Salt-tolerance gene GhSHOU4 of wild upland cotton strains and its application

Abstract

This invention discloses a salt-tolerance gene GhSHOU4 from wild-type upland cotton and its application. The gene ID of the cotton gene GhSHOU4 is GH_A05G1741, and its nucleotide sequence is shown in Sequence 1. This invention cloned the upland cotton wild-type gene GhSHOU4 from wild-type upland cotton. Subcellular localization results showed that the GhSHOU4 protein is located on the cell membrane. Gene-silenced cotton was obtained using VIGS technology. Phenotypic identification and physiological and biochemical index determination after salt stress treatment showed that the malondialdehyde content of gene-silenced plants was significantly increased, confirming that silencing this gene significantly reduced the cotton's tolerance to salt stress. This invention provides a good gene resource for molecular breeding and genetic improvement of cotton salt tolerance.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to a wild upland cotton germplasm salt-tolerant gene GhSHOU4 and an application thereof. Background Art

[0002] Salt stress has gradually become an important factor restricting the normal growth and development of cotton. Soil salinization seriously affects the yield and quality of cotton. Salt stress can affect the photosynthesis of cotton and the absorption of nutrients, resulting in adverse performances such as senescence and damage of leaves, short plants, delayed and shortened flowering and boll period, reduced fiber quality, etc. It can also cause cotton bolls to fall off, greatly affecting cotton yield. The damage of salt stress to cotton is mainly water stress, ion toxicity and nutritional imbalance, which in turn has an adverse effect on its physiological and biochemical metabolic processes, and ultimately hinders the normal development of the plant. Therefore, the development and utilization of saline-alkali land has become an important means to solve this production problem.

[0003] Wild strains of upland cotton, also known as semi-wild cotton, originated in Central America and mainly include seven geographical strains: broadleaf cotton ( G.hirsutum race latifolium )、Palmier Cotton( G.hirsutum race palmeri )、Spiky cotton( G.hirsutum race punctatum ), Yucatan cotton ( race yucatanese )、Richmond Cotton( G.hirsutum race richmondi )、Molier Cotton( G.hirsutum race morrilli )、Marigalante Cotton( G.hirsutum race marie-galante ). Wild upland cotton strains contain rich genetic variation and good resistance, and have great utilization value. They are important germplasm resources for cotton genetic breeding research. They can grow normally in saline-alkali environments. Mining and utilizing their salt-tolerant genes is an important way to improve cotton's salt tolerance and enhance its salt tolerance.

[0004] Studies have shown that SHOU4 protein and its homologous protein SHOU4L can regulate the transport of cellulose synthase complex to the plasma membrane and negatively regulate cellulose synthesis by inhibiting the exocytosis of CESA. However, the function of SHOU4 in abiotic stress has not been clearly studied. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention studies and explores the function of the GhSHOU4 gene, provides a good gene resource for cotton salt-tolerant molecular breeding, expands the application scope of the gene GhSHOU4, and provides a new application of a wild cotton salt-tolerant gene GhSHOU4 in improving plant salt tolerance. The gene ID of the cotton gene GhSHOU4 is GH_A05G1741, and the full length of the CDS of the gene is 1317 bp.

[0006] The technical solution of the present invention is:

[0007] The present invention provides a salt-tolerant gene GhSHOU4, and the CDS sequence of the GhSHOU4 is shown in Sequence 1.

[0008] The salt tolerance gene GhSHOU4 comes from cotton, which is the wild upland cotton line Gossypium hirsutum 85.

[0009] The invention also provides a protein encoded by the salt-tolerant gene.

[0010] The present invention also provides a primer pair for amplifying the above-mentioned salt-tolerant gene GhSHOU4, characterized in that the primer pair comprises an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in Sequence 3; the nucleotide sequence of the downstream primer is shown in Sequence 4.

[0011] The present invention also provides the use of the salt-tolerant gene GhSHOU4 described in the above technical solution or the primer pair described in the above technical solution in preparing one or more of a recombinant expression vector, an expression cassette, a transformant and a recombinant bacterium.

[0012] The present invention also provides a recombinant expression vector, which comprises the salt-tolerant gene GhSHOU4 fragment according to claim 1 and a basic vector.

[0013] The present invention also provides a transformant of the above recombinant expression vector, wherein the host bacteria of the transformant is Agrobacterium GV3101.

[0014] The present invention also provides the use of the salt-tolerant gene GhSHOU4 described in the above technical solution or the protein described in the above technical solution in improving the salt tolerance of plants.

[0015] Preferably, the plant is cotton.

[0016] Preferably, the tolerance of cotton to salt stress is reduced by silencing the salt tolerance gene GhSHOU4.

[0017] The advantages and positive effects of the present invention are:

[0018] The present invention provides a salt-tolerant gene GhSHOU4, whose CDS sequence is shown in Sequence 1. The present invention proves that GhSHOU4 is located on the cytoplasmic membrane through a tobacco transient transformation experiment. Gene-silenced cotton is obtained by using VIGS technology. After salt stress treatment, physiological and biochemical indicators are measured. The malondialdehyde content of the gene-silenced plant is significantly increased, which confirms that the tolerance of cotton under salt stress is significantly reduced after silencing the gene. The above results show that GhSHOU4 plays an important role in the salt tolerance of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only illustrate the embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is the subcellular localization result of GhSHOU4 provided in the embodiment of the present invention;

[0021] Figure 2 TRV: GhSHOU4 silenced cotton plants show albino phenotype and gene expression level provided in the embodiments of the present invention;

[0022] Figure 3 The phenotypic results of TRV:GhSHOU4 silenced plants subjected to salt stress provided by the embodiments of the present invention;

[0023] Figure 4 These are the results of measuring physiological and biochemical indicators of TRV:GhSHOU4 silenced plants subjected to salt stress, as provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0024] Unless otherwise specified, the methods used in the following examples are all conventional methods.

[0025] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0026] Example 1 Cloning of cotton GhSHOU4 gene

[0027] Total RNA was extracted from leaf tissue of wild upland cotton line Marigarante 85 (preserved by the Wild Cotton Research Group of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences). After gDNA was digested with DNase I, the total RNA was reverse transcribed into first-strand cDNA using reverse transcriptase to obtain cDNA of leaf tissue of Marigarante 85.

[0028] Using this cDNA as a template, the specific primers (sequence 3 and sequence 4):

[0029] GFP_GhSHOU4_PF: 5'-GAGAACACGGGGGAC TCTAGA ATGGGTTCACGGGTTCAGTCT -3' (underlined is the recognition site of restriction endonuclease XbaI) GFP_GhSHOU4_PR: 5'-TCTCCTTTACCCATG TTAATTAACACTGGGACAACATCATCATTCTG-3' (the underline is the recognition site of the restriction endonuclease PacI) was used as a primer for PCR amplification to obtain a PCR amplification product of about 1317 bp, and fragment 1 was recovered; the plant expression vector pCAMBIA2300-35S-eGFP was double-digested with restriction endonucleases Xba I and Pac I, and the vector backbone 2 of about 11k was recovered, and the fragment 1 and the vector backbone 2 were connected using a homologous recombinase to obtain a recombinant plasmid pCAMBIA2300-35S: GhSHOU4-eGFP; the recombinant plasmid was sequenced, and the sequencing results showed that the recombinant plasmid contained the DNA molecule shown in sequence 1 in the sequence table (hereinafter named GhSHOU4 gene).

[0030] Sequence 1:

[0031]

[0032] Sequence 2:

[0033] MGSRVQSHQLSNGLIVSGRPEQLKERQPSMPLRAVPYTGGNVKKSGELGKIFDIPVLDHSPSSAPPSNPNSNPNSKQQPQLLQPSRFSSSSQPNSGSMRSGSNSGPIRK SSGTMPLQPTGLITSGPLSSGSPSRSGQLGQAGQSGAVSGKAGYGPAVTSLGEGVGFGFRLSKAVVWVVMVVVAMGLLVGAFLMVAIKKMVVLGAVGALVAPMGLGLVWNC IWGRKGLFGFVKMYPDAELRGAVDGQYIKVTGVVTCGSIPLESSYQREPRCVYVSTELYEYKGWGGKSANSKQRCFSWGCKHSEKFVADFYISDFQSGLRALVKAGYGSK VAPMIKPATVLDVTKENRDSFPSLLRWLAERNLSNDDCIMRLKEGCIKEGSTVSVMGVVRRHDNVLMIVPPSEPISTGCQWSRCLLPTYVEGLILTCDDTQNDDVVPV*.

[0034] Example 2 Instantaneous transformation and fluorescence observation of tobacco leaves

[0035] The recombinant vector pCAMBIA2300-35S: GhSHOU4-eGFP obtained in Example 1 was transformed into Agrobacterium GV3101 competent cells for subcellular localization observation, and the steps were as follows: (1) Bacterial liquid activation: The Agrobacterium bacterial liquid containing the target plasmid was added to 5 mL LB liquid culture medium containing 50 ng / μL rifampicin and 50 ng / μL kanamycin at a ratio of 1:1000, and cultured at 28°C, 200 rpm, and shaken for 24 to 36 h until the OD 600Reach 0.8~1.0. (2) Prepare resuspension: Add the activated bacterial solution to 50 mL LB liquid culture medium (containing rifampicin and kanamycin) at a ratio of 1:100, culture at 28℃, 200r / min shaking for 6h, then centrifuge at 5000r / min for 10min to collect the bacteria. Resuspend the bacteria in a resuspension containing 100μM AS to make OD600 reach 1.0. (3) Infection: Use a 1mL sterile syringe to draw up the resuspended bacterial solution and fill the entire tobacco leaf with the resuspended solution. Use the bacterial solution containing the 35S: eGFP empty vector as a positive control. The infected tobacco plants are cultured at 28℃, 16h light / 8h dark for 24h. (4) Observation: Take a 0.5cm 2 After preparing slices of tobacco leaves of different sizes, the expression position of the target gene in tobacco mesophyll cells was observed under a laser confocal microscope (Leica, Germany). The subcellular localization results showed that GhSHOU4 was located on the plasma membrane, such as Figure 1 shown.

[0036] Example 3 VIGS verification of the role of GhSHOU4 in cotton salt tolerance

[0037] Using the cDNA of Marigold 85 as a template, specific primers were used for PCR amplification to construct the VIGS gene silencing vector, using the primer pair (sequence 5 and sequence 6):

[0038] VIGS_SHOU4_PF:GTGAGTAAGGTTACC GAATTC CAACCCTCACGCTTTTCCTCT (the underline is the recognition site of restriction endonuclease EcoR I)

[0039] VIGS_SHOU4_PR:TCCCCATGGAGGCCT TCTAGA TCTAAACCCAAATCCCACCCC (the underline is the recognition site of restriction endonuclease Xba I)

[0040] The VIGS fragment of the GhSHOU4 gene was amplified, and the VIGS fragment was connected to the TRV vector double-digested with EcoR I and Xba I using homologous recombinase. The construct was verified to be correct by sequencing, and the TRV:GhSHOU4 gene silencing vector was successfully constructed. The gene silencing vector was transferred into Agrobacterium GV3101 competent cells, and TRV:00 Agrobacterium was used as a negative control and TRV: PDS Agrobacterium was used as a positive control for VIGS injection.

[0041] The wild strain of upland cotton, Marigalande 85, was used as the VIGS injection material. After germination of cotton seeds for 4 days using the filter paper roll method, cotton seedlings of uniform size were selected and transferred to hydroponic boxes and placed in a greenhouse at 28 °C, 16h light / 8h dark conditions for growth. When the two cotyledons of the seedlings were flat, VIGS injection was performed, the back of the cotyledons was gently scratched with a syringe needle, and the bacterial solution was injected into the entire cotyledon. After the injection, the plants were cultured in the dark for 24 hours, and then transferred to normal conditions for culture. Salt stress treatment was performed after the plants grew three true leaves.

[0042] Example 4 Phenotypic Identification of Gene Silenced Cotton under Salt Stress

[0043] The TRV:00 empty-loaded plants and TRV:GhSHOU4 gene silenced plants at the three-leaf and one-heart stage were subjected to salt stress (250mM NaCl) and the phenotypes were observed 48h after salt treatment. The results showed that compared with the TRV:00 empty-loaded plants, the TRV:GhSHOU4 gene silenced plants had more severe leaf curling and wilting, and their tolerance to salt stress decreased. Figure 2 and Figure 3 shown.

[0044] Example 5 Determination of physiological and biochemical indicators of gene-silenced cotton under salt stress

[0045] Leaf samples were collected from TRV:00 empty-loaded plants and TRV:GhSHOU4 gene-silenced plants at 0h and 48h after salt treatment, and the leaf relative water content (RLWL) and ion permeability (IL) of TRV:00 and TRV:GhSHOU4 plants were measured. The superoxide dismutase (SOD) kit (Solabo) was used to measure the SOD enzyme activity, and the malondialdehyde (MDA) content determination kit (Solabo) was used to measure the MDA content. The determination was performed according to the specific steps in the instructions of the kit. The experimental results showed that the relative leaf water content of TRV: GhSHOU4 gene-silenced plants was significantly reduced, while the ion permeability was significantly higher than that of TRV:00 control plants; the SOD enzyme activity of TRV:GhSHOU4 gene-silenced plants was significantly reduced, while the MDA content was significantly increased. Figure 4 shown.

[0046] The present invention cloned the wild lineage gene GhSHOU4 of upland cotton from the wild lineage of upland cotton, and the subcellular localization results showed that the GhSHOU4 protein was localized on the cytoplasmic membrane. The present invention compared the tolerance of TRV:00 empty-loaded plants and TRV:GhSHOU4 gene-silenced plants to salt stress, and measured their physiological and biochemical indicators after salt stress treatment. The experimental results showed that the relative leaf water content of the gene-silenced plants was significantly reduced, while the ion permeability was significantly higher than that of the TRV:00 control plants; the SOD enzyme activity of the TRV:GhSHOU4 gene-silenced plants was significantly reduced, and the malondialdehyde content of the TRV:GhSHOU4 gene-silenced plants was significantly increased, confirming that the tolerance of the gene-silenced plants under salt stress was significantly reduced. It shows that the GhSHOU4 gene plays an enhancing role in plant salt stress.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of silencing salt-tolerance gene GhSHOU4 in reducing tolerance of plants to salt stress, characterized in that: The plant is cotton, and the CDS sequence of the salt-tolerant gene GhSHOU4 is shown in Sequence 1.

2. The use according to claim 1, characterized in that The amino acid sequence of the protein encoded by the salt-tolerant gene GhSHOU4 is shown in Sequence 2.

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