Arginyl-tRNA-protein transferase gene for improving fiber length of upland cotton and application of arginyl-tRNA-protein transferase gene

Abstract

The invention relates to an arginyl-tRNA-protein transferase gene capable of improving the fiber length of upland cotton and application of the arginyl-tRNA-protein transferase gene, and the arginyl-tRNA-protein transferase gene related to the fiber length of the upland cotton is excavated through upland cotton variety population re-sequencing and whole genome correlation analysis, namely, ATE (arginyl-tRNA-protein transferase). Whole genome association analysis results show that the gene is closely associated with the fiber length character. By detecting that the genotype of the 1283bp site of the arginyl-tRNA-protein transferase gene is A, it can be judged that cotton is a cotton plant with high fiber length.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to an arginyl-tRNA-protein transferase gene for improving upland cotton fiber length and an application thereof. Background Art

[0002] Cotton (Gossypium spp.), as one of the key economic crops in my country, has a significant impact on the development of agriculture and the national economy. Cotton fiber originates from a single cell differentiated from the epidermal cells of the outer integument of the ovule. Its development goes through four stages: initial differentiation, rapid elongation, secondary wall synthesis, and mature dehydration, which ultimately determine the quality of the fiber. As the core raw material of the textile industry, the quality of cotton fiber, especially the fiber length, plays a decisive role in the spinning process, yarn properties, and the texture, gloss, and durability of the final textile. Although my country ranks first in global cotton production, consumption, textile production, and export, the quality of domestic cotton cannot fully meet the diversified needs of the textile industry, resulting in some cotton textile companies relying on imported cotton to produce high-grade cotton yarn. Therefore, improving the length and quality of domestic cotton fibers is crucial to ensuring the supply of raw materials for the textile industry and enhancing international competitiveness. By discovering excellent genetic variations related to cotton fiber length and breeding high-quality cotton varieties, it is of great significance to enhance the competitiveness and economic benefits of my country's cotton industry, which provides a clear direction and research value for cotton molecular breeding.

[0003] Initially, the research on cotton quality traits focused on quantitative trait loci (QTL) analysis. Genome-wide association analysis (GWAS) is a method that uses whole genome resequencing technology to perform high-throughput identification of molecular markers (such as SNPs and CNVs) in populations with rich genetic diversity, and combines phenotypic data for association analysis. This method uses statistical means to determine the association between mutation sites and traits, and quickly locates chromosome segments or gene loci that affect the trait phenotype. With the advancement of sequencing technology and computational methods, GWAS has become an important tool for identifying natural variation in complex traits of crops. It can mine genes related to agronomic traits on a large scale without the need to preset candidate genes. It has high detection capabilities and accuracy, and has become the focus of molecular breeding. GWAS has been successfully applied in a variety of crops such as rice, mustard, soybeans, and cotton, and has achieved remarkable results.

[0004] Li et al. (2024) studied the phenotypic variation of defoliation traits in 383 upland cotton (Gossypium hirsutum L.) germplasms and conducted genome-wide association studies (GWAS), linkage disequilibrium (LD) interval genotyping and functional identification. The study found that defoliants significantly increased the defoliation rate and had little effect on yield and fiber quality. The study identified 174 significant SNPs and located two loci significantly associated with relative defoliation rate (RDR7 on chromosome A02 and RDR13 on chromosome A13), and identified the key candidate genes GhLRR and GhCYCD3;1. The study found that the combination of two favorable haplotypes increased sensitivity to defoliants, and the frequency of these favorable haplotypes generally increased in high-latitude regions of China to adapt to the local environment. Tian et al. (2024) identified a major locus (PH1) controlling plant height in upland cotton (Gossypium hirsutum) through a genome-wide association study (GWAS), which encodes gibberellin 2-oxidase 1A (GhPH1) and discovered a structural variation (PAVPH1) associated with it. The study revealed that the GhGARF transcription factor recognizes and binds to specific sequences in the GhPH1 promoter and PAVPH1, thereby downregulating the expression of GhPH1. In addition, a direct interaction between DWARF53 (D53) and GhGARF was found, inhibiting its binding to the target site. These findings reveal a new gibberellin-brassinolide cross-regulatory mechanism, regulating the plant height of upland cotton through the GhD53-GhGARF-GhPH1 / PAVPH1 module, providing valuable insights for the breeding of semi-dwarf cotton varieties. Zhao et al. (2021) resequenced 336 sea island cotton (Gossypium barbadense), identified 16 million SNPs, and conducted a genome-wide association study (GWAS) based on 15 agronomic traits, including fiber quality, yield, disease resistance, maturity, and plant architecture. The study revealed two major gene pools and a mixed subpopulation, as well as the results of geographical dissemination and hybridization. Through gene expression analysis and VIGS transgenic experiments, five candidate genes were confirmed to regulate four key traits, namely disease resistance, fiber length, fiber strength, and seed cotton percentage. The study also demonstrated the geographical and temporal considerations for the selection and improvement of sea island cotton in China, as well as increased resistance to wilt and improved fiber quality and yield through pedigree selection breeding. Li et al. (2020) used the automated phenotyping platform (APP) to systematically evaluate 119 image-based digital traits (i-traits) under drought stress in 200 representative upland cotton varieties at the seedling stage. A total of 390 genetic loci were identified by genome-wide association studies (GWAS) combining 56 morphological and 63 textural i-traits.The study identified candidate genes associated with drought response, including GhRD2, GhNAC4, GhHAT22, and GhDREB2. Further analysis showed that Gh_A04G0377 and Gh_A04G0378 were involved in cotton drought response as negative regulators. Combining digital phenotyping, GWAS analysis, and transcriptome data, the study concluded that the phenotypic dataset provides an excellent resource for characterizing key genetic loci, which can provide information for future genome-based improvement of cotton drought resistance. Du et al. (2018) resequenced 243 cotton plants (including tree cotton and grass cotton) to generate a genomic variation map and found that they were as differentiated as Raymond cotton. Through a genome-wide association study (GWAS) of tree cotton, 98 significant association peaks associated with 11 important agronomic traits were identified, including non-synonymous mutations in the GaKASIII gene that regulates fatty acid composition and expression activation of the GaGSTF9 gene associated with cotton Verticillium wilt resistance. These findings provide an important step towards understanding the evolution of the cotton A genome and improving key agronomic traits for cotton fiber production. The above research results fully demonstrate that genome-wide association analysis (GWAS) has a high positioning accuracy and can be accurate to the level of a single gene. By utilizing the obtained functional markers associated with the target trait, the target trait can be effectively screened, thereby significantly accelerating the breeding process and improving breeding efficiency, and promoting the rapid development of crop precision breeding. This method provides new strategies and tools for crop improvement and has important application prospects.

[0005] Arginine-tRNA-protein transferase is a key enzyme involved in post-translational modification of proteins. It can transfer arginine from arg-tRNA to the N-terminus of the acceptor protein. This process is essential for protein degradation and various metabolic pathways in cells. This transferase usually contains one or more additional domains, especially the C-terminal domain, which may be involved in the regulation of enzyme activity and interaction with other proteins (Shrader TE et al., 1993). Arabidopsis ATE1 (AT5G05700) has an arginine-tRNA-protein transferase (PF04377) domain and is believed to encode an arginyl-tRNA protein transferase that plays a key role in the N-end rule pathway of proteins. This gene is highly expressed in Arabidopsis roots and leaves, and its loss of function leads to a variety of growth defects, such as abnormal root development and blocked seed germination (Zhang et al., 2015; Willi Riber et al., 2015). In addition, mutations in the ATE1 gene lead to delayed leaf senescence, indicating its important role in regulating plant senescence (Yoshida S et al., 2002). In moss (Physcomitrella patens), the loss of the ATE1 gene leads to severe developmental defects, affecting the morphogenesis and physiological functions of the plant (Schuessele C et al., 2016). ATE1 has an important effect on seed germination and seedling growth in wheat (Shao et al., 2017). Therefore, we speculate that the homologous gene of AT5G05700 in cotton may be involved in cotton fiber development. In-depth research on the function and molecular mechanism of ATEs in cotton fiber development may provide theoretical support for improving cotton fiber length and promote the progress of cotton breeding. Summary of the invention

[0006] The purpose of the present invention is to mine an arginine-tRNA-protein transferase gene associated with the fiber length of upland cotton through resequencing of upland cotton variety populations and whole genome association analysis. The results of the whole genome association analysis showed that the gene was closely associated with the fiber length trait.

[0007] Specifically, an arginyl-tRNA-protein transferase gene for improving the fiber length of upland cotton, the arginyl-tRNA-protein transferase gene sequence is shown in SEQ ID NO.2.

[0008] And, the use of the aforementioned arginyl-tRNA-protein transferase gene for improving upland cotton fiber length in identifying high fiber length upland cotton.

[0009] The application of the aforementioned arginyl-tRNA-protein transferase gene for improving upland cotton fiber length in improving the fiber length trait of upland cotton.

[0010] The application of the aforementioned arginyl-tRNA-protein transferase gene for improving the fiber length of upland cotton in breeding new varieties of upland cotton with high fiber length through genetic engineering means.

[0011] A method for screening high-fiber-length upland cotton varieties, specifically, detecting the genotype at the 1283bp position of the upland cotton arginyl-tRNA-protein transferase gene, and the upland cotton with genotype A is the upland cotton with high fiber length.

[0012] The primers for detecting genotypes are: the upstream primer is shown in SEQ ID NO.5, and the downstream primer is shown in SEQ ID NO.6.

[0013] The advantages of the present invention are:

[0014] (1) The genome of allotetraploid cultivated cotton is relatively complex, and the research on resource mining and breeding of upland cotton is not in-depth enough. Based on the high-quality upland cotton genome sequence, the present invention uses population genome resequencing and genome-wide association analysis technology to identify the gene GhATE that is closely related to the fiber length trait of upland cotton. The arginyl-tRNA-protein transferase gene GhATE of the present invention was identified in the genome-wide association analysis ( Figure 1 ), expression level analysis of different cotton tissues and developmental stages ( Figure 2 ) are closely related to the cotton fiber length traits.

[0015] (2) The SNP genotype of GhATE in the population of relatively high fiber length varieties was verified by PCR technology, which is easy to operate, highly sensitive and accurate.

[0016] (3) According to the different SNP genotypes of GhATE, the variety population can be divided into two categories, and there is a significant difference in cotton fiber length between the two groups. This result further proves the correlation between this gene and the cotton fiber length trait ( Figure 3 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0018] Figure 1 This is the result of GWAS association analysis of cotton fiber length traits of the present invention;

[0019] Among them, the highest point of chromosome A10 represents the SNP site associated with the quality trait; the horizontal axis represents the position on the chromosome (Mb), and the vertical axis represents the significance of the association of the SNP site, expressed as -log10 (P value);

[0020] Figure 2 The expression level of GhATE in different tissues and developmental stages of cotton;

[0021] The horizontal axis represents different tissues, including root, stem, leaf, ovule and fiber; the ovule tissue includes 3 days before flowering, 1 day before flowering, the day of flowering, and 1 to 35 days after flowering; the fiber tissue includes 5 to 25 days after flowering; the vertical axis represents the relative expression level FPKM, which is a standard for measuring the relative expression level of genes;

[0022] Figure 3 The sequence information of GhATE of the present invention and the identification of different haplotypes;

[0023] Among them, a non-synonymous mutation SNP site was detected in the GhATE sequence in the variety population; the base of the genomic SNP site changed from A to G; the variety population was divided into different haplotypes, marked as GhATE L and GhATE S; L represents long fiber and S represents short fiber;

[0024] Figure 4 It is a comparative analysis of fiber length between different haplotypes of GhATE of the present invention;

[0025] Among them, the box plot represents the distribution of fiber length in the variety population; there are 190 and 22 varieties containing haplotypes AA and GG respectively; the gray box plot represents the fiber length trait distribution of haplotypes AA and GG; the horizontal line in the box represents the median of the trait distribution; * indicates a difference at the 0.05 level. DETAILED DESCRIPTION

[0026] The technical scheme in the embodiment of the present invention is described clearly and completely below in conjunction with the accompanying drawings in the embodiment of the present invention. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0027] 1. Mining of Arginyl-tRNA-Protein Transferase Genes Associated with Cotton Fiber Length Traits:

[0028] From 2011 to 2013, a detailed survey of cotton fiber length traits was conducted in Xinxiang, Henan, Anyang, Henan, and Korla, Xinjiang for 234 modern varieties or lines. At the same time, the whole genome of these 234 cotton varieties was resequenced to obtain 4.54Tb of sequencing data. These sequences were aligned to the genome sequence of TM-1 (V2.1), the genetic standard line of upland cotton, and the whole genome single nucleotide polymorphism markers (SNPs) were identified using Samtools software. A total of 5,566,352 high-quality SNPs (minimum gene frequency>0.05) were mined for subsequent analysis. The EMMAx software was used to perform genome-wide association analysis with phenotypes, and then the P<1×10 -6 SNP-associated signal sites were screened, and 111 cotton yield and fiber quality trait-associated sites were finally obtained. Among them, a trait-associated site (A10Gh:114152259) was identified on chromosome A10, which was significantly associated with cotton fiber length trait ( Figure 1 ). The candidate gene in the LD block region where the site is located is the arginyl-tRNA-protein transferase gene GhATE (GH_A10G2601), and its cDNA sequence and genomic sequence are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0029] 2. Acquisition of the Arginyl-tRNA-Protein Transferase Gene GhATE:

[0030] GhATE (GH_A10G2601) was obtained from the genome sequence of upland cotton. The full-length primers of the gene were designed according to the two ends of cDNA for PCR amplification. The primers are shown in Table 1. The PCR reaction program is as follows: 98℃ pre-denaturation for 5min; 98℃ denaturation for 10sec, 58℃ annealing for 5sec, 68℃ extension for 25sec, 34 cycles; and finally 68℃ extension for 5min. The PCR amplification product was sequenced and compared with cDNA to determine the accuracy of the sequence. The arginyl-tRNA-protein transferase gene GhATE was obtained.

[0031] Table 1 PCR amplification primer sequences

[0032]

[0033]

[0034] 3. Analysis of GhATE expression levels in different tissues and developmental stages of cotton:

[0035] In this experiment, RNA samples from tissues such as roots, stems, leaves, ovules, and fibers at different developmental stages were used for transcriptome sequencing. The average sequencing read length for each sample reached 25,530,023. The reads obtained by sequencing were aligned with the upland cotton genome TM-1 using TopHat2.1.1 software, and the transcriptome data were quantified using Cufflinks 2.2.1 software. The expression level was expressed as FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) value ( Figure 2 ). The experimental results showed that the gene GhATE was highly expressed at different stages of cotton ovule development, indicating that the gene was related to the formation of fiber length traits. This result shows that GhATE is indeed closely related to the factors affecting cotton fiber length and has a significant effect on improving cotton fiber length.

[0036] 4. Application of Arginyl-tRNA-Protein Transferase Gene in Identification of High Fiber Length Cotton:

[0037] GhATE sequences have SNP sites with non-synonymous mutations in the population, such as Figure 3 As shown, the base changed from A to G at the 1283bp position of the genomic sequence, and the corresponding position on the transcribed cDNA changed from A to G, causing the amino acid to change from Asp to Ser. Based on the location of the SNP site on chromosome A10 (A10Gh:114152259), amplification primers (Table 2) were designed at both ends for PCR amplification and sequencing. The PCR reaction procedure was as follows: 98℃ pre-denaturation for 5min; 98℃ denaturation for 10sec, 57℃ annealing for 5sec, 68℃ extension for 10sec, 34 cycles; and finally 68℃ extension for 5min.

[0038] Table 2 PCR amplification primer sequences

[0039]

[0040] Based on the base information and sequencing results of this SNP site (A10:114152259), the genotype of each variety population at this SNP site was analyzed, and 190 haplotype AA materials and 22 haplotype GG materials were identified (Table 2). Combining the GWAS association results and phenotypic survey data, the variety materials with AA haplotype were marked as GhATE L, and the variety materials with GG haplotype were marked as GhATE S ( Figure 3 ). L represents high fiber length; S represents low fiber length.

[0041] At the same time, the correlation between the two haplotypes in cotton fiber length was calculated using the t-test method. Figure 4). The results showed that the cotton fiber length of haplotype GhATEL(A) increased by 2.90% compared with GhATES(G);

[0042] From the above results, it can be seen that the gene GhATE has important research value in improving new cotton varieties with improved cotton fiber length. On the one hand, molecular markers can be designed based on the two haplotypes of the gene GhATE, which can effectively identify the cotton fiber length trait and have great application value in the research of breeding cotton varieties with high fiber length. On the other hand, taking the breeding of new high-fiber-length cotton varieties as an example, genes containing the high-fiber-length haplotype GhATE (A) can be transferred into cotton varieties through genetic engineering to improve cotton fiber length, and SNP sites in the low-fiber-length haplotype GhATE (G) can also be site-directed mutated to transform it into a high-fiber-length haplotype to breed new high-fiber-length cotton varieties.

[0043] Table 2 Distribution of two genotypes in various varieties

[0044]

[0045]

[0046] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. An arginyl-tRNA-protein transferase gene for improving upland cotton fiber length, characterized in that: The arginyl-tRNA-protein transferase gene sequence is shown in SEQ ID NO.

2.

2. Use of the arginyl-tRNA-protein transferase gene for improving upland cotton fiber length according to claim 1 in identifying high fiber length upland cotton.

3. Use of the arginyl-tRNA-protein transferase gene for improving upland cotton fiber length according to claim 1 in improving the fiber length trait of upland cotton.

4. Use of the arginyl-tRNA-protein transferase gene for improving upland cotton fiber length according to claim 1 in breeding new upland cotton varieties with high fiber length by genetic engineering means.

5. A method for screening high fiber length upland cotton varieties, characterized in that: The genotype at the 1283bp position of the arginyl-tRNA-protein transferase gene of upland cotton was detected, and the upland cotton with genotype A was high-fiber-length upland cotton.

6. The method according to claim 5, characterized in that The primers for detecting the genotype are: the upstream primer is shown in SEQ ID NO.5, and the downstream primer is shown in SEQ ID NO.6.

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