Ghc48 gene and application thereof in improving fiber strength of upland cotton

Abstract

The application discloses a GHCDC48 gene and application thereof in improving fiber strength of Gossypium hirsutum. The gene GHCDC48 is from Gossypium hirsutum, and a nucleotide sequence in a genetic standard strain TM-1 of Gossypium hirsutum is shown as SEQ ID NO. 1. The gene expression level is positively correlated with the fiber strength of cotton. Overexpression of the GHCDC48 gene in Gossypium hirsutum can significantly improve the ATP enzyme activity in cotton fiber cells, increase the secondary wall thickness of the fiber and the fiber strength. Decreasing the expression of the GHCDC48 gene in Gossypium hirsutum reduces the growth rate of the Gossypium hirsutum plants, the ATP enzyme activity in the cells and the secondary wall thickness. Therefore, the GHCDC48 gene can be used to improve the fiber strength of Gossypium hirsutum. The research of the application lays a foundation for establishing new germplasm of Gossypium hirsutum with high fiber strength, and has important practical significance for basic scientific research or future agricultural industry application.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology application, and particularly relates to a GHCDC48 gene derived from Gossypium hirsutum and application thereof in improving fiber strength. BACKGROUND

[0002] Cotton (Gossypium spp.) is an important economic crop in China, providing more than 85% of natural fibers for the textile industry, and is an important raw material for the textile industry. Although different uses of cotton fibers require different varieties to be cultivated, fiber quality is the only important selection condition that does not change. The measurement standards of cotton fiber mainly include fiber length, breaking specific strength, elongation, micronaire value, etc. Among them, the breaking specific strength of cotton fiber, i.e. fiber strength, is an important indicator for evaluating cotton fiber quality, and is also a major condition for determining whether cotton fiber can be processed. Fiber strength is defined as the breaking load of a single fiber divided by the cross-sectional area of a single fiber, i.e. the maximum force that can be withstood per unit fiber cross-sectional area, which is a standard for measuring the relative force of cotton fiber. Fiber materials with good fiber strength are firm and durable, so with the innovation of textile technology, the requirements for cotton fiber quality are also getting higher and higher, especially for the strength of cotton fiber. Therefore, how to improve the strength of cotton fiber has become the main goal of current breeding work.

[0003] Many studies have reported the influence of cell secondary wall on fiber strength. During the dynamic thickening of fiber secondary wall, related genes work together to form a complex regulatory network, which controls the development process of secondary wall. For example, the expression of GhADF1 (encoding an actin depolymerization factor) is down-regulated in transgenic cotton plants, and the secondary cell wall is thicker than that of the non-transgenic control group. GhFIM2 is a member of the actin filament protein family, which has actin binding and bundling ability. It positively regulates fiber elongation and secondary wall synthesis, thereby enhancing fiber strength (Zhang et al., 2017). WLIM1a is preferentially expressed during the elongation and secondary wall synthesis stages of fiber development, and it is both an actin filament binding factor that promotes fiber cell elongation and a transcription factor that enhances secondary wall synthesis (Han et al., 2016). Recent studies have shown that a β-1,3-glucanase, GhGLU18, can be directly activated by the NAC transcription factor GhFSN1 associated with secondary cell wall synthesis. This activation enhances polysaccharide metabolism and promotes cellulose synthesis (Fang et al., 2023). Overall, existing research has clearly established a close link between the enhancement of cotton fiber strength and the thickening of secondary wall. SUMMARY

[0004] The application aims at solving the problems of the prior art, and provides a GHCDC48 gene and potential and application thereof in improving fiber strength of Gossypium hirsutum and cultivating new germplasm of high fiber strength cotton.

[0005] To achieve the above-mentioned object, the application provides the following technical scheme: in a first aspect, the application provides a cotton fiber strength-related GHCDC48 gene, the nucleotide sequence of which in Gossypium hirsutum standard line TM-1 is shown in SEQ ID NO. 1. The expression amount of the gene is positively correlated with the secondary wall thickness, intracellular ATPase activity and fiber strength of cotton. Overexpression of the GHCDC48 gene in cotton can significantly improve the secondary wall thickness, intracellular ATPase activity and fiber strength of cotton. Decreasing or silencing the expression of the GHCDC48 gene in cotton can significantly reduce the secondary wall thickness and intracellular ATPase activity of cotton.

[0006] In a second aspect, the application provides application of the GHCDC48 gene in improving fiber strength of Gossypium hirsutum. The expression of the GHCDC48 gene is increased in Gossypium hirsutum, and the fiber strength of Gossypium hirsutum is improved. For example, overexpression of the GHCDC48 gene shown in SEQ ID NO. 1 is applied in improving fiber strength of Gossypium hirsutum or cultivating new cotton varieties with high fiber strength.

[0007] In a third aspect, the application provides application of biological materials for increasing the expression of the GHCDC48 gene in improving fiber strength of Gossypium hirsutum and / or cultivating new germplasm of high fiber strength cotton. Further, the biological materials comprise at least one of the following: a) an expression cassette capable of significantly up-regulating the GHCDC48 gene; b) a recombinant vector having the function of a); and c) a recombinant bacterium having the function of a) or the recombinant vector of b).

[0008] In a fourth aspect, the application provides a method for improving fiber strength of Gossypium hirsutum. The expression of the GHCDC48 gene shown in SEQ ID NO. 1 is up-regulated or overexpressed in cotton, and the fiber strength of Gossypium hirsutum is improved.

[0009] Further, the GHCDC48 gene is used as a template gene, and the cotton fiber strength is improved or new cotton varieties with improved fiber strength are cultivated by overexpression of the GHCDC48 gene in cotton through Agrobacterium-mediated plant transformation.

[0010] The application has the beneficial effect that up-regulation or overexpression of the GHCDC48 gene in cotton can enhance the fiber strength of mature fibers. The application lays a foundation for establishing new germplasm of Gossypium hirsutum with high fiber strength, and has important practical significance for basic scientific research and future agricultural industry application. BRIEF DESCRIPTION OF DRAWINGS

[0011] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings required by the embodiments or prior art description will be briefly introduced as follows. Obviously, the accompanying drawings in the following description only only the embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of the provided drawings.

[0012] Figure 1 The transgenic cotton phenotype analysis after overexpression of the GHCDC48 gene in the present application; wherein, Figure 1 A in the above table is the PCR identification of the overexpression transgenic line, and the numbers 1-11 represent 11 independent transgenic lines; Figure 1 B in the above table is the relative expression amount of the GHCDC48 gene in the 6 selected positive lines of the transgenic GHCDC48 gene, and the detection method is qRT-PCR; Figure 1 C in the above table is the overhead view of the mature fiber of the transgenic receptor TM-1 and the overexpression of the GHCDC48 gene; Figure 1 D in the above table is the paraffin section of the mature fiber of the transgenic receptor TM-1 and the overexpression of the GHCDC48 gene, and the photograph is taken after the fast green staining; Figure 1 E in the above table is the secondary cell wall thickness statistics of the mature fiber of the transgenic receptor TM-1 and the overexpression of the GHCDC48 gene; Figure 1 F in the above table is the ATPase activity statistics of the fiber of the transgenic receptor TM-1 and the overexpression of the GHCDC48 gene; **, p<0.01, t-test;

[0013] Figure 2 The growth conditions and ATPase activity detection of the gene silencing group (TRV:GHCDC48) and the control group (TRV:00) cotton plants 16 days after injection in the present application; wherein, Figure 2 A in the above table is the growth condition of the gene silencing group and the control group cotton plants 16 days after injection, and the white leaf of TRV:CLA represents that the silencing efficiency of this experiment is good; Figure 2 B in the above table is the height statistics of the gene silencing group and the control group cotton plants; Figure 2 C in the above table is the qRT-PCR analysis of the expression amount of the GHCDC48 in the gene silencing group and the control group cotton; Figure 2 D in the above table is the cross section of the stem of the gene silencing group and the control group cotton, and the photograph is taken after the toluidine blue staining; Figure 2 E in the above table is the secondary cell wall thickness statistics of the gene silencing group and the control group cotton plants; Figure 2 F in the above table is the ATPase activity statistics of the gene silencing group and the control group cotton plants; **, p<0.01, t-test. DETAILED DESCRIPTION

[0014] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0015] In the following examples, the standard line of upland cotton TM-1 is preserved in the laboratory.

[0016] In the following examples, the VIGS vectors pTRV2 and pTRV1 can be purchased through commercial channels, and TRV::CLA1 is a vector for silencing the CLA1 gene constructed in pTRV2, which is a commonly used positive control group in the art, and causes the positive plants to turn white after silencing.

[0017] Example 1: Obtaining of the sequence of the GhCDC48 gene

[0018] Acala Prema, a high-fiber-strength representative variety of Acala cotton, and another low-fiber-strength upland cotton popular variety 86-1 are used as parent hybrids to prepare a recombinant inbred line population, and a major gene GhUBX regulating fiber strength is obtained by map-based cloning technology, and a protein interacting with GhUBX, GhCDC48, is screened by molecular biology means. The GhCDC48 gene contains an AAA-ATP domain and belongs to the AAA-ATP (p97) family.

[0019] The GhCDC48 gene is cloned as follows:

[0020] (1) Cotton planting

[0021] The seeds of the standard line TM-1 of upland cotton are sown in a seedling tray, and the nutrient medium is mixed at a ratio of 1:1:1 of nutrient soil:vermiculite:perlite; and the seedling tray is placed in a light culture room with 16h light / 8h darkness for growth, and water is poured in time to keep the soil moist.

[0022] (2) Extraction of RNA

[0023] The root tissue is sampled as described above. The grinding rod is placed in a mortar and 3-4 mL of anhydrous ethanol is added to ignite for RNAse inactivation. The mortar is rapidly cooled with liquid nitrogen, and then the root sample is rapidly ground in liquid nitrogen. When the sample is ground into powder without particles, it is quickly loaded into a 1.5 mL RNase-free centrifuge tube, and then placed in liquid nitrogen or a -80°C ultra-low temperature refrigerator for long-term storage. The extraction of total RNA of cotton is completed according to the operation instructions of Spectrum Plant Total RNA Extraction Kit (Sigma-Aldrich, DE). The total RNA is stored in a -80°C ultra-low temperature refrigerator for subsequent analysis.

[0024] (3) Reverse transcription reaction to obtain cDNA

[0025] The reverse transcription reaction was performed using reverse transcriptase from ABclonal. RNA template denaturation helps to open the secondary structure, which can greatly improve the yield of first-strand cDNA. The reverse transcription reaction system is shown in Table 1 below, and the reverse transcription reaction conditions are as follows: 37°C for 2 min, 55°C for 15 min, and 85°C to terminate the reaction. The reverse transcription product can be used immediately for subsequent experiments or stored in a -20°C refrigerator and used within six months to avoid repeated freezing and thawing.

[0026] Table 1 Reverse transcription reaction system

[0027] Component Amount 4x AB Script NeoRT Master Mix 5 μL 20x gDNA Remover Mix 1 μL Total RNA 1 μg Nuclease-free H2O Supplemented to 20 μL

[0028] (4) Amplification of the full-length of the GHCDC48 gene

[0029] According to the principle of homologous recombination, primer 5.0 was used to design amplification primers to connect the target gene between the Xba I-Sma I enzyme digestion sites of the vector pBI121. The 5' end of the upstream and downstream primers was added with an enzyme digestion site and a corresponding 15-base vector fragment before or after the enzyme digestion site in the corresponding vector to ensure the correct direction of the target fragment inserted into the vector. The length of the primer is generally about 40 bp, and the GC content is 40%-60%. The primers designed in this example are as follows:

[0030] Upstream primer (SEQ ID NO. 2):

[0031] 5'-gagaacacgggggactctagaATGTCTAACCAAGCAGAATCATCTGA-3'

[0032] Downstream primer (SEQ ID NO. 3):

[0033] 5'-ataagggactgaccacccgggACTGTAGAGGTCATCTTCATCGGC-3'

[0034] The diluted primers and the cDNA obtained in step (3) were used for fragment PCR amplification, and the PCR amplification system is shown in Table 2.

[0035] Table 2 PCR amplification system

[0036] Component Amount Upstream primer 1.5 μL Downstream primer 1.5 μL cDNA cDNA ~ 500 ng 2x KOD Mix Buffer 25 μL ddH2O Supplemented to 50 μL

[0037] PCR program: 98°C pre-denaturation 3 min; 98°C denaturation 10 sec, 59°C annealing 5 sec, 68°C extension 1-10 sec / kb, 25-35 cycles; 68°C final extension 5 min.

[0038] (5) PCR amplification product recovery and purification

[0039] The PCR product was separated by 1% agarose electrophoresis, and the product recovery step referred to the Tianxi Gel / PCR Purification kit instruction manual. The recovered product was detected for concentration and quality using a NanoDrop One 2000 spectrophotometer.

[0040] (6) Homologous recombination reaction

[0041] The linearized pBI121 vector and the recovered PCR product were used to prepare a homologous recombination reaction system using the homologous recombination enzyme ClonExpress II One Step Cloning Kit from Nanjing Novizen Biological Company, as shown in Table 3. The reaction was performed at 37°C for 30 min, and the recombination product was immediately used or stored in a -20°C refrigerator.

[0042] Table 3 Homologous recombination reaction system

[0043] Component Amount Linearized vector 50-200 ng Purified DNA of interest 10-100 ng Exnase 1 μL 5x CE Buffer 2 μL ddH2O Supplemented to 10 μL

[0044] (7) Recombination product transformation of E. coli competent DH5a, sequencing and sequence analysis

[0045] (7.1) 5-10 μL of the recombination product to be transformed was added to 50-100 μL of competent cells, gently mixed, and then placed in an ice bath for 30 min.

[0046] (7.2) The centrifuge tube was placed in a 42°C water bath for heat shock for 90 sec.

[0047] (7.3) 700 μL of antibiotic-free LB liquid medium was added to the centrifuge tube, and the mixture was incubated at 37°C on a shaker at 200 rpm for 45 min / h.

[0048] (7.4) The incubated bacterial solution was centrifuged at 3500 rpm for 5 min, the supernatant was discarded, 100 μL of antibiotic-free LB liquid medium was used to resuspend the bacterial pellet, and the mixture was transferred to LB solid medium containing the corresponding antibiotic and evenly spread.

[0049] (7.5) After the bacterial solution on the surface of the plate was absorbed, the culture dish was sealed and inverted and incubated at 37°C for 12-16 h.

[0050] (7.6) Use a sterilized toothpick to pick a single colony and place it in 600 μL of resistant liquid LB medium. Incubate at 37°C in a shaker at 200 rpm for 6-8 h. Then, aspirate 1 μL of the culture medium for PCR. Bacteria that are positive by PCR are sent to Qingke Biotechnology for Sanger sequencing. The reference nucleotide sequence of the GHCDC48 gene is shown in SEQ ID NO. 1. Compare the sequence to the sequence. Completely identical plasmids and recombinant bacterial cultures can be stored in an ultra-low temperature freezer for subsequent experiments.

[0051] Example 2: Application of silencing the GHCDC48 gene in improving fiber strength

[0052] Virus-induced gene silencing (VIGS) is a technique for studying gene function that can induce endogenous gene silencing in modern plants. This example uses VIGS to investigate the function of the GHCDC48 gene, verifying its role in the thickening of cotton cell secondary walls. This provides a scientific basis for cotton breeding and genetic improvement, as follows:

[0053] (1) Construction of GHCDC48 gene VIGS vector

[0054] Based on the CDS sequence information of the target gene GHCDC48, a specific silencing target sequence was designed, and appropriate restriction sites (BamHI and SmaⅠ) were added according to the target sequence and the information on the pTRV2 vector. Specific amplification was performed using the root cDNA of Prema as an amplification template. The specific amplification system is shown in Table 4, and the primers used are as follows:

[0055] Forward primer (SEQ ID NO.4):

[0056] 5'-agaaggcctccatggggatccCAGCCTTTTAATTCTCCTTATAAATAACC-3'

[0057] Reverse primer (SEQ ID NO.5):

[0058] 5'-tgtcttcgggacatgcccgggCTTTCTTTCCAAGATCGCAGTACTAA-3'

[0059] Table 4 Specific amplification system

[0060]

[0061] The PCR product, linearized pTRV2 vector, recombinant transformed E. coli and sequencing were recovered according to the method of Example 1. The plasmid with correct sequencing was TRV:GHCDC48, which was stored in a refrigerator at -20°C for standby use.

[0062] (2) Transformation of Agrobacterium

[0063] The plasmid transformation was performed using the Agrobacterium competent LBA3101 of the unique geographical organism, and the specific operation steps were as follows:

[0064] a) The Agrobacterium competent stored at -80°C was placed on ice to be thawed.

[0065] b) 0.01-1 μg of TRV:GHCDC48 plasmid was added to each 100 μL of Agrobacterium competent, the bottom of the tube was gently stirred to mix, and it was placed on ice for 5 min, frozen in liquid nitrogen for 5 min, 37°C water bath for 5 min, and ice bath for 5 min.

[0066] c) 600 μL of LB liquid medium without resistance was added, and it was cultured at 28°C for 2-4 h with shaking.

[0067] d) Centrifugation was performed at 3500 rpm for 5 min, the supernatant was discarded, 100 μL of LB liquid medium without resistance was used to resuspend the bacterial pellet, and it was plated on LB solid medium containing Kan / Rif resistance, and cultured at 28°C for 2-3 days with inversion.

[0068] (3) Agrobacterium-mediated VIGS infection of cotton

[0069] The recombinant bacteria containing TRV:GHCDC48, TRV:CLA1 and pTRV2 and pTRV1 plasmids were cultured in liquid LB medium containing Kan / Rif, and cultured at 28°C at 220 rpm until the OD600 reached 2.0, centrifuged at 4000 rpm for 10 min, and the supernatant was discarded; the bacterial body was suspended with the same volume of suspension (10 mM MgCl2, 100 mM MES, 200 μM AS) to OD600 of 2.0, and placed at 28°C for 3 h, then the suspensions of TRV:GHCDC48, TRV:CLA1 and pTRV2 empty vector were mixed with the suspension of pTRV1 at a volume ratio of 1:1. Then the mixed bacteria were injected into the cotyledons of two-leaf-one-heart size cotton, wherein the cotton injected with TRV:GHCDC48 bacteria was the gene silencing group, the cotton injected with TRV:CLA1 bacteria was the positive control group, and the cotton injected with pTRV2 empty bacteria was the control group; after injection, the cotton seedlings were cultured at 22°C in the dark overnight, and then cultured normally at 23°C with 16 h light / 8 h dark. After two weeks, the newly born true leaves of the positive control group turned white, proving that the silencing efficiency of this batch of VIGS injection was good and the effectiveness of the technical system of this batch was good.

[0070] (4) Detection of the silencing efficiency of the GHCDC48 gene and the secondary wall thickness

[0071] The RNA of the control group and the experimental group was extracted according to Example 1, and cDNA was obtained by reverse transcription. RT-qPCR was performed using quantitative primers (SEQ ID NO. 6 and SEQ ID NO. 7) to detect the silencing of the GHCDC48 gene.

[0072] Design specific primers:

[0073] SEQ ID NO. 6: 5'-GGAGGCCTGGAGAATGTTAAA-3';

[0074] SEQ ID NO. 7: 5'-CTGGAGGGCCATAGAACAATAC-3'

[0075] Quantitative PCR detection was performed, and the results of RT-qPCR are shown in C of Figure 2 The expression amount of the GHCDC48 gene of the cotton plants in the gene silencing group was significantly lower than that in the control group, indicating that the silencing effect was good.

[0076] To analyze whether the GHCDC48 gene is involved in the secondary wall thickening of cotton cells and the growth and development of plants. Photographs were taken after 16 days of silencing, and the plant height was counted. The results are shown in B of Figure 2 The plant height of the silencing group was significantly lower than that of the control group, indicating that the growth and development of the plant were affected after silencing the GHCDC48 gene; the ATPase activity of the silencing group and the control group was detected, and the results are shown in F of Figure 2 The ATPase activity of the silencing group was also significantly lower than that of the control group, indicating that the ATPase activity of the plant cells was directly affected after silencing the GHCDC48 gene, and the GHCDC48 gene was positively correlated with the ATPase activity.

[0077] Next, the stems of the same position of the silencing group and the control group were paraffin-embedded, sectioned and stained, and the secondary wall thickness of the cells was counted. The results are shown in D of Figure 2 and E of Figure 2 After 16 days of silencing the GHCDC48 gene, the secondary wall of the control group cotton cells grew normally, the secondary wall of the gene silencing group cotton cells developed slowly, and the thickness was less than that of the control group, indicating that after silencing the GHCDC48 gene, the secondary wall thickness of the cotton cells was significantly reduced.

[0078] Example 3: Application of overexpression of the GHCDC48 gene in improving fiber strength

[0079] The fiber strength (cN / tex) parameter of TM-1 and GhCDC48-OE transgenic cotton plants harvested in Jingshan, Hangzhou was compared, as shown in Table 5, wherein GhCDC48-OE is a transgenic cotton plant obtained by overexpressing the GhCDC48 gene.

[0080] Table 5 Transgenic cotton fiber detection data after overexpression of the GhCDC48 gene

[0081]

[0082] The data is shown as the average of three experimental repeats (± standard deviation), *, p < 0.05; **, p < 0.01, t-test. The fiber quality traits include upper half mean length (UHML), fiber length uniformity (UI), fiber micronaire value (Mic), fiber strength (FS) and fiber elongation (Elg). The test standard is GB / T 20392-2006 HVI Cotton Fiber Physical Property Test Method. The fiber samples were measured by the Cotton Quality Supervision and Inspection Test Center of the Ministry of Agriculture and Rural Affairs. It can be known that overexpression of the GhCDC48 gene in Gossypium hirsutum can significantly improve the fiber strength of cotton.

[0083] As shown in Figure 1 , the secondary cell wall thickness of mature fibers and ATPase activity in the fibers of the transgenic receptor TM-1 and the overexpression of the GhCDC48 gene were compared, and the results showed that overexpression of the GhCDC48 gene in Gossypium hirsutum can significantly improve the ATPase activity in the cotton fiber cells and increase the fiber secondary wall thickness.

[0084] The above description of the disclosed embodiments enables one skilled in the art to make or use the application. Numerous modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Therefore, the present application is not intended 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. Application of the GHCDC48 gene in improving the fiber strength of upland cotton, characterized in that: The nucleotide sequence of the GHCDC48 gene is shown in SEQ ID NO. 1, and the application is: over-expressing the GHCDC48 gene in upland cotton.

2. Use of the biomaterial for improving the expression of the GHCDC48 gene according to claim 1 in improving the fiber strength of upland cotton, characterized in that: The biological material comprises at least one of the following: a) an expression cassette for overexpressing the GHCDC48 gene; b) a recombinant vector containing the expression cassette in a); c) a recombinant bacterium containing the expression cassette in a) or the recombinant vector in b) .

3. Use of the biomaterial for improving the expression of the GHCDC48 gene according to claim 1 in cultivating new cotton germplasm with high fiber strength, characterized in that: The biological material comprises at least one of the following: a) an expression cassette for overexpressing the GHCDC48 gene; b) a recombinant vector containing the expression cassette in a); c) a recombinant bacterium containing the expression cassette in a) or the recombinant vector in b) .

4. A method for improving the strength of upland cotton fibers, characterized in that: The GHCDC48 gene is overexpressed in upland cotton to improve the fiber strength of upland cotton. The nucleotide sequence of the GHCDC48 gene is shown in SEQ ID NO.1.

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