CN112143736A - BnTLP1 gene for regulating and controlling cabbage type rape sclerotiniose resistance and application thereof - Google Patents

Abstract

The invention discloses a BnTLP1 gene for regulating and controlling cabbage type rape sclerotinia sclerotiorum resistance and application thereof. The invention obtains a gene BnTLP1 for regulating and controlling cabbage type rape sclerotinia rot by separating and cloning from double-11 in a cabbage type rape sclerotinia rot high-resistance variety, and the nucleotide sequence of the gene BnTLP1 is shown as SEQ ID NO: 1, the protein sequence coded by the gene is shown as SEQ ID NO: 2, respectively. The function of the BnTLP1 gene is verified. The genetic engineering technology verifies that the cabbage type rape defense related gene BnTLP1 positively regulates sclerotinia, provides a new gene source for the excavation and molecular breeding of rape sclerotinia functional genes, and has important application prospect for enhancing the resistance of cabbage type rape to sclerotinia.

Description

BnTLP1 gene for regulating and controlling cabbage type rape sclerotiniose resistance and application thereof

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a BnTLP1 gene for regulating and controlling cabbage type rape sclerotinia sclerotiorum disease resistance and application thereof.

Background

Rape is the oil crop with the largest planting area in China, and nearly 50% of domestic edible oil is provided in China. Sclerotinia sclerotiorum is a fungal disease caused by Sclerotinia sclerotiorum (Lib.) de bark of Sclerotinia sclerotiorum of Hymenochaetaceae of Ascomycotina, subgenomycetes, and is also the first major disease in oilseed rape production in China (Boland G J, Hall R.index of Plant hosts of Sclerotinia sclerotiorum,1994, Canadian Journal of Plant Pathology,16(2): 93-108). How to effectively prevent and control sclerotinia sclerotiorum is a problem which needs to be solved urgently in the rape industry at present, the prevention and control measures of sclerotinia sclerotiorum are mainly chemical prevention and control, but the chemical prevention and control has high cost and is easy to cause secondary pollution, and the physical prevention and control and the biological prevention and control have limitations. Comprehensive evaluation of the current measures for preventing and treating the sclerotinia sclerotiorum, and the cultivation of disease-resistant varieties is the most economic and effective way. However, due to the quantitative inheritance of the sclerotinia sclerotiorum, no complete immune varieties and no identification of major disease-resistant genes, the breeding process of sclerotinia sclerotiorum disease resistance is greatly limited.

Pathogenesis-related Proteins (PR) are an important class of defense proteins that are widely found in plants, animals, and fungi, and comprise 17 subfamilies. PR5 is a family 5 of disease-associated proteins, mainly including thaumatin, osmotin and zeamantin. The thaumatin-like proteins (TLPs) are named because their amino acid sequences are highly similar to those of thaumatin (thaumatin) which is a plant of Africa, West non-bamboo yam, but TLPs are not sweet and have antifungal activity (Van Loon L C et al, Significance of specimen transformed protein in infected plants,2006, Annu Rev phytopathohol, 44(1): 135-62). TLP proteins are classified into L (Large) type and S (Small) type, and they are distinguished by the size of the protein, with the L-type protein molecule having a size of 21-26KD and the S-type about 16 KD. The L form has 16 conserved cysteine residues, 6 more than the S form, but they are all Induced by pathogenic bacteria to express antifungal activity (Reimmann C et al, cDNA Cloning and Sequence Analysis of a Pathogen-Induced Thaumatin-Like Protein from Rice (Oryza sativa),1993, Plant Physiology,101(3): 1113-. Typical TLP proteins form a stable disulfide structure with 16 cysteines, which ensures proper folding of the protein and is resistant to thermal denaturation, acid, base and protease degradation (Smole U et al, Mal d 2, the Thaumatic-Like Allergy from Apple, is high purity resistant to organic differentiation and thermal processing,2008, International Archives of Allergy and Immunology,147 (298); 289).

The plant TLP gene exists in the form of gene family, and the rice, model plant Arabidopsis thaliana and poplar genomes have 31, 26 and 55 genes respectively. With current research, their biological functions are mainly focused on antifungal activity, beta-1, 3-glucanase activity, antifreeze activity, enzyme inhibitor activity, plant allergen activity, and the like. Datta et al, resistant to the rice sheath blight pathogen Rhizoctonia solani and the leaf sheath rot pathogen Sarocladium oryzae by rice constitutively expressing TLPs genes (Datta K et al, Over-expression of the closed rice sheath protein-like protein (PR-5) gene in transgenic plant expression to Rhizoctonia solani consuming fertilizer release 1999, Applied & limited Genetics,98(6-7): 1138) 1145). Kuwabara et al clones winter wheat TaTLPs gene, the expressed protein inhibits the growth of wheat snow mold blight germ at low temperature environment, enhances the snow mold resistance of winter wheat (Kuwabara C et al, Abscisic acid-and cold-induced thaumatin-like protein in winter wheat snow mold complex, Microdochium nivale,2002, Physiologia Plantarum,115(1): 101-110).

Fungal diseases caused by sclerotinia sclerotiorum are important diseases in rape production in China, and the yield and quality loss of the rape caused by sclerotinia sclerotiorum in the current production are huge, so that the breeding of disease-resistant varieties is particularly important, but the cognition of genes participating in sclerotinia sclerotiorum defense in the rape and a gene regulation network is not clear at present. TLPs, as a class, possess a significant number of defense protein families whose biological functions in the defence against sclerotinia rot of colza are not fully and deeply resolved.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a cabbage type rape defense related gene BnTLP1 and application thereof in sclerotiniose resistance. The invention mainly aims at the cabbage type rape BnTLP1 gene, the expression of the gene is regulated by molecules, and the function of BnTLP1 gene on sclerotinia sclerotiorum resistance is researched by an arabidopsis transformation system.

A cabbage type rape sclerotinia sclerotiorum resistance related gene BnTLP1, wherein the nucleotide sequence of the gene BnTLP1 is shown as SEQ ID NO. 1:

ATGATTTATCAAAAAACACTTCTCACAGTCTTTTTCTTTGCATTTATCACCATATACTTTGTAATCTTAGCAGATGCGACTACGTTCACAGTAAGGAACAATTGTCCATATGTTGTGTGGGCTGCCACATCTGCTCCGGGAAAGCCTGGCGGTGGGAAGCGACTCAATCAAGGCGAAACGTGGATCGTTACTGGTGATCCAGGTACCACCCAGGCTCGGATTTGGGGTCGTACCAACTGCAACTTCGATGTCTCTGGAAGAGGTGGATGCCAAACTGGAGATTGTAATGGTGTACTTGAGTGCAAATCTTATGGACGAGCACCAAATACATTGGCAGAATATTCTCTTAAACAATATGCAGACCAAGATTTCATCGATATTTCTGTGATCGATGGATTCAATATTCCAATGGAATTCAGTTCTGCATCTGGACAATGCACCCGCAAAATTAGGTGTACGGGAGATATTATAGCTCAATGTCCAGCCCAACTAAGAATGGACGGCGCTTGCAACGGACCGTGTCCGGTGTTGAAGACGGAGGAACATTGTTGCAACTCTGGTAATTGTGGACCGACCCCACTCTCTATGTTTTTCAAGCAACGTTGTCCAGATGCCTATAGTTATCCTAAGGATGATCCCACCAGCCTTTTCACTTGCCCTAGCGGAACCAACTACAATGTCATTTTCTGTCCGTGA。

a cabbage type rape sclerotinia sclerotiorum resistance related protein BnTLP1, wherein the protein BnTLP1 is coded by the nucleotide sequence.

Further, the amino acid sequence of the protein BnTLP1 is shown in SEQ ID NO. 2:

MIYQKTLLTVFFFAFITIYFVILADATTFTVRNNCPYVVWAATSAPGKPGGGKRLNQGETWIVTGDPGTTQARIWGRTNCNFDVSGRGGCQTGDCNGVLECKSYGRAPNTLAEYSLKQYADQDFIDISVIDGFNIPMEFSSASGQCTRKIRCTGDIIAQCPAQLRMDGACNGPCPVLKTEEHCCNSGNCGPTPLSMFFKQRCPDAYSYPK DDPTSLFTCPSGTNYNVIFCP。

the invention also provides application of a cabbage type rape sclerotinia sclerotiorum resistance related gene BnTLP1, wherein the nucleotide sequence of the gene BnTLP1 is shown as SEQ ID No. 1.

The invention also provides application of the cabbage type rape sclerotinia sclerotiorum resistance related gene BnTLP1 in the aspect of rape disease resistance improvement, wherein the nucleotide sequence of the gene BnTLP1 is shown in SEQ ID No. 1.

The invention also provides application of a cabbage type rape sclerotinia sclerotiorum resistance related gene BnTLP1 in molecular breeding of rape, wherein the nucleotide sequence of the gene BnTLP1 is shown in SEQ ID No. 1.

The invention also provides a recombinant expression vector which comprises the brassica napus sclerotinia sclerotiorum resistance related gene BnTLP1 in claim 1.

The invention also provides a recombinant strain comprising the brassica napus sclerotinia resistance-related gene BnTLP1 of claim 1.

Furthermore, the invention also provides application of the recombinant expression vector in sclerotinia rot resistance of rape, the recombinant expression vector is transformed into microorganisms for culture and expression, and a target plant is transformed by a floral dip method to obtain a resistant transgenic strain.

The invention also provides a method for improving the resistance of rape to sclerotinia rot, which comprises the step of over-expressing a sclerotinia rot resistance related gene BnTLP1 in rape, wherein the nucleotide sequence of the gene BnTLP1 is shown in SEQ ID No. 1.

The invention also provides application of the cell location and possible participation of the cabbage type rape defense related gene BnTLP1 in defense signal paths.

Cloning of a defense related gene BnTLP1 in the middle double 11 of the cabbage type rape sclerotiniose high-resistance variety, and constructing an overexpression vector 35S based on a homologous recombination method, wherein the overexpression vector comprises BnTLP 1; an agrobacterium GV 3101-mediated genetic transformation method is adopted, an overexpression vector is transferred into wild arabidopsis Col-0, resistance regeneration plants are obtained through hygromycin resistance screening, and then positive transgenic plants are finally determined through PCR analysis; identifying and obtaining an arabidopsis thaliana orthologous gene T-DNA homozygous mutant tlp1-1 of BnTLP1 by a three-primer method; the sclerotinia sclerotiorum resistance identification result shows that the lesion extension of the overexpressed BnTLP1 is obviously smaller than that of the wild type, and the lesion of the mutant tlp1-1 is larger than that of the wild type; subcellular localization indicates that the BnTLP1 is simultaneously localized in cell nucleus and cell membrane, and different defense signal pathway marker gene expression analysis shows that jasmonic acid/ethylene pathway probably mediates the sclerotiniose resistance function of the BnTLP1 in the early stage of the sclerotinia sclerotiorum invasive plant.

In summary, compared with the prior art, the invention achieves the following technical effects:

1. although a plurality of TLP family genes are cloned in model plants such as rice, arabidopsis thaliana and the like, and some disease resistance functions of the TLP family genes are verified by researchers, the functions of the TLP genes are not deeply studied in the sclerotinia sclerotiorum disease resistance aspect, and the BnTLP1 gene cloned by the invention enriches the research on the genes in the rape.

2. The BnTLP1 gene cloned in the invention provides a new genetic resource for breeding disease resistance of other crops, and has guidance and reference functions for improving the resistance of other crops to sclerotinia rot.

3. The BnTLP1 overexpression strain obtained after the rape is transformed by the plant recombinant expression vector constructed by the invention is researched from the aspect of function acquisition of BnTLP1, provides a raw material for the research of the disease resistance function of TLP, and has important significance.

4. A series of researches on BnTLP1 overexpression strains in terms of sclerotinia resistance prove that the strains can positively regulate and control the resistance of the plant sclerotinia, and BnTLP1 is proved to participate in the resistance response of the plant to the sclerotinia and play an important role. The expression of resistance related genes in plants can be regulated by regulating the expression of the BnTLP1 gene, so that the resistance of the plants to sclerotinia sclerotiorum is improved. Has important significance for explaining the biological function of the BnTLP1 gene.

The discovery of BnTLP1, a sclerotinia sclerotiorum resistance gene, has important theoretical guiding significance for discovering and identifying the sclerotinia sclerotiorum resistance gene of crops and discussing the molecular action mechanism of the sclerotinia sclerotiorum resistance gene.

6. The breeding of the rape material with the sclerotinia rot resistance is always regarded by rape breeders, and the development and the utilization of the BnTLP1 are beneficial to solving the problem of sclerotinia rot resistance of the rape in production. The method has the advantages that the yield and quality loss of the rape caused by sclerotinia rot in the current production are huge, so that the method is particularly important for breeding disease-resistant varieties, the cloning of the BnTLP1 gene is beneficial to breeding high-resistance rape varieties, and theoretical and practical basis is provided for the application of TLP in the aspect of sclerotinia rot of main oil crops (rape); provides a new breeding material for further cultivating the rape variety with sclerotinia sclerotiorum resistance; has important practical guidance value in the breeding practice, variety improvement and variety popularization of the crop anti-sclerotinia rot.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is the cloning of the Brassica napus BnTLP1 gene; m is DL2000 DNA marker; 1, the size of a BnTLP1 gene fragment;

FIG. 2 shows PCR identification of BnTLP1 transgenic plants; m is DL2000 DNA marker; 1, Col-0 wild type arabidopsis; 2:35S, BnTLP1 recombinant plasmid positive control; 3-7:35S, wherein BnTLP1 transgenic positive seedlings; 35S, BnTLP1-1, -2, -3, -4 and-5 are 5 BnTLP1 overexpression transgenic lines. An arabidopsis thaliana Actin gene (AT1G13180) is used as a quantitative internal reference gene;

FIG. 3 is a qRT-PCR analysis of BnTLP1 transgenic plants. Col-0 is wild type Arabidopsis thaliana; 35S, BnTLP1 is a positive control of the recombinant plasmid; 35S, wherein the BnTLP1-1, -2, -3, -4 and-5 are respectively 5 BnTLP1 overexpression transgenic lines. An arabidopsis thaliana Actin gene (AT1G13180) is used as a quantitative internal reference gene;

FIG. 4 shows the screening of Arabidopsis homozygous mutant tlp 1-1. M: DL2000 DNA marker; no. 1-24, mutant strain;

FIG. 5 shows the sclerotinia disease resistance analysis of Brassica napus BnTLP1 overexpression and Arabidopsis thaliana mutant tlp 1-1; the left panel shows the lesion status at different times (35S:: BnTLP1 is the average of 3 transgenic lines); the right picture shows the lesion condition after inoculation of arabidopsis thaliana with 36 hpi; 35S: BnTLP 1: an overexpression strain; col-0 is wild type Arabidopsis; tlp 1-1: T-DNA homozygous mutant of Arabidopsis BnTLP 1;

FIG. 6 shows the subcellular localization of BnTLP1-GFP protein; a is a fluorescence channel; b, a white light channel; c, a composite channel;

FIG. 7 shows quantitative analysis of different defense signaling pathway marker genes of BnTLP 1.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, shall fall within the scope of protection of the present invention.

The principles and features of the present invention are described below by way of example with reference to the sclerotiniose resistance of BnTLP1, and materials, reagents, vectors, agrobacterium, etc., used in the following examples, which are commercially available from companies, unless otherwise specified.

Example 1: TA cloning and sequence determination of BnTLP1 gene

The method comprises the steps of taking double 11 young leaf parts in brassica napus, extracting total RNA by using TriZol Reagent (Invitrogen, product number 15596026), detecting the content and purity of the total RNA by using Nanodrop, taking 2.0 mu g of the total RNA for reverse transcription reaction, wherein the adopted reverse transcriptase is M-MLV (Promega, product number M1701), and referring to the use instruction of the reverse transcriptase in the reverse transcription reaction steps. First strand cDNA synthesized by reverse transcription was used as a template, and primers:

5’-ATGATTTATCAAAAAACACTTCTC-3’；

5’-TCACGGACAGAAAATGACAT-3’；

carrying out conventional PCR amplification; pThe CR reaction system was (20. mu.L): mu.L of cDNA, 2. mu.L of 10 XBuffer, 1.6. mu.L of dNTP (2.5mM), 1. mu.L each of forward/reverse primers (10. mu.M), 0.4. mu.L of Taq enzyme (5U/. mu.L) and 13. mu.L of ddH₂O (set 8 tube system, large amplification of target gene). Sample was added to ice and mixed well. The PCR reaction conditions are as follows: 5min at 94 ℃; 94 ℃ for 30sec, 52 ℃ for 30 sec; 30 cycles at 72 ℃ for 30 sec; 10min at 72 ℃. The PCR amplification obtained a fragment of 696bp in length. The fragment is recovered by 1% agarose gel electrophoresis, and the DNA quality is detected by a Nanodrop instrument, in which

TA Cloning was performed by Simple Cloning Vector (Takara Shuzo Co., Cat. No. CT111-01) using the reaction system: mu.L of the recovered product, 1. mu.L

Simple Cloning Vector, 25 ℃ incubator reaction for 5 min. The ligation products were transformed into E.coli DH 5. alpha. competent cells by heat shock method, added with 400. mu.L of fresh liquid LB medium, recovered for 20min, spread on LB plate of ampicillin, and cultured overnight at 37 ℃ by inversion. And selecting white monoclonal colonies, performing amplification culture in a liquid LB culture medium containing aminobenzene antibiotics, and sequencing. Sequencing analysis shows that the sequence contains a complete open reading frame and 696 bases in the full length, the sequence is shown as SEQ ID NO.1, the coded protein contains 231 amino acid residues, and the sequence is shown as SEQ ID NO. 2.

Example 2: construction of BnTLP1 Gene overexpression vector

Using the TA cloning plasmid of the BnTLP1 gene of example 1 as a template, the BnTLP1 gene was cloned using the primer BnTLP 1-F/R. The primers are as follows:

BnTLP1-F:

5’-CCATCGATAGTACTGTCGACATGATTTATCAAAAAACACTTCTC-3’；

BnTLP1-R:

5’-TCCATCCCGGGAGCGGTACCCGGACAGAAAATGACAT-3’；

the BnTLP1 gene is connected with a laboratory modified vector PGTV II-3FLAG by a homologous recombination method, and a connecting system (20 mu L): PGTV II-3FLAG linear vector 12. mu.L, BnTLP1 purified product 1. mu.L, enzyme (Exnase II) 2. mu.L, 5 × CE II Buffer 5. mu.L. In this example, the PGTV II-3FLAG vector was used, and other vectors were used to construct a recombinant expression vector for BnTLP 1. The size of the target gene fragment in the recombinant vector is verified by PCR, as shown in FIG. 1, lane "M" in the figure is DL2000 DNA marker, lane "1" in the figure is BnTLP1 gene fragment, and the size of the BnTLP1 gene fragment is 696bp, which is in line with the expectation. The recombinant plasmid is transformed into escherichia coli DH5 alpha by a heat shock method, and is subjected to kanamycin resistance screening to obtain a monoclonal and is sequenced. Coli DH 5. alpha. is a strain commonly used in the laboratory for transformation, in this example DH 5. alpha. is taken as an example, and other strains that can be used for transformation can be used to prepare a recombinant strain containing BnTLP 1. Sequencing analysis shows that the gene sequence of BnTLP1 contains a complete open reading frame and 696 bases in the full length, the sequence is shown as SEQ ID NO.1, the coded protein contains 231 amino acid residues, and the sequence is shown as SEQ ID NO. 2.

Example 3: genetic transformation of BnTLP1 Gene

A DH5 alpha recombinant strain plasmid of a BnTLP1 gene is extracted by an alkaline lysis method, and is named 35S:: BnTLP 1. Competent cells of Agrobacterium GV 3101: adding 10 mu L of recombinant plasmid after the competent cells are frozen and thawed, uniformly mixing, and carrying out ice bath for 5 min; freezing in liquid nitrogen for 5 min; water bath in a constant temperature water bath kettle at 37 deg.C for 5 min; adding fresh liquid LB culture medium without antibiotics, mixing uniformly, activating for 2h at 28 ℃ and 220 rpm/min. The cells were plated on LB plates containing kanamycin and rifampicin, cultured in inverted dark for 2 days at 28 ℃ and positive single clones were detected using the primer BnTLP1-F/R of example 2, and the amplified culture was carried out in a liquid LB medium resistant to kanamycin and rifampicin for genetic transformation of the wild type Arabidopsis thaliana Col-0.

The agrobacterium-mediated wild type arabidopsis Col-0 genetic transformation method comprises the following steps: amplifying the cultured agrobacterium, centrifuging at 4000rpm/min at room temperature for 15min, discarding the supernatant, adding sucrose suspension, and mixing (300mL ddH)₂O15 g sucrose and 70. mu.L Silwet L-77) were added. Soaking the inflorescence of the wild Col-0 in the agrobacterium suspension for 45sec by an inflorescence infection method, and carrying out moisture-preserving culture for 24h in a dark condition. The amplified BnTLP1 gene was identified by PCR, and the result is shown in FIG. 2, lane M is DL2000 DNA marker; lane 1 is Col-0 wild type Arabidopsis thaliana(ii) a Lane 2 is 35S: BnTLP1 recombinant plasmid positive control; lanes 3-7 are 35S-BnTLP 1 transgenic positive shoots; 35S, BnTLP1-1, -2, -3, -4 and-5 are 5 BnTLP1 overexpression transgenic lines.

Example 4: molecular identification of transgenic plants

Taking the tender leaf tissue of the identified transgenic positive plant, extracting total RNA by using a TriZol (Invitrogen company, product number 15596026) method, synthesizing first strand cDNA by using reverse transcriptase (Promega company, product number M1701), using an Arabidopsis thaliana Actin gene as an internal reference gene, and detecting the expression quantity of the positive transgenic plant of the trans BnTLP1 gene by using primers BnTLP1-qF/qR, wherein the primer sequences are as follows:

BnTLP1-qF：5’-ATGATTTATCAAAAAACACTTCTCAC-3’；

BnTLP1-qR：5’-CTTTCCCGGAGCAGATGT-3’；

AtActin-qF：5’-CCCGCTATGTATGTCGCCA-3’；

AtActin-qR：5’-AACCCTCGTAGATTGGCACA-3’；

as shown in FIG. 3, Col-0 is wild type Arabidopsis thaliana; 35S, BnTLP1 is a positive control of the recombinant plasmid; 35S, wherein the BnTLP1-1, -2, -3, -4 and-5 are respectively 5 BnTLP1 overexpression transgenic lines. The expression level of the BnTLP1 gene in the transgenic plant 35S, namely BnTLP1-1, -2, -3, -4 and-5, is obviously higher than that of the wild type, which shows that the target gene BnTLP1 is successfully transferred into the wild type Arabidopsis.

Example 5: screening of homozygous mutant tlp1-1

The orthologous gene of BnTLP1 in Arabidopsis thaliana was AtTLP1(AT4G11650), and a mutant of this gene was purchased from TAIR website (WiscDsLoxHs168_05C, insertion site 3' to CDS sequence). Obtaining the primer information of LP, RP and a universal primer LBb1 according to the website information of http:// signal.salk.edu/tdnaprimers.2.html, wherein the primers are as follows:

LP:5’-TCATATTTTCCGCACTTTTGC-3’；

RP:5’-CGTACACCCTCACACACACAC-3’；

LBb1：5’-GCGTGGACCGCTTGCTGCAACT-3’；

screening 24 homozygous mutants by a three-primer method (Limin et al, PCR identification of Arabidopsis thaliana T-DNA insertion mutant ATSUC3, 2006, plant physiology communication, (1):91-94), and as shown in FIG. 4, a Lane M is DL2000 DNA marker; lanes 1-24 are mutant strains No. 1-24, respectively, and 1 of them was selected for disease resistance identification.

Example 6: sclerotinia disease resistance analysis of cabbage type rape BnTLP1

3 transgenic positive lines highly expressing the BnTLP1 gene in example 4 and the homozygous mutant tlp1-1 in example 5 were selected as the sclerotiniose identification material, respectively. The experimental material was cultured for 5 weeks in the growth room (16 h/light, 8 h/dark) and the living body was inoculated with Sclerotinia sclerotiorum. As shown in FIG. 5, the left panel shows the lesion status at different times (35S:: BnTLP1 is the average of 3 transgenic lines); the right picture shows the lesion condition after inoculation of arabidopsis thaliana with 36 hpi; 35S, BnTLP1 is an over-expression strain; col-0 is wild type Arabidopsis thaliana; tlp1-1 is a T-DNA homozygous mutant of Arabidopsis BnTLP 1; from the results of the left panel of FIG. 5, it can be seen that the onset of the transgenic Arabidopsis thaliana 24 hours after inoculation (hops inoculation, hpi) is more obvious, and the onset of the wild type Col-0 is more obvious; after inoculation with 48hpi, the lesion size of transgenic Arabidopsis is enlarged, and the lesion size of wild type Col-0 is enlarged to the edge of the leaf. The results of lesion size statistics show that the lesion extension of the BnTLP1 overexpression material is significantly smaller than that of the wild-type Col-0, and the lesion extension of the tlp1-1 mutant is larger than that of the wild-type Col-0. Single-factor anova analysis of variance was performed on BnTLP1 overexpression material, the tlp1-1 mutant and the lesion sizes of 24hpi, 36hpi and 48hpi after inoculation of wild type Col-0, and it was found that BnTLP1 overexpression material has a very significant resistance to Sclerotinia sclerotiorum compared with the mutant tlp1-1, when the wild type Col-0 is used as a control. The above results indicate that up-regulation of the expression of the BnTLP1 gene can enhance sclerotiniose resistance.

Example 7: subcellular localization of BnTLP1

In order to clarify the possible cell positions of the function of the BnTLP1, the invention expresses the fusion protein of BnTLP1-GFP by means of a tobacco transient transformation system. The BnTLP1 gene and the subcellular localization vector P2300-GFP were ligated by homologous recombination as described in example 2, and then the Agrobacterium GV3101 was transferred after the correct sequencing. After 2 days of the tobacco transfer experiment, the fluorescence of the BnTLP1-GFP in the green fluorescence channel in the cell nucleus and the cell membrane is observed under the observation of a laser confocal microscope, as shown in FIG. 6, A is a green fluorescence channel, B is a white light channel, C is a composite channel, the fluorescence of the BnTLP1-GFP in the green fluorescence channel is observed in the cell nucleus and the cell membrane, the expression of the BnTLP1-GFP in the cell nucleus and the cell membrane can be clearly seen by combining white light and composite light, and the BnTLP1 gene can be positioned.

Example 8: quantitative analysis of each genetic material signal path marker gene of BnTLP1

The result of subcellular localization in example 7 shows that BnTLP1 gene is localized not only on the cell membrane but also in the nucleus, therefore, the invention further studies the possible signal path of the gene in the nucleus for regulating the resistance of sclerotic disease, and samples are taken at different time points after BnTLP1 overexpression strain, tlp1-1 and wild type Col-0 are inoculated to detect the expression changes of marker genes PDF1.2, PR1 and RbohB of JA (jasmonic acid)/ET (ethylene), SA (salicylic acid) and ROS (reactive oxygen species) pathways. The results are shown in FIG. 7, the expression level of the defense gene PDF1.2 of JA pathway is in an increasing trend in each material along with the inoculation time, which shows that JA/ET can positively respond to the invasion of Sclerotinia sclerotiorum; at the 24hpi time point, the expression level of PDF1.2 is higher than that of a wild type in an overexpression material, while the expression level of tlp1-1 is lower than that of the wild type, which suggests that the JA/ET pathway may be involved in the sclerotiniose resistance regulated by BnTLP1 in the early stage of sclerotinia infection; however, after 24hpi, the expression of PDF1.2 did not change regularly in the respective material, and it was speculated that the resistance mediated by BnTLP1 did not have a clear link to the JA/ET pathway at the late stage of infection. The expression of PR1 was significantly inhibited in all materials, indicating that the SA pathway plays a negative regulatory role in the sclerotinia sclerotiorum-induced plant signal defense pathway, while the TLP1 genetic materials were not significantly changed from the wild type Col-0, indicating that the TLP1 function in sclerotic disease does not pass through the SA pathway. The expression of RbohB shows similar rules in BnTLP1 overexpression transgenic lines and mutant tlp1-1 compared with wild type Col-0, and also indicates that the ROS signaling pathway is not involved in mediating the function of BnTLP 1.

In conclusion, the overexpression of the BnTLP1 in Arabidopsis thaliana can obviously improve the resistance of Arabidopsis thaliana sclerotinia sclerotiorum disease through a genetic engineering technology, preliminarily reveals that the BnTLP1 has a positive effect in the resistance of sclerotinia sclerotiorum disease, and the analysis of tobacco subcellular localization and the expression of different hormone pathway marker genes shows that the BnTLP1 not only resists germ invasion on a cell membrane, but also has a possible signal transduction function in a cell nucleus. The cabbage type rape defense related gene BnTLP1 positively regulates the sclerotinia rot, provides a new target gene source molecule for the excavation of the functional gene of the sclerotinia rot of the rape and the breeding for disease resistance of the sclerotinia rot, also provides direction guidance and feasibility basis for the research of a resistance regulation network of the sclerotinia rot of the rape, and has important application prospect for enhancing the resistance of the cabbage type rape to the sclerotinia rot.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

SEQUENCE LISTING

<110> institute of oil crop of academy of agricultural sciences of China

<120> BnTLP1 gene for regulating cabbage type rape sclerotiniose resistance and application thereof

<130> 2020

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 696

<212> DNA

<213> Brassica napus (Brassica napus)

<400> 1

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gctgccacat ctgctccggg aaagcctggc ggtgggaagc gactcaatca aggcgaaacg 180

tggatcgtta ctggtgatcc aggtaccacc caggctcgga tttggggtcg taccaactgc 240

aacttcgatg tctctggaag aggtggatgc caaactggag attgtaatgg tgtacttgag 300

tgcaaatctt atggacgagc accaaataca ttggcagaat attctcttaa acaatatgca 360

gaccaagatt tcatcgatat ttctgtgatc gatggattca atattccaat ggaattcagt 420

tctgcatctg gacaatgcac ccgcaaaatt aggtgtacgg gagatattat agctcaatgt 480

ccagcccaac taagaatgga cggcgcttgc aacggaccgt gtccggtgtt gaagacggag 540

gaacattgtt gcaactctgg taattgtgga ccgaccccac tctctatgtt tttcaagcaa 600

cgttgtccag atgcctatag ttatcctaag gatgatccca ccagcctttt cacttgccct 660

agcggaacca actacaatgt cattttctgt ccgtga 696

<210> 2

<211> 231

<212> PRT

<213> Brassica napus (Brassica napus)

<400> 2

Met Ile Tyr Gln Lys Thr Leu Leu Thr Val Phe Phe Phe Ala Phe Ile

1 5 10 15

Thr Ile Tyr Phe Val Ile Leu Ala Asp Ala Thr Thr Phe Thr Val Arg

20 25 30

Asn Asn Cys Pro Tyr Val Val Trp Ala Ala Thr Ser Ala Pro Gly Lys

35 40 45

Pro Gly Gly Gly Lys Arg Leu Asn Gln Gly Glu Thr Trp Ile Val Thr

50 55 60

Gly Asp Pro Gly Thr Thr Gln Ala Arg Ile Trp Gly Arg Thr Asn Cys

65 70 75 80

Asn Phe Asp Val Ser Gly Arg Gly Gly Cys Gln Thr Gly Asp Cys Asn

85 90 95

Gly Val Leu Glu Cys Lys Ser Tyr Gly Arg Ala Pro Asn Thr Leu Ala

100 105 110

Glu Tyr Ser Leu Lys Gln Tyr Ala Asp Gln Asp Phe Ile Asp Ile Ser

115 120 125

Val Ile Asp Gly Phe Asn Ile Pro Met Glu Phe Ser Ser Ala Ser Gly

130 135 140

Gln Cys Thr Arg Lys Ile Arg Cys Thr Gly Asp Ile Ile Ala Gln Cys

145 150 155 160

Pro Ala Gln Leu Arg Met Asp Gly Ala Cys Asn Gly Pro Cys Pro Val

165 170 175

Leu Lys Thr Glu Glu His Cys Cys Asn Ser Gly Asn Cys Gly Pro Thr

180 185 190

Pro Leu Ser Met Phe Phe Lys Gln Arg Cys Pro Asp Ala Tyr Ser Tyr

195 200 205

Pro Lys Asp Asp Pro Thr Ser Leu Phe Thr Cys Pro Ser Gly Thr Asn

210 215 220

Tyr Asn Val Ile Phe Cys Pro

225 230

Claims (10)

1. The brassica napus sclerotinia resistance related gene BnTLP1 is characterized in that the nucleotide sequence of the gene BnTLP1 is shown as SEQ ID NO. 1.

2. The brassica napus sclerotinia resistance associated protein BnTLP1, wherein the protein BnTLP1 is encoded by the nucleotide sequence of claim 1.

3. The brassica napus sclerotinia resistance-associated protein BnTLP1 according to claim 2, wherein the amino acid sequence of the protein BnTLP1 is as shown in SEQ ID No. 2.

4. The application of a cabbage type rape sclerotinia sclerotiorum resistance related gene BnTLP1 is characterized in that the nucleotide sequence of the gene BnTLP1 is shown as SEQ ID No. 1.

5. The application of a cabbage type rape sclerotinia sclerotiorum resistance related gene BnTLP1 in the aspect of rape disease resistance improvement is characterized in that the nucleotide sequence of the gene BnTLP1 is shown as SEQ ID No. 1.

6. The application of a cabbage type rape sclerotinia sclerotiorum resistance related gene BnTLP1 in the aspect of rape molecular breeding is characterized in that the nucleotide sequence of the gene BnTLP1 is shown as SEQ ID No. 1.

7. A recombinant expression vector comprising the Brassica napus sclerotinia resistance-associated gene BnTLP1 of claim 1.

8. A recombinant strain comprising the brassica napus sclerotinia resistance-associated gene bntlpl 1 of claim 1.

9. The application of the recombinant expression vector in the sclerotinia sclerotiorum resistance of rape as claimed in claim 7, wherein the recombinant expression vector is transformed by microorganism culture expression, and the target plant is transformed by floral dip method to obtain the resistant transgenic line.

10. A method for improving the resistance of rape to sclerotinia rot is characterized by comprising the step of over-expressing a sclerotinia rot resistance related gene BnTLP1 in rape, wherein the nucleotide sequence of the gene BnTLP1 is shown as SEQ ID No. 1.

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