Cotton s-locus protein kinase gene and application thereof in regulating high temperature stress response

Abstract

The application belongs to the technical field of plant genetic engineering, and particularly relates to a cotton S-locus protein kinase gene and application thereof in regulating high-temperature stress response. The application clones a coding sequence of cotton S-locus protein kinase, i.e. a GhHRKs gene, mines important gene resources of cotton, and breaks a bottleneck of cotton breeding. Expression of the GhHRKs gene is induced to change by high-temperature stress, and the GhHRKs gene is involved in high-temperature stress response of a cotton anther. By inhibiting expression of the GhHRKs gene in a plant, the ability of the plant to resist high-temperature stress can be improved, and technical support is provided for breeding of germplasm materials resistant to high-temperature stress.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the cotton S-locus protein kinase gene and its application in regulating the response to high temperature stress. Background Technology

[0002] Plants contain a class of proteases that catalyze the activity of specific proteins; these are called protein kinases. Based on their function and specific structure, they can be broadly classified into receptor-like protein kinases (RLKs), calcium-dependent protein kinases (CDPKs), calmodulin-interacting protein kinases (CIPKs), mitogen-activated protein kinases (MAPKs), and sucrose-non-fermentative protein kinases (SnRKs). The RLK family in plants is primarily composed of serine / threonine kinases, and is extremely diverse. Typical RLK proteins contain an extramembrane ligand domain, a transmembrane domain, and an intramembrane catalytic domain, performing signal transduction and amplification functions. Based on function and genotype phenotype, the RLK family can be further divided into leucine-rich RLKs, S-domain RLKs, and lectin-like RLKs, etc. However, because different types of RLKs often have similar functions, the classification within the family is not absolute. Lectins are proteins that can bind to energy substances such as carbohydrates in organisms to form complexes. Kinases possessing the coding sequence for this type of protein are called lectin-like RLKs, or LecRLKs. Among LecRLKs, there is a class of RLK gene members that participate in the development of plant reproductive organs and specifically regulate self-incompatibility in species such as Brassicaceae, and are called S-locus LecRLKs.

[0003] Currently, S-locus LecRLK has been found to regulate self-incompatibility in a few species, such as Brassicaceae, and existing technologies indicate that the function of S-locus LecRLK has diverged to some extent in other species. Its function in cotton needs to be explored and applied. Summary of the Invention

[0004] The purpose of this invention is to provide the cotton S-locus protein kinase gene GhHRKs and its application. By inhibiting the expression of the GhHRKs gene, the plant's ability to resist high temperature stress can be improved, the cotton gene resource library can be enriched, and technical support can be provided for the breeding of cotton germplasm resistant to high temperature.

[0005] The present invention provides a cotton S-locus protein kinase encoding gene GhHRKs, wherein the GhHRKs gene includes at least one of GhHRK1, GhHRK2, GhHRK3, GhHRK4 and GhHRK5, and the nucleotide sequences of GhHRK1, GhHRK2, GhHRK3, GhHRK4 and GhHRK5 are shown as SEQ ID NO.35-SEQ ID NO.39, respectively.

[0006] The present invention also provides primers for cloning the GhHRKs genes in the above-mentioned technical solutions, including a first primer pair, a second primer pair, a third primer pair, a fourth primer pair and a fifth primer pair for cloning GhHRK1, GhHRK2, GhHRK3, GhHRK4 and GhHRK5;

[0007] The nucleotide sequences of the forward and reverse primers of the first primer pair are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively;

[0008] The nucleotide sequences of the forward and reverse primers of the second primer pair are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively;

[0009] The nucleotide sequences of the forward and reverse primers of the third primer pair are shown in SEQ ID NO.23 and SEQ ID NO.24, respectively;

[0010] The nucleotide sequences of the forward and reverse primers of the fourth primer pair are shown in SEQ ID NO.25 and SEQ ID NO.26, respectively;

[0011] The nucleotide sequences of the forward and reverse primers of the fifth primer pair are shown in SEQ ID NO.27 and SEQ ID NO.28, respectively.

[0012] This invention also provides the application of the GhHRKs gene or the primers described in the above-mentioned technical solutions in regulating the plant's response to high temperature stress.

[0013] Preferably, the regulation of plant high-temperature stress response includes improving the plant's tolerance to high-temperature stress.

[0014] Preferably, the plants include monocotyledonous plants and / or dicotyledonous plants.

[0015] The present invention also provides the application of biomaterials that inhibit the expression of the GhHRKs gene described in the above-mentioned technical solutions in improving the heat stress resistance of plants and / or cultivating heat stress resistant plants.

[0016] Preferably, the biomaterial comprises at least one of the following A1 to A8:

[0017] A1: sgRNA used to knock out GhHRKs genes;

[0018] A2: Nucleic acid molecules obtained by amplification of the sgRNA described in A1;

[0019] A3: A recombinant expression vector containing the nucleic acid molecules described in A2;

[0020] A4: Recombinant microorganisms containing the nucleic acid molecules described in A2;

[0021] A5: Recombinant microorganisms containing the recombinant expression vector described in A3;

[0022] A6: Transgenic plant cell lines containing the nucleic acid molecules described in A2;

[0023] A8: Transgenic plant cell lines containing the recombinant expression vector described in A3.

[0024] Preferably, the nucleotide sequence of the sgRNA used to knock out the GhHRKs gene meets the following four conditions: a) specifically targets one exon of the GhHRKs gene; b) the nucleotide sequence does not contain consecutive A or T bases; c) the GC ratio in the nucleotide sequence is 40-60%; and d) there are fewer than 5 off-target sites on the genome.

[0025] The present invention also provides a biomaterial comprising at least one of the following B1 to B5:

[0026] B1: sgRNA used to knock out GhHRKs genes;

[0027] B2: Nucleic acid molecules obtained by amplification of the sgRNA described in B1;

[0028] B3: A recombinant expression vector containing the nucleic acid molecules described in B2;

[0029] B4: Recombinant microorganisms containing the nucleic acid molecules described in B2;

[0030] B5: Recombinant microorganisms containing the recombinant expression vector described in B3.

[0031] The present invention also provides a method for cultivating heat-resistant plants, comprising: reducing the expression of the GhHRKs gene in the target plant, or reducing the content of the protein encoded by the GhHRKs gene in the target plant, to obtain the heat-resistant plant.

[0032] Beneficial effects:

[0033] This invention provides the cotton S-locus protein kinase encoding gene GhHRKs, which includes at least one of GhHRK1, GhHRK2, GhHRK3, GhHRK4, and GhHRK5. The nucleotide sequences of GhHRK1, GhHRK2, GhHRK3, GhHRK4, and GhHRK5 are shown in SEQ ID NO.35-SEQ ID NO.39, respectively. This invention cloned the cotton S-locus protein kinase encoding gene GhHRKs, uncovering an important genetic resource in cotton and breaking through a bottleneck in cotton breeding. The expression of the GhHRKs gene changes under high-temperature stress and participates in the high-temperature stress response of cotton anthers. By inhibiting the expression of the GhHRKs gene in plants, the ability of the target plant to resist high-temperature stress can be improved, providing technical support for cultivating germplasm materials resistant to high-temperature stress. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0035] Figure 1 Annotation of the protein domains of the Arabidopsis thaliana S-locus LecRLK gene in Example 1;

[0036] Figure 2 The phylogenetic tree of 66 S-locus LecRLKs in the genome of upland cotton, 19 S-locus LecRLKs in Arabidopsis thaliana, and SRKb in Arabidopsis thaliana in Example 1;

[0037] Figure 3 The results of QTL identification for heat resistance in the upland cotton genome in Example 2;

[0038] Figure 4 This is the expression pattern of 10 S-locus LecRLK genes in anthers of upland cotton Jin668 at different developmental stages in Example 2 under normal temperature and high temperature stress conditions.

[0039] Figure 5 This shows the gene model read coverage of the five S-locus LecRLK genes on chromosome A01 in Example 2.

[0040] Figure 6 This shows the gene model read coverage of the five S-locus LecRLK genes on chromosome D01 in Example 2.

[0041] Figure 7This section provides transcript clones of the five S-locus LecRLK genes on chromosome A01 in Example 3, along with their corresponding primer information.

[0042] Figure 8 This section provides transcript clones of the five S-locus LecRLK genes on chromosome D01 in Example 3, along with their corresponding primer information.

[0043] Figure 9 This is the named phylogenetic tree from Example 3;

[0044] Figure 10 The image shows the pYES-DEST52 plasmid vector from Example 4.

[0045] Figure 11 This is a sequence diagram illustrating the key elements of the pYES-DEST52 plasmid in Example 4;

[0046] Figure 12 This is a map of the pYES-GFP plasmid vector from Example 4;

[0047] Figure 13 This is a sequence diagram illustrating the key elements of the pYES-GFP plasmid in Example 4;

[0048] Figure 14 This is a map of the pYES-GhHRKs-GFP plasmid vector from Example 4;

[0049] Figure 15 This is a map of the pYES-GhHRKs-GFP plasmid vector from Example 4;

[0050] Figure 16 This is a growth experiment of different yeast transformants in Example 4 in solid culture media with glucose as carbon source (A) and galactose as carbon source (B);

[0051] Figure 17 The edited progeny of the five GhHRK genes in cotton in Example 4 showed a heat-resistant phenotype. Detailed Implementation

[0052] This invention provides a cotton S-locus protein kinase encoding gene, GhHRKs, which includes at least one of GhHRK1, GhHRK2, GhHRK3, GhHRK4, and GhHRK5. The nucleotide sequences of GhHRK1, GhHRK2, GhHRK3, GhHRK4, and GhHRK5 are shown in SEQ ID NO. 35-SEQ ID NO. 39, respectively. The nucleotide sequences shown in SEQ ID NO. 35-SEQ ID NO. 39 of this invention are illustrated in the examples section and will not be described again.

[0053] This invention also provides primers for cloning the GhHRKs genes in the above-mentioned technical solutions, including a first primer pair, a second primer pair, a third primer pair, a fourth primer pair, and a fifth primer pair for cloning GhHRK1, GhHRK2, GhHRK3, GhHRK4, and GhHRK5; the nucleotide sequences of the forward and reverse primers of the first primer pair are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively; the nucleotide sequences of the forward and reverse primers of the second primer pair are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively; the nucleotide sequences of the forward and reverse primers of the third primer pair are shown in SEQ ID NO.23 and SEQ ID NO.24, respectively; the nucleotide sequences of the forward and reverse primers of the fourth primer pair are shown in SEQ ID NO.25 and SEQ ID NO.26, respectively; and the nucleotide sequences of the forward and reverse primers of the fifth primer pair are shown in SEQ ID NO.27 and SEQ ID NO.28, respectively. The specific nucleotide sequences of the primers described in this invention are shown in the Examples section and will not be repeated here. This invention does not impose any special limitations on the PCR amplification reagents and procedures used in the cloning process; commercially available PCR reagents and corresponding PCR amplification procedures commonly used in the art can be employed.

[0054] This invention also provides the application of the GhHRKs gene or the primers described in the above-mentioned technical solutions in regulating the plant's high-temperature stress response. The regulation of the plant's high-temperature stress response according to this invention preferably includes improving the plant's tolerance to high-temperature stress. The plants mentioned in this invention preferably include monocotyledonous plants and / or dicotyledonous plants, more preferably dicotyledonous plants, and even more preferably cotton.

[0055] This invention also provides the application of biological materials that inhibit the expression of the GhHRKs gene described in the above-mentioned technical solutions in improving the heat stress tolerance of plants and / or in cultivating heat stress-tolerant plants. The biological materials of this invention preferably include at least one of the following A1 to A8: A1: sgRNA for knocking out the GhHRKs gene; A2: nucleic acid molecule amplified from the sgRNA described in A1; A3: a recombinant expression vector containing the nucleic acid molecule described in A2; A4: a recombinant microorganism containing the nucleic acid molecule described in A2; A5: a recombinant microorganism containing the recombinant expression vector described in A3; A6: a transgenic plant cell line containing the nucleic acid molecule described in A2; A8: a transgenic plant cell line containing the recombinant expression vector described in A3. The plants of this invention preferably include monocotyledonous plants and / or dicotyledonous plants, more preferably dicotyledonous plants, and more preferably cotton. The nucleotide sequence of the sgRNA for knocking out the GhHRKs gene described in this invention preferably meets the following four conditions: a) it specifically targets one exon of the GhHRKs gene; b) the nucleotide sequence does not contain consecutive A or T bases; c) the GC ratio in the nucleotide sequence is 40-60%; d) the number of off-target sites on the genome is less than 5. Specifically, the sgRNA for knocking out the GhHRKs gene described in this invention includes sgRNA1, sgRNA2, sgRNA3, sgRNA4, and sgRNA5 for knocking out GhHRK1, GhHRK2, GhHRK3, GhHRK4, and GhHRK5, and the nucleotide sequences of sgRNA1, sgRNA2, sgRNA3, sgRNA4, and sgRNA5 are shown in SEQ ID NO. 55 to SEQ ID NO. 59, respectively.

[0056] The preferred method for preparing the nucleic acid molecule of the present invention includes: adding the nucleotide sequences shown in SEQ ID NO. 52 and SEQ ID NO. 53 to the 5' and 3' ends of sgRNA, respectively, to obtain a single-stranded nucleotide sequence; performing PCR amplification on the single-stranded nucleotide sequence using the primer pair shown in SEQ ID NO. 52 and SEQ ID NO. 54 to obtain a PCR amplification product; sequencing the PCR amplification product to obtain the nucleotide molecule; the nucleotide sequence shown in SEQ ID NO. 54 is the reverse complementary sequence of the nucleotide sequence shown in SEQ ID NO. 53. The present invention does not specifically limit the reagents and procedures for PCR amplification; reagents commonly used in the art for PCR amplification can be used.

[0057] The initial vector for the recombinant expression vector of the present invention preferably includes a CRISPR-Cas9 plasmid vector, and more preferably a pRGEB32-U6-HtKt vector. The recombinant expression vector of the present invention is preferably prepared by introducing the nucleic acid molecule described in the above technical solution into the initial vector. The present invention does not have any particular limitation on the process of introducing the nucleic acid molecule into the initial vector; the conventional recombinant expression vector construction process in the art can be used.

[0058] The initial microorganism in the recombinant microorganisms of the present invention preferably includes Agrobacterium, and more preferably GV3101.

[0059] The transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs described in this invention do not include propagation material. Preferably, the transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs described in this invention are obtained by transferring the recombinant microorganisms into the target plant. This invention does not specify a particular method for transferring the recombinant microorganisms into the target plant; conventional transgenic methods in the art are acceptable.

[0060] This invention also provides a biological material comprising at least one of the following B1 to B4: B1: sgRNA for knocking out the GhHRKs gene; B2: a nucleic acid molecule amplified from the sgRNA described in B1; B3: a recombinant expression vector containing the nucleic acid molecule described in B2; B4: a recombinant microorganism containing the nucleic acid molecule described in B2; and B5: a recombinant microorganism containing the recombinant expression vector described in B3. The defining features of the biological material of this invention have been specified in the above technical solutions and will not be repeated hereafter.

[0061] This invention also provides a method for cultivating heat-stress-tolerant plants, comprising: reducing the expression of the GhHRKs gene in a target plant, or reducing the content of the protein encoded by the GhHRKs gene in the target plant, to obtain the heat-stress-tolerant plant. The plant described in this invention preferably includes monocotyledonous plants and / or dicotyledonous plants, more preferably dicotyledonous plants, and even more preferably cotton. The nucleotide sequence of the GhHRKs gene described in this invention has been described in the above technical solutions and will not be repeated here.

[0062] The heat-resistant plant of this invention is obtained by introducing a nucleic acid molecule that knocks out the GhHRKs gene into the target plant. The sequence characteristics and process of the nucleic acid molecule used to knock out the GhHRKs gene have been defined in the above technical solutions and will not be repeated here.

[0063] The expression of the GhHRKs gene described in this invention changes under high temperature stress and participates in the high temperature stress response of cotton anthers. By inhibiting the expression of the GhHRKs gene in plants, the ability of the target plant to resist high temperature stress can be improved, providing technical support for the cultivation of germplasm materials resistant to high temperature stress.

[0064] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0065] Example 1

[0066] Identification of S-locus protein kinase and its function in response to high temperature stress in the genome of upland cotton

[0067] Since the concept of S-locus LecRLK was first proposed in the type species of Brassicaceae, this embodiment first consults reports on S-locus LecRLK in the type species of Brassicaceae, Arabidopsis thaliana (Nasrallah et al., 2002). Studies show that the S-locus LecRLK gene SRK in the Arabidopsis genome has mutated into a truncated ψSRK genotype, thus losing its ability to control self-incompatibility (Sherman-Broyles et al., 2007). The Arabidopsis lyrata, a closely related species of Arabidopsis, contains a complete and functional S-locus LecRLK gene, SRKb (Strickler et al., 2013). Therefore, in this embodiment, the SRKb protein sequence from Arabidopsis lyrata was obtained and used as a guide sequence for sequence alignment in the Arabidopsis Genome Web Database (https: / / www.arabidopsis.org / ). A total of 20 genes were identified and annotated as potential S-locus LecRLKs. Among them, the protein sequence of the AT1G61665 gene was relatively short, and its length and difference from the protein sequences of the other 19 S-locus LecRLK genes were large, failing to meet the basic conditions for LecRLK, and therefore it was excluded. After protein domain annotation using the SMART web tool, the 19 S-locus LecRLK genes in Arabidopsis showed a lectin domain at the N-terminus and a serine / threonine protein kinase at the C-terminus, meeting the conditions for LecRLK. Figure 1 ).

[0068] Furthermore, in this embodiment, the conserved protein sequences of the lectin and kinase domains of the aforementioned 19 Arabidopsis LecRLK genes were used as queries for alignment in the upland cotton TM-1 genome database using a computer command: blastall-p blastp-m 8-b 10-v 10-e 1e-10-d cotton_db-i query.fa. Using this command, 94 potential S-locus LecRLKs were identified in the upland cotton TM-1 genome. Among them, 28 genes had a short total protein length (<150 amino acids), which did not conform to the normal amino acid composition and length of LecRLKs; therefore, these sequences were excluded, ultimately identifying 66 potential LecRLK genes in upland cotton.

[0069] This embodiment then merges the protein sequences of the S-locus LecRLK gene from the aforementioned 66 cotton genomes, the 19 S-lcous LecRLK genes from Arabidopsis thaliana, and the S-lcous LecRLK gene from Arabidopsis thaliana, and constructs a phylogenetic tree in MEGA11 software. Figure 2 The study identified overlapping branching patterns for 48 cotton S-locus LecRLK genes and 8 Arabidopsis S-locus LecRLK genes, suggesting potential functional similarities among these genes. Furthermore, 18 cotton S-locus LecRLK genes and 11 Arabidopsis S-locus LecRLK genes clustered on independent branching patterns within their respective species, indicating sequence differentiation of S-locus LecRLK between cotton and Arabidopsis. Additionally, SRKb was located on a relatively independent branching pattern, consistent with reports of its specific regulatory function on self-incompatibility. Figure 2 Pink-colored branches).

[0070] Example 2

[0071] Model and expression level analysis of S-locus protein kinase gene in cotton heat resistance QTLs

[0072] In previous studies, three heat-resistant QTLs were identified on chromosomes A01, D01, and D05 of the upland cotton genome. Among the QTL candidate genes, 10 are S-locus LecRLK kinase genes. Figure 3 ; Figure 2 (Blue clade). All 10 S-locus LecRLK genes mentioned above are located on the same phylogenetic clade as the Arabidopsis AT4G27290 gene, indicating that their sequences and functions may be highly similar.

[0073] Firstly, this embodiment utilizes RNA-seq results obtained by our research group at different developmental stages of cotton anthers under both ambient and high temperatures (https: / / www.ncbi.nlm.nih.gov / bioproject / PRJNA1010459 / ). By aligning transcriptome reads to the cotton genome and calculating expression levels to create an expression heatmap, we analyzed the changes in the aforementioned 10 S-locusLecRLKs induced by high temperatures at different developmental stages of cotton anthers. Figure 4 Except for Ghir_A01G006190, which showed no expression under both normal temperature control and high temperature stress, the other nine S-locus LecRLKs were detected at different developmental stages of cotton anthers. Based on the expression patterns, these nine S-locus LecRLKs function in the relatively early stages of anther development, and their expression increased to varying degrees after high temperature stress, indicating that these genes do indeed respond to high temperature stress. Furthermore, in this embodiment, gene models and mRNA coverage data corresponding to the above 10 S-locus LecRLKs were retrieved from the NCBI BioProject database (accession number: PRJNA1010459, https: / / www.ncbi.nlm.nih.gov / bioproject / PRJNA1010459 / ) submitted by our research group, and primers were designed to clone these S-locus LecRLKs.

[0074] During the analysis of gene models and coverage, it was found that some of the gene models of the aforementioned S-locus LecRLK contained gene model annotation errors and abnormal enrichment of mRNA. Figure 5 and Figure 6The Ghir_A01G006170 genome annotation includes three transcripts of different lengths: Ghir_A01G006170.1, Ghir_A01G006170.2, and Ghir_A01G006170.3. mRNA enrichment indicates that the longest transcript, Ghir_A01G006170.3, is the true transcript. Meanwhile, the other two shorter transcripts cannot complete a normal reading, therefore Ghir_A01G006170.1 and Ghir_A01G006170.2 are considered incorrectly annotated. The Ghir_A01G006180 transcript is very long; in the model annotation, the two genes were linked together as Ghir_A01G006180. mRNA enrichment indicates that transcription occurs in the first seven exons, while the transcript abundance in the last seven exons is not significant. In this embodiment, to distinguish the correct gene, Ghir_A01G006180 was split into two genes at the 5' and 3' ends, namely Ghir_A01G006180.1 and Ghir_A01G006180.2 transcripts. The Ghir_A01G006190 gene model is normal, with a small amount of mRNA enriched in the introns of the Ghir_A01G006190.1 transcript, while no mRNA enrichment was found in the other exons, explaining why no expression level of the Ghir_A01G006190 gene was detected. The Ghir_A01G006210 gene model is normal, but a significant amount of mRNA is enriched in the third exon, the first intron, and the second intron, making the probability of producing a normal transcript relatively low. The Ghir_A01G006220 gene model is normal, but the mRNA is mainly enriched in the fourth intron, also making the probability of producing a normal transcript relatively low. The gene model of Ghir_D01G006460 is similar to that of Ghir_A01G006180, with instances where the individual models of the two genes are combined, resulting in four transcripts: Ghir_D01G006460.1, Ghir_D01G006460.2, Ghir_D01G006460.3, and Ghir_D01G006460.4. The mRNA is mainly enriched at the 3' end of the Ghir_D01G006460.4 transcript, indicating a high probability that the 3' end of Ghir_D01G006460.4 is the correct transcript of Ghir_D01G006460. The models Ghir_D01G006470, Ghir_D01G006490, and Ghir_D01G006510 are normal, and the mRNA enrichment is also normal.The Ghir_D01G006520 gene model is similar to the Ghir_A01G006180 and Ghir_D01G006460 gene models, also exhibiting instances where the two gene models are linked together, with mRNA enrichment occurring in the 3' end of the Ghir_D01G006520 gene model. Similarly, in this example, the Ghir_D01G006520 gene model was segmented, corresponding to the 5' end Ghir_D01G006520.1 and the 3' end Ghir_D01G006520.2.

[0075] Based on the above analysis and preliminary correction of the gene model, the corrected sequence obtained in this embodiment will be used for subsequent cloning.

[0076] Example 3

[0077] Cloning of the S-locus protein kinase gene sequence in cotton heat-resistant QTLs

[0078] Based on the gene model analysis results of Example 2, in this example, 17 pairs of primers were first designed (Table 1, SEQ ID NO.1-SEQ ID NO.34) to clone some short transcripts corresponding to different S-locus LecRLKs. In Table 1, the forward and reverse primer sequences of Ghir_A01G006170.1 are numbered as SEQ ID NO.1 and SEQ ID NO.2, respectively, and the forward and reverse primer sequences of Ghir_A01G006170.2 are numbered as SEQ ID NO.3 and SEQ ID NO.4, respectively, and so on.

[0079] Table 1 Primer sequences used for cloning different transcripts

[0080]

[0081]

[0082] To avoid false positives / negatives from transcriptome sequencing and to clone all possible transcripts, thereby improving the cloning success rate, primers were designed under the following conditions:

[0083] 1) Annealing temperature above 58℃; 2) Primer 3' end overlaps with the transcriptome mRNA-enriched region and conforms to the correct reading frame; 3) If mRNA enrichment is not present, design a complete transcript primer containing start / stop codons.

[0084] In this example, using the primer pairs described above, and based on cotton anther cDNA templates (cDNA templates extracted from a mixture of anthers at different developmental stages), PCR amplification was performed on 17 transcripts of 10 S-locusLecRLKs on chromosomes A01 and D01 according to the PCR system in Table 2 and the PCR program in Table 3. The results are as follows: Figure 7 and Figure 8 As shown.

[0085] Table 2 PCR amplification system (Vazyme Phanta DNA polymerase kit#P505-d1)

[0086]

[0087] Table 3 PCR amplification program

[0088]

[0089] Figure 7 The eight lanes of the image represent the amplification results of eight S-locus LecRLK transcripts on chromosome A01. Lanes 2, 3, 4, and 7 showed distinct bands, with a total of six PCR product band patterns. Cloning and sequencing yielded the following results: Lane 2 contained the complete Ghir_A01G006170.2 transcript sequence; Lane 3 contained a smaller PCR product representing the Ghir_A01G006170.2 transcript, with its 3' end being a non-specific annealing sequence from the reverse primer; the larger PCR product fragment did not match any S-locus LecRLK and was therefore disregarded; Lane 4 contained a smaller PCR product fragment representing the complete Ghir_A01G006180.1 transcript sequence; the larger PCR product fragment did not match any S-locus LecRLK and was therefore disregarded; LecRLK sequences could not be matched and were no longer considered. The PCR product fragment in lane 7 was small, and sequencing revealed it to be an overlap extension of intron 3 of Ghir_A01G006210 after annealing with an unknown sequence, not the correct sequence of Ghir_A01G006210. Therefore, the correct transcripts of the two S-locus LecRLK genes, Ghir_A01G006170 and Ghir_A01G006180, were successfully cloned on chromosome A01.

[0090] Figure 8The nine lanes of the image show the amplification results of nine S-locus LecRLK transcripts on chromosome D01. Lanes 3, 4, 5, and 6 showed distinct bands, with a total of five different PCR product band patterns. Cloning and sequencing yielded the following results: the product in lane 3 did not match any of the S-locus LecRLK transcripts; the small fragment PCR product in lane 4 was the complete transcript sequence of Ghir_D01G006460.4, while the large fragment PCR product was similar to the PCR product in lane 3; the PCR product in lane 5 was the complete ORF sequence of Ghir_D01G006470; and the PCR product in lane 6 was the complete ORF sequence of Ghir_D01G006490. Through the above embodiments, the correct transcripts of the three S-locus LecRLK genes, namely Ghir_D01G006460, Ghir_D01G006470 and Ghir_D01G006490, were successfully cloned on chromosome D01.

[0091] In summary, five S-locus LecRLK genes were successfully cloned from cotton anther cDNA using 17 primer pairs. Based on their phylogenetic relationship and heat-related kinase phenotypes, they were named GhHRK1-GhHRK5 (SEQ ID NO.35-SEQ ID NO.39), respectively. Their positions on the phylogenetic tree are as follows: Figure 9 As shown;

[0092]

[0093]

[0094]

[0095]

[0096]

[0097] Example 4

[0098] Verification of cotton S-locus kinase's function in high-temperature stress and creation of materials involved in high-temperature stress response

[0099] In Example 3, five S-locus LecRLK genes expressed in cotton anthers and responsive to high-temperature stress were successfully cloned. In this example, the heat resistance function of these five S-locus LecRLK genes was verified in *Saccharomyces cerevisiae*. In this example, the protein expression rate was controlled by transforming the *Saccharomyces cerevisiae* protein expression strain iNVSc1 with the commercially available galactose-inducible plasmid pYES-DEST52. Figure 10 The specific implementation steps are as follows:

[0100] A) Following the restriction enzyme digestion system in Table 4, pYES-DEST52 was double-digested using Pvu II and Pme I restriction endonucleases to remove the V5 and 6×His protein tags and the T7 promoter sequence that may induce endogenous initiation on the pYES-DEST52 vector, thus exposing the corresponding sticky ends. Figure 11 ).

[0101] B) Using the nucleotide sequences shown in SEQ ID NO.40 and SEQ ID NO.41 as primers, the green fluorescent protein (GFP) gene with the recombination adapter was amplified according to the PCR system in Table 2 and the PCR procedure in Table 3 (Zhao et al., 2023). The enzyme digestion products from A) and the PCR products from B) were mixed according to the recombination system in Table 5 to construct the pYES-GFP vector expressing the empty GFP protein control. Figure 12 , Figure 13 );

[0102] Among them, SEQ ID NO.40: This sequence is the forward primer sequence for cloning the GFP gene fragment with an EcoRI restriction site and homologous recombination sequence at the 5' end. The bolded sequence is consistent with the 5' end sequence of the pYES-DEST52 digestion product; the underlined sequence is the EcoRI restriction site sequence, used to replace the original PvuII restriction site and provide space for the introduction of a new sequence; the italicized sequence is consistent with the 5' end sequence of the GFP gene and is used for cloning the GFP gene.

[0103] SEQ ID NO.41: This sequence is a reverse primer sequence used to clone the GFP gene, which contains a Pme I restriction site and homologous recombination sequence at the 3' end. The bolded sequence is reverse complementary to the 3' end sequence of the pYES-DEST52 digestion product, the underlined sequence is the Pme I restriction site sequence, and the italicized sequence is reverse complementary to the 3' end sequence of the GFP gene, used for cloning the GFP gene.

[0104] C) Digest the pYES-GFP plasmid with EcoRI according to the enzyme digestion system in Table 4.

[0105] D) Using the nucleotide sequences shown in SEQ ID NO.42-SEQ ID NO.51 as primers, the GhHRK gene with the recombination adapter was amplified according to the PCR system in Table 2 and the PCR procedure in Table 3. The enzyme digestion products in C) and the PCR products in D) were mixed according to the recombination system in Table 5 to carry out the recombination reaction, and the pYES-GhHRKs-GFP vector expressing the empty GFP protein control was constructed. Figure 14 , Figure 15 );

[0106] SEQ ID NO.42: GhHRK1-F SEQ ID NO.43: GhHRK1-R

[0107] SEQ ID NO.44: GhHRK2-F SEQ ID NO.45: GhHRK2-R

[0108] SEQ ID NO.46: GhHRK3-F SEQ ID NO.47: GhHRK3-R

[0109] SEQ ID NO.48: GhHRK4-F SeqID49: GhHRK4-R

[0110] SEQ ID NO.50: GhHRK5-F SEQ ID NO.51: GhHRK5-R

[0111] The primers described above were used to amplify the GhHRKs gene with the recombination adapter. All F primers were forward primers; the bolded sequence is the 5' homologous sequence of the pYES-GFP vector after EcoRI digestion, the underlined sequence is the EcoRI restriction site sequence, and the italicized sequence coincides with the 5' end sequence of the GhHRKs gene coding sequence. All R primers were reverse primers; the bolded italicized sequence is inversely complementary to the 3' homologous sequence of the pYES-GFP vector after EcoRI digestion, the underlined sequence is the EcoRI restriction site sequence, and the lowercase sequence is inversely complementary to the 3' end sequence of the GhHRKs gene coding sequence, with the corresponding stop codon removed.

[0112] E) Transform the plasmid into the iNVSc1 yeast strain, select positive single clones, and first propagate them in SC liquid medium with glucose as the carbon source to OD=0.6. Then, according to the dilution ratio of 1×, 10×, 100×, and 1000×, inoculate the bacterial solution into SC solid medium with glucose as the carbon source and galactose as the carbon source respectively for growth experiments at 30℃ control, 37℃ for 3 days, and 37℃ for 3 days followed by recovery for 5 days.

[0113] like Figure 16 As shown, in SC solid medium with glucose as the carbon source, the growth rates of iNVSc1 yeast clones transformed with different plasmids were similar at 30°C; the growth rates after 37°C high-temperature treatment for 3 days and recovery for 5 days were also similar. Since glucose inhibits protein expression in the iNVSc1 strain, there was no significant difference in high-temperature resistance among transformants of different plasmids in the glucose-based carbon source medium. The slower growth of colonies after high-temperature stress only reflects the high-temperature resistance of the iNVSc1 strain itself. Figure 16 (A). In SC solid medium with galactose as the carbon source, the growth rate of transformants converted to the S-locus LecRLK gene was already delayed compared to GFP-expressing transformants at 30℃. The growth rate after treatment at 37℃ and recovery at 30℃ was significantly slower than that of GFP-expressing transformants, indicating that the S-locus LecRLK gene plays a negative regulatory role in high-temperature resistance in yeast. Figure 16 (B)

[0114] Table 4 Plasmid digestion system

[0115]

[0116] Table 5. Homologous recombination reaction system (Vazyme ClonExpress II Cloning Kit#C112-01)

[0117]

[0118] Furthermore, in this embodiment, the CRISPR-Cas9 gene editing technology established in our laboratory (Wang et al., 2018, 10.1111 / pbi.12755, entry vector name pRGEB32-U6-HtKt, both the vector and the technology have been disclosed) was used to create gene-edited mutants corresponding to the above-mentioned five S-locus LecRLK genes in the cotton cultivar Jin668. Figure 17 The main steps are as follows:

[0119] 1. Using the CRISPR-P v2 web tool (http: / / crispr.hzau.edu.cn / CRISPR2 / ), based on the upland cotton genome, design sgRNAs that specifically target the above 5 S-locus LecRLK genes. The above sgRNAs must meet the following conditions: a) specifically target one exon; b) have no consecutive A / T bases; c) have a GC ratio of 40-60%; d) have fewer than 5 off-target sites on the genome. The specific 5 sgRNA sequences are shown in SEQ ID NO.55-SEQ ID NO.59: SEQ ID NO.55: 5'-GTTGCATCAGGAAAGCTGAG-3', SEQ ID NO.56: 5'-CGTTTGGATAGCCAACCGCG-3', SEQ ID NO.57: 5'-CCATCTCTGATAGATCGGCC-3', SEQ ID NO.58: 5'-ACTTTGTCACCGGTTCCGAG-3', SEQ ID NO.59: 5'-ACAAATTTAGATATCCGAGA-3'.

[0120] 2. Add the sequences TTCCCGGCTGGTGCA (SEQ ID NO.52) and GTTTTTAGAGCTAGAA (SEQ ID NO.53) to the 5' and 3' ends of the sgRNA, respectively, to synthesize a 50 bp single-stranded nucleotide. Simultaneously synthesize 15 bp primers 5'-TTCCCGGCTGGTGCA-3' (SEQ ID NO.52) and 5'-TTCTAGCTCTAAAAC-3' (SEQ ID NO.54). Using the PCR system in Table 2 and the PCR program in Table 3, amplify the 50 bp single-stranded sgRNA into a double-stranded DNA sequence.

[0121] 3. Using Bsa I restriction endonuclease, digest the pRGEB32-U6-HtKt vector according to the system in Table 4. After exposing the corresponding sticky ends, use the recombination system in Table 5 to recombine the PCR product from the previous step into the endonuclease-digested pRGEB32-U6-HtKt vector. Heat shock the recombination system into *E. coli* DH5α, select positive single clones for propagation, and extract the plasmid. Electroporate the corresponding plasmid into *Agrobacterium* GV3101, select positive single clones for propagation, and perform *Agrobacterium*-mediated genetic transformation on the hypocotyls of the upland cotton variety Jin668 to create mutant materials.

[0122] Boll setting and pollen staining results of self-pollinated offspring of gene-edited mutants showed that the S-locus LecRLK gene also plays a negative regulatory role in heat resistance in cotton. In summary, it can be confirmed that the above five S-locus LecRLK genes negatively regulate heat resistance, and their functions are broad-spectrum across different species (Ma et al., 2021).

[0123] References:

[0124] Yizan Ma, Ling Min, Junduo Wang, Yaoyao Li, Yuanlong Wu, Qin Hu, YuanhaoDing, Maojun Wang, Yajun Liang, Zhaolong Gong, Sai Xie, Xiaojun Su, Yanlong Li, Huabin Chi, Miao Chen, Aamir Hamid Khan, Keith Lindsey, Longfu Zhu, Xueyuan Li, Xianlong Zhang (2021). A combination of genome-wide and transcriptome-wide association studies reveals genetic elements leading to male sterility during high temperature stress in cotton. New Phytologist 231:165-181.

[0125] Nasrallah ME,Liu P,Nasrallah JB(2002)Generation ofself-incompatibleArabidopsis thaliana by transfer oftwo S locus genes fromA.lyrata.Science297:247-249.

[0126] Sherman-Broyles S,Boggs N,Farkas A,Liu P,Vrebalov J,Nasrallah ME,Nasrallah JB(2007)S locus genes and the evolution ofself-fertilityinArabidopsis thaliana.Plant Cell 19:94-106.

[0127] Strickler SR,Tantikanjana T,Nasrallah JB(2013)Regulation ofthe S-locus receptor kinase and self-incompatibility inArabidopsis thaliana.G3(Bethesda)3:315-322.

[0128] Wang,P.,J.Zhang,L.Sun,Y.Ma,J.Xu,S.Liang,J.Deng,J.Tan,Q.Zhang,L.Tu,H.Daniell,S.Jin&X.Zhang(2018)High efficient multi-sites genome editing inallotetraploid cotton(Gossypium hirsutum)using CRISPR / Cas9system.PlantBiotechnology Journal,16,137-150.

[0129] Zhao R,Li N,Lin Q,Li M,Shen

[0130] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of inhibiting GhHRK5 gene expression in improving the heat stress resistance of cotton; the nucleotide sequence of the GhHRK5 gene is shown in SEQ ID NO.

39.

2. The application according to claim 1, characterized in that, The primers for cloning the GhHRK5 gene include a forward primer and a reverse primer, the nucleotide sequences of which are shown in SEQ ID NO.27 and SEQ ID NO.28, respectively.

3. Application of biomaterials that inhibit GhHRK5 gene expression in improving the heat stress resistance of cotton and / or cultivating heat stress-resistant cotton; the nucleotide sequence of the GhHRK5 gene is shown in SEQ ID NO.39; The biomaterial includes at least one of the following A1 to A6: A1: sgRNA used to knock out the GhHRK5 gene; A2: A recombinant expression vector containing the sgRNA described in A1; A3: Recombinant microorganisms containing the sgRNA described in A1; A4: Recombinant microorganisms containing the recombinant expression vector described in A2; A5: Transgenic plant cell lines containing the sgRNA described in A1; A6: Transgenic plant cell lines containing the recombinant expression vector described in A2.

4. The application according to claim 3, characterized in that, The nucleotide sequence of the sgRNA used to knock out the GhHRK5 gene is shown in SEQ ID NO.

59.

5. A method for cultivating cotton resistant to high-temperature stress, characterized in that, include: The expression of the GhHRK5 gene in the target cotton or the content of the protein encoded by the GhHRK5 gene in the target cotton are reduced to obtain the high-temperature stress resistant cotton; the nucleotide sequence of the GhHRK5 gene is shown in SEQ ID NO.39.

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