WO2023227137A1 - TaPDIL4-1B基因在植物抗赤霉病中的应用及其转基因植株的构建方法 - Google Patents

TaPDIL4-1B基因在植物抗赤霉病中的应用及其转基因植株的构建方法 Download PDF

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WO2023227137A1
WO2023227137A1 PCT/CN2023/101955 CN2023101955W WO2023227137A1 WO 2023227137 A1 WO2023227137 A1 WO 2023227137A1 CN 2023101955 W CN2023101955 W CN 2023101955W WO 2023227137 A1 WO2023227137 A1 WO 2023227137A1
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tapdil4
gene
plant
wheat
constructing
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PCT/CN2023/101955
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French (fr)
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刘易科
宋婧含
卫波
朱展望
邹娟
佟汉文
陈泠
高春保
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湖北省农业科学院粮食作物研究所
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Publication of WO2023227137A1 publication Critical patent/WO2023227137A1/zh
Priority to US18/611,679 priority Critical patent/US20240218389A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • A01H6/4678—Triticum sp. [wheat]
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0051—Oxidoreductases (1.) acting on a sulfur group of donors (1.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y108/00—Oxidoreductases acting on sulfur groups as donors (1.8)
    • C12Y108/01—Oxidoreductases acting on sulfur groups as donors (1.8) with NAD+ or NADP+ as acceptor (1.8.1)
    • C12Y108/01008—Protein-disulfide reductase (1.8.1.8), i.e. thioredoxin
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to the technical field of genetic engineering, specifically the application of TaPDIL4-1B gene in plant resistance to scab and its construction method of transgenic plants.
  • Wheat head blight (Fusarium Head Blight, FHB) is a global fungal disease caused by Fusarium. With global climate warming and changes in farming systems, the frequency of head blight continues to increase and the scope of occurrence continues to expand. . Wheat scab not only seriously affects wheat yields, but also produces a large amount of mycotoxins during the process of infecting wheat. If the wheat and its products contaminated by toxins are eaten by humans and animals, it will cause vomiting, diarrhea, abortion and other problems, seriously harming humans and animals. healthy. Wheat scab seriously affects grain production and food safety, and has become the focus of widespread attention of the international community.
  • ROS homeostasis reactive oxygen species stabilization
  • programmed cell death programmed cell death
  • thioredoxin Trx
  • glutaredoxin Grx
  • PDI Protein disulfide isomerase
  • the purpose of the present invention is to provide an application of TaPDIL4-1B gene in plant resistance to scab and a method for constructing transgenic plants, so as to provide candidate genes for the research and cultivation of new varieties of crops resistant to scab, so as to cultivate more resistant varieties. New varieties of scab.
  • TaPDIL4-1B gene in plant resistance to scab, the cDNA sequence of the TaPDIL4-1B gene is shown in SEQ ID NO: 1.
  • the gDNA sequence of the TaPDIL4-1B gene is shown in SEQ ID NO: 2, and its gDNA consists of 11 exons and 10 introns; starting from the 5' end, the length of the exons is 224bp and 85bp. , 98bp, 56bp, 118bp, 28bp, 92bp, 157bp, 125bp, 100bp, 488bp, and the intron lengths are 1131bp, 143bp, 85bp, 422bp, 96bp, 115bp, 216bp, 86bp, 122bp, 88bp.
  • the amino acid sequence of the protein encoded by the TaPDIL4-1B gene is shown in SEQ ID NO: 3.
  • This protein is a thiodisulfide bond oxidoreductase located in the endoplasmic reticulum.
  • any of the exogenous genes can be Any vector for introducing genes into plants for expression can be used in the present invention.
  • TaPDIL4-1B gene in the breeding of scab-resistant plant varieties.
  • the cDNA sequence of the TaPDIL4-1B gene is shown in SEQ ID NO: 1. Wherein, the plant is wheat.
  • the specific application method is: introducing the TaPDIL4-1B gene or the recombinant plasmid containing the TaPDIL4-1B gene into the cells, tissues or organs of the host wheat, and cultivating a new wheat variety with resistance to scab.
  • the recombinant plasmid is pMWB110-TaPDIL4-1B.
  • pMWB110-TaPDIL4-1B is processed and selectable markers, such as GUS, etc. are added.
  • the present invention applies plant genetic engineering technology and obtains gene TaPDIL4-1B from wheat Chinese spring for the first time.
  • the gene is transferred into plants (wheat) through Agrobacterium-mediated methods.
  • This transgene can make plants (wheat) ) has strong resistance to scab, which provides good candidate genes for the research and breeding of new varieties of crops resistant to scab, and is of great significance for the cultivation of new varieties of plants resistant to scab.
  • the new scab resistance gene TaPDIL4-1B discovered in the present invention is of great significance to the genetic improvement of wheat scab resistance and is suitable for popularization and application.
  • Figure 1 shows the PCR detection results of transgenic TaPDIL4-1B wheat positive plants.
  • #1-#8 are transgenic lines, Fielder is wild type, and NC is the negative control in which the amplification template is water.
  • Figure 2 shows the comparison of scab resistance between the overexpression strain and wild-type Fielder 21 days after inoculation.
  • A is the phenotypic identification result of head blight resistance in TaPDIL4-1B gene-transgenic wheat (the incidence of head blight in single-flower infusion inoculation);
  • B is the incidence of head blight in spikelets of TaPDIL4-1B gene-transgenic wheat.
  • Rate data statistics results.
  • T is the transgenic strain
  • CK is the wild-type Fielder.
  • the wheat variety used in the present invention Chinese Spring (English name: Chinese Spring), is a very important local wheat variety, and Chinese Spring is widely used in wheat genetic research.
  • the wheat variety used for genetic modification is Fielder, which is a commonly used model variety for wheat genetic modification and has the characteristics of high transformation efficiency.
  • RNA is precipitated on a sterile operating table and left to dry for about 10-15 minutes. When the RNA becomes slightly transparent, add 50 ⁇ l of RNase-free water to fully dissolve it and store it at -80°C for long-term use.
  • RNAPCRKit AMV Ver.3.0 kit (TaKaRa, DRR019A); first perform the first step of the reverse transcription reaction, use 500ng of RNA as a template for reverse transcription, and follow the instructions in the reaction system sequentially.
  • the reaction program was: 42°C for 30 min, 99°C for 5 min, and 5°C for 5 min.
  • Upstream primer F1 5’-GAGCTCGCAGCAAAACAGAT-3’, downstream primer R1: 5’-CCGCTAAACTTTCACTGCCA-3’;
  • the recovered PCR product was connected to pMT18-T (Bao Biotechnology Co., Ltd.) for sequencing.
  • the nucleotide sequence of the amplified PCR product was 1104bp from the start codon to the stop codon (shown in SEQ ID NO: 1 ), the gene of the PCR product is named TaPDIL4-1B, and the amino acid sequence of the protein encoded by the gene TaPDIL4-1B is shown in SEQ ID NO: 3.
  • gDNA extraction Extract Chinese spring wheat gDNA according to the instructions of the plant gDNA extraction kit (Tiangen Biochemical Technology Co., Ltd., Beijing);
  • Upstream primer F2 5’-TAGGAAGCCAAAGCGTTCGT-3’
  • Downstream primer R2 5’-TACTCGTGGCGATCCATTCG-3’;
  • PCR amplification was performed, and the amplification conditions were the same as the steps for amplifying cDNA in Example 1.
  • the obtained specific amplified band was connected to the pMT18-T vector and sequenced, and the full-length gDNA sequence of the gene TaPDIL4-1B in Chinese Spring was obtained, as shown in SEQ ID NO: 2, from the start codon to the stop codon. Sub-size is 4075bp.
  • gDNA consists of 11 exons and 10 introns; starting from the 5' end, the lengths of the exons are 224bp, 85bp, 98bp, 56bp, 118bp, 28bp, 92bp, 157bp, 125bp, 100bp, 488bp , the intron lengths are 1131bp, 143bp, 85bp, 422bp, 96bp, 115bp, 216bp, 86bp, 122bp, 88bp.
  • Example 1 Use the cDNA obtained in Example 1 as a template and use KOD FX DNA polymerase (Cat. No.: KFX-101) to perform PCR amplification.
  • the reaction system is (20 ⁇ l): KOD FX (1 unit/ ⁇ l), 0.4 ⁇ l, 2 ⁇ PCR buffer 10 ⁇ l, dNTP 2.4 ⁇ l, 126100EF and 126100ER primers (10 ⁇ M) 0.6 ⁇ l, cDNA template 1 ⁇ l, deionized water 5 ⁇ l.
  • the PCR product containing the TaPDIL4-1B gene of the pCUB vector homologous recombination arm was obtained.
  • Use Axygen gel recovery kit to recover PCR products.
  • the carrier is Peasy-Basic Seamless cloning and assembly kit from Beijing Quanshijin Company.
  • the reaction system is: 2 ⁇ Basic assembly mix 5 ⁇ l, the molar ratio of pCUB vector and PCR template is 1:2. Reaction conditions are: 50°C, 15 minutes. After the reaction is completed, place the centrifuge tube on ice for a few seconds.
  • PCR reaction system 2 ⁇ TaqPCRMasterMix 10 ⁇ L, upstream primer 126100EF 1 ⁇ L, downstream primer 126100ER 1 ⁇ l, ddH 2 O 8 ⁇ L, PCR reaction program: 94°C pre-denaturation 5min; 94°C denaturation 15sec, 55°C annealing 15sec, 72°C extension 1min15s, 30 cycles /min; extend for 7 minutes at 72°C; store at 20°C;
  • the recombinant plasmid was sent for sequencing.
  • the sequencing primers were: 126100EF and 126100ER.
  • the correctly sequenced vector was named pCUB-TaPDIL4-1B.
  • Agrobacterium C58C1 Pick a single colony of Agrobacterium and inoculate it into 3ml LYEB (60mg/L rif) liquid medium, shake culture at 28°C overnight; take 500 ⁇ L and inoculate it into 50ml LYEB (60mg/L rif) liquid medium.
  • the process is done by biotech.
  • the TaPDIL4-1B gene was transferred into the wheat variety Fielder using Agrobacterium-mediated method, so that the gene can be overexpressed in wheat (this process was completed by a commercial biological company).
  • the transgenic lines were tested for positive plants using the following PCR system: 2 ⁇ TaqPCR MasterMix 10 ⁇ L, upstream primer 126100EF 1 ⁇ L, downstream primer 126100ER 1 ⁇ l, ddH 2 O 8 ⁇ L.
  • the PCR reaction program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 15 sec, annealing at 55°C for 15 sec, and 72 Extend at °C for 1 min for 15 seconds, 30 cycles/min; extend at 72°C for 7 min; store at 20°C; the amplified product is subjected to gel electrophoresis. If a specific band of 1155 bp appears, it is proved to be a positive plant ( Figure 1).
  • the T2 generation transgenic lines overexpressing the TaPDIL4-1B gene were used to identify the resistance to scab. Phenotypic identification results showed that 21 days after single-flower instillation inoculation, the scab resistance of the transgenic line was significantly improved compared to the wild-type Fielder (Figure 2A). The diseased spikelet rate of the transgenic line was 23.0%, while that of the wild-type Fielder The diseased spikelet rate was 78.1%, the difference was extremely significant (Figure 2B), indicating that overexpression of TaPDIL4-1B gene can significantly improve wheat scab resistance.
  • transforming the gene TaPDIL4-1B into plants can make the plants have strong resistance to scab.

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Abstract

本发明提供了一种TaPDIL4-1B基因在植物抗赤霉病中的应用及其转基因植株的构建方法，所述TaPDIL4-1B基因的cDNA序列如SEQ ID NO:1所示。本发明从小麦中获得基因TaPDIL4-1B，通过农杆菌介导的方法将该基因转入植物中，该转基因能够使植物具有较强的抗赤霉病能力，这为农作物抗赤霉病新品种的研究和培育提供了良好的候选基因。

Description

TaPDIL4-1B基因在植物抗赤霉病中的应用及其转基因植株的构建方法 技术领域
本发明涉及基因工程技术领域，具体为TaPDIL4-1B基因在植物抗赤霉病中的应用及其转基因植株的构建方法。
背景技术
小麦赤霉病(Fusarium Head Blight，FHB)是由镰刀菌引起的一种全球性真菌病害，随着全球气候变暖以及耕作制度的变化，赤霉病的发生频率不断增加，发生范围也不断扩大。小麦赤霉病不仅严重影响小麦产量，而且镰刀菌在侵染小麦过程中产生大量真菌毒素，受毒素污染的小麦及其产品若被人畜食用，会引起呕吐、腹泻、流产等问题，严重危害人畜健康。小麦赤霉病严重影响粮食生产和食品安全，已成为国际社会普遍关注的焦点。
小麦赤霉病遗传改良因主效基因较少以及抗病机理尚不明晰等原因，导致抗性改良进展较慢，难以满足生产需求。因此，克隆能够抵抗赤霉病的新基因并研究其分子机制，进行小麦抗赤霉病改良具有重要意义。
目前有研究结果表明，活性氧稳定(ROS homeostasis)和细胞程序性死亡(programmed cell death)与小麦赤霉病抗性相关。硫氧还蛋白(Trx)和谷氧还蛋白(Grx)两个并行的还原酶系统可以相互调控，起到清除生物体内ROS和维持生物体内氧化还原平衡等多种功能。蛋白质二硫键异构酶(protein disulfide isomerase，PDI)含有数量和种类不等的Trx结构域，属硫氧还蛋白超家族，植物中的PDI家族基因一般被命名为PDI类蛋白基因(protein disulfide isomerase-like，PDIL)，目前该类基因的功能研究主要集中在小麦品质形成方面。截止目前，尚没有小麦PDIL基因参与小麦赤霉病抗性调控的报道。
发明内容
本发明的目的在于提供一种TaPDIL4-1B基因在植物抗赤霉病中的应用及其转基因植株的构建方法，为农作物抗赤霉病新品种的研究和培育提供候选基因，以培育更多抗赤霉病新品种。
为实现上述目的，本发明提供如下技术方案：
TaPDIL4-1B基因在植物抗赤霉病中的应用，所述TaPDIL4-1B基因的cDNA序列如SEQ ID NO:1所示。
其中，所述TaPDIL4-1B基因的gDNA序列如SEQIDNO:2所示，其gDNA由11个外显子和10个内含子组成；从5’端开始，外显子的长度依次为224bp、85bp、98bp、56bp、118bp、28bp、92bp、157bp、125bp、100bp、488bp，内含子长度依次为1131bp、143bp、85bp、422bp、96bp、115bp、216bp、86bp、122bp、88bp。
其中，TaPDIL4-1B基因的编码蛋白的氨基酸序列如SEQIDNO:3所示。该蛋白是一种定位于内质网的硫代二硫键氧化还原酶。
一种重组质粒，所述重组质粒所述的小麦抗赤霉病基因TaPDIL4-1B；该质粒的载体优选为pMWB110，即重组质粒优选为pMWB110-TaPDIL4-1B，此外，任何一种可以将外源基因导入植物中表达的载体都可以用于本发明。
TaPDIL4-1B基因在选育抗赤霉病植物品种中的应用，所述TaPDIL4-1B基因的cDNA序列如SEQ ID NO:1所示。其中，所述植物为小麦。
具体应用方法为：将TaPDIL4-1B基因或者将含有TaPDIL4-1B基因的重组质粒导入宿主小麦的细胞、组织或器官中，培育得到具有抗赤霉病小麦新品种。
其中，所述重组质粒为pMWB110-TaPDIL4-1B。
为了便于对转基因植物或细胞系进行筛选，对含有pMWB110-TaPDIL4-1B进行加工，加入选择标记，如GUS等。
与现有技术相比，本发明的有益效果是：
本发明应用了植物基因工程技术，首次从小麦中国春中获得基因TaPDIL4-1B，通过实验证明，通过农杆菌介导的方法将该基因转入植物(小麦)中，该转基因能够使植物(小麦)具有较强的抗赤霉病能力，这为农作物抗赤霉病新品种的研究和培育提供了良好的候选基因，对实现培育抗赤霉病植物新品种具有十分重要的意义。本发明发现的抗赤霉病新基因TaPDIL4-1B对小麦抗赤霉病遗传改良具有重要意义，适于推广应用。
附图说明
图1为转基因TaPDIL4-1B小麦阳性植株的PCR检测结果。#1-#8为转基因株系，Fielder为野生型，NC为扩增模板为水的阴性对照。
图2为接种21天后过表达株系和野生型Fielder的赤霉病抗性比较。其中，(A)为转TaPDIL4-1B基因小麦赤霉病抗性的表型鉴定结果(单花滴注接种穗部发病情况)；(B)为转TaPDIL4-1B基因小麦赤霉病发病小穗率数据统计结果。T为转基因株系，CK为野生型Fielder。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。实施例中所用的试验材料、试剂等，如无特殊说明，均可从商业途径得到。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。文中的M表示mol/L。
本发明所用小麦品种中国春(英文名称Chinese Spring)，是一个非常重要的小麦地方品种，中国春被广泛应用于小麦遗传学研究。转基因所用小麦品种为Fielder，为小麦转基因常用的模式品种，具有转化效率高的特点。
实施例1 小麦TaPDIL4-1B基因的克隆
1、提取小麦总RNA
(1)将小麦品种中国春的组织材料放入液氮预冷的研钵中，在液氮中充分研磨成粉末；
(2)待液氮挥发干，立即转移到2ml的离心管中，每100mg材料约加入1ml的Invitrogen公司的Trizol提取液，融化后，用加样枪反复吸吹，剧烈振荡混匀样品，使其充分裂解，室温放置5分钟；
(3)加入0.2ml氯仿(chloroform)，剧烈振荡混匀15秒，室温放置10分钟；在4℃ 下，12000rpm离心15分钟；
(4)小心吸出上层水相，加入到干净的1.5ml的离心管中，加入500μl的异丙醇(上层水相与异丙醇的体积比为1:1)，充分混匀，在-20℃下，沉淀30min；在4℃下，12000rpm离心10min，小心弃去上清液，留沉淀；
(5)将沉淀用1ml的体积百分比浓度为75％的乙醇溶液洗涤，在4℃下，8000rpm离心10min，收集RNA沉淀；
(6)RNA沉淀于无菌操作台上晾干约10-15分钟，当RNA略显透明，加入50μl的RNase-free水充分溶解，可放于-80℃下长期保存，备用；
(7)用紫外分光光度计及质量百分比浓度为1％的Agrose凝胶电泳检测RNA浓度及质量。
2、cDNA反转录
按照RNAPCRKit(AMV)Ver.3.0试剂盒(TaKaRa,DRR019A)的说明书进行；首先进行第一步反转录反应，以500ng的RNA为模板进行反转录，按照所述说明书指示在反应体系中依次加入由MgCl2、10×RT buffer、RNase Freed H2O、dNTP Mixture、RNase Inhibitor、AMV Reverse Transcriptase、Oligo dT Primer和Total RNA组成的反应体系共10μL。反应程序为：42℃ 30min，99℃ 5min，5℃ 5min。
3、克隆和序列测定
(1)以cDNA为模板，在5’UTR和3’UTR设计基因特异引物，引物序列(如SEQ ID NO：4-5所示)分别为：
上游引物F1：5’-GAGCTCGCAGCAAAACAGAT-3’，下游引物R1：5’-CCGCTAAACTTTCACTGCCA-3’；
(2)PCR反应体系(共20μL)：
2×TaqPCR MasterMix  10μL
上游引物F1  0.5μL
下游引物R1  0.5μL
模板cDNA  1μL
ddH2O     8μL
(3)PCR反应程序：94℃预变性5min；94℃变性30sec，58℃退火30sec，72℃延伸1min15s，30循环/min；72℃延伸7min；20℃保存，扩增得到的特异扩增条带。
回收PCR产物连接pMT18-T(宝生物技术有限公司)进行测序，扩增得到的PCR产物的核苷酸序列，其起始密码子到终止密码子的大小为1104bp(SEQ ID NO：1所示)，该PCR产物的基因命名为TaPDIL4-1B，该基因TaPDIL4-1B的编码蛋白的氨基酸序列如SEQ ID NO：3所示。
实施例2 TaPDIL4-1B基因组gDNA全长克隆
(1)gDNA提取：按照植物gDNA提取试剂盒说明书(天根生化科技有限公司，北京)方法提取中国春小麦gDNA；
(2)以中国春的gDNA为模板，在5’UTR和3’UTR设计基因特异引物，上下游引物如下(SEQ ID NO：6-7所示)：
上游引物F2：5’-TAGGAAGCCAAAGCGTTCGT-3’，
下游引物R2：5’-TACTCGTGGCGATCCATTCG-3’；
进行PCR扩增，扩增条件同实施例1扩增cDNA的操作步骤。将得到的特异扩增条带，连接pMT18-T载体并测序，得到了基因TaPDIL4-1B在中国春中的全长gDNA序列，如SEQ ID NO：2所示，从起始密码子到终止密码子大小为4075bp。其gDNA由11个外显子和10个内含子组成；从5’端开始，外显子的长度依次为224bp、85bp、98bp、56bp、118bp、28bp、92bp、157bp、125bp、100bp、488bp，内含子长度依次为1131bp、143bp、85bp、422bp、96bp、115bp、216bp、86bp、122bp、88bp。
实施例3 重组质粒pCUB-TaPDIL4-1B的获得
(1)合成包含pCUB载体上下游同源臂(下划线为同源臂)的引物： (126100OEF:TTGGTGTTACTTCTGCAGGTCGACTATGGCGACCCCTCAGATCTAC
126100OER:ATCGGGGAAATTCGAGCTCGGTACCCTTAAGAGGAGAAGGCTGAAAG)，如SEQ ID NO：8-9所示。
以实例1中获得的cDNA作为模板，用KOD FX DNA聚合酶(货号为：KFX-101)进行扩增酶进行PCR扩增。反应体系为(20μl)：KOD FX(1单位/μl)，0.4μl，2×PCR buffer 10μl,dNTP 2.4μl,126100EF和126100ER引物(10μM)个0.6μl，cDNA模板1μl，去离子水5μl。得到含有pCUB载体同源重组臂的TaPDIL4-1B基因的PCR产物。用Axygen胶回收试剂盒进行PCR产物回收。
(2)用上一部分所获得的含有同源臂的PCR模板和pCUB载体通过同源重组方式进行载体构建。载体为全北京全式金公司的Peasy-Basic Seamless cloning and assembly kit。反应体系为：2×Basic assembly mix 5μl，pCUB载体和PCR模板的摩尔比为1:2。反应条件为：50℃，15分钟。反应结束后，将离心管置于冰上数秒。
(3)重组质粒的鉴定：用菌落PCR法筛选有插入片段的克隆，具体操作如下：
①挑取转化后白色菌落在平板上划短线，37℃培养至菌线可见，进行菌落PCR反应；
②用牙签刮取少量的菌体转入20μl含所述引物的PCR体系中，进行PCR反应。PCR反应体系：2×TaqPCRMasterMix10μL、上游引物126100EF 1μL、下游引物126100ER 1μl、ddH2O8μL，PCR反应程序为：94℃预变性5min；94℃变性15sec，55℃退火15sec，72℃延伸1min15s，30循环/min；72℃延伸7min；20℃保存；
③PCR产物于0.8％琼脂糖凝胶电泳，检测是否含有1155bp分子量大小的片段(即SEQ ID NO：1序列与引物126100OEF、126100OER结合后的片段)，验证载体为正确的构建，鉴定重组质粒后即得到带有目的基因的植物表达载体。
将重组质粒送去测序，测序引物分别为：126100EF和126100ER，将测序正确的载体命名该重组质粒为pCUB-TaPDIL4-1B。
实施例4 将重组质粒转入农杆菌C58C1感受态
(1)培养农杆菌C58C1：挑取农杆菌单菌落接种于3mLYEB(60mg/L rif)液体培养基中，28℃摇培过夜；取500μL接种于50mLYEB(60mg/L rif)液体培养基中，28℃摇培至OD600为0.6；将菌液转入50mL离心管中，冰浴30min；4℃，5000g，离心5min；弃上清，沉淀用10mL0.15M的NaCl重悬；4℃，5000g，离心5min；弃上清，沉淀用1mL20mM的CaCl2重悬；每管100μL/管分装；液氮冷冻5min；-70℃保存；
(2)重组质粒的转入：将50ng实施例3制备的重组质粒pCUB-TaPDIL4-1B加入30μL农杆菌C58C1感受态中，混匀；依次冰浴30min，液氮冷冻3-5min，37℃水浴5min；加入1mLYEB液体培养基，28℃缓慢摇培2-4h；4℃，5000rpm，离心5min；弃部分上清，剩余上清重悬菌体后涂于YEB(60mg/Lrif)固体培养基上，28℃培养2-3天；
(3)菌体PCR鉴定，挑阳性克隆得到重组农杆菌，其具体鉴定步骤同实施例3中步骤(4)，得到含有重组质粒的农杆菌C58C1。
实施例5 转基因小麦的筛选及获得
该过程由生物公司完成。
利用农杆菌介导法将TaPDIL4-1B基因转入小麦品种Fielder中，使该基因在小麦中可以过量表达(该过程由商业化生物公司完成)。转基因株系利用下列PCR体系进行阳性植株检测：2×TaqPCRMasterMix10μL、上游引物126100EF1μL、下游引物126100ER1μl、ddH2O8μL，PCR反应程序为：94℃预变性5min；94℃变性15sec，55℃退火15sec，72℃延伸1min15s，30循环/min；72℃延伸7min；20℃保存；扩增产物经凝胶电泳，若出现为1155bp的特异条带，则证明其为阳性植株(图1)。
实施例6 转基因TaPDIL4-1B小麦抗赤霉病功能鉴定
为了验证该基因功能，利用过表达TaPDIL4-1B基因的T2代转基因株系进行抗赤霉病功能鉴定。表型鉴定结果表明，单花滴注接种21天后，相对于野生型Fielder，转基因株系的赤霉病抗性明显提高(图2A)。转基因株系的病小穗率为23.0％，而野生型Fielder 的病小穗率为78.1％，差异极显著(图2B)，说明过表达TaPDIL4-1B基因可显著提高小麦赤霉病抗性。
综上所述，将基因TaPDIL4-1B转入植物中能够使植物具有较强的赤霉病抗性。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。

Claims (8)

  1. TaPDIL4-1B基因在植物抗赤霉病中的应用，其特征在于：所述TaPDIL4-1B基因的cDNA序列如SEQ ID NO:1所示。
  2. 根据权利要求1所述的TaPDIL4-1B基因在植物抗赤霉病中的应用，其特征在于：所述TaPDIL4-1B基因的gDNA序列如SEQIDNO:2所示，其gDNA由11个外显子和10个内含子组成；从5’端开始，外显子的长度依次为224bp、85bp、98bp、56bp、118bp、28bp、92bp、157bp、125bp、100bp、488bp，内含子长度依次为1131bp、143bp、85bp、422bp、96bp、115bp、216bp、86bp、122bp、88bp。
  3. 根据权利要求1所述的TaPDIL4-1B基因在植物抗赤霉病中的应用，其特征在于：TaPDIL4-1B基因的编码蛋白的氨基酸序列如SEQIDNO:3所示。
  4. 一种TaPDIL4-1B转基因植株的构建方法，其特征在于：所述TaPDIL4-1B基因的cDNA序列如SEQ ID NO:1所示。
  5. 根据权利要求4所述的转基因植株的构建方法，其特征在于：所述植物为小麦。
  6. 根据权利要求5所述的转基因植株的构建方法，其特征在于：将TaPDIL4-1B基因或者将含有TaPDIL4-1B基因的重组质粒导入宿主小麦的细胞、组织或器官中，培育得到具有抗赤霉病小麦新品种。
  7. 根据权利要求6所述的转基因植株的构建方法，其特征在于：所述重组质粒为pMWB110-TaPDIL4-1B。
  8. 根据权利要求7所述的转基因植株的构建方法，其特征在于：对含有pMWB110-TaPDIL4-1B进行加工，加入选择标记。
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