GhGAUT9 gene for regulating and controlling length of cotton fiber and application of GhGAUT9 gene

Abstract

The invention belongs to the technical field of cotton fibers, and particularly relates to a GhGAUT9 gene for regulating and controlling the length of cotton fibers and application of the GhGAUT9 gene. It is found for the first time that GhGAUT9 is highly expressed in the fiber elongation period of a short fiber variety and is hardly expressed in the fiber elongation period of a long fiber variety. After GhGAUT9 is silenced in cotton, it is found that the pectin content of cotton fibers of 15DPA is reduced, the fiber length is remarkably increased, and the fiber yield is not affected. The GhGAUT9 gene for regulating and controlling the length of the cotton fiber, provided by the invention, can provide new gene resources and theoretical support for improving the quality of the cotton fiber.

Description

Technical Field

[0001] The invention relates to the technical field of cotton fibers, in particular to a GhGAUT9 gene for regulating cotton fiber length and an application thereof. Background Art

[0002] Cotton is an important fiber economic crop. Pectin plays a vital role in the growth and development of cotton, of which upland cotton accounts for 95% of the world's cotton production, but its fiber quality still needs to be continuously improved to meet human needs and the development of the textile industry.

[0003] Cotton fiber comes from the epidermal cells of the ovule. It is the most important natural fiber and provides raw materials for the textile industry. Cotton fiber development includes four overlapping periods: fiber initiation, primary wall elongation, secondary wall thickening and dehydration maturity. The transition period between primary wall elongation and secondary wall thickening is 16-20 days post anthesis, DPA. The cell wall of cotton fiber determines the fiber quality characteristics such as fiber length, industrial processing and fiber performance. The composition of cotton fiber cell wall can affect the fiber morphology and quality. Therefore, cotton fiber has become an ideal model for studying cell elongation and cell wall biosynthesis.

[0004] The cell wall of cotton fiber is mainly formed during the synthesis of primary cell wall (PCW) and secondary cell wall (SCW). Cell wall polysaccharides are essential in the elongation of primary wall and thickening of secondary wall of fiber cells. The composition of cotton fiber cell wall changes continuously throughout the development process, among which non-cellulose polysaccharides (pectin and hemicellulose, etc.) account for 30% to 50% of the total sugar content during the fiber elongation period, but only 3% of the total sugar content during the secondary wall thickening period. Mature cotton fiber is composed of more than 90% cellulose and about 3% non-cellulose polysaccharides. The amount and molecular size of pectin polysaccharides show great changes in cotton fiber development, thus playing an important regulatory role in fiber elongation and the synthesis and deposition of secondary wall. Therefore, the synthesis of pectin polysaccharides is necessary for both the primary wall formation and the secondary wall thickening period of normal fiber development. The biosynthesis of cotton cellulose has always been widely concerned, mainly focusing on the identification and functional analysis of cellulose synthase. In contrast, little is known about the mechanism of pectin synthesis in cotton fiber. Summary of the invention

[0005] The present invention provides a GhGAUT9 gene for regulating cotton fiber length and its application. The present invention experimentally confirms that cotton fiber length is related to the total pectin content of the fiber, but has no correlation with the degree of pectin methyl esterification. The present invention proposes a new research idea for the mechanism of cotton fiber biosynthesis.

[0006] Based on the research results of the present invention, in a first aspect, the present invention provides the use of the GhGAUT9 gene in negatively regulating the length of plant epidermal hairs. The use of the GhGAUT9 gene in negatively regulating the length of plant hypocotyls and their cells.

[0007] In a second aspect, the present invention provides the use of the GhGAUT9 gene in negatively regulating cotton fiber length.

[0008] In a third aspect, the present invention provides application of the GhGAUT9 gene in regulating the pectin content of cotton fiber.

[0009] When the GhGAUT9 gene is used to regulate the pectin content of cotton fiber, the GhGAUT9 gene is overexpressed in cotton to increase the pectin content of cotton fiber. Conversely, the GhGAUT9 gene is silenced in cotton to reduce the pectin content of cotton fiber.

[0010] In a fourth aspect, the present invention provides an application of the GhGAUT9 gene in increasing cotton seed weight. Specifically, silencing the GhGAUT9 gene in cotton can increase cotton seed weight to a certain extent.

[0011] In the above four applications provided by the present invention, the nucleotide sequence of the GhGAUT9 gene is shown as SEQ ID NO.1.

[0012] In a fifth aspect, the present invention also provides a method for increasing cotton fiber length, wherein the GhGAUT9 gene as shown in SEQ ID NO.1 is silenced in the cotton genome.

[0013] In a sixth aspect, the present invention provides a method for breeding long-fiber cotton varieties, which detects the expression level of the GhGAUT9 gene as shown in SEQ ID NO.1 in the tested cotton, and selects a cotton variety with a low expression level as a long-fiber cotton variety.

[0014] The beneficial effects of the present invention are:

[0015] The present invention first discovered that GhGAUT9 is highly expressed in the fiber elongation period of short-fiber varieties, but is almost not expressed in the fiber elongation period of long-fiber varieties. It was further discovered that GhGAUT9 is located in the Golgi apparatus and participates in galacturonic acid metabolism and promotes pectin synthesis.

[0016] The present invention has confirmed through experiments that the length of cotton fibers is related to the total pectin content of the cotton fibers, but has no correlation with the degree of pectin methyl esterification. The present invention provides a new research idea for the mechanism of action of cotton fiber length.

[0017] Overexpression of GhGAUT9 homologous genes in Arabidopsis significantly reduced epidermal hair density and epidermal hair length, while the mutant gaut9 showed the opposite phenotype. After silencing GhGAUT9 in cotton, the pectin content of cotton fiber at 15DPA was reduced, the fiber length was significantly increased, and the fiber yield was not affected. The research results of the present invention will provide new gene resources and theoretical support for improving cotton fiber quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 It is the tissue-specific expression analysis result of GhGAUT9 in Example 2 of the present invention. A is the difference in fiber length between Daizi cotton short fruit branches and Nongda cotton No. 13, B is the expression level of GhGAUT9 in different fiber development stages of Daizi cotton short fruit branches and Nongda cotton No. 13; C is the expression level of GhGAUT9 in different tissues.

[0020] Figure 2 It is the identification result of the gaut9 mutant in Example 3 of the present invention. A is the detection of SALK_040287.41.40.x (gaut9-1) by three primers; B: the detection of SALK_115588.20.55.x (gaut9-2) by three primers; M in A and B is DL 2000 DNA marker; C is the expression level of endogenous AtGAUT9 in Arabidopsis thaliana detected by qRT-PCR.

[0021] Figure 3 It is the detection and screening result of the overexpression Arabidopsis strain in Example 4 of the present invention. Among them, A is the PCR detection result of the T1 generation positive plants, M: DL 2000DNA marker; B is the result of herbicide spraying; C is the expression level of GhGAUT9 detected by qRT-PCR in different Arabidopsis materials; D is the protein immunoblotting detection of GFP.

[0022] Figure 4 This is the subcellular localization result of GhGAUT9 in Arabidopsis hypocotyl in Example 5 of the present invention.

[0023] Figure 5It is the functional identification result of GhGAUT9 in Arabidopsis in Example 6 of the present invention. Among them, A is the detection of pectin content in GhGAUT9 overexpression strains; B is the action model of GAUT consuming Gal; C is a schematic diagram of planting different Arabidopsis materials in a square culture dish; D is the germination of Arabidopsis in a culture medium without adding Gal (-Gal); E is the germination of Arabidopsis in a culture medium with added Gal (+Gal); F is the statistics of germination in different culture media.

[0024] Figure 6 This is a graph showing the change in the length of Arabidopsis epidermal hairs in Example 7 of the present invention, wherein A to E: microscopic observation of epidermal hairs; F: measurement of epidermal hair length (n≥100).

[0025] Figure 7 This is the result of microscopic observation of the cell length of the Arabidopsis hypocotyl in Example 8 of the present invention. Wherein, A is PI staining and microscopic observation of the hypocotyl; B is the measurement of the cell length of the hypocotyl.

[0026] Figure 8 This is the result diagram of using VIGS to verify the role of GhGAUT9 in fiber elongation in Example 9 of the present invention. Among them, A is a schematic diagram of the RNAi target site; B is the PCR detection result of TRV:GhGAUT9 vector; C is the albino control; D is the silencing efficiency detection in 15DPA fiber; E is the total pectin content detection in 15DPA fiber; F is the pectin methyl esterification degree detection in 15DPA fiber; G is the fiber length observation by combing method; H is the seed morphology observation; I is the fiber length measurement; J is the fiber weight per seed; K is the seed weight. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] In the embodiments of the present invention, the methods used are:

[0029] 1. Real-time quantitative analysis of reverse transcription

[0030] The plant tissue was wrapped in tin foil, frozen in liquid nitrogen, and then stored in a -80°C freezer. Plant Total RNA Isolation Kit instructions; refer to the HiScript II Q SelectRT SuperMix for qPCR (+gDNA wiper) kit instructions to configure the reaction system and reverse transcribe RNA into cDNA; refer to the PerfectStart Green qPCR SuperMix kit instructions to configure the reaction system and set the program for qRT-PCR reaction in Roche LightCycler 96, and the results are obtained by 2 －Δ The relative expression was calculated by CT method.

[0031] 2. Gene cloning and vector construction

[0032] The protein coding sequence (CDS) sequence of the gene was downloaded from the CottonFGD (https: / / cottonfgd.net / browse / ) website. Primers were designed by seamless connection based on the gene sequence and vector sequence. The primer design software was Primer Premier 5 (http: / / www.premierbiosoft.com / primerdesign / ). The fiber samples stored at -80°C were ground into powder in a mortar, and liquid nitrogen was added during the process to prevent sample degradation; RNA was then extracted and reversely transcribed into cDNA according to the instructions of the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit, which was stored in a -80°C refrigerator for subsequent use. The designed specific amplification primers were further used as templates to perform polymerase chain reaction (PCR) to amplify the target gene. The reaction system and procedures were referred to the instructions of PrimeSTAR Max DNA Polymerase. The PCR program is: 95°C, 3min; 95°C, 5sec, 58°C, 10sec, 72°C, 15sec, 35 cycles; 72°C, 5min; 12°C storage. The above PCR reaction solution was subjected to agarose gel electrophoresis detection. If the band is single, refer to the instructions of the DiaSpin column PCR product purification kit to obtain the purified product. If there are multiple bands, refer to the instructions of the agarose gel DNA recovery kit to recover the target fragment. Use restriction endonucleases to digest the vector, XhoI and SpeI digest pGreen-35Spro-GFP, EcoRI and SmaI digest the VGIS empty vector, refer to the instructions of the endonucleases to configure the best reaction system, react at 37°C for 30 minutes; then gel recover the linear vector fragment; the present invention adopts a seamless connection method, and the amplification primer carries the homology arm, and there is no need to digest the target gene. The recovered vector is further connected to the target fragment. The reaction system refers to the instructions of the In-Fusion HD CloningKit kit and incubated at 50°C for 15 minutes. Refer to the instructions for TOP10 E. coli competence, transfer the above recombinant vector into E. coli, culture at 37°C overnight, use the universal primers of the vector to detect monoclonal plaques, and send the correct monoclonal bacterial solution to Genewise Biotech for Sanger sequencing to determine the base sequence of the target gene. Extract the monoclonal E. coli plasmid with the correct sequencing results and transfer it into GV3101 Agrobacterium.

[0033] 3. Identification of Arabidopsis mutants

[0034] The mutant numbers of Arabidopsis homologous genes were queried in TAIR (https: / / www.arabidopsis.org / ). Arabidopsis mutants were purchased from AraShare Science (www.arashare.cn). The two mutants of AT3G02350 homologous to GhM_D07G2211 (SALK_040287.41.40.x and SALK_115588.20.55.x) were both exon T-DNA insertion types. The mutants were identified by the three-primer method (LP, BP and RP), and the mutant detection primers were designed by T-DNA Primer Design (http: / / signal.salk.edu / tdnaprimers.2.html). The obtained mutants and Columbia wild-type Arabidopsis seeds (wild tip, WT) were planted in a 23°C climate chamber. After Arabidopsis thaliana bolted, the CTAB method was used to extract DNA from Arabidopsis thaliana leaves. The PCR reaction system was configured according to the instructions of the Flash PCR MasterMix kit. The DNA of WT and mutants was used as templates, and the combination of LP+RP and BP+RP was used for detection until homozygous mutant strains were screened. The PCR program was: 95℃, 3min; 95℃, 10sec, 55℃, 10sec, 72℃, 10sec, 32 cycles; 72℃, 5min; 12℃ storage.

[0035] 4. Arabidopsis genetic transformation and screening and identification

[0036] The Agrobacterium solution overexpressing the recombinant vector was suspended in 5% sucrose containing 0.03% silwet L-77 and transferred into Columbia wild-type Arabidopsis by the floral dip method. The early generation Arabidopsis was sprayed with BASTA and the universal primers of the transformation vector were used for PCR amplification technology to identify and purify the transgenic strains. The RNA and protein of the rosette leaves were extracted for qRT-PCR and Western blotting to screen the strains with higher expression levels.

[0037] 5. Measurement of Arabidopsis epidermal hair length

[0038] Plant pure transgenic Arabidopsis, and strictly take insect and moisture prevention measures to ensure the normal growth and development of Arabidopsis; use tweezers to take the fifth true leaf of Arabidopsis with consistent growth before bolting (about 3 weeks after sowing), put it in a 10mL centrifuge tube, add 5mL 95% ethanol to dechloro, overnight, and repeat this process until the leaves are no longer green; finally, remove the ethanol, add 3-5mL 50% glycerol for preservation, and observe under a microscope. Choose the appropriate magnification and adjust the brightness until the tip of the leaf epidermal hair is clear and take pictures; use ImageJ software (https: / / imagej.nih.gov / ij / ) to measure the length of epidermal hair.

[0039] 6. Observation and subcellular localization of Arabidopsis hypocotyl

[0040] Place an appropriate amount of Arabidopsis seeds (about 0.1 mL) in a 1.5 mL centrifuge tube, first add 1 ml of sterile water with a pipette, vortex, centrifuge for a moment and then aspirate; add 1 ml of anhydrous ethanol to the centrifuge tube, gently blow and mix, let stand at room temperature for 1 minute, centrifuge for a moment and then aspirate; add 1 ml of 25% sodium hypochlorite to the centrifuge tube, gently blow and mix, let stand at room temperature for 6 minutes, centrifuge for a moment and then aspirate; add 1 ml of sterile water to the centrifuge tube, gently blow and mix, centrifuge for a moment and then aspirate, and repeat this step 5 times; place the above-disinfected Arabidopsis seeds in a 4°C refrigerator for vernalization for 2 to 3 days, then sow on a 1 / 2MS culture medium containing sucrose, and culture in the dark at about 23 degrees Celsius for 5 to 6 days. Pour PI staining solution (15 mM, 10 mg / ml) into the cap of a 50 ml centrifuge tube, place the hypocotyl therein, and incubate in the dark for 13 minutes; observe under an intelligent laser scanning confocal microscope, and simultaneously observe the subcellular localization of GhGAUT9-GFP.

[0041] 7. Cotton VIGS and phenotypic observation

[0042] Zhongmianso 50 was planted in a greenhouse and injected with Agrobacterium after budding, including the bacterial solution of empty vector control, albino control and target gene silencing vector, once a month to ensure the silencing efficiency. Fiber RNA was extracted 15 days after flowering, and the silencing efficiency was detected by qRT-PCR technology, and the fiber length was measured by combing method.

[0043] 8. Determination of pectin content and degree of pectin methylation

[0044] The rosette leaves of Arabidopsis thaliana before bolting (about 3 weeks after sowing) and with uniform growth were placed in an oven to dry to constant weight, and then ground into powder using a mortar; 50 mg of sample was weighed, 1 mL of extracting solution A was added, and the mixture was quickly homogenized at room temperature, and extracted in a 90°C water bath for 30 min (strictly sealed to prevent water loss), cooled to room temperature, centrifuged at 5000g for 10 min at room temperature, the supernatant was discarded, and the precipitate was retained; 1 mL of extracting solution A was added to the precipitate, and the mixture was centrifuged at 5000g for 10 min at room temperature, the supernatant was discarded, and the precipitate was retained; 1 mL of extracting solution B was added to the precipitate, and the mixture was hydrolyzed at 90°C for 1 h (strictly sealed to prevent water loss), and the mixture was mixed and inverted every 10 minutes to ensure sufficient hydrolysis, cooled to room temperature, and centrifuged at 8000g for 15 min at room temperature, and the supernatant was diluted 3 to 5 times as the sample to be tested. Subsequently, the standard curve was drawn and the pectin content was determined by referring to the instructions of the total pectin content detection kit, and the absorbance value was measured at 530 nm using a visible light spectrophotometer; the degree of methyl esterification was detected by referring to the instructions of the pectin methyl esterification degree test kit.

[0045] Example 1 Isolation and sequence analysis of GhGAUT9

[0046] Through GWAS screening, a GAUT family member GhM_D07G2211 related to fiber length was identified, and the reference genome functional annotation showed that this gene encodes galacturonosyltransferase 9 (GAUT9).

[0047] This embodiment is based on the sequence design of the reference genome to isolate the gene. Through PCR reaction, the protein coding sequence of GhGAUT9 was successfully isolated from the cDNA of the short fruit branch fiber elongation period of Daizi cotton. Agarose gel electrophoresis showed that the fragment length was about 1700bp, which was consistent with the 1674bp shown in the reference genome; the fragment was further obtained by gel recovery, and the Sanger sequencing analysis results showed that the base sequence of the amplified fragment was consistent with the reference genome, the CDS length was 1674bp (the nucleotide sequence was shown in SEQ ID NO.1), and the length of the encoded protein was 557aa. The obtained sequence was used to construct a phylogenetic tree with Arabidopsis GAUT family members. The results showed that the gene was homologous to AtGAUT9, and the amino acid sequence alignment similarity was 75.4%. The conservative domain analysis of the protein encoded by this gene showed that the protein contained a GT8 conservative domain, which was located at amino acids 293 to 531.

[0048] Example 2 Expression Analysis of GhGAUT9

[0049] In order to further clarify the expression level of GhM_D07G2211 in fiber development, long-fiber varieties (Nongda cotton No. 13, 31.17 mm) and short-fiber varieties (Daizi cotton short fruit branch, 24.69 mm) were selected ( Figure 1 qRT-PCR analysis was performed on fibers of 0, 5, 10, 15, 20 and 25 DPA of A). The results showed that the expression level of GhM_D07G2211 in short-fiber varieties reached a peak at 15 DPA; while the expression level of Nongda Cotton No. 13 was generally low at all stages of fiber development ( Figure 1 B). The high expression of GhM_D07G2211 in short-fiber varieties, especially reaching a peak during the fiber elongation period, suggests that it may negatively regulate fiber elongation. At the same time, in order to explore whether GhM_D07G2211 is specifically expressed in cotton fibers, its expression levels in the calyx, petals, roots, stems and leaves of cotton were analyzed by qRT-PCR. The results showed that the expression levels of GhM_D07G2211 in different tissues were lower than those in the fiber elongation period of the short fruit branches of Daizi cotton, but the expression level in the roots was relatively high compared with other tissues ( Figure 1 C).

[0050] Example 3 Screening of mutants of AtGAUT9, a homologous gene of GhGAUT9. Two mutants of AtGAUT9 were selected in the present invention: SALK_040287.41.40.x (gaut9-1) and SALK_115588.20.55.x (gaut9-2), both of which were T-DNA insertion types. In order to use pure mutant strains for subsequent experiments, multiple individual strains were randomly selected, their DNA was extracted, and the mutants were identified by the three-primer method. The results showed that when BP and RP primers were used and the DNA of the mutant was used as a template, most of them had only one band; when LP and RP primers were used and the DNA of the mutant was used as a template, there was no band, and when the DNA of the WT was used as a template, there was only one band; the sizes of the bands amplified using BP and RP as primers were all smaller than WT ( Figure 2 A and B), indicating that these strains are homozygous mutants. qRT-PCR results showed that compared with WT, the expression level of endogenous AtGAUT9 in the mutants was completely knocked out ( Figure 2 C).

[0051] Example 4 Identification and expression analysis of GhGAUT9 overexpressing transgenic Arabidopsis

[0052] In order to verify the functional role of GhGAUT9, the isolated GhGAUT9 was connected to a plant overexpression vector driven by the 35s promoter and carrying the encoding green fluorescent protein (GFP) by seamless connection. The pGreen 35sPro-GhGAUT9-GFP vector was obtained and then transferred into GV3101 Agrobacterium. It was then transformed into Columbia wild-type Arabidopsis by the floral dip method, and the seeds (T1 generation) were harvested. DNA of the T1 generation plants was extracted and tested by PCR. The results of agarose gel electrophoresis showed that there were no amplified bands in the wild type and mutants, and the target bands were successfully detected in multiple transgenic plants, which were consistent with the size of the positive control (recombinant plasmid), indicating that the transformation was successful ( Figure 3 A). Based on the characteristics of the herbicide-resistant (basta) gene carried by the vector, the pure lines were further screened by spraying herbicides. All wild-type and mutants died after spraying herbicides, while no seedlings of the T3 generation overexpression lines died, indicating that the overexpression Arabidopsis thaliana was screened to homozygous lines ( Figure 3 B). Two strains with higher expression levels (OE-3 and 4) were further selected by qRT-PCR. Their absolute expression levels were 4 and 2 times that of the Arabidopsis thaliana internal reference Atactin ( Figure 3 C), Western blotting results also showed that GFP protein could be detected in the transgenic lines ( Figure 3 D).

[0053] Example 5 Subcellular localization of GhGAUT9

[0054] After obtaining transgenic Arabidopsis, in order to explore the cellular localization of the GhGAUT9 protein carrying the GFP tag, the present invention observed the hypocotyls of the stable Arabidopsis overexpression strain (Arabidopsis hypocotyl is a typical model for studying cell expansion, and found that the green fluorescence was distributed in a dot-like manner (this is similar to the reported localization of the Golgi apparatus in the Arabidopsis hypocotyl, and is mainly distributed near the cell wall, especially concentrated at both ends of the hypocotyl polar elongation ( Figure 4 A and B), indicating that GhGAUT9 acts on the site of polar extension of the cell wall through the Golgi apparatus in cells undergoing polar elongation.

[0055] Example 6 Effect of GhGAUT9 on Pectin Content and Galactose Tolerance in Arabidopsis

[0056] After clarifying the subcellular localization of GhGAUT9, the present invention first explored whether GhGAUT9 has galacturonosyltransferase activity and participates in the GalA pathway of Arabidopsis thaliana by applying galactose Gal exogenously. GAUT transfers GalA to the non-reducing end of endogenous or exogenous HG receptors, which can indirectly consume Gal ( Figure 5 A). The present invention adds an excessive amount (75 mM) of Gal (+Gal) to 1 / 2MS medium, and uses no Gal as a control (-Gal) to observe the germination of different Arabidopsis materials ( Figure 5 B, C and D), the results showed that compared with wild-type Arabidopsis, the relative germination rate of Arabidopsis overexpressing GhGAUT9 increased significantly, while the relative germination rate of gaut9 decreased ( Figure 5 E). Compared with the wild type, the overexpression strain was less affected by the toxicity of Gal, while the mutant was more affected. GhGAUT9 can participate in the metabolism of GalA.

[0057] In order to further verify whether GhGAUT9 affects pectin synthesis, the present invention detected the total pectin content of Arabidopsis rosette leaves. It is worth mentioning that reported studies have shown that the galacturonic acid content of Arabidopsis mutant gaut9 is lower than that of the wild type. Therefore, the present invention first determined the pectin content of overexpressed GhGAUT9. The results showed that the average total pectin content of wild-type Arabidopsis rosette leaves was about 0.195 mmol / g, and the average total pectin content of the two overexpression lines was 0.225 and 0.221 mmol / g, respectively. Figure 5 F), indicating that high expression of GhGAUT9 can promote pectin synthesis.

[0058] Example 7 Effect of GhGAUT9 on the Length of Arabidopsis Epidermal Hairs

[0059] In order to preliminarily verify whether GhGAUT9 affects the elongation of Arabidopsis leaf epidermal hairs, the fifth whorl leaf of Arabidopsis material before bolting (about 20 days after sowing) was selected for microscopic observation ( Figure 6 The results showed that the average length of the epidermal hairs of wild-type Arabidopsis was 232 μm, that of the two overexpression lines was 204 and 209 μm, and that of the mutant lines was 248 and 247 μm. After overexpression of GhGAUT9, the length of the epidermal hairs was significantly shortened, while that of the mutant was significantly longer ( Figure 6 F), indicating that GhGAUT9 has the function of negatively regulating epidermal hair length.

[0060] Example 8 Effect of GhGAUT9 on the Length of Arabidopsis Hypocotyl Cells

[0061] In this example, the hypocotyl cell wall was stained with propidium iodide stain, and the results of laser scanning confocal microscopy showed that the length of the hypocotyl cells of the overexpressed Arabidopsis thaliana was shorter than that of the wild type, while the length of the hypocotyl cells of the mutant was longer than that of the wild type ( Figure 7 A); the average length of wild-type hypocotyl cells was 614.5 μm, the average length of hypocotyls of the two overexpression lines OE-3 and 4 was 479.5 and 395 μm, respectively, and the average length of hypocotyls of the two mutants gaut9-1 and gaut9-2 was 696 μm and 747 μm, respectively ( Figure 7 B). The above results indicate that GhGAUT9 negatively regulates the length of hypocotyl and its cells.

[0062] Example 9 Effect of GhGAUT9 on Cotton Fiber Length

[0063] In order to accurately silence GhGAUT9 in cotton, the present invention selects the region located in the gene UTR and the first exon as the silencing target fragment ( Figure 8 A). The target fragment was connected to the pTRV2 vector (tobaccorattle virus, TRV) by seamless ligation. The correctness of the fragment sequence was confirmed by Sanger sequencing. Then, GV3101 Agrobacterium was transformed and detected using universal primers for the vector. The results of agarose gel electrophoresis showed that the amplified band was consistent with the expected ( Figure 8 B), which can be used for subsequent VIGS experiments.

[0064] Further, the bacterial solution was injected when the cotton buds appeared, including the pTRV-CLA1 albino control, the pTRV-00 empty vector and the PTRV-GhGAUT9 silenced strain, and then injected once a month to ensure the silencing efficiency. The albino control strain showed obvious albino phenomenon ( Figure 8C), indicating that the silencing system is efficient. The silencing efficiency, pectin content and degree of methylation were measured in the fibers of the gene-silenced plants and the control plants 15 days after flowering. The results showed that the VIGS system significantly reduced the expression level of GhGAUT9 during the fiber elongation period by about 13% ( Figure 8 D), the average total pectin content after silencing GhGAUT9 was about 15.33 μmol / g, which was significantly reduced by about 19% compared with the total pectin content of the control (average 18.94 μmol / g) ( Figure 8 E), the degree of pectin methylation did not change significantly ( Figure 8 After the plants finished boll opening, the results of measuring the cotton fiber length by combing method showed that the average fiber length of cotton plants with GhGAUT9 silenced was 31.75 mm, while the average fiber length of cotton plants with control was 29.7 mm. After silencing GhGAUT9, the fiber length increased significantly by 2 mm (about 6.7%) ( Figure 8 G and I). Considering whether GhGAUT9 would increase fiber at the expense of yield, we observed seed length and found no significant changes in seed morphology ( Figure 8 The fiber weight per seed did not change significantly ( Figure 8 However, interestingly, seed weight was significantly increased after silencing GhGAUT9 ( Figure 8 In summary, silencing GhGAUT9 can significantly promote fiber elongation, and this promoting effect is mainly derived from the accumulation of pectin rather than the cross-linking effect related to methyl esterification between pectins, and silencing GhGAUT9 will not reduce the yield.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of GhGAUT9 gene in negatively regulating the length of plant epidermal hairs.

2. Application of GhGAUT9 gene in negative regulation of cotton fiber length.

3. Application of GhGAUT9 gene in regulating pectin content in cotton fiber.

4. The use according to claim 3, characterized in that: The GhGAUT9 gene is overexpressed in cotton to increase the pectin content of cotton fiber.

5. The use according to claim 3, characterized in that: Silencing the GhGAUT9 gene in cotton reduces the pectin content of cotton fiber.

6. Application of GhGAUT9 gene in increasing cotton seed weight, characterized in that: Silencing of the GhGAUT9 gene in cotton.

7. The use according to any one of claims 1 to 6, characterized in that: The nucleotide sequence of the GhGAUT9 gene is shown in SEQ ID NO.

1.

8. A method for increasing the length of cotton fibers, characterized in that: In the cotton genome, the GhGAUT9 gene shown in SEQ ID NO.1 was silenced.

9. A method for breeding long-fiber cotton varieties, characterized in that: The expression level of the GhGAUT9 gene shown in SEQ ID NO.1 in the cotton to be tested is detected, and a cotton variety with a low expression level is selected as a long-fiber cotton variety.

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