Application of GhGRP protein and coding gene thereof in improvement of drought resistance of cotton

Abstract

The invention discloses application of GhGRP protein and a coding gene thereof in improvement of drought resistance of cotton, and relates to the technical field of genetic engineering, and the amino acid sequence of the GhGRP protein is shown as SEQ ID NO.2. Overexpression of the gene can significantly enhance the drought resistance of cotton, and a mutant with the gene knocked out shows high sensitivity to drought stress. The cloned drought-resistant gene and the encoded protein thereof can be widely applied to improvement of the drought stress resistance of cotton, so that not only is a solid theoretical support provided for research of a crop drought-resistant mechanism, but also an important material foundation is laid for drought-resistant genetic improvement and molecular breeding of crops; the method has profound significance in the field of drought-resistant biological breeding of crops.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of genetic engineering, and more particularly to application of GhGRP protein and a coding gene thereof in improving drought resistance of cotton. BACKGROUND

[0002] Cotton is an important economic crop. Drought is a core disaster affecting cotton production and can cause a chain of damage throughout the whole growth period. Drought at the seedling stage can inhibit root development and seedling growth, leading to uneven emergence, weak and thin seedlings, and decreased stress resistance. The period from the squaring stage to the boll opening stage is a critical period for water requirement, and water deficiency can cause a large number of bolls to drop, reduce boll weight, and hinder fiber development, thereby directly reducing yield and quality. Drought at the boll opening stage can also cause the cotton bolls to crack, affecting picking efficiency, and causing the fiber length and strength to decrease. In addition, drought can exacerbate soil salinization, affect nutrient uptake, and induce secondary disasters such as insect pests. In the case of severe drought, cotton can prematurely senesce and wilt, resulting in a significant reduction in yield or even complete loss. Therefore, under the extremely severe situation of water resources, breeding drought-resistant cotton varieties to improve the comprehensive drought tolerance of cotton is of great significance to the sustainable development of the cotton industry and the protection of food security.

[0003] As a rapid and effective method for genetic improvement of cotton, transgenic technology has brought great development space for cotton genetic breeding. Research and identification of drought-resistant genes in cotton not only provide a theoretical basis for exploring the drought regulation mechanism of cotton, but also provide target genes and material resources for improving the drought resistance of cotton by biological breeding methods.

[0004] Plant drought resistance response involves multiple drought resistance mechanisms and signal transduction pathways, and the molecular mechanism is complex. Studies have shown that some genes encoding protective proteins, drought response signal molecules, and drought-related transcription factors can improve the drought tolerance of plants by regulating defense mechanisms through a series of cellular and molecular signal adjustments such as signal perception, physiological response, and expression regulation of stress-related genes. Although some progress has been made in the excavation of drought-resistant genes in cotton, the number of functional genes is still very limited.

[0005] Therefore, it is of great significance to plant drought resistance molecular breeding to deeply excavate drought-related key genes and create drought-resistant germplasm resources of cotton. SUMMARY

[0006] Therefore, the application provides application of GhGRP protein and a coding gene thereof in improving drought resistance of cotton.

[0007] To achieve the above-mentioned purpose, the application adopts the following technical solutions: The application of enhancing the content and / or activity of GhGRP protein in improving the drought resistance of cotton, wherein the amino acid sequence of the GhGRP protein is shown as SEQ ID NO. 2.

[0008] It is another object of the present application to provide a method for improving drought resistance of cotton GhGRP The application of the expression amount of the gene in improving drought resistance of cotton GhGRP The amino acid sequence encoded by the gene is shown in SEQ ID NO. 2.

[0009] As a preferred technical solution, the GhGRP The nucleotide sequence of the gene is shown in SEQ ID NO. 1 or its complementary sequence.

[0010] It is another object of the present application to provide a method for improving drought resistance of cotton GhGRP The application of the biological material for improving the expression amount of the gene in improving drought resistance of cotton, wherein the biological material is one of the following: a. a nucleic acid molecule capable of encoding an amino acid sequence as shown in SEQ ID NO. 2; b. an expression cassette capable of overexpressing the nucleic acid molecule of a; c. a recombinant vector containing the expression cassette of a; d. a recombinant microorganism containing the expression cassette of a or the recombinant vector of b; e. a non-regenerative plant part containing the expression cassette of a, the recombinant vector of b or the recombinant microorganism of c.

[0011] It is another object of the present application to provide a breeding method for improving drought resistance of cotton, which overexpresses the gene in a target plant body by using genetic engineering means, so as to increase the gene expression amount. GhGRP

[0012] Beneficial effects: the present application provides a gene for improving drought resistance of cotton GhGRP Overexpression of the gene can significantly enhance the drought resistance of cotton, and the mutant with the gene knocked out shows high sensitivity to drought stress. The cloned drought resistance gene and the encoded protein can be widely applied to improve the drought stress resistance of cotton, which not only provides a solid theoretical support for the research on the drought resistance mechanism of crops, but also lays an important material foundation for the genetic improvement and molecular breeding of crops, and has a profound significance in the field of drought resistance biological breeding of crops. BRIEF DESCRIPTION OF DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and those skilled in the art can obtain other drawings according to the provided drawings without creative labor.

[0014] Figure 1 GhGRP protein sequence signal peptide prediction.​

[0015] Figure 2 Results of GhGRP gene expression levels in overexpressed and wild-type plants.

[0016] Figure 3 This is a first-generation sequencing detection for dual-target gene editing; where A represents the target detection of gene A subgroup copies; and B represents the target detection of gene D subgroup copies.

[0017] Figure 4 This study aims to predict gene-edited knockout mutant proteins. A represents the GhGRP protein mutation status of different representative strains in subgroup A, B represents the GhGRP protein mutation status of different representative strains in subgroup D, and CE represent the three-dimensional structures of GhGRP and mutant proteins in wild-type, ms2, and ms3 mutant strains, respectively.

[0018] Figure 5 The phenotypes of transgenic cotton plants under drought stress are shown; where A represents the plants before drought stress treatment and B represents the plants 8 days after drought stress treatment.

[0019] Figure 6 Analysis of redox-related physiological and biochemical indicators in different transgenic strains under drought stress. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 are within the scope of protection of the present invention.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art described herein.

[0022] The term "expression cassette" generally refers to a nucleic acid construct containing sufficient nucleic acid elements to express a target gene. A typical expression cassette includes a promoter, a multiple cloning site (MCS), and a terminator. Expression cassettes may also include the target gene, marker genes (such as TK, DHFR, CAT, and NEO genes), ribosome recognition and binding sites (SDs), transcription factor binding sites (TFBSs), enhancers, silencers, repressors, introns, poly(A) signal sequences, and / or mRNA splicing signal sequences. Elements within an expression cassette can be directly linked or indirectly linked through adapters.

[0023] The term "vector" generally refers to a vector capable of delivering exogenous DNA or a target gene into host cells for amplification and / or expression. This vector can be a cloning vector or an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material they carry to be amplified and / or expressed within the host cells. Those skilled in the art can select appropriate vectors based on the purpose of genetic engineering and the properties of the recipient cells. The vectors include, but are not limited to: plasmids, phages (such as λ phage or M13 phage), cosmids (i.e., Cosmids), phagemids, shuttle vectors (such as yeast expression vectors), Ti plasmids, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), P1 artificial chromosomes (PAC), or Ti plasmid artificial chromosomes (TAC)), and viral vectors (such as baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, poxviruses, papillomaviruses, papillomaviruses (such as SV40), and herpesviruses (such as herpes simplex virus)). A vector may contain multiple elements controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain a replication origin site.

[0024] The term "microorganism" typically includes bacteria, viruses, fungi, actinomycetes, rickettsiae, mycoplasma, chlamydiae, spirochetes, algae, etc.

[0025] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by linking a foreign target gene to a vector in vitro. It can be constructed in any suitable way, as long as the constructed recombinant vector can carry the foreign target gene into the recipient cell and provide the foreign target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0026] The term "recombinant microorganism" generally refers to a recombinant microorganism whose genes have been manipulated and modified to obtain a functionally altered microorganism. This can be achieved by introducing a foreign target gene or recombinant vector into the target microorganism, or by directly editing the endogenous genes of the target microorganism.

[0027] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0028] Example 1 GRP Gene cloning and bioinformatics analysis Primers GhGRP-F (5'-ATGGCTTTCAAAGCTCTGCT-3') and GhGRP-R (5'-TCAAGGACATTTGTGCTTG-3') were designed, and cDNA from Jimian 262 leaf samples was used as a template for homologous cloning via RT-PCR to obtain... GRP Gene.

[0029] In this study, total RNA was extracted using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Tiangen Biotech (Beijing) Co., Ltd., DP441) and the instructions were followed. RNA was extracted from the leaves of Jimian 262 and reverse transcribed into cDNA according to the instructions of the PrimeScript™ RT reagent Kit (TaKaRa).

[0030] The PCR amplification program was as follows: 95℃ for 5 min; 95℃ for 30 sec, 56℃ for 30 sec, 72℃ for 1 min, 32 cycles; 72℃ for 10 min; incubate at 4℃.

[0031] The total volume of the PCR amplification system is 20 μl, including: template (cDNA) 1 μL, GhGRP-F 0.5 μL, GhGRP-R 0.5 μL, 2×Taq PCR master Mix 10 μL, and ddH2O 8 μL.

[0032] Cloning GRP The full-length CDS sequence of the gene is 795 bp, and its nucleotide sequence is shown in SEQ ID NO.1: ATGGCTTTCAAAGCTCTGCTGCTGTTACTATTGGCCACTTTTCTGCTTGTCTCAACAACAGTTGCTTCCAATGAAGTGGGAGAGAAGACTGAGATTAAGTATGCTGCTCCTGTTCCAGTGAAGGCACCTATCCCTGCTCCACCCGTTAAGCCTCCCACCACTCCGGCACCGCCGTACAAGGCCCCAACTCCAGCACCCCCAACCAAGGCCCCCACTCCACCATATAAACCACCAGCTCCTGCGCCACCAACCAAGGCTCCTACTCCACCATATAAGCCCCCAGCTCCTGCGCCACCAACCAAGGCTCCTACTCCACCATATAAGCCCCCAGCACCAGCACCACCAACCAAGGCTCCTACTCCCCCATATAAACCCCCTACTCCCGCACCGGCACCTCCAGTCAAGGCCCCTACACCCCCATATAAGCCCCCAACACCTGCTCCAGCACCCCCAACCAAAGCACCAACTCCAGCACCAGCACCACCAACTAAAGCACCAACTCCCCCATATAAGCCCCCAGTTCCTACACCTCCAGTTAAGCCACCAACAACTCCAGCACCGCCTTACAAGCCACCAAGTCCACCATTGCCGCCTGTTAGGACAAAAAAGGATTGCATCCCATTATGTGGACAAAGGTGCAAATTACACTCAAGGACTAACCTATGCTTGAGAGCTTGCATGACATGCTGTGACAGATGCAAATGTGTCCCACCAGGGACATATGGCAACAGAGAAATGTGTGGCAAATGTTACACTGATATGAGAACCCACCGCAACAAGCACAAATGTCCTTGA, SEQ ID NO.1。

[0033] The encoded protein contains 264 amino acids, and the amino acid sequence is shown in SEQ ID NO.2: MAFKALLLLLLATFLLVSTTVASNEVGEKTEIKYAAPVPVKAPIPAPPVKPPTTPAPPYKAPTPAPPTKAPTPPYKPPAPAPPTKAPTPPYKPPAPAPPTKAPTPPYKPPAPAPPTKAPTPPYKPPTPAPAPPV KAPTPPYKPPTPAPAPPTKAPTPAPAPPTKAPTPPYKPPVPTPPVKPPTTPAPPYKPPSPPLPPVRTKKDCIPLCGQRCKLHSRTNLCLRACMTCCDRCKCVPPGTYGNREMCGKCYTDMRTHRNKHKCP*, SEQ ID NO.2.

[0034] SignalP 5.0 analysis revealed that the protein contains a 22-amino acid signal peptide at its N-terminus, suggesting it is a secretory protein (see appendix). Figure 1 ).

[0035] Example 2: Genetic transformation to obtain transgenic cotton Using pCAMBIA3101 as a backbone vector, a whole-genome synthesis technique was employed to construct... GRP Gene overexpression vector.

[0036] Using pRGEB32-GhU6.9-NPT II (Wang P, et al. (2018) High efficient multisites genome editing in allotetraploid cotton ( Gossypium hirsutum (using CRISPR / Cas9 system. Plant Biotechnology Journal 16.) as the scaffold vector, using CRISPRP 2.0 online software, in GRPTwo sgRNAs [sgRNA1: 5'-CGGAGTGGTGGGAGGCTTAA-3' (SEQ ID NO.3) and sgRNA2: 5'-GTGGTTTATATGGTGGAGTG-3' (SEQ ID NO.4)] were designed at the 5' end of the third exon region of the gene, simultaneously targeting two homologous genes in subgroups A and D of upland cotton. The sequences were inserted between two Bsa I restriction sites in the backbone vector using gene synthesis technology. Based on the vector and the inserted sequence, detection primers U6.9-F (5'-TGTGCCACTCCAAAGACATCAG-3', SEQ ID NO.5) and GRP-T2-R (5'-ttctagctctaaaacGTGGTTTATATGGTGGAGTG-3', SEQ ID NO.6) were designed. Positive clones were identified by PCR, and a CRISPR gene editing knockout vector was constructed.

[0037] Following the method described by Jin Shuangxia (Optimization of Cotton Genetic Transformation System and Creation of Mutants [D]. Huazhong Agricultural University, 2006), Agrobacterium-mediated genetic transformation of cotton was performed, and the aforementioned vectors were introduced into upland cotton Jin668. The obtained overexpression and gene-edited regenerated plants were subjected to PCR positive detection and editing result testing. Seeds from the overexpression positive and gene-edited plants were harvested, which were designated as T0 seeds. After planting the T0 seeds, PCR and editing result testing were performed again to screen for target lines. This process was repeated up to T3, resulting in 5 lines. GRP Overexpressing transgenic lines (OE1, OE2, OE6, OE7, and OE9), and 5 gene-editing knockout mutant lines (ms1-ms5).

[0038] The primers used for the detection of overexpression plant specificity were 35S-F (5'-GACGCACAATCCCACTATCC-3', SEQ ID NO.7) and GhGRP-seq-R (5'-ATGGTCTTTGTAGTCCATAGGAC-3', SEQ ID NO.8). The primers used for the detection of gene expression levels in overexpression plants were GhGRP-qF (5'-GTGAAGGCACCTATCCCTGC-3', SEQ ID NO.9) and GhGRP-qR (5'-GGGTTTATATGGAGTGGGGC-3', SEQ ID NO.10). Histone3 was used as an internal reference gene, and the detection primers were Histon3-F (5'-TCAAGACTGATTTGCGTTTCCA-3', SEQ ID NO.11) and Histon3-R (5'-GCGCAAAGGTTGGTGTCTTC-3', SEQ ID NO.12). The results showed that the relative expression level of the target gene GhGRP in the overexpressing plants was 53.11–89.96 times that of the control wild type (see Appendix). Figure 2 ).

[0039] Genomic DNA was extracted from gene-edited plants. Primers GhGRP-sanger-F (5'-GCCATTGTGGTGCATAAGAC-3', SEQ ID NO.13) and GhGRP-sanger-R (5'-TGGTGGACTTGGTGGCTTG-3', SEQ ID NO.14) were designed to amplify sequences covering two gene-editing target sites. The PCR products were TA cloned and Sanger sequenced, and the results were compared with wild-type reference sequences. The mutated base sequences (see Appendix) were then analyzed. Figure 3 The mutated protein sequence was translated into amino acids and analyzed using Snapgene alignment. The structure of the mutated protein was predicted using the online software Swiss-model (https: / / swissmodel.expasy.org / ). Results showed significant editing mutations at both target sites. Primary structure prediction of the amino acid sequences translated from the mutated base sequences revealed that, except for ms3_D, all other editing mutation types resulted in frameshift mutations in the coding amino acid sequence, along with premature termination of the coding sequence (see Appendix). Figure 4 (A and B in the text). Modeling and predicting the three-dimensional structure of the mutant protein revealed that the mutant protein altered the original amino acid arrangement, folding, and spatial conformation (see appendix). Figure 4 The gene editing process (CE) resulted in the loss of the original protein's structure, function, and activity. Therefore, it can be concluded that the gene editing results in different strains all achieved effective knockout of this gene.

[0040] Example 3: Identification of drought resistance in transgenic cotton Transgenic overexpression lines (OE6 and OE9), gene-edited knockout lines (ms1 and ms2), and wild-type recipient seeds were sown in nutrient pots with a 1:1 ratio of nutrient soil to vermiculite and cultured in a culture room at 27°C with 8 hours of light and 16 hours of darkness. Ten pots were planted for each line, with three seedlings per pot.

[0041] Natural drought treatment was applied to cotton seedlings at the three-leaf stage. Two pots of cotton from each line were watered normally as a control, while the remaining eight pots were left unwatered. Phenotypic changes were observed and recorded, and photographs were taken. The first true leaf of cotton was harvested at 0, 2, 4, 6, and 8 days after watering was stopped, and quickly frozen in liquid nitrogen. RNA was extracted to detect the expression of the target gene. Three plants and three true leaves from each pot served as three biological replicates. The second and third true leaves of plants 6 days after watering was stopped were harvested, immediately torn and mixed, and the material was accurately weighed. Redox indicators such as proline content, MDA content, and SOD activity were determined according to the relevant kit instructions from Suzhou Keming Biotechnology Co., Ltd.

[0042] As attached Figure 5 As shown, before drought stress treatment, there were no significant phenotypic differences among the different transgenic lines. After drought stress treatment, all plants began to lose water and wilt, with the drying and severity gradually increasing with the duration of treatment. Compared with the wild type, the overexpression lines showed less water loss phenotype in their leaves, with only some older leaves exhibiting edge curling and slight water loss bending in the upper part of the stem. Compared with the wild type and the overexpression lines, the gene-edited mutants showed more pronounced water loss, wilting, and wrinkling. This indicates... GhGRP Plants overexpressing the gene showed increased tolerance to drought stress, while gene-edited mutants were more sensitive to drought stress.

[0043] As attached Figure 6 As shown, after 6 days of drought stress treatment, the effect on... GhGRP The results of proline (PRO), malondialdehyde (MDA), and superoxide dismutase (SOD) activities were measured in overexpression plants, gene-edited mutants, and wild-type plants, respectively. GhGRP The accumulation of PRO and the activity of SOD in the leaves of the gene-overexpressing plants were significantly higher than those of the wild type, while the MDA content was significantly lower (P<0.05). Conversely, the MDA content in the leaves of the gene-edited mutant was significantly higher than that of the control, while the PRO content and SOD activity were significantly lower than those of the control plant leaves (P<0.05). These results indicate that... GhGRP Overexpression enhances the plant's tolerance to drought, while gene-edited mutants become more sensitive to drought stress.

[0044] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0045] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of enhancing the content and / or activity of GhGRP protein in improving the drought resistance of cotton, characterized in that, The amino acid sequence of the GhGRP protein is shown in SEQ ID NO.

2.

2. Improve GhGRP The application of gene expression levels in improving the drought resistance of cotton is characterized by, The GhGRP The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

3. The application according to claim 2, characterized in that, The GhGRP The nucleotide sequence of the gene is shown in SEQ ID NO.1 or its complementary sequence.

4. Improve GhGRP The application of biomaterials with high gene expression levels in improving the drought resistance of cotton is characterized by, The biomaterial is one of the following: a. Nucleic acid molecules capable of encoding amino acid sequences such as SEQ ID NO.2; b. An expression cassette capable of overexpressing the nucleic acid molecules described in a; c. A recombinant vector containing the expression cassette described in a; d. Recombinant microorganisms containing the expression cassette described in a or the recombinant vector described in b; e. A non-renewable plant portion containing the expression cassette described in a, the recombinant vector described in b, or the recombinant microorganism described in c.

5. A breeding method for improving the drought resistance of cotton, characterized in that, Using genetic engineering techniques, overexpression in target plants GhGRP Genes, causing their gene expression levels to increase.

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