Cotton high-oil gene GhGYP and method for creating cotton high-oil germplasm

Abstract

The invention discloses a cotton high oil content gene GhGYP and a method for creating cotton high oil germplasm, and belongs to the technical field of biotechnology application, the cotton high oil content gene GhGYP is a gene GhCYP93A2, and the CDS sequence of the gene is a nucleotide sequence shown in Seq ID No.1; or a protein which is derived from Seq ID No.1 and is related to the plant seed oil content, wherein the nucleotide sequence shown in Seq ID No.1 is subjected to substitution and / or deletion and / or addition of one or more amino acid residues; or a nucleotide sequence hybridized with the nucleotide sequence shown as Seq ID No.1 under strict conditions. The cotton high-oil gene GhCYP93A2 is beneficial to acceleration of creation of excellent high-oil materials of cotton.

Description

Technical Field

[0001] The invention relates to the technical field of biotechnology application, and in particular to a cotton high-oil gene GhGYP and a method for creating cotton high-oil germplasm. Background Art

[0002] Upland cotton is the world's main fiber crop and one of the most important oil crops along with soybeans, rapeseed, sunflowers and peanuts. In 2022, my country's imported edible vegetable oil production was 48.8187 million tons, and the demand was 55.128 million tons. my country's own edible vegetable oil is not enough to meet the total demand. Many edible vegetable oils still rely on imports, which seriously threatens the safety of my country's edible vegetable oil. However, in 2022, my country's peanut, rapeseed, sesame, soybean and sunflower seed production accounted for 33.89%, 17.81%, 6.49%, 5.81% and 5.40% of the world's total production respectively. In the same year, my country's cottonseed oil production was 1.484 million tons, accounting for 29.62% of the world's total production, which can provide rich oil resources for edible vegetable oil.

[0003] Cotton is an important source of income for cotton farmers. In addition to being used for seed preservation, more than 80% of cottonseed is used for oil extraction. It is the second most important source of edible oil in the world after soybeans. Due to its fairly neutral taste, cottonseed oil is often needed by the food industry because it does not mask the natural flavor of food used for cooking or processing. Cotton main and by-products have high yields and utilization values. Although my country's cotton planting area has decreased in recent years, cotton can still maintain a high yield per unit area. Cottonseed meal is also the main by-product of cottonseed processing. Cottonseed meal contains rich protein and amino acids and can be used as human food (after removing toxic gossypol) and animal feed. In fact, the protein in cottonseed meal is also the second largest potential source of plant protein after soybeans. It is estimated that the annual output of cottonseed protein in the world is about 11 million tons, and my country's cottonseed meal output in 2022 is 4.093 million tons. With the development of domestic breeding and feed industries, my country's demand for cottonseed meal has remained at a high level, with a demand of 4.115 million tons in the same year. In addition, Xinjiang has a huge amount of cotton stalk resources, with an annual output of more than 10 million tons, which can be used for fuel, compost, feed, etc. The entire cotton plant can generate high economic benefits for cotton farmers every year.

[0004] With the growth of my country's population, the improvement of people's living standards, the acceleration of urbanization and the continuous growth of industrial oil consumption, my country's edible oil consumption demand will continue to maintain a rigid growth trend in terms of total volume. At the same time, people have also put forward higher requirements for cottonseed derivatives. Therefore, it is particularly urgent to breed cotton varieties that meet current production needs and can give full play to the value of cottonseed.

[0005] It should be noted that the information disclosed in the background technology section of the invention is only intended to deepen the understanding of the general background technology of the invention, and should not be regarded as an admission or suggestion in any form that the information constitutes prior art already known to those skilled in the art. Summary of the invention

[0006] The purpose of the present invention is to provide a cotton high-oil gene GhGYP and a method for creating cotton high-oil germplasm to solve the problem of cotton oil content.

[0007] In order to solve the above technical problems, the first aspect of the present invention provides a cotton oil-related gene GhCYP93A2, wherein the cotton high oil gene GhGYP is the gene GhCYP93A2, and its CDS sequence is:

[0008] The nucleotide sequence as shown in Seq ID No.1; or,

[0009] A protein derived from Seq ID No. 1, wherein the nucleotide sequence shown in Seq ID No. 1 is substituted and / or deleted and / or added with one or more amino acid residues and is related to plant seed oil; or

[0010] A nucleotide sequence that hybridizes with the nucleotide sequence shown in Seq ID No. 1 under stringent conditions.

[0011] Preferably, the gene GhCYP93A2 encodes a protein.

[0012] Preferably, the protein encoded by the gene GhCYP93A2 has a protein sequence as shown in Seq ID No.2.

[0013] The second aspect of the present invention provides an application in identifying high-oil content cotton germplasm.

[0014] In a third aspect, the present invention provides an sgRNA for gene editing mutation of the cotton high oil gene GhGYP as described in the first aspect above, and the sgRNA sequence of the gene GhCYP93A2 is shown in Seq ID No.3.

[0015] In a fourth aspect, the present invention provides a CRISPR / Cas9 system for gene editing of the cotton high oil gene GhGYP as described above, comprising the sgRNA described in the second aspect.

[0016] In a fifth aspect of the present invention, based on the cotton high-oil gene GhGYP described in the first aspect, the application of the gene GhCYP93A2 in breeding new cotton varieties with high oil content by genetic engineering means.

[0017] In a sixth aspect, the present invention provides an application of a reagent for gene editing the cotton oil-related gene GhCYP93A2 described in the first aspect in breeding new cotton varieties with high oil content by genetic engineering means, wherein the reagent is the sgRNA described in the third aspect or the CRISPR / Cas9 system described in the fourth aspect.

[0018] Preferably, according to the applications described in the second, fifth and sixth aspects above, the cotton includes upland cotton.

[0019] In a sixth aspect, the present invention provides a method for creating high-oil cotton germplasm, comprising the following steps:

[0020] CRISPR / Cas 9 is used to construct the sgRNA as described in the third aspect to perform gene editing on the gene GhCYP93A2 to disable its protein function, obtain transgenic plants of the cotton high-oil gene GhGYP as described in the first aspect, and harvest T1 generation mutant seeds.

[0021] The cotton high-oil gene GhGYP and the method for creating cotton high-oil germplasm provided by the present invention provide a cotton high-oil gene GhCYP93A2, which is conducive to accelerating the creation of excellent cotton high-oil materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the distribution and trend of cottonseed oil content under four environments;

[0023] Figure 2 is the genome-wide LD attenuation distance of 168 upland cotton material populations;

[0024] Figure 3a to Figure 6a These are the Manhattan plots of cottonseed oil content in the environments of Korla in 2018, Shihezi in 2018, Korla in 2019, and Shihezi in 2019;

[0025] Figure 3b to Figure 6b They represent the QQ graphs of cottonseed oil content under the environments of Korla in 2018, Shihezi in 2018, Korla in 2019, and Shihezi in 2019;

[0026] Figure 7 It is a statistical graph of the number of differentially expressed genes;

[0027] Figure 8 It is the expression heat map of 47 genes at different periods;

[0028] Fig. 9 It is the genetic structure and mutation site of CYP93A2;

[0029] Fig.10 is the base sequence alignment between the mutant material and Jin668;

[0030] Fig.11 is the amino acid sequence alignment of the mutant material and Jin668;

[0031] Fig.12 is the haplotype analysis of GhCYP93A2;

[0032] Fig.13 is the oil content of mutant materials and Jin668;

[0033] Fig.14 is the protein content of mutant materials and Jin668.

[0034] In the attached figure:

[0035] ** indicates a significant difference at the p < 0.005 level. DETAILED DESCRIPTION

[0036] The following is a further detailed description of the cotton high oil gene GhGYP and the method for creating cotton high oil germplasm proposed by the present invention in conjunction with the accompanying drawings and specific embodiments. According to the following description, the advantages and features of the present invention will be clearer. It should be noted that the drawings are all in a very simplified form and use non-precise proportions, which are only used to conveniently and clearly assist in explaining the purpose of the embodiments of the present invention. It should be understood that the drawings in the specification do not necessarily show the specific structure of the present invention in proportion, and the illustrative features used to illustrate certain principles of the present invention in the drawings in the specification will also adopt a slightly simplified drawing method. The specific design features of the present invention disclosed herein include, for example, specific dimensions, directions, positions and shapes, which will be determined in part by the specific environment to be applied and used. And, in the embodiments described below, sometimes the same figure mark is used in common between different drawings to represent the same part or a part with the same function, and its repeated description is omitted. In this specification, similar numbers and letters are used to represent similar items, so once an item is defined in one drawing, it does not need to be further discussed in subsequent drawings.

[0037] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0038] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0039] The inventors found that cottonseed oil content is a complex quantitative trait controlled by multiple genes. Previous studies have mainly focused on cotton fiber and quality, while research on cottonseed is relatively limited. This has led to a lack of cotton varieties with high oil content and good fiber quality, affecting the overall economic value of cottonseed.

[0040] Based on this, the cottonseed oil content of a group of upland cotton materials was measured in four different environments by near-infrared analysis, and the loci significantly associated with cottonseed oil content were screened out by combining the results of genome association analysis. At the same time, transcriptome sequencing analysis was performed on upland cotton materials with significant differences in cottonseed oil content, combined with GWAS results to dig out key candidate genes for cottonseed oil content. This study helps to reveal the genetic mechanism of cottonseed oil accumulation, provides a theoretical basis for breeding new varieties with high oil content, and is of great significance to further improve the overall economic benefits of cotton.

[0041] The core idea of ​​the present invention is that by providing a specific cotton high-oil gene GhCYP93A2, it is beneficial to accelerate the creation of excellent high-oil cotton materials, and provide a CRISPR / Cas9 system for mutating the cotton high-oil gene GhCYP93A2, which can site-directedly mutate the gene GhCYP93A2, thereby causing its protein function to lose. By knocking out the GhCYP93A2 gene, new high-oil materials of upland cotton are created, which is of great significance to cottonseed oil utilization and population improvement.

[0042] Specifically, the present invention is implemented through the following technical solutions, and the specific implementation steps are as follows:

[0043] S1, through genome-wide association analysis and extreme material transcriptome analysis, a major QTL controlling the oil content of upland cotton seeds was detected on chromosome D12 of upland cotton, and it was named as gene GhCYP93A2. The cotton high oil gene GhGYP is gene GhCYP93A2, and its CDS sequence is: the nucleotide sequence shown in Seq ID No.1; or, the nucleotide sequence shown in Seq ID No.1 is substituted and / or deleted and / or added with one or several amino acid residues and the protein derived from Seq ID No.1 related to plant seed oil; or, the nucleotide sequence hybridized with the nucleotide sequence shown in Seq ID No.1 under stringent conditions.

[0044] Specifically, the protein encoded by the gene GhCYP93A2 has a protein sequence as shown in Seq ID No.2.

[0045] Among them, the specific exploration of gene GhCYP93A2 is as follows.

[0046] First, relevant experimental materials are provided, including upland cotton varieties Xinluzao 26, Xinluzao 29, Xinluzhong 9 and Xinluzhong 10, as well as genetic line materials such as GhCYP93A2, for example, a total of 168 upland cotton germplasm resources materials are set up.

[0047] Secondly, mature cotton seeds of 168 upland cotton varieties grown in four different environments were harvested and, after delinting with sulfuric acid, 100 healthy seeds with shells were randomly selected and measured using near-infrared spectroscopy to measure the seed oil content of each variety in the four environments, with two replicates.

[0048] Exemplarily, the instrument for measuring seed oil content is a PERTEN near infrared analyzer.

[0049] Furthermore, 168 upland cotton materials were sequenced for the whole genome, and the average sequencing depth was 12.4×. The upland cotton TM-1 genome of Huazhong Agricultural University was used as the reference genome and aligned to the reference genome using BWA software. SNPs and InDels were identified using GATK3.1 software, and SNP markers with a minimum allele frequency of less than 0.05 and a missing rate of less than 0.1 were filtered using vcftools software to obtain high-quality SNP sites for subsequent analysis.

[0050] In one embodiment, based on the above seed oil content results, the seeds are classified into low-oil varieties: Xinlu Zao 26 and Xinlu Zao 29; high-oil varieties: Xinlu Zhong 9 and Xinlu Zhong 10. Among them, Xinlu Zhong 9 and Xinlu Zhong 10 cotton seeds have a higher oil content, with an average oil content of 19.38% and 19.92%; Xinlu Zao 26 and Xinlu Zao 29 cotton seeds have a lower oil content, with an average oil content of 15.48% and 15.04%. These four materials were then planted in the experimental field of Shihezi University in April 2023 and subjected to conventional field management. The day of flowering was recorded as 0 DPA. At 10, 15, 20, and 25 DPA, 2-3 plants were selected from each material at each period, and 2-3 bolls were selected from each plant. After picking, they were mixed and three biological replicates were taken. After removing the long fibers on the surface of the ovules, they were stored in liquid nitrogen and kept in a -80°C refrigerator. Tissue RNA was extracted using the Invitrogen TRIzol extraction kit.

[0051] Table 1 Descriptive statistics of cottonseed oil content traits

[0052]

[0053] Table 2 Analysis of variance of cottonseed oil content under four environments

[0054] sum of squares Mean Square F-number Pr(>F) Significance genotype 1939.659 11.61473 1.29411 0.0000 *** environment 179.6277 59.87592 3.796211 0.0000 *** Genotype × environment 385.4781 0.769417 1.184481 0.0000 ***

[0055] Note: *** indicates significant difference at p < 0.001 level.

[0056] Based on the above experimental data, according to the phenotypic data analysis results of 168 upland cotton germplasm resources, it was found that the variation range of cottonseed oil content in four different environments was between 14.06% and 21.41%, indicating that the genetics of cottonseed oil content is basically stable, but is still affected by environmental factors to a certain extent (Table 1).

[0057] As shown in Table 2, two-way ANOVA further confirmed that environment, genotype, and the interaction between genotype and environment had significant effects on cottonseed oil content (p < 0.001). Overall, the phenotypic data between different locations varied greatly, while the phenotypic data at the same location were relatively stable, such as Figure 1 , which provides an important reference for further research.

[0058] GWAS analysis was performed based on genotype and phenotype data, where the number of effective SNPs was 3707559, and the phenotypes were the cottonseed oil content phenotype values ​​and BLUP values ​​in each environment. When the threshold was -log10(p)≥6.57, (p≤1 / 3707559), 2018KEL was used as the phenotype value, and on chromosome D12 ( Figure 3a, 3b) detected 109 significantly associated SNP markers; taking 2018SHZ as the phenotype value, in A01 ( Figure 4a , 4b) 32 significantly associated SNP markers were detected on chromosomes; 2019SHZ was used as the phenotype value ( Figure 6a , 6b), 18 significantly associated SNP markers were detected on chromosome D02, and 3 significantly associated SNP markers were detected on chromosome D11; 2019KEL was used as the phenotypic value ( Figure 5a , 5b), two significantly associated SNP markers were detected on chromosome D12. According to the significantly associated SNP and LD value ( Figure 2 ), a total of 325 candidate genes were detected.

[0059] Four varieties with significant differences in cottonseed oil content were selected from 168 population materials. Low-oil varieties: Xinlu Zao 26 (Z26) and Xinlu Zao 29 (Z29); high-oil varieties: Xinlu Zhong 9 (ZH9) and Xinlu Zhong 10 (ZH10). Then, the differential expression of ovules at 10, 15, 20, and 25 DPA was analyzed at the transcriptome level to study the expression levels of these differentially expressed genes at different developmental stages: using the absolute value of expression difference > 1, significance P < 0.01, a total of 9593 differentially expressed genes ( Figure 7 ). Comparison with 325 candidate genes in the candidate interval of whole genome association analysis revealed 47 identical genes, combined with heat map analysis of differential gene expression in ovules at different stages after flowering ( Figure 8 ), the above results showed that the expression level of GhCYP93A2 was higher in the early stage of cottonseed development, and it was inferred that it was involved in the early carbon accumulation process. The expression of gene GhCYP93A2 was strongly correlated with cottonseed oil content.

[0060] S2. Based on the gene GhCYP93A2 obtained above, transgenic plants are obtained by gene editing. Specifically, CRISPR / Cas 9 is used to construct sgRNA to edit the candidate gene, and finally transgenic plants of the gene GhCYP93A2 are obtained, and T1 generation mutant seeds are harvested.

[0061] Based on the above scheme, the oil content of mutant cotton seeds was measured, and the oil content of cotton seeds was relatively high. The applicant transformed the cotton seeds through gene editing methods and finally successfully created cotton germplasm materials with high oil content.

[0062] like Figure 9-11, the gene has a six base deletion (AGTGCC) at 3684-3689bp in the second exon of the mutant, resulting in the deletion of the 463rd and 464th amino acids V (leucine) and P (proline). This gene has two haplotypes in the population, Hap1 (GG) and Hap2 (TT), and there is a significant difference in cottonseed oil content between the two haplotypes (p < 0.005). By measuring the oil and protein content of the mutant T1 generation seeds, it was found that the mutant seeds had an oil content of 18.10% and a protein content of 21.34%; the wild-type seeds had an oil content of 15.78% and a protein content of 21.22%. The mutant cottonseed oil content increased by 2.33% ( Fig.13 ), but the cottonseed protein content remained almost unchanged ( Fig.14 ). The results preliminarily indicate that the key candidate genes for cottonseed oil content identified in this study have high reliability. At the same time, the research results provide good research materials for breeding high-oil cotton varieties.

[0063] The mutation of the gene GhCYP93A2 causes an increase in the oil content of cotton seeds. These results not only confirm the regulatory effect of the candidate gene on the oil content of cotton seeds, but also provide an important research basis and genetic material for breeding high-oil cotton varieties.

[0064] It can be understood that the gene GhCYP93A2 can be used in the identification of high-oil cotton germplasm, in the breeding of new cotton varieties with high oil content by genetic engineering, and in the breeding of new cotton varieties with high oil content by genetic engineering. The reagent for gene editing of the cotton oil-related gene GhCYP93A2 described in the first aspect is provided, and the reagent is the above-mentioned sgRNA or the above-mentioned CRISPR / Cas9 system in the fourth aspect. Specifically, according to the above-mentioned application, the cotton includes upland cotton.

[0065] The present invention provides an sgRNA for gene editing mutation of the cotton high oil gene GhGYP as described in the first aspect above, and the sgRNA sequence of the gene GhCYP93A2 is shown in Seq ID No.3.

[0066] The present invention also provides a CRISPR / Cas9 system for gene editing such as the above-mentioned cotton high-oil gene GhGYP, comprising the sgRNA described in the second aspect.

[0067] Based on the application of the above gene GhCYP93A2, the present invention also provides a method for creating high-oil cotton germplasm, comprising the following steps:

[0068] CRISPR / Cas 9 is used to construct the sgRNA as described in the third aspect to perform gene editing on the gene GhCYP93A2 to disable its protein function, obtain transgenic plants of the cotton high-oil gene GhGYP as described in the first aspect, and harvest T1 generation mutant seeds.

[0069] In summary, in the cotton high-oil gene GhGYP and the method for creating cotton high-oil germplasm provided in the embodiments of the present invention, by providing a cotton high-oil gene GhCYP93A2, it is beneficial to accelerate the creation of excellent high-oil materials in cotton, and provide a CRISPR / Cas9 system for mutating the cotton high-oil gene GhCYP93A2, which can site-directedly mutate the gene GhCYP93A2, thereby causing the loss of its protein function.

[0070] The above description is only a description of the preferred embodiments of the present invention, and is not intended to limit the scope of the present invention. Any changes or modifications made by a person skilled in the art in the field of the present invention based on the above disclosure shall fall within the scope of protection of the claims.

[0071] CDS sequence of 1GhCYP93A2

[0072]

[0073] Protein sequence of 2GhCYP93A2

[0074] MALLFTFVIASSILWLTWMYLKSKEGKPPPPPGPLGLPLVGNLPFLRPDLHSYFSELAHIYGPVVKLQLGSKLGILITSSSTAREVLKDQDMAFANCDVPVVAKVLTGGRDIVWNPYGPEWRMLRKVCVIKMLSNATLDKVYSLRRQEVRETLCYLYSKAGSPVNVGEQMFLTILNVVTNMLWGGSMEGEARASLGAEFRQVI SEVTELLGLPNVSDFFPALAPLDLQGAVKRIRKPTEKLDAIIDKIINQRSEEKQSGQTVEEFKDFLQFLLKLKDEDDSKTPMTMTHIKALLLDMVVGGSDTSSNSIEFTMAEIINKPEVMRKAQQELDEVVGKDNFVEESHIHKLPYLL AVMKESLRLHPVLPLLVPHCPSEPNTVGGYAIPKGSRVFVNAWAIHRDPSIWENPLEFNPDRFLNSQWDFSGSDFRYFPFGSGRRICAGIAMAERMVLYSVASLLHSFDWQVPGGEKLDLSEKFGIVLKLQNPLVAIPTPRLSDPALYQP

[0075] sgRNA sequence of 3GhCYP93A2

[0076] TTTGACTGGCAAGTGCCAGG.

Claims

1. A cotton high oil gene GhGYP, characterized in that: The cotton high oil gene GhGYP is gene GhCYP93A2, and its CDS sequence is: The nucleotide sequence as shown in Seq ID No.1; or, A protein derived from Seq ID No. 1, wherein the nucleotide sequence shown in Seq ID No. 1 is substituted and / or deleted and / or added with one or more amino acid residues and is related to plant seed oil; or A nucleotide sequence that hybridizes with the nucleotide sequence shown in Seq ID No. 1 under stringent conditions.

2. The cotton high oil gene GhGYP according to claim 1, characterized in that: The gene GhCYP93A2 encodes a protein.

3. The cotton high oil gene GhGYP according to claim 2, characterized in that: The protein encoded by the gene GhCYP93A2 has a protein sequence as shown in Seq ID No.

2.

4. The cotton high oil gene GhGYP according to any one of claims 1 to 3, characterized in that: Application in identification of high-oil content cotton germplasm.

5. A sgRNA for gene editing mutation of the cotton high oil gene GhGYP according to any one of claims 1 to 3, characterized in that: The sgRNA sequence of the gene GhCYP93A2 is shown in Seq ID No.

3.

6. A CRISPR / Cas9 system for gene editing of the cotton high oil gene GhGYP as claimed in any one of claims 1 to 3, characterized in that: Comprising the sgRNA of claim 5.

7. The cotton high oil gene GhGYP according to any one of claims 1 to 3, characterized in that: The gene GhCYP93A2 is used in breeding new cotton varieties with high oil content through genetic engineering.

8. Use of a reagent for gene editing the cotton high-oil gene GhGYP described in any one of claims 1-3 in breeding new cotton high-oil varieties by genetic engineering means, wherein the reagent is the sgRNA described in claim 5 or the CRISPR / Cas9 system described in claim 6.

9. The use according to any one of claims 4, 7 and 8, characterized in that: The cotton includes upland cotton.

10. A method for creating high-oil cotton germplasm, characterized in that: The following steps are involved: The sgRNA as claimed in claim 5 is constructed using CRISPR / Cas 9 to perform gene editing on the gene GhCYP93A2 to lose its protein function, thereby obtaining a transgenic plant of the cotton high-oil gene GhGYP as claimed in any one of claims 1 to 3, and harvesting T1 generation mutant seeds.

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