Cotton GhbZIP18 gene related to oil synthesis and application of cotton GhbZIP18 gene

Abstract

The invention discloses a cotton GhbZIP18 gene related to oil synthesis and application of the cotton GhbZIP18 gene, and belongs to the technical field of plant genetic engineering. The GhbZIP18 gene comprises a pair of homologous genes GhbZIP18D04 and GhbZIP18A05, and the nucleotide sequences of the homologous genes GhbZIP18D04 and GhbZIP18A05 are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 3. Transcriptome analysis shows that the gene pair maintains a relatively high expression level at the early development stage of cotton seeds, namely 4-26 days of flowering; physical evolution analysis proves that the gene is closest to Arabidopsis thaliana AtbZIP18 gene genetic relationship and is a positive regulatory factor for cottonseed grease synthesis. According to the application, the significant correlation between the expression level of the GhbZIP18 gene and the oil content and the fatty acid component of the cottonseed is determined by further analyzing the agronomic and grease traits of the GhbZIP18 gene edited cotton strain. According to the established auxiliary identification method, excellent cotton strains with high oil content or specific components can be rapidly screened out only by detecting the expression quantity of the GhbZIP18 gene, and an efficient means is provided for cultivating special cotton varieties meeting differentiated market requirements.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the cotton GhbZIP18 gene related to oil synthesis and its application. Background Technology

[0002] Cotton (Gossypium spp.), the world's fifth largest oilseed crop, is not only an excellent source of renewable fiber but also an important source of vegetable oil and protein feed. Although cottonseed is a byproduct of cotton processing, it is incredibly valuable, with every part playing a vital role from oil extraction and protein processing to fiber utilization and hull applications. Currently, my country's annual consumption of edible vegetable oil exceeds 45 million tons, with soybean oil import dependence exceeding 80%. Addressing this critical bottleneck, diversifying oilseed sources is crucial.

[0003] In long-term cotton breeding work, research has focused primarily on improving fiber quality and yield, while relatively little attention has been paid to traits such as cottonseed size, fatty acid content, and protein content. This has led to a decline in cottonseed quality despite continuous increases in fiber yield. Cottonseed oil, as a high-quality vegetable oil, can further expand its market value even with minor improvements in its composition. Studies show that the kernel of hulled cottonseed contains approximately 30%-40% oil, and cottonseed oil is composed of 65-70% unsaturated fatty acids and 26-35% saturated fatty acids. Among the unsaturated fatty acids, linoleic acid accounts for the majority (55%), followed by oleic acid (15%) and linolenic acid (less than 1%). Saturated fatty acids include palmitic acid (26%) and stearic acid (2%). Cottonseed oil does not contain harmful trans fatty acids, has high nutritional value, and contains unsaturated fatty acids that are very beneficial to human cardiovascular health, making it an important source of edible oil. Furthermore, vegetable-based blended oils for frying, formulated with cottonseed oil as the base oil, have extended their frying life, improved stability by 37%, and exhibited superior sensory effects during frying, making them a promising new type of frying blended oil. Palmitic acid in cottonseed oil is a saturated higher fatty acid that can be used to produce chemical reagents, precipitating agents, and waterproofing agents. Stearic acid can be used as a solvent for oil-soluble pigments, a polishing agent for waxed paper, and an emulsifier. However, existing cotton varieties generally suffer from low oil content, hindering the extension of the deep processing industry chain. Therefore, increasing the oil content of cottonseed and improving the fatty acid composition of cottonseed are important goals in cotton breeding.

[0004] The biosynthesis and accumulation of plant oils is a highly conserved process, which can be divided into four continuous and highly coordinated stages: the conversion of sucrose to pyruvate, the de novo synthesis of fatty acids (FA), the synthesis of triacylglycerols (TAG) in the endoplasmic reticulum, and the assembly of oil bodies. TAG is the main storage form of cottonseed oil, consisting of glycerol esters formed from glycerol and three fatty acid molecules under the catalysis of various enzymes. Its biosynthetic pathway is synergistically regulated by a series of functional genes. Therefore, systematically studying the key genes and transcription factors in the cottonseed fatty acid synthesis pathway is of great significance for improving the fatty acid composition of cottonseed and increasing its oil content. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide the cotton GhbZIP18 gene related to oil synthesis and its applications.

[0006] This application provides the cotton GhbZIP18 gene related to oil synthesis, the nucleotide sequence of which is shown in SEQ ID NO:1 or SEQ ID NO:3.

[0007] This application found that a pair of homologous genes of the cotton GhbZIP18 gene are GhbZIP18_D04 and GhbZIP18_A05, located on chromosome 4 of the D subgenome and chromosome 5 of the A subgenome of upland cotton, respectively. The nucleotide sequence of the GhbZIP18_D04 gene is shown in SEQ ID NO:1, and the nucleotide sequence of the GhbZIP18_A05 gene is shown in SEQ ID NO:3.

[0008] This application provides a cotton GhbZIP18 protein related to oil synthesis, the amino acid sequence of which is shown in SEQ ID NO:2 or SEQ ID NO:4.

[0009] That is, the amino acid sequence of the GhbZIP18_D04 protein is shown in SEQ ID NO:2, and the amino acid sequence of the GhbZIP18_A05 protein is shown in SEQ ID NO:4.

[0010] This application provides the use of the cotton GhbZIP18 gene and protein related to oil synthesis in any of the following: (a1) Application in regulating cotton plant height; (a2) Application in regulating cottonseed development; (a3) Application in regulating the oil content of cotton and cottonseed; (a4) Application in regulating the oil composition of cotton and cottonseed.

[0011] This application provides a specific primer pair for detecting the expression level of the cotton GhbZIP18 gene, wherein the nucleotide sequence of the primer pair is selected from one or more of the following groups: (1) The forward primer shown in SEQ ID NO: 7 and the reverse primer shown in SEQ ID NO: 8; (2) The forward primer shown in SEQ ID NO: 9 and the reverse primer shown in SEQ ID NO: 10.

[0012] This application provides a detection kit comprising the specific primer pairs described above.

[0013] Preferably, the detection kit further comprises PCR amplification buffer, reverse transcriptase, DNA polymerase, dNTPs and / or fluorescent dye.

[0014] More preferably, the test kit also includes instructions for use.

[0015] This application provides reagents for detecting the cotton GhbZIP18 gene related to oil synthesis, reagents for detecting the cotton GhbZIP18 protein related to oil synthesis, the specific primer pairs described above, or the detection kits described above, in any of the following applications, including: (b1) To assist in the screening or identification of cotton germplasm materials with high oil content; (b2) To assist in the screening or identification of cotton germplasm materials with high palmitic acid (C16:0), high oleic acid (C18:1) and / or high linoleic acid (C18:2) content; (b3) To assist in the screening or identification of cotton germplasm materials with large-seed cotton; (b4) To assist in the screening or identification of tall cotton germplasm materials; (b5) Molecular identification or fingerprinting of cotton varieties.

[0016] This application provides a method for regulating the oil content in cotton seeds, comprising the following steps: By altering the expression levels of the GhbZIP18 gene or GhbZIP18 protein in cotton, cotton germplasm materials with target oil content can be obtained. The GhbZIP18 gene is as described above, or the GhbZIP18 protein is as described above.

[0017] Preferably, the target oil includes at least one of palmitic acid, oleic acid, and linoleic acid.

[0018] This application provides a method for assisting in the identification of cotton varieties with high oil content, comprising the following steps: S1. RNA extraction and reverse transcription: Total RNA was extracted from the ovules of the cotton plants 4-45 days after flowering and reverse transcribed to synthesize cDNA. S2. Real-time quantitative PCR amplification: Using the cDNA as a template, real-time quantitative PCR (qRT-PCR) amplification was performed using the specific primer pairs and internal reference gene primer pairs described above. S3. Expression level analysis: Based on the amplification cycle number (Ct value) in S2, the expression level of the GhbZIP18 gene was calculated by normalization with the internal reference gene as the standard. S4. Result determination: Compare the relative expression level of the GhbZIP18 gene obtained in step S3 with the preset threshold, low oil content control, or population average level; if the relative expression level of the GhbZIP18 gene in the cotton to be tested is significantly higher than the control or population average level, then the cotton to be tested is determined to be a high oil content cotton variety or a cotton germplasm resource with high oil content potential.

[0019] Preferably, the internal reference gene is the cotton UBQ7 gene, and the primer pair for the internal reference gene includes the forward primer shown in SEQ ID NO: 5 and the reverse primer shown in SEQ ID NO: 6.

[0020] This application provides a CRISPR / Cas9 gene editing vector for editing the GhbZIP18 gene, wherein the gene editing vector contains a nucleotide sequence encoding a Cas9 protein and a single guide RNA encoding sequence specifically targeting the GhbZIP18 gene.

[0021] Preferably, the unidirectional guide RNA coding sequence is as shown in SEQ ID NO: 11 and / or SEQ ID NO: 12.

[0022] In summary, compared with the prior art, this application achieves the following technical effects: This study found that the expression level of the GhbZIP18 gene was high in the early stage of seed development (4-26DPA) of the upland cotton standard genetic reference line TM-1, indicating that the gene plays an important regulatory role in the early stage of seed development.

[0023] Technicians in this industry can obtain cotton varieties with different oil compositions by adjusting the transcription level or protein level of the GhbZIP18 gene in cotton, thereby meeting the market demand for functional or specialized cottonseed products.

[0024] This application provides an auxiliary identification method for high-oil-content cotton varieties, offering significant advantages in early prediction and breeding efficiency. Breeders only need to detect the expression level of the GhbZIP18 gene in the ovules during the early developmental stage (within 45 days after flowering) to accurately and quickly predict the oil content potential of mature cotton seeds. This technological breakthrough allows for a significant advancement in the screening of cotton varieties targeting high oil content traits, helping breeders to promptly eliminate low-oil-content plants during the field growing season. This avoids the later management and propagation of ineffective lines, thus significantly shortening the breeding cycle for new high-oil-content cotton varieties, reducing breeding costs, and accelerating the commercialization of functional cotton varieties that meet specific market demands. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Expression patterns of GhbZIP18_D04 and GhbZIP18_A05 in ovules of upland cotton from day 4 to 40. Figure 1 A is a scatter plot based on principal component analysis (PCA); Figure 1 B is a heatmap of the expression of GhbZIP18_D04 (GH_D04G0139) and GhbZIP18_A05 (GH_A05G4237) in ovules from day 4 to 40.

[0027] Figure 2 Phylogenetic tree of the GhbZIP18 gene.

[0028] Figure 3 To verify the relative expression level of the GhbZIP18 gene in high-oil and low-oil cotton varieties. Figure 3 A represents the relative expression level of GhbZIP18_D04 in high-oil and low-oil cotton varieties; Figure 3 B represents the relative expression level of GhbZIP18_A05 in high-oil and low-oil cotton varieties.

[0029] Figure 4 PCR detection results for T0 generation plants of GhbZIP18 gene editing.

[0030] Figure 5 Analysis of the GhbZIP18 gene editing type.

[0031] Figure 6Functional validation of T1 generation plants with GhbZIP18 gene editing. Figure 6 A is a comparison of cotton plants of Jin668, Ghbzip18-1 and Ghbzip18-2; Figure 6 B represents the measurement results of cotton plant height for Jin668, Ghbzip18-1, and Ghbzip18-2 cotton varieties. Figure 6 C represents the measurement results of single boll weight of cotton using Jin668, Ghbzip18-1, and Ghbzip18-2 cotton varieties. Figure 6 D represents the measurement results of the number of cotton seeds per boll in Jin668, Ghbzip18-1, and Ghbzip18-2 cotton varieties.

[0032] Figure 7 To validate the function of cottonseed in T1 generation plants with GhbZIP18 gene editing. Figure 7 A is a phenotypic diagram of the length and width of cottonseeds from Jin668, Ghbzip18-1, and Ghbzip18-2; Figure 7 B represents the measured 100-seed weight of cotton seeds from Jin668, Ghbzip18-1, and Ghbzip18-2. Figure 7 C represents the measurement results of cottonseed length using Jin668, Ghbzip18-1, and Ghbzip18-2 cottonseeds. Figure 7 D represents the measurement results of cottonseed width using Jin668, Ghbzip18-1, and Ghbzip18-2 methods.

[0033] Figure 8 Determination of cottonseed oil content in T1 generation plants of GhbZIP18 gene editing. Figure 8 A represents the results of the determination of crude protein content in cottonseed from Jin668, Ghbzip18-1, and Ghbzip18-2. Figure 8 B represents the results of the determination of crude fat content in cottonseeds from Jin668, Ghbzip18-1, and Ghbzip18-2. Figure 8 C represents the determination results of the fatty acid component content of cottonseed from Jin668, Ghbzip18-1, and Ghbzip18-2. Detailed Implementation

[0034] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0035] The cotton varieties used in the embodiments of this application include: The standard genetic reference line for upland cotton is "TM-1," the high-oil cotton material is "Jiangsu Cotton No. 2," the low-oil cotton material is "Junmian No. 1," and the cotton variety used for genetic modification is "Jin668." All of the above cotton varieties are commercially available products.

[0036] Example 1: Identification of highly expressed genes during the early stage of lipid synthesis (4-26 DPA) Ovule RNA was extracted from the standard genetic reference line TM-1 of upland cotton on days 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40 after flowering. The extraction process was strictly carried out in accordance with the instructions of the centrifuged column-type polysaccharide and polyphenol plant total RNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd.

[0037] After RNA extraction, the concentration and purity of the extracted RNA samples were determined using a Nanodrop ND-2000 micro-spectrophotometer (assessed by A260 / A280 and A260 / A230 ratios). Subsequently, 1.2% agarose gel electrophoresis was performed at 120 V for 15 minutes to check RNA integrity. The electrophoresis results, observed under UV light, showed two clear rRNA bands, representing 28S and 18S rRNA respectively, with a brightness ratio of approximately 2:1, indicating good RNA integrity. RNA samples that passed quality testing were stored at -80℃ for subsequent experiments.

[0038] cDNA libraries were constructed using 3 μg of high-quality RNA, and library preparation was performed according to the manufacturer's instructions for the Illumina TruSeq Stranded RNA Kit (Illumina, California, USA). All libraries were sequenced by Novogene (Tianjin, China) using the Illumina HiSeq sequencing system.

[0039] The raw sequencing data obtained from the Illumina HiSeq sequencing platform underwent preliminary quality assessment. FastQC and TrimGalore software were used to perform quality checks, adapter sequence removal, and low-quality fragment filtering on the transcriptome sequencing data. Hisat2 was used to align the filtered sequencing sequences to the upland cotton reference genome, and FeatureCounts was used to quantify gene expression levels. After standardizing the generated expression matrix, a sample clustering tree was constructed based on hierarchical clustering. In the clustering tree, if the distance between a specific sample and other samples in the same group (same cotton species, same developmental stage) was significantly greater than the average distance between samples within the group, the sample was identified as a potential outlier. Principal component analysis (PCA) was further used to perform dimensionality reduction analysis on the standardized expression matrix, and the PCA results were used to comprehensively validate outlier samples. After removing confirmed outlier samples, the expression matrix was reconstructed to ensure the accuracy and reliability of subsequent differentially expressed gene analysis results.

[0040] Based on transcriptome sequencing data of seed development in the upland cotton standard genetic line TM-1, the gene expression profiles during key stages of lipid synthesis were systematically analyzed.

[0041] The results are as follows Figure 1 As shown, GH_D04G0139 and GH_A05G4237 are significantly highly expressed in the early stage of lipid synthesis (4-26 DPA).

[0042] The evolutionary relationships between GH_D04G0139 and GH_A05G4237 and bZIP transcription factors identified in Arabidopsis were analyzed by constructing a phylogenetic tree.

[0043] The results are as follows Figure 2 As shown, the sequences GH_D04G0139 and GH_A05G4237 exhibit the highest homology with the AtbZIP18 gene of Arabidopsis thaliana. Therefore, in this application, the gene was named GhbZIP18_D04 and GhbZIP18_A05.

[0044] Previous studies have reported that AtbZIP18 in Arabidopsis thaliana plays an important role in pollen development. Specific evidence includes: high expression of the AtbZIP18 gene in male gametophytes and its interaction with other bZIP proteins expressed in other pollen; mutations in its alleles moderately reduce plant dispersal via male gametophytes. This gene family is primarily associated with abiotic stress and growth and development, but no functional reports have been found related to oil formation.

[0045] By querying COTTONOMICS (http: / / cotton.zju.edu.cn / ), the CDS and protein sequences corresponding to GhbZIP18_D04 and GhbZIP18_A05 can be obtained, as follows: (1) CDS sequence of GhbZIP18_D04 gene:

[0046] (2) GhbZIP18_D04 protein sequence: MQDPRNLNPNPDPAFPSFTQNMPSFSNPAQYRGSYHRRAQSEVQFRIPDDLDLVSDPFEGVGSEDDMLCSYMDIEMPGESAKGVEAAAGSWSQNPKGEEVSGGSGIGEKYNGGGKGRHRYSNSVDGCSIMESIEAKKAMAPDKLAELWTIDPKRAKRIIANRRSATRSKEKKALYMSELERKVQTLQTEATTLCAHLTLFQRDTTGLTTENAELKLRLQAMEQQAQLSDALNEALKKEVERLKTATGEITTPTDTFNLGMHHISYAQSSFFPPQNTQLPPFHPFHSNLLTSSLNTNSHVLADMMQQDPLGLLQGLDTSSRGSPFVKSESPSICAAERSGTV (SEQ ID NO:2).

[0047] (3) GhbZIP18_A05 gene CDS sequence:

[0048] (4) GhbZIP18_A05 protein sequence: MQDPRNLNPNPDPAFPSFTQNMPSFSNPIQYRGSYHRRAQSEVQFRIPDDLDLVSDSFEGVGSEDDMFCSYMDIEMPGESAKGVEVAAGSWSQNRKGEEVSGGSGIGEKYNGGGKGRHRYSNSVDGCSIMESIEAKKAMAPDKLAELWTIDPKRAKRIIANRRSATRSKEKK ALYMSELERKVQTLQTEATTLSAHLTLFQRDTTGLTTENAELKLRLQAMEQQAQLCDALNEALKKEVERLKTATGEITTPTDTFNLGMHHISYAQSSFFPPQNTQLPPFHPFHSNLLTSASNTNSHALADMMQQDPLGLLQGLDTSSRGSPFVKSESPSICAAESSGTV(SEQ ID NO:4).

[0049] Example 2: Verification of the relative expression levels of GhbZIP18 in high-oil and low-oil materials To verify the relationship between the expression level of the GhbZIP18 gene and the oil content of cottonseed, this application used the high-oil upland cotton variety "Jiangsu Cotton No. 2" and the low-oil upland cotton variety "Jun Cotton No. 1" as experimental materials.

[0050] RNA was extracted from ovules of upland cotton varieties “Jiangsu Cotton No. 2” and “Jun Cotton No. 1” on days 10, 15, 20, 25, 30, 35, 40, and 45 after flowering. The extraction process was strictly carried out in accordance with the instructions of the centrifuged column-type polysaccharide and polyphenol plant total RNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd.

[0051] After RNA extraction, cDNA was synthesized via reverse transcription using TransScript® One-Step gDNA Removal and cDNASynthesis SuperMix (TransGen Biotech, Beijing) for subsequent experiments.

[0052] qRT-PCR was performed using the PerfectStart Green qPCR SuperMix kit. The reaction mixture consisted of 20 ng of template cDNA, 10 μL of 2×PerfectStart Green qPCR SuperMix, and 400 nmol / L each of forward and reverse primers (Table 1). -1Add ddH2O to a final volume of 20 μL. Perform qRT-PCR on a LightCycler 480 II instrument under the following conditions: 95℃ pre-denaturation for 10 s; 95℃ for 15 s, 60℃ for 20 s, for 40 cycles. Utilize 2 -ΔΔCt The method calculates the relative expression level of genes, with each sample repeated three times, using the cotton UBQ7 gene as an internal reference gene.

[0053] Table 1. qRT-PCR primers used in this example The results are as follows Figure 3 As shown, during the critical period of cottonseed oil synthesis (20 DPA), the expression of GhbZIP18_D04 and GhbZIP18_A05 genes in the high-oil variety "Jiangsu Cotton 2" was significantly higher than that in the low-oil variety "Junmian 1" (p<0.01).

[0054] Figure 3 The results showed that the GhbZIP18 gene was positively correlated with the natural oil content of cottonseed, proving that the gene has a positive regulatory role in the process of oil accumulation.

[0055] Example 3: Functional analysis of GhbZIP18 in cotton 1. Identification of the function of GhbZIP18 in the formation of cotton and cottonseed oil using gene editing technology. Sequence-specific SgRNAs were designed using the online tool CRISPR-P2.0 (http: / / crispr.hzau.edu.cn / CRISPR2 / ). Suitable SgRNAs were selected based on factors such as off-target score, gene location, GC% content, and homologous gene similarity. The primer sequences are as follows: sgRNA1-22KN113T1:GTTTCGGATTCCAGACGACT (SEQ ID NO: 11); sgRNA2-22KN113T2:CATGGACATTGAGATGCCAG (SEQ ID NO: 12).

[0056] The target fragment was amplified using the 2×TranStart® FastPfu Fly PCR SuperMix (Pfu) kit from Beijing TransGen Biotech Co., Ltd. The PCR reaction system is as follows: Table 2. PCR amplification system After the PCR amplification system was prepared, amplification was performed at 62℃. The amplification reaction system and procedure are as follows: Table 3. PCR amplification reaction system Table 4. PCR Amplification Procedure This application describes gene editing of GhbZIP18 using CRISPR-Ca9 gene editing technology. Using P7N-K as the backbone vector, PCR amplification was performed on cotton samples using primers sgRNA1-22KN113T1 and sgRNA2-22KN113T2. The vector was then transferred into the cotton variety Jin668 via Agrobacterium-mediated genetic transformation.

[0057] T0 generation seedlings of GhbZIP18 gene-edited plants obtained from tissue culture were transplanted to the experimental field. Specific PCR primers were designed based on the GhbZIP18 gene sequence to identify the target site editing type in T1 generation plants. The primer sequences used are shown below: 22KN113T1:ggagtgagtacggtgtgcCCCTCCTTCACCCAAAATATG (SEQ ID NO: 13); 22KN113T2: gagttggatgctggatggGTTCTGACTCCAAGACCCAGCA (SEQ ID NO: 14).

[0058] The results are as follows Figure 4 As shown in the figure, 22 positive seedlings were obtained by PCR detection. Hitom assay was performed on these 22 positive plants, yielding two stable double knockout lines. These two different knockout lines in this application are named Ghbzip18-1 and Ghbzip18-2, respectively. Figure 5 As shown.

[0059] pass Figure 5 and Figure 6 Further analysis of the experimental results on cotton plants showed that, compared with the control plant Jin668, both GhbZIP18 gene knockout lines showed varying degrees of base deletion at both target sites. Figure 5 ), exhibiting a significant dwarfing phenotype ( Figure 6 Both the weight per boll and the number of cotton seeds per boll decreased (AB). Figure 6 CD).

[0060] More specifically, the reduction in plant height, boll weight, and number of seeds per boll is reflected in the following ways: the average plant height of Ghbzip18-1 is about 28.37% lower than that of Jin668, and the average plant height of Ghbzip18-2 is about 16.67% lower than that of Jin668; the average boll weight of Ghbzip18-1 is about 23.35% lower than that of Jin668, and the average boll weight of Ghbzip18-2 is about 25.79% lower than that of Jin668; the average number of seeds per boll of Ghbzip18-1 is about 17.66% lower than that of Jin668, and the average number of seeds per boll of Ghbzip18-2 is about 30.24% lower than that of Jin668.

[0061] To better analyze the effects of GhbZIP18 on cotton fatty acids, this application also verified the function of the GhbZIP18 gene in cottonseed.

[0062] Figure 7 To verify the function of cotton seeds from GhbZIP18 gene-edited plants, compared with the control plant Jin668, it can be seen that the cotton seeds of Ghbzip18-1 and Ghbzip18-2 are significantly smaller overall. Figure 7 A), the total length and total width of cottonseeds decreased significantly ( Figure 7 CD), the weight of 100 grains decreased significantly ( Figure 7 B).

[0063] More specifically, the reduction in cottonseed width, length, and 100-seed weight is reflected in the following: the average width of Ghbzip18-1 is about 4.3% lower than that of Jin668, and the average width of Ghbzip18-2 is about 3.5% lower than that of Jin668; the average length of Ghbzip18-1 is about 12.07% lower than that of Jin668, and the average length of Ghbzip18-2 is about 12.3% lower than that of Jin668; the average 100-seed weight of Ghbzip18-1 is about 6.7% lower than that of Jin668, and the average 100-seed weight of Ghbzip18-2 is about 8.8% lower than that of Jin668.

[0064] The crude protein content of cottonseed was measured using near-infrared scanning. Compared with the control plant Jin668, the crude protein content of Ghbzip18-1 and Ghbzip18-2 was significantly increased. Figure 8 A), Crude fat content decreased ( Figure 8 B).

[0065] More specifically, the significant increase in crude protein content and decrease in crude fat content are reflected in the following: the average crude protein content of Ghbzip18-1 is about 10.27% higher than that of Jin668, and the average crude protein content of Ghbzip18-2 is about 6.08% higher than that of Jin668; the average crude fat content of Ghbzip18-1 is about 5.7% lower than that of Jin668, and the average crude fat content of Ghbzip18-2 is about 6.16% lower than that of Jin668.

[0066] The fatty acid composition of cottonseed was determined. Compared with the control plant Jin668, the levels of palmitic acid (C16:0), oleic acid (C18:1), and linoleic acid (C18:2) in Ghbzip18-1 and Ghbzip18-2 were significantly decreased. Figure 8 C).

[0067] More specifically, the significant decreases in palmitic acid (C16:0), oleic acid (C18:1), and linoleic acid (C18:2) are reflected in the following: the average palmitic acid (C16:0) in Ghbzip18-1 was approximately 8.7% lower than that in Jin668, and the average palmitic acid (C16:0) in Ghbzip18-2 was approximately 5.6% lower than that in Jin668; the average oleic acid (C18:1) in Ghbzip18-1 was approximately 13.54% lower than that in Jin668, and the average oleic acid (C18:1) in Ghbzip18-2 was approximately 10.49% lower than that in Jin668; the average linoleic acid (C18:2) in Ghbzip18-1 was approximately 4.4% lower than that in Jin668, and the average linoleic acid (C18:2) in Ghbzip18-2 was approximately 2.3% lower than that in Jin668.

[0068] In cotton plants, knocking out the Ghbzip18 gene leads to a decrease in fatty acid composition, and it is considered a transcription factor positively correlated with oil formation.

[0069] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A cotton GhbZIP18 gene related to oil synthesis, characterized in that, The nucleotide sequence of the GhbZIP18 gene is shown in SEQ ID NO: 1 or SEQ ID NO:

3.

2. A cotton GhbZIP18 protein associated with oil content synthesis, characterized in that, The amino acid sequence of the GhbZIP18 protein is shown in SEQ ID NO: 2 or SEQ ID NO:

4.

3. The use of the cotton GhbZIP18 gene of claim 1 or the cotton GhbZIP18 protein of claim 2 in any one of the following, characterized in that, The application comprises: (a1) application in regulating the height of cotton plants; (a2) application in regulating the development of cotton seeds; (a3) application in regulating the oil content of cotton seeds; (a4) application in regulating the oil composition of cotton seeds.

4. A primer pair for detecting the expression amount of GhbZIP18 gene in cotton, characterized in that, The nucleotide sequence of the primer pair is selected from one or more of the following groups: (1) a forward primer shown in SEQ ID NO: 7 and a reverse primer shown in SEQ ID NO: 8; (2) a forward primer shown in SEQ ID NO: 9 and a reverse primer shown in SEQ ID NO:

10.

5. A test kit characterized in that, The detection kit comprises the primer pair of claim 4; preferably, the detection kit further comprises PCR amplification buffer, reverse transcriptase, DNA polymerase, dNTPs and / or fluorescent dye; more preferably, the detection kit further comprises an instruction manual.

6. Use of the reagent for detecting the cotton GhbZIP18 gene according to claim 1, the reagent for detecting the cotton GhbZIP18 protein according to claim 2, the primer pair according to claim 4, or the detection kit according to claim 5 in any one of the following, characterized in that, The application comprises: (b1) assisting in screening or identifying cotton germplasm materials with high oil content; (b2) assisting in screening or identifying cotton germplasm materials with high palmitic acid, high oleic acid and / or high linoleic acid content; (b3) assisting in screening or identifying cotton germplasm materials with large cotton seeds; (b4) assisting in screening or identifying cotton germplasm materials with high plant height; (b5) molecular identification or fingerprinting of cotton varieties.

7. A method of modulating oil content in cotton seeds, characterized in that, The application comprises the following steps: By changing the expression amount of GhbZIP18 gene or GhbZIP18 protein in cotton, cotton germplasm materials with target oil content are obtained; wherein the GhbZIP18 gene is as described in claim 1, or the GhbZIP18 protein is as described in claim 2; the target oil includes at least one of palmitic acid, oleic acid and linoleic acid.

8. A method of assisting in the identification of high oil cotton varieties, comprising: The application comprises the following steps: S1. RNA extraction and reverse transcription: total RNA of ovules of the cotton to be tested at 4-45 days after flowering is extracted and reverse transcribed into cDNA; S2. Real-time fluorescent quantitative PCR amplification: using the cDNA as a template, the primer pair of claim 4 and a primer pair of an internal reference gene are used for real-time fluorescent quantitative PCR amplification; S3. Expression amount analysis: according to the number of amplification cycles in S2, the relative expression amount of GhbZIP18 gene is calculated by normalizing the internal reference gene as a standard; S4. Result determination: the relative expression amount of GhbZIP18 gene obtained in step S3 is compared with a preset threshold, a low oil content control or a population average level; if the relative expression amount of GhbZIP18 gene of the cotton to be tested is significantly higher than the control or the population average level, the cotton to be tested is determined as a cotton variety with high oil content or a cotton germplasm resource with high oil content potential.

9. A CRISPR / Cas9 gene editing vector for editing GhbZIP18 gene, characterized in that, The gene editing vector comprises a nucleotide sequence encoding a Cas9 protein, and a single guide RNA coding sequence specifically targeting the GhbZIP18 gene of claim 1.

10. The gene editing vector of claim 9, wherein, The one-way guide RNA coding sequence is shown in SEQ ID NO: 11 and / or SEQ ID NO:

12. The one-way guide RNA coding sequence is shown in SEQ ID NO: 11 and / or SEQ ID NO: 12.

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