特异sgRNA的CRISPR/Cas9载体的构建1. Construction of CRISPR/Cas9 vector containing specific sgRNA

首先，在SGN网站https://solgenomics.net/上找到番茄SlBZR1基因的DNA全长序列如SEQ ID NO.4所示，将其输入http://crispr.hzau.edu.cn/CRISPR2/网站，找出onscore得分高，且GC含量>40％，位于蛋白编码区的一段PAM结构前的20bp碱基序列作为特异性靶向基因蛋白编码区的sgRNA，如SEQ ID NO.5所示。First, find the full-length DNA sequence of the tomato SlBZR1 gene on the SGN website https://solgenomics.net/ as shown in SEQ ID NO.4, and enter it into the website http://crispr.hzau.edu.cn/CRISPR2/ , find out the 20bp base sequence with high onscore score and GC content>40%, located in front of a PAM structure in the protein coding region as the sgRNA specifically targeting the protein coding region of the gene, as shown in SEQ ID NO.5.

合成单链sgRNA序列后退火形成双链sgRNA，且其两端具有BbsⅠ限制性内切酶酶切位点，上述合成片段插入到AtU6-sgRNA-AtUBQ-Cas9载体的BbsⅠ位点，得到中间载体。The single-stranded sgRNA sequence was synthesized and then annealed to form a double-stranded sgRNA with BbsI restriction endonuclease cleavage sites at both ends. The above-mentioned synthetic fragment was inserted into the BbsI site of the AtU6-sgRNA-AtUBQ-Cas9 vector to obtain an intermediate vector.

利用HindⅢ和KpnⅠ限制性内切酶对上述中间载体及pCAMBIA1301载体进行双酶切并连接转化大肠杆菌DH5α感受态，挑单克隆进行测序，测序正确的质粒电击转化GV3101农杆菌感受态，获得可用于构建CRISPR/Cas9基因编辑材料的农杆菌菌株。The above intermediate vector and pCAMBIA1301 vector were double-enzyme digested with HindIII and KpnI restriction enzymes and ligated to transform E. coli DH5α competent. Single clones were picked for sequencing, and the correct plasmid was electroporated to transform GV3101 Agrobacterium-competent to obtain a plasmid that can be used for Construction of Agrobacterium strains for CRISPR/Cas9 gene editing materials.

2、SlBZR1基因突变体材料的制备与鉴定2. Preparation and identification of SlBZR1 mutant materials

番茄种子经消毒后播种于1/2MS培养基中，黑暗培养至种子发芽后，移至光下培养。待子叶长出后，将子叶切下置于看护培养基中，黑暗培养12-24h。将准备好的用于构建CRISPR/Cas9基因编辑材料的农杆菌培养至对数期，离心去上清，加入MS0.2重悬浮，用于侵染子叶外植体，暗下侵染2.5min后，吸干子叶外植体表面剩余菌液后置于看护培养基上，黑暗下共培养两天。共培养后，子叶外植体转移至2Z培养基中诱导愈伤组织，2-3周后转入0.2Z培养基中诱导发芽，每2周转入新的0.2Z培养基，直至长出小苗后，转入R培养基诱导生根，约2-3周后待根系发育良好，将其转移至基质中并进行保湿，成活后对T₀代基因编辑材料进行验证。Tomato seeds were sterilized and sown in 1/2 MS medium, cultured in the dark until the seeds germinate, and then moved to light for culture. After the cotyledons grow, the cotyledons are cut off and placed in a care medium for 12-24h in the dark. The prepared Agrobacterium for constructing CRISPR/Cas9 gene editing materials was cultured to log phase, centrifuged to remove the supernatant, and resuspended by adding MS0.2 for infecting cotyledon explants. After 2.5 min of infection in the dark, The remaining bacterial liquid on the surface of the cotyledon explants was blotted dry and placed on the care medium, and co-cultured for two days in the dark. After co-cultivation, the cotyledon explants were transferred to 2Z medium to induce callus, transferred to 0.2Z medium after 2-3 weeks to induce germination, and transferred to new 0.2Z medium every 2 weeks until seedlings grew. Then, it was transferred to R medium to induce rooting. After about 2-3 weeks, when the root system was well developed, it was transferred to the substrate and moisturised. After survival, the T₀ generation gene editing material was verified.

取少量T₀代植株叶片提取基因组DNA并以其为模板，进行PCR扩增含有sgRNA的DNA片段，然后对PCR产物进行测序比对，选取sgRNA序列发生碱基突变、缺失或插入的植株进行自交繁种，获得T₀代种子。Take a small amount of T₀ generation plant leaves to extract genomic DNA and use it as a template, carry out PCR amplification of DNA fragments containing sgRNA, then sequence and compare the PCR products, and select plants with base mutations, deletions or insertions in the sgRNA sequence to carry out self-assembly. Cross-breed the seeds to obtain T₀ generation seeds.

验证引物如下：Verify primers are as follows:

验证前引物：5’-AGTAAGCAAAACCCACCTGC-3’(SEQ ID NO.6)；Primer before verification: 5'-AGTAAGCAAAACCCACCTGC-3' (SEQ ID NO.6);

验证后引物：5’-TTCATAATCAATTCTAGATC-3’(SEQ ID NO.7)。Primer after verification: 5'-TTCATAATCAATTCTAGATC-3' (SEQ ID NO. 7).

将T₀代的种子播种后得到T₁代植株，利用上述方法检测T₁代植株的sgRNA序列碱基编辑情况及Cas9的存在与否，选取不含Cas9，但sgRNA发生变异的植株进行繁种，自交获得不含外源基因Cas9，且稳定遗传的T₁代纯合突变体植株1个株系，其基因编辑位点如图1所示。bzr1突变体比对照缺少10个碱基。After sowing the seeds of the T₀ generation, the T₁ generation plants were obtained. The above method was used to detect the base editing situation of the sgRNA sequence of the T₁ generation plants and the presence or absence of Cas9, and the plants without Cas9 but with sgRNA variation were selected for breeding. , a line of T₁ generation homozygous mutant plants without exogenous gene Cas9 and stable inheritance was obtained by selfing, and its gene editing site is shown in Figure 1. The bzr1 mutant is 10 bases shorter than the control.

对上述株系进一步自交繁种，种子播种后获得不含外源基因Cas9，且sgRNA发生变异的稳定遗传的T₂代植株。The above lines were further self-bred, and after sowing the seeds, a stable genetic T₂ generation plant without the exogenous gene Cas9 and sgRNA mutated was obtained.

以下实施例均以T₂代纯合株系作为材料进行实验。The following examples all use T₂ generation homozygous strains as materials for experiments.

实施例2番茄SlBZR1基因抗棉铃虫表型的鉴定Example 2 Identification of tomato SlBZR1 gene resistance to cotton bollworm phenotype

棉铃虫采用河南省济源白云实业有限公司购买的3龄棉铃虫，挑选大小一致的棉铃虫至24孔板中，进行饥饿一晚的处理。The cotton bollworms were the 3rd instar cotton bollworms purchased by Jiyuan Baiyun Industrial Co., Ltd. in Henan Province, and the cotton bollworms of the same size were selected into 24-well plates and starved for one night.

待番茄幼苗长至六叶一心时，选取从上往下第2-4节位叶片，称取相同质量叶片(1±0.05g)置于装有1％琼脂的培养皿中，以防止离体叶片失水，并对接虫前番茄叶片进行拍照记录。然后将两头经饥饿处理的棉铃虫接种到一个培养皿的番茄叶片上，盖上培养皿盖并用皮筋固定，防止棉铃虫离开培养皿。弱光下接虫处理3天后，检测棉铃虫重量，然后用70％甲醇浸泡棉铃虫至其不再活动，按体型大小排列后进行拍照记录。对接虫后番茄叶片也相应进行拍照记录及称重。When the tomato seedlings grow to six leaves and one heart, select the 2nd-4th node leaves from top to bottom, weigh the leaves of the same mass (1±0.05g) and place them in a petri dish with 1% agar to prevent in vitro The leaves were dehydrated, and the tomato leaves before the docking were photographed and recorded. Two starved bollworms were then inoculated on tomato leaves in a petri dish, and the petri dish was covered and secured with rubber bands to prevent bollworms from leaving the petri dish. After 3 days of inoculation treatment under low light, the weight of cotton bollworms was detected, and then the cotton bollworms were soaked in 70% methanol until they were no longer active, and were arranged according to body size and then photographed and recorded. The tomato leaves were also photographed and weighed accordingly after docking.

结果如图2和图3所示，饲喂bzr1突变体叶片的棉铃虫相比饲喂野生型叶片的棉铃虫体型较小、重量较轻，且bzr1突变体叶片经接虫处理后受到的损失相比野生型叶片更轻，受损后叶片重量较重，说明通过敲除SlBZR1基因可以提高番茄的棉铃虫抗性。The results are shown in Figures 2 and 3. The bollworms fed on bzr1 mutant leaves were smaller in size and lighter in weight than those fed on wild-type leaves, and the bzr1 mutant leaves suffered losses after inoculation treatment. Compared with the wild-type leaves, the weight of the damaged leaves is heavier, indicating that the knockout of the SlBZR1 gene can improve the resistance of tomato bollworm.

实施例3番茄SlBZR1基因对抗性激素JA的影响Example 3 The effect of tomato SlBZR1 gene against the anti-hormone JA

待番茄幼苗长至六叶一心时，选取从上往下第3-4节位叶片进行W+OS模拟棉铃虫取食处理，并于0、1h采集叶片样品分析JA激素水平，每个时间点设置4个重复。When the tomato seedlings grew to six leaves and one heart, the 3rd to 4th node leaves from top to bottom were selected for W+OS simulated cotton bollworm feeding treatment, and leaf samples were collected at 0 and 1 h to analyze JA hormone levels. Set 4 repetitions.

W+OS模拟棉铃虫取食处理方式如下：用止血钳在小叶主叶脉两侧叶面各夹一次制造机械伤，然后在创伤处涂抹稀释五倍的棉铃虫口器分泌物。The W+OS simulated H. armigera feeding treatment method was as follows: use a hemostatic forceps to make mechanical wounds on both sides of the leaflet's main vein, and then apply five-fold diluted H. armigera mouthpart secretions on the wounds.

棉铃虫口器分泌物的获取方式为：棉铃虫饥饿12h后，饲喂番茄叶片，12h后吸取棉铃虫口器分泌物为原液。The method of obtaining the mouthpart secretions of the cotton bollworm is as follows: after the cotton bollworm is starved for 12 hours, the tomato leaves are fed, and the mouthpart secretions of the cotton bollworm are absorbed as the stock solution after 12 hours.

JA激素测定方式为：称取0.1g番茄叶片，经液氮研磨后，加入1ml色谱级乙酸乙酯，避光加入25μl激素内标，涡旋混匀后，包上一层锡箔纸以避光，置于4℃摇床过夜，摇床频率为150rpm。4℃、12000rpm离心10min后，吸取850μl上清于新的10ml管中，4℃避光保存。剩下的沉淀中再加入1ml色谱级乙酸乙酯，重悬后置于4℃摇床震荡1h，再次4℃、12000rpm离心10min后，吸取900μl上清。合并两次上清，利用氮气吹干。加入500μl 70％色谱级甲醇，涡旋30s，转移液体至1.5ml管中，瞬离后吸取200μl上清至进样瓶内衬中，利用高效液相色谱串联质谱法测定(Varian320-MSLC/MS，Agilent Technologies，Amstelveen，Netherlands)。The JA hormone determination method is as follows: weigh 0.1 g of tomato leaves, grind them in liquid nitrogen, add 1 ml of chromatographic grade ethyl acetate, add 25 μl of the hormone internal standard in the dark, vortex and mix evenly, and wrap a layer of tin foil to protect from light. , placed on a shaker at 4°C overnight with a shaking frequency of 150 rpm. After centrifugation at 12000 rpm for 10 min at 4°C, 850 μl of the supernatant was pipetted into a new 10ml tube, and stored at 4°C in the dark. 1 ml of chromatographic grade ethyl acetate was added to the remaining precipitate, resuspended, placed on a shaker at 4°C for 1 hour, centrifuged again at 4°C and 12000 rpm for 10 min, and 900 μl of the supernatant was aspirated. The two supernatants were combined and blown dry with nitrogen. Add 500 μl of 70% chromatographic grade methanol, vortex for 30 s, transfer the liquid to a 1.5 ml tube, pipette 200 μl of the supernatant into the liner of the injection vial, and measure it by high performance liquid chromatography tandem mass spectrometry (Varian320-MSLC/MS , Agilent Technologies, Amstelveen, Netherlands).

结果如图4所示，W+OS处理后1h，bzr1突变体植株的JA含量显著高于野生型植株，说明敲除SlBZR1基因可以提高番茄植株在棉铃虫取食后的JA含量，从而提高番茄的棉铃虫抗性。The results are shown in Figure 4. 1h after W+OS treatment, the JA content of bzr1 mutant plants was significantly higher than that of wild-type plants, indicating that knocking out the SlBZR1 gene can increase the JA content of tomato plants after H. armigera feeding, thereby increasing tomato plants. of cotton bollworm resistance.

实施例4番茄SlBZR1蛋白负调控Acx1基因的转录验证Example 4 Transcription verification of tomato SlBZR1 protein negatively regulates Acx1 gene

1、构建含SlBZR1基因的pGreenII 0029 62-SK载体：以番茄cDNA为模板，进行PCR扩增，得到SlBZR1 CDS序列。引物如下：1. Construction of pGreenII 0029 62-SK vector containing SlBZR1 gene: using tomato cDNA as a template, PCR amplification was performed to obtain the SlBZR1 CDS sequence. The primers are as follows:

SlBZR1-SK-F：SlBZR1-SK-F:

5’-cgctctagaactagtggatccATGTGGGAAGGTGGAGGGTT-3’(SEQ ID NO.8)；SlBZR1-SK-R：5'-cgctctagaactagtggatccATGTGGGAAGGTGGAGGGTT-3' (SEQ ID NO. 8); SlBZR1-SK-R:

5’-gataagcttgatatcgaattcTCACATCCGAGCAGTCCCAC-3’(SEQ ID NO.9)；5'-gataagcttgatatcgaattcTCACATCCGAGCAGTCCCAC-3' (SEQ ID NO. 9);

利用BamHⅠ和EcoRⅠ限制性内切酶对上述载体及PCR产物进行双酶切，并连接转化大肠杆菌DH5α感受态。The above vector and PCR products were double digested with BamHI and EcoRI restriction enzymes, and then ligated and transformed into E. coli DH5α competent.

2、构建含Acx1基因启动子序列的pGreen II 0800-LUC载体：方法同上，以番茄基因组DNA为模板，酶切位点为NotⅠ和NcoⅠ，所用引物如下：2. Construct the pGreen II 0800-LUC vector containing the Acx1 gene promoter sequence: the method is the same as above, with tomato genomic DNA as the template, the restriction sites are NotI and NcoI, and the primers used are as follows:

pAcx1-LUC-F：pAcx1-LUC-F:

5’-tccactagttctagagcggccgcTTTTTTTCATGTTGGACGGTCTG-3’(SEQ ID NO.10)；5'-tccactagttctagagcggccgcTTTTTTTTCATGTTGGACGGTCTG-3' (SEQ ID NO. 10);

pAcx1-LUC-R：pAcx1-LUC-R:

5’-tgtttttggcgtcttccatggAATAAAATGTTCGTCCACTTTAATATATTC-3’(SEQ IDNO.11)。5'-tgtttttggcgtcttccatggAATAAAATGTTCGTCCACTTTAATATATTC-3' (SEQ ID NO. 11).

3、将测序正确的质粒电击转化农杆菌GV3101感受态，获得可用于双荧光素酶报告基因检测实验的农杆菌菌株。3. Transform the correctly sequenced plasmid into the competent Agrobacterium GV3101 by electric shock to obtain an Agrobacterium strain that can be used for the dual luciferase reporter gene detection experiment.

4、将上述两种农杆菌菌液在含有150μmol/L乙酰丁香酮的侵染液中重悬并调至最佳浓度(OD₆₀₀＝0.75)，置于28℃培养箱活化3h，按SlBZR1-SK：pAcx1-LUC＝10:1比例混合后共侵染本氏烟(Nicotiana benthamiana)叶片，3天后采用美国Promega公司的双荧光素酶报告基因检测系统(Dual-Luciferase Reporter Assay System)检测萤火虫荧光素酶和海肾荧光素酶活性，计算两者荧光比值与pGreenII 0029 62-SK空载体(Empty Vector)加Acx1启动子(设为1)的值的关系。4. Resuspend the above two kinds of Agrobacterium bacteria solutions in the infection solution containing 150 μmol/L acetosyringone and adjust to the optimal concentration (OD₆₀₀ =0.75), place them in a 28°C incubator for activation for 3 hours, press SlBZR1- The leaves of Nicotiana benthamiana were co-infected after mixing SK:pAcx1-LUC=10:1. After 3 days, the firefly fluorescence was detected by the Dual-Luciferase Reporter Assay System from Promega, USA. The luciferase and Renilla luciferase activities were calculated, and the relationship between the fluorescence ratio of the two and the value of the pGreenII 0029 62-SK empty vector (Empty Vector) plus the Acx1 promoter (set to 1) was calculated.

结果如图5所示，相比空载体，BZR1的表达使得Acx1启动子受到抑制，LUC/REN相对值较低，说明BZR1抑制了Acx1的转录。The results are shown in Figure 5. Compared with the empty vector, the expression of BZR1 inhibited the Acx1 promoter and the relative value of LUC/REN was lower, indicating that BZR1 inhibited the transcription of Acx1.

序列表sequence listing

<110> 浙江大学<110> Zhejiang University

浙江大学山东（临沂）现代农业研究院Shandong (Linyi) Institute of Modern Agriculture, Zhejiang University

<120> BZR1基因在调控植物对虫害胁迫抗性中的应用<120> Application of BZR1 gene in regulating plant resistance to insect pests

<160> 11<160> 11

<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0

<210> 1<210> 1

<211> 987<211> 987

<212> DNA<212> DNA

<213> 番茄(Solanum lycopersicum L.)<213> Tomato (Solanum lycopersicum L.)

<400> 1<400> 1

atgatgtggg aaggtggagg gttgccggtg gagggtggtg gtggtgttgg tgaaggtggt 60atgatgtggg aaggtggagg gttgccggtg gagggtggtg gtggtgttgg tgaaggtggt 60

ggtgttggtg gtggtggagg tggtggtagt gggaggagga agccatcatg gagggaaagg 120ggtgttggtg gtggtggagg tggtggtagt gggaggagga agccatcatg gagggaaagg 120

gagaataata ggaggaggga aaggaggaga agggcaatag cagctaagat ttatagtgga 180gagaataata ggaggaggga aaggaggaga agggcaatag cagctaagat ttatagtgga 180

ttaagagcac aggggaatta taatcttcct aaacattgtg ataacaatga ggttttgaag 240ttaagagcac aggggaatta taatcttcct aaacattgtg ataacaatga ggttttgaag 240

gctctttgtg ttgaagctgg atggattgtt gagcctgatg gaactactta tagaaaggga 300gctctttgtg ttgaagctgg atggattgtt gagcctgatg gaactactta tagaaaggga 300

tgcaggccaa ctccaatgga gattggaggc acttcagcca acattacgcc aagttcttca 360tgcaggccaa ctccaatgga gattggaggc acttcagcca acattacgcc aagttcttca 360

cgaaatccaa gtcctccctc ttcatacttt gctagcccga ttccatctta ccaagttagt 420cgaaatccaa gtcctccctc ttcatacttt gctagcccga ttccatctta ccaagttagt 420

ccaacatcct cgtctttccc aagtccatct cgtggtgatg ctaacatgtc gtcacatcca 480ccaacatcct cgtctttccc aagtccatct cgtggtgatg ctaacatgtc gtcacatcca 480

tttgcatttc tccatagttc cattcccttg tcgctaccac cattacgaat atcaaacagt 540tttgcatttc tccatagttc cattcccttg tcgctaccac cattacgaat atcaaacagt 540

gcccctgtaa caccacctct ttcatcacca actagagtcc ctaagcagat atttaatctt 600gcccctgtaa caccacctct ttcatcacca actagagtcc ctaagcagat atttaatctt 600

gagactttgg ctagagagtc tatgtctgct ctaaatatcc ctttctttgc tgcttcagcc 660gagactttgg ctagagagtc tatgtctgct ctaaatatcc ctttctttgc tgcttcagcc 660

ccaactagcc caactcgagg tcagcgattc actcctgcta caataccaga gtgtgacgaa 720ccaactagcc caactcgagg tcagcgattc actcctgcta caataccaga gtgtgacgaa 720

tctgattcat ccaccattga ttctggccag tggatgagct ttcaaaagta cgcagccaat 780tctgattcat ccaccattga ttctggccag tggatgagct ttcaaaagta cgcagccaat 780

gggatcccta cttctccgac ttttaatctt attaagcctg tagctcagag aattccttct 840gggatcccta cttctccgac ttttaatctt attaagcctg tagctcagag aattccttct 840

aatgatatga tcatcgacaa gggtaagagc attgaatttg actttgagaa tgtatcagtt 900aatgatatga tcatcgacaa gggtaagagc attgaatttg actttgagaa tgtatcagtt 900

aaggcagcat gggaaggtga aaagattcat gaggttggtt tagatgatct ggagctcact 960aaggcagcat gggaaggtga aaagattcat gaggttggtt tagatgatct ggagctcact 960

ctcggaagtg ggactgctcg gatgtga 987ctcggaagtg ggactgctcg gatgtga 987

<210> 2<210> 2

<211> 328<211> 328

<212> PRT<212> PRT

<213> 番茄(Solanum lycopersicum L.)<213> Tomato (Solanum lycopersicum L.)

<400> 2<400> 2

Met Met Trp Glu Gly Gly Gly Leu Pro Val Glu Gly Gly Gly Gly ValMet Met Trp Glu Gly Gly Gly Leu Pro Val Glu Gly Gly Gly Gly Val

1 5 10 151 5 10 15

Gly Glu Gly Gly Gly Val Gly Gly Gly Gly Gly Gly Gly Ser Gly ArgGly Glu Gly Gly Gly Gly Val Gly Gly Gly Gly Gly Gly Gly Gly Ser Gly Arg

20 25 30 20 25 30

Arg Lys Pro Ser Trp Arg Glu Arg Glu Asn Asn Arg Arg Arg Glu ArgArg Lys Pro Ser Trp Arg Glu Arg Glu Asn Asn Arg Arg Arg Glu Arg

35 40 45 35 40 45

Arg Arg Arg Ala Ile Ala Ala Lys Ile Tyr Ser Gly Leu Arg Ala GlnArg Arg Arg Ala Ile Ala Ala Lys Ile Tyr Ser Gly Leu Arg Ala Gln

50 55 60 50 55 60

Gly Asn Tyr Asn Leu Pro Lys His Cys Asp Asn Asn Glu Val Leu LysGly Asn Tyr Asn Leu Pro Lys His Cys Asp Asn Asn Glu Val Leu Lys

65 70 75 8065 70 75 80

Ala Leu Cys Val Glu Ala Gly Trp Ile Val Glu Pro Asp Gly Thr ThrAla Leu Cys Val Glu Ala Gly Trp Ile Val Glu Pro Asp Gly Thr Thr

85 90 95 85 90 95

Tyr Arg Lys Gly Cys Arg Pro Thr Pro Met Glu Ile Gly Gly Thr SerTyr Arg Lys Gly Cys Arg Pro Thr Pro Met Glu Ile Gly Gly Thr Ser

100 105 110 100 105 110

Ala Asn Ile Thr Pro Ser Ser Ser Arg Asn Pro Ser Pro Pro Ser SerAla Asn Ile Thr Pro Ser Ser Ser Arg Asn Pro Ser Pro Pro Ser Ser

115 120 125 115 120 125

Tyr Phe Ala Ser Pro Ile Pro Ser Tyr Gln Val Ser Pro Thr Ser SerTyr Phe Ala Ser Pro Ile Pro Ser Tyr Gln Val Ser Pro Thr Ser Ser

130 135 140 130 135 140

Ser Phe Pro Ser Pro Ser Arg Gly Asp Ala Asn Met Ser Ser His ProSer Phe Pro Ser Pro Ser Arg Gly Asp Ala Asn Met Ser Ser His Pro

145 150 155 160145 150 155 160

Phe Ala Phe Leu His Ser Ser Ile Pro Leu Ser Leu Pro Pro Leu ArgPhe Ala Phe Leu His Ser Ser Ile Pro Leu Ser Leu Pro Pro Leu Arg

165 170 175 165 170 175

Ile Ser Asn Ser Ala Pro Val Thr Pro Pro Leu Ser Ser Pro Thr ArgIle Ser Asn Ser Ala Pro Val Thr Pro Pro Leu Ser Ser Pro Thr Arg

180 185 190 180 185 190

Val Pro Lys Gln Ile Phe Asn Leu Glu Thr Leu Ala Arg Glu Ser MetVal Pro Lys Gln Ile Phe Asn Leu Glu Thr Leu Ala Arg Glu Ser Met

195 200 205 195 200 205

Ser Ala Leu Asn Ile Pro Phe Phe Ala Ala Ser Ala Pro Thr Ser ProSer Ala Leu Asn Ile Pro Phe Phe Ala Ala Ser Ala Pro Thr Ser Pro

210 215 220 210 215 220

Thr Arg Gly Gln Arg Phe Thr Pro Ala Thr Ile Pro Glu Cys Asp GluThr Arg Gly Gln Arg Phe Thr Pro Ala Thr Ile Pro Glu Cys Asp Glu

225 230 235 240225 230 235 240

Ser Asp Ser Ser Thr Ile Asp Ser Gly Gln Trp Met Ser Phe Gln LysSer Asp Ser Ser Thr Ile Asp Ser Gly Gln Trp Met Ser Phe Gln Lys

245 250 255 245 250 255

Tyr Ala Ala Asn Gly Ile Pro Thr Ser Pro Thr Phe Asn Leu Ile LysTyr Ala Ala Asn Gly Ile Pro Thr Ser Pro Thr Phe Asn Leu Ile Lys

260 265 270 260 265 270

Pro Val Ala Gln Arg Ile Pro Ser Asn Asp Met Ile Ile Asp Lys GlyPro Val Ala Gln Arg Ile Pro Ser Asn Asp Met Ile Ile Asp Lys Gly

275 280 285 275 280 285

Lys Ser Ile Glu Phe Asp Phe Glu Asn Val Ser Val Lys Ala Ala TrpLys Ser Ile Glu Phe Asp Phe Glu Asn Val Ser Val Lys Ala Ala Trp

290 295 300 290 295 300

Glu Gly Glu Lys Ile His Glu Val Gly Leu Asp Asp Leu Glu Leu ThrGlu Gly Glu Lys Ile His Glu Val Gly Leu Asp Asp Leu Glu Leu Thr

305 310 315 320305 310 315 320

Leu Gly Ser Gly Thr Ala Arg MetLeu Gly Ser Gly Thr Ala Arg Met

325 325

<210> 3<210> 3

<211> 480<211> 480

<212> DNA<212> DNA

<213> 番茄(Solanum lycopersicum L.)<213> Tomato (Solanum lycopersicum L.)

<400> 3<400> 3

tttttttcat gttggacggt ctgcctactt tatttttttt tgttcatttt ccgttagtga 60tttttttcat gttggacggt ctgcctactt tatttttttt tgttcatttt ccgttagtga 60

aatctgatta aatttaaata atagagcatg aaatttattt ggagtgatat atttaacaca 120aatctgatta aatttaaata atagagcatg aaatttattt ggagtgatat atttaacaca 120

attttttcta tatcatgtgt ttgaatttga aattttttat taaagatgaa ataatttctc 180atttttttcta tatcatgtgt ttgaatttga aattttttat taaagatgaa ataatttctc 180

cgttgcacga taactcattt tagaagtacc attattgatc aaaatactat taaatattca 240cgttgcacga taactcattt tagaagtacc attattgatc aaaatactat taaatattca 240

aataaaattt attgacaaat tgatgtatcg ctctttttaa aaaaaacata ttaggagtac 300aataaaattt attgacaaat tgatgtatcg ctctttttaa aaaaaacata ttaggagtac 300

tgaaaataga aaaatatggg ctaaatatcc tcttatcgtc actataccga ctgttcacac 360tgaaaataga aaaatatggg ctaaatatcc tcttatcgtc actataccga ctgttcacac 360

tgcggaccac acgaagcaaa aaaaaaaagg ctataattat actcttaata tactatgagc 420tgcggaccac acgaagcaaa aaaaaaaagg ctataattat actcttaata tactatgagc 420

attttttttt aaaaaaaata tatatactat gaatatatta aagtggacga acattttatt 480attttttttt aaaaaaaata tatatactat gaatatatta aagtggacga acattttatt 480

<210> 4<210> 4

<211> 3493<211> 3493

<212> DNA<212> DNA

<213> 番茄(Solanum lycopersicum L.)<213> Tomato (Solanum lycopersicum L.)

<400> 4<400> 4

taaaataaac ttattcattt attatttata aacaagttat attataattt ttgataatca 60taaaataaac ttattcattt attatttata aacaagttat attataattt ttgataatca 60

aactttctga ttcaacctca ttataaaata tccttgttat tgccttatcg ctttttctat 120aactttctga ttcaacctca ttataaaata tccttgttat tgccttatcg ctttttctat 120

ctataaaatt tatcacaatt atacaaaaaa aaaattactt taattttatc tatgtgtcta 180ctataaaatt tatcacaatt atacaaaaaa aaaattactt taattttatc tatgtgtcta 180

tcataactaa cctcgattta agaccagaaa acttataata acatagctat taccccatta 240tcataactaa cctcgattta agaccagaaa acttataata acatagctat taccccatta 240

ataaaatttt aaaaagcagt caaacattaa aaactttata tttaaattcc aaaatataaa 300ataaaatttt aaaaagcagt caaacattaa aaactttata tttaaattcc aaaatataaa 300

ctagtaaata acaattatta tcttgcttca tttgataagt aaaaaattat ttcaactctt 360ctagtaaata acaattatta tcttgcttca tttgataagt aaaaaattat ttcaactctt 360

aaataacagt caaaaaaatt aatttagtag tctaatcatg taatttgtaa aatctaaaaa 420aaataacagt caaaaaaatt aatttagtag tctaatcatg taatttgtaa aatctaaaaa 420

agtagtcaaa gttttttttt ctttttgcca tttttaaaaa atagaatgaa aaacgcttac 480agtagtcaaa gttttttttt ctttttgcca ttttttaaaaa atagaatgaa aaacgcttac 480

acatgataat tctgaagctg taacaaactt tatctcttca attattataa ttttgctaaa 540acatgataat tctgaagctg taacaaactt tatctcttca attattataa ttttgctaaa 540

aaacaaaaca cacaaaacac catttcatca tcattttttt tttatatata tatatatata 600aaacaaaaca cacaaaacac catttcatca tcattttttt tttatatata tatatatata 600

acacaaacac acagagagaa aagtaaagaa gaaaattctt cagattgttt gtagtagtat 660acacaaacac acagagagaa aagtaaagaa gaaaattctt cagattgttt gtagtagtat 660

aaactttaga gcgtatttct cgtgagtagt cccaaaaaaa tgctttatag agtggcaccc 720aaactttaga gcgtatttct cgtgagtagt cccaaaaaaa tgctttatag agtggcaccc 720

ctagtaccta tacaacacag taagcaaaac ccacctgctg tctgaacaca cccacctggg 780ctagtaccta tacaacacag taagcaaaac ccacctgctg tctgaacaca cccacctggg 780

accagccccc ctttttcttc ttctttcttc tttggattca tcatataaaa aaaacccatt 840accagcccccc ctttttcttc ttctttcttc tttggattca tcatataaaa aaaacccatt 840

ttcttaattt tgagaaatct tgaaaaaaat cgattctttt tggggagaaa atgatgtggg 900ttcttaattt tgagaaatct tgaaaaaaat cgattctttt tggggagaaa atgatgtggg 900

aaggtggagg gttgccggtg gagggtggtg gtggtgttgg tgaaggtggt ggtgttggtg 960aaggtggagg gttgccggtg gagggtggtg gtggtgttgg tgaaggtggt ggtgttggtg 960

gtggtggagg tggtggtagt gggaggagga agccatcatg gagggaaagg gagaataata 1020gtggtggagg tggtggtagt gggaggagga agccatcatg gagggaaagg gagaataata 1020

ggaggaggga aaggaggaga agggcaatag cagctaagat ttatagtgga ttaagagcac 1080ggaggaggga aaggaggaga agggcaatag cagctaagat ttatagtgga ttaagagcac 1080

aggggaatta taatcttcct aaacattgtg ataacaatga ggttttgaag gctctttgtg 1140aggggaatta taatcttcct aaacattgtg ataacaatga ggttttgaag gctctttgtg 1140

ttgaagctgg atggattgtt gagcctgatg gaactactta tagaaaggtt agtaaaattc 1200ttgaagctgg atggattgtt gagcctgatg gaactactta tagaaaggtt agtaaaattc 1200

atcttctttt atgaattgat gtatggttaa agtagtgttt gtgtcttagt ttttgtttgt 1260atcttctttt atgaattgat gtatggttaa agtagtgttt gtgtcttagt ttttgtttgt 1260

ttggattgaa ggggtgatgt gtttttggtt ggagttttgc ttagatctat agatttggaa 1320ttggattgaa ggggtgatgt gtttttggtt ggagttttgc ttagatctat agatttggaa 1320

aatggggttg ttgaatttgg atcaaagatt gtgtttttat ctgttttcag ttggtttttg 1380aatggggttg ttgaatttgg atcaaagatt gtgtttttat ctgttttcag ttggtttttg 1380

actgttttat ggttttctga tctagaattg attatgaaca gcttctgagt ttgtgttttc 1440actgttttat ggttttctga tctagaattg attatgaaca gcttctgagt ttgtgttttc 1440

tttagttcaa ttgttctttt atgtacttat aatctgttca tgttctcatt tctggataat 1500tttagttcaa ttgttctttt atgtacttat aatctgttca tgttctcatt tctggataat 1500

tggtatgaat gtatgtagtt taatttgtct gagaatggga gttgtttctg gaaaaaacaa 1560tggtatgaat gtatgtagtt taatttgtct gagaatggga gttgtttctg gaaaaaacaa 1560

gaaggaagaa ttgacttgtt cttaatatag aaatggataa gcagggtgtt ttggtgtcta 1620gaaggaagaa ttgacttgtt cttaatatag aaatggataa gcagggtgtt ttggtgtcta 1620

aggtggggtt tgatgcattt ggttgtggtg tttttagttt atcaactact tttcgagatt 1680aggtggggtt tgatgcattt ggttgtggtg tttttagttt atcaactact tttcgagatt 1680

caaagttgca atctgttgaa gttgttatct cagattacag agccttttga tcatttatgt 1740caaagttgca atctgttgaa gttgttatct cagattacag agccttttga tcatttatgt 1740

tcactagttt gatttgaaaa tgtcctcttt tactatgttt tcagggatgc aggccaactc 1800tcactagttt gatttgaaaa tgtcctcttt tactatgttt tcagggatgc aggccaactc 1800

caatggagat tggaggcact tcagccaaca ttacgccaag ttcttcacga aatccaagtc 1860caatggagat tggaggcact tcagccaaca ttacgccaag ttcttcacga aatccaagtc 1860

ctccctcttc atactttgct agcccgattc catcttacca agttagtcca acatcctcgt 1920ctccctcttc atactttgct agcccgattc catcttacca agttagtcca acatcctcgt 1920

ctttcccaag tccatctcgt ggtgatgcta acatgtcgtc acatccattt gcatttctcc 1980ctttcccaag tccatctcgt ggtgatgcta acatgtcgtc acatccattt gcatttctcc 1980

atagttccat tcccttgtcg ctaccaccat tacgaatatc aaacagtgcc cctgtaacac 2040atagttccat tcccttgtcg ctaccaccat tacgaatatc aaacagtgcc cctgtaacac 2040

cacctctttc atcaccaact agagtcccta agcagatatt taatcttgag actttggcta 2100cacctctttc atcaccaact agagtcccta agcagatatt taatcttgag actttggcta 2100

gagagtctat gtctgctcta aatatccctt tctttgctgc ttcagcccca actagcccaa 2160gagagtctat gtctgctcta aatatccctt tctttgctgc ttcagcccca actagcccaa 2160

ctcgaggtca gcgattcact cctgctacaa taccagagtg tgacgaatct gattcatcca 2220ctcgaggtca gcgattcact cctgctacaa taccagagtg tgacgaatct gattcatcca 2220

ccattgattc tggccagtgg atgagctttc aaaagtacgc agccaatggg atccctactt 2280ccattgattc tggccagtgg atgagctttc aaaagtacgc agccaatggg atccctactt 2280

ctccgacttt taatcttatt aagcctgtag ctcagagaat tccttctaat gatatgatca 2340ctccgacttt taatcttatt aagcctgtag ctcagagaat tccttctaat gatatgatca 2340

tcgacaaggg taagagcatt gaatttgact ttgagaatgt atcagttaag gcagcatggg 2400tcgacaaggg taagagcatt gaatttgact ttgagaatgt atcagttaag gcagcatggg 2400

aaggtgaaaa gattcatgag gttggtttag atgatctgga gctcactctc ggaagtggga 2460aaggtgaaaa gattcatgag gttggtttag atgatctgga gctcactctc ggaagtggga 2460

ctgctcggat gtgattttca tgattaaaag aactgctcag tgtggattta cagattgaga 2520ctgctcggat gtgattttca tgattaaaag aactgctcag tgtggattta cagattgaga 2520

ttatccgagc ttgctagctg atgattcact caagcagtcc agtggatgtt cgttacagag 2580ttatccgagc ttgctagctg atgattcact caagcagtcc agtggatgtt cgttacagag 2580

atagatcttg catgttgctg tttaccttat atacagacat tttttttttc cttttcgcct 2640atagatcttg catgttgctg tttaccttat atacagacat tttttttttc cttttcgcct 2640

tatctgatga ggcctttaag tttagtttgg gtcttgtcat aagcatttag gtatagatag 2700tatctgatga ggcctttaag tttagtttgg gtcttgtcat aagcatttag gtatagatag 2700

ttgatggttt ctatctcctt gtggttgaga attgtttcaa gtcattgtag actagcagga 2760ttgatggttt ctatctcctt gtggttgaga attgtttcaa gtcattgtag actagcagga 2760

gttctcctta aacctgaact atccacagtt atattttcat gattatagta gatccctttg 2820gttctcctta aacctgaact atccacagtt atattttcat gattatagta gatccctttg 2820

ttgttatgca actggatttc ttgttttatg atctagcgtc aattcaaaat caatggatgt 2880ttgttatgca actggatttc ttgttttatg atctagcgtc aattcaaaat caatggatgt 2880

gctattcttt aagtctcttt gatgctttat gtccagatca aaacattagt actagttctc 2940gctattcttt aagtctcttt gatgctttat gtccagatca aaacattagt actagttctc 2940

tttgtttaca cacactttca ctataccatt ttaactcata tgtccttaaa ggacaagcaa 3000tttgtttaca cacactttca ctataccatt ttaactcata tgtccttaaa ggacaagcaa 3000

tggtgggcca gaaaagactt tgacgactca agggttcata agtgagtgaa agtaagatag 3060tggtgggcca gaaaagactt tgacgactca agggttcata agtgagtgaa agtaagatag 3060

ccctctaaag gaagatctat tcagctcgat catctaattc actccaacag acatgcatga 3120ccctctaaag gaagatctat tcagctcgat catctaattc actccaacag acatgcatga 3120

ttccgtaatc gaaataactt ccatagggct ggccaatggc aagatggatg tcaagaagtt 3180ttccgtaatc gaaataactt ccatagggct ggccaatggc aagatggatg tcaagaagtt 3180

ggaatttttt gacaatcaaa tcaaagttgt tgaattttct ttgtcactca tttggctcca 3240ggaatttttt gacaatcaaa tcaaagttgt tgaattttct ttgtcactca tttggctcca 3240

ttagtaaact accagtagtc atattaataa taatagcatg ttcatgcaca cgtttgttat 3300ttagtaaact accagtagtc atattaataa taatagcatg ttcatgcaca cgtttgttat 3300

caacacaagc acagacagag aagaagaaag ataaaacaaa tggtgtttaa ctgatttaat 3360caacacaagc acagacagag aagaagaaag ataaaacaaa tggtgtttaa ctgatttaat 3360

gtacaagacc acggcctaat gtctggatgt ggggttctgt actactgtat tacacatgtt 3420gtacaagacc acggcctaat gtctggatgt ggggttctgt actactgtat tacacatgtt 3420

cctctacagt tactttttca ttattttttt ctcataacgc ggttattgtc acatgtcttg 3480cctctacagt tactttttca ttatttttttt ctcataacgc ggttattgtc acatgtcttg 3480

cttgctgcta tat 3493cttgctgcta tat 3493

<210> 5<210> 5

<211> 20<211> 20

<212> DNA<212> DNA

<213> 番茄(Solanum lycopersicum L.)<213> Tomato (Solanum lycopersicum L.)

<400> 5<400> 5

ggaagccatc atggagggaa 20ggaagccatc atggaggggaa 20

<210> 6<210> 6

<211> 20<211> 20

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 6<400> 6

agtaagcaaa acccacctgc 20agtaagcaaa acccacctgc 20

<210> 7<210> 7

<211> 20<211> 20

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 7<400> 7

ttcataatca attctagatc 20ttcataatca attctagatc 20

<210> 8<210> 8

<211> 41<211> 41

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 8<400> 8

cgctctagaa ctagtggatc catgtgggaa ggtggagggt t 41cgctctagaa ctagtggatc catgtgggaa ggtggagggt t 41

<210> 9<210> 9

<211> 41<211> 41

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 9<400> 9

gataagcttg atatcgaatt ctcacatccg agcagtccca c 41gataagcttg atatcgaatt ctcacatccg agcagtccca c 41

<210> 10<210> 10

<211> 46<211> 46

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 10<400> 10

tccactagtt ctagagcggc cgcttttttt catgttggac ggtctg 46tccactagtt ctagagcggc cgcttttttt catgttggac ggtctg 46

<210> 11<210> 11

<211> 51<211> 51

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 11<400> 11

tgtttttggc gtcttccatg gaataaaatg ttcgtccact ttaatatatt c 51tgtttttggc gtcttccatg gaataaaatg ttcgtccact ttaatatatt c 51

Claims (9)

1.BZR1The application of the gene in regulating and controlling insect pest stress resistance of the tomato is characterized in that the application comprises the following steps: making use of biotechnological meansBZR1The gene function is deleted, so that the resistance of the plant to insect pest stress is improved; the above-mentionedBZR1The coding sequence of the gene is shown in SEQ ID NO. 1.

2. Use according to claim 1, characterized in that said method comprises the step of determining the absolute value of the absolute valueBZR1The amino acid sequence of the protein coded by the gene is shown in SEQ ID NO. 2.

3. The use of claim 1, wherein the insect pest stress is cotton bollworm.

4. Use according to claim 1, characterized in that saidBZR1The gene-encoded protein negatively regulates the synthesis of jasmonic acid.

5. The use of claim 4, wherein saidBZR1The gene codesProtein binding to jasmonic acid synthesis related geneAcx1On the promoter of (1), negative regulationAcx1Transcription of the gene reduces the content of jasmonic acid.

6. Use according to claim 5, characterized in that saidAcx1The nucleotide sequence of the gene promoter is shown as SEQ ID NO. 3.

7. The application of claim 1, wherein the application comprises: the gene mutation, gene knockout, gene interference or gene silencing technology is used to causeBZR1The expression of the gene is reduced or deleted, so that mutant plants with enhanced resistance to insect pest stress are obtained.

8. The method for cultivating the cotton bollworm-resistant tomatoes is characterized by comprising the following steps of:

(1) in thatSlBZR1Selecting a target fragment containing a PAM structure from a protein coding region of the gene, designing a primer based on the first 20 bases of the PAM structure of the target fragment, and constructing a CRISPR/Cas9 vector; the above-mentionedSlBZR1The nucleotide sequence of the gene is shown as SEQ ID NO. 4;

(2) constructing agrobacterium gene engineering bacteria containing the CRISPR/Cas9 vector in the step (1);

(3) transforming the genetically engineered bacteria in the step (2) into tomato cotyledons to obtain a stably inherited homozygous mutant strain which does not contain exogenous Cas9 protein and has a target sequence with variation.

9. The method for cultivating tomato with resistance to cotton bollworm as claimed in claim 8, wherein the nucleotide sequence of the first 20bp of the target fragment PAM structure is shown as SEQ ID No. 5.

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