阅读说明：本技术 一种岷江百合WRKY转录因子基因LrWRKY2及应用 (Lilium regale WRKY transcription factor gene LrWRKY2 and application thereof ) 是由 刘迪秋 普丽梅 陈虹均 郑锂蕾 李珊 王自娥 葛锋 于 2019-11-13 设计创作，主要内容包括：本发明公开了一种岷江百合WRKY转录因子基因<I>LrWRKY2</I>,其核苷酸序列如SEQ ID NO：1所述,编码如SEQ ID NO：2所示氨基酸序列的蛋白质,本发明通过功能基因组学相关技术研究证实<I>LrWRKY2</I>基因具有提高植物抗真菌的功能,将本发明抗真菌的<I>LrWRKY2</I>基因构建到植物表达载体上并转入烟草中过量表达,转基因烟草植株具有很强的抗真菌侵染能力,实验结果显示超表达<I>LrWRKY2</I>的转基因烟草对稻黑孢霉、茄腐镰刀菌、轮枝镰刀菌、葡萄座腔菌、人参链格孢的侵染具有高水平的抗性。(The invention discloses a Lilium regale WRKY transcription factor gene LrWRKY2 The nucleotide sequence is shown as SEQ ID NO:1, encoding the polypeptide shown as SEQ ID NO:2, the invention is proved by related technical research of functional genomics LrWRKY2 The gene has the function of improving the antifungal effect of plants, and the invention is antifungal LrWRKY2 The gene is constructed on a plant expression vector and is transferred into tobacco for over-expression, and the transgenic tobacco plant has strong true resistanceThe bacteria infection ability and the experimental result show that the over-expression is realized LrWRKY2 The transgenic tobacco has high-level resistance to infection of nigrospora oryzae, fusarium solani, fusarium verticillium, botrytis cinerea and alternaria ginseng.)

1. Lilium regale WRKY transcription factor geneLrWRKY2The method is characterized in that: the nucleotide sequence is shown as SEQ ID NO. 1, and the protein of the amino acid sequence shown as SEQ ID NO. 2 is coded.

2. Lilium regale WRKY transcription factor gene as claimed in claim 1LrWRKY2In improving tobacco to Nicotiana oryzae (Nigrospora oryzae) Fusarium solani (F.solani) (II)Fusarium solani) Fusarium verticillatum (A)Fusarium verticillioides) Staphylococus viticola (A. vinifera)Botryosphaeria dothidea) Ginseng, alternaria alternata (Alternaria panax) Use in resistance.

Technical Field

The invention relates to the field of research of related technologies of molecular biology and genetic engineering, in particular to a Lilium regale WRKY transcription factor gene with antifungal infection capacityLrWRKY2And application thereof.

Background

The WRKY transcription factor is a regulatory protein gene family in a Plant defense reaction related transcription factor network, is mainly involved in a Plant immune system to deal with various biological and abiotic stresses (PandeY SP, Somsich IE. of role of WRKY transcription factors in Plant immunity mechanism, 2009, 150 zinc finger (4): 1648 + E +.

Promoter regions of target genes regulated by WRKY mostly contain W-box (TTGACC/T), wherein TGAC is the core sequence of W-box and is highly conserved, and once a certain nucleotide is changed, the WRKY protein is reduced or even lost in binding capacity (Schwann, Wangjun, Chengjin, etc.. Plant WRKY transcription factor and biological function research progress. Chinese agricultural science and technology guide, 2016, 18(3): 46-54.). WRKY domain is composed of four β folds, conserved KYGQK residues correspond to β folds (strand β -1) at the N-terminal, can enter into DNA groove and interact with W cassettes on DNA (Ciolkowski I, WanD, Birkenbihl RP, et al. Studies on DNA-binding of selection genes) (strain 92, 2008-insert 92).

The WRKY transcription factor is reprogrammed to deal with the invasion of different pathogens by regulating a plant transcriptome, and the transcriptional regulation of the WRKY transcription factor on genes related to disease resistance response is a key component of the defense response of plants to pathogens and plays an important role in the defense response of plants. The WRKY gene family in plants contains many members that regulate plant growth and development and various stress responses (Wu ZJ, Li XH, Liu ZW, et alCamellia sinensisWRKY transformation factors in stress to temperature stress MolGenomics, 2016, 291(1): 255-69.). Overexpression in Populus diversifoliaPtrWRKY18 andPtrWRKY35can enhance the activity of rust fungus: (Melampsora) Simultaneously increasing the resistance in transgenic plantsPRExpression of The Gene (Jiang Y, Guo L, Ma X, et al, The WRKY transcription factors PtrWRKY18 and PtrWRKY35 promoterMelampsoraresistance inPopulusTree Physiol, 2017, 37(5): 665-. Strawberry FaWRKY1 regulating fruit pair Colletotrichum gloeosporioides negatively ((B))Colletotrichum acutatum) Resistance of (1) (Higura JJ, Garrido-Gala J, Lekhbou A, et al, The strain face WRKY1 transformation effects regulation resistance toColletotrichum acutatumin fruit uponInfection. Front Plant Sci, 2019, 10: 480.)。PtWRKY14Tobacco can be enhanced by overexpressionNicotiana tabacum) Resistance to TMV infection (wang xing, forest branches.PtWRKY14Research on the influence of gene transformation on tobacco and resistance to TMV, Guangdong agricultural science, 2014, 41(7): 130-. In addition, Salicylic Acid (SA) -mediated Reactive Oxygen Species (ROS) signaling enhancesJcWRKYResistance of transgenic tobacco to pathogenic bacteria of alfalfa carbon rot (Agarwal P, Patel K, Agarwal PK. Ectopic expression ofJcWRKYconfers enhanced resistancein transgenic tobacco againstMacrophomina phaseolina. DNA Cell Biol, 2018,37(4): 298-307.)。

The Bulbus Lilii is Liliaceae (Liliaceae) Lilium (Lilium)Lilium) The plant is a general term of perennial root flower. During the processes of seed ball propagation and fresh cut flower production, lily is vulnerable to various pathogenic bacteria such as fungi, viruses and bacteria. At present, the methodThe lily diseases found are more than dozens, among which, the lily is of the genus Fusarium (A)Fusariumspp.) blight caused by fungi (also known as basal rot, stem rot) is the most serious disease in lily production. After the fusarium infects the lily seed balls, the basal disc is necrotic, the scales are rotted and fall off, and the quality of the seed balls is reduced; after the plants are infected by fusarium, the leaves turn yellow and droop wilting, and the plants die in advance, so that the yield and the quality of the cut lily flowers are seriously influenced. Wherein Fusarium oxysporum (F.), (F. oxysporum) The pathogenicity is strongest, the separation frequency is highest, and the lily is the main pathogenic bacterium of lily wilt. Lilium regale (Lilium regale)L. regaleWilson) is a unique species in China, is only distributed in rock cracks from valleys with the altitude of 800-2700 m in Minjiang river basin to the mountains, has strong blight resistance, and is an important germplasm resource for modern lily breeding. WRKYs participate in responding to various biotic and abiotic stresses and are an important component of a plant defense system, so that important research and application values of the WRKY transcription factor gene in Lilium regale are provided.

Disclosure of Invention

The invention aims to provide a Lilium regale WRKY transcription factor geneLrWRKY2And application thereof, namely improving the effect of tobacco on the black sporotrichum oryzae (B)Nigrospora oryzae) Fusarium solani (F.solani) (II)Fusarium solani) Fusarium verticillatum (A)Fusarium verticillioides) Staphylococus viticola (A. vinifera)Botryosphaeria dothidea) Ginseng, alternaria alternata (Alternaria panax) Use in resistance.

The invention relates to a full-length gene of a WRKY transcription factor with antifungal activity, which is cloned from Lilium regaleLrWRKY2The nucleotide sequence is shown as SEQ ID NO. 1, the gene cDNA full length sequence is 1302bp, comprises a 1032 bp open reading frame, a 45 bp 5 'untranslated region and a 225 bp 3' untranslated region, and encodes protein of amino acid sequence shown as SEQ ID NO. 2.

In the inventionLrWRKY2The coding region of the gene is a nucleotide sequence shown in 46 th-1077 th sites in a sequence table SEQ ID NO. 1.

The invention separates and clones an antibody of Lilium regaleThe complete cDNA fragment of a fungus-related gene using Agrobacterium tumefaciens (A. tumefaciens) (A. tumefaciens)Agrobacterium tumefaciens) The target gene is transferred into a receptor plant and is overexpressed, whether the gene has antifungal activity is verified through further experiments, a foundation is laid for the capability of improving tobacco and other plants to resist fungal diseases by utilizing the gene in the later period, and the inventor names the gene asLrWRKY2。

As described aboveLrWRKY2The gene can be applied to improving the antifungal property of tobacco, and the specific operation is as follows:

(1) using amplificationLrWRKY2The specific primer is used for extracting total RNA from the root of Lilium regale after inoculating fusarium oxysporum and amplifying the total RNA by reverse transcription-polymerase chain reaction (RT-PCR)LrWRKY2Then connecting the full-length coding region to a pGEM-T vector, and obtaining a clone with a target gene through sequencing;

(2) using restriction endonucleasesBamHI andXbai enzyme digestion pGEM-T-LrWRKY2Recovering the carrier by glue to obtain target gene segment, using the same endonuclease to enzyme-cut plant expression carrier pCAMBIA2300s, recovering the glue to obtain the required carrier large segment, and recovering the obtained carrier large segmentLrWRKY2Connecting the gene fragment with the pCAMBIA2300s fragment to construct a plant overexpression vector, and then transferring the constructed recombinant vector into tobacco to express through the mediation of agrobacterium tumefaciens;

(3) screening transformants by using a resistance marker on the recombinant vector T-DNA, obtaining a real transgenic plant through PCR and RT-PCR detection, analyzing the capability of the transgenic plant leaf against fungal infection, and finally screening out the transgenic plant with obviously enhanced fungal resistance.

The invention provides a new method for improving the resistance of plants to fungal diseases, the defects of traditional breeding can be overcome by cultivating disease-resistant plants by means of genetic engineering, the breeding period is shortened, the operation is simple, and high-resistance materials are easy to obtain. The invention is derived from Lilium regaleLrWRKY2The gene can enhance the resistance of plants to fungi, and can be introduced into tobacco to produce new varieties and new materials with fungal resistance.The cultivation of resistant plant varieties and materials by utilizing the genetic engineering technology has obvious advantages and irreplaceable importance; the invention not only can provide convenience for large-scale production of crops, medicinal materials, horticultural plants and the like, greatly reduces the use of chemical pesticides, but also can save the cost for agricultural production and reduce the environmental pollution, thereby having wide market application prospect.

Drawings

FIG. 1 is a drawing of the present inventionLrWRKY2PCR detection result graph of transgenic tobacco genome DNA, in the graph: the Marker is DL2000 DNA Marker (Dalibao biology); the positive control is plasmid pGEM-T-LrWRKY2PCR products as templates; WT is the product of PCR using total DNA of non-transgenic tobacco (wild type) as template;

FIG. 2 shows the positivity of the present inventionLrWRKY2In transgenic tobaccoLrWRKY2A graph of the results of expression analysis at the transcriptional level; in the figure: marker is DL2000 DNA Marker (Dalibao biology); WT is a PCR product with non-transgenic tobacco total RNA reverse transcription cDNA as a template; the positive control was plasmid pGEM-T-LrWRKY2A PCR product as a template;

FIG. 3 is a drawing of the present inventionLrWRKY2A result graph of transgenic tobacco resistance identification; in the figure, a, b, c, d and e are respectively inoculated with Fusarium oxysporum, Fusarium verticillarum, Staphylotrichum botrytis, Fusarium solani and alternaria ginsengLrWRKY2Transgenic tobacco leaf; WT is leaf of wild tobacco, 1, 8, 17, 27 areLrWRKY2Leaves of transgenic tobacco.

Detailed Description

The present invention is further illustrated by the following figures and examples, but the scope of the present invention is not limited to the above description, and the examples are conventional methods unless otherwise specified, and reagents used are conventional commercially available reagents or reagents formulated according to conventional methods unless otherwise specified.