CN114920812A - GhERF9 protein related to low potassium stress response and its related biomaterials and applications - Google Patents

Abstract

The invention discloses a GhERF9 protein related to low potassium stress response and its related biological materials and applications. The GhERF9 protein can specifically be the protein of the following A1), A2) or A3): A1) The amino acid sequence is the protein of SEQ ID No. 1 in the sequence listing; A2) The protein of A1) is replaced by amino acid residues and/or The protein obtained by deletion and/or addition has more than 75% identity with the protein shown in A1) and has the activity of regulating low potassium stress; A3) Connect the protein at the N-terminus or/and C-terminus of A1) or A2). Tag the resulting fusion protein. GhERF9 protein and its related biomaterials can be used to regulate the response of plants to low potassium stress.

Description

GhERF9 protein related to low potassium stress response and its related biomaterials and applications

technical field

The present invention relates to a GhERF9 protein related to low potassium stress response in the field of biotechnology and its encoding gene and application.

Background technique

Potassium is the most abundant cation in plant cells, and it is one of a large number of mineral nutrients necessary for plant growth and development. It plays a key role in the growth and development of plants at all stages, and determines the final yield and quality of crops. called the quality element of the crop.

Cotton is an economic crop that likes potassium, and needs a lot of potassium nutrition during its growth and development. Potassium can promote the growth and development of cotton shoots and roots, and increase dry matter accumulation. However, in recent years, with the gradual improvement of cotton multiple cropping index and yield, and the popularization of transgenic insect-resistant cotton, the phenomenon of potassium deficiency and the degree of potassium deficiency in cotton production have become more and more common and serious. In addition, due to unreasonable fertilizer management, potassium deficiency in cotton production is further exacerbated, which often leads to premature aging and seriously affects yield and quality improvement.

Since the 1990s, people have carried out systematic and in-depth research on the molecular genetic mechanism of plant potassium nutritional traits. Numerous potassium channels, potassium transporters and related regulatory proteins in plants have been cloned successively. Their roles in potassium affinity, selectivity and energy coupling are all different. According to the difference in structure and function of potassium transport and channel proteins, they can be divided into three potassium channel families and three potassium transporter families. They are: Shaker potassium channel, TPK potassium channel, Kir-like potassium channel and KUP/HAK/KT potassium transporter, TPK/HKT transporter, CPA cation proton antiporter.

Cotton is an important cash crop and is of great significance to economic development. Potassium deficiency leads to premature senescence of cotton, which seriously affects the improvement of cotton yield and quality. For many years, traditional methods such as conventional breeding have been used to solve the problem of potassium deficiency in cotton varieties, but they are often costly, labor-intensive and inefficient in selection. With the continuous development of molecular biology, it is possible to cultivate low-potassium tolerant cotton varieties by means of biotechnology. Although the current cotton gene cloning work has achieved certain results, the cotton gene cloning work is far behind cotton, corn, wheat and other food crops.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is how to improve plant yield under low potassium conditions.

The present invention provides a protein named GhERF9, which is the following A1), A2) or A3) protein:

A1) the amino acid sequence is the protein of SEQ ID No. 1 in the sequence listing;

A2) The protein of A1) is obtained by substitution and/or deletion and/or addition of amino acid residues, which is more than 75% identical to the protein shown in A1) and has the activity of regulating low potassium stress response;

A3) A fusion protein obtained by linking a protein tag to the N-terminus or/and C-terminus of A1) or A2).

Among them, SEQ ID No.1 consists of 222 amino acid residues.

The above protein can be derived from cotton.

In the above-mentioned proteins, the identity refers to the identity of the amino acid sequence. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as the Matrix, and set the Gap existence cost, Per residue gap cost and Lambda ratio to be respectively 11, 1 and 0.85 (default value) and search for the identity of a pair of amino acid sequences to calculate the identity value (%).

In the above proteins, the identity of more than 75% may be at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical.

Among the above proteins, the protein-tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology, so as to facilitate the expression, detection, tracking and/or purification of the target protein. The protein tags can be Flag tags, His tags, MBP tags, HA tags, myc tags, GST tags and/or SUMO tags, etc. The amino acid sequences of some of the available tags are shown in Table 1.

Table 1 Sequences of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

The above proteins can be artificially synthesized or obtained by first synthesizing their coding genes and then carrying out biological expression.

Biomaterials related to the protein GhERF9 also belong to the protection scope of the present invention.

The biological material related to the protein GhERF9 provided by the present invention is any one of the following B1) to B5):

is any of the following B1) to B5):

B1) a nucleic acid molecule encoding the protein GhERF9;

B2) an expression cassette containing the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2);

B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

B5) Transgenic plant cell line, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the nucleic acid molecule of B1), or transgenic plant cell line, transgenic plant cell line, transgenic plant tissue containing the expression cassette of B2) or transgenic plant organs.

Wherein, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

In the above-mentioned biological material, the nucleic acid molecule in B1) is the gene shown in any of the following b1)-b2):

b1) The coding sequence of the coding strand is the cDNA molecule or DNA molecule of SEQ ID No. 2;

b2) The nucleotide is a DNA molecule of SEQ ID No.2.

In the above-mentioned biological material, the expression cassette (GhERF9 gene expression cassette) described in B2) containing the DNA molecule refers to a DNA molecule capable of expressing GhERF9 in a host cell, and the DNA molecule can not only include a start-up that starts the transcription of the GhERF9 gene , and may also include a terminator that terminates transcription of GhERF9. Further, the expression cassette may also include enhancer sequences. Promoters useful in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: the constitutive promoter 35S of cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiology 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiol acid S-methyl ester)); tomato Protease inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoter (US Pat. No. 5,187,267); tetracycline-inducible promoter (US Pat. No. 5,057,422 ); seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 2007 1 0099169.7)), seed storage protein-specific promoters (for example, promoters of bean protein, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO J. 4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine Synthase terminators (see, eg: Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; ^Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acids Res., 15:9627).

A recombinant vector containing the protein GhERF9-encoding gene or the expression cassette of the protein GhERF9-encoding gene can be constructed by using an existing plant expression vector. The plant expression vector can be a Gateway system vector or a binary Agrobacterium vector, such as pHBT-GFP, pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pGWB18, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb . When using GhERF9 to construct a recombinant vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription initiation nucleotide, such as cauliflower mosaic virus (CAMV) 35S Biotin gene Ubiqutin promoter (pUbi), etc., which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct plant expression vectors, enhancers can also be used, including translation enhancers or transcriptional enhancers Enhancers, these enhancer regions can be ATG initiation codons or contiguous region initiation codons, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The translation control signals and initiation codons can be derived from a wide variety of sources, either natural or synthetic. The translation initiation region can be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding a gene (GUS gene, luciferase gene, luciferase gene) that can be expressed in plants encoding an enzyme that can produce color change or a luminescent compound. Gene, etc.), antibiotic markers with resistance (gentamycin marker, kanamycin marker, etc.) or anti-chemical reagent marker gene (such as herbicide resistance gene) and so on.

In the above biological materials, the recombinant microorganism can be specifically yeast, bacteria, algae and fungi; for example, the bacteria can be Agrobacterium strain GV3101.

The application in any one of the following C1-C2 of the above-mentioned protein or the above-mentioned biological material also belongs to the protection scope of the present invention:

C1) Application in regulating plant response ability under low potassium stress;

C2) Application in the preparation of a product for regulating the response ability of plants under low potassium stress.

In the present invention, the regulation can be up-regulation or enhancement or improvement. The modulation can also be down-regulated or attenuated or decreased.

The present invention also provides a method for improving the response ability of plants under low potassium stress, the method comprises step M, and the step M is to enhance, improve or up-regulate the activity and/or content of the GhERF9 protein in the target plant, or/and, Enhance, increase or up-regulate the expression of the gene encoding the GhERF9 protein to improve the response ability of plants to low potassium stress.

In the above method, the target plant may be a dicotyledonous plant. The dicotyledonous plant can be a Malvaceae plant, further can be a cotton plant, specifically cotton.

In the above method, the response ability under low potassium stress can be specifically expressed as an increase in the chlorophyll content of leaves, an increase in the biomass of order plants, and an increase in the content of potassium ions in various parts of the plant. The biomass can refer to dry weight of shoots, leaf weight, root weight and the like.

In order to solve the above technical problems, the present invention also provides plant reagents, the function of which is to regulate low potassium stress response ability.

The plant reagent provided by the present invention contains the protein or/and the protein-related biological material.

The active ingredient of the above-mentioned plant agent may be the protein or/and the protein-related biological material, the active ingredient of the above-mentioned plant agent may also contain other biological ingredients or/and non-biological components, and other active ingredients of the above-mentioned plant agent. Those skilled in the art can determine the effect of response ability under low potassium stress.

The plant of interest may be a dicotyledonous plant. The dicotyledonous plant can be a Malvaceae plant, further can be a cotton plant, specifically cotton.

The experiment of GhERF9 gene silencing in cotton showed that compared with the control, the transgenic cotton with silenced GhERF9 gene was more sensitive to low potassium stress, and its K ⁺ absorption capacity was significantly reduced, which in turn affected the accumulation of K ⁺ in plants. Shows more severe symptoms of potassium deficiency.

Description of drawings

Figure 1 is a homologous alignment of the protein sequences of cotton GhERF9 and Arabidopsis AtERF9 in Example 1 of the present invention.

Figure 2 shows the results of subcellular localization of cotton GhERF9 in Example 1 of the present invention.

Figure 3 is the tissue-specific expression analysis of cotton GhERF9 gene in Example 1 of the present invention. SCRC22 is "Lu Mianyan No. 22", and CCRI41 is "China Cotton Research Institute 41". In the figure, * represents the significant difference analysis result of P<0.05, ** represents the significant difference analysis result of P<0.01, and *** represents the significant difference analysis result of P<0.001.

Figure 4 is the expression level change of cotton GhERF9 genome under low potassium stress in Example 1 of the present invention. Different lowercase letters represent significant differences between treatments, P<0.05.

Figure 5 shows the detection of silencing efficiency and low potassium stress phenotype of VIGS-GhERF9 plants in Example 2 of the present invention. Panel A of Figure 5 is the silencing detection of GhERF9 and GhHAK5 in VIGS plants, Panel B of Figure 5 is the phenotype photo of the whole plant, and Panel C of Figure 5 is the phenotype photo of the plant's bottom-up leaves. In the figure, * represents the result of significant difference analysis, P<0.05.

Figure 6 shows the physiological indicators of VIGS-GhERF9 plants in Example 2 of the present invention. In the figure, * represents the result of significant difference analysis, P<0.05.

Figure 7 is the measurement of K ⁺ uptake rate of VIGS plants in Example 2 of the present invention. In the figure, * represents the result of significant difference analysis, P<0.05.

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and are not intended to limit the present invention in any way.

Quantitative experiments in the following examples are all set up to repeat the experiments three times, and the results are averaged.

The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The pHBT-GFP vectors in the following examples are all described in the non-patent literature "Bai L, Ma XN, Zhang GZ, Song SF, Zhou L, Gao LJ, Miao YC, Song CP. A receptor-like kinase mediatesammonium homeostasis and is important for the polar growth of root hairs in Arabidopsis. The Plant Cell, 2014, 26(4): 1497-1511.”, which can be obtained by the public from China Agricultural University (i.e. the applicant) to replicate the experiments in this application.

The pTRV1, pTRV2, pTRV2-GhCLA1, pTRV2-GhHAK5 and pTRV2-GFP vectors in the following examples are all described in the non-patent literature "Wang YR, Wang Y, Li B, Xiong CM, Eneji AE, Zhang MC, Li FJ, TianXL, Li ZH. The cotton high-affinity K ⁺ transporter, GhHAK5a, is essential for shoot regulation of K ⁺ uptake in root under potassium deficiency. Plant and Cell Physiology, 2019, 60(4):888-899", public available from China Agricultural University (ie the applicant) to repeat the experiment of this application.

The cotton varieties in the following examples "China Cotton Institute 41" (potassium low-efficiency type variety) and "Lu Mianyan No. 22" (potassium high-efficiency type variety) are described in the non-patent documents "Yang DD, Li FJ, Yi F, Eneji AE, Tian XL, LiZH. Transcriptome analysis unravels key factors involved in response topotassium deficiency and feedback regulation of K ⁺ uptake in cottonroots. International Journal of Molecular Sciences, 2021, 22(6): 3133", public available from China Agricultural University (ie from the applicant) to repeat the experiments of the present application.

The following examples use SPSS 21.0 (SPSS Inc., Chicago, IL, USA) statistical software to process the data, the experimental results are expressed as mean ± standard deviation, using Duncan's multiple comparison (Duncan multiple comparison procedure) test, P < 0.05 (*) indicates a significant difference, P<0.01 (**) indicates a very significant difference.

Example 1. Cloning and localization of GhERF9 protein and its encoding gene

The transcription factors bound to the cotton potassium transporter GhHAK5 promoter were screened from the low potassium stress yeast one-hybrid library, and the protein sequence of Gh_D02G1817 was compared with the cotton database, and the Arabidopsis thaliana protein homologous gene sequence was searched. Sequence alignment results were generated using neighbor joining method to generate phylogenetic tree. The phylogenetic tree showed the highest homology with Arabidopsis AtERF9 (AT5G44210), so the gene was named GhERF9 (see Figure 1 for the protein sequence alignment results). A new protein was obtained from the cotton variety "Lumianyan 22" and named GhERF9. details as follows:

According to the GhERF9 gene sequence obtained from the cotton database, two specific primers were designed, which are F1 and R1:

F1: 5'-ATGGCTCCCCAAGACAAAA-3';

R1: 5'-TCAAGCATCGACTGGAGG-3'.

The leaf RNA of cotton "Lumianyan No. 22" was extracted using the Adlai RNA kit, and the first-strand cDNA was synthesized with the M-MLV reverse transcription kit. The obtained first-strand cDNA was used to amplify the full length of the GhERF9 gene.

20μL PCR reaction system includes template 10xBuffer 2μL, 10mmol/L dNTPs 2μL, MgSO ₄ 1.4μL, cDNA 1.2μL, KOD-Plus enzyme 0.4μL, upper primer 0.3μL, lower primer 0.3μL, ddH ₂ O 12.4μL; PCR amplification program The program was: 94°C, 2 min; 30 cycles of the program were 94°C, 15s; 50°C, 30s; 68°C, 3min; and a final extension at 68°C for 10min.

The PCR products were electrophoresed on a 1% agarose gel. After electrophoresis, the target band was excised under ultraviolet light, and purified with agarose gel DNA recovery kit (purchased from Tiangen Biochemical Technology Co., Ltd.), and the operation steps were referred to the instruction manual of the kit.

The end of the recovered fragment needs to be added with A. The 10 μL reaction system is: 10xBuffer 1 μL, dATP 1 μL, Taq enzyme 0.5 μL, recovered fragment 7.5 μL, and react at 72°C for half an hour to obtain the recovered fragment after adding A. The recovered fragment after adding A was connected to the PMD18-T vector (purchased from TaKaRa company), and the operation was carried out according to the instructions of TaKaRa company. The PCR tube was added in sequence: 4.5 μL of the recovered fragment after adding A, 0.5 μL of PMD18-T, and 5 μL of Solution I. , the total volume was 10 μL; ligated overnight at 16°C.

Take 5 μL of the ligation product, and transform Escherichia coli DH5α into Escherichia coli DH5α by heat shock method (refer to J. Sambrook, et al., translated by Huang Peitang, etc., Molecular Cloning Experiment Guide (Third Edition), Science Press, 2002 Edition). The positive clones were screened on the LB solid plate of L ampicillin, and 5 clones were picked and sequenced (the sequencing work was done by Shanghai Invitrogen Company) to obtain the desired full-length gene cDNA. The sequencing results show that the full-length cDNA sequence of GhERF9 gene is 669bp, and the nucleotide sequence is shown in SEQ ID No. 2, encoding a complete ORF reading frame of 222 amino acids, which encodes the protein GhERF9, and the amino acid sequence of the protein is SEQ ID No. 1 . The coding sequence (CDS) of GhERF9 is SEQ ID No.2.

2. Subcellular localization of GhERF9 protein

The root total RNA of "Lu Mianyan No. 22" was extracted and reverse transcribed into cDNA, and PCR amplification was carried out with F2 and R2 as primers. The sequences of F2 and R2 are as follows:

F2: 5'-CGGGATCCATGGCTCCCCAAGACAAAA-3';

R2: 5'-GAAGGCCTAGCATCGACTGGAGG-3'.

The obtained PCR amplification product was double digested with restriction enzymes BamHI and StuI, and pHBT-GFP vector was double digested with restriction enzymes BamHI and StuI at the same time, and the recovered product was subjected to homologous recombination. The cDNA sequence of GhERF9 was Constructed on the pHBT-GFP vector, the cDNA sequence (SEQ ID No. 2) of GhERF9 was obtained to replace the fragment between the restriction endonuclease BamHI and StuI recognition sites of pHBT-GFP vector (including the recognition site of BamHI and A small fragment including the StuI recognition site), keeping the other sequences of the pHBT-GFP vector unchanged, to obtain a recombinant expression vector of the GhERF9 protein, named pHBT-GFP-GhERF9.

The recombinant expression vector pHBT-GFP-GhERF9 was transiently transformed in Arabidopsis leaf protoplasts. Microscopic observation showed that GhERF9 co-localized with nuclear localization markers (see Figure 2 for localization results), indicating that GhERF9 protein is a nuclear localized protein.

3. Tissue-specific expression of GhERF9 gene

The expression site of GhERF9 gene in cotton varieties "China Cotton Research Institute 41" and "Lu Mianyan No. 22" was analyzed by real-time quantitative PCR: the instrument used for real-time quantitative PCR was ABI 7500Fast (Applied Biosystem), and the primer pairs used were For F3 and R3, the sequence is as follows:

F3: 5'-GCGTGAGTTTCGTGGACCTA-3';

R3: 5'-TGTTGACTCCACGCTCCAC-3'.

The samples used to detect tissue-specific expression were cDNAs obtained by reverse transcription of total RNAs from different parts of cotton at the three-leaf stage under normal nutrient levels. The PCR program was as follows: denaturation at 94°C for 30s; cycle; the relative expression was calculated by the 2- ^ΔΔCt method, and the cotton Actin9 gene was used as a control. The primer pairs used to identify the cotton Actin9 gene were F-Actin9 and R-Actin9, and the sequences were as follows:

F-Actin9: 5'-GCCTTGGACTATGAGCAGGA-3';

R-Actin9: 5'-AAGAGATGGCTGGAAGAGGA-3'.

The relative expression of GhEF9 gene in each part of cotton at the three-leaf stage under normal nutrient levels is shown in Figure 3. GhERF9 is constitutively expressed in cotton plants, and expressed in stems, terminal buds, leaves and roots. Except for stems, GhERF9 is expressed in cotton. The expression levels in the potassium-high-efficiency variety "Lumianyan 22" were higher than those in the potassium-low-efficiency variety "Zhongmiansuo 41".

4. Tissue-specific expression of GhERF9 gene in different potassium treatments

Taking "Lu Mianyan No. 22" as the test material, the seedlings were treated with low potassium stress of 0.03 mM K ⁺ at the three-leaf stage, and the treatment was treated with low potassium Hoagland's nutrient solution (LK, 0.03 mM K ⁺ ) hydroponic culture, and the control was treated with Normal Hoagland's nutrient solution (CK, 2.5mM K ⁺ ) was cultured in water, and the changes of GhERF9 expression levels in roots and leaves were detected by RT-qPCR. The results are shown in Figure 4. The transcription level of GhERF9 in roots was rapidly up-regulated by low potassium stress, and reached a significant level within 3 h of stress (Figure A in Figure 4); the expression level of GhERF9 in leaves was induced to a certain extent (Figure 4). 4, panel B), peaked at 1 d under low potassium stress, indicating that GhERF9 also plays a role in leaves.

Example 2. Phenotype of VIGS silenced plants

1. Construction of VIGS-GhERF9 silencing vector

1. The total RNA of the leaves of the cotton variety "Lu Mianyan 22" was extracted and reverse transcribed into cDNA.

2. Using the cDNA obtained in step 1 as a template, perform PCR amplification with a primer pair composed of F4 and R4 to obtain a PCR amplification product.

F4: 5'-CCG GAATTC TACTTTCGATACGGCTGAGGAAGC-3' (EcoRI recognition site is underlined);

R4: 5'-CGG GGTACC GGCCGCGACCGGGTCA-3' (KpnI recognition site is underlined).

(Please check the primer sequences)

3. The PCR amplification product obtained in step 2 was double digested with restriction enzymes EcoRI and KpnI, and the digested product was recovered.

4. The pTRV2 vector was digested with restriction enzymes EcoRI and KpnI, and the vector backbone was recovered.

5. Connect the enzyme digestion product of step 3 and the vector backbone of step 4 to obtain a restriction endonuclease that replaces the pTRV2 vector with the cDNA fragment of GhERF9 (nucleotide sequence is the 165-467th position of SEQ ID No. 2) The fragment between the recognition sites of the enzymes EcoRI and KpnI (a small fragment including the recognition site of EcoRI and the recognition site of KpnI) keeps other sequences of the pTRV2 vector unchanged, and the resulting recombinant plasmid is named pTRV2-GhERF9.

2. Acquisition of VIGS-GhERF9 silenced plants

1. The recombinant plasmid pTRV2-GhERF9 was introduced into Agrobacterium GV3101 to obtain recombinant Agrobacterium. Then, the recombinant Agrobacterium was suspended with VIGS solution to obtain a bacterial solution with OD _600nm =1.5.

2. The pTRV1 vector was introduced into Agrobacterium GV3101 to obtain recombinant Agrobacterium. Then, the recombinant Agrobacterium was suspended with VIGS solution to obtain a bacterial solution with OD _600nm =1.5.

3. Mix equal volumes of the bacterial solution obtained in step 1 and the bacterial solution obtained in step 2 to obtain mixed solution A.

4. The pTRV2-GhCLA1, pTRV2-GhHAK5 and pTRV2-GFP were respectively introduced into Agrobacterium GV3101 to obtain recombinant Agrobacterium. Then, the recombinant Agrobacterium was suspended with VIGS solution to obtain a bacterial solution with OD _600nm =1.5.

5. The pTRV2-GhCLA1, pTRV2-GhHAK5 and pTRV2-GFP bacterial solutions obtained in step 4 are mixed with equal volumes of the bacterial solutions obtained in step 2, respectively, to obtain mixed solutions B, C and D, respectively.

6. Group injection

Test group (VIGS-GhERF9, namely GhERF9 gene silencing group): Operation on the cotyledon stage "Lu Mianyan 22" plant: inject mixed liquid A on the lower surface of the cotyledon (each plant is injected with two cotyledons, and the injection until full of cotyledons).

Positive control group 1 (VIGS-GhCLA1, namely GhCLA1 gene silencing group): operation on the cotyledon stage "Lu Mianyan 22" plant: inject mixture B on the lower surface of the cotyledons (two cotyledons for each plant were injected , injected until the cotyledons are filled).

Positive control group 2 (VIGS-GhHAK5, namely GhHAK5 gene silencing group): operation on the cotyledon stage "Lu Mianyan 22" plant: inject mixed solution C on the lower surface of the cotyledons (two cotyledons for each plant were injected , injected until the cotyledons are filled).

Negative control group (VIGS-Ctrl): operate on the cotyledon stage "Lu Mianyan No. 22" plant: inject the mixed solution D on the lower surface of the cotyledons (two cotyledons are injected into each plant until the cotyledons are filled) .

Plants in each group were hydroponically cultured in Hoagland's nutrient solution and replaced with new nutrient solution every week.

3. Phenotype of VIGS-GhERF9 silenced plants

Cotton plants with silenced GhERF9 gene were obtained according to the above method, and the plants of positive control group 1 (VIGS-GhCLA1) injected with pTRV2-GhCLA1 bacterial solution showed an albino phenotype after 14 days of injection.

The gene silencing efficiency of the plants in the test group (VIGS-GhERF9) was detected (Panel A of Figure 5). The test group (VIGS-GhERF9) silenced plants, positive control group 2 (VIGS-GhHAK5) silenced plants and negative control group (VIGS-Ctrl) were treated with normal nutrient solution (CK, 2.5mM K ⁺ ) and low potassium nutrient solution ( LK, 0.03mM K ⁺ ) after culturing for 24 days, the phenotype was observed and the dry weight, chlorophyll content and potassium ion content were detected.

Chlorophyll content determination method: Tang DQ, Qian HM, Zhao LX, Huang DF, TangKX. 2005. Transgenic tobacco plants expressing BoRS1 gene from Brassicaoleracea var. acephala show enhanced tolerance to water stress. J. Biosci 30, 647–655. The chlorophyll content of the first leaf (1L), the second leaf (2L), the third leaf (3L) and the fourth leaf (4L) from bottom to top was detected. A sample of 12 plants was taken from each group of plants under each treatment, and the results were averaged.

Test method for dry weight: 80°C (dry to constant weight), weigh. The dry weights of leaves/stems/roots were detected. A sample of 12 plants was taken from each group of plants under each treatment, and the results were averaged.

Potassium content determination method: Xu J, Tian XL, Eneji AE, Li ZH. 2014. Functional characterization of GhAKT1, a novel Shaker-like K ⁺ channel gene involved in K ⁺ uptake from cotton(Gossypium hirsutum).Gene 545,61-71 .. The potassium content of the first leaf (1L), the second leaf (2L), the third leaf (3L), the fourth leaf (4L), the stem (Stem) and the root (Root) from bottom to top was detected. A sample of 12 plants was taken from each group of plants under each treatment, and the results were averaged.

Panel B of Figure 5 is a photo of the whole plant phenotype. Panel C of Figure 5 is a phenotypic photograph of the plant's bottom-up leaves. Under low potassium conditions, the leaves in the experimental group were more yellow than those in the negative control group.

The results of chlorophyll content, potassium ion content and dry weight are shown in Figure 6. Under low potassium conditions, compared with the negative control group (VIGS-Ctrl) plants, the experimental group (VIGS-GhERF9) plants had lower chlorophyll content in leaves, lower shoot dry weight, lower potassium ion content in each part, leaf weight and Root weight is lower.

The above results indicated that the plants in the test group (VIGS-GhERF9, ie GhERF9 gene silenced plants) were more sensitive to low potassium.

4. Root K uptake rate of VIGS-GhERF9 silenced plants

VIGS-GhERF9-silenced plants, VIGS-GhHAK5 ^- silenced plants and control VIGS-Ctrl were grown to the three-leaf stage under the culture conditions with sufficient potassium supply. Cultured for 8 days under the condition of low potassium stress (LK, 0.03mM K ⁺ ). Plants with similar plant sizes were selected for the determination of K ⁺ uptake rate (Fig. 7A). VIGS-GhERF9 plants were all subjected to K ⁺ starvation treatment for 2 days, and then replaced with depletion solution with an initial K ⁺ concentration of 0.08 mM. The K ⁺ content in the depleted solution was measured at 55 min and 115 min of depletion, and converted into K per unit fresh weight and unit time. ⁺ Absorption rate.

The cotton seedlings of VIGS-GhERF9 had a significantly reduced K ⁺ absorption capacity, which in turn affected the K ⁺ accumulation in the plants, showing more severe symptoms of potassium deficiency (see Figure 7).

Cotton is an important cash crop and occupies a very important position in the national economy. However, with the popularization of Bt gene-resistant cotton and the unreasonable fertilizer and water management, the potassium deficiency in cotton has become more and more serious, and cotton is a potassium-loving crop. Seriously affected cotton yield and quality. Solving the problem of potassium deficiency in cotton is of great significance for the improvement of cotton yield and quality. With the continuous development of molecular biology, it is possible to cultivate low-potassium tolerant cotton varieties by means of biotechnology. Although the current cotton gene cloning work has achieved certain results, the cotton gene cloning work is far behind rice, corn, wheat and other food crops. Virus-induced gene silencing (VIGS), as an effective reverse genetics technique, is widely used in the identification of plant genome functions. Virus-induced gene silencing can successfully silence endogenous genes in different parts of the plant. , which provides a practical means for studying gene function in different growth stages. In this study, cotton GhERF9 gene was discovered and cloned, GhERF9 was silenced in cotton by VIGS technology, and the biological function of GhERF9 gene was explored. In response to the regulatory pathway in the process of low potassium stress, study the expression and regulation of genes under low potassium stress at the molecular level, improve the signal transmission and gene expression regulatory network under stress conditions, and further study the response of plants to low potassium stress signals This mechanism lays a good molecular basis for effectively improving plant tolerance to low potassium stress.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experimentation, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. While the invention has been given particular embodiments, it should be understood that the invention can be further modified. In conclusion, in accordance with the principles of the present invention, this application is intended to cover any alterations, uses or improvements of the invention, including changes made using conventional techniques known in the art, departing from the scope disclosed in this application. The application of some of the essential features can be made within the scope of the following appended claims.

sequence listing

<110> China Agricultural University

<120> GhERF9 protein related to low potassium stress response and its related biomaterials and applications

<130> GNCSY220301

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 222

<212> PRT

<213> Upland cotton (Gossypium hirsutum)

<400> 1

Met Ala Pro Gln Asp Lys Asn Ala Ser Lys Ile Leu Lys Lys Ala Asn

1 5 10 15

Val Thr Gly Ser Thr Ser Ser Gln Glu Val His Phe Arg Gly Val Arg

20 25 30

Lys Arg Pro Trp Gly Arg Tyr Ala Ala Glu Ile Arg Asp Pro Gly Lys

35 40 45

Lys Ser Arg Val Trp Leu Gly Thr Phe Asp Thr Ala Glu Glu Ala Ala

50 55 60

Arg Ala Tyr Asp Ala Ala Ala Arg Glu Phe Arg Gly Pro Lys Ala Lys

65 70 75 80

Thr Asn Phe Pro Leu Pro Asp Glu Thr Asn Cys Tyr Lys Gly Gln Asn

85 90 95

Gln Gln Ser Pro Ser Gln Ser Ser Thr Val Glu Glu Ser Gly Ser Pro

100 105 110

Thr Val Glu Arg Gly Val Asn Thr Leu Ser Gly Ala Val Gly Arg Phe

115 120 125

Pro Phe Ala Cys His Gln Gln Leu Ala Leu Gly Gly Gly Val Ala Asn

130 135 140

Gly Gly Ile Ser Gly Val Thr Arg Ser Arg Pro Val Leu Phe Phe Glu

145 150 155 160

Ala Leu Gly Gly Ala Gly Val Val Gly Gln Val Tyr Pro Val Arg Phe

165 170 175

Asp Pro Val Gly Val Gln Leu Gly Met Gly Phe Ala Ser Val Val Arg

180 185 190

Ser Glu Pro Asp Ser Ser Ser Ser Ala Ile His Cys Lys Ala Arg Arg Pro

195 200 205

Gly Leu Ala Leu Asp Leu Asn Leu Pro Pro Pro Val Asp Ala

210 215 220

<210> 2

<211> 669

<212> DNA

<213> Artificial Sequence

<400> 1

atggctcccc aagacaaaaa tgcgagcaaa atcttgaaga aagctaacgt tactggaagt 60

acgagcagcc aagaggtgca tttcagggga gtaaggaaga ggccatgggg taggtacgct 120

gccgaaatca gagatcccgg caagaaaagc cgtgtttggc ttggtacttt cgatacggct 180

gaggaagctg ccagagccta cgacgcggcg gcgcgtgagt ttcgtggacc taaggctaag 240

accaacttcc ctttaccgga tgaaaccaac tgttacaagg gccagaacca gcagagccct 300

agccaaagca gcacggtgga ggaatctgga agcccaacgg tggagcgtgg agtcaacacc 360

ttgtccgggg cagtagggag attccctttc gcgtgccacc agcagctggc tctgggtggt 420

ggagtcgcta atggtgggat tagcggggtg acccggtcgc ggccggttct atttttcgaa 480

gcgttggggg gagctggcgt tgttggtcag gtttatccgg ttcggttcga tccggtggga 540

gtacagttgg gtatgggatt tgcaagtgta gtccgaagtg aaccggactc ttcatcggcc 600

attcattgca aggcaaggag acctggcctt gccctcgatc ttaaccttcc tcctccagtc 660

gatgcttga 669

Claims (8)

1. A protein, characterized in that the protein is the protein of the following A1), A2) or A3): A1) the amino acid sequence is the protein of SEQ ID No. 1 in the sequence listing; A2) The protein of A1) is obtained by substitution and/or deletion and/or addition of amino acid residues, which is more than 75% identical to the protein shown in A1) and has the activity of regulating low potassium stress response; A3) A fusion protein obtained by linking a protein tag to the N-terminus or/and C-terminus of A1) or A2). 2 . The protein according to claim 1 , wherein the protein is derived from cotton. 3 . 3. The biological material related to the protein of claim 1 or 2 is any one of the following B1) to B5): B1) a nucleic acid molecule encoding the protein of claim 1; B2) an expression cassette containing the nucleic acid molecule of B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) Transgenic plant cell line, transgenic plant cell line, transgenic plant tissue or transgenic plant organ containing the nucleic acid molecule of B1), or transgenic plant cell line, transgenic plant cell line, transgenic plant tissue containing the expression cassette of B2) or transgenic plant organs. 4. biological material according to claim 3 is characterized in that: B1) described nucleic acid molecule is the gene shown in any one of following b1)-b2): b1) The coding sequence of the coding strand is the cDNA molecule or DNA molecule of SEQ ID No. 2; b2) The nucleotide is a DNA molecule of SEQ ID No.2. 5. The application of the protein according to claim 1 or 2 in regulating the response ability of plants under low potassium stress. 6. The application of the protein of claim 1 or 2 in the preparation of a product that regulates the response ability of plants under low potassium stress. 7. The application of the biological material of claim 3 or 4 in regulating the response ability of plants under low potassium stress. 8. The application of the biological material of claim 3 or 4 in the preparation of a product for regulating the response ability of plants under low potassium stress.

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