Application of GhRIC6b gene in regulation of yield and quality of cotton

Abstract

The invention discloses application of a GhRIC6b gene in regulation of cotton yield and quality, and belongs to the technical field of gene engineering. Through overexpression of the GhRIC6b gene in cotton, fiber development is promoted, cotton lint percentage is increased, and quality of fiber length, strength, uniformity index, elongation and the like are improved. When the method is used for improving the variety of cotton, a feasible strategy is provided for synchronously improving the yield and the quality of cotton fibers.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to the application of the GhRIC6b gene in regulating cotton yield and quality. Background Art

[0002] Cotton is an important cash crop, providing natural fiber raw materials for the textile industry. High-quality cotton fibers have always been the main demand for imported raw cotton in China, and are also the focus of domestic cotton breeding work. Therefore, improving cotton yield and fiber quality is of great significance for the sustainable development of China's cotton industry and cotton textile industry.

[0003] Traditional genetic breeding methods have a long cycle and a large workload. Moreover, both cotton fiber yield and quality are controlled by quantitative traits, with a strong negative correlation in genetics, making it difficult to improve them simultaneously, and being easily affected by many environmental factors. It is very difficult to rapidly and efficiently improve cotton quality only relying on traditional breeding techniques. The development of modern molecular biology techniques and the successful application of transgenic techniques in cotton have provided new ideas and methods for cotton quality improvement. Summary of the Invention

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

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

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

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

[0008] Further, the application is to overexpress the GhRIC6b gene to improve the yield and fiber quality of crops.

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

[0010] Further, the dicotyledonous plant is cotton.

[0011] Further, the yield traits include lint percentage; the fiber quality includes fiber length, upper half mean length, fiber strength, evenness index, and elongation rate.

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

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

[0014] The present invention also discloses a method for obtaining a cotton variety with high yield and fiber quality, which overexpresses the GhRIC6b gene, incorporates it into Agrobacterium, then infects cotton explants, and after cultivation, obtains a cotton variety with high yield and fiber quality.

[0015] The beneficial effects of the present invention are as follows: By overexpressing the GhRIC6b gene in cotton, it is found that it can promote the early elongation of fiber cells, initiate fiber cells faster, and at the same time increase the length of mature fibers, significantly improving the cotton fiber length. At the same time, the upper half mean length, fiber strength, uniformity index, elongation rate, and lint percentage are all improved synchronously. Using the method of the present invention to improve cotton can break the incompatibility between cotton yield and quality in the breeding process, balance the yield and quality of cotton, and provide a feasible strategy for the synchronous genetic improvement of cotton fiber yield and quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram for constructing the GhRIC6b plant expression vector;

[0017] Figure 2 Comparison of GhRIC6b expression levels;

[0018] Figure 3 Physical map of fibers 5 days after cotton flowering;

[0019] Figure 4 Fiber length statistics 5 days after cotton flowering;

[0020] Figure 5 Physical map of mature cotton fibers;

[0021] Figure 6 Fiber length statistics of mature cotton fibers;

[0022] Figure 7 Measurement results of cotton lint percentage. DETAILED DESCRIPTION OF THE INVENTION

[0023] The specific embodiments of the present invention will be described below to facilitate those skilled in the art of the present technology to understand the present invention. For those conditions not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those of ordinary skill in the art of the present technology, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions and creations using the concept of the present invention are within the scope of protection.

[0024] Unless otherwise specified, all reagent drugs in the following examples are commercially available, and all materials and methods not specifically described refer to "Molecular Cloning: A Laboratory Manual" (Sambrook and Russell, 2001).

[0025] GhRIC6b is a class of proteins that can interact with the unique small G protein ROP in plants. All such proteins contain a highly conserved CRIB (Cdc42 / Rac-interactive binding) domain, and this domain is an important domain for the interaction between ROP protein and RIC dependent on GTP. RIC is one of the important downstream effector factors of the small G protein ROP. RIC interacts with different members of ROP, controls various ROP-dependent signal pathways, and performs different functions in different parts of plants.

[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] TRKPKDANQTTETTTKDPKKSRRKKVKDMGGEGSSRRSRTTQESDTLSEAGSLISCDSEFV

[0041] EGEAN*(SEQ ID NO.2)。

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

[0043] Table 1 Primer Sequence Table

[0044]

[0045]

[0046] Example 1 Construction of the Vector

[0047] 1. Plant RNA Extraction

[0048] Approximately 1 g of fresh cotton material was selected and thoroughly ground into fine powder in a mortar with liquid nitrogen. RNA was extracted using the EASYspin Plant RNA Kit from Aidlab Biotechnologies Co., Ltd., and the extraction steps were carried out strictly according to the enclosed instructions. After extraction, 2 μL 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 PrimeScript RT reagent Kit with gDNAEraser from TaKaRa, and the operation steps were carried out according to the instructions.

[0051] 3. Obtaining the Cotton Gene GhRIC6b

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

[0053] 4. Construction of the GhRIC6b Overexpression Vector

[0054] The construction process is as Figure 1 shown. The backbone vector used for constructing the plant expression vector includes the pLN vector. For the convenience of transgenic plant identification, a fluorescent protein gene GFP was added between the constitutive promoter UBQ10 and polyA (which has the function of transcriptional termination) for fusion with the target gene GhRIC6b. Among them, 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 terminator of the nopaline synthase gene; 35S Terminator is the terminator of the 35S gene; LB is the left border of T-DNA; RB is the right border of T-DNA.

[0055] The pLN plant expression vector modified from the pCambia2300 vector was digested and linearized with BamHⅠ and EcoRⅠ. All kinds of restriction endonucleases were products of Thermo Scientific and were digested according to the instructions. The nucleotide sequence of GFP was SEQ ID NO.5, and the stop codon was deleted. 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 were designed to amplify the GhRIC6b sequence primer (SEQ ID NO.8 and SEQ ID NO.9). The linearized pLN vector, GFP, and the amplified product of GhRIC6b were mixed at a molecular ratio of 1:3:3. After reacting at 37°C for 30 min under the catalysis of homologous recombinase, Escherichia coli transformation was carried out, thereby 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, and the cotton experimental material used was upland cotton (Gossypium hirsutum L).

[0056] Example 2 Transformation of Cotton

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

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

[0059] The steps of Agrobacterium electroporation transformation were as follows: Take out the Agrobacterium competent cells and melt them on ice. The electroporation cuvette used for electroporation transformation was washed 5 - 6 times with sterile water and placed on ice for later use. Pipette 20 - 50 ng of plasmid and mix it with the competent cells, then add it to the electroporation cuvette. Turn on the electroporation instrument of Bio-RAD company and select the Agr / Bacteria mode for electroporation. Immediately after electroporation, add 800 μL of YEB liquid medium (including 0.5% sucrose (W / V), 0.1% yeast extract for bacteria (W / V), 1% tryptone for bacteria (W / V), 0.05% MgSO 4 ·7H 2 O (W / V) and 1.5% agar powder (W / V), pH = 7.0), and culture it with shaking at 28°C and 200 rpm for 3 h. Then centrifuge at 10000 rpm for 1 min, discard the supernatant, and leave about 100 μL of the medium to resuspend the bacteria. The bacterial solution was evenly spread on the YEB plate containing 50 μg / mL Kan (kanamycin) and 50 μg / mL Rif (rifampicin), and cultured inverted at 28°C for 48 h.

[0060] 2. Integration of the constitutive expression vector of GhRIC6b into the cotton genome

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

[0062] (1) Cotton seed germination: The seeds of the upland cotton cultivar were shelled, and the large and plump cotyledons were placed in a triangular flask. First, they were sterilized with 75% alcohol for 1 - 2 min and rinsed twice with sterile water; then they were sterilized with 0.1% mercuric chloride (HgCl 2 ) for 10 min and rinsed six times with sterile water. They were cultured on a shaker at room temperature and 120 rpm for 12 h. The germinated cotyledons with the radicles facing down were inoculated into the seed germination medium and germinated under dark conditions at 28°C for 2 - 3 d to obtain sterile cotton seedlings.

[0063] (2) Culture of transformed Agrobacterium: The Agrobacterium strain containing the above plant expression vector was streaked on a YEB solid medium containing 50 mg / L kanamycin and 125 mg / L streptomycin. A single colony of Agrobacterium activated from the newly streaked plate was inoculated into 5 mL of YEB liquid medium containing the same antibiotics and cultured overnight at 28°C and 200 rpm. 1 mL of the bacterial solution was taken and sub-cultured in 20 - 25 mL of YEB liquid medium containing the same antibiotics until the OD 600 reached 0.8 - 1.0. Finally, it was centrifuged at 10000 rpm for 1 min to collect the bacteria, and the bacteria were resuspended with an equal volume of liquid co-culture medium (containing 40 mg / L acetosyringone, AS) as the infection solution for standby.

[0064] (3) Infection and co-culture: The cotton sterile hypocotyls were cut into small segments about 0.5 cm long and placed in a triangular flask containing the infection solution. They were infected on a shaker at 28°C and 100 rpm for 45 min. The bacterial solution was discarded, and the infected hypocotyl segments were inoculated onto the solid co-culture medium (containing 40 mg / L acetosyringone, AS) and cultured in the dark at 28°C for 2 d.

[0065] (4) Screening of transformants: After co-cultivation, transfer the hypocotyl segments to the screening medium for bacteria elimination and selective culture. Incubate them under light at 28 °C for 2 weeks, then transfer the hypocotyls to the subculture medium and subculture once every 2 weeks. When the hypocotyls swell and a large amount of callus grows, transfer the callus together with the hypocotyl segments to the embryogenic callus induction medium and subculture once every 3 weeks. After fine granular embryogenic callus grows on the surface of the callus, transfer the embryogenic callus to the liquid suspension medium and culture at 150 rpm for 2 weeks to obtain somatic embryos. Then rinse the somatic embryos 3 - 4 times with sterile water, transfer the washed and sedimented somatic embryos to the somatic embryo maturation, elongation and plating medium, and incubate them under light at 28 °C. Transfer the growing young shoots to a new somatic embryo elongation and plating medium for culture and subculture once every 3 weeks. When the seedlings grow to about 5 cm in height, take out the seedlings for hydroponics, harden them off and then transfer them to flower pots for growth.

[0066] Table 2 Media for Agrobacterium tumefaciens-mediated genetic transformation of cotton

[0067]

[0068]

[0069] 3. Obtaining cotton with improved fiber traits

[0070] The obtained T0 generation transgenic cotton is cultured in the greenhouse, and the self-pollinated cotton seeds are collected and sown. Plant the T1 generation cotton in the field (Chongqing), collect the fibers for observation, and use the non-transgenic T1 generation cotton self-segregated from the T0 generation transgenic cotton as the control group (WT).

[0071] Example 3 Identification of the expression level of GhRIC6b overexpressing transgenic cotton

[0072] Extract the RNA of cotton fibers at 2 - 3 days after flowering, and use primers SEQ ID NO.10 and SEQ ID NO.11 for RT-PCR expression level identification (the internal reference gene is GhHIS3, and the primers are SEQ ID NO.12 and SEQ ID NO.13 respectively).

[0073] The results are as Figure 2 shown. Compared with the wild type, the expression levels of GhRIC6b in UBQ10::GFP::GhRIC6b transgenic cotton (#0 and #3 are two independent transgenic lines of UBQ10::GFP::GhRIC6b transgenic cotton) are all significantly up-regulated, indicating that GhRIC6b is overexpressed in transgenic cotton.

[0074] Example 4 Measurement of fiber length of transgenic cotton 5 days after flowering

[0075] By counting the fiber length 5 days after cotton flowering, the developmental status of cotton fiber initiation was judged. Ten ovules each of UBQ10::GFP::GhRIC6b transgenic and wild-type cotton 5 days after flowering were taken. After combing the fibers on the ovules with a writing brush, the fiber length was measured. The results are as Figure 3 and Figure 4 shown.

[0076] Figure 3 It shows that 5 days after flowering, the fiber lengths of two transformants of transgenic cotton overexpressing GhRIC6b were both longer than those of the wild type, indicating that GhRIC6b promoted fiber elongation during cotton fiber initiation. As Figure 4 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 increased by 1.65 mm compared with the wild type, and the fiber length of #3 transgenic cotton increased by 1.86 mm compared with the wild type. It can be seen from the statistical results that the fiber length of transgenic cotton overexpressing GhRIC6b 5 days after flowering was significantly elongated compared with the wild type, indicating that overexpressing the GhRIC6b gene can increase the fiber length of cotton 5 days after flowering.

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

[0078] By counting the mature fiber length of transgenic cotton, the developmental status of fiber elongation of transgenic cotton was judged. Fifteen mature cotton seeds each of UBQ10::GFP::GhRIC6b transgenic and wild-type cotton were taken. After combing the fibers on the cotton seeds with a comb, the fiber length was measured. The results are as Figure 5 and Figure 6 shown.

[0079] Figure 5 It shows that the mature fiber lengths of two transformants of transgenic cotton overexpressing GhRIC6b were both longer than those of the wild type, indicating that GhRIC6b promoted cotton fiber elongation. As Figure 6 shown, the mature fiber length of wild-type cotton was 24.1 mm, while the mature fiber lengths of UBQ10::GFP::GhRIC6b transgenic cotton were 27.0 mm (#0) and 26.4 mm (#3) respectively. The mature fiber length of #0 transgenic cotton increased by 2.9 mm compared with the wild type, and the mature fiber length of #3 transgenic cotton increased by 2.4 mm compared with the wild type. This indicates that the mature fiber length of UBQ10::GFP::GhRIC6b transgenic cotton was significantly increased, indicating that overexpressing the GhRIC6b gene can promote the elongation of mature cotton fibers.

[0080] In summary, overexpression of the GhRIC6b gene can promote early elongation of fiber cells, initiate fiber cells faster, increase the length of mature fibers at the same time, and significantly improve the cotton fiber length.

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

[0082] At the maturity stage of cotton fibers, the seed cotton was harvested and naturally air-dried. The cotton was ginned, and then fiber quality was detected. Each 12 g was taken as a replicate, and each line had 3 replicates. A high-volume instrument (HVI) was used to read the fiber quality of each cotton lint sample, including the upper half mean length (Length), fiber strength (Strength), micronaire value (Micronaire), uniformity index (Uniformity), and elongation rate (Elongation rate). The results are shown in Table 3.

[0083] Table 3 Data of Cotton Fiber Quality Determination

[0084]

[0085] As can be seen from Table 3, compared with wild-type cotton, the upper half mean length, fiber strength, uniformity index, and elongation rate of cotton fibers overexpressing the GhRIC6b gene were all improved, and there was no significant difference in the micronaire value, indicating that overexpression of the GhRIC6b gene can improve cotton fiber quality.

[0086] Example 7 Determination of Lint Percentage of Transgenic Cotton

[0087] The lint percentage is the weight percentage of cotton fibers in cotton seeds, showing the contribution rate of cotton fibers and being an important indicator of cotton yield. At the maturity stage of cotton fibers, the seed cotton was harvested and naturally air-dried. The cotton was ginned, and the seed weight and fiber weight were measured. The formula for calculating the lint percentage is:

[0088] Lint percentage = lint weight / seed cotton weight * 100%

[0089] The results are as Figure 7 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 #0 transgenic cotton increased by 6.7% compared with that of wild-type cotton, and the lint percentage of #3 transgenic cotton increased by 5.6% compared with that of wild-type cotton, indicating that overexpression of the GhRIC6b gene can significantly increase the lint percentage of cotton.

Claims

1. Application of GhRIC6b gene in regulating cotton yield and quality, characterized in that: The nucleotide sequence of the GhRIC6b gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The application is to improve the yield and fiber quality of crops by overexpressing the GhRIC6b gene.

3. The use according to claim 2, characterized in that: The crop is a dicotyledonous plant.

4. The use according to claim 3, characterized in that: The dicotyledonous plant is cotton.

5. The use according to claim 2, characterized in that: The yield traits include lint percentage; the fiber quality traits include fiber length, upper half mean length, fiber strength, uniformity index and elongation.

6. An overexpression vector, characterized in that: Comprising the GhRIC6b gene according to claim 1.

7. A preparation for regulating crop yield and fiber quality, characterized in that: Comprising the overexpression vector according to claim 6.

8. A method for obtaining a cotton variety with high yield and fiber quality, characterized in that: The GhRIC6b gene of claim 1 is overexpressed and loaded into Agrobacterium, which is then infected into cotton explants and cultivated to obtain a cotton variety with high yield and fiber quality.

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