Cotton GhNFD4 gene and application thereof in drought resistance

Abstract

The invention discloses a cotton GhNFD4 gene and application thereof in drought resistance, and belongs to the technical field of biology. The coding amino acid sequence of the GhNFD4 gene is as shown in SEQ ID NO. 1, and the nucleotide sequence of the GhNFD4 gene is as shown in SEQ ID NO. 2. According to the invention, a target gene is silenced in cotton by using a gene silencing technology, and after drought stress treatment, the drought resistance of the cotton is obviously reduced after the silent GhNFD4 gene is found; the GhNFD4 gene is capable of positively regulating and controlling the cotton to respond to the drought resistance reaction and is an important gene resource available for the drought-resistant breeding of the cotton, and the water loss rate, the relative conductivity, the malondialdehyde content, the hydrogen peroxide content and the total antioxidant enzyme activity of a cotton plant are increased, so that the cotton shows the intolerance to the drought stress, and the GhNFD4 gene is capable of positively regulating and controlling the cotton to respond to the drought resistance reaction and is an important gene resource available for the drought-resistant breeding of the cotton.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to an upland cotton GhNFD4 gene and an application thereof in drought resistance. Background Art

[0002] Cotton is an important economic crop in my country. Suitable soil moisture is a necessary prerequisite for high cotton yields. However, with global warming and the shortage of irrigable water resources, the harm of cotton to drought stress is becoming increasingly serious. Continuous drought and water loss seriously affect the growth and development of cotton during the bud and boll stages, causing a sharp decline in cotton quality and yield. Upland cotton (Gossypium hirsutum L.), as the main cultivated cotton species in production, accounts for 90% of the global cotton production and has a very broad application prospect. Therefore, the present invention silences the GhNFD4 gene through VIGS technology, verifies the role of the GhNFD4 gene in cotton response to drought, and provides important gene resources for drought-resistant breeding of upland cotton. Summary of the invention

[0003] The purpose of the present invention is to provide a cotton GhNFD4 gene and its application in drought resistance. The gene positively regulates the cotton response to drought stress to improve the drought resistance of cotton.

[0004] To achieve the above object, the present invention uses the following scheme:

[0005] A cotton GhNFD4 gene, comprising:

[0006] (1) The protein whose amino acid sequence is shown in SEQ ID NO. 1 comprises 238 amino acids.

[0007] (2) A protein obtained by gene mutation with SEQ ID NO.1, or having an amino acid sequence similarity of more than 80% with SEQ ID NO.1, and being associated with drought resistance.

[0008] The GhNFD4 gene sequence is SEQ ID NO. 2, and the full length of its coding region is 1653 bp.

[0009] The invention contains a gene expression frame, a recombinant vector and a recombinant bacterium of SEQ ID NO.2 or a nucleotide sequence having a similarity of more than 80% with SEQ ID NO.2, and affects the drought tolerance of cotton.

[0010] The cotton GhNFD4 gene of the present invention can be directly synthesized, or produced by an organism after obtaining the coding gene by molecular biological technology.

[0011] The present invention also provides a gene expression frame, expression vector and recombinant bacteria containing cotton GhNFD4 gene for use in cotton drought resistance. The amino acid sequence of the cotton GhNFD4 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the cotton GhNFD4 gene is shown in SEQ ID NO.2.

[0012] The present invention also provides an application of the cotton GhNFD4 gene in plant drought resistance breeding, the amino acid sequence of the cotton GhNFD4 gene is shown in SEQ ID NO.1, the nucleotide sequence of the cotton GhNFD4 gene is shown in SEQ ID NO.2, and the plant is cotton.

[0013] Furthermore, the drought resistance of the plant can be adjusted by regulating the expression level of the GhNFD4 gene in the plant.

[0014] The present invention also provides an application of the cotton GhNFD4 gene in cotton drought resistance. The amino acid sequence of the cotton GhNFD4 gene is shown in SEQ ID NO.1.

[0015] Furthermore, the nucleotide sequence of the cotton GhNFD4 gene is shown in SEQ ID NO.2.

[0016] The technical solution is to explore the drought resistance of plants by silencing the expression of the above-mentioned gene GhNFD4.

[0017] Specifically, gene-silenced plants were obtained by silencing the expression of the above-mentioned gene GhNFD4 or the activity of the above-mentioned protein through the VIGS technology. Since the GhNFD4 gene-silenced plants showed greater sensitivity to drought stress, the relevant physiological indexes of the target gene-silenced plants were measured, which is helpful to illustrate the physiological effect of the GhNFD4 gene in drought resistance, and preliminarily proved that the GhNFD4 gene positively responded to cotton's response to drought stress.

[0018] Compared with the existing technology, the present invention has the following beneficial effects: the present invention clones the GhNFD4 gene related to drought stress. The GhNFD4 gene in cotton is silenced by VIGS technology, and the drought resistance of cotton with silenced GhNFD4 gene is analyzed. The phenotype and physiological indicators of the obtained cotton plants with silenced GhNFD4 gene show that they are more sensitive to drought stress, indicating that the cotton GhNFD4 gene is related to cotton drought resistance. The present invention provides important gene resources for drought resistance breeding of upland cotton. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1:GhNFD4 gene interference fragment cloning result diagram; M. Marker D3000; Among them, 1. Negative control; 2, 3, 4. GhNFD4 interference fragment PCR products;

[0020] Figure 2 :Double enzyme digestion identification result of pTRV2-GhNFD4 recombinant plasmid; M. MarkerⅢ; 1, 3. Plasmid control; 2, 4. Enzyme digestion product;

[0021] Figure 3 : Positive control phenotype of TRV2:GhCLA1;

[0022] Figure 4 : Target gene silencing efficiency test results, asterisks (*) represent significant differences (*P<0.05, **P<0.01);

[0023] Figure 5 :Phenotypic analysis of cotton plants before and after drought stress;

[0024] Figure 6 : Comparison of water loss rate (A), relative conductivity (B) and malondialdehyde content (C) between control plants and target gene silenced plants; data are the mean ± SD of three replicates; asterisks (*) indicate significant differences between control plants and target gene silenced plants (*P < 0.05, **P < 0.01);

[0025] Figure 7 : Determination of reactive oxygen levels and total antioxidant enzyme activity in control plants and target gene silenced plants before and after drought stress; (A) NBT and DAB staining; (B) Determination of hydrogen peroxide content; (C) Determination of total antioxidant enzyme activity; Data are the mean ± SD of three replicates; Asterisks (*) indicate significant differences between control plants and target gene silenced plants; * represents P < 0.05, ** represents P < 0.01. DETAILED DESCRIPTION

[0026] Example 1

[0027] Acquisition of GhNFD4 gene

[0028] (1) Total RNA was extracted from cotton leaves using an RNA extraction kit and cDNA synthesis kit (Vazyme, Nanjing, China) and reverse transcribed into cDNA;

[0029] (2) The coding sequence of the GhNFD4 gene was downloaded from the cotton database (https: / / cottonfgd.net / ), and the specific segment of the GhNFD4 gene was obtained through the SGN VIGS online website. Gene-specific primers GhNFD4-F (SEQ ID NO. 3): cggaattcGACAAAGAGGGCCAAAGATGC; GhNFD4-R (SEQ ID NO. 4): cgggatccTCACCCCACTCTCCATTTTGT were designed using NCBI;

[0030] (3) Using cDNA as a template and utilizing PCR amplification technology, the PCR reaction system is shown in Table 1, and the PCR reaction procedure is shown in Table 2 to amplify a target gene fragment of 266 bp in size;

[0031] (4) The amplified product was detected by 2% agarose gel electrophoresis. Figure 1 , there is a bright and correct bright band above 200bp, while the negative control has no band.

[0032] The correct electroporation bands were excised and recovered and connected to the cloning vector. The connection system is shown in Table 3;

[0033] Table 1 Gene cloning PCR reaction system

[0034]

[0035] Table 2 Gene cloning PCR program

[0036]

[0037] Table 3 T-vector connection system

[0038]

[0039]

[0040] Example 2

[0041] Construction of gene silencing expression vector

[0042] The PMD19-T recombinant plasmid with the GhNFD4 gene that was sequenced correctly was double-digested with EcoRI and BamHI, and the interference fragment was recovered from the gel after enzyme digestion. The pTRV2 empty vector plasmid stored at -80°C was transformed into Escherichia coli DH5α, and a single colony was picked and shaken to culture the plasmid, and the pTRV2 linearized fragment was double-digested with EcoRI and BamHI to recover the pTRV2 linearized fragment. The GhNFD4 interference fragment after enzyme digestion was connected to the pTRV2 linearized vector. The connection reaction system and procedure are shown in Table 4.

[0043] Table 4 Connection reaction system and procedure table

[0044]

[0045] The ligation product was transformed into competent E. coli DH5a, and the positive single colony was selected for bacterial liquid PCR detection. The correct bacterial liquid was shaken to extract the plasmid and double enzyme digestion was performed for identification. The enzyme digestion system is shown in Table 5. EcoRI and BamHI were used to double enzyme digestion to verify the pTRV2-GhNFD4 recombinant plasmid. Figure 2 The double enzyme digestion and PCR identification results were consistent with expectations, indicating that the GhNFD4 target fragment had been successfully connected to the pTRV2 vector.

[0046] Table 5 Enzyme digestion reaction system and conditions

[0047]

[0048]

[0049] The recombinant plasmid pTRV2-GhNFD4 was transformed into Agrobacterium by freeze-thaw method, and a single colony was picked for shaking culture. The bacterial solution was identified by PCR, and the identified bacterial solution was preserved as a strain.

[0050] Example 3

[0051] VIGS silencing of cotton GhNFD4 gene

[0052] Experimental materials: Select seeds of Upland Cotton 'TM-1' with full grains and plant them in pots containing 70% moisture nutrient soil (matrix: vermiculite = 2:1). In order to ensure uniform emergence of the germinated seeds, the planting depth was maintained at 1.5 cm. Then, they were cultured in a constant temperature box at 22 / 25℃ (night / day 14h) for about 12 days. Cotton seedlings with good emergence and uniform growth were selected for VIGS infection experiment.

[0053] (1) Transfer 1 ml of pTRV1, pTRV2-GhCLA1, and pTRV2 Agrobacterium mother liquids at -80°C to 50 ml of LB liquid medium (Kan + 50μg / ml, Rif 25μg / ml) at 180rpm, 28℃ shaking culture for 12h, and after activation, the cells were propagated according to the same ratio;

[0054] (2) The OD value of the overnight culture should be between 0.8 and 1.0;

[0055] (3) Collect the cells at 5000 rpm for 10 min, resuspend them in an equal volume of resuspension solution, and place them at 28°C in the dark for 3 h.

[0056] (4) The Agrobacterium resuspension containing pTRV1 was mixed evenly with the Agrobacterium resuspensions containing pTRV2-GhCLA1, pTRV2-GhNFD4 and pTRV2 empty vector in equal proportions, and the mixture was injected into cotton cotyledons 12 days after seedling emergence.

[0057] (6) Use a 1 ml syringe needle to make a needle-tip-sized wound on the back of the cotyledon. Remove the needle, use the syringe to absorb the mixed infectious solution, and inject it into the wound. The amount of injected infectious solution should fill the entire cotyledon as much as possible.

[0058] (7) In order to achieve a better infection effect, the infected cotton plants were placed in a dark environment at 25°C for 24 h, and then cultured under normal light.

[0059] Example 4

[0060] Detection of GhNFD4 gene silencing efficiency

[0061] The results are as follows Figure 3 As shown in the figure, after the positive control GhCLA1 cotton leaves showed albino phenotype, the wild type, blank group and experimental group cotton leaves were selected to detect the gene silencing efficiency. The third true leaf of cotton was taken for RNA extraction and reverse transcribed into cDNA. Specific primers for GhNFD4 fluorescence quantitative PCR were designed, and real-time fluorescence quantitative PCR was performed with GhUBQ7 as the internal reference gene. The RT-PCR reaction parameters are shown in Table 6, and then 2 -ΔΔCt Methods to analyze the data. Figure 4 As shown, compared with the wild-type and blank group (pTRV2-00) plants, the expression level of GhNFD4 in the experimental group (pTRV2-GhNFD4) plants was significantly decreased, indicating that the GhNFD4 gene in cotton seedlings was successfully silenced, which can be used for further exploration of the function of the GhNFD4 gene.

[0062] Table 6 RT-PCR reaction parameters

[0063]

[0064] Example 5

[0065] Drought treatment of cotton

[0066] The cotton plants injected with pTRV2-00 empty vector bacterial solution were used as the blank control group, and the cotton plants (TRV2-NFD4) of 1 month old with significant decrease in GhNFD4 gene expression as shown by real-time fluorescence quantitative PCR were selected for study. After giving these plants sufficient water, they were no longer watered. After drought culture for 15 days, the phenotypes of the plants were observed and photographed. After 15 days of continuous water shortage, if Figure 5As shown, the leaves of VIGS plants (TRV2-NFD4) began to droop, wilt and lose water very seriously, while the leaves of control plants only slightly wilted and turned yellow. This confirmed that the sensitivity of cotton plants with silenced GhNFD4 gene to drought stress was enhanced.

[0067] Example 6

[0068] Determination of relevant physiological indicators

[0069] (1) Determination of water loss rate

[0070] The water loss of detached leaves largely reflects the water status of the whole plant. Therefore, the weighing method was used to determine the water loss rate of cotton leaves of control plants and VIGS plants. The leaves from the same part of the control plants and VIGS plants were weighed and the initial fresh weight was recorded as W0. The weight was then measured every 1 hour and recorded until the 8th hour. According to the water loss rate formula: Water loss rate % = (W0h-Wxh) / W0h (i.e. W0h: the weight of the detached leaves before water loss; Wxh: the weight of the detached leaves after water loss, xh = 1, 2, 3, 4, 5, 6, 7, 8), the water loss rate of the leaves per hour was calculated. The results are as follows: Figure 6 (A) The water loss rate of detached leaves of VIGS plants was higher than that of control plants, indicating that silencing the GhNFD4 gene can accelerate water loss in cotton plants.

[0071] (2) Determination of relative conductivity and malondialdehyde content

[0072] Determination of membrane damage: MDA is a lipid peroxidation product produced by oxidative stress in plant cells. The degree of damage to the plasma membrane can be reflected by measuring the MDA content and relative conductivity of plant cells. The lipid peroxidation level of the control plants and the experimental group TRV2-NFD4 plants was determined by the thiobarbituric acid (TBA) method to determine the accumulation of malondialdehyde (MDA). The relative electrolyte leakage (REL) was then measured using an EC 215 thermal conductivity meter. Figure 6 (B) (C) show that in the absence of stress, the contents of MDA and REL in the control plants and VIGS plants were basically similar. After drought stress, the levels of MDA and REL in VIGS plants were significantly higher than those in the control plants, 1.52 times and 1.91 times higher than those in the control plants, respectively. The relative conductivity and malondialdehyde of cotton plants with GhNFD4 gene silenced were higher than those of control plants, indicating that silencing GhNFD4 gene after drought treatment deepened the damage of cotton plasma membrane.

[0073] (3) Determination of reactive oxygen species levels and total antioxidant enzyme activity

[0074] Detection of reactive oxygen levels: NBT and DAB staining can directly reflect the two main ROS products under drought stress.2- and H2O2 accumulation. Take cotton leaves from the same part of the control plant and VIGS plant after drought stress and soak them in NBT reaction solution (0.5mg / ml) and DAB reaction solution (1mg / ml, pH5.5, 50mM Tris-HCl,) and stain for 16h in the dark. Then use 95% ethanol to decolorize in a hot water bath at 80℃ for 2h, observe and take pictures. Figure 7 (A) (B) The results showed that the detached leaves of VIGS plants under drought stress were darker blue and brown, indicating that the O 2- and H2O2 accumulation levels were higher than those in the control group, and drought stress enhanced ROS-related oxidative damage in VIGS plants. Figure 7 (C) shows that the T-AOC activity of VIGS plants under drought stress is 0.44 times that of control plants. GhNFD4 gene silenced plants accumulated higher ROS than control plants after drought stress, while T-AOC activity decreased, indicating that silencing the GhNFD4 gene can lead to a decrease in the antioxidant capacity of cotton plants in drought environments.

[0075] All the above indicate that the GhNFD4 gene is positively involved in the drought resistance response of cotton.

Claims

1. An application of cotton GhNFD4 gene in cotton drought resistance, characterized in that: The amino acid sequence of the cotton GhNFD4 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the cotton GhNFD4 gene is shown in SEQ ID NO.

2.

3. Application of a gene expression cassette, expression vector and recombinant bacteria containing cotton GhNFD4 gene in cotton drought resistance, characterized in that: The amino acid sequence of the cotton GhNFD4 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the cotton GhNFD4 gene is shown in SEQ ID NO.

2.

4. An application of cotton GhNFD4 gene in plant drought resistance breeding, characterized in that: The amino acid sequence of the cotton GhNFD4 gene is shown as SEQ ID NO.1, the nucleotide sequence of the cotton GhNFD4 gene is shown as SEQ ID NO.2, and the plant is cotton.

5. The use according to claim 4, characterized in that: By up-regulating the expression level of the GhNFD4 gene in the plant, the drought resistance of the plant is improved.

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