CN113549639B - Regulatory gene for reducing contents of total proteins and phenol in flue gas of tobacco leaves - Google Patents

Abstract

Translated fromChinese

本发明属于烟草基因工程领域，具体涉及一种降低烟叶总蛋白及烟气苯酚含量的调控基因烟草番茄红素ε‑环化酶基因Ntε‑LCY2，该基因碱基序列如SEQ ID NO.1所示。烟草番茄红素ε‑环化酶Ntε‑LCY2由498个氨基酸残基组成，其中第105‑287和290‑475位氨基酸是保守的转运蛋白结构域。该蛋白与植物叶片中总蛋白质含量相关，降低该基因表达后，叶片中总蛋白质含量明显降低。本发明通过对特定Ntε‑LCY2的初步研究，发现其与烟草总蛋白质含量高度相关，在将该基因突变后，烟草中总蛋白质含量明显降低。基于这一特性，可为低苯酚含量烟草新品种培育提供一定的应用基础和参考借鉴。

The invention belongs to the field of tobacco genetic engineering, and in particular relates to a regulatory gene tobacco lycopene ε-cyclase geneNtε-LCY2 that reduces the total protein and phenol content of tobacco leaves, and the base sequence of the gene is as shown in SEQ ID NO.1 Show. Tobacco lycopene ε-cyclase Ntε-LCY2 consists of 498 amino acid residues, of which amino acids 105-287 and 290-475 are conserved transporter domains. This protein is related to the total protein content in plant leaves. After reducing the expression of this gene, the total protein content in leaves is significantly reduced. Through preliminary research on specificNtε-LCY2 in the present invention, it is found that it is highly correlated with the total protein content of tobacco, and the total protein content in tobacco is significantly reduced after the gene is mutated. Based on this characteristic, it can provide a certain application basis and reference for the cultivation of new tobacco varieties with low phenol content.

Description

Translated fromChinese

一种降低烟叶总蛋白及烟气苯酚含量的调控基因A regulatory gene that reduces the total protein and phenol content of tobacco leaves

技术领域technical field

本发明属于烟草基因工程领域，具体涉及烟草番茄红素ε-环化酶基因Ntε-LCY2应用。The invention belongs to the field of tobacco genetic engineering, in particular to the application of tobacco lycopene ε-cyclase geneNtε-LCY2 .

背景技术Background technique

苯酚被国家烟草专卖局列为重点控制的7种代表性有害成分之一，其随烟气吸入后会快速分布到所有组织中，具有显著的黏膜渗透性，导致组织坏死和腐蚀脱落。长期吸入含苯酚气体会导致上呼吸道哮鸣、呼吸窘迫甚至衰竭，另外还会导致头痛、晕眩、肾脏损伤和心脏系统疾病、以及癌症等^[1-7]。苯酚还具有致突变和基因毒性等毒性作用，被列入EPA有害成分名单（US.EPA, 2002）。Phenol is listed as one of the seven representative harmful components under key control by the State Tobacco Monopoly Administration. It is rapidly distributed into all tissues after inhalation with smoke, and has significant mucosal permeability, leading to tissue necrosis and corrosion and shedding. Long-term inhalation of phenol-containing gas can lead to upper airway wheezing, respiratory distress and even failure, as well as headache, dizziness, kidney damage and cardiac system diseases, as well as cancer^[1-7] . Phenol also has toxic effects such as mutagenicity and genotoxicity, and is included in the EPA list of harmful ingredients (US.EPA, 2002).

烟气苯酚的来源和前体物已经明确，因此，应用生物技术干扰相关前体物含量是有效降低烟气苯酚含量的策略。烟叶蛋白质、游离酪氨酸（Tyr）和绿原酸（chlorogenicacid, CGA）是烟气苯酚的主要前体物。The sources and precursors of phenol in smoke have been clarified. Therefore, the application of biotechnology to interfere with the content of related precursors is an effective strategy to reduce the content of phenol in smoke. Tobacco leaf protein, free tyrosine (Tyr) and chlorogenic acid (CGA) are the main precursors of smoke phenol.

番茄红素ε-环化酶（lycopene ε-cyclase, ε-LCY）基因是植物类胡萝卜素合成通路中的关键基因，能够催化α-胡萝卜素和黄体素的合成。多数植物的ε-LCY基因在沉默或者缺失后能够提高植物β-胡萝卜素的合成及含量，与类胡萝卜素合成通路其他基因比较有着独特的生物学特性。ε-LCY基因是植物类胡萝卜素基因工程的优良靶标基因，对于提升植物的类胡萝卜素含量、抗逆性及品质有着较大的潜力。类胡萝卜素C40碳氢链末端的环化是类胡萝卜素合成通路中的第一个重要分支点。番茄红素环化酶基因由ε-番茄红素环化酶和β-番茄红素环化酶组成。ε-LCY酶只能催化番茄红素分子的一端，形成一个δ环，生成δ-胡萝卜素，而δ-胡萝卜素还可以进一步在β-LCY酶的催化下在另一端形成β环，最终生成α-胡萝卜素，然后α-胡萝卜素经过羟基化等作用生成黄体素，这一分支，被称为类胡萝卜素合成的α分支。而β-LCY酶可以催化番茄红素的两端，形成两个β环，生成β-胡萝卜素，后经羟基化、环氧化等作用生成β-隐黄质、玉米黄质、花药黄质、紫黄质和新黄质等各类胡萝卜素，这一分支被称为类胡萝卜素合成的β分支。因此，ε-LCY 和β-LCY两个酶的活性直接决定了底物α-胡萝卜素和β-胡萝卜素的比例，其合成流向对植物体内类胡萝卜素的组成有重要的影响The lycopene ε-cyclase (ε-LCY) gene is a key gene in the plant carotenoid synthesis pathway, which can catalyze the synthesis of α-carotene and lutein. Silencing or deletion of theε-LCY gene in most plants can increase the synthesis and content of β-carotene in plants, and has unique biological characteristics compared with other genes in the carotenoid synthesis pathway.ε-LCY gene is an excellent target gene for plant carotenoid genetic engineering, and has great potential to improve the carotenoid content, stress resistance and quality of plants. The cyclization of the end of the carotenoid C40 hydrocarbon chain is the first important branch point in the carotenoid synthesis pathway. The lycopene cyclase gene consists of ε-lycopene cyclase and β-lycopene cyclase. ε-LCY enzyme can only catalyze one end of the lycopene molecule to form a delta ring to generate delta-carotene, and delta-carotene can further form a beta ring at the other end under the catalysis of β-LCY enzyme, and finally generate Alpha-carotene, and then alpha-carotene undergoes hydroxylation and other actions to generate lutein, this branch is called the alpha branch of carotenoid synthesis. The β-LCY enzyme can catalyze the two ends of lycopene to form two β rings to generate β-carotene, and then undergo hydroxylation, epoxidation, etc. to generate β-cryptoxanthin, zeaxanthin, and antheraxanthin. Carotenoids such as violaxanthin, violaxanthin and neoxanthin, this branch is called the beta branch of carotenoid synthesis. Therefore, the activities of ε-LCY and β-LCY directly determine the ratio of substrates α-carotene and β-carotene, and their synthetic flow has an important impact on the composition of carotenoids in plants.

烟草Ntε-LCY基因的突变体在大田种植后品质性状有明显提升，其农艺性状与云烟87无显著差异，类胡萝卜素主成分叶黄素和β-胡萝卜素含量提高（大于20%）。Ntε-LCY基因的突变影响烟草叶片中叶绿素和类胡萝卜素的含量；并且与Ntε-LCY1基因突变相比，Ntε-LCY2基因的突变更有利于提高类胡萝卜素的含量，特别是在叶绿素、叶黄素和β-胡萝卜素等色素含量方面。Ntε-LCY基因的突变影响类胡萝卜素生物合成酶相关基因的表达量，在强光胁迫条件下，与Ntε-lcy1-1相比，Ntε-lcy2-1更有利于PSY、PDS、ZDS、CRTISO、β-LCY、β-OHase、VDE、NXS等基因的表达。Ntε-LCY基因的突变可以提高烟草植株的光合能力和热耗散能力，在强光胁迫条件下，烟草植株的光合能力有所降低，热耗散水平显著提高，与Ntε-LCY1基因的突变相比，Ntε-LCY2基因的突变更有助于提高烟草植株的光合能力和光保护能力。在正常光照和强光胁迫条件下，Ntε-LCY同源基因的突变均降低了O^2-和H₂O₂的含量，并且Ntε-LCY2基因的突变更有利于降低活性氧的积累量，从而对烟草植株起到保护作用。但是Ntε-LCY2基因在苯酚方面的研究在并无相关报道。The mutants ofNtε-LCY gene of tobacco had significantly improved quality traits after planting in the field, and their agronomic traits were not significantly different from those of Yunyan 87, and the content of the main carotenoid components lutein and β-carotene was increased (more than 20%). The mutation ofNtε-LCY gene affects the content of chlorophyll and carotenoids in tobacco leaves; and compared with the mutation ofNtε-LCY1gene , the mutation of Ntε-LCY2 gene is more beneficial to increase the content of carotenoids, especially in chlorophyll, In terms of pigment content such as lutein and beta-carotene. The mutation ofNtε-LCY gene affects the expression of carotenoid biosynthesis related genes. Compared withNtε-lcy1-1 ,Ntε-lcy2-1 is more beneficial toPSY ,PDS ,ZDS ,CRTISO under strong light stress conditions. ,β-LCY ,β-OHase ,VDE ,NXS and other genes expression. The mutation ofNtε-LCY gene can improve the photosynthetic capacity and heat dissipation capacity of tobacco plants. Under the condition of strong light stress, the photosynthetic capacity of tobacco plants decreases and the level of heat dissipation increases significantly, which is consistent with the mutation ofNtε-LCY1 gene. Compared with that, the mutation ofNtε-LCY2 gene is more helpful to improve the photosynthetic capacity and photoprotective capacity of tobacco plants. Under normal light and strong light stress conditions, the mutation of theNtε-LCY homologous gene reduces the content of O^2- and H₂ O₂ , and the mutation of theNtε-LCY2 gene is more conducive to reducing the accumulation of reactive oxygen species, thereby reducing the amount of reactive oxygen species. Protects tobacco plants. However, there is no relevant report on the study ofNtε-LCY2 gene in phenol.

发明内容SUMMARY OF THE INVENTION

本发明目的在于提供一种烟草番茄ε-环化酶基因Ntε-LCY2及其在烟草总蛋白质物质含量调节方面的应用，从而为低苯酚含量烟叶的培育提供基础。The purpose of the invention is to provide a tobacco tomato ε-cyclase geneNtε-LCY2 and its application in regulating the content of total protein substances in tobacco, so as to provide a basis for the cultivation of tobacco leaves with low phenol content.

Ntε-LCY2基因的突变体分析表明类胡萝卜素的提高是提高烟叶品质的重要途径，类胡萝卜素含量对于烟叶品质非常关键。那么类胡萝卜素含量的升高，是否会同时引起蛋白质含量的变化，从而实现降低烟叶有害物质前体物的目标。本发明项目组进一步检测了Ntε-LCY2基因突变体的叶片蛋白质总量，结果显示，Ntε-LCY2突变体的叶片总蛋白含量（单位mg/mL）比对照株系要降低了22.74%，同时该突变体烤后烟叶苯酚含量也有一定降低，证明该基因能够可以作为减害调控靶标基因。The mutant analysis ofNtε-LCY2 gene showed that the improvement of carotenoids is an important way to improve the quality of tobacco leaves, and the content of carotenoids is very important for the quality of tobacco leaves. So whether the increase of carotenoid content will cause changes in protein content at the same time, so as to achieve the goal of reducing the precursors of harmful substances in tobacco leaves. The project team of the present invention further detected the total protein content of the leaves of theNtε-LCY2 gene mutants. The results showed that the total protein content (unit mg/mL) of the leaves of theNtε-LCY2 mutants was 22.74% lower than that of the control line. The phenol content of tobacco leaves after curing was also reduced in the mutant, which proved that this gene can be used as a target gene for harm reduction regulation.

本申请所采取的技术方案详述如下：The technical solution adopted in this application is described in detail as follows:

本发明的第一目的是提供烟草番茄ε-环化酶基因Ntε-LCY2，其碱基序列如SEQ IDNO.1所示，其中特异性核酸片段为315-861和870-1425位碱基。The first object of the present invention is to provide the tobacco tomato ε-cyclase geneNtε-LCY2 , the base sequence of which is shown in SEQ ID NO.1, wherein the specific nucleic acid fragments are bases 315-861 and 870-1425.

烟草番茄红素ε-环化酶基因Ntε-LCY2，其氨基酸序列如SEQ ID NO.2所示，其由498个氨基酸残基组成，其中第105-287和290-475位氨基酸是保守的结构域。Tobacco lycopene ε-cyclase gene Ntε-LCY2, the amino acid sequence of which is shown in SEQ ID NO.2, which consists of 498 amino acid residues, wherein amino acids 105-287 and 290-475 are conserved structures area.

本发明的第二目的是提供上述烟草番茄ε-环化酶基因Ntε-LCY2的应用，具体如下：The second object of the present invention is to provide the application of above-mentioned tobacco tomato ε-cyclase geneNtε-LCY2 , specifically as follows:

1.所述烟草番茄ε-环化酶基因Ntε-LCY2在叶片总蛋白含量调控中的应用，利用基因沉默技术、或者基因超表达方法，通过调节烟草Ntε-LCY2蛋白表达量，来调节控制烟叶中总蛋白质物质含量情况。1. the application of described tobacco tomato ε-cyclase geneNtε-LCY2 in the regulation of total protein content in leaves, using gene silencing technology or gene overexpression method, by regulating the expression of tobaccoNtε-LCY2 protein, to regulate and control tobacco leaves total protein content.

所述烟草番茄红素ε-环化酶Ntε-LCY2在叶片总蛋白质含量调控中的应用，该蛋白与植物叶片中总蛋白质含量相关，降低该蛋白表达后，叶片中总蛋白质含量明显降低。The application of the tobacco lycopene ε-cyclaseNtε-LCY2 in the regulation of total protein content in leaves is related to the total protein content in plant leaves. After the expression of the protein is reduced, the total protein content in leaves is significantly reduced.

2.所述烟草番茄ε-环化酶基因Ntε-LCY2在烟草中苯酚含量调控中的应用。2. The application of the tobacco tomato ε-cyclase geneNtε-LCY2 in the regulation of phenol content in tobacco.

3.所述烟草番茄ε-环化酶基因Ntε-LCY2在烟草新品种培育中的应用。尤其是在总蛋白质含量降低的烟草新品种培育中的应用。尤其更进一步是在低苯酚含量烟叶的新品种培育中的应用。3. The application of the tobacco tomato ε-cyclase geneNtε-LCY2 in the cultivation of new tobacco varieties. Especially the application in the cultivation of new tobacco varieties with reduced total protein content. In particular, it is further applied in the cultivation of new varieties of tobacco leaves with low phenol content.

利用所述编码基因Ntε-LCY2的烟草新品种培育方法，通过转基因技术、瞬时表达技术或基因组编辑技术，构建含有Ntε-LCY2基因的病毒诱导沉默载体、RNAi干涉载体、超表达载体或基因组编辑载体，转化烟草，筛选获得总蛋白质含量降低的烟草新品种；Using the method for cultivating new tobacco varieties encoding the geneNtε-LCY2 , through transgenic technology, transient expression technology or genome editing technology, a virus-induced silencing vector, RNAi interference vector, overexpression vector or genome editing vector containing theNtε-LCY2 gene is constructed , transforming tobacco, and screening to obtain new tobacco varieties with reduced total protein content;

具体例如：利用病毒诱导的基因沉默（VIGS）的技术，干扰Ntε-LCY2基因的表达使其沉默，Ntε-LCY2基因沉默植株中总蛋白质含量显著下降，进而获得总蛋白质含量下降的植物新品种。Specifically, for example: using virus-induced gene silencing (VIGS) technology to interfere with the expression of Ntε-LCY2 gene to silence it, the total protein content inNtε-LCY2 gene silenced plants is significantly reduced, and a new plant variety with reduced total protein content is obtained.

本发明还提供一种低苯酚含量烟草新品种的培育方法，通过对烟草番茄红素ε-环化酶基因Ntε-LCY2进行干涉。The invention also provides a method for cultivating a new tobacco variety with low phenol content by interfering with the tobacco lycopene ε-cyclase geneNtε-LCY2 .

本发明通过对特定烟草番茄红素ε-环化酶基因Ntε-LCY2的初步研究，发现其与烟草总蛋白质含量高度相关，在将该基因沉默后，烟草中总蛋白质含量发生了明显降低。基于这一特性，可为烟草低苯酚含量烟叶的新品种培育提供一定的应用基础和参考借鉴。Through preliminary research on the specific tobacco lycopene ε-cyclase geneNtε-LCY2 , the present invention finds that it is highly correlated with the total protein content of tobacco. After the gene is silenced, the total protein content in tobacco is significantly reduced. Based on this characteristic, it can provide a certain application basis and reference for the cultivation of new varieties of tobacco leaves with low phenol content.

附图说明Description of drawings

图1为本发明烟草TRV2-PDS、TRV2-GFP及TRV2-Ntε-LCY2载体转化组的表型对比图；Fig. 1 is the phenotype comparison diagram of the tobacco TRV2-PDS, TRV2-GFP and TRV2-Ntε-LCY2 vector transformation groups of the present invention;

图2 为与对照植株相比Ntε-LCY2基因沉默植株中该基因的相对表达量；Figure 2 shows the relative expression of this gene inNtε-LCY2 gene silenced plants compared with control plants;

图3 为病毒诱导基因沉默的烟叶及对照烟叶中的总蛋白质含量比较。Figure 3 is a comparison of total protein content in tobacco leaves with virus-induced gene silencing and control tobacco leaves.

图4为病毒诱导基因沉默的烟叶及对照烤后烟叶烟气中苯酚含量比较。Figure 4 is a comparison of the phenol content in the flue gas of the virus-induced gene silencing tobacco leaves and the control tobacco leaves after curing.

具体实施方式Detailed ways

下面结合实施例对本申请做进一步解释说明，在介绍具体实施例前，就下述实施例中具体实验背景资料情况简要介绍如下。The present application will be further explained below with reference to the embodiments. Before introducing the specific embodiments, the specific experimental background information in the following embodiments will be briefly introduced as follows.

生物材料：biomaterials:

本氏烟草，一种常用烟草材料，下述实施例中烟草种植于郑州烟草院种植基地，育苗钵中育苗，待发芽后两周进行分苗，种于塑料钵（10cm×10cm）中，22℃、16h光/8h暗条件下进行日常肥水管理等；Tobacco benthamiana, a commonly used tobacco material, in the following examples, the tobacco was planted in the planting base of Zhengzhou Tobacco Institute, and the seedlings were raised in the seedling pot. ℃, 16h light/8h dark conditions for daily fertilizer and water management, etc.;

下述实施例中所采用的VIGS载体是一种来自烟草脆裂病毒的病毒载体（tobaccorattle virus，TRV)， 所具体利用的TRV2由郑州烟草研究院基因中心保存，其带有卡那筛选标记和35S启动子，同时TRV2带有EcoR I和BamH I等多克隆位点，可以用来携带和转化外源基因；The VIGS carrier adopted in the following examples is a kind of viral vector (tobaccorattle virus, TRV) from tobacco crisp virus, and the TRV2 specifically utilized is preserved by the Gene Center of Zhengzhou Tobacco Research Institute, and it has kana screening marker and 35S promoter, and TRV2 has multiple cloning sites such asEcoR I andBamH I, which can be used to carry and transform foreign genes;

实验试剂：Experimental reagents:

LB液体培养基，1L含量中含有：10 g细菌蛋白胨（bacteriological peptone）；10g氯化钠（NaCl）；5 g酵母抽提物（yeast extract），高温高压灭菌；LB liquid medium, 1L content contains: 10 g bacterial peptone (bacteriological peptone); 10 g sodium chloride (NaCl); 5 g yeast extract (yeast extract), high temperature and high pressure sterilization;

YEB液体培养基，1L含量中含有：5g 牛肉浸膏（beef extract）；5 g细菌蛋白胨（bacteriological peptone）；5 g 蔗糖（sucrose）；1 g酵母抽提物（yeast extract）；2 mL1M硫酸镁（MgSO4），高温高压灭菌；YEB liquid medium, 1L content contains: 5g beef extract; 5g bacteriological peptone; 5g sucrose; 1g yeast extract; 2mL 1M magnesium sulfate (MgSO4), high temperature and high pressure sterilization;

1M 2-(N-吗啉)乙磺酸（MES）储备液：ddH₂O溶解，过滤灭菌，-20℃储存备用；1M 2-(N-morpholine)ethanesulfonic acid (MES) stock solution: dissolve in ddH₂ O, filter sterilize, and store at -20°C for later use;

200 mM乙酰丁香酮（Acetosyringone，As）储备液：二甲基亚砜（DSMO）溶解，-20℃储存备用；200 mM acetosyringone (Acetosyringone, As) stock solution: dissolve in dimethyl sulfoxide (DSMO) and store at -20°C for later use;

MMA（100 mL）：1 mL（1 M）MgCl2；1 mL（1 M,pH5.6）MES；75 μL（200 mM）As。MMA (100 mL): 1 mL (1 M) MgCl2; 1 mL (1 M, pH 5.6) MES; 75 μL (200 mM) As.

实施例1Example 1

本实施例就烟草Ntε-LCY2基因克隆及沉默载体的构建过程简要介绍如下。In this example, the process of cloning the tobaccoNtε-LCY2 gene and constructing the silencing vector is briefly introduced as follows.

（1）烟草Ntε-LCY2基因克隆(1) Cloning of tobaccoNtε-LCY2 gene

根据前期对于烟草基因组及相关Ntε-LCY2基因研究，选择特异编码序列为目标片段，设计PCR扩增用引物序列如下：According to the previous research on the tobacco genome and relatedNtε-LCY2 gene, the specific coding sequence was selected as the target fragment, and the primer sequences for PCR amplification were designed as follows:

Ntε-LCY2-F：5′-TCTTAGGTGTGTGGAGGCAG- 3′，Ntε-LCY2-F: 5′-TCTTAGGTGTGTGGAGGCAG-3′,

Ntε-LCY2-R：5′-AGAATTTCCCTGAGGCAGCA- 3′。Ntε-LCY2-R: 5'-AGAATTTCCCTGAGGCAGCA-3'.

以烟草K326叶片的cDNA为模板，进行PCR扩增获得Ntε-LCY2基因；TheNtε-LCY2 gene was obtained by PCR amplification with the cDNA of tobacco K326 leaves as the template;

PCR扩增程序为：95℃预变性3 min；95℃变性15s，55℃退火15s，72℃延伸30s，34个循环后，72℃彻底延伸 5min；The PCR amplification program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15s, annealing at 55°C for 15s, extension at 72°C for 30s, after 34 cycles, complete extension at 72°C for 5min;

对PCR扩增产物进行琼脂糖凝胶电泳检测，并回收电泳产物备用。The PCR amplification products were detected by agarose gel electrophoresis, and the electrophoresis products were recovered for future use.

（2）构建重组TRV2-Ntε-LCY2载体(2) Construction of recombinant TRV2-Ntε-LCY2 vector

将步骤（1）中的PCR扩增产物进行EcoR I、BamH I双酶切，同时对空载体TRV2进行EcoR I、BamH I双酶切，分别回收酶切产物，利用T4 DNA连接酶进行连接；The PCR amplification product in step (1) was double digested withEcoR I andBamH I, and the empty vector TRV2 was double digested withEcoR I andBamH I, respectively, and the digested products were recovered and ligated with T4 DNA ligase;

将连接产物转化大肠杆菌感受态DH5α，转化操作结束后将转化产物涂布在含50mg/L Kan的LB固体培养基上，在37℃过培养夜；The ligation product was transformed into E. coli competent DH5α, and after the transformation operation, the transformed product was spread on LB solid medium containing 50 mg/L Kan, and cultured at 37°C overnight;

挑选阳性单菌落扩增后进一步进行PCR鉴定， 并结合测序验证，确保获得构建正确的重组载体TRV2-Ntε-LCY2。Positive single colonies were selected and amplified, and further identified by PCR, combined with sequencing verification, to ensure that the correctly constructed recombinant vector TRV2-Ntε-LCY2 was obtained.

烟草Ntε-LCY2基因，包括1497个碱基，碱基序列如SEQ ID NO.1所示，具体如下：TobaccoNtε-LCY2 gene, including 1497 bases, the base sequence is shown in SEQ ID NO.1, as follows:

> SEQ ID NO.1> SEQ ID NO.1

ATGGATTGTATTGGAGCTCGAAATTTTGCTACAATGGCGGTTTTTACGTGTCCGAGATTCAAATCATTAGGAAGAAGGAGAATTATGCCAAGAAAAAAGCAACCAATTTGGCCTATACATATGCAAGTGAAGTGTAGTGGAAATGAGAGTTGTGTAGTAGTTAAAGAAGATTTTGCCGATGAAGAGGATTATATAAAAGCTGGTGGTTCAGAACTTGTTTTTGTTCAAATGCAGCAGAATAAAGACATGGATCTGCAGTCTAAGCTTTCTGATAAGTTGCGACAAATATCATCAGCTGGACAAACTATACTGGATTTGGTGGTCATAGGCTGTGGTCCTGCTGGTCTTGCTCTTGCTGCGGAGTCTGCTAAACTCGGATTGAACGTTGGGCTCGTTGGTCCTGATCTTCCTTTCACAAATAACTATGGTGTTTGGGAGGATGAGTTCAAAGATCTTGGGCTTCAAGCGTGCATTGAACATGTTTGGAGGGATACCATAGTATATCTTGACGATGCCGATCCAATTCTTATCGGTCGTGCTTATGGAAGAGTTAGTCGCCATTTACTGCACGAGGAGTTACTCAAAAGGTGTGTGGAGGCAGGTGTTTTATATCTTAACTCGAAAGTGGATAGGATCGTTGAGTCCACAAGTGGCCACAGTCTTGTAGAGTGCGAGGGCGACATTGTCATTCCTTGCAGGTTTGTCACTGTTGCATCTGGTGCTGCCTCAGGGAAATTCTTGCAGTATGAGTTGGGAGGTCCTCGGGTTTCTGTTCAAACAGCTTATGGAGTGGAAGTTGAGGTCGATAACAATCCGTATGATCCAAGCCTGATGGTTTTCATGGATTATAGAGACTATGTCAGACACGAAACTTGTTTGGCTTCAAAAGATGCAATGCCATTTGATTTGTTAAAGAAAAAACTGATGTTACGATTGAACACACTGGGTGTAAGAATTAAGCAAATCTACGAGGAGGAATGGTCATACATACCAGTTGGTGGATCTTTACCAAATACCGAGCAAAAAACACTTGCATTTGGTGCTGCTGCTAGCATGGTTCATCCAGCTACAGGTTATTCAGTTGTCAGATCACTGTCCGAGGCACCAAAATGCGCCTCCGTACTTGCTAATATTTTACGACAAAATCATGTCAAGAACATGCTAACCAGTTCAAGTACCACAAGTATCTCAACTCAAGCTTGGAACACCCTTTGGCCACAAGAACGAAAAAGGCAACGATCGTTTTTCCTATTTGGATTGGCACTCATATTGCAGTTGGATATTGAGGGGATTAGGTCATTTTTCCGCGCATTCTTCCGTGTGCCAAAATGGATGTGGCAAGGATTTCTTGGCTCTAGTCTTTCATCAGCAGACCTCATGTTATTTGCCTTCTACATGTTTATTATTGCACCAAATGACATGAGAAAAGGCCTAATCAGACATTTGTTATCTGATCCAACTGGTGCAACCATGATAAGAACTTATCTTACATTTTAGATGGATTGTATTGGAGCTCGAAATTTTGCTACAATGGCGGTTTTTACGTGTCCGAGATTCAAATCATTAGGAAGAAGGAGAATTATGCCAAGAAAAAAGCAACCAATTTGGCCTATACATATGCAAGTGAAGTGTAGTGGAAATGAGAGTTGTGTAGTAGTTAAAGAAGATTTTGCCGATGAAGAGGATTATATAAAAGCTGGTGGTTCAGAACTTGTTTTTGTTCAAATGCAGCAGAATAAAGACATGGATCTGCAGTCTAAGCTTTCTGATAAGTTGCGACAAATATCATCAGCTGGACAAACTATACTGGATTTGGTGGTCATAGGCTGTGGTCCTGCTGGTCTTGCTCTTGCTGCGGAGTCTGCTAAACTCGGATTGAACGTTGGGCTCGTTGGTCCTGATCTTCCTTTCACAAATAACTATGGTGTTTGGGAGGATGAGTTCAAAGATCTTGGGCTTCAAGCGTGCATTGAACATGTTTGGAGGGATACCATAGTATATCTTGACGATGCCGATCCAATTCTTATCGGTCGTGCTTATGGAAGAGTTAGTCGCCATTTACTGCACGAGGAGTTACTCAAAAGGTGTGTGGAGGCAGGTGTTTTATATCTTAACTCGAAAGTGGATAGGATCGTTGAGTCCACAAGTGGCCACAGTCTTGTAGAGTGCGAGGGCGACATTGTCATTCCTTGCAGGTTTGTCACTGTTGCATCTGGTGCTGCCTCAGGGAAATTCTTGCAGTATGAGTTGGGAGGTCCTCGGGTTTCTGTTCAAACAGCTTATGGAGTGGAAGTTGAGGTCGATAACAATCCGTATGATCCAAGCCTGATGGTTTTCATGGATTATAGAGACTATGTCAGACACGAAACTTGTTTGGCTTCAAAAGATGCAATGCCATTTGATTTGTTAAAGAAAAAACTGATGTTACGATTGAACACACTGGGTGTAAGAATTAAGCAAATCTACGAGGAGGAATGGTCATACATACCAGTTGGTG GATCTTTACCAAATACCGAGCAAAAAACACTTGCATTTGGTGCTGCTGCTAGCATGGTTCATCCAGCTACAGGTTATTCAGTTGTCAGATCACTGTCCGAGGCACCAAAATGCGCCTCCGTACTTGCTAATATTTTACGACAAAATCATGTCAAGAACATGCTAACCAGTTCAAGTACCACAAGTATCTCAACTCAAGCTTGGAACACCCTTTGGCCACAAGAACGAAAAAGGCAACGATCGTTTTTCCTATTTGGATTGGCACTCATATTGCAGTTGGATATTGAGGGGATTAGGTCATTTTTCCGCGCATTCTTCCGTGTGCCAAAATGGATGTGGCAAGGATTTCTTGGCTCTAGTCTTTCATCAGCAGACCTCATGTTATTTGCCTTCTACATGTTTATTATTGCACCAAATGACATGAGAAAAGGCCTAATCAGACATTTGTTATCTGATCCAACTGGTGCAACCATGATAAGAACTTATCTTACATTTTAG

烟草番茄红素ε-环化酶Ntε-LCY2，包括498个氨基酸，氨基酸序列如SEQ ID NO.2所示，具体如下：Tobacco lycopene ε-cyclase Ntε-LCY2, including 498 amino acids, the amino acid sequence is shown in SEQ ID NO.2, and the details are as follows:

> SEQ ID NO.2> SEQ ID NO.2

MDCIGARNFATMAVFTCPRFKSLGRRRIMPRKKQPIWPIHMQVKCSGNESCVVVKEDFADEEDYIKAGGSELVFVQMQQNKDMDLQSKLSDKLRQISSAGQTILDLVVIGCGPAGLALAAESAKLGLNVGLVGPDLPFTNNYGVWEDEFKDLGLQACIEHVWRDTIVYLDDADPILIGRAYGRVSRHLLHEELLKRCVEAGVLYLNSKVDRIVESTSGHSLVECEGDIVIPCRFVTVASGAASGKFLQYELGGPRVSVQTAYGVEVEVDNNPYDPSLMVFMDYRDYVRHETCLASKDAMPFDLLKKKLMLRLNTLGVRIKQIYEEEWSYIPVGGSLPNTEQKTLAFGAAASMVHPATGYSVVRSLSEAPKCASVLANILRQNHVKNMLTSSSTTSISTQAWNTLWPQERKRQRSFFLFGLALILQLDIEGIRSFFRAFFRVPKWMWQGFLGSSLSSADLMLFAFYMFIIAPNDMRKGLIRHLLSDPTGATMIRTYLTFMDCIGARNFATMAVFTCPRFKSLGRRRIMPRKKQPIWPIHMQVKCSGNESCVVVKEDFADEEDYIKAGGSELVFVQMQQNKDMDLQSKLSDKLRQISSAGQTILDLVVIGCGPAGLALAAESAKLGLNVGLVGPDLPFTNNYGVWEDEFKDLGLQACIEHVWRDTIVYLDDADPILIGRAYGRVSRHLLHEELLKRCVEAGVLYLNSKVDRIVESTSGHSLVECEGDIVIPCRFVTVASGAASGKFLQYELGGPRVSVQTAYGVEVEVDNNPYDPSLMVFMDYRDYVRHETCLASKDAMPFDLLKKKLMLRLNTLGVRIKQIYEEEWSYIPVGGSLPNTEQKTLAFGAAASMVHPATGYSVVRSLSEAPKCASVLANILRQNHVKNMLTSSSTTSISTQAWNTLWPQERKRQRSFFLFGLALILQLDIEGIRSFFRAFFRVPKWMWQGFLGSSLSSADLMLFAFYMFIIAPNDMRKGLIRHLLSDPTGATMIRTYLTF

实施例2Example 2

在实施例1基础上，利用农杆菌介导的VIGS技术，发明人进一步将所构建的重组TRV2-Ntε-LCY2载体转化了烟草植株，并就相关植物表型变化情况做了验证分析，具体实验过程简介如下。On the basis of Example 1, using the VIGS technology mediated by Agrobacterium, the inventor further transformed the constructed recombinant TRV2-Ntε-LCY2 vector into tobacco plants, and made a verification analysis on the phenotypic changes of the relevant plants. A brief introduction to the process is as follows.

（1）转化农杆菌(1) Transformation of Agrobacterium

需要说明的是，参考实施例1操作及现有技术，发明人同时制备了TRV2-GFP、TRV2-PDS重组载体作为转基因正负对照，具体转化过程为：It should be noted that, with reference to the operation of Example 1 and the prior art, the inventors simultaneously prepared TRV2-GFP and TRV2-PDS recombinant vectors as transgenic positive and negative controls, and the specific transformation process is as follows:

将TRV2-GFP（载体对照）、TRV2-PDS（VIGS效率对照）及TRV2-Ntε-LCY2的阳性克隆质粒，分别通过电击转化方式转化进入农杆菌GV3101感受态细胞中，利用含50mg/L Kan 和50mg/L Rif的YEB平板进行培养筛选，在28℃倒置培养2d后，利用菌落PCR筛选带有目的基因的农杆菌。The positive cloned plasmids of TRV2-GFP (vector control), TRV2-PDS (VIGS efficiency control) and TRV2-Ntε-LCY2 were transformed into Agrobacterium GV3101 competent cells by electroporation, respectively. 50mg/L Rif YEB plate was cultured and screened, and after inversion culture at 28°C for 2 days, colony PCR was used to screen Agrobacterium with the target gene.

（2）制备转染用菌液(2) Preparation of bacterial solution for transfection

将步骤（1）中筛选所得阳性农杆菌克隆在5 mL的YEB液体培养基（含50 mg/L Kan和50 mg/L Rif）中，28℃、250 rpm条件下培养过夜；The positive Agrobacterium clones screened in step (1) were cultured in 5 mL of YEB liquid medium (containing 50 mg/L Kan and 50 mg/L Rif) at 28°C and 250 rpm overnight;

取50uL 过夜培养物接种至50 mL的YEB液体培养基（含50 mg/L Kan）中，培养至OD₆₀₀ =1.0 ~ 1.5左右，然后4000g 离心5 min，收集菌体，再用MMA(1 mL（1 M）MgCl₂；1 mL（1 M, pH5.6）MES；75 μL（200 mM）As)重悬，调节OD₆₀₀=1.0 左右；Take 50uL of the overnight culture and inoculate it into 50 mL of YEB liquid medium (containing 50 mg/L Kan), culture to OD₆₀₀ =1.0 ~ 1.5, then centrifuge at 4000g for 5 min, collect the bacteria, and then use MMA (1 mL (1 M) MgCl₂ ; 1 mL (1 M, pH5.6) MES; 75 μL (200 mM) As) resuspend, adjust OD₆₀₀ = about 1.0;

最后室温放置3 h左右后，作为转染用菌液。Finally, after standing at room temperature for about 3 hours, it was used as a bacterial solution for transfection.

（3）瞬时转化(3) Instantaneous transformation

以3~4 w苗龄的本氏烟草叶片为实验材料，利用1 mL规格注射器，将步骤（2）中所制备转染用菌液注射至烟草叶片中，注射后的烟草继续在人工培养箱内培养，观察表型变化。Using 3-4 w seedlings of N. benthamiana leaves as experimental materials, the bacterial solution for transfection prepared in step (2) was injected into tobacco leaves using a 1 mL syringe, and the injected tobacco continued to be placed in an artificial incubator. cultured and observed phenotypic changes.

注射3周后的烟草表型变化情况如图1所示。可以看出，含TRV2-PDS的农杆菌浸染植株新生叶有漂白现象，说明侵染成功；而TRV2-GFP组则无明显变化，相对应的TRV2-Ntε-LCY2组烟草植株无显著变化，表明Ntε-LCY2基因对烟草其他基本生理状态无明显影响。The phenotypic changes oftobacco 3 weeks after injection are shown in Figure 1. It can be seen that the new leaves of Agrobacterium-infected plants containing TRV2-PDS have bleaching phenomenon, indicating that the infection is successful; while the TRV2-GFP group has no significant change, and the corresponding TRV2-Ntε-LCY2 group tobacco plants have no significant changes, indicating that the infection is successful. The Ntε-LCY2 gene had no significant effect on other basic physiological states of tobacco.

进一步通过qRT-PCR对Ntε-LCY2基因表达情况进行了检测，结果如图2所示，可以看出，TRV2- Ntε-LCY2的侵染植株中，Ntε-LCY2的表达量显著降低。The expression ofNtε-LCY2 was further detected by qRT-PCR. The results are shown in Figure 2. It can be seen that the expression ofNtε-LCY2 was significantly reduced in TRV2-Ntε-LCY2-infected plants.

进一步地，发明人对实验组（TRV2- Ntε-LCY2浸染植株）和对照组（TRV2-GFP浸染植株）中的植物总蛋白质含量情况进行了检测，结果如图3，结果可以看出，沉默植株的生长表型正常；基因表达量略有下降，不足15%；总蛋白质含量降低为对照的25%左右，烤后烟叶烟气苯酚含量降低30%左右（如图4所示）。这一结果表明Ntε-LCY2基因是一个很好地调控烟叶总蛋白质以及烟叶苯酚释放量的靶标基因，可以在不影响烟株表型的情况下有效降低烟叶中苯酚前体物总蛋白质含量以及苯酚释放量。Further, the inventors tested the total protein content of plants in the experimental group (TRV2-Ntε-LCY2-infected plants) and the control group (TRV2-GFP-infected plants). The growth phenotype was normal; the gene expression decreased slightly, less than 15%; the total protein content was reduced to about 25% of the control, and the phenol content of flue-cured tobacco leaves was reduced by about 30% (as shown in Figure 4). This result indicates thatNtε-LCY2 gene is a target gene that can well regulate the total protein and phenol release of tobacco leaves, and can effectively reduce the total protein content of phenol precursors and phenol in tobacco leaves without affecting the phenotype of tobacco leaves. release amount.

序列表sequence listing

<110> 云南中烟工业有限责任公司<110> Yunnan China Tobacco Industry Co., Ltd.

<120> 一种降低烟叶总蛋白及烟气苯酚含量的调控基因<120> A regulatory gene that reduces the total protein and phenol content of tobacco leaves

<141> 2021-07-17<141> 2021-07-17

<160> 2<160> 2

<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0

<210> 1<210> 1

<211> 1497<211> 1497

<212> DNA<212> DNA

<213> Nicotiana debneyi<213> Nicotiana debneyi

<220><220>

<221> iDNA<221> iDNA

<222> (1)..(1497)<222> (1)..(1497)

<223> Ntε-LCY2碱基序列<223> Ntε-LCY2 base sequence

<400> 1<400> 1

atggattgta ttggagctcg aaattttgct acaatggcgg tttttacgtg tccgagattc 60atggattgta ttggagctcg aaattttgct acaatggcgg tttttacgtg tccgagattc 60

aaatcattag gaagaaggag aattatgcca agaaaaaagc aaccaatttg gcctatacat 120aaatcattag gaagaaggag aattatgcca agaaaaaagc aaccaatttg gcctatacat 120

atgcaagtga agtgtagtgg aaatgagagt tgtgtagtag ttaaagaaga ttttgccgat 180atgcaagtga agtgtagtgg aaatgagagt tgtgtagtag ttaaagaaga ttttgccgat 180

gaagaggatt atataaaagc tggtggttca gaacttgttt ttgttcaaat gcagcagaat 240gaagaggatt atataaaagc tggtggttca gaacttgttt ttgttcaaat gcagcagaat 240

aaagacatgg atctgcagtc taagctttct gataagttgc gacaaatatc atcagctgga 300aaagacatgg atctgcagtc taagctttct gataagttgc gacaaatatc atcagctgga 300

caaactatac tggatttggt ggtcataggc tgtggtcctg ctggtcttgc tcttgctgcg 360caaactatac tggatttggt ggtcataggc tgtggtcctg ctggtcttgc tcttgctgcg 360

gagtctgcta aactcggatt gaacgttggg ctcgttggtc ctgatcttcc tttcacaaat 420gagtctgcta aactcggatt gaacgttggg ctcgttggtc ctgatcttcc tttcacaaat 420

aactatggtg tttgggagga tgagttcaaa gatcttgggc ttcaagcgtg cattgaacat 480aactatggtg tttgggagga tgagttcaaa gatcttgggc ttcaagcgtg cattgaacat 480

gtttggaggg ataccatagt atatcttgac gatgccgatc caattcttat cggtcgtgct 540gtttggaggg ataccatagt atatcttgac gatgccgatc caattcttat cggtcgtgct 540

tatggaagag ttagtcgcca tttactgcac gaggagttac tcaaaaggtg tgtggaggca 600tatggaagag ttagtcgcca tttactgcac gaggagttac tcaaaaggtg tgtggaggca 600

ggtgttttat atcttaactc gaaagtggat aggatcgttg agtccacaag tggccacagt 660ggtgttttat atcttaactc gaaagtggat aggatcgttg agtccacaag tggccacagt 660

cttgtagagt gcgagggcga cattgtcatt ccttgcaggt ttgtcactgt tgcatctggt 720cttgtagagt gcgagggcga cattgtcatt ccttgcaggt ttgtcactgt tgcatctggt 720

gctgcctcag ggaaattctt gcagtatgag ttgggaggtc ctcgggtttc tgttcaaaca 780gctgcctcag ggaaattctt gcagtatgag ttgggaggtc ctcgggtttc tgttcaaaca 780

gcttatggag tggaagttga ggtcgataac aatccgtatg atccaagcct gatggttttc 840gcttatggag tggaagttga ggtcgataac aatccgtatg atccaagcct gatggttttc 840

atggattata gagactatgt cagacacgaa acttgtttgg cttcaaaaga tgcaatgcca 900atggattata gagactatgt cagacacgaa acttgtttgg cttcaaaaga tgcaatgcca 900

tttgatttgt taaagaaaaa actgatgtta cgattgaaca cactgggtgt aagaattaag 960tttgatttgt taaagaaaaa actgatgtta cgattgaaca cactgggtgt aagaattaag 960

caaatctacg aggaggaatg gtcatacata ccagttggtg gatctttacc aaataccgag 1020caaatctacg aggaggaatg gtcatacata ccagttggtg gatctttacc aaataccgag 1020

caaaaaacac ttgcatttgg tgctgctgct agcatggttc atccagctac aggttattca 1080caaaaaacac ttgcatttgg tgctgctgct agcatggttc atccagctac aggttattca 1080

gttgtcagat cactgtccga ggcaccaaaa tgcgcctccg tacttgctaa tattttacga 1140gttgtcagat cactgtccga ggcaccaaaa tgcgcctccg tacttgctaa tattttacga 1140

caaaatcatg tcaagaacat gctaaccagt tcaagtacca caagtatctc aactcaagct 1200caaaatcatg tcaagaacat gctaaccagt tcaagtacca caagtatctc aactcaagct 1200

tggaacaccc tttggccaca agaacgaaaa aggcaacgat cgtttttcct atttggattg 1260tggaacaccc tttggccaca agaacgaaaa aggcaacgat cgtttttcct atttggattg 1260

gcactcatat tgcagttgga tattgagggg attaggtcat ttttccgcgc attcttccgt 1320gcactcatat tgcagttgga tattgagggg attaggtcat ttttccgcgc attcttccgt 1320

gtgccaaaat ggatgtggca aggatttctt ggctctagtc tttcatcagc agacctcatg 1380gtgccaaaat ggatgtggca aggatttctt ggctctagtc tttcatcagc agacctcatg 1380

ttatttgcct tctacatgtt tattattgca ccaaatgaca tgagaaaagg cctaatcaga 1440ttatttgcct tctacatgtt tattattgca ccaaatgaca tgagaaaagg cctaatcaga 1440

catttgttat ctgatccaac tggtgcaacc atgataagaa cttatcttac attttag 1497catttgttat ctgatccaac tggtgcaacc atgataagaa cttatcttac attttag 1497

<210> 2<210> 2

<211> 498<211> 498

<212> PRT<212> PRT

<213> Nicotiana debneyi<213> Nicotiana debneyi

<220><220>

<221> CONFLICT<221> CONFLICT

<222> (1)..(498)<222> (1)..(498)

<223> Ntε-LCY2氨基酸序列<223> Ntε-LCY2 amino acid sequence

<400> 2<400> 2

Met Asp Cys Ile Gly Ala Arg Asn Phe Ala Thr Met Ala Val Phe ThrMet Asp Cys Ile Gly Ala Arg Asn Phe Ala Thr Met Ala Val Phe Thr

1 5 10 151 5 10 15

Cys Pro Arg Phe Lys Ser Leu Gly Arg Arg Arg Ile Met Pro Arg LysCys Pro Arg Phe Lys Ser Leu Gly Arg Arg Arg Ile Met Pro Arg Lys

20 25 30 20 25 30

Lys Gln Pro Ile Trp Pro Ile His Met Gln Val Lys Cys Ser Gly AsnLys Gln Pro Ile Trp Pro Ile His Met Gln Val Lys Cys Ser Gly Asn

35 40 45 35 40 45

Glu Ser Cys Val Val Val Lys Glu Asp Phe Ala Asp Glu Glu Asp TyrGlu Ser Cys Val Val Val Lys Glu Asp Phe Ala Asp Glu Glu Asp Tyr

50 55 60 50 55 60

Ile Lys Ala Gly Gly Ser Glu Leu Val Phe Val Gln Met Gln Gln AsnIle Lys Ala Gly Gly Ser Glu Leu Val Phe Val Gln Met Gln Gln Asn

65 70 75 8065 70 75 80

Lys Asp Met Asp Leu Gln Ser Lys Leu Ser Asp Lys Leu Arg Gln IleLys Asp Met Asp Leu Gln Ser Lys Leu Ser Asp Lys Leu Arg Gln Ile

85 90 95 85 90 95

Ser Ser Ala Gly Gln Thr Ile Leu Asp Leu Val Val Ile Gly Cys GlySer Ser Ala Gly Gln Thr Ile Leu Asp Leu Val Val Ile Gly Cys Gly

100 105 110 100 105 110

Pro Ala Gly Leu Ala Leu Ala Ala Glu Ser Ala Lys Leu Gly Leu AsnPro Ala Gly Leu Ala Leu Ala Ala Glu Ser Ala Lys Leu Gly Leu Asn

115 120 125 115 120 125

Val Gly Leu Val Gly Pro Asp Leu Pro Phe Thr Asn Asn Tyr Gly ValVal Gly Leu Val Gly Pro Asp Leu Pro Phe Thr Asn Asn Tyr Gly Val

130 135 140 130 135 140

Trp Glu Asp Glu Phe Lys Asp Leu Gly Leu Gln Ala Cys Ile Glu HisTrp Glu Asp Glu Phe Lys Asp Leu Gly Leu Gln Ala Cys Ile Glu His

145 150 155 160145 150 155 160

Val Trp Arg Asp Thr Ile Val Tyr Leu Asp Asp Ala Asp Pro Ile LeuVal Trp Arg Asp Thr Ile Val Tyr Leu Asp Asp Ala Asp Pro Ile Leu

165 170 175 165 170 175

Ile Gly Arg Ala Tyr Gly Arg Val Ser Arg His Leu Leu His Glu GluIle Gly Arg Ala Tyr Gly Arg Val Ser Arg His Leu Leu His Glu Glu

180 185 190 180 185 190

Leu Leu Lys Arg Cys Val Glu Ala Gly Val Leu Tyr Leu Asn Ser LysLeu Leu Lys Arg Cys Val Glu Ala Gly Val Leu Tyr Leu Asn Ser Lys

195 200 205 195 200 205

Val Asp Arg Ile Val Glu Ser Thr Ser Gly His Ser Leu Val Glu CysVal Asp Arg Ile Val Glu Ser Thr Ser Gly His Ser Leu Val Glu Cys

210 215 220 210 215 220

Glu Gly Asp Ile Val Ile Pro Cys Arg Phe Val Thr Val Ala Ser GlyGlu Gly Asp Ile Val Ile Pro Cys Arg Phe Val Thr Val Ala Ser Gly

225 230 235 240225 230 235 240

Ala Ala Ser Gly Lys Phe Leu Gln Tyr Glu Leu Gly Gly Pro Arg ValAla Ala Ser Gly Lys Phe Leu Gln Tyr Glu Leu Gly Gly Pro Arg Val

245 250 255 245 250 255

Ser Val Gln Thr Ala Tyr Gly Val Glu Val Glu Val Asp Asn Asn ProSer Val Gln Thr Ala Tyr Gly Val Glu Val Glu Val Asp Asn Asn Pro

260 265 270 260 265 270

Tyr Asp Pro Ser Leu Met Val Phe Met Asp Tyr Arg Asp Tyr Val ArgTyr Asp Pro Ser Leu Met Val Phe Met Asp Tyr Arg Asp Tyr Val Arg

275 280 285 275 280 285

His Glu Thr Cys Leu Ala Ser Lys Asp Ala Met Pro Phe Asp Leu LeuHis Glu Thr Cys Leu Ala Ser Lys Asp Ala Met Pro Phe Asp Leu Leu

290 295 300 290 295 300

Lys Lys Lys Leu Met Leu Arg Leu Asn Thr Leu Gly Val Arg Ile LysLys Lys Lys Leu Met Leu Arg Leu Asn Thr Leu Gly Val Arg Ile Lys

305 310 315 320305 310 315 320

Gln Ile Tyr Glu Glu Glu Trp Ser Tyr Ile Pro Val Gly Gly Ser LeuGln Ile Tyr Glu Glu Glu Trp Ser Tyr Ile Pro Val Gly Gly Ser Leu

325 330 335 325 330 335

Pro Asn Thr Glu Gln Lys Thr Leu Ala Phe Gly Ala Ala Ala Ser MetPro Asn Thr Glu Gln Lys Thr Leu Ala Phe Gly Ala Ala Ala Ser Met

340 345 350 340 345 350

Val His Pro Ala Thr Gly Tyr Ser Val Val Arg Ser Leu Ser Glu AlaVal His Pro Ala Thr Gly Tyr Ser Val Val Arg Ser Leu Ser Glu Ala

355 360 365 355 360 365

Pro Lys Cys Ala Ser Val Leu Ala Asn Ile Leu Arg Gln Asn His ValPro Lys Cys Ala Ser Val Leu Ala Asn Ile Leu Arg Gln Asn His Val

370 375 380 370 375 380

Lys Asn Met Leu Thr Ser Ser Ser Thr Thr Ser Ile Ser Thr Gln AlaLys Asn Met Leu Thr Ser Ser Ser Thr Thr Ser Ile Ser Thr Gln Ala

385 390 395 400385 390 395 400

Trp Asn Thr Leu Trp Pro Gln Glu Arg Lys Arg Gln Arg Ser Phe PheTrp Asn Thr Leu Trp Pro Gln Glu Arg Lys Arg Gln Arg Ser Phe Phe

405 410 415 405 410 415

Leu Phe Gly Leu Ala Leu Ile Leu Gln Leu Asp Ile Glu Gly Ile ArgLeu Phe Gly Leu Ala Leu Ile Leu Gln Leu Asp Ile Glu Gly Ile Arg

420 425 430 420 425 430

Ser Phe Phe Arg Ala Phe Phe Arg Val Pro Lys Trp Met Trp Gln GlySer Phe Phe Arg Ala Phe Phe Arg Val Pro Lys Trp Met Trp Gln Gly

435 440 445 435 440 445

Phe Leu Gly Ser Ser Leu Ser Ser Ala Asp Leu Met Leu Phe Ala PhePhe Leu Gly Ser Ser Leu Ser Ser Ala Asp Leu Met Leu Phe Ala Phe

450 455 460 450 455 460

Tyr Met Phe Ile Ile Ala Pro Asn Asp Met Arg Lys Gly Leu Ile ArgTyr Met Phe Ile Ile Ala Pro Asn Asp Met Arg Lys Gly Leu Ile Arg

465 470 475 480465 470 475 480

His Leu Leu Ser Asp Pro Thr Gly Ala Thr Met Ile Arg Thr Tyr LeuHis Leu Leu Ser Asp Pro Thr Gly Ala Thr Met Ile Arg Thr Tyr Leu

485 490 495 485 490 495

Thr PheThr Phe

Claims (4)

Translated fromChinese

1.一种调控基因的应用，其特征在于，用于烟草叶片总蛋白质含量调控，该调控基因的碱基序列如SEQ ID NO.1所示，其中特异性核酸片段为315-861和870-1425位碱基。1. an application of regulation gene, it is characterized in that, for tobacco leaf total protein content regulation, the base sequence of this regulation gene is as shown in SEQ ID NO.1, and wherein specific nucleic acid fragment is 315-861 and 870- 1425 bases.2.一种调控基因的应用，其特征在于，用于降低烟草的烟气中苯酚含量，该调控基因的碱基序列如SEQ ID NO.1所示，其中特异性核酸片段为315-861和870-1425位碱基。2. an application of regulation gene, it is characterized in that, for reducing phenol content in the smoke of tobacco, the base sequence of this regulation gene is as shown in SEQ ID NO.1, and wherein specific nucleic acid fragment is 315-861 and 870-1425 bases.3.一种调控基因的应用，其特征在于，用于烟草新品种培育，该调控基因的碱基序列如SEQ ID NO.1所示，其中特异性核酸片段为315-861和870-1425位碱基。3. the application of a kind of regulation gene, it is characterised in that, for the cultivation of new tobacco varieties, the base sequence of this regulation gene is as shown in SEQ ID NO.1, and wherein specific nucleic acid fragment is 315-861 and 870-1425 positions base.4.一种低苯酚含量烟草新品种的培育方法，通过一种调控基因进行干涉，该调控基因的碱基序列如SEQ ID NO.1所示，其中特异性核酸片段为315-861和870-1425位碱基。4. A method for cultivating a new variety of tobacco with low phenol content, by interfering with a regulatory gene whose base sequence is as shown in SEQ ID NO.1, wherein the specific nucleic acid fragments are 315-861 and 870- 1425 bases.

Images