Application of GhRIC6b gene in regulating yield and quality of cotton

Abstract

The application discloses application of a GhRIC6b gene in regulating cotton yield and quality and belongs to the technical field of genetic engineering. The application promotes fiber development, improves cotton lint percentage, and improves fiber length, strength, regularity index, elongation rate and other qualities by overexpressing the GhRIC6b gene in cotton. The application provides a feasible strategy for simultaneously improving the yield and quality of cotton fiber by improving cotton varieties.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of the GhRIC6b gene in regulating cotton yield and quality. Background Technology

[0002] Cotton is an important economic crop, providing natural fiber raw materials for the textile industry. High-quality cotton fiber has always been a major demand for my country's raw cotton imports, and it is also a key focus of domestic cotton breeding efforts. Therefore, increasing cotton yield and improving fiber quality are of great significance to the sustainable development of my country's cotton industry and cotton textile industry.

[0003] Traditional genetic breeding methods are time-consuming and labor-intensive. Moreover, cotton fiber yield and quality are controlled by quantitative traits, which are strongly negatively correlated genetically, making simultaneous improvement difficult. Furthermore, cotton is easily affected by many environmental factors. Relying solely on traditional breeding techniques makes it difficult to quickly and efficiently improve cotton quality. However, the development of modern molecular biology techniques and the successful application of transgenic technology in cotton have provided new ideas and methods for improving cotton quality. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide an application of the GhRIC6b gene in regulating cotton yield and quality, so as to solve the existing technical problem that cotton yield and quality cannot be improved simultaneously.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is to provide the application of the GhRIC6b gene in regulating cotton yield and quality, and the nucleotide sequence of the GhRIC6b gene is shown in SEQ ID NO.1.

[0006] Based on the above technical solution, the present invention can be further improved as follows:

[0007] Furthermore, the amino acid sequence of the protein encoded by the GhRIC6b gene is shown in SEQ ID NO.2.

[0008] Furthermore, it can be applied to improve crop yield and fiber quality by overexpressing the GhRIC6b gene.

[0009] Furthermore, the crop is a dicotyledonous plant.

[0010] Furthermore, the dicotyledonous plant is cotton.

[0011] Furthermore, yield characteristics include lint percentage; fiber quality includes fiber length, upper half average length, fiber strength, uniformity index, and elongation.

[0012] The present invention also discloses an overexpression vector containing the GhRIC6b gene.

[0013] The present invention also discloses a formulation for regulating crop yield and fiber quality, comprising an overexpression vector.

[0014] The present invention also discloses a method for obtaining cotton varieties with high yield and fiber quality, which involves overexpressing the GhRIC6b gene, loading it into Agrobacterium, infecting cotton explants, and cultivating the cotton varieties to obtain high yield and fiber quality.

[0015] The beneficial effects of this invention are as follows: By overexpressing the GhRIC6b gene in cotton, it was found that early fiber cell elongation and faster fiber cell initiation are promoted, while the length of mature fibers is increased, resulting in a significant improvement in cotton fiber length. Simultaneously, the average upper half length, fiber strength, uniformity index, elongation, and lint percentage are all increased. Using the method of this invention to improve cotton can overcome the incompatibility between cotton yield and quality during breeding, balancing cotton yield and quality, and providing a feasible strategy for the simultaneous genetic improvement of cotton fiber yield and quality. Attached Figure Description

[0016] Figure 1 A schematic diagram of the construction of the GhRIC6b plant expression vector;

[0017] Figure 2 Comparison of GhRIC6b expression levels;

[0018] Figure 3 A photograph of cotton fibers 5 days after flowering;

[0019] Figure 4 Statistics on fiber length of cotton 5 days after flowering;

[0020] Figure 5 A photograph of mature cotton fibers;

[0021] Figure 6 Statistics on the length of mature cotton fibers;

[0022] Figure 7 This is the measurement result of cotton lint percentage. Detailed Implementation

[0023] The specific embodiments of the present invention are described below to facilitate understanding of the invention by those skilled in the art. Unless otherwise specified, specific conditions are applied according to conventional conditions or the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various modifications are obvious as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims. All inventions utilizing the concept of this invention are protected.

[0024] Unless otherwise specified, all reagents and pharmaceuticals used in the following examples are commercially available, and all materials and methods used are based on Molecular Cloning: A Laboratory Manual (Sambrook and Russell, 2001).

[0025] GhRIC6b is a class of proteins that interact with plant-specific small G proteins (ROPs). These proteins all contain a highly conserved CRIB (Cdc42 / Rac-interactive binding) domain, which is a crucial domain for GTP-dependent ROP-RIC interactions. RICs are important downstream effectors of small G protein ROPs. RICs interact with different ROP members, controlling various ROP-dependent signaling pathways and performing different functions in different parts of the plant.

[0026] The nucleotide sequence of the GhRIC6b gene is as follows:

[0027] ATGAAAGGATTGCTAAAAGGTTTTAGATACATTACTCAATTATTTGAAAGTGACAAA

[0028] GAGCCAGAAATGCAGATTAGTATGCCAACAGACGTAAAGCATGTGGCTCATATAGGATG

[0029] GGATGGTCCATCAGCTGTAAATTCTACACCCAGCTGGATGAATGAGTTCAAAACACCAG

[0030] CAGGAGGGCTTCAATCAACACCTTTAGCTCAAGCAGGACAAGAGAGGTTGAGCCGAAA

[0031] AGGTTCGAGGGCTCAAAGTTCTTCAACAAGAGACATGCCCGACCTGCCTAAATCATCAA

[0032] AGCGCACTTCATCCACTAAAGAAAATGCTTCTTCAACAAAGCAATCGAAGAAGCCTTCC

[0033] AAGTCGACTAGAAAACCCAAGGATGCAAACCAAACAACCGAGACAACCACAAAAGAC

[0034] CCAAAGAAAAGTAGGCGAAAGAAGGTGAAAGACATGGGTGGTGAAGGAAGCTCTAGG

[0035] CGGTCCAGAACCACCCAGGAGTCAGATACTCTGTCCGAGGCAGGGTCCCTAATCAGTTG

[0036] TGATTCAGAGTTTGTAGAAGGGGAGGCTAATTGA (SEQ ID NO. 1).

[0037] The amino acid sequence of the protein encoded by the GhRIC6b gene is as follows:

[0038] MKGLLKGFRYITQLFESDKEPEMQISMPTDVKHVAHIGWDGPSAVNSTPSWMNEFKT

[0039] PAGGLQSTPLAQAGQERLSRKGSRAQSSSTRDMPDLPKSSKRTSSTKENASSTKQSKKPSKS

[0040] TRKPKDANQTTETTTKDPPKSRRKKVKDMGGEGSSRRSRTTQESDTLSEAGSLISCDSEFV

[0041] EGEAN*(SEQ ID NO.2).

[0042] The sequences of the primers used in this invention are shown in Table 1.

[0043] Table 1 Primer sequence list

[0044]

[0045]

[0046] Example 1: Construction of the carrier

[0047] 1. Plant RNA extraction

[0048] Approximately 1g of fresh cotton material was selected and ground into a fine powder in a mortar with liquid nitrogen. RNA was extracted using the EASYspin Plant RNA Kit from Adley Biotechnology Co., Ltd. The extraction steps were strictly performed according to the included instructions. After extraction, 2μL was taken for agarose gel electrophoresis to detect the quality of the extracted RNA.

[0049] 2. cDNA synthesis

[0050] After RNA extraction, cDNA was synthesized using the TaKaRa PrimeScript RT reagent Kit with gDNAEraser, following the instructions.

[0051] 3. Obtaining the cotton gene GhRIC6b

[0052] Primers SEQ ID NO.3 and SEQ ID NO.4 were designed based on the nucleotide sequence of GhRIC6b (SEQ ID NO.1) to directly amplify the GhRIC6b gene from the cDNA of ovules on the day of flowering in upland cotton. After recovering the GhRIC6b fragment, the fragment was ligated into the pTOPO-Blunt cloning vector, and after sequencing verification, it was used for subsequent vector construction.

[0053] 4. Construction of GhRIC6b overexpression vector

[0054] The build process is as follows Figure 1 As shown, the backbone vector used to construct the plant expression vector includes the pLN vector. For ease of identification of transgenic plants, a fluorescent protein gene GFP is added to the constitutive promoters UBQ10 and polyA (which terminates transcription) for fusion with the target gene GhRIC6b. NPTII is the neomycin phosphotransferase gene; UBQ10 is a plant constitutive promoter derived from Arabidopsis thaliana; 35S is a plant constitutive promoter derived from cauliflower mosaic virus; nos Terminator is the crown gall synthase gene terminator; 35S Terminator is the 35S gene terminator; LB is the left boundary of T-DNA; and RB is the right boundary of T-DNA.

[0055] The pLN plant expression vector, modified from the pCambia2300 vector, was linearized by digestion with BamHI and EcoRII. Various restriction endonucleases were products of Thermo Scientific, and digestion was performed according to the manufacturer's instructions. The nucleotide sequence of GFP is SEQ ID NO.5, with the stop codon removed. Primers with homologous arms were designed to amplify the mCherry sequence (SEQ ID NO.6 and SEQ ID NO.7) and primers with homologous arms to amplify the GhRIC6b sequence (SEQ ID NO.8 and SEQ ID NO.9). The linearized pLN vector, GFP, and GhRIC6b amplification products were mixed at a molecular ratio of 1:3:3 and reacted at 37°C for 30 min under the catalysis of homologous recombinase, followed by transformation into *E. coli*, thus completing the construction of the pLN::proUBQ10::GFP::GhRIC6b plant expression vector. GhRIC6b was constructed in the pLN plant expression vector for cotton genetic transformation. The cotton experimental material used was upland cotton (Gossypium hirsutum L).

[0056] Example 2: Conversion of cotton

[0057] 1. The constructed plant expression vector plasmid was introduced into Agrobacterium GV3101 using electroporation.

[0058] Referring to the Bio-RAD MicroPulser user manual, the GhRIC6b overexpression vector constructed in Example 1 was introduced into Agrobacterium GV3101 via electroporation.

[0059] The Agrobacterium electroporation transformation procedure is as follows: Remove competent Agrobacterium cells and thaw them on ice. Rinse the electroporation cuvette 5-6 times with sterile water and place it on ice for later use. Add 20-50 ng of plasmid to the competent cells and mix thoroughly. Turn on the Bio-Rad electroporator and select the Agr / Bacteria mode for electroporation. Immediately after electroporation, 800 μL of YEB liquid medium (containing 0.5% sucrose (W / V), 0.1% yeast extract for bacteria (W / V), 1% tryptic peptone for bacteria (W / V), 0.05% MgSO4·7H2O (W / V), and 1.5% agar powder (W / V), pH=7.0) was added. The culture was incubated at 28℃ and 200 rpm for 3 h with shaking. Then, the culture was centrifuged at 10000 rpm for 1 min, the supernatant was discarded, and about 100 μL of medium was left to resuspend the bacterial cells. The bacterial suspension was evenly spread on YEB plates containing 50 μg / mL Kan (caramycin) and 50 μg / mL Rif (rifampicin), and incubated at 28℃ inverted for 48 h.

[0060] 2. Constitutive expression of the GhRIC6b vector was integrated into the cotton genome.

[0061] Genetic transformation of cotton was performed using Agrobacterium tumefaciens-mediated transformation (the culture media used in the transformation process are shown in Table 2). The T-DNA segment of the above-mentioned plant expression vector was introduced into cotton using Agrobacterium-mediated embryogenic callus. The specific method is as follows:

[0062] (1) Cotton seed germination: For upland cotton cultivars, remove the outer shell from the seeds, select large and plump kernels, place them in an Erlenmeyer flask, sterilize with 75% alcohol for 1-2 minutes, rinse twice with sterile water; then sterilize with 0.1% mercuric chloride (HgCl2) for 10 minutes, rinse six times with sterile water. Incubate with sterile water at room temperature and 120 rpm on a shaker for 12 hours. Inoculate the kernels with the radicle facing down into the seed germination medium and germinate in the dark at 28℃ for 2-3 days to obtain sterile cotton seedlings.

[0063] (2) Culture of Agrobacterium transformation: Agrobacterium strain containing the above-mentioned plant expression vector was inoculated onto YEB solid medium containing 50 mg / L kanamycin and 125 mg / L streptomycin and streaked. A single colony of newly streaked and activated Agrobacterium was then inoculated into 5 mL of YEB liquid medium containing the same antibiotics and cultured overnight at 28°C with shaking at 200 rpm. 1 mL of the bacterial culture was then added to 20–25 mL of YEB liquid medium containing the same antibiotics for secondary activation and cultured at 28°C with shaking at 200 rpm until OD (Organic Degrees Per Minute) was reached. 600 The concentration was 0.8–1.0. Finally, the cells were centrifuged at 10,000 rpm for 1 min, collected, and resuspended in an equal volume of liquid co-culture medium (containing 40 mg / L acetylsalicylic acid, AS) as the infection solution for later use.

[0064] (3) Inoculation and co-culture: Cut sterile cotton hypocotyls into small segments of about 0.5 cm and place them in Erlenmeyer flasks containing the inoculation solution. Inoculate at 28°C and 100 rpm for 45 min on a shaker. Discard the bacterial solution and inoculate the inoculated hypocotyl segments onto solid co-culture medium (containing 40 mg / L acetylsylgenone, AS). Incubate at 28°C in the dark for 2 days.

[0065] (4) Screening of transformants: After co-culture, the hypocotyl segments were transferred to a selection medium for sterilization and selection culture, and cultured under light at 28°C for 2 weeks. The hypocotyls were then transferred to a subculture medium and subcultured every 2 weeks. After the hypocotyls swelled and produced a large amount of callus, the callus, along with the hypocotyl segments, was transferred to an embryogenic callus induction medium and subcultured every 3 weeks. Once fine, granular embryogenic callus appeared on the callus surface, the callus was transferred to a liquid suspension medium and cultured at 150 rpm for 2 weeks to obtain somatic embryos. The somatic embryos were then rinsed 3-4 times with sterile water. The washed and settled somatic embryos were transferred to a somatic embryo maturation and elongation medium and cultured under light at 28°C. The resulting buds were further transferred to a new somatic embryo elongation and elongation medium and subcultured every 3 weeks. When the seedlings reached approximately 5 cm in height, they were removed and hydroponically cultured. After hardening off, they were transplanted into flowerpots for further growth.

[0066] Table 2. Culture media for Agrobacterium tumefaciens-mediated genetic transformation of cotton.

[0067]

[0068]

[0069] 3. Obtain cotton with improved fiber properties

[0070] Transgenic cotton of generation T0 was cultivated in a greenhouse, and the self-pollinated cotton seeds were collected and sown. T1 generation cotton was planted in the field (Chongqing), and the fibers were collected for observation. Non-transgenic T1 generation cotton that was self-pollinated from T0 generation transgenic cotton was used as the control group (WT).

[0071] Example 3: Identification of GhRIC6b overexpression level in transgenic cotton

[0072] RNA was extracted from cotton fibers 2-3 days after flowering, and its expression level was identified by RT-PCR using primers SEQ ID NO.10 and SEQ ID NO.11 (internal reference gene was GhHIS3, and primers were SEQ ID NO.12 and SEQ ID NO.13, respectively).

[0073] The results are as follows Figure 2 As shown, compared with the wild type, the expression level of GhRIC6b in UBQ10::GFP::GhRIC6b transgenic cotton (#0 and #3 are two independent transformant lines of UBQ10::GFP::GhRIC6b transgenic cotton) was significantly upregulated, indicating that GhRIC6b was overexpressed in transgenic cotton.

[0074] Example 4: Fiber length determination of transgenic cotton 5 days after flowering

[0075] The developmental status of cotton fibers at the onset of flowering was determined by statistically analyzing fiber length 5 days after flowering. Ten ovules each from UBQ10::GFP::GhRIC6b transgenic and wild-type cotton plants were collected 5 days after flowering. The fibers on the ovules were combed with a brush, and the fiber length was measured. The results are as follows: Figure 3 and Figure 4 As shown.

[0076] Figure 3 Five days after flowering, the fibers of both transformants of the GhRIC6b-overexpressing transgenic cotton were longer than those of the wild type, indicating that GhRIC6b promoted fiber elongation at the initiation stage of cotton fiber growth. Figure 4 As shown, the fiber length of wild-type cotton 5 days after flowering was 3.15 mm, while the fiber lengths of UBQ10::GFP::GhRIC6b transgenic cotton were 4.8 mm (#0) and 5.01 mm (#3), respectively. The fiber length of #0 transgenic cotton was 1.65 mm longer than that of wild-type, and the fiber length of #3 transgenic cotton was 1.86 mm longer than that of wild-type. The statistical results show that the fiber length of GhRIC6b-overexpressing transgenic cotton 5 days after flowering was significantly longer than that of wild-type, indicating that overexpression of the GhRIC6b gene can increase the fiber length of cotton 5 days after flowering.

[0077] Example 5: Determination of mature fiber length in transgenic cotton

[0078] The developmental status of transgenic cotton fiber elongation was assessed by statistically analyzing the mature fiber length. Fifteen mature cotton seeds each from UBQ10::GFP::GhRIC6b transgenic and wild-type cotton were taken. After combing the fibers from the cotton seeds, the fiber length was measured. The results are as follows: Figure 5 and Figure 6 As shown.

[0079] Figure 5 The transgenic cotton overexpressing GhRIC6b showed that both transformants had mature fibers longer than the wild type, indicating that GhRIC6b promotes cotton fiber elongation. Figure 6 As shown, the mature fiber length of wild-type cotton is 24.1 mm, while the mature fiber lengths of UBQ10::GFP::GhRIC6b transgenic cotton are 27.0 mm (#0) and 26.4 mm (#3), respectively. The mature fiber length of #0 transgenic cotton is 2.9 mm longer than that of wild-type, and the mature fiber length of #3 transgenic cotton is 2.4 mm longer than that of wild-type. This indicates that the mature fiber length of UBQ10::GFP::GhRIC6b transgenic cotton is significantly increased, suggesting that overexpression of the GhRIC6b gene can promote the elongation of mature cotton fibers.

[0080] In summary, overexpression of the GhRIC6b gene can promote early elongation of fibroblasts, accelerate fibroblast initiation, and increase the length of mature fibers, thus significantly improving the length of cotton fibers.

[0081] Example 6: Determination of Fiber Quality in Transgenic Cotton

[0082] Seed cotton was harvested at the cotton fiber maturity stage and air-dried naturally. The cotton was then ginned, and fiber quality was tested, with each 12g sample constituting a replicate, and three replicates per strain. High-volume volume (HVI) instruments were used to read the fiber quality of each cotton sample, including average upper half length, fiber strength, micronaire value, uniformity index, and elongation rate. The results are shown in Table 3.

[0083] Table 3. Data on cotton fiber quality determination

[0084]

[0085] As shown in Table 3, compared with wild-type cotton, cotton fibers overexpressing the GhRIC6b gene showed improved upper half average length, fiber strength, uniformity index, and elongation, while the micronaire value showed no significant difference. This indicates that overexpressing the GhRIC6b gene can improve the quality of cotton fibers.

[0086] Example 7: Determination of lint content in transgenic cotton

[0087] Lining percentage is the weight percentage of cotton fiber in cottonseed, indicating the contribution of cotton fiber and serving as an important indicator of cotton yield. Seed cotton is harvested at fiber maturity and naturally air-dried. The cotton is then ginned, and the weight of the seed and fiber are measured. The formula for calculating lining percentage is:

[0088] Lining percentage = (weight of lint cotton / weight of seed cotton) * 100%

[0089] The results are as follows Figure 7 As shown, through 8 repeated measurements, the lint percentage of wild-type cotton was 37.1%, while the lint percentages of UBQ10::GFP::GhRIC6b transgenic cotton were 43.9% (#0) and 42.7% (#3), respectively. The lint percentage of transgenic cotton #0 was 6.7% higher than that of wild-type, and the lint percentage of transgenic cotton #3 was 5.6% higher than that of wild-type, indicating that overexpression of the GhRIC6b gene can significantly increase the lint percentage of cotton.

Claims

1. The application of the GhRIC6b gene in regulating cotton yield and quality, characterized by, The nucleotide sequence of the GhRIC6b gene is shown in SEQ ID NO.1; the application is to improve crop yield and fiber quality by overexpressing the GhRIC6b gene; the crop is cotton; the yield trait includes lint percentage; the fiber quality includes fiber length, average upper half length, fiber strength, uniformity index, and elongation.

2. A method for obtaining cotton varieties with high yield and fiber quality, characterized in that, An overexpression vector containing the GhRIC6b gene as described in claim 1 is transferred into Agrobacterium, and then cotton explants are infected. After cultivation, a cotton variety with high yield and fiber quality is obtained. The yield trait includes lint percentage. The fiber quality includes fiber length, average upper half length, fiber strength, uniformity index, and elongation.

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