CN110564762A

CN110564762A - Elongation factor BnELP4 gene for regulating cabbage type rape sclerotinia sclerotiorum resistance and application thereof

Info

Publication number: CN110564762A
Application number: CN201910911403.4A
Authority: CN
Inventors: 杜雪竹; 胡慧贞; 汤依唯; 金先达; 陈飞志; 杨一丹
Original assignee: Hubei University
Current assignee: Hubei University
Priority date: 2019-09-25
Filing date: 2019-09-25
Publication date: 2019-12-13

Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to an elongation factor BnELP4 gene for regulating and controlling cabbage type rape sclerotinia sclerotiorum resistance and application thereof. The invention separates and clones a gene BnELONGATOR4 (BnELP 4 gene for short) for controlling cabbage type rape sclerotinia sclerotiorum from cabbage type rape webtar, and the nucleotide sequence is shown as SEQ ID NO:1 is shown in the specification; the protein sequence coded by the gene is shown as SEQ ID NO:2, respectively. The function of the BnELP4 gene is verified. Through the gene engineering technology, the improvement of the expression quantity of the BnELP4 gene in the brassica napus can activate a jasmonic acid disease-resistant signal path, improve the accumulation of active oxygen and the expression of disease-resistant related genes, reduce the sclerotinia sclerotiorum disease spot area of transgenic rape leaves by 67.54 percent, and the plant shows more resistance to sclerotinia sclerotiorum disease, thereby having important application prospect for enhancing the resistance of the brassica napus to the sclerotinia sclerotiorum disease.

Description

elongation factor BnELP4 gene for regulating cabbage type rape sclerotinia sclerotiorum resistance and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to an elongation factor BnELP4 gene for regulating and controlling cabbage type rape sclerotinia sclerotiorum resistance and application thereof.

Background

Rape is the most important oil crop in China and is the first major source of domestic edible vegetable oil. The growth of the rape is influenced by a plurality of environmental factors, and both biotic stress (including pathogenic bacteria, insect pests and the like) and abiotic stress (including drought, heavy metal, salt damage, low temperature, high temperature and the like) cause great loss on the yield and the quality of the rape, so that the stress resistance related genes are transferred into the rape varieties by utilizing the genetic engineering technology, and the cultivation of high-quality new varieties with high yield and stress resistance is one of effective ways for coping with food and energy resource shortage.

sclerotinia sclerotiorum (Sclerotinia sclerotiorum) caused by Sclerotinia sclerotiorum is the most main disease of rape main producing areas in China, especially the Yangtze river basin and the southeast coastal area are the most common and serious, and the rape yield is seriously influenced (plum ball, etc., rape Sclerotinia sclerotiorum resistance identification, resistance mechanism and resistance genetic breeding research progress, crop research [ J ], 2001, 15 vol, 85-92, 3), economic benefit (Liu Sheng Jiang, analysis of Sclerotinia sclerotiorum disease resistance reaction molecule network, Chinese plant pathology society academic annual meeting literature [ C ], 2010) and Rapeseed oil quality (McCarney H, Doughty K, Noaon G.A sty of the infection of disease quality specific parameter index. in:10th natural index, cancer, 1999). The existing prevention and treatment method of sclerotinia rot of colza is mainly chemical prevention and treatment technology and breeding of disease-resistant varieties, but chemical prevention and treatment has the problems of low prevention effect, difficult elimination of pathogen, difficult pesticide spraying in flowering phase, pesticide residue, environmental pollution and the like; the conventional breeding has the defects of difficult finding of effective resistant materials, very slow speed and the like, and the disease resistance problem is difficult to effectively solve. Therefore, the molecular improvement of the sclerotinia sclerotiorum variety and the rapid cultivation of the sclerotinia sclerotiorum variety are important strategies for improving the yield of the rapeseed in unit area. Research shows that the cabbage type rape sclerotinia sclerotiorum resistance has medium heritability, is quantitative and is controlled by a plurality of micro-effective resistance sites, and the generalized heritability is about 70 percent (Wujian, etc., research progress of the interaction molecular mechanism of rape and sclerotinia sclerotiorum, Chinese oil crop academy [ J ], 2018, Vol. 40, 5 th, 721-729 pages). No effective source of resistance is found in crops such as rape, soybean and sunflower, the identified sclerotinia sclerotiorum QTL has small effect and large positioning range, and no key regulatory gene of sclerotinia sclerotiorum resistance in rape (ZHao XM, She XP, Yu W, et al. effects of oligonucleotides on tobacancecells and role of oligonucleotides nitride burst of tobacanceto microbial virus. the Plant Patholojournal, 2007,89(1):55-65) is found. Therefore, although the utilization of quantitative resistance is the main way to improve sclerotinia sclerotiorum resistance of rape, the current functional genomics research can only provide a rough framework and direction for fully understanding the rape-sclerotinia sclerotiorum interaction mechanism, and the gene resources and molecular mechanism are still to be deeply excavated. The genetic engineering method is a technique for improving varieties at a molecular level by genetic manipulation according to a previously designed target and plan. Particularly, diseases such as sclerotinia rot of colza which are difficult to find antigens in host plant species, and a genetic engineering method provides a way to solve the problems.

the expression of the protein involved in the transformation of the genome-specific transcriptional reprogramming process (Molecular K, Levine AT, Morgan A, et al. the expression of the genome-specific transcriptional system of the microorganism-specific transcriptional gene of the genome-specific genetic, Nature genetic, 2000,26(4): 403-410; Tao Y, Xie Z, Chen W, et al. the expression of expression vector with the expression of the genome-specific expression with the genome of the microorganism-specific expression of the genome-specific transcriptional gene of the microorganism-specific expression of the genome-specific expression of the strain of the genome-specific transcriptional gene of the microorganism-specific transcriptional gene of the microorganism of the genome of the strain of the microorganism, 2003,15 (2-317; Katageri of the genome of the expression of the genome-specific expression of the microorganism of the genome-specific transcriptional gene of the microorganism-specific expression of the microorganism of the genome-specific transgenic microorganism of the strain of the microorganism of the genome of the microorganism of the strain of the microorganism of the strain.

At present, only the function of the ELP protein (AtELP1-6) of a model plant Arabidopsis thaliana is reported, and the result shows that the ELP plays a positive regulation role in the immunity of the plant. When Arabidopsis AtELP3(ELO3) and AtELP4(ELO1) mutant plants were inoculated with wild-type, fully-immunized pathogens, it was found that the mutant strains lost resistance to these pathogens, suggesting that elongation factors play a critical role in plant non-host resistance. Specifically, the expression shows that ELP is involved in the non-host resistance of two pathogens, namely citrus canker pathogen Xanthomonas citribsp.Citri (Xcc) and bean bacterial blight pathogen Pseudomonas syringae pv.PhaseolicolaNPS3121(Psp NPS3121) in Arabidopsis thaliana. After inoculation of Arabidopsis mutant elp1-5 with the pathogen described above, the sensitivity of the elp4 mutant to the Xcc pathogen was significantly increased, and the elp3 mutant to the Psp NPS3121 pathogen was significantly increased, whereas wild type Arabidopsis was fully immunized against the pathogen as not being host for the pathogen described above (An C, Wang C, Mou Z.the Arabidopsis generator complex for non-pathogen resistance against the bacterial pathogen induced mutation the bacterial pathogen Xanthomonas cir. subcsp. trici and Pseudomonas aeruginosa pv. phaseolicola NPS3121.the New phytologist,2017,214(3): 1245-1259). In Arabidopsis thaliana, the single mutations ELP2 and NPR1 (Atelp2 and NPR1) are more sensitive to Pseudomonas syringae PstDC3000/avrRpt2, whereas the double mutation Atelp2NPR1 is more sensitive to the above pathogens (Defraia C T, Zhang X, MouZ. Elongator subbunit 2is an accelerator of immune responses in Arabidopsis thaliana. plant Journal,2010,64(3): 511-523). Wang et al further discovered that ELP2 was able to interact with PAD 4. Mutations in ELP2 block pathogen-induced changes in DNA methylation levels of NPR1 and PAD4 and delay the induction of defense genes (Wang Y, An C, Zhang X, et al. the Arabidopsis reactor complex × Subunit2 epidemic ligands plants responses. the Plant Cell,2013,25(2): 762-776). In addition, the researchers found that AtELP2 also has a better resistance to necrotic fungal pathogens such as Botrytis cinerea and northern leaf spot of Brassicaceae (Wang C, Ding Y, Yao J, Zhang Y, Sun Y, Colee J, Mouz. Arabidopsis thaliana Fabry 2 bacterial microorganisms to resistance to the novel bacterial fungal pathogen and Alternaria solanacearus the Plant Journal 2015,83: 1019-1033). In strawberry, heterologous overexpression of AtELP 3and AtELP4 genes can improve the resistance of plants to crown rot, powdery mildew and angular leaf spot (Silva KJP, Brunengs AM, Pereira JA, et al. the Arabidopsis ELP3/ELO3and ELP4/ELO1genes engineering disease resistance in Fragaria vesca L. BMC Plant Biology,2017,17(1): 230). Recent research shows that the heterologous overexpression of AtELP 3and AtELP4 genes in tomato can obviously enhance the resistance of plants to tomato bacterial spot disease caused by Pseudomonas syringae PV, does not have adverse effect on the growth and development of the plants, and has good potential of coordinating the growth and disease resistance of the plants. Further RNA-sequencing and analysis of AtELP4 transgenic tomato plants revealed that the levels of defense-related genes were significantly higher than controls after pathogen infection, indicating that ELP4 could significantly induce activation of the plant defense response (Pereira JA, Yu F, Zhang Y, et al. the Arabidopsis reactor subenit ELP3and ELP4 control resistance to Bacterial Speck in tomato in tomato. frontiers in plant science 2018,24(9): 1066). In conclusion, the Arabidopsis ELP family can positively regulate plant immunity, but the research on the disease resistance function of the ELP is not deep at present, the ELP functions of other plants, particularly crops, are rarely reported, and the effect of the ELP on main diseases of the crops, such as sclerotinia rot of colza, rice blast, cotton verticillium wilt and the like, is not clear.

The homology between the genomes of rape and Arabidopsis Brassica plants is high, and the sequence alignment analysis shows that the nucleotide sequence similarity of the rape and Arabidopsis ELP4 genes is 90%, the protein sequence similarity is 78%, and Siliton et al find that Arabidopsis genes usually show similar functions in rape (Siliton D, Parkin IA, Mayerhofer R, et al. Arabidopsis thaliana: a source of candidate disease-resistance genes for Brassica napus. genome.2000,43(3): 452-60); in addition, the ELP proteins are highly conserved in both yeast and plants (Kolaj-Robin O, S raphin B. structures and activities of the elongator complex and its genes. enzymes,2017,41: 117-149); therefore, a molecular mechanism of rape BnELP for regulating sclerotinia sclerotiorum resistance is further explored, theoretical guidance and new materials are provided for rape sclerotinia sclerotiorum resistance breeding, and no relevant literature and patent literature reports exist at present for the action of rape BnELP 4. In the invention, the applicant finds that the expression level of BnELP4 is obviously improved after the sclerotinia rot is subjected to stress treatment on the cabbage type rape, and then the resistance of the transgenic over-expression plant of the cabbage type rape BnELP4 to the sclerotinia rot is obviously enhanced, so that the result has important application prospect in enhancing the resistance of the sclerotinia rot and increasing the yield of the rapeseed oil.

disclosure of Invention

The invention aims to overcome the defects and shortcomings of the prior art, separates an elongation factor complex-Elongator complex member BnELP4 gene for regulating and controlling sclerotinia sclerotiorum disease resistance of Brassica napus, and performs functional verification and primary transformation application on the gene. According to an elongation factor compound subunit AtELP4 sequence which has been reported in a model plant Arabidopsis and plays a key role in plant non-host resistance, sequence comparison is carried out in Brassica napus of Brassicaceae with high homology, a BnELP4 sequence with the highest homology is screened out, the gene is cloned and overexpressed in Brassica napus Westar, sclerotinia is inoculated, the function of the gene in sclerotinia sclerotiorum resistance of rape is rapidly identified, and new genetic resources are provided for sclerotinia sclerotiorum resistant breeding of rape.

The technical scheme of the invention is as follows:

The applicant found, by analyzing the reported subunit disease resistance function of the Arabidopsis elongation factor complex, that AtELP4 plays a key role in Plant non-host resistance (An C, Wang C, Mou Z. the Arabidopsis elongatus complex required for non-host resistance, the antibiotic resistance of the Bacterial strain Xanthomonas minor, strain p v. phaseolola NP3121. the New phytologist,2017,214(3): 1245-1259; Silva Kyu JP, Brunengs AM, Pereira JA, et al. the Arabidopsis ELP 3/O3 and ELP 4/O1 genetic resistance of the Plant strain in Fragaria, strain P2018. the bacterium resistance of the Plant strain P1069. the strain F8, the bacterium resistance of the Plant strain P23, strain F8. the bacterium resistance of the Plant strain P23, strain F7, strain F8. the bacterium resistance of the Plant strain P1069, strain F8. the strain No. 12B, strain F3. the strain F3, strain F8, strain F, strain No. 8, however, no homologous gene function has been reported in crops. The homology between the genomes of the rape and the arabidopsis crucifer brassica plants is high, and the ELP protein is highly conserved in the plants. Through sequence alignment, the nucleotide sequence similarity of the rape BnELP4 and the Arabidopsis Paxneb protein-like protein (ELO1)/AtELP4 gene is found to be 90 percent and the protein sequence similarity is found to be 78 percent (figure 1). Extracting leaf RNA from Brassica napus webstar (usingsuper total RNA extraction kit, available from Promega, USA), using reverse transcriptase [ seeII Q RT Supermix for qRNA (+ gDNA wrapper) kit, purchased from Vazyme, China, which was reverse transcribed into cDNA, the reaction conditions were: 42 ℃ for 2min, 50 ℃ for 15min, 85 ℃ for 5 sec. Using genome information of Brassica napus, TA clone amplification primers BnELP4-F (5 'CGAGTTCCTTGTTGTGAT 3') and BnELP4-R (5 'CTACGAAGAAAATGAGA 3') were synthesized to amplify the full length cDNA (1143bp) of BnELP4 gene. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min; 30sec at 94 ℃, 30sec at 60 ℃, 1min at 72 ℃ for 10sec, 32 cycles; extension at 72 ℃ for 5 min. Connecting the PCR product obtained by amplification into a pMD-18T vector (purchased from Takara Bio-engineering Co., Ltd.), screening positive clones and sequencing to obtain the required gene ORF (1170bp), wherein the nucleotide sequence of the gene ORF is shown as a sequence table SEQ ID NO:1, the 358-amino acid protein sequence corresponding to ORF is determined by BlastX (http:// www.ncbi.nlm.nih.gov) to be consistent with BnELP4 protein, and the protein sequence coded by the gene is shown in SEQ ID NO:2, respectively.

Furthermore, EcoRI and KpnI enzyme cutting sites and corresponding protection bases are respectively added at two ends of a BnELP4TA cloning amplification primer, the designed primers are respectively named as OE-BnELP4-F (5'CGGAATTCCGAGTTCCTTGTTGTGAT3') and OE-BnELP4-R (5'GGGGTACCGCTACGAAGAAAATGAGA3'), a TA positive cloning colony plasmid of BnELP4 is used as a template for PCR amplification, the length of an obtained PCR product is 1159bp, and the PCR product is constructed on a pCAMBIA2301-1300 intermediate vector through double enzyme cutting connection, so that a recombinant plant expression vector pCAMBIA2301-1300-D35s-BnELP4-NOS (figure 2) is obtained.

The constructed over-expression recombinant vector pCAMBIA2301-1300-D35s-BnELP4-NOS is transformed into the hypocotyl of Brassica napus Westar by utilizing agrobacterium-mediated plant transformation through a rape hypocotyl infection method until a regenerated differentiated seedling is obtained (figure 3). Wild type cabbage type rape is extracted by a 2x CTAB method to serve as control and leaf genome DNA of transgenic rape strains, primers BnGUS-F (5 'ACGACTCGTCCGTCCTGTAG 3') and BnGUS-R (5 'TTGCAAAGTCCCGCTAGTGC 3') are designed according to GUS sequences on a carrier to carry out PCR amplification, and positive strains such as OE-1, OE-3, OE-11, OE-20, OE-29, OE-32 and OE-33 are screened out (shown in a picture in figure 4). The transcription level of the BnELP4 gene in the positive seedlings was analyzed by using the primers Q-BnELP4-F (5 'CCGCATCGCCATCCAGTCATTC 3') and Q-BnELP4-R (5 'CGCCATGTGCTGCAGTCTTGTTGAG 3'), and the results showed that the expression amount of the BnELP4 gene in four positive seedling lines of OE-1, OE-3, OE-32 and OE-33 was significantly increased compared with the control (see B diagram in FIG. 4).

Wild cabbage type rape (WT) control and BnELP4 gene overexpression strain BnELP4(OE) are respectively inoculated with Sclerotinia sclerotiorum (sclerotiorum) mycelium blocks in a seedling stage and a seedling stage excised leaf, the Sclerotinia sclerotiorum (sclerotiorum) is generally most obviously attacked 36 hours (36hpi) after inoculation, and the disease index of Sclerotinia sclerotiorum is counted when the Sclerotinia sclerotiorum is obviously attacked, and the specific method refers to a cabbage type rape Sclerotinia sclerotiorum resistance identification method of Wujian et al (Wu J, Zhao Q, Yang Q, et al. The disease index statistical result shows that the resistance of the BnELP4(OE) plant is obviously higher than that of the WT, the spread area of the disease spot is obviously reduced (figure 5), and the BnELP4 gene positively regulates the resistance reaction of rape leaves to sclerotinia rot. In the period of significant difference, leaf tissues of the same parts around the lesions of WT and BnELP4(OE) are taken, RNA of the two is extracted and is reversely transcribed into cDNA, and the expression quantity analysis is carried out on ROS related genes, jasmonic acid signal pathway genes and resistance related genes, and the result shows that the expression quantity of the genes in the BnELP4(OE) strain is significantly higher than that of wild-type genes (WT) (figure 6). Further taking leaf tissue of the consistent part around the lesion at 0 hour (0hpi) and 36 hours (36hpi) after inoculation of WT and BnELP4(OE) plants inoculated with sclerotinia sclerotiorum, and grinding and extracting hydrogen peroxide (H) in the leaf₂O₂) And quantitatively analyzing H₂O₂content, results are in agreement with graph A in FIG. 6, H in BnELP4(OE) strain₂O₂The content was significantly higher than WT (fig. 7). Therefore, the overexpression of the BnELP4 gene is probably caused by that more hydrogen peroxide (H) is accumulated by the plant₂O₂) Enhances natural immune signals and regulates the resistance of the cabbage type rape to sclerotinia sclerotiorum through a jasmonic acid signal path. The above results have important significance for cultivating rape disease-resistant strains.

The technical scheme of the invention is summarized as follows:

The applicant prepares and obtains a plant recombinant vector pCAMBIA2301-1300s-D35s-BnELP4-NOS, and is characterized in that the plant recombinant vector comprises SEQ ID NO:1, and the nucleotide sequence of the BnELP4 gene.

Applicants provide a polypeptide comprising the sequence set forth as SEQ ID NO:1, the preparation method of the plant recombinant vector pCAMBIA2301-1300s-D35s-BnELP4-NOS of the nucleotide sequence of the BnELP4 gene shown in the figure, which is characterized in that: adding a Double CaMV 35S strong promoter into the upstream EcoRI/HidIII enzyme cutting site of the plant recombinant vector pCAMBIA1300S, and transforming to obtain a pCAMBIA2301-1300S vector; and then the nucleotide sequence shown in SEQ ID NO: the nucleotide sequence shown in1, namely the gene BnELP4, is cloned to the restriction enzyme sites EcoRI/KpnI of pCAMBIA2301-1300s to obtain the nucleotide sequence.

the method comprises the following specific steps: constructing a recombinant vector pCAMBIA2301-1300s-D35s-BnELP 4-NOS; transferring the obtained recombinant vector into agrobacterium GV3101, transforming cabbage type rape by the following hypocotyl infection method, and identifying by a PCR method to obtain a transgenic strain; the expression level of the BnELP4 gene is further identified by a qPCR method, so that the rape leaves of a BnELP4 overexpression transgenic strain transfected by a recombinant plant vector pCAMBIA2301-1300s-D35s-BnELP4-NOS show the resistance to the sclerotinia rot of rape.

the more detailed application steps are as follows:

(1) According to SEQ ID NO:1 to obtain a recombinant plant vector pCAMBIA2301-1300s-D35s-BnELP 4-NOS;

(2) transferring the recombinant vector obtained in the step (1) into agrobacterium GV3101, transforming the cabbage type rape by using the hypocotyl infection method, and identifying and obtaining a transgenic plant by using a PCR method;

(3) identifying the expression level of the BnELP4 gene by a qPCR method;

(4) carrying out rape leaf in vitro inoculation on the overexpression transgenic strain with the remarkably improved BnELP4 gene expression level in the step (3), taking fresh leaves at the consistent positions around bacterial plaques after 36h inoculation, extracting total RNA of the leaves, identifying active oxygen accumulation related genes BnCAT1, BnPER21 and BnPER54 by a qPCR method,Jasmonic acid signal path and expression levels of downstream disease-resistant related genes BnPGIP, BnChitinase1, BnGSL05, BnPDF1.2, BnERF1 and BnORA 59; (5) carrying out in-vitro inoculation on sclerotinia sclerotiorum on the overexpression transgenic strain with the remarkably improved BnELP4 gene expression level in the step (3), respectively inoculating 0H and 36H, taking fresh leaves at the consistent positions around the bacterial plaque, and grinding and extracting H in the leaves₂O₂And quantitatively analyzing H₂O₂And (4) content.

The plant recombinant vector pCAMBIA2301-1300s-D35s-BnELP4-NOS is applied to the resistance of rape sclerotinia rot.

the BnELP4 gene cloned by the invention can be applied to the resistance to sclerotinia rot of rape.

The invention has the following advantages:

1. although a plurality of elongation factor complex ELP genes are cloned in Arabidopsis thaliana and researchers verify the disease resistance functions of the ELP genes, the functions of the ELP genes are not reported in rape so far, and the cloned BnELP4 gene enriches the research on the genes in the rape.

2. The BnELP4 gene cloned in the invention provides a new genetic resource for breeding other crops for disease resistance, and has guidance and reference functions for improving the resistance of other crops to sclerotinia rot.

3. The BnELP4 over-expression strain obtained after the plant expression vector pCAMBIA2301-1300-D35s-BnELP4-NOS transforms rape is researched from the aspect of function acquisition of BnELP4, provides a raw material for disease resistance research of ELP4, and has important significance.

4. Through a series of researches on BnELP4 overexpression strains on the resistance to sclerotinia, the invention fully proves that BnELP4 is involved in the resistance response of plants to sclerotinia and plays an important role. The accumulation of resistance factors in plants can be regulated by regulating the expression of the BnELP4 gene, and the resistance of the plants to sclerotinia sclerotiorum is improved. Has important significance for illustrating the biological function of the BnELP4 gene.

The discovery of BnELP4, a sclerotinia sclerotiorum resistance gene, has important theoretical guiding significance for discovering and identifying the sclerotinia sclerotiorum resistance gene of crops and deeply 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 BnELP4 are helpful to solve the sclerotinia rot resistance problem 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 selective breeding of disease-resistant varieties is particularly important, the cloning of the BnELP4 gene is beneficial to the cultivation of high-resistance rape varieties, and theoretical and practical basis is provided for the application of the ELP4 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

FIG. 1: and (3) the sequenced rape BnELP4 gene is aligned with the known amino acid sequence of the Arabidopsis AtELP4 gene. Description of reference numerals: bn (Brassica napus L.): (ii) brassica napus; at (Arabidopsis thaliana): arabidopsis thaliana.

FIG. 2: the construction process of plant transformation vector pCAMBIA2301-1300-D35s-BnELP4-NOS is shown in the figure. Description of reference numerals: LB: the T-DNA left border; 35S: cauliflower virus 35S promoter; MCS: a multiple cloning site; NOS: a terminator; RB: right border of T-DNA.

FIG. 3: agrobacterium-mediated cabbage type rape hypocotyl genetic transformation system. Description of reference numerals: FIG. 3A is a photograph showing the germination of the brassica napus webstar seeds sterilized with 0.1% mercuric chloride on a 1/2MS medium; FIG. 3B is a photograph showing the etiolated hypocotyl grown in dark culture at 24 ℃ for about 5 days; FIG. 3C is a diagram showing that the etiolated hypocotyl is cut into small pieces of about 0.8cm under aseptic conditions and is infected with Agrobacterium GV3101 containing a transformation vector; FIG. 3D is a photograph showing the hypocotyls after infection after transfer to M1 medium and dark culture for 48 hours; FIG. 3, Panel E is a photograph of explants after co-cultivation placed in M2 medium for 15-20d selection culture with 16h light/8 h dark culture period; panel F of FIG. 3 is a photograph of explants after selection culture placed in M3 medium and subjected to differential culture for more than 15 days in 16h light/8 h dark culture period until budding; FIG. 3G is a graph G showing that explants after differentiation culture budding are placed in fresh M3 medium and selectively differentiated for more than 15 days in 16h light/8 h dark culture period until differentiation into seedlings; in FIG. 3, H is a graph showing the rooting of seedlings on M4 medium.

FIG. 4: and (3) identifying positive seedlings of rape BnELP4 overexpression transgenic lines and analyzing expression quantity. Description of reference numerals: FIG. 4A is the identification of positive seedlings of BnELP4 overexpression transgenic lines, GUS gene fragment on PCR amplification carrier, target size is 750 bp; FIG. 4, panel B, shows the detection of the BnELP4 gene expression level in transgenic over-expressed lines and non-transgenic wild-type lines (WT) using the qPCR method; WT is wild type control; OE-1, OE-3, OE-11, OE-20, OE-29, O-32 and OE-33 are all independent over-expressed transgenic lines.

FIG. 5: identification of resistance of BnELP4 overexpression transgenic lines to sclerotinia sclerotiorum. Description of reference numerals: FIG. 5A is a graph showing the phenotype of WT disease after 36 hours of in vitro inoculation with BnELP4(OE) plant leaves at seedling stage; panel B of FIG. 5 corresponds to the lesion size of the WT and BnELP4(OE) leaves of FIG. 5 after 36h of sclerotinia infection. 36hpi, inoculated for 36 hours; WT + s.sclerotomum and BnELP4(OE) + s.sclerotomum indicate inoculation of sclerotinia with wild type and BnMED16 overexpression lines, respectively. The results show that: the hypha expansion area of the leaf of the BnELP4(OE) strain is obviously smaller than that of a control, which shows that the resistance of the plant to the sclerotinia sclerotiorum is enhanced after the BnELP4 is over-expressed.

FIG. 6: analyzing the expression quantity of disease resistance related genes after the seedling stage leaves of Wild Type (WT) and transgenic BnELP4(OE) are inoculated with sclerotinia sclerotiorum in vitro for 36 hours. Description of reference numerals: FIG. 6A is a graph showing the measurement of the expression level of a critical gene related to ROS using the material of FIG. 5 as a template; FIG. 6B is a diagram showing the detection of the expression level of a disease-resistance-associated key gene; FIG. 6C is a diagram showing the detection of the expression level of a key gene in the jasmonic acid signaling pathway. The results show that: after the BnELP4 is over-expressed, the key genes are all activated, which shows that the BnELP4 can enhance natural immune signals through more hydrogen peroxide accumulated by plants and regulate the resistance of the Brassica napus to sclerotinia sclerotiorum through a jasmonic acid signal path.

FIG. 7: seedling-stage leaves of wild-type (WT) and transgenic BnELP4(OE) plants are inoculated with sclerotinia sclerotiorum in vitro for 0 hour (0hpi) and 36 hours (36hpi) and then the hydrogen peroxide content (H) in the leaves is increased₂O₂) Quantitative analysis of (3). The results show that: h in BnELP4(OE) strain after overexpression of BnELP4₂O₂The content is obviously higher than that of WT, and the further verification shows that the overexpression of the BnELP4 gene causes the plants to accumulate more hydrogen peroxide to enhance natural immune signals.

Detailed Description

description of the sequence listing

SEQ ID NO. 1 is the nucleotide sequence of the isolated and cloned rape BnELP4 gene.

SEQ ID NO. 2is the protein sequence encoded by the BnELP4 gene.

The following examples define the invention and describe the method of the invention in which a cDNA fragment containing the entire coding segment of the BnELP4 gene is isolated and cloned, and the function of the BnELP4 gene is verified. From the following description and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Example 1: BnELP4 gene isolation clone

(1) Isolation and cloning of the BnELP4 Gene

The cabbage type rape webstar variety (provided by rape research laboratory of Huazhong university of agriculture) is used as an experimental materialThe Super total RNA extraction kit (purchased from Promega, USA) extracts the total RNA of the leaf blade of the rape of the brassica napus webstar variety, after the RNA extraction is completed, DNaseI (purchased from Promega kit) is used for processing, and the RNA integrity is detected by 1.2% (w/V) agarose gel (EtBr) electrophoresis (5V/cm). The determination of the nucleic acid concentration was carried out on an IMPLENNANOPHOTOMETER-N50 series ultramicro UV spectrophotometer (Germany). RNA 260/280 ratio between 1.9 and 2.1, 260/230 ratio greater than 2.0, concentration greater than 500 ng/. mu.L RNA for further analysis. Of cDNAThe synthesis is byII Q RT SuperMix for qRNA (+ gDNA wiper) kit (from Vazyme, China), 1. mu.g total RNA as template, 4. mu.L 4 XgDNA wiper Mix, DEPC-water mixed well, total volume 16. mu.L, 42 ℃ 2min, ice quench 2-3 min; then adding 4 mu L of 5 XHiscript II qRT Super Mix II and mixing evenly, wherein the total volume is 20 mu L; then, each cDNA was diluted to 200. mu.L at 50 ℃ for 15min and 85 ℃ for 5sec, and stored at-20 ℃ until use.

The target band was amplified using the TA cloning primers BnELP4-F (5 'CGAGTTCCTTGTTGTGAT 3') and BnELP4-R (5 'CTACGAAGAAAATGAGA 3'), amplified using TransTaq HiFi DNA Polymerase (Beijing Omega gold Biotech) under the PCR conditions: pre-denaturation at 94 ℃ for 3 min; 30sec at 94 ℃, 30sec at 60 ℃, 1min at 72 ℃ for 10sec, 32 cycles; extension at 72 ℃ for 5 min. The PCR product was then cloned into the pMD18-T vector (Takara Bio engineering, Inc.). The recovery, ligation and transformation of the target fragment were performed with reference to a UNIQ-10 column DNA gel recovery kit (Biotechnology engineering (Shanghai) Co., Ltd.). The connecting body of the target fragment and the T vector is as follows: mu.l of the target fragment, 0.5. mu.l of pMD-18T vector, 5. mu.l of Solution I (Takara Bio Inc.) were ligated overnight at 16 ℃. The ligation product is transformed into DH5 alpha competence by thermal excitation, bacterial liquid is smeared on an LB solid plate containing 100mg/L Amp antibiotic, after about 10-12h of growth, a single colony is selected as a colony for PCR, and the primer is the universal primer M13F/R. Positive colonies were sent to Biotechnology engineering (Shanghai) GmbH for sequencing.

(2) BnELP4 gene sequence analysis

The amplified nucleotide sequence of this gene was found to be 1159bp in length and to contain an open reading frame of 1170bp in full length by sequencing, and alignment of the amino acid sequence using the ClustalX (Thompson JD, Gibson TJ, Plewniak F, et al. the ClustalXwindows interface: flexible protocols for multiple sequence alignment information analysis techniques. nucleic Acids Research,1997,25:4876-82) program revealed that this band of interest had 78% homology at the amino acid level with Arabidopsis ELP4, consistent with the results of alignment at NCBI (FIG. 1).

Example 2 acquisition of transgenic rape plants overexpressing BnELP4

(1) Construction of plant overexpression vectors

Based on the CaMV 35S sequence (Gene ID: AJ007626) information published on NCBI, the following primers were designed using Primer Premier5.0 software:

5'-GAATTCTTAATTAAGAGCTCGCATGCC-3' and

5'-GGTACCGTCCCCGTGTTCTCTCCAA-3', obtaining Double CaMV 35S fragment from pCAMBIA1300S vector (purchased from Changsheng biotechnology Limited liability company in Beijing ancient kingdom) by adopting PCR method, then connecting the Double CaMV 35S fragment to EcoRI/HidIII enzyme cutting site of pCAMBIA2301 vector (offered by Wangjingshen university of Huazhong agriculture), and finally obtaining final vector pCAMBIA2301-1300S (11634 bp).

EcoRI and KpnI restriction sites and corresponding protective bases are respectively added at two ends of a BnELP4TA cloning amplification primer, the primers are named as OE-BnELP4-F (5'CGGAATTCCGAGTTCCTTGTTGTGAT3') and OE-BnELP4-R (5'GGGGTACCGCTACGAAGAAAATGAGA3'), PCR amplification is carried out by taking a TA positive cloning colony plasmid of BnELP4 as a template, the length of an obtained PCR product is 1159bp, EcoRI/KpnI double restriction enzyme digestion is carried out, and meanwhile, pCAMBIA2301-1300s vector is also subjected to double restriction enzyme digestion. The reaction system is as follows: 10 ug of gene fragment or vector; EcoRI 2.5. mu.L; KpnI 2.5. mu.L; 10 × M buffer 15 μ L; ddH₂0 to total volume of 150 mu L, mixing, standing at 37 ℃ for 8-12h, and respectively recovering the double enzyme digestion gene fragment and the pCAMBIA2301-1300s plasmid fragment. The BnELP4 double enzyme cutting gene fragment is connected by T4 ligase and is constructed on an EcoRI/KpnI enzyme cutting site of a pCAMBIA2301-1300s vector, thereby obtaining a cabbage type rape BnELP4 transgenic over-expression vector pCAMBIA2301-1300s-D35s-BnELP4-NOS, a T-DNA region of the vector contains a gene sequence resisting kanamycin, and a promoter excessively expressed by BnELP4 is a D35s promoter (the structure is shown in figure 2).

(2) Genetic transformation of oilseed rape

The recombinant plasmid (i.e. the overexpression vector) pCAMBIA2301-1300s-D35s-BnELP4-NOS is transferred into agrobacterium GV3101 by a conventional hypocotyl infection method, and is screened and differentiated into seedlings, and the specific steps are as follows (figure 3):

a. Sowing: cleaning the brassica napus webstar seeds for 1min by using 75% alcohol, cleaning the seeds for 5min by using 0.1% mercury bichloride solution, and finally cleaning the seeds for 5 times by using sterile water; the seeds were put in M0 solid medium (4.404 g/L of MS inorganic salts and trace elements formulated in 1962, pH was adjusted to 5.8-5.9, agar was added at 7g/L, and sterilization was performed by conventional autoclaving) using sterilized tweezers, and the inoculated seeds were cultured in the dark at 24 ℃ for 5 days.

b. activating agrobacterium: after 3 days of sowing, Agrobacterium strain GV3101, which had been stored at-80 ℃ was streaked on LB solid medium containing rifampicin (50mg/mL), gentamicin (50mg/mL) and kanamycin (50mg/mL), and cultured at 28 ℃ for 16 hours. A single colony was selected and cultured in 5mL of LB liquid medium containing rifampicin (50mg/mL), gentamicin (50mg/mL) and kanamycin (50mg/mL) at 28 ℃ for 20-24h with shaking at 200rpm, and Agrobacterium was grown to the logarithmic phase. And (3) taking 500uL of the cultured bacterial liquid to perform amplification culture on 50mL of LB liquid culture medium (containing three antibiotics) for about 12 hours.

c. Preparing infection bacterial liquid: pouring the activated agrobacterium liquid into 2 sterile centrifuge tubes with the volume of 50mL, centrifuging at 3500rpm for 15min, removing supernatant, placing on ice, sterilizing by using 1mL of DM liquid culture medium (the formula of MS inorganic salt and trace elements 4.404g/L and sucrose 30g/L in 1962, adjusting the pH of the culture medium to 5.8-5.9,121 ℃ for 20min, adding 2, 4-D1 mg/L, AS 100mmol/L and KT 0.3mg/L in a sterile operating platform after sterilization is finished, cleaning bacterial liquid, centrifuging, removing supernatant, adding 2-3mL of DM liquid culture medium, and re-suspending the bacterial liquid to ensure that the OD600 of the infected bacterial liquid is about 0.6.

d. infecting the explant: and (c) placing the infected bacterial liquid in the step (c) on ice, cutting hypocotyls (the length of each hypocotyl is 0.8-1.0cm is proper) of the seedlings of the brassica napus cultured in the step (a) in a dark mode by using a scalpel, transferring the cut hypocotyls to a dish filled with the infected bacterial liquid by using sterile forceps, and infecting for 15-30min (shaking every 3 min) according to the concentration of the bacterial liquid.

e. co-culturing: transferring the infected explants to a plate with filter paper (which should be sterilized in advance), sucking off the visible infection liquid on the surfaces of the explants, transferring to an M1 solid medium (MS inorganic salt and trace elements of 4.404g/L, mannitol 18g/L, sucrose 30g/L,2, 4-D2 mg/L and KT 0.3mg/L in the formula of 1962, adjusting the pH of the medium to 5.8-5.9, adding agar 7g/L, sterilizing and adding 100mmoL/L AS), and dark culturing at 24 ℃ for 40-48 h.

f. screening: the explants after the co-culture are transferred to an M2 solid medium (4.404 g/L of MS inorganic salts and trace elements, 18g/L of mannitol, 30g/L of cane sugar, 2, 4-D2 mg/L of 2, 0.3mg/L of KT, pH of the medium is adjusted to 5.8-5.9, 7g/L of agar) for 20 days of screening culture under the conditions that: culturing at 24 deg.C for 16h light/8 h dark.

g. differentiation culture: transferring the screened explants to an M3 solid medium (4.404 g/L of MS inorganic salts and trace elements in the formula of 1962, 10g/L of glucose, 0.25g/L of xylose, 0.6g/L of yeast extract (MES), adjusting the pH of the medium to 5.8-5.9, adding 7g/L of agar, sterilizing, adding 2mg/L of Zeatin (ZT) and 0.1mg/L of IAA (Chinese name), TMT250mg/L and Kan25mg/L), starting differentiation culture, and subculturing once every 15-20 days until buds are differentiated (the culture conditions are consistent with the screening conditions in step f).

h. Rooting culture: when the obvious growing point can be found on the differentiated and germinated seedling, a scalpel is used for sticking the callus and the bud at the connecting position, the seedling is cut off cautiously (care must be taken not to damage the growing point in the operation process), then the seedling is transferred to an M4 solid culture medium (2.202 g of MS inorganic salt and trace elements in the formula in 1962, 2.202g of sucralose 10g/L and IBA0.5mg/L, the pH value of the culture medium is adjusted to 5.8-5.9, 7g/L of agar is added, 250mg/L of TMT and Kan25mg/L are added after sterilization for rooting.

(3) Identification of transgenic plants

a. Extraction of genome DNA of cabbage type rape leaf

The extraction of DNA was carried out by the conventional CTAB method. The method comprises the following specific steps: taking 1-2 cm long tender cabbage type rape leaves, placing the leaves in a mortar pre-cooled in advance, adding liquid nitrogen for 2-3 times in the process, grinding the leaves into fine slurry, transferring the slurry into a 1.5mL centrifuge tube, and adding 700 mu L of 2x CTAB solution. Incubation at 70 ℃ for 30min, gentle shaking once every 6min, incubation at 70 ℃ for 30min and gentle shaking once every 10 min. Cooling to room temperature, adding 700. mu.L of water at a volume ratio of 2524:1 Tris saturated phenol: chloroform: the isoamyl alcohol is mixed by repeated inversion and then is gently shaken for about 40 times. Centrifuge at 3100rpm at room temperature for 15 min. The supernatant was aspirated at about 500. mu.L, and an equal volume of 24:1 chloroform: isoamyl alcohol. After shaking up, centrifuge at 3100rpm for 15min at room temperature. Removing supernatant, adding frozen-20 deg.C anhydrous ethanol 1mL, ice-cooling at-20 deg.C for 30min, centrifuging at 12000rpm, and centrifuging at room temperature for 10 min. Soaking in 75% ethanol, washing, and repeatedly blowing to precipitate for 3min to remove salt. Pouring off the alcohol, air-drying, and adding 30-50 μ L ddH to each sample₂And dissolving the O. The extracted cabbage type rape genome DNA is detected in the concentration detecting instrument with Nanodrop micro nucleic acid detecting instrument.

b. positive transgenic plant detection

Taking young and tender leaves of a transformed plant, extracting DNA by adopting a CTAB method, and identifying positive seedlings by using GUS genes on a PCR amplification carrier, wherein the used primers are as follows:

GUS-F: 5'-ACGACTCGTCCGTCCTGTAG-3' and

GUS-R:5′-TTGCAAAGTCCCGCTAGTGC-3′，

The control was a non-transgenic wild type plant (WT).

The detection result shows (A picture in figure 4), all 7 transformed plants of OE-1, -3, -11, -20, -29, -32 and-33 can amplify electrophoretic bands (750bp) with expected sizes, while the wild type control has no electrophoretic band, which indicates that the genome of the transgenic rape already contains the DNA fragment of the exogenous gene.

(4) qPCR identification of transgenic rape overexpressing BnELP4

a. Extraction of genome RNA of cabbage type rape leaf

By usingThe Super total RNA extraction kit (purchased from Promega, USA) extracts total RNA of the leaves of BnELP4 over-expression positive strain identified in WT and step (3). And useII Q RT Supermix for qRNA (+ gDNA wrapper) kit (Vazyme Co., China) reverse transcribes RNA into cDNA

b. Real-time fluorescent quantitative PCR

In order to determine whether the BnELP4 is over-expressed in the rape, the transgenic plants identified in the step (3) are analyzed by a quantitative fluorescence PCR (qPCR) method. Use of qPCRGreen Realtime PCR Master Mix-Plus-kit (Bao bioengineering (Dalian) Co., Ltd.) uses rape housekeeping gene BnActin7(Brassica napusactin-7) as internal standard gene, and its primer is synthesized by Nanjing Kingsry Biotechnology Co., Ltd. The qPCR primers for BnActin7(Gene ID:106418315) were:

BnActin7-F1: 5'-TCTTCCTCACGCTATCCTCCG-3' and

BnActin7-R1:5’-AGCCGTCTCCAGCTCTTGC-3’

The qPCR primers for the BnELP4 gene were:

Q-BnELP 4-F5 'CCGCATCGCCATCCAGTCATTC 3' and

Q-BnELP4-R:5’CGCCATGTGCTGCAGTCTTGTTGAG 3’

(the pair of primers are qPCR-specific primers designed from the nucleotide sequence sequenced in example 1)

PCR procedure: pre-denaturation at 95 ℃ for 30sec, followed by 40 cycles (95 ℃ 10sec, 60 ℃ 10sec, 72 ℃ 26 sec). Transgenic plants identified in step (3) are analyzed for over-expression and are preceded by an OE (abbreviation for overexpression).

As shown in Panel A of FIG. 4, the expression levels of BnELP4 were significantly higher for lines OE-1, -3, -32, and-33 than for the non-transgenic wild-type (WT) control, where the expression levels of OE-1, -3, and-32 were approximately 2-fold higher than for the wild-type. It was shown that these several lines are independent BnELP4 overexpression transgenic lines.

Example 3: evaluation of BnELP4 overexpression transgenic rape sclerotinia sclerotiorum disease resistance

(1) Immune response of BnELP4 overexpression strains to Sclerotinia sclerotiorum

Performing sclerotinia sclerotiorum (sclerotinia sclerotiorum, life science college of China university of agriculture) experiment on rape seedlings (four leaves and one heart period) with two leavesRoom preservation, laboratory activation) by taking two inverted leaves of wild type and the positive line with significantly increased expression in example 2, cutting the leaves and placing them in a transparent square box, placing each transgenic plant leaf in a box side by side with non-transgenic control, laying several layers of filter paper sheets and wet gauze under the leaf, taking freshly preparedThe hypha blocks, with the hypha facing downwards, were inoculated in the middle of the leaves at a position slightly off the main vein, and two hypha blocks were inoculated per leaf. Covering with a cover, keeping moisture, and culturing at 22 deg.C in dark. The leaves are continuously observed from 0h to 72h of inoculation, and the growth area of sclerotinia sclerotiorum lesion spots in a BnELP4 overexpression transgenic line is much smaller than that of a non-transgenic line, the area of damaged tissues is small, and the resistance difference is most remarkable after 36h of inoculation (A picture in figure 5); the sclerotinia sclerotiorum lesion area of the transgenic rape leaves was reduced by 67.54% compared to the wild type (panel B in fig. 5). Therefore, the BnELP4 gene can effectively enhance the resistance of rape leaves to sclerotinia sclerotiorum.

(2) extraction of RNA from inoculated rape leaves

By usingthe Super total RNA extraction kit (Promega corporation, USA) extracts the total RNA of leaves after the treatment of the sclerotinia sclerotiorum for 36h in the step (1). And useII Q RT Supermix for qRNA (+ gDNA wrapper) kit (Vazyme Co., China) reverse transcribes RNA into cDNA

(3) qPCR detection of disease resistance related gene expression

Use of qPCRGreen Realtime PCR Master Mix-Plus-kit, the disease resistance related gene primers are as follows:

The qPCR primers for the BnCAT 1gene were:

Q-BnCAT1-F, 5'-TACAGACACGAAACAGCA-3' and

Q-BnCAT1-R:5’-GACAGAAACTAGCAAGCC-3’

The qPCR primers of the BnPER21 gene are as follows:

Q-BnPER21-F, 5'-TTGCCCAAGTCCAAACCC-3' and

Q-BnPER21-R:5’-GACCAGGAGCCCTCTATG-3’

the qPCR primers of the BnPER54 gene are as follows:

Q-BnPER54-F, 5'-TTCTCCCACCATCCCTAT-3' and

Q-BnPER54-R:5’-TCAGCAGAGCCAGACCTT-3’

the qPCR primers for the BnPGIP gene were:

Q-BnPGIP-F: 5'-CCAGTTCATAGACCTTTCCTTCAAC-3' and

Q-BnPGIP-R:5’-GCTGGAAACGACCCAAATGACT-3’

The qPCR primers of the BnChitinase 1gene are as follows:

Q-BnChitinase1-F, 5'-GTAAAGGCTACTACGGTCGTGGA-3' and

Q-BnChitinase1-R:5’-CCTGTCCTGAAAGCCACAGTTG-3’

The qPCR primers of the BnGSL05 gene are as follows:

Q-BnGSL05-F, 5'-CTGGGAGATATGGTGGTATGAGG-3' and

Q-BnGSL05-R:5’-ACTTGTCACGGGCGTATTGG-3’

The qPCR primers for the BnPDFF 1.2 gene are as follows:

Q-BnPDF1.2-F5'-CATCACCCTTCTCTTCGCTGC-3' and

Q-BnPDF1.2-R:5’-ATGTCCCACTTGACCTCTCGC-3’

the qPCR primers for the BnERF 1gene were:

Q-BnERF1-F, 5'-GTTCCGAGCCAAACCCTAATACC-3' and

Q-BnERF1-R:5’-GGAACATCAAGAAAGCCAGAGAAGT-3’

The qPCR primers for the BnPR 1gene were:

Q-BnORA59-F, 5'-TAGCCCTTCCTACTACTCTGAC-3' and

Q-BnORA59-R:5’-TCCTCACTCCTCTGTATGACCTCTC-3’

Specific primers were designed for the coding region of the gene using Primer Premier5.0 software, and Primer-BLAST (NCBI) was used to perform Primer-specific alignment analysis in the brassica gene database (BRAD) and the rape EST database to verify Primer specificity.

The qPCR reaction system was 20 μ L: contains 10. mu.L of SYBR Mix, 0.8. mu.L of each forward and reverse primer (10. mu. mol/L), 2. mu.L of template and DEPC-treated sterile water. The amplification conditions were: 95 ℃ for 30 sec; then, the temperature is 95 ℃ for 5s, 60 ℃ for 30s, 72 ℃ for 27s, and 40 cycles are carried out; fluorescence detection was performed at 72 ℃ renaturation end for each cycle. After the reaction is finished, heating to 95 ℃, then cooling to 72 ℃, slowly heating to 95 ℃, and recording the change of the fluorescence signal to obtain the melting curve of the amplification product. Each set of experiments completed three biological replicates, with at least three technical replicates per biological replicate. The PCR was carried out using the primer pair Q-BnCAT1-F/R, Q-BnPER21-F/R, Q-BnPER54-F/R, Q-BnPGIP-F/R, Q-BnChitinase1-F/R, Q-BnGSL 05-F/05-BnPDF1.2-F/05-BnERF 05-F/R and Q-BnORA 05-F/R to amplify the genes BnGSL05 (BnCAT 07g 05), BnPER 05 (BnC 03g20530 05), BnPER 05(BnA 10g24230 05), BnPGIP (BnPGIP 09g48930 05), BnChitinase 05 (BnC 04g49090 05), BnGSL05 (BnPDA 09g 05), BnF1.2 (BnC 09g 2372), BnCAT F05 (BnC 23F) and Q-BnGST 05-BnGST F05 (BnC 2372) F72-F01-F2372-F72-F14-F05-F01-. Meanwhile, a primer pair BnActin7-F1/R1 is used for specifically amplifying the rape BnActin7 gene to be used as an internal reference, and the internal reference is used for carrying out relative quantitative analysis. Detecting the expression change of the disease-resistant related gene in the rape leaves after 36h of induction of the sclerotinia sclerotiorum.

The results show that when the sclerotinia sclerotiorum is infected for 36h, the transcription levels of active oxygen accumulation related genes (BnCAT1, BnPER21 and BnPER54), downstream disease resistance related genes and jasmonic acid signal pathways (BnPGIP, BnChitinase1, BnGSL05, BnPDF1.2, BnERF1 and BnORA59) are obviously increased on average (figure 6), which indicates that the overexpression of the BnELP4 gene can enhance natural immune signals by accumulating more hydrogen peroxide in plants and regulate the resistance of the brassica napus to the sclerotinia sclerotiorum through the jasmonic acid signal pathway.

(4) Quantitative analysis of hydrogen peroxide content before and after inoculation

The method for inoculating sclerotinia in vitro in the same step (1) is to take Wild Type (WT) and transgenic BnELP4(OE)the leaves at the seedling stage of the plant are inoculated with the sclerotinia sclerotiorum in vitro for 0 hour (0hpi) and 36 hours (36hpi) and then the fresh leaves at the consistent positions around the lesion spots are obtained. Respectively taking 0.1g of fresh leaves, adding 800 mu L of precooled 80% acetone, and fully grinding at low temperature until homogenate is obtained; mixed extraction is carried out for 1h at 4 ℃; centrifuging at 12000rpm at 4 deg.C for 10min, transferring the supernatant into a new centrifuge tube, adding 200 μ L pre-cooled 80% acetone, and mixing; centrifuging at 12000rpm at 4 deg.C for 10min, and transferring the supernatant (hydrogen peroxide extract) into a new centrifuge tube for quantitative analysis of hydrogen peroxide. According to kit H₂O₂A calibration curve was prepared by the Quantitative Assay Kit (Water-Compatible) method, and the hydrogen peroxide extract was quantitatively measured. The results show that: h in BnELP4(OE) strain after overexpression of BnELP4₂O₂The content is significantly higher than that of WT (figure 7), and further verifies that the overexpression of the BnELP4 gene causes the plants to accumulate more hydrogen peroxide to enhance natural immune signals.

Sequence listing

<110> university of Hubei

<120> elongation factor BnELP4 gene for regulating cabbage type rape sclerotinia sclerotiorum resistance and application

<141> 2019-09-12

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1170

<212> DNA

<213> Brassica napus (Brassica napus)

<220>

<221> gene

<222> (1)..(1170)

<220>

<221> CDS

<222> (1)..(1170)

<400> 1

atg gct gca ccc aac gtt cgt act agc agc ttc tct cgc aac ata tca 48

Met Ala Ala Pro Asn Val Arg Thr Ser Ser Phe Ser Arg Asn Ile Ser

1 5 10 15

gtt gtc tcc tca tcc cct caa ata cct ggt ctc aaa tgc ggt cct aat 96

Val Val Ser Ser Ser Pro Gln Ile Pro Gly Leu Lys Cys Gly Pro Asn

20 25 30

ggc act act ttc ata tcc act ggg att cgt gat ctc gac agg ata cta 144

Gly Thr Thr Phe Ile Ser Thr Gly Ile Arg Asp Leu Asp Arg Ile Leu

35 40 45

ggt ggt ggg tat cct ttg gga agc tta gtg atg gtg atg gaa gat cct 192

Gly Gly Gly Tyr Pro Leu Gly Ser Leu Val Met Val Met Glu Asp Pro

50 55 60

gaa gcg ccg cat cat atg gat ctc ctg agg act ttt atg tct caa ggg 240

Glu Ala Pro His His Met Asp Leu Leu Arg Thr Phe Met Ser Gln Gly

65 70 75 80

ctt gtt aac aac cag cca ctt ctt tac gct agt cct tcc aaa gat cct 288

Leu Val Asn Asn Gln Pro Leu Leu Tyr Ala Ser Pro Ser Lys Asp Pro

85 90 95

aga ggg ttt ctt ggt act ttg ccg aat ccc gga tcg tcc aaa gaa gat 336

Arg Gly Phe Leu Gly Thr Leu Pro Asn Pro Gly Ser Ser Lys Glu Asp

100 105 110

aag cct act gcc cca gac ctt gat cag gga gag agc ttg agg att gct 384

Lys Pro Thr Ala Pro Asp Leu Asp Gln Gly Glu Ser Leu Arg Ile Ala

115 120 125

tgg cag tat cgg aag tat atg gaa aac cag aag agt tct att gat gat 432

Trp Gln Tyr Arg Lys Tyr Met Glu Asn Gln Lys Ser Ser Ile Asp Asp

130 135 140

tac tcc aat gac ttt gat atg aga aag cca ttg gag agg cag ttt ctt 480

Tyr Ser Asn Asp Phe Asp Met Arg Lys Pro Leu Glu Arg Gln Phe Leu

145 150 155 160

gct gga cgg ccg att gat tgt gtc agt ctg cta gat tct tcc gat ctt 528

Ala Gly Arg Pro Ile Asp Cys Val Ser Leu Leu Asp Ser Ser Asp Leu

165 170 175

tct gtt gct gag gac cat tgc gct act ttt tta tca aaa ttt ccg aga 576

Ser Val Ala Glu Asp His Cys Ala Thr Phe Leu Ser Lys Phe Pro Arg

180 185 190

aac agc agt aac att gca tcc att ggc cgc atc gcc atc cag tca ttc 624

Asn Ser Ser Asn Ile Ala Ser Ile Gly Arg Ile Ala Ile Gln Ser Phe

195 200 205

tgc tct ccc ctt tgt gag tat tct gat aag gaa tca gaa atg ctt tca 672

Cys Ser Pro Leu Cys Glu Tyr Ser Asp Lys Glu Ser Glu Met Leu Ser

210 215 220

ttc ata aga ttg ctg aaa agt atg ctg atg gtt tca aat gcg gta gcc 720

Phe Ile Arg Leu Leu Lys Ser Met Leu Met Val Ser Asn Ala Val Ala

225 230 235 240

att gtt act ttc ccg cct tcc atg ctc tcc ccg tct tcc tca aca aga 768

Ile Val Thr Phe Pro Pro Ser Met Leu Ser Pro Ser Ser Ser Thr Arg

245 250 255

ctg cag cac atg gcg gat acc tta ctc tct atc aaa gcg atc cca gat 816

Leu Gln His Met Ala Asp Thr Leu Leu Ser Ile Lys Ala Ile Pro Asp

260 265 270

ggt gac aag gaa ttg gag aag ctt ctc act ggc tat aag gac ata aat 864

Gly Asp Lys Glu Leu Glu Lys Leu Leu Thr Gly Tyr Lys Asp Ile Asn

275 280 285

gga ttc ctc aac gta cac aag gtt gcg cgt atc aac aca cag gtg cct 912

Gly Phe Leu Asn Val His Lys Val Ala Arg Ile Asn Thr Gln Val Pro

290 295 300

gta att cta gag gca aag acc ttc tca atg agc tta aag aag aga agg 960

Val Ile Leu Glu Ala Lys Thr Phe Ser Met Ser Leu Lys Lys Arg Arg

305 310 315 320

ttc ttg gct cta gag tgt ctg aat caa gcc cct gta gac gga tcc agt 1008

Phe Leu Ala Leu Glu Cys Leu Asn Gln Ala Pro Val Asp Gly Ser Ser

325 330 335

gga aca tcg tat ggc aca tct ggg agc tgc tct gga tca tcc aaa tcc 1056

Gly Thr Ser Tyr Gly Thr Ser Gly Ser Cys Ser Gly Ser Ser Lys Ser

340 345 350

gga gca ctc gat ttc tga gtt aat gag tta gtt ttt tct cat ttt ctt 1104

Gly Ala Leu Asp Phe Val Asn Glu Leu Val Phe Ser His Phe Leu

355 360 365

cgt agc ggt acc cca agg gcc aat tcg ttt aaa cct gca gga cta gtc 1152

Arg Ser Gly Thr Pro Arg Ala Asn Ser Phe Lys Pro Ala Gly Leu Val

370 375 380

cct tta gtg aag ggt taa 1170

Pro Leu Val Lys Gly

385

<210> 2

<211> 357

<212> PRT

<213> Brassica napus (Brassica napus)

<400> 2

Met Ala Ala Pro Asn Val Arg Thr Ser Ser Phe Ser Arg Asn Ile Ser

1 5 10 15

Val Val Ser Ser Ser Pro Gln Ile Pro Gly Leu Lys Cys Gly Pro Asn

20 25 30

Gly Thr Thr Phe Ile Ser Thr Gly Ile Arg Asp Leu Asp Arg Ile Leu

35 40 45

Gly Gly Gly Tyr Pro Leu Gly Ser Leu Val Met Val Met Glu Asp Pro

50 55 60

Glu Ala Pro His His Met Asp Leu Leu Arg Thr Phe Met Ser Gln Gly

65 70 75 80

Leu Val Asn Asn Gln Pro Leu Leu Tyr Ala Ser Pro Ser Lys Asp Pro

85 90 95

Arg Gly Phe Leu Gly Thr Leu Pro Asn Pro Gly Ser Ser Lys Glu Asp

100 105 110

Lys Pro Thr Ala Pro Asp Leu Asp Gln Gly Glu Ser Leu Arg Ile Ala

115 120 125

Trp Gln Tyr Arg Lys Tyr Met Glu Asn Gln Lys Ser Ser Ile Asp Asp

130 135 140

Tyr Ser Asn Asp Phe Asp Met Arg Lys Pro Leu Glu Arg Gln Phe Leu

145 150 155 160

Ala Gly Arg Pro Ile Asp Cys Val Ser Leu Leu Asp Ser Ser Asp Leu

165 170 175

Ser Val Ala Glu Asp His Cys Ala Thr Phe Leu Ser Lys Phe Pro Arg

180 185 190

Asn Ser Ser Asn Ile Ala Ser Ile Gly Arg Ile Ala Ile Gln Ser Phe

195 200 205

Cys Ser Pro Leu Cys Glu Tyr Ser Asp Lys Glu Ser Glu Met Leu Ser

210 215 220

Phe Ile Arg Leu Leu Lys Ser Met Leu Met Val Ser Asn Ala Val Ala

225 230 235 240

Ile Val Thr Phe Pro Pro Ser Met Leu Ser Pro Ser Ser Ser Thr Arg

245 250 255

Leu Gln His Met Ala Asp Thr Leu Leu Ser Ile Lys Ala Ile Pro Asp

260 265 270

Gly Asp Lys Glu Leu Glu Lys Leu Leu Thr Gly Tyr Lys Asp Ile Asn

275 280 285

Gly Phe Leu Asn Val His Lys Val Ala Arg Ile Asn Thr Gln Val Pro

290 295 300

Val Ile Leu Glu Ala Lys Thr Phe Ser Met Ser Leu Lys Lys Arg Arg

305 310 315 320

Phe Leu Ala Leu Glu Cys Leu Asn Gln Ala Pro Val Asp Gly Ser Ser

325 330 335

Gly Thr Ser Tyr Gly Thr Ser Gly Ser Cys Ser Gly Ser Ser Lys Ser

340 345 350

Gly Ala Leu Asp Phe

355

<210> 3

<211> 31

<212> PRT

<213> Brassica napus (Brassica napus)

<400> 3

Val Asn Glu Leu Val Phe Ser His Phe Leu Arg Ser Gly Thr Pro Arg

1 5 10 15

Ala Asn Ser Phe Lys Pro Ala Gly Leu Val Pro Leu Val Lys Gly

20 25 30

Claims

1. A plant recombinant vector pCAMBIA2301-1300s-D35s-BnELP4-NOS, characterized in that the plant recombinant vector comprises SEQ ID NO:1, and the nucleotide sequence of the BnELP4 gene.

2. The use of the plant recombinant vector pCAMBIA2301-1300s-D35s-BnELP4-NOS in the resistance of rape sclerotinia sclerotiorum as claimed in claim 1.

3. A polypeptide comprising the amino acid sequence of claim 1 as set forth in SEQ ID NO:1, the preparation method of a plant recombinant vector pCAMBIA2301-1300S-D35S-BnELP4-NOS of the nucleotide sequence of the BnELP4 gene is characterized in that a Double CaMV 35S strong promoter is added at the upstream EcoRI/HidIII enzyme cutting site of the plant recombinant vector pCAMBIA1300S, and the pCAMBIA2301-1300S vector is obtained by modification; and then the nucleotide sequence shown in SEQ ID NO: the nucleotide sequence shown in1, namely the gene BnELP4, is cloned to the restriction enzyme sites EcoRI/KpnI of pCAMBIA2301-1300s to obtain the nucleotide sequence.

4. use according to claim 2, characterized in that it comprises the following steps: constructing a recombinant vector pCAMBIA2301-1300s-D35s-BnELP 4-NOS; transferring the obtained recombinant vector into agrobacterium GV3101, transforming cabbage type rape by the following hypocotyl infection method, and identifying by a PCR method to obtain a transgenic strain; the expression level of the BnELP4 gene is further identified by a qPCR method, so that the rape leaves of a BnELP4 overexpression transgenic strain transfected by a recombinant plant vector pCAMBIA2301-1300s-D35s-BnELP4-NOS show the resistance to the sclerotinia rot of rape.

5. The application according to claim 4, characterized in that said application comprises in particular the steps of:

(3) identifying the expression level of the BnELP4 gene by a qPCR method;

(4) Carrying out in-vitro inoculation of sclerotinia sclerotiorum on the overexpression transgenic strain with the remarkably improved BnELP4 gene expression level in the step (3), taking fresh leaves of consistent positions around bacterial plaques after inoculation for 36h, extracting total RNA of leaves, and identifying the expression levels of active oxygen accumulation related genes BnCAT1, BnPER21 and BnPER54, jasmonic acid signal pathway genes and downstream disease-resistant related genes BnPGIP, BnChitinase1, BnGSL05, BnPDF1.2, BnERF1 and BnORA59 by a qPCR method;

(5) Carrying out in-vitro inoculation on sclerotinia sclerotiorum on the overexpression transgenic strain with the remarkably improved BnELP4 gene expression level in the step (3), respectively inoculating 0H and 36H, taking fresh leaves at the consistent positions around the bacterial plaque, and grinding and extracting H in the leaves₂O₂and quantitatively analyzing H₂O₂And (4) content.

The application of the BnELP4 gene in resisting sclerotinia rot of rape is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO:1 is shown.