CN106148351B - Gene g61 for improving disease resistance of ginseng and encoding protein thereof - Google Patents

Abstract

The invention relates to a gene g61 for improving disease resistance of ginseng and a coding protein thereof, belongs to the field of biotechnology and medicinal plant genetic engineering, and mainly relates to a method for analyzing ginseng transcriptome data by utilizing bioinformatics and molecular biology, cloning a high-expression disease-resistant gene in ginseng under forest, a coding product and application thereof. Specifically, the invention separates and obtains a protein G61 which is highly expressed in wild type Changbai forest ginseng and is related to disease resistance and a gene G61 thereof by comparing the difference of gene transcripts in wild type Changbai forest ginseng (CBA) with disease resistance and cultivated variety Changbai cultivated ginseng (CBB), Ji' an cultivated ginseng (JAB) and Fusong cultivated ginseng (FSB) with poor disease resistance.

Description

gene g61 for improving disease resistance of ginseng and encoding protein thereof

Technical Field

The invention belongs to the field of biotechnology and medicinal plant genetic engineering, and mainly relates to a method for analyzing ginseng transcriptome data by utilizing bioinformatics and molecular biology means, cloning a high-expression disease-resistant gene in ginseng under forest, and a coding product and application thereof.

Background

The invention aims to provide a protein G61 related to disease resistance of ginseng and a gene G61 thereof, which are used for improving the disease performance of plants.

Ginseng (Panax ginseng c.a.meyer) is a species of Panax in Araliaceae (Araliaceae), is a famous and precious Chinese medicinal material, is known as the king of various grasses, and has a long history of application. Researches show that the ginsenoside has good effects in regulating immunity, resisting pressure, resisting fatigue, resisting oxidation, resisting inflammation, resisting tumor, reducing blood sugar, protecting liver, enhancing memory and the like, so the ginsenoside has wide application prospect and considerable economic value. However, the growth of ginseng is slow, and wild ginseng is increasingly rare along with the development of wild resources by people.

The ginseng cultivation method can be divided into ginseng under forest and ginseng garden according to different ginseng cultivation methods, wherein the ginseng under forest is obtained by artificially sowing ginseng seeds in a forest suitable for ginseng growth, and the growth and development of the ginseng seeds are completely completed under natural conditions; the garden ginseng is sown, grown and developed completely under artificial condition. Under the condition of artificial cultivation, the garden ginseng has sufficient fertilizer and water and fast growth, can grow to 15-20cm in 5 years generally and has the weight of 100-200 g; while the ginseng grows for 5 years under the forest, the main root only grows for 3-4cm, and the weight is about 5-15 g. Although garden ginseng grows faster, its medicinal components are less than those of ginseng under forest, and thus its value is low. Since the planting density of the garden ginseng is high, the garden ginseng has more diseases and pests, and the garden ginseng must be harvested in 5 years generally, otherwise, the accumulation of the diseases can cause the death of plants, and destructive loss is brought to ginseng farmers. The forest ginseng has strong disease resistance, can grow for decades, and the content and the economic value of the nutrient components are increased along with the growth of the growth years.

With the advent of the post-genome era, various omics technologies such as transcriptomics, proteomics, metabolomics and the like have appeared in succession, wherein transcriptomics is the technology which is developed first and has the most extensive application. The rules of the genetic center indicate that genetic information is transmitted from DNA to proteins through messenger RNA (mRNA) under precise control. Thus, mRNA is considered to be a "bridge" between the transmission of biological information between DNA and protein, and the identity of all expressed genes, and their transcription levels, are collectively referred to as the Transcriptome (Transcriptome) [2 ]. The transcriptome is the sum of all RNAs transcribed from a particular tissue or cell at a certain developmental stage or functional state, and mainly comprises mRNA and non-coding RNA (ncRNA). Transcriptome studies are the basis and starting point for gene function and structure studies, understanding the transcriptome is essential for interpreting genomic functional elements and revealing molecular composition in cells and tissues, and has important roles in understanding body development and disease. With the commercialization of Next-generation sequencing (NGS) platforms, the use of RNA-Seq technology has revolutionized the way transcriptomics is thought. RNA-Seq, also known as transcriptome sequencing, is a recently developed technology for transcriptome analysis using deep sequencing technology, which can detect the overall transcriptional activity of any species at the single nucleotide level, and can find unknown transcripts and rare transcripts while analyzing the structure and expression level of transcripts.

The invention discovers the disease-resistant related genes highly expressed in the ginseng under forest by analyzing transcriptome of 6 samples of 5 producing areas.

Disclosure of Invention

According to the invention, through comparing the difference of gene transcripts in wild type Changbai forest ginseng (CBA) with disease resistance and cultivated species Changbai cultivated ginseng (CBB), Ji' an cultivated ginseng (JAB) and Fusong cultivated ginseng (FSB) with poor disease resistance, a protein G61 which is highly expressed in the wild type Changbai forest ginseng and is related to disease resistance and a gene G61 thereof are obtained by separation, and the gene can be used for improving the disease resistance of plants.

The g61 gene related to disease resistance provided by the invention is derived from Chinese ginseng (Panax ginseng C.A.Mey) of Panax of Araliaceae, and encodes protein with the following amino acid sequence:

SEQ ID NO:1 consists of 246 amino acid residues and is protein G61.

The encoding gene of the disease resistance related GTLP of ginseng in the invention can be a cDNA sequence of the gene, a genome DNA sequence of the gene, or a DNA sequence which has more than 90% of homology with the gene and encodes the same functional protein. Has the sequence shown in SEQ ID NO:1 can have the amino acid sequence shown in SEQ ID NO: 2.

The expression vector, the transgenic cell line and the host bacterium containing the gene belong to the protection scope of the invention.

Primer pairs for amplifying any fragment of g61 are also within the scope of the invention.

The invention provides a gene g61 related to disease resistance. The homology of the amino acid sequence of the gene with the plant disease-resistant gene database PRGDB00213092 gene reaches 48.8, and the E value is 2E-58. The homology of the gene with the fungal disease resistance related gene thaumatin-like protein [ Cucumis melo ] of the European cork oak is up to 90 percent, and the E value is 5E-149.

another object of the present invention is to provide a method for improving disease resistance of plants.

The method for improving the disease resistance of the plant provided by the invention is to introduce the gene which encodes the disease resistance-related gene of the invention into plant tissues, cells or organs, so as to improve the disease resistance of the plant.

In the method for improving disease resistance of plants, the g61 gene related to disease resistance of tobacco in the invention can be a cDNA sequence of the gene or a genome gene sequence of the gene; the DNA sequence which has more than 90% of homology with the gene and encodes the same functional protein is obtained by separating and/or modifying and/or designing cDNA or genome gene sequence of the gene by a known method. It will be appreciated by those skilled in the art that minor changes in nucleotide identity in a particular gene sequence may result in a reduction or enhancement in the efficacy of the gene, and that in some applications (e.g., antisense or cosuppression techniques), partial sequences will often function as effectively as full-length sequences. Methods for altering or shortening gene sequences, and methods for testing the effectiveness of such altered genes, are well known to those skilled in the art.

The g61 gene related to disease resistance or homologous sequence thereof can be introduced into plant tissues, cells or organs through a plant expression vector; the starting vector used for constructing the plant expression vector can be any binary vector which can be used for transforming plants by agrobacterium tumefaciens or agrobacterium rhizogenes or a vector which can be used for plant microprojectile bombardment, such as a pBin series vector (such as pBin 19) or a pBI series vector (such as pBI 101) or a gateway TW series vector (such as pH2GW 7) or the like), a pCAMBIA series vector (such as pCAMBIA 3301 or the like), per8 or pX6 or other derivative plant expression vectors, and the starting vector can also be a vector which can be replicated in prokaryotes, such as pENTER-TOPO, pUCppUCCUscript series vector or the like.

When the plant expression vector is constructed by using the disease resistance related g61 gene of ginseng or its homologous sequence, any enhanced, constitutive, tissue-specific or inducible (ABA, drought, saline alkali or chemical induction, etc.) promoter can be added before the transcription initiation nucleotide. The constitutive expression promoter can be a cauliflower mosaic virus (CAMV)35S promoter, a maize Ubiquitin promoter or a rice actin1 promoter and the like; the tissue-specific expression promoter can be a root-specific expression promoter, a leaf-specific expression promoter, a vascular-specific expression promoter, a seed-specific expression promoter, a flower-specific expression promoter or a pollen-specific expression promoter, such as a 2S1 promoter (GenBank No.: NM-118848.2, GI:30687489) and a NapinA (GenBank No.: M64633.1, GI:349405) promoter; the inducible promoter can be a promoter induced by low temperature, drought, ABA, ethylene, saline alkali or chemistry and the like. The above promoters may be used alone or in combination with other plant promoters. In addition, when the gene of the present invention is used to construct plant expression vectors, enhancers, including translational enhancers and/or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codons or initiation codons of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound which can produce a color change (GUS gene, GFP gene, luciferase gene, etc.), an antibiotic marker having resistance (neomycin phosphotransferase (NPTII) gene, Hygromycin phosphotransferase (Hygromycin phosphotransferase) gene, gentamycin marker, kanamycin marker, etc.), or a chemical agent resistance marker gene (e.g., herbicide resistance gene), etc., which can be expressed in plants. The host plant cell, tissue or organ containing the Neomycin Phosphotransferase (NPTII) gene may be selected by kanamycin or its alternative derivatives such as G418, etc., and the host plant cell, tissue or organ containing the Hygromycin phosphotransferase (Hygromycin phosphotransferase) gene may be selected by Hygromycin. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene. After the screening by the method, molecular detection means such as Southern, PCR or dot hybridization can be adopted to detect the transgenic plant so as to determine whether the transgenic plant is transformed with a target gene.

Wherein, the plant expression vector which is constructed by taking pCAMBIA1300 as a starting vector and contains the g61 gene related to stress tolerance of the inventor is named as pCactF-g 61. The plant expression vector carrying the g61 gene or its homologous sequence involved in stress tolerance of the present inventors can be used to transform plant cells, tissues or organs by using any one or a combination of several of conventional biological methods such as protoplast-chemical mediated method (Ca2+, PEG), Ti plasmid, Ri plasmid, plant viral vector, direct DNA transformation, pollen tube introduction, microinjection, electric stimulation, gene gun, agrobacterium mediation, etc., and culture the transformed plant cells, tissues or organs into plants; the tissues and organs may include pods, callus, stem tips, leaves, seeds, etc. of the host plant.

In addition, after the transgenic plant transformed with the g61 gene or the homologous sequence thereof involved in stress tolerance correlation of the present inventors is subcultured, a transgenic plant homozygous for the gene can be further selected therefrom. In addition, the transgenic plant can be propagated, so that the disease resistance of the transgenic plant can be further improved and enhanced. The propagation of the transgenic plant includes asexual propagation and/or seed propagation.

The method of the present invention is applicable to both dicotyledonous plants and monocotyledonous plants, and therefore, the transformed plant cell, tissue or organ may be derived from dicotyledonous plants such as tobacco, rape, cotton, soybean, poplar, pine, eucalyptus, potato or pasture, or may be derived from monocotyledonous plants such as rice, corn, wheat, barley, sorghum, millet or turf grass.

The invention provides a GTLP sweet egg gene g61 related to disease resistance. Experiments prove that the gene of the invention can improve the fungal disease resistance of tobacco when being transformed into the tobacco, and has no obvious influence on the normal growth and economic traits of the tobacco. The protein and the coding gene thereof have important theoretical and practical significance for researching the disease resistance mechanism of plants, improving the disease resistance of the plants and improving the related characters, play an important role in improving the disease resistance genetic engineering of the plants and have wide application prospect.

The present invention will be described in further detail with reference to specific examples.

drawings

FIG. 1 is a total RNA electrophoresis of ginseng samples, wherein 1 is Changbai ginseng under forest, 2 is Changbai ginseng, 3 is Jian ginseng, 4 is Customia ginseng, 5 is Xunke ginseng and 6 is Pandamia oblongata.

FIG. 2 shows that realtimePCR verifies the relative expression of gene g61 in each material; RQ is relative value of gene expression, CBA is Changbai Linnaeus ginseng, CBB is Changbai cultivated ginseng, JAB is Jian cultivated ginseng, FSB is Fusong cultivated ginseng, XKB is Xunke cultivated ginseng, and KDC is Pantianus pulchrus.

FIG. 3 is ORF prediction.

FIG. 4 functional domain prediction analysis.

FIG. 5 map of the T-DNA region of expression vector pCactF. LB and RB are the left and right borders of the T-DNA, respectively; hyg indicates hygromycin resistance; p35S denotes the promoter of the 35S gene; T35S represents the terminator of the 35S gene; pAct represents the promoter of the Actin gene; 3flag represents 3 times flag tag sequence; OCS represents the terminator of the OCS gene; HindIII, KpnI, SpeI, XbaI, SalI and PstI respectively represent the restriction sites of the restriction enzymes.

Detailed Description

The methods used in the following examples are conventional unless otherwise specified, and the primers and sequencing used were synthesized by Shanghai Yingjun Biotechnology, PCR kit was purchased from Toyobo Biotech Co., Ltd, endonuclease and T4DNA ligase in vector construction was purchased from Neuroy Biotechnology Co., Ltd (NEB), and pEASY-T1 ligation kit was purchased from Beijing Quanjin Biotechnology, all methods were performed according to the methods provided in the kits. The vector pHPG used in the experiment is obtained by modifying the experiment, and the basic framework is derived from pCAMBIA1303 of CAMBIA company.

Example 1 sample Collection

Collecting 5-year-old ginseng under forest and cultivated ginseng in 9 months of 2012, wherein the production area of the ginseng under forest is Changbai, and the production area of the cultivated ginseng is Changbai, and the ginseng is collected, pacified and pacified, and pacified. Collecting Ginseng radix, cleaning, and quick freezing with liquid nitrogen for low temperature storage. Wherein CBA is Changbai ginseng under forest, CBB, JAB, FSB, XKB and KDC are Changbai, Ji' an, Fu Song, Xunke and Guangdian ginseng respectively. Example 2 RNA extraction

Samples stored at-80 ℃ were first ground well in liquid nitrogen, total RNA was extracted using Qiagen's RNeasy plant mini kit, RNA purification was accomplished by removing residual DNA using DNase I (NEB), RNA integrity was checked by 1% agarose electrophoresis (FIG. 1), and A260, A280 ratios and concentrations were determined using a Nanodrop2000 nucleic acid quantifier.

Example 3 RNA-Seq and denovo splicing

mRNA was enriched from total RNA using oligo (dT) magnetic beads, followed by mRNA fragmentation into short fragments, first cDNA strand synthesis using hexabase random primers (random hexamers) using mRNA as template, and then construction of sequencing library for RNA sequencing using second generation Solexa HiSeq 2000. By removing the linker and low quality sequences, a total of 1.364X 10 was obtained⁸High quality reads, totaling to 2.08 × 10⁷base pairs. And carrying out De novo splicing on the sequencing result by using Trinity software to obtain 82,448 genes in total.

Example 4 Gene annotation and Gene differential expression

The resulting transcriptome was aligned with the NCBI non-redundant protein database (NR), swissprot and TAIR10 databases for a total of 102395 transcripts (about 60.7%) with functional annotation. Gene expression analysis was carried out using the programs Tophat-HTq-Seq in turn according to the methods in the literature (Tranell Cet al., Differential gene and transcript expression analysis of RNA-seqexences with TopHat and Cufflinks. Nature protocols 2012,7(3): 562-578; Anders S et al., HTSeq-A copy frame to work with high-throughput prediction data. bioRxiv prediction 2014, doi: 10.1101/002824; Anders S et al., Differential expression analysis for sequence correlation data. genome biology 2010,11(10): R106), with 822 transcripts having a significant difference (the average transcript value of the transcripts with a significant difference: 352 is greater than or less than the average transcript value of 352, 84. log2, 84. or less than the average transcript value of Fongel 2). The plant disease-resistant gene database PRGDB (http:// PRGDB. org) is compared by using BlastX, 5482 transcripts are totally obtained to obtain disease-resistant gene annotations, wherein the expression of the gene g61 in Changbai ginseng under forest (CBA) is obviously higher than that of other materials, namely, the ginseng under forest is higher than that of cultivated ginseng materials in various regions, and specific results are detailed in Table 1. The log2(Fold _ change) of the expression values of the gene g61 in CBA, KDC, CBB, FSB, JAB and XKB is calculated to be 2.5213,6.0443,7.288,3.2616 and 6.3752 which are more than 2 in sequence, which indicates that the expression of the gene g61 in CBA is obviously higher than that of other materials.

TABLE 1 relative expression of g61 in each material

Name of breed	CBA	KDC	CBB	FSB	JAB	XKB
							Relative expression value of g61	677.1476	117.9495	10.26568	4.331766	70.60526	8.214193

Example 5 verification of expression of disease-resistant Gene

According to the bioinformatic data, real time PCR primers (61realF 5-GCAAGCCGACAGCATACT-3, SEQ ID NO: 4; 61realR 5-TGAGGACAGAAGGTGAGGAA-3, SEQ ID NO:5) were designed using the spliced g61 transcript (the sequence of which is shown in SEQ ID NO:3 in the sequence Listing) as a template. cDNA extracted from CBA, KDC, CBB, FSB, JAB, XKB and other materials are taken as templates, diluted by 10 times, and subjected to quantitative analysis by 7500Fast Real-Time PCR System (applied biosystems), wherein the reaction System is as follows:

The PCR program was 95 ℃ 30sec

95℃ 5sec

60℃ 34sec

40 cycles

relative quantitation of GAPDH was performed with internal reference (F5-CTGTGGATGTCTCTGTGGTA-3, SEQ ID NO: 6; R5-CTCCGACTCCTCCTTGATAG-3, SEQ ID NO:7) 2^-ΔΔCt method statistics of gene expression. From the experimental results, it can be seen that the gene g61 is expressed in CBA higher than other materials, consistent with bioinformatics analysis results. FIG. 2 shows the relative expression level of gene g61 in each material, the ordinate shows the relative value of gene expression, CBA shows a forest ginseng with strong disease resistance, the g61 gene is 1, the values of other materials are relative values divided by CBA, and the abscissa shows each experimental material.

Example 6 functional prediction

The sequence of the spliced transcript of gene g61 was aligned with the plant disease resistance gene database PRGDB (http:// PRGDB. org) using blastx, and the results are shown in Table 2. The gene has homology from 154-810bp with 55-276bp of a presumed disease-resistant gene PRGDB00213092, and the value is 2.00E-58, which shows that the similarity of the two genes is very high. Based on the transcript sequence, 6 open reading frames are predicted. FIG. 3 shows that the open reading frame of the gene with disease resistance function is 85-822bp of SEQ ID NO 3, with 154-810bp as reading frame and ATG found at 5' end based on the homologous alignment result.

TABLE 2 Gene blastx results in disease-resistant database

Example 7 cloning of the Gene

According to the predicted ORF sequence, namely the sequence of D85-822 bp of the sequence SEQ ID NO. 3, a 5 'end primer is designed from the coding start site ATG of the gene, and a 3' end primer is designed at the position of a termination code:

ORF-F：5'-ATGGAAGTAATGCTGCTCAG-3'(SEQ ID NO:8)

ORF-R：5'-CTAAGCTAGACTAATGGTGAGGA-3'(SEQ ID NO:9)

Extracting total RNA of ginseng root, reverse transcription to obtain cDNA, and RT-PCR to obtain full-length gene.

The specific reaction is as follows: mu.l of 10 XDnase buffer, 1. mu.l of DNase, and DEPC-treated water were added to a 10. mu.l system, mixed, incubated at 37 ℃ for 30min, then 1. mu.l of RQ DNase stop solution, incubated at 65 ℃ for 10min to stop the reaction, then 2. mu.l of Oligo (dT)18primer (0.1. mu.g/. mu.l), 4. mu.l of 5 XFirst-strand buffer, 1. mu.l of Ribonucleae inhibitor (40U/. mu.l), 2. mu.l of 4 XdNTPs (10 mM each), 1. mu.l of MMLVREverse Transcriptase (200U/. mu.l), mixed carefully, and incubated at 37 ℃ for 1 hour. Then treated at 90 ℃ for 5 minutes, cooled on ice, and centrifuged to collect the corresponding reverse transcription product cDNA. The cDNA thus obtained was diluted 10-fold, and 1. mu.l of each of the upstream and downstream primers (10. mu.M), 1. mu.l of each of LA Taq enzyme (5U/. mu.l), 1. mu.l of 4 XDNTPs (10 mM each), 25. mu.l of 2 XBuffer (Mg2+), and 20.5. mu.l of H2O 20.5 were used as a template for PCR. The PCR reaction conditions were preheating at 95 ℃ for 5min, denaturation at 94 ℃ for 1min, annealing at 56 ℃ for 30sec, extension at 72 ℃ for 2min for 10sec, and 35 cycles. After the reaction is finished, carrying out 1% agarose gel electrophoresis detection on the PCR amplification product, and obtaining a DNA fragment with the size consistent with the expected result through amplification. The above fragments were recovered and purified, ligated into the vector pEASY-T1 (all-open gold Co.), transformed into E.coli (E.coli) TOP10 strain by heat shock method, positive colonies were selected and added to 5ml LB liquid medium containing 50mg/L kanamycin, and cultured in a shaker at 37 ℃ and 200rpm for 12-16 hours to extract plasmids, and recombinant plasmids containing the desired fragments were obtained.

After sequencing, the whole length of the gene is 738bp, and the nucleotide sequence is shown as SEQ ID NO:2, the amino acid sequence of the encoded protein is shown as SEQ ID NO:1 is shown.

Example 8 plant expression vector construction

Designing a 5 'primer containing a SalI enzyme cutting site from the coding start site ATG of the gene, and designing a 3' primer of a PstI enzyme cutting site at the termination code:

CDS-F：5'-gtcgac ATGGAAGTAATGCTGCTCAG-3'(SEQ ID NO:10)

CDS-R：5'-ctgcagCTAAGCTAGACTAATGGTGAGGA-3'(SEQ ID NO:11)

The gtcgac sequence in CDS-F is the restriction site of restriction enzyme SalI, and the underlined sequence is the coding sequence of the g61 gene; the ctgcag sequence in CDS-R is the restriction site of restriction enzyme PstI, and the underlined sequence is the coding sequence of the g61 gene.

Using a cloning vector containing g61 correctly sequenced as a template, 1. mu.l of each of the upstream and downstream primers (10. mu.M) was used as a template for PCR, 1. mu.l of KOD-Plus-Neo enzyme (1U/. mu.l), 5. mu.l of 4 XDNTPs (2 mM each), 5. mu.l of 10 XPCR Buffer for KOD-Plus-Neo, 5. mu.l of 25mM MgSO 45. mu.l, H₂O30. mu.l. The PCR conditions were preheating at 94 ℃ for 2min, denaturation at 94 ℃ for 10sec, annealing at 60 ℃ for 30sec, and elongation at 68 ℃ for 30sec, for 30 cycles. After the reaction is finished, carrying out 1% agarose gel electrophoresis detection on the PCR amplification product, and obtaining a DNA fragment with the size consistent with the expected result through amplification. Recovering and purifying the fragment, PCR cloning the primerAnd g61DNA fragment containing the enzyme cutting site is subjected to double enzyme cutting by SalI and PstI, a target fragment is cut off, the target fragment is connected with a pCactF plant expression vector subjected to double enzyme cutting to obtain pCactF-g61, and a colony identified as positive by PCR is selected for sequencing verification. The T-DNA region map of the plant expression vector pCactF is shown in FIG. 5.

Example 9 obtaining transgenic tobacco

The gene g61 containing the restriction enzyme cutting site obtained in the embodiment 4 is transformed into tobacco by agrobacterium-mediated method, which comprises the following steps:

The recombinant vector pCactF-g61 was transformed into Agrobacterium AGL0 strain (strain conservation Co., Ltd.) by heat shock method, and tobacco was co-transformed with Agrobacterium.

Extracting total DNA from partial leaves of the obtained tobacco transgenic plant by a conventional method, amplifying hygromycin phosphotransferase gene under the guide of a forward primer 5'-ACTCACCGCGACGTCTGT-3' (SEQ ID NO:12) and a reverse primer 5'-TTCCTTTGCCCTCGGACG-3' (SEQ ID NO:13), obtaining a 1009bp DNA fragment which is a positive transgenic plant by amplification, and detecting results show that the transgenic tobacco transformed with pCactF-g61 is obtained by the method.

Example 10 detection of disease resistance

T1 transgenic tobacco seeds are harvested, and 12 positive plants are obtained by screening with MS culture medium containing 30mg/L hygromycin. When the seedlings grow to 4 leaves, transplanting the seedlings into seedling pots. And when the seedlings grow to 6 leaves, performing disease resistance detection.

Taking tobacco leaves with blight, cutting with scissors, placing in 200ml sterile water, shaking with a shaking table at 200rpm for 20min, filtering with sterile gauze, and spraying to plant. Non-transgenic tobacco was used as a control. Disease condition investigation is carried out after 10 days, 1 true leaf of the transgenic seedling is obviously wilted, 1 true leaf of the transgenic seedling is slightly wilted, 2 leaves of the transgenic seedling are yellowed or wilted, 3 leaves of the transgenic seedling are yellowed, and 1 true leaf of the transgenic seedling is not diseased. And 7 non-transgenic tobaccos die, 3 tobacco leaves have 2 wilting leaves, and 2 tobacco leaves have 1 mild wilting leaf. The disease resistance of the tobacco with the transferred g61 gene is stronger than that of a wild control. The specific results are shown in Table 4.

The disease grading criteria in this example were: level 0: no symptoms; level 1: 1, yellowing of leaf of cotyledon; and 2, stage: 2 yellowing or wilting of the leaves; and 3, level: 1 true leaf slight wilting; 4, level: 1-2 leaves are obviously wilted; and 5, stage: the whole plant wilted or died severely. Wherein, the disease resistance is below 2, and the infection type is above 3.

Table 4: statistical table of disease resistance detection results of g61 transgenic tobacco

	Level 0	Level 1	Stage 2	Grade 3	4 stage	Grade 5
							Transgenosis	1	3	5	2	1	0
Wild type	0	0	0	2	3	7

The result is combined, the ginseng g61 gene and the coding protein thereof are related to disease resistance, and can improve the disease resistance of plants.

SEQUENCE LISTING

<110> Ming Xingwang System crop design front laboratory (Beijing) Co., Ltd

<120> Gene g61 for improving disease resistance of ginseng and protein encoded by same

<130>

<160> 13

<170> PatentIn version 3.3

<210> 1

<211> 245

<212> PRT

<213> Chinese ginseng (Panax ginseng C.A. Mey)

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Leu His Leu Pro Pro Ala Trp Ser Gly Arg Ile Trp Gly Arg His Gly

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Leu Ala Glu Ile Thr Leu Gly Ser Asp Gln Asp Phe Tyr Asp Val Ser

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atccaacccg gcgcaggcaa gccaatcctc gcccggggcg gcttcaagct tcggccgaag 180

aaatcctaca ctctgcatct tccccctgcg tggtccggcc ggatctgggg ccgtcacggc 240

tgtgcatttg atgcctccgg tcgtggaaag tgcgccaccg gtgattgtgg cggagcccta 300

ttctgcaacg gcattggcgg cactcctccg gccactcttg ccgaaatcac cctcggcagc 360

gaccaggact tctatgatgt cagccttgtt gatgggtaca acttggccat ctccattacc 420

cctttcaaag gctccggcaa atgcacctat gccggctgtg ttagtgatct gaacatgatg 480

tgtccagtgg gacttcaggt gcggtctcat gacaatcggc gagtggtggc gtgcaagagt 540

gcttgctttg ccttcaactc cccgagatat tgctgtacgg gaagctttgg gagtccgcaa 600

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ctctttatct tcctcttgac actacacatt tcagccacaa caataaccat atacaacaag 180

tgcacccacc cagtgtggcc cggaatccaa cccggcgcag gcaagccaat cctcgcccgg 240

ggcggcttca agcttcggcc gaagaaatcc tacactctgc atcttccccc tgcgtggtcc 300

ggccggatct ggggccgtca cggctgtgca tttgatgcct ccggtcgtgg aaagtgcgcc 360

accggtgatt gtggcggagc cctattctgc aacggcattg gcggcactcc tccggccact 420

cttgccgaaa tcaccctcgg cagcgaccag gacttctatg atgtcagcct tgttgatggg 480

tacaacttgg ccatctccat tacccctttc aaaggctccg gcaaatgcac ctatgccggc 540

tgtgttagtg atctgaacat gatgtgtcca gtgggacttc aggtgcggtc tcatgacaat 600

cggcgagtgg tggcgtgcaa gagtgcttgc tttgccttca actccccgag atattgctgt 660

acgggaagct ttgggagtcc gcaatcgtgc aagccgacag catactcgag gatattcaag 720

tccgcgtgtc caaaggctta ttcttatgct tatgatgatc ccactagtat tgctacttgc 780

actggtggta gttatttcct caccttctgt cctcaccatt agtctagctt agcttagtta 840

tattttctgt ctcttgctag tattatacta ttattactgt ttgatgagca tgttgttaga 900

tttccttgat ttgattttgg atttggctac aagtttaatg ggtagatgaa tgaatatgat 960

cggaattggc attttctttt gaagtacttt tggtagtttt aggttttgtt cgatttttag 1020

tgtttggagt ggagcgttta atgtatttag atcattaaac gggattcg 1068

<210> 4

<211> 18

<212> DNA

<213> Artificial Synthesis

<400> 4

gcaagccgac agcatact 18

<210> 5

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 5

tgaggacaga aggtgaggaa 20

<210> 6

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 6

ctgtggatgt ctctgtggta 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 7

ctccgactcc tccttgatag 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 8

atggaagtaa tgctgctcag 20

<210> 9

<211> 23

<212> DNA

<213> Artificial Synthesis

<400> 9

ctaagctaga ctaatggtga gga 23

<210> 10

<211> 26

<212> DNA

<213> Artificial Synthesis

<400> 10

gtcgacatgg aagtaatgct gctcag 26

<210> 11

<211> 29

<212> DNA

<213> Artificial Synthesis

<400> 11

ctgcagctaa gctagactaa tggtgagga 29

<210> 12

<211> 18

<212> DNA

<213> Artificial Synthesis

<400> 12

actcaccgcg acgtctgt 18

<210> 13

<211> 18

<212> DNA

<213> Artificial Synthesis

<400> 13

ttcctttgcc ctcggacg 18

Claims (2)

1. A method for improving the anti-blight capability of plants is characterized by comprising the step of transferring anti-blight genes into tobacco to obtain transgenic positive plants with improved anti-blight capability, wherein the amino acid sequence of the anti-blight genes is shown as SEQ ID NO. 1.

2. The method of claim 1, wherein the nucleotide sequence of the fusarium wilt resistance gene is set forth in SEQ ID No. 2.