CN113897375B - C6-like zinc finger protein encoding gene of Aspergillus extreme halophilus and its application - Google Patents

Abstract

The invention discloses an extreme halophilic aspergillus C6-like zinc finger protein coding gene and application thereof, the coding gene is transferred into plants, under NaCl simulated salt stress, plants over-expressing the extreme halophilic aspergillus C6-like zinc finger protein coding gene are obviously superior to wild plants in length, weight and seed dry weight, and salt tolerance is obviously enhanced, so that the extreme halophilic aspergillus C6-like zinc finger protein coding gene provided by the invention has an important role in improving the saline-alkali tolerance of plants.

Description

C6-like zinc finger protein encoding gene of Aspergillus extreme halophilus and its application

Technical field

The invention belongs to the technical field of plant genetic engineering, and more specifically, relates to a C6-like zinc finger protein encoding gene of extreme halophilic Aspergillus and its application in improving the salt stress tolerance of plants.

Background technique

Soil salinization is a gradual geological environmental problem, mainly caused by arid climate, topography and irrational use of water and soil resources by humans. It is reported that there are 950 million hectares of saline-alkali land in the world, about 100 million hectares of saline-alkali land in my country, of which 9.2 million hectares of saline-alkali arable land cause direct economic losses of more than 100 billion US dollars each year. Soil salinization causes physiological drought in plants, affects nutrient absorption, and harms crop growth and yield. Using genetic engineering technology to improve plant salt tolerance is an important way to ecologically restore saline-alkali soil.

At present, research on improving plant salt tolerance using genetic engineering methods has made certain progress, and plant salt tolerance has been improved to a certain extent through resistance gene transformation. For example, Zhai Honghong used AhCMO-AhBADH double genes and AhCMO and AhBADH single genes to construct transgenic tobacco. The transgenic plants were treated with NaCl. The results showed that the growth of transgenic plants was better than that of wild-type plants. The root length, number of lateral roots and fresh weight of transgenic plants were all increased compared with wild-type plants.

Zinc finger proteins are a class of proteins that contain short, self-folding structures that are stabilized by binding to Zn ²⁺ to form a "finger" structure. This protein plays an important role in gene expression regulation, cell differentiation regulation, and plant stress response. plays an important role. At present, such research mainly focuses on obtaining zinc finger protein expression genes with tolerance from plants growing under lower salinity. For example, the C2H2 zinc finger protein ZjZFN1 isolated from Zoysia by Teng et al. Overexpression in Arabidopsis. Although compared with the wild type, the seed germination rate of transgenic Arabidopsis is increased, the percentage of green cotyledons is increased, and the plant's salt stress tolerance is enhanced, but because it is extracted from plants grown under lower salinity gene, its ability to improve salt stress tolerance is limited.

Rapeseed is an important oil crop in my country and the main source of edible vegetable oil. At present, the supply of edible oils and fats in my country is seriously insufficient. How to obtain new salt-tolerant transgenic rape plants and develop and utilize saline-alkali land resources are of great significance for increasing rapeseed yields.

Contents of the invention

In view of the problems existing in the prior art, the present invention provides an extreme halophilic Aspergillus C6-like zinc finger protein encoding gene and its application. By transferring the extreme halophilic Aspergillus C6-like zinc finger protein encoding gene into plants, it can Improve plants' ability to tolerate salt stress.

The present invention is realized through the following technical solutions:

An extremely halophilic Aspergillus C6-like zinc finger protein encoding gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

A protein encoded by the C6-like zinc finger protein encoding gene of Aspergillus extrema, the amino acid sequence of which is shown in SEQ ID NO.2.

An expression vector containing the gene encoding the C6-like zinc finger protein of Aspergillus extreme halophilia.

Preferably, the expression vector is obtained by connecting the C6-like zinc finger protein encoding gene and the vector pBWA(V)HS.

A cell containing the expression vector.

A host bacterium containing the C6-like zinc finger protein encoding gene of extreme halophilic Aspergillus.

Preferably, the host bacterium is Agrobacterium.

The application of the extremely halophilic Aspergillus C6-like zinc finger protein encoding gene in improving plant salt tolerance.

Preferably, the application includes the following steps:

1) Construct an expression vector containing the gene encoding the C6-like zinc finger protein of Aspergillus extrema;

2) Transform the constructed expression vector into plants or plant cells;

3) Cultivate the plants or plant cells obtained in step 2), and screen to obtain transgenic plants with improved salt tolerance.

Preferably, the plant is rapeseed.

Compared with the existing technology, the present invention has the following beneficial technical effects:

The present invention uses Aspergillus montevidensis ZYD4 as material, extracts and amplifies an Aspergillus montevidensis C6-like zinc finger protein encoding gene, and transfers the encoding gene into plants. Because the salt-tolerant gene of the present invention is obtained from extremophile microorganisms that grow in high-salt conditions, its transgenic plants are more able to adapt to the environment of high-salt conditions. The research of the present invention shows that under NaCl simulated salt stress, plants that overexpress the gene encoding the C6-like zinc finger protein of Aspergillus extreme halophilus are significantly better than wild-type plants in length, weight and seed dry weight, and their salt tolerance is significantly enhanced. It shows that the extremely halophilic Aspergillus C6-like zinc finger protein encoding gene provided by the present invention plays an important role in improving plant salt-alkali tolerance, provides important genetic resources for plant stress resistance improvement, and is useful in ecological restoration of saline-alkali soil using genetically engineered plants. aspect is of great significance.

Description of drawings

Figure 1 shows the phylogenetic tree constructed based on cDNA sequences;

Figure 2 is a phylogenetic tree constructed based on amino acid sequences;

Figure 3 is a photo of the PCR reaction amplification of the gene encoding the C6-like zinc finger protein; M is the DL6000DNAmarker, and C6-like is the PCR product of the gene encoding the C6-like zinc finger protein;

Figure 4 is a picture of mature plants of transgenic rapeseed;

Figure 5 is an analysis chart of the plant height of transgenic rapeseed plants under different salt concentrations;

Figure 6 shows the biomass analysis chart of transgenic rapeseed plants under different salt concentrations;

Figure 7 is a graph showing the dry matter weight analysis of transgenic rapeseed seeds.

Detailed ways

The present invention will be further described in detail below with reference to specific examples, which are explanations rather than limitations of the present invention.

The present invention uses Aspergillus montevidensis ZYD4 as the material, extracts RNA, and uses RT-PCR to amplify the gene fragment. The length of the target cDNA fragment is 1896 bp, as shown in SEQ ID NO. 1, and encodes 631 amino acids, as shown in SEQ Shown as ID NO.2.

According to the analysis of online biology software, the length of the target protein corresponding to the C6-like zinc finger protein encoding gene of Aspergillus extrema is 631, the molecular weight is 73KDa, and the predicted isoelectric point is 6.45.

Comparative analysis of its cDNA sequence with GenBank database data showed that the gene sequence is homologous to the C6 zinc finger protein genes of A.glaucus CBS 516.65, A.sclerotioniger CBS 115572, A.ibericus CBS 121593, and A.steynii IBT 23096 strains. The sex is between 87.6% and 71.72%. Download the sequence data with high homology to construct a diagram of the developmental tree. Refer to Figure 1 for the results. Among them, the C6-like zinc finger protein coding gene of the target strain A. montevidensis ZYD4 formed an independent branch with A. glaucus CBS 516.65, with high homology, with a step value of 96%.

Comparison of the amino acid sequence corresponding to the C6 zinc finger protein gene of Aspergillus extrema and GenBank database data shows that the amino acid sequence of the gene is the same as that of A. ruber CBS135680, A. glaucus CBS 516.65, A.wentii DTO134E9, A. phoenicis ATCC 13157 and other strains The amino acid sequence homology of C6 zinc finger protein is high. Download the sequence data with high homology to construct a diagram of the developmental tree. Refer to Figure 2 for the results. The C6-like zinc finger protein encoding gene of the target strain A. montevidensis ZYD4 is most closely related to A. ruber CBS 135680 and A. glaucus CBS 516.65, forming an independent branch with a step length of 88%, and the three together constitute a relatively For large branches, the step value reaches 100%.

In the present invention, the obtained extreme halophilic Aspergillus C6-like zinc finger protein encoding gene is connected to the expression vector pBWA(V)HS to construct the prokaryotic expression vector pBWA(V)HS-C6-LIKE, which is transferred into E. coli and verified through screening. , then transform the overexpression vector into Agrobacterium GV3101, and then infect the rapeseed explants to obtain transgenic rapeseed plants.

The present invention conducts cultivation tests on rapeseed overexpressing the C6-like zinc finger protein encoding gene at different salt concentrations (0mM, 60mM, 120mM, 240mM), and measures the rapeseed plant length (root + stem), weight and seed dry weight. , the results show that overexpression of the gene encoding the C6-like zinc finger protein of extreme halophilic Aspergillus can significantly improve the salt tolerance of rapeseed in saline-alkali soil, as shown in Figures 5-7 respectively.

Example 1 Construction of the overexpression vector pBWA(V)HS-C6-LIKE of the gene encoding the C6-like zinc finger protein of Aspergillus extreme halophilus

1. Amplify the target fragment

(1) Amplify the primer sequence of the C6-like zinc finger protein encoding gene of Aspergillus extreme halophilus:

C6-F1:

5’-ATATACACACATTTACAATGCTTATTAAGAACAGACGTCTACA-3’

C6-R1:

5’-GGCGAAGAAGTCCAAAGCTTTATCCAGGCATGCATGAGTTAT-3’

The synthesized primers were diluted into a stock solution with a final concentration of 10 μmol/L.

(2) The template cDNA is delivered to the company for synthesis. The amplification system is as follows:

cDNA 1ul Premix ^TaqTM 12.5ul C6-F1 1ul C6-R1 1ul H ₂ O 9.5ul

Add the above materials into the thin-walled tube, mix well, tap it out, and put it into the PCR machine. Select the appropriate annealing temperature and extension temperature to start PCR amplification.

(3) After PCR, agarose gel electrophoresis is performed. After gel imaging, the results are shown in Figure 3. The amplification product appears as a band at about 1900 bp, where M is the standard molecular weight of DNA (M is the standard molecular weight of DNA: 6000, 4000, 3000, 2000, 1500, 1000, 750, 500, 250, 100bp). Cut the gel containing the cDNA fragment of interest, collect it, and use a DNA gel recovery kit to recover and purify it for use.

2. Double enzyme digestion of C6-like target fragment and vector pBWA(V)HS

(1) Double digestion with restriction endonucleases BsaI and Eco31I.

The vector pBWA(V)HS enzyme digestion system is as follows:

carrier 6ul Buffer 2ul BsaI 1ul Eco31I 1ul H ₂ O 10ul

The C6-like target fragment enzyme digestion system is as follows:

Digest at 37°C for about 1 hour.

(2) Purify the vector digestion product and the target fragment digestion product using PCR purification kits respectively.

3. Recombinant construction of overexpression vector pBWA(V)HS-C6-LIKE

The system is as follows:

Incubate in a PCR machine at 37°C for 30 minutes and cool in an ice bath.

4.Conversion

(1) Place the competent cells DH5α on ice and wait for them to thaw naturally. Add 10 μL of the overexpression vector pBWA(V)HS-C6-LIKE to the competent cells and place on ice for 15 minutes.

(2) Then heat shock in a 42°C water bath for 45 seconds, heat shock transformation, and then quickly place on ice (4°C) for 5 minutes.

(3) Add 300 μL of LB liquid culture medium without antibiotics, and incubate for 1 hour at 37°C and 180 rpm with shaking.

(4) Inoculate the competent cells after activation culture treatment on an LB solid medium plate containing kanamycin (30 μg/ml), place the culture dish upside down in a 37°C environment and culture it for 12 hours to obtain the overexpression vector pBWA (V)HS-C6-LIKE-osgfp.

Example 2 Transformation of overexpression vector pBWA(V)HS-C6-LIKE-osgfp into Agrobacterium (1). The Agrobacterium strain used was GV3101. The liquid nitrogen freeze-thawing method was used to transform the constructed overexpression vector pBWA(V)HS-C6-LIKE-osgfp into Agrobacterium tumefaciens.

The specific operations are as follows:

1) Take the Agrobacterium competent cells stored at -80°C and thaw them on ice.

2) Add 0.01-1μg plasmid DNA (overexpression vector pBWA(V)HS-C6-LIKE-osgfp) to each 100μL competent state, stir the bottom of the tube by hand to mix, then let stand on ice for 5min, liquid nitrogen for 5min, 37 ℃ water bath for 5 minutes, ice bath for 5 minutes.

3) Add 700 μL of antibiotic-free LB liquid culture medium and incubate with shaking at 28°C for 2 to 3 hours.

4) Centrifuge at 6000 rpm for one minute to collect the bacteria. Leave about 100 μL of the supernatant and gently pipette to resuspend the bacterial mass and spread it on an LB plate containing the corresponding antibiotics. Place it upside down in a 28°C incubator for 48 hours.

5) Positive clones detected by PCR should be stored at 4°C for later use.

Example 3 Agrobacterium infects rapeseed

1. Preparation of rapeseed explants

Select rapeseed seeds for disinfection, inoculate the sterilized rapeseed seeds on MS medium, and grow for 7 days to obtain sterile rapeseed seedlings. When the hypocotyl of the seedlings reaches the mouth of the bottle, use tweezers to take out the sterile seedlings and remove the cotyledons and cotyledons. The hypocotyl was cut into 1 to 2cm segments as explants and placed on the pre-culture medium.

2. Dyeing

The positive cloned Agrobacterium detected by PCR was shaken to OD ₆₀₀ 0.8. The rapeseed explants pre-cultured for 2-3 days were placed in the Agrobacterium suspension and infected for 10min. The infected rapeseed explants were placed in Dry on the filter paper, place on the co-culture medium, and culture for 2 days. Then transfer the explants to the sieve medium and culture for 7 days.

3. Induction and screening of callus

Select the effective callus and transfer it to the selection medium containing hygromycin, and screen for about 15 days, for a total of 2 to 3 times.

4. Differentiation and rooting

Transfer the vigorously growing positive callus to the differentiation medium, wait until it differentiates into seedlings, transfer the differentiated seedlings to the rooting culture to take root for 7-10 days, and obtain positive plants.

5.Detection

Label the seedlings that have grown roots, take 0.5cm ² of each rape leaf, grind the sample, use the grinding liquid as a DNA template for PCR amplification, and run agarose gel electrophoresis to determine the positive seedlings and positive rate. The positive seedlings were continuously cultured to obtain transgenic plants containing the C6-like zinc finger protein encoding gene. As shown in Figure 4, the plants were in good growth status, with many root branches, thick stems, and enlarged leaves.

Example 3 Research on the Salt Stress Tolerance Function of Transgenic Rapeseed

Transgenic rapeseed and wild-type rapeseed were cultivated in soil with salt concentrations of 0mM, 60mM, 120mM, and 240mM NaCl respectively and cultured under 20°C light (18000Lx) for 60 days. The plant height and weight of each plant were measured respectively, and the rapeseed was finally collected. seeds and determine the dry matter content of the seeds.

A column chart was drawn based on the plant height of rapeseed under different soil salt stresses. The results are shown in Figure 5. The plant height of transgenic rapeseed is higher than that of wild-type rapeseed under different salt concentrations, and the transgenic plants grow under high salt concentrations of 240mM. The advantages are obvious.

A column chart was drawn based on the fresh weight of rapeseed plants under different soil salt stresses. The results are shown in Figure 6. The growth status of transgenic rapeseed remained stable under different salt concentrations, while the plant weight of wild-type rapeseed decreased significantly under high salt concentrations.

A column chart was drawn based on the dry matter content of rapeseed seeds under different soil salt stresses. The results are shown in Figure 7. It can be seen that the seed quality of transgenic rapeseed is significantly better than that of wild-type rapeseed.

sequence list

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<120>Extreme halophilic Aspergillus C6-like zinc finger protein encoding gene and its application

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Claims (8)

1. The encoding gene of the extreme halophilic aspergillus C6-like zinc finger protein is characterized in that the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 1.

2. The protein encoded by the gene encoding the C6-like zinc finger protein of extreme halophilic aspergillus of claim 1, which is characterized in that the amino acid sequence is shown in SEQ ID NO. 2.

3. An expression vector comprising the gene encoding the C6-like zinc finger protein of aspergillus extremely halophilus according to claim 1.

4. An expression vector containing the C6-like zinc finger protein coding gene of Aspergillus extreme halophilus according to claim 3, wherein said expression vector is obtained by ligating the C6-like zinc finger protein coding gene with vector pBWA (V) HS.

5. A host bacterium comprising the gene encoding the C6-like zinc finger protein of aspergillus extremely halophilus of claim 1.

6. The host bacterium containing a gene encoding a C6-like zinc finger protein of aspergillus terreus according to claim 5, wherein the host bacterium is agrobacterium.

7. The use of the C6-like zinc finger protein coding gene of aspergillus extremely halophilus according to claim 1 for improving salt tolerance of plants, characterized in that said plants are canola.

8. The use according to claim 7, characterized by the steps of:

1) Constructing an expression vector containing an extreme halophilic aspergillus C6-like zinc finger protein coding gene;

2) Transforming the constructed expression vector into a plant or plant cell;

3) Cultivating the plant or plant cell obtained in the step 2), and screening to obtain the transgenic plant with improved salt tolerance.