CN118910096A - Application of StMybHv-like gene in improving cold resistance of potatoes - Google Patents

Abstract

The present invention relates to the field of biotechnology, and in particular to the application of StMybHv1-like genes in improving the cold resistance of potatoes. The present invention discloses a StMybHv1-like gene for regulating the cold resistance of potatoes, wherein the nucleotide sequence thereof is as shown in (a), (b) or (c), wherein (a) is a nucleotide sequence as shown in SEQ ID NO. 1; (b) is a nucleotide sequence that hybridizes with and encodes the nucleotide sequence shown in SEQ ID NO. 1; (c) is a nucleotide sequence that has more than 80% homology with the nucleotide sequence shown in SEQ ID NO. 1 and encodes the nucleotide sequence. The present invention uses StMybHv1-like or its transgenic biological material to regulate the cold resistance of potato plants, provides important theoretical support for the screening of genetically transformed positive plants and the study of the mechanism of resistance to low temperature stress of potatoes, and has broad application prospects.

Description

Application of StMybHv1-like gene in improving cold resistance of potato

Technical Field

The invention relates to the field of biotechnology, and in particular to application of StMybHv1-like gene in improving cold resistance of potato.

Background Art

Potato ( Solanum tuberosum L.) is the world's most important tuber crop, widely grown around the world, and plays an important role in ensuring global food security. Frost damage greatly affects plant growth, development, production efficiency and geographical distribution. Common potato cultivars are almost not frost-resistant, and agricultural growth will be seriously affected once a low temperature cold wave occurs.

Most of the cold-responsive gene regulatory mechanisms that have been analyzed so far are regulated through CBFs-related pathways. The ICE-CBF-COR signaling pathway is believed to play a key role in plant cold resistance. Genes such as HOS1 , SIZ1 , and OST1 are upstream regulatory factors of ICE1. He et al. found that the expression level of the galactitol synthase gene ( ScGols1 ) increased after cold stress, and then found that ScGols1 changed the sugar composition and improved the freezing resistance of potatoes by inducing the expression of ethylene signaling pathways and CBF family genes. CBFs are mainly genes that respond during the domestication process above 0°C, and there are few reports on the mechanism of action of responsive genes under potato freezing stress. Therefore, it is very necessary to explore the resistance genes of wild potatoes and cultivate new potato varieties with cold resistance.

Summary of the invention

In view of the shortcomings of the prior art, the present invention provides a StMybHv1-like gene for regulating cold resistance of potato and

The protein it encodes has been shown to significantly enhance the cold resistance of potatoes and can be applied to potato low-temperature resistance breeding and variety improvement, providing more options for potato resistance breeding.

To achieve the above object, the present invention provides a protein, wherein the protein is (a) or (b);

(a) a protein consisting of the amino acids shown in SEQ ID NO. 2;

(b) a derivative protein having the same function as the amino acid sequence shown in SEQ ID NO. 2, wherein one or more amino acid residues are substituted and/or deleted and/or added;

The protein of one of the above (a) or (b), which improves the survival rate of potato under low temperature stress compared with wild-type plants.

In some specific embodiments, the present invention provides a protein having an amino acid sequence with 80% identity to the sequence shown in SEQ ID NO.1, and having an amino acid sequence with enhanced cold resistance; preferably, 85% identity, more preferably, 90% identity, more preferably, 95% identity, and most preferably, 99% identity.

The present invention also provides a gene encoding the above-mentioned protein, wherein the nucleotide sequence of the gene is (a), (b) or (c);

(a) the nucleotide sequence shown in SEQ ID NO. 1;

(b) a nucleotide sequence that hybridizes with and encodes the nucleotide sequence shown in SEQ ID NO. 1 under stringent conditions;

(c) a nucleotide sequence encoding a gene having 80% or more homology to the nucleotide sequence shown in SEQ ID NO. 1.

In some specific embodiments, the StMybHv1-like gene can regulate the cold resistance of potato.

It is well known to those skilled in the art that, since the same amino acid may be determined by a variety of different codons, the nucleotide sequence encoding the above-mentioned protein is not limited to just one type. It can be a nucleotide sequence that can also encode the mutant amino acid sequence of the present invention by mutating one or more nucleotides of the mutant nucleotide sequence shown in SEQ ID NO. 1 to form a synonymous mutation, or a nucleotide sequence that can encode the mutant amino acid sequence of the present invention can be designed based on codon optimization.

In some specific embodiments, the present invention provides a protein whose gene nucleotide sequence has 80% identity with the sequence shown in SEQ ID NO. 1; preferably, it has 85% identity, more preferably, it has 90% identity, more preferably, it has 95% identity, and most preferably, it has 99% identity.

The recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing the above gene also fall within the protection scope of the present invention.

The use of any of the above proteins, genes, recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria in the resistance of potato cultivars to low temperature stress also falls within the protection scope of the present invention.

Furthermore, the cold resistance of the crop is improved by increasing the expression level and/or activity of the StMybHv1-like gene in the crop.

A method for preparing transgenic plants also falls within the scope of protection of the present invention, comprising the following steps: introducing the coding gene of the above protein into a recipient plant to obtain a transgenic plant; compared with wild-type plants, the cold resistance of the transgenic plant is improved; the plant is potato.

Furthermore, the coding gene is introduced into the plant via a recombinant expression vector; the recombinant expression vector is obtained by inserting the coding gene into the multiple cloning site of the initial vector pH7lic9.0.

Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID No. 1.

Beneficial effects: The present invention provides a StMybHv1-like gene with the function of regulating the cold tolerance of potatoes. The application of StMybHv1-like or its transgenic biological materials can regulate the cold tolerance of potato plants, which provides important theoretical support for the screening of genetic transformation-positive plants and the study of the mechanism of potato resistance to low temperature stress, and has broad application prospects.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is qRT-PCR validation of the expression pattern of StMybHv1-like in DM and CMM;

Figure 2 is a schematic diagram of the pH7lic9.0-MybHV1-like overexpression vector;

Figure 3 shows the identification of StMybHv1-like overexpression positive strains;

Among them, 3A is the amplification diagram of the full-length cDNA of StMybHv1-like , 3B is the expression level detection diagram of the StMybHv1-like overexpressing plants, and 3C is the plant phenotypic difference diagram of the StMybHv1-like overexpressing plants;

FIG4 shows the effect of overexpression of StMybHv1-like on cold resistance of potato;

Among them, 4A is a phenotypic difference diagram of StMybHv1-like overexpressing plants under low temperature stress, 4B is an expression level detection diagram of StMybHv1 - like overexpressing plants, 4C is a fresh plant quantity determination diagram of StMybHv1-like overexpressing plants under low temperature stress, 4D is a plant survival rate determination diagram of StMybHv1-like overexpressing plants under low temperature stress, and 4E is a damage value determination diagram of StMybHv1-like overexpressing plants under low temperature stress;

FIG5 is a diagram showing the yeast two-hybrid interaction verification and identification of StMybHv1-like.

DETAILED DESCRIPTION

In order to make those skilled in the art better understand the technical scheme of the present invention, the present invention is described in detail below in conjunction with specific embodiments. The experimental methods for which specific conditions are not indicated in the following examples are usually carried out under normal conditions or according to the conditions recommended by the manufacturer. The test materials used in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent stores. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used in the text have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar to or equal to the recorded content can all be applied to the present invention. The preferred implementation methods and materials described in the text are only for demonstration purposes.

Example 1 Study on StMybHv1-like gene and expression pattern

The plant materials used in this study were the freeze-resistant material CMM and the low-temperature sensitive material DM. They were grown on MS medium supplemented with 3% sucrose and 0.35% agar at 20±1°C for 3 weeks. After that, the 3-week-old tissue culture seedlings were transplanted into plastic pots (10×10 cm) and planted in a plant growth climate chamber (22±2°C, 16 h light/8 h dark photoperiod). After 4-week-old plants grown under normal conditions were moved into a 4°C cold room for cold acclimation for 3 days, the acclimated plants were treated at −4°C for 0 h, 3 h, 6 h, and 12 h. Leaves were taken from the treated plants, RNA was extracted, and reverse transcribed into cDNA for qRT-PCR. The amplification primers were as follows. SEC was used as the internal reference gene, and 2 ^–ΔΔCT was used to calculate the relative expression of genes (see Figure 1). The error bars represent SE (n = 3, ∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001;Student's t-test).

The reaction system is as follows:

The reaction program was: 95°C, 30 s; 95°C, 5 s; 58°C, 30 s; 40 cycles

The results showed that the expression of StMybHv1-like gene decreased sharply in the low-temperature sensitive material DM under low-temperature induction, while the expression level was significantly upregulated in the frost-resistant material CMM, and the expression level reached a higher value when treated at -4℃ for 3 h and 6 h (Figure 1). Therefore, the present invention takes StMybHv1-like gene as the research object to study whether it can improve the low-temperature resistance of potato cultivars.

Example 2 Cloning of StMybHv1-like gene and construction of overexpression vector

StMybHv1-like

Gene cloning was carried out by reverse transcription of total RNA from leaf tissues of potato frost-resistant material CMM and non-freeze-resistant material DM. Specific amplification primers were designed based on the cDNA sequence of SEQ ID NO. 1.

StMybHv1-like-F: 5'-CATTTGGAGAGAACAGAGCTCATGGCTAATTGTTGCTCTAAAAGG-3'

StMybHv1-like-R:

5'-GGTGTCGACTCTAGAGGATCC TGCAGAAGAACTTTCAGCATCAG-3'

The reaction system is as follows:

The reaction procedure is as follows:

The target band of the PCR product obtained by 1% agarose gel electrophoresis was recovered using TIANGEN's agarose gel DNA recovery kit.

Overexpression vector construction (1) Digest the vector plasmid with Stu I to pH 7lic9.0. The reaction system is as follows:

Place in a 37°C oven overnight;

(2) Purify the PCR product ^a and the vector plasmid ^b after restriction digestion, and connect them using the ClonExpress ^® II One Step Cloning Kit instructions. The specific steps are as follows:

The following connection system is configured on ice:

37°C, 30 min;

(3) Conversion

Use the instructions of the Weidi Biotech DH5α Chemically Competent Cell product to transform E. coli. The specific steps are as follows:

a. Take out the DH5α competent cells from −80°C and quickly put them in ice. After 5 min, wait for the bacterial block to melt, add 5 μL of the above ligation product and gently mix it by hand at the bottom of the EP tube, and let it stand in ice for 25 min;

b. Heat shock in a 42°C water bath for 45 seconds, quickly put back on ice and let stand for 2 minutes (shaking will reduce transformation efficiency);

c. Add 700 μL of LB without antibiotics, mix well and recover at 37°C, 200 rpm for 60 min;

d. Centrifuge at 5000 rpm for 1 min to collect the cells, take about 100 μL of the supernatant, gently blow to resuspend the cells and spread on the LB plate containing Kan;

e. Place the plate upside down in a 37°C incubator overnight.

(4) Identification of positive clones

Use the specific primers on the vector to amplify and sequence the positive clones. The plasmid extracted from the positive clones with correct sequencing is named pH7lic9.0-MybHV1-like for future use.

Specific primers are shown below

Gene forward primer: StMybHv1-like-F: CATTTGGAGAGAACAGAGCTCATGGCTAATTGTTGCTCTAAAAGG;

lic vector rear primer: GTGATTTTTGCGGACTCTAGCAT

Example 3 Agrobacterium-mediated genetic transformation of potato

Agrobacterium transformation The plasmid pH7lic9.0-MybHV1-like was transformed with Agrobacterium using the instructions for the GV3101 Chemically Competent Cell product of Weidi Biotechnology. The specific steps are as follows:

(1) Thaw the GV3101 Agrobacterium competent cells stored at −80°C on ice;

(2) Add 1 μL of the plasmid DNA to be transformed into every 50 μL of competent cells, mix gently, and place on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min;

(3) Add 700 μL of LB liquid medium without antibiotics and culture at 28°C with shaking for 2 h;

(4) Pipette about 100 μL of bacterial solution and spread it on an LB plate containing Kan. Invert the plate and incubate at 28°C for 2-3 days.

(5) Colony PCR identification.

2. Agrobacterium-mediated genetic transformation of potato

The recipient material for genetic transformation was the potato variety "Atlantic" and the following process was involved:

(1) Pre-culture: Take four-week-old Atlantic sterile seedlings, cut the stem segments without lateral buds and place them on A1 pre-culture medium, and culture them under light for 48 h;

(2) Co-cultivation: The above pH7lic9.0-MybHv1-like vector was transformed into Agrobacterium GV3101 by freeze-thaw method for activation and shaking, so that the final _OD600 of the bacterial solution was 0.5. After centrifugation to collect the bacteria, it was poured into MS30 liquid culture medium so that the _OD600 was 0.5, and 1/1000 of 40 mg/L acetosyringone (Acetosyringone, AS) was added for later use. The pre-cultured stem segments were placed in the infection solution, and then placed on a low-speed shaker for infection at room temperature for 10 min.

(3) Regeneration culture: Pour out the infection solution, place the stem segments on sterile filter paper to dry, transfer the stem segments to A2 co-culture medium with sterile filter paper, and culture in the dark for 48 h. After the dark culture, place the stem segments flat on the differentiation medium and continue to culture. Replace the differentiation medium SIM every 2 weeks until the callus differentiates and sprouts. Then cut the buds and transfer them to the rooting medium for culture, and finally obtain complete plants. The transgenic culture medium is shown in Table 1.

Table 1

3. Detection of transgenic positive strains

(1) Cut the leaves of transgenic plants and extract DNA using the CTAB method;

(2) Using the extracted DNA as a template for PCR amplification;

Gene forward primer: StMybHv1-like-F: CATTTGGAGAGAACAGAGCTCATGGCTAATTGTTGCTCTAAAAGG;

lic vector rear primer: GTGATTTTTGCGGACTCTAGCAT;

The PCR reaction system is as follows:

PCR amplification conditions are as follows:

The positive strains were obtained by 1% agarose gel electrophoresis.

(3) RNA from transgenic plants and control groups was extracted and reverse transcribed to generate cDNA. Real-time fluorescence quantitative PCR was performed using the TB Green® Premix Ex TaqTM II kit. The results are shown in Figure 3.

As can be seen from Figure 3, PCR amplification was performed using genomic DNA as a template using the gene front primer StMybHv1-like-F and the vector rear primer lic-R, and 18 positive transgenic plants were found (Figure 3A); among them, in three overexpression transgenic plants (numbered OE#4, OE#6, and OE#8), the expression level of the StMybHv1-like gene was significantly increased (Figure 3B), while there was no significant difference in the growth phenotype between the transgenic plants and the control plants (Figure 3C).

Example 4 Overexpression of StMybHv1-like enhances cold resistance of potato

The positive transgenic plants and control plants were subjected to cold acclimation (4°C) and low temperature stress (−2.5°C) treatments as follows:

(1) The transgenic plants and control plants were watered with the same amount of water and lighting conditions and grown in a plant growth climate chamber for 3 weeks, during which they were watered every 3 days;

(2) After 3 weeks, move them into a 4°C climate chamber for acclimatization for one week, and then move them into a -2.5°C climate chamber for 6 hours of low temperature treatment;

(3) Return to cold acclimatization at 4°C for 24 h. Repeat this process three times.

As can be seen from Figure 4, the three overexpression transgenic lines were significantly better than the control in terms of the number of frozen plants, survival rate, and final scores, indicating that the cold resistance of StMybHv1-like overexpression transgenic plants was significantly enhanced.

Example 5 Investigating whether StMybHv1-like can form homodimers

In order to explore whether StMybHv1-like needs to form homodimers to function, yeast two-hybridization and yeast point-to-point verification were performed.

Yeast carrying StMybHv1-like and empty PGBKT7, empty PGADT7 and empty PGBKT7 and known positive controls were cultured in two-deficient (SD/-Leu-Trp) medium and four-deficient (SD/-Leu-Trp-His -Ade) plates, respectively.

As can be seen from Figure 5, co-transformation of PGADT7-StMybHv1-like and PGBKT7-StMybHv1

-like plasmid, empty PGADT7 and PGBKT7-StMybHv1-like, PGADT7-StMybHv1-like and empty PGBKT7, empty PGADT7 and empty PGBKT7 and known positive control yeast can grow on two-deficiency (SD/-Leu-Trp) medium, but on four-deficiency (SD/-Leu-Trp-His-Ade) plates, except for the positive control, the others cannot grow on the plate, indicating that potato StMybHv1-like cannot form homodimers, and StMybHv1-like does not need to form homodimers to function in regulating potato cold resistance.

Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Under the guidance of the present invention, those skilled in the art can make a variety of similar expressions without violating the purpose and claims of the present invention, and such changes all fall within the protection scope of the present invention.

Claims (9)

1. A protein encoded by the StMybHv1-like gene 1 for regulating potato cold resistance, characterized in that the amino acid sequence of the protein is shown in SEQ ID NO. 2. 2. A recombinant vector, expression cassette, transgenic line or recombinant bacterium containing the StMybHv1-like gene, characterized in that the nucleotide sequence of the gene is (a), (b) or (c); (a) the nucleotide sequence shown in SEQ ID NO. 1; (b) a nucleotide sequence that hybridizes with and encodes the nucleotide sequence shown in SEQ ID NO. 1 under stringent conditions; (c) a nucleotide sequence encoding a gene having 80% or more homology to the nucleotide sequence shown in SEQ ID NO. 1. 3. A use of StMybHv1-like gene in potato cultivars to resist low temperature stress, characterized in that the nucleotide sequence of the gene is (a), (b) or (c); (a) the nucleotide sequence shown in SEQ ID NO. 1; (b) a nucleotide sequence that hybridizes with and encodes the nucleotide sequence shown in SEQ ID NO. 1 under stringent conditions; (c) a nucleotide sequence encoding a gene having 80% or more homology to the nucleotide sequence shown in SEQ ID NO. 1. 4. Use of the protein according to claim 1 in protecting potato cultivars from low temperature stress. 5. Use of the recombinant vector, expression cassette, transgenic line or recombinant bacteria according to claim 2 in the resistance of potato cultivars to low temperature stress. 6. The use according to any one of claims 3 to 5, characterized in that the cold resistance of the crop is improved by increasing the expression level and/or activity of the StMybHv1-like gene in the crop. 7. A method for preparing a transgenic plant, comprising the following steps: introducing the coding gene of the protein according to claim 1 into a recipient plant to obtain a transgenic plant; compared with wild-type plants, the cold resistance of the transgenic plant is improved; and the plant is potato. 8. The method according to claim 7 is characterized in that: the coding gene is introduced into the plant via a recombinant expression vector; the recombinant expression vector is obtained by inserting the coding gene into the multiple cloning site of the initial vector pH7lic9.0. 9. The method according to claim 7 or 8, characterized in that the nucleotide sequence of the coding gene is as shown in SEQ ID No. 1.