CN112725367B - Sweet potato sucrose invertase gene IbINV and its application - Google Patents

Abstract

The invention discloses a sweet potato sucrose invertase gene IbINV and an application thereof. The sweet potato sucrose invertase gene is a DNA molecule shown in SEQ ID NO: 1; the protein encoded by the gene is composed of an amino acid sequence shown in SEQ ID NO: 2 protein; the recombinant vector containing the coding gene is the recombination obtained by inserting the fragment of the DNA molecule shown in SEQ ID NO. vector. The IbINV gene and the recombinant vector of the present invention can regulate the resistance of the plant to the black spot fungus, and the DNA molecule encoding the IbINV protein can be introduced into the target plant to obtain a transgenic plant with significantly enhanced black spot fungus resistance. The IbINV gene and the encoded protein thereof provided by the invention can improve the resistance of plants to the fungus of sweet potato black spot, and lay a foundation for the directional improvement of the resistance of plants to the fungus of sweet potato black spot.

Description

Sweet potato sucrose invertase gene IbINV and its application

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a sweet potato sucrose invertase gene IbINV and an application thereof.

Background technique

Sweet potato black spot disease, also known as black spot disease, occurs in all sweet potato producing areas in the world. In 1937, the disease was introduced into Gai County, Liaoning Province, my country from Kagoshima, Japan, and gradually spread from north to south. There are currently 26 provinces, Cities and autonomous regions have reported the occurrence and harm of the disease one after another. Sweet potato black spot is common in all potato regions in China, and the damage is more serious in North China, the Huanghuaihai Basin, the Yangtze River Basin, and the southern potato region. The annual yield loss caused by sweet potato black spot disease is about 5% to 10%, and the loss caused by serious damage can reach 20% to 50%, or even higher. In addition, the potato pieces infected by the black spot fungus can produce toxic substances such as sweet potato nigra ketone, which can cause poisoning or even death after human and animal consumption. Therefore, improving the resistance to sweet potato black spot has gradually become a key work in the field of scientific research.

Traditional breeding methods can improve the resistance of plants to black spot fungus to a certain extent, but traditional breeding has the problem of long time and heavy workload. With the rapid development of molecular biology technology, it has become a reality to use genetic engineering technology such as transgenic to improve plant stress resistance. Therefore, it is an effective way to improve the resistance of plants to black spot fungus by excavating new genes with resistance to black spot fungus of sweet potato and utilizing them by means of bioengineering.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is how to improve the resistance of plants to black spot fungus of sweet potato.

In order to solve the above-mentioned technical problems, the nucleotides of the sweet potato sucrose invertase gene IbINV are shown in SEQ ID NO: 1.

As a preferred solution of the sweet potato sucrose invertase gene IbINV of the present invention: hybridize with the DNA molecule defined by SEQ ID NO: 1 under stringent conditions, and the amino acid sequence is the protein shown in SEQ ID NO. 2.

As the preferred solution of the sweet potato sucrose invertase gene IbINV of the present invention: the protein is shown as: B1) or B2):

The B1) is a fusion protein obtained by linking the N-terminus or/and the C-terminus of the amino acid sequence shown in SEQ ID NO.2 with a protein tag;

The B2) is a sequence with more than 90% of the amino acid sequence SEQ ID NO.2 obtained by the substitution and/or deletion and/or addition of one or several amino acid residues to the amino acid sequence shown in SEQ ID NO.2 Consistent and functionally identical proteins.

As the preferred version of the sweet potato sucrose invertase gene IbINV of the present invention: the relevant biological material of the protein is any one of the following C1) to C12):

C1) is the nucleic acid molecule of the amino acid sequence SEQ ID NO.2 of the protein;

C2) an expression cassette containing the nucleic acid molecule of C1);

C3) a recombinant vector containing the nucleic acid molecule described in C1);

C4) a recombinant vector containing the expression cassette of C2);

C5) a recombinant microorganism containing the nucleic acid molecule of C1);

C6) a recombinant microorganism containing the expression cassette of C2);

C7) a recombinant microorganism containing the recombinant vector described in C3);

C8) a recombinant microorganism containing the recombinant vector described in C4);

C9) a transgenic plant cell line containing the nucleic acid molecule of C1);

C10) a transgenic plant cell line containing the expression cassette of C2);

C11) a transgenic plant cell line containing the recombinant vector described in C3);

C12) A transgenic plant cell line containing the recombinant vector described in C4).

The application of sweet potato sucrose invertase gene IbINV in plant resistance to sweet potato black spot.

The present invention has beneficial technical effects. The IbINV gene and its encoded protein provided by the present invention can improve the resistance of plants to S. japonica, and lay a foundation for directional improvement of the resistance of plants to S. spp. A recombinant vector obtained by inserting a fragment of the DNA molecule shown in SEQ ID NO. 1 with the stop codon removed and fused with the GFP gene sequence between the two attR sites of pGWB5. The IbINV gene and the recombinant vector of the present invention can regulate the resistance of the plant to S. spp., the gene is introduced into the sweet potato plant to obtain a transgenic sweet potato plant that overexpresses IbINV, and the transgenic plant is inoculated with S. spp. Compared with the transgenic control plants (named NT lines), the resistance of the transgenic lines (named OEV lines) to S. spp. was significantly enhanced, specifically in that the disease index was significantly lower than that of the control, indicating that the IbINV protein and its encoding Genes play an important role in the regulation of resistance to black spot fungus. The IbINV protein and its encoding gene provided by the present invention have important theoretical significance and application value in improving the resistance of plants to S. spp. The invention will have broad application space and market prospect in the agricultural field.

Description of drawings

Figure 1 shows IbINV gene fragment electrophoresis detection (A) and INV protein phylogenetic tree construction (B)

Figure 2 is a map of pGWB5:IbINV plant overexpression vector

Fig. 3 is the electrophoresis picture of PCR detection of IbINV transgenic sweet potato plants

Figure 4 shows the screening of IbINV transgenic sweet potato lines by real-time PCR

Figure 5 shows the black spot resistance phenotype of IbINV transgenic sweet potato plants

Detailed ways

The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1 Acquisition of sweet potato sucrose invertase IbINV gene

1. Extraction of total RNA from sweet potato

The total RNA of the fourth fully expanded leaf of Xushu 29 plants with a transplanting period of about 5w was extracted using a plant total RNA extraction kit (Beijing Tiangen Company), the RNA concentration and quality were detected by Nanodrop, and the RNA integrity was detected by electrophoresis. For specific operations, refer to the instruction manual of the total RNA extraction kit from Tiangen plants.

2. First-strand cDNA synthesis

Genomic DNA was removed and reverse transcribed using DNaseI and AMV reverse transcriptase from Takara, and the total RNA extracted from Xushu 29 was used as a template. The removal of genomic DNA was carried out with reference to the system ratio in Table 1. After mixing, 1 μL of EDTA was added in a water bath at 37 °C for 30 min, and the reaction was terminated at 65 °C for 10 min, and then quickly cooled on ice.

Table 1. Removal system ratio table of genomic DNA

For the reverse transcription reaction, prepare the mixed solution according to the reverse transcription system in Table 2. After mixing evenly, place it in a PCR machine for reverse transcription. The reaction conditions are: 50 °C for 1 h, 95 °C for 5 min, and reverse transcription to obtain cDNA Store at -20°C.

Table 2. Reverse transcription system configuration table

3. Cloning of cDNA coding sequence of IbINV protein

Using the cDNA of Xushu 29 as a template, forward and reverse primers IbINV_F and IbINV_R (sequences are SEQ ID NO: 3 and SEQ ID NO: 4, respectively) were designed, and the above-mentioned forward and reverse primers were used for PCR amplification. The total volume of the amplification reaction system was 50 μL, including 25 μL of KOD buffer, 10 μL of dNTP, 10 μL of ddH ₂ O, 1 μL of KOD enzyme, 1.5 μL of forward and reverse primers, and 1 μL of genomic DNA template. The PCR amplification conditions were pre-denaturation at 94 °C for 2 min, denaturation at 98 °C for 10 s, annealing at 55 °C for 30 s, extension at 68 °C for 60 s, cycle 35 times, and extension at 68 °C for 6 min to terminate the reaction. PCR products were detected by 0.8% agarose gel electrophoresis at 100V, and an amplified fragment with a size of about 1983 bp was obtained;

SEQ ID NO: 3 in the above is: ATGGCCGCCACCACTTCTTCCG;

SEQ ID NO: 4 is: TTACAATTGATTGATGAAAGAG.

PCR amplification conditions are as follows:

4. Recovery and purification of IbINV gene fragments

Refer to the instruction manual for the recovery of fragments from agarose gel electrophoresis, and follow the steps below:

The first step, under the UV lamp of the gel imaging system, use a clean blade to cut the amplified band with a length of about 1983bp, and try to cut off the gel without DNA, and the smaller the gel volume, the better;

The second step is to put the excised gel containing DNA bands into a 1.5ml centrifuge tube and weigh;

The third step, adding 2 times the volume of sol/binding solution DB;

The fourth step, put it in a 56 ℃ water bath for about 5 minutes until the glue is completely dissolved, and vortex every 1-2 minutes to help accelerate the dissolution;

The fifth step, put the adsorption column into the collection tube, add the above solution to the micro-adsorption column, centrifuge at 12000rpm for 30-60s, and discard the waste liquid;

The sixth step, add 900 μL of rinsing solution WB (absolute ethanol has been added), centrifuge at 12000 rpm for 1 min, and discard the waste liquid;

The seventh step, centrifuge again at 12000rpm for 2min, try to remove the rinse solution;

The eighth step, take out the micro-adsorption column, put it into a new centrifuge tube, add 35 μL of elution buffer to the middle part of the adsorption membrane, put it at room temperature for 2 minutes, and then centrifuge at 12,000 rpm for 1 minute.

5. Construction of T-IbINV vector

After purification, connect the T vector. The ligation system is 6 μl. The system includes: All in one buffer 6 μl, T4 ligase 1 μl, and purified product 4 μl.

Specific steps are as follows:

In the first step, add 6 μl of the ligation product to a 50 μl E. coli DH5α competent cell tube, mix well, and let stand for 30 minutes;

In the second step, the above mixture was transferred to a 42°C water bath, and rapidly cooled for 2min after thermal shock for 30s;

The third step, add 900μl LB liquid medium, shake at 37°C, 200rpm for 1h;

The fourth step, take out 10000rpm centrifugation for 30s, discard the supernatant, and leave 150-200μl LB medium;

Step 5: After mixing, transfer the transformed cells to LB plates containing 100 μg/ml carbenicillin, spread evenly, and place them in a 37°C incubator for 12-16 hours;

The sixth step, pick a single clone from the resistant plate, add it to 500 μl of LB liquid medium with a concentration of 100 μg/ml carbenicillin, and shake at 37 ° C and 200 rpm for about 3 hours;

The seventh step is to take the shake culture solution for PCR identification. The PCR 20μl system includes: 10μl of premix, 1.0μl of forward and reverse primers, 2μl of bacteria solution and supplemented with 6μl of DDH ₂ O to 20μl, and then carry out the PCR reaction. After the reaction is completed, use 0.8 Band size was detected by 100V agarose gel electrophoresis.

PCR amplification conditions are as follows:

6. T-IbINV plasmid extraction and sequencing

Take a single clone with the correct band size, add it to 5-7ml of LB liquid medium with a concentration of 100μg/ml carbenicillin, 37°C, 200rpm overnight shaking culture, use Tiangen plasmid mini-extraction kit for plasmid extraction, Refer to the manual for specific operations. The extracted plasmids were sent to Shanghai Sangon for sequencing to verify the sequence, and bioinformatics software such as Bioedit was used for sequence comparison analysis (Figure 1).

Example 2 The application of IbINV protein in regulating plant resistance to sweet potato black spot

1. Construction of pGWB5-IbINV overexpression vector

The T-IbINV plasmid was used as a template, and the volume ratio of the plasmid and water was 1:49 as a PCR amplification template. The primers were specific attb-IbINV forward and reverse primers, and the primer sequences were SEQ ID NO: 5: AAAAAGCAGGCTGCATGGCCCGCCACCACTTCT ; SEQ ID NO: 6 is: AGAAAGCTGGGTCCAATTGATTGATGAAAGAG; Prepare according to the following reaction system:

Table 3. attb-IbINV PCR reaction system preparation table

The configured system was flicked and mixed, and then dissociated. PCR amplification conditions were: pre-denaturation at 94 °C for 2 min, denaturation at 98 °C for 10 s, annealing at 60 °C for 60 s, extension at 68 °C for 30 s, cycle 20 times, and then extend at 68 °C for 6 min Then the reaction was terminated.

Using the PCR1 product as a template and attb-adaptor as a primer, the forward and reverse primer sequences of SEQ ID NO: 7 are: GGGGACAAGTTTGTACAAAAAAGCAGGCT; SEQ ID NO: 8 is: GGGGACCACTTTGTACAAGAAAGCTGGGT.

The system configuration is the same as above, and the PCR amplification conditions are as follows:

After the PCR, the electrophoresis detected the target band and it was very bright. The BP reaction was carried out after the gel was recovered and purified. The BP reaction system of 10 μL is shown in Table 4. Mix well and centrifuge at 25°C for 3h, add 1 μL of Proteinase K Solution, mix well and incubate at 37°C for 10min to terminate the reaction.

After the ligation product was transformed into E. coli DH5α, the transformed cells were transferred to LB-resistant plates containing gentamicin. The identified single clones were shaken and cultured in LB liquid medium containing gentamicin overnight, and the plasmid pDONOR207-IbINV was extracted, and part of it was used for sequencing verification. The remaining plasmids were subjected to the next LR reaction. Show:

Table 4. BP reaction system preparation table

Table 5. LR reaction system preparation table

Mix well and centrifuge at 25°C for 5h, add 1 μL of Proteinase K Solution, mix well and incubate at 37°C for 10min to terminate the reaction. The transformation steps and PCR identification methods were the same as above, and the transformed cells were transferred to LB resistant plates containing kanamycin. The single clone identified by PCR was shaken overnight in LB liquid medium containing kanamycin, and the plasmid pGWB5-IbINV was extracted (Fig. 2).

2. Introduction of pGWB5-IbINV overexpression vector into Agrobacterium tumefaciens

The IbINV recombinant plasmid is imported into Agrobacterium tumefaciens, including the preparation of Agrobacterium tumefaciens competent cells and the transformation of Agrobacterium tumefaciens by liquid nitrogen freeze-thaw method, wherein the preparation steps of Agrobacterium tumefaciens competent cells are as follows:

The first step is to pick a single colony of Agrobacterium tumefaciens EHA105 in 5 mL of YEP liquid medium containing 40 μg/mL rifamycin (Rif) at 28 °C and 230 rpm to OD ₆₀₀ ≈0.6;

In the second step, 1.5 mL of bacterial liquid was collected, centrifuged at 4°C, 8000 rpm for 1 min, and the supernatant was discarded;

In the third step, the cells were fully resuspended with 200 μL ddH ₂ O, centrifuged at 4°C, 8000 rpm for 1 min, and the supernatant was discarded;

The fourth step, repeat the third step three times, discard the supernatant;

In the fifth step, the cells were fully resuspended with 200 μL of ddH ₂ O, which was the competent cells of Agrobacterium tumefaciens.

The liquid nitrogen freeze-thaw method is used to transform Agrobacterium tumefaciens, and the specific operation steps are as follows:

The first step is to thaw 100 μL of Agrobacterium EHA105 competent cells in an ice bath;

In the second step, 20 μL of the recombinant plasmid was added to Agrobacterium tumefaciens competent cells, mixed gently, and placed on ice for 10 min;

In the third step, the mixture after the ice bath was immediately placed in liquid nitrogen for quick freezing for 1 min;

The fourth step, after 37 ℃ water bath for 5 minutes, add 900mL YEP liquid medium, 28 ℃, 200rpm shaking culture for 2h;

Step 5: Centrifuge at 8000 rpm for 2 min, leave 100 μL of bacterial liquid at the bottom, suspend and precipitate and spread evenly on YEP solid culture containing 100 μg/mL kanamycin (Km) or spectinomycin (Spec) and 40 μg/mL rifampicin On the base, incubate at 28°C upside down for 2 days;

The sixth step, bacterial liquid PCR detection of positive clones.

3. Acquisition of IbINV Transgenic Sweet Potato Plants

First step, infection and co-cultivation

The cDNA coding sequence of IbINV was introduced into the embryogenic callus of Xushu 29 by the method of genetic transformation of embryogenic callus mediated by Agrobacterium. ₆₀₀ = 1.0 Agrobacterium tumefaciens liquid, infect for 10 minutes, during which the triangular flask is gently shaken several times to make the bacterial liquid and callus fully contact. Then the bacterial liquid was poured out, and the excess bacterial liquid was sucked out with sterile filter paper. The infected embryogenic suspension cells were transferred to MS solid medium containing 2.0mg/l 2,4-D, 20mg/l AS for co-cultivation, and a layer of sterile filter paper was placed on the solid medium to inhibit the growth of Bacillus growth, 28 ℃, dark culture for 3d. At the same time, untransformed callus was used as a control.

The second step, bacteriostatic and selective culture of transformants

The embryogenic callus cells co-cultured for 3 days were washed with sterile water for 5 to 6 times, and then treated with MS liquid medium containing 600 mg/l cephalosporin for bacteriostatic treatment for 1 h. During the period, placed on a horizontal shaking table and shaken at 100 rpm. , to more effectively remove residual Agrobacterium tumefaciens. Dark incubation was then carried out with medium containing 200 mg/l cephalosporin and 100 mg/l hygromycin.

The third step, induction of somatic embryos

The screened resistant calli were transferred to somatic embryo induction medium, and somatic embryogenesis was induced by light culture at 28°C.

The fourth step, the regeneration of the pseudo-transgenic plants

Whole plants were regenerated by light cultivation with MS medium containing 200 mg/l cephalosporin and 100 mg/l hygromycin.

The fifth step, PCR identification

Extract the genomic DNA of the above-mentioned regeneration sweet potato strain, detect whether the IbINV gene is integrated into the wild-type sweet potato plant by PCR technology, and the concrete steps are as follows:

①Extracting the genomic DNA of sweet potato plants, the specific steps are as follows:

(1) The CTAB extract was preheated in a 65°C water bath;

(2) Weigh 1.0 g of fresh leaves with the main vein removed, place them in a pre-cooled mortar, pour in liquid nitrogen, and grind the leaves as soon as possible;

(3) Add 1 g of powder directly to 8 mL of preheated CTAB extract, and then add 160 μL of β-mercaptoethanol (final concentration of 2%). After the CTAB extract is slightly dissolved, the CTAB extract is mixed with the ground The samples were mixed evenly and transferred to a 50mL centrifuge tube;

(4) Keep the sample in a water bath at 65°C for 60min, shake the centrifuge tube every 10-20min, be careful not to close the cap of the centrifuge tube tightly to prevent the sample from being ejected;

(5) Add an equal volume of 24:1 chloroform/isoamyl alcohol, gently invert and mix;

(6) After equilibration, centrifuge at 10000rpm for 5min at room temperature;

(7) Take the upper aqueous phase and carefully transfer it into a new tube, add 2 times the volume of pre-cooled absolute ethanol, invert and mix, and centrifuge at 10,000 rpm for 5 min at room temperature to discard the supernatant;

(8) add 2mL of 75% ethanol to wash 2 times and then place it to dry at room temperature;

(9) adding ddH ₂ O and helping to dissolve at 37°C;

(10) Add 2 μL (10 μg/μL) Rnase, digest at 37°C for 30 min, and remove RNA in the DNA sample;

(11) Store at -20°C after dispensing.

② Design primer 8 and primer 9 for PCR identification (primer 8 is SEQ ID NO: 3, primer 9 is SEQ ID NO: 9), primer 10 and primer 11 (primer 10 is SEQ ID NO: 10, and primer 11 is SEQ ID NO: 9) SEQ ID NO: 9);

In the above, SEQ ID NO: 9 is: TTGCTTTGAAGACGTGGTTG; SEQ ID NO: 10 is: GATTCAAGGCTTGCTTCACA.

3. Take the genomic DNA extracted above as a template, carry out PCR detection, and the PCR amplification system is as shown in Table 6,

Table 6. Preparation of PCR reaction system for identification of transgenic plants

The PCR amplification procedure is:

The amplified products were detected by 0.8% agarose gel electrophoresis. It can be seen from Figure 3 that both target fragments can be amplified into IbINV transgenic positive sweet potato plants, indicating that the IbINV gene has been integrated into the sweet potato genome. The transgenic line was named OEV line.

4. Fluorescence quantitative PCR screening of IbINV transgenic sweet potato lines

Use the plant total RNA extraction kit (Beijing Tiangen Company) to extract the total RNA of the IbINV transgenic line, and complete the first-strand cDNA synthesis. Take 10 μL of reverse transcribed cDNA and add 40 μL of DW to dilute it into a template, and the fluorescence quantitative PCR reaction system is 20 μL. As shown in Table 7, the primer sequences of RT-INV-Primer-F and RT-INV-Primer-R are SEQ ID NO. 11 and SEQ ID NO. 12 respectively; wherein, SEQ ID NO: 11 is: GGGGCCGTTCGGACTTCT; SEQ ID NO: 12 is: ACCGTGCTTCCATAAAACCTCTT. The prepared 20 μL system was shaken, mixed and centrifuged, and the reaction was carried out. The reaction conditions were: pre-denaturation at 95 °C for 15 min, denaturation at 95 °C for 20 s, annealing at 58 °C for 40 s, extension at 72 °C for 20 s, cycle 45 times, and then react at 95 °C for 5 s , dissolved at 65°C for 5s. After the reaction, the dissolution curve was observed and the Ct value was recorded. The quantitative calculation method was carried out using 2 ^-ΔΔt .

Table 7. Quantitative PCR reaction system configuration table

The results are shown in Figure 4. OEV lines generally showed up-regulated expression of IbINV gene. The average up-regulated expression of IbINV in OEV7 was about 7.5 times that of NT, and the up-regulated expression of IbINV gene in OEV8 was about 14.4 times that of NT. In the experiment, OEV7 and OEV8 were selected for further study;

V. Identification of the resistance of IbINV transgenic plants to S. spp.

The phenotype analysis of sweet potato black spot resistance identification refers to the following steps:

The first step, collecting sweet potato black spot samples, separating and purifying to obtain sweet potato black spot bacteria, and after being verified by Koch's formula, cultured in PDA plate for 7 days, for use;

The second step is to weigh 20g of potatoes, add 1L of ultrapure water, boil until the potatoes are completely rotten and soft, filter, add 15g of sucrose and dilute to 1L, then autoclave at 121°C for 15min for later use;

The third step, take out the activated sweet potato black spot bacteria, use a hole punch with a diameter of 6mm to punch out the bacterial plates, inoculate 20 bacterial plates in each triangular flask, and place the inoculated triangular flask on a constant temperature shaker at 28°C medium, shake at 120rpm/min for 48h;

The fourth step, the filtrate is collected by filtration, and the concentration of the spore suspension is adjusted to about 1×10 ⁵ CFU/ml;

The 5th step, cut each 3 strains of OEV7 and OEV8 with consistent growth, and plant them in flowerpots filled with sterilized soil respectively, and the prepared spore suspension is treated for root irrigation, and the amount of root irrigation in each pot is 50ml, Moisturize regularly afterwards;

The sixth step, 15d after inoculation, observe and record the incidence. The disease index was divided into 6 grades according to the severity of the disease.

The grading standard of black spot disease index of potato seedlings is as follows: grade 0: the whole plant is healthy and disease-free; grade 1: the plant takes root normally, the stem has sporadic black spots on the ground, the leaves at the base of the stem are yellow or wilted, and the shoots grow normally. ;Grade 3: The plant takes root but there are few roots, the underground stem has continuous black spots, the leaves at the base of the stem are yellow, and the potato seedlings grow slowly; Grade 5: The plant hardly takes root, the leaves at the base of the stem are yellow, and the potato seedlings are completely indistinguishable. Branch; Grade 7: Underground necrosis and rot, leaves of the whole plant turn yellow, and the plant wilts; Grade 9: Underground necrosis and rot, potato seedling heart leaves rot, and the whole plant dies. The average disease index was calculated according to the following formula, average disease index=(repetition 1 disease index + repeat 2 disease index + repeat 3 disease index)/3.

The results are shown in Figure 5. It can be seen that the resistance of the IbINV transgenic plant OEV line to the black spot disease is significantly improved than that of the wild-type sweet potato plant NT. As shown in Table 8, the average disease index of OEV7 and OEV8 were 0.67 and 1.00 after 15 days of black spot infection, and the average disease index of NT was 3.67. The results of significance analysis showed that the average disease index of OEV7 and OEV8 was significantly lower than that of NT.

Table 8. Disease index of OEV strains after inoculation with S. nigricans for 15 days

The above experimental results indicated that the IbINV protein and its encoding gene could regulate the resistance of plants to S. zeae.

Claims (1)

1. The application of sweet potato sucrose invertase gene IbINV in sweet potato black spot pathogen resistance, wherein the nucleotide of the sweet potato sucrose invertase gene IbINV is obtained by taking cDNA of Xushu 29 as a template and performing PCR amplification through a primer IbINV _ F and a primer IbINV R;

the primer IbINV F is as follows: ATGGCCGCCACCACTTCTTCCG, respectively;

the primer IbINV R is as follows: TTACAATTGATTGATGAAAGAG are provided.

Images