CN101698838B - Uridine diphosphate glucuronate isomerase and its coding gene and application - Google Patents

Abstract

The invention discloses a uridine diphosphate glucuronosyl isomerase, and a coding gene and application thereof. Uridine diphosphate glucuronosyltransferase isomerase, being the protein of a) or b) as follows: a) a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table; b) protein which is derived from a) and related to the synthesis of uridine diphosphate galacturonic acid by substituting and/or deleting and/or adding one or more amino acids in the amino acid sequence shown as the sequence 2 in the sequence table. The invention also discloses a coding gene of the protein. The encoding gene of the uridine diphosphate glucuronosyl acid isomerase is introduced into cotton, so that the length of cotton fibers is increased, and the quality and the yield of the cotton fibers are improved. The uridine diphosphate glucuronosyl isomerase and the coding gene thereof have great economic value and application prospect.

Description

Uridine diphosphate glucuronate isomerase and its coding gene and application

technical field

The invention relates to uridine diphosphate glucuronate isomerase and its coding gene and application.

Background technique

Cotton fiber is a unicellular structure differentiated from the outer skin cells of the ovule. Cotton fiber is an important raw material for the textile industry and has important economic value. Fiber quality determines its final length and strength. At the same time, cotton fiber cells are also an ideal system for studying important biological phenomena such as cell elongation, differentiation and cell wall synthesis. Therefore, the study of fiber elongation has important economic value and theoretical significance.

The development process of cotton fiber cells is a process of supernormal cell elongation and supernormal cell wall thickening. The length of upland cotton is about 3.0cm, and the diameter of the upland cotton cell wall is 11-22μm, that is to say, the aspect ratio is 1000-3000. Cotton fiber cell formation can generally be divided into four overlapping stages: fiber initiation, cell elongation (primary wall formation), secondary wall deposition, and maturation.

The cell wall is one of the main organs that distinguish plants from animals. The primary cell wall is mainly composed of polysaccharides such as cellulose, pectin, and hemicellulose. Some special cells, such as cotton fiber, xylem and sclerenchyma cells, will continue to deposit secondary cell walls inside the primary wall after stopping growth, mainly composed of cellulose, hemicellulose and a small amount of lignin. The deposition and changes of cell walls play an important role in the growth, development and response of plants to the external environment. It ultimately determines the size and shape of the cell.

Plant cell wall pectin is mainly composed of a mixture of three types of polysaccharides: homogalacturonic acid (HGA), rhamnogalacturonic acid I (RGI) and rhamnogalacturonic acid II (RGII). Homogalacturonic acid is a homopolymer of galacturonic acid. Polyrhamnogalacturonic acid I is a heteromer composed of repeating rhamnose and galacturonic acid disaccharide units, in which the rhamnose residues are also linked with arabinan, galactan and arabinogalactan sugar. Polyrhamnogalacturonic acid II is a modified homogalacturonic acid, which is a polysaccharide with diverse connection structures, and its content in the cell wall is very low.

Plant cell wall-rich polysaccharides are synthesized by uridine diphosphate glucose through a series of conversions into various nucleoside sugars, and then synthesized by glycosyltransferases. Galacturonic acid is the main sugar unit of pectin polysaccharides. UDP-galacturonic acid (UDP-GalA) is an activated precursor for the synthesis of polysaccharides containing galacturonic acid. UDP-GalA is isomerized from uridine diphosphate glucuronic acid (UDP-GlcA) under the catalysis of UDP-D-glucuronic acid4-epimerase (GAE). GAE is a reversible enzyme that can also catalyze the conversion from UDP-GalA to UDP-GlcA.

In 2002, Tokumoto et al. found that during the fiber elongation period, cell wall matrix polysaccharides (mainly pectin and hemicellulose) accounted for 30-50% of the total cell wall sugars, and the proportion decreased rapidly during the secondary wall thickening period. to 3%. In 1999, Chanliaud and Gidley confirmed that pectin polysaccharides can affect the properties of cell walls by participating in the deposition of cellulose. In the same year, Wen et al. found that inhibiting the expression of pectin methylesterase could change the traits of pea root cells, resulting in shorter roots. In 2003, Jones et al. used arabinanase to hydrolyze pectin RGI to lose the opening and closing function of stomata in isolated Commelina leaves. In 2006, Moore et al. studied the leaf cell wall structure of Myrothamnus flabellifolius (which can still be revived with water after several long-term drought dehydration) and found that the presence of arabinan-rich cell wall polysaccharides endowed it with structural properties of multiple dehydration and hydration. All the above studies have shown that pectin polysaccharides are very important for cell elongation and cell wall flexibility.

Contents of the invention

The purpose of the present invention is to provide a uridine diphosphate glucuronate isomerase and its coding gene and application.

The uridine diphosphate glucuronate isomerase provided by the present invention is a protein related to the synthesis of uridine diphosphate galacturonic acid, and the protein is named GhGAE1, which is the protein of a) or b) as follows:

a) a protein consisting of the amino acid sequence shown in Sequence 2 in the sequence listing;

b) A protein derived from a) after the amino acid sequence of Sequence 2 in the sequence listing is substituted and/or deleted and/or one or several amino acids are added and related to the synthesis of uridine diphosphogalacturonic acid.

Among them, the sequence 2 in the sequence listing consists of 431 amino acid residues.

The amino acid sequence of the above b) is shown in the 22-431 position of sequence 2.

In order to facilitate the purification of the GhGAE1 protein in a), tags shown in Table 1 can be attached to the amino-terminus or carboxy-terminus of the protein consisting of the amino acid sequence shown in Sequence 2 in the Sequence Listing.

Table 1. Sequence of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

The GhGAE1 protein in b) above can be synthesized artificially, or its coding gene can be synthesized first, and then biologically expressed. The gene encoding the GhGAE1 protein in the above b) can be obtained by deleting the codon of one or several amino acid residues from the DNA sequence shown in the 169-1464 position of the 5' end of the sequence 1 in the sequence listing, and/or performing a Or a missense mutation of a few base pairs, and/or the coding sequence of the tag shown in Table 1 is attached to its 5' end and/or 3' end.

The gene encoding the protein also belongs to the protection scope of the present invention.

The coding gene of the protein is the gene described in any one of the following 1) to 5):

1) Its nucleotide sequence is sequence 1 in the sequence listing;

2) Its coding sequence is the 169th-1464th position from the 5' end of sequence 1 in the sequence listing;

3) Its nucleotide sequence is the 233-1536 position from the 5' end of sequence 1 in the sequence listing;

4) A DNA molecule that hybridizes to the DNA fragment defined in 1) or 2) or 3) under stringent conditions and encodes a protein related to the synthesis of uridine diphosphogalacturonic acid;

5) A DNA molecule having more than 90% homology with the gene defined in 1) or 2) or 3) and encoding a protein related to the synthesis of uridine diphosphate galacturonic acid.

The gene in step 5) preferably has more than 95% homology with the gene in 1).

Sequence 1 in the Sequence Listing consists of 1584 nucleotides, and the 169th-1464th position from the 5' end is a coding sequence, encoding the protein shown in Sequence 2 in the Sequence Listing.

The above-mentioned stringent conditions can be hybridization at 68° C. in a solution of 6×SSC, 0.5% SDS, and then wash the membrane once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS respectively.

The primer pair for amplifying the full length or any fragment of the GhGAE1 gene also belongs to the protection scope of the present invention.

Recombinant vectors, transgenic cell lines and recombinant bacteria containing the above-mentioned GhGAE1 gene, and primer pairs for amplifying the full length of the gene and any fragment thereof also belong to the protection scope of the present invention.

The recombinant expression vector containing GhGAE1 gene can be constructed by existing plant expression vector. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, such as pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other derived plant expression vectors. The plant expression vector carrying the GhGAE1 gene of the present invention can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conduction, and Agrobacterium-mediated.

When using the GhGAE1 gene to construct a recombinant plant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription initiation nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter promoter, ubiquitin gene Ubiquitin promoter (pUbi), etc., they can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancement These enhancer regions can be ATG initiation codons or adjacent region initiation codons, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding genes (GUS genes, luciferase genes, etc.) that can express enzymes or luminescent compounds that can produce color changes in plants. ), antibiotic markers with resistance (gentamicin markers, kanamycin markers, etc.), or chemical-resistant marker genes (such as herbicide resistance genes), etc.

Another object of the present invention is to provide the application of any one of the above-mentioned proteins as uridine diphosphate glucuronate isomerase.

Another object of the present invention is to provide the application of any of the above-mentioned proteins or any of the above-mentioned genes in converting uridine diphosphate glucuronic acid (UDP-GlcA) into uridine diphosphate galacturonic acid (UDP-GalA) Or the application in converting UDP-GalA to UDP-GlcA.

Another object of the present invention is to provide a method for cultivating transgenic plants with high uridine diphosphate galacturonic acid content.

The method for cultivating transgenic plants with high uridine diphosphate galacturonic acid content provided by the present invention is to introduce the gene into plant cells to obtain transgenic plants with increased uridine diphosphate galacturonic acid content.

Wherein, the plant can specifically be cotton.

The uridine diphosphate glucuronate isomerase and its encoding gene of the invention can be used to breed cotton with increased fiber length.

The uridine diphosphate glucuronate isomerase encoding gene of the present invention has the highest expression abundance in the rapid fiber elongation period (10 days after flowering), and the rapidly elongated fiber primary cell wall contains a large amount of galacturonic acid, indicating that GhGAE1 is A key step in the synthesis of galacturonic acid in pectin polysaccharides, which is very important for fiber elongation. The uridine diphosphate glucuronide isomerase coding gene of the present invention is introduced into cotton, thereby increasing the length of cotton fibers and improving the quality and yield of cotton fibers. The uridine diphosphate glucuronate isomerase and its coding gene of the invention have great economic value and application prospect.

Description of drawings

Figure 1 shows the expression level analysis of GhGAE1 in different developmental stages of cotton.

Fig. 2 is a gas chromatographic analysis of primary walluronic acid sugar components in cotton fibers and ovules.

Figure 3 is the effect of UDP-galacturonic acid on fiber elongation.

Figure 4 shows the effect of ethylene treatment on the expression level of GhGAE1 gene.

Fig. 5 is the in vitro expression of GhGAE1 protein.

Figure 6 is the activity assay of GhGAE1 protein.

Detailed ways

All reagents used in the following examples can be obtained from commercial sources.

The experimental methods in the following examples are conventional methods unless otherwise specified.

The percentages in the following examples are mass percentages unless otherwise specified.

Embodiment 1, cloning of uridine diphosphate glucuronate isomerase coding gene

1. Cloning of GhGAE1 gene

Select wild-type upland cotton variety Xuzhou 142 (WT) (National Mid-term Cotton Seed Bank of Cotton Research Institute of Chinese Academy of Agricultural Sciences) fibers 10 days after flowering, extract total RNA, and extract total RNA according to the RNAgents Total RNA Isolation System kit of Promega Company conduct. Take 5g of total RNA and reverse transcribe to obtain one strand of cDNA. The reaction system is as follows:

Total RNA 10 μL (5 μg), Oligo (dT) (20 μmol/L) 1.5 μL, 10× buffer 2.5 μL, dNTPmix (2.5 mmol/L) 2 μL, sterile water 8 μL.

After bathing in water at 42°C for 1 min, add 1 μL (200 U) of SuperScript ^™ IIRT to the reaction system, mix gently, and keep warm at 42°C for 50 min; bath in water at 70°C for 15 min to terminate the reaction; obtain the first strand of cDNA.

Primers were designed for PCR amplification, and the primer sequences were as follows:

P1: 5′AATAAAAACTCCCCTCCCTTTTTTTC 3′;

P2: 5′TACGGAATCCTCGTCTTTCTCTTG 3′.

The PCR amplification conditions were as follows: first 94°C for 3 min; then 94°C for 1 min, 59°C for 45 s, 72°C for 2 min, 30 cycles; finally 72°C for 10 min.

A fragment of about 1584bp was amplified by PCR, and after recovery, it was connected to the pGEM-T Easy vector to construct the recombinant plasmid pT-GhGAE1, and transformed into Escherichia coli DH5α, and the plasmid of the positive clone identified by restriction enzyme digestion was extracted and sequenced. The sequencing results showed that, The cloned cDNA sequence is shown as sequence 1 in the sequence listing, and its coding sequence is the 169-1464 position from the 5' end of sequence 1 in the sequence listing, encoding the protein shown in sequence 2 in the sequence listing.

2. Relationship between expression level of GhGAE1 and fiber elongation

The wild-type upland cotton variety Xuzhou 142 (WT) and the fluff-free and flocculent-free mutant (FL) cotton (National Mid-term Cotton Seed Bank of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences) were selected at different developmental stages, and the expression level of GhGAE1 was analyzed by real-time quantitative PCR. .

Specific steps are as follows:

Extraction of RNA: according to the operation manual Micro-TO-Midi Total RNA Purification System (Invitrogen, USA) from the ovules of the wild-type upland cotton variety Xuzhou 142 on the day of flowering and 3 days after flowering, 5, 10, 15, 20 and 25 days after flowering Total RNA was extracted from the fiber, and the ovules 10 days after flowering of the fuzzless and flocculent-free mutant and the wild-type upland cotton variety Xuzhou 142.

Preparation of cDNA template: Using 5 μg of total RNA as a template, the genomic DNA was digested with DNase I, and then the first strand of cDNA was synthesized with the SUPERSCRIPT ^TM first strand synthesis system according to the operation manual.

Real-time quantitative PCR: GhUBQ7 and GhGAE1 gene-specific primers were designed respectively, the housekeeping gene cotton ubiquitin (Ubiquitin, UBQ) was selected as an internal standard, and real-time quantitative PCR was performed to analyze GhGAE1 according to the DyNAmo ^TM SYBR Green Qpcr Kit (Finnzymes, USA) operating manual. Express.

GhUBQ7 primer 5' sequence: 5'-GAAGGCATTCCACCTGACCAAC-3';

GhUBQ7 primer 3' sequence: 5'-CTTGACCTTTCTTCTTCTTGTGCTTG-3'.

GhGAE1 primer 5' sequence: 5'-ACGCCAGGGAAGTTCAAGGTCG-3';

GhGAE1 primer 3' sequence: 5'-GCCGTGGCTGTTAAGCAGGGAT-3'.

The reaction system is as follows:

Reaction parameters: pre-denaturation at 95°C for 20s, 42 cycles of amplification (each cycle includes denaturation at 94°C for 10s, annealing at 58°C for 20s, and extension at 72°C for 30s), then read the fluorescence data collected at 78-80°C; finally extend for 5min .

The results of real-time quantitative PCR are shown in Figure 1. The expression of GhGAE1 was very low in the ovules of the wild-type upland cotton variety Xuzhou 142 10 days after flowering and in the ovules of the lintless and flocculent-free mutant 10 days after flowering. The fiber of the wild-type upland cotton variety Xuzhou 142 had the highest abundance, which indicated that the expression level of GhGAE1 was significantly up-regulated in fiber cells and may play an important role in fiber development. In Fig. 1, WT-F represents the fibers of the wild type upland cotton variety Xuzhou 142, WT-O represents the ovules of the wild type upland cotton variety Xuzhou 142, FL-O represents the ovules of the lint-free and flocculent-free mutant, and the numbers that follow represent after flowering number of days.

3. Gas chromatographic analysis of primary walluronic acid sugar components in cotton fibers and ovules

Specific steps are as follows:

1. Polysaccharide decomposition: Weigh 10 mg of cell wall, add 1.25 ml of 2M trifluoroacetic acid (TFA), add 50 μl of 5 mg/ml inositol as internal standard, 120 ° C, 2 h; centrifuge.

2. Take 250μl TFA polysaccharide lysate, transfer it to a 15ml centrifuge tube, and dry it with nitrogen in a 40-degree water bath.

3. Add 100ul of pyridine solution containing 20mg/ml methoxyamine hydrochloride, and react at 37°C for 2 hours.

4. Add 100 ul of silylating reagent [bis(trimethylsilyl)fluoroacetamide (BSTFA) + trimethylsilane (TMS)], and react at 37° C. for 0.5 hour.

5. Take 10 μl of the reaction solution for GC-MS analysis, DB-5MS column, program: 160°C, 1min, 10°C/min to 172°C, 5°C/min to 208°C, 10sec to 200°C, hold 2min, 30sec to 160°C, keep 2min. Each sugar component was first identified according to the retention time of the standard, and then confirmed by mass spectrometry.

The results are shown in Figure 2. No glucuronic acid was detected in the cell walls of fibers and ovules ((WT-F-10, FL-O-10 and WT-O-10) after flowering for 10 days by gas chromatography-mass spectrometry analysis The galacturonic acid content (galacturonic acid 1 and galacturonic acid 2) in the cell wall of the fiber of flowering 10 days is more than two times higher than in the ovule.In Fig. 2, WT-F represents the wild type upland cotton variety Xuzhou 142 fibers, WT-O indicates the ovules of the wild-type upland cotton variety Xuzhou 142, FL-O indicates the ovules of the fuzzless and flocculent-free mutant, and the following numbers indicate the days after flowering.

4. Effect of uridine diphosphate galacturonic acid (UDP-GalA) on fiber elongation

The material composition of the ovule culture medium is shown in Table 1.

Table 1. Composition of cotton ovule tissue culture medium

Element The concentration of each component in the culture medium (mmol/L) KH ₂ PO ₄ 2.00 H ₃ BO ₃ 0.10 Na ₂ MoO ₄ 0.001 CaCl ₂ 2H ₂ O 3.00 KI 0.005 CoCl ₂ 6H ₂ O 0.0001 MgSO ₄ 7H ₂ O 2.00 MnSO ₄ ·H ₂ O 0.10 ZnSO ₄ 7H ₂ O 0.03 CuSO ₄ 5H ₂ O 0.0001

KNO ₃ 50.00 FeSO ₄ 7H ₂ O 0.030 Na ₂ EDTA 0.030 Nicotinic acid 0.004 Vitamin B6 (Pyridoxine·HCl) 0.004 Vitamin B1 (Thiamine·HCl) 0.0040 inositol 1.00 D-glucose 100.00 D-Fructose 20.00

Cotton ovules 1 day after flowering were used for culturing experiments, and the steps were as follows:

1) After picking the cotton bolls of the wild type upland cotton variety Xuzhou 142 one day after flowering, soak them in 10% sodium hypochlorite for 10-15 minutes, and then wash them with ovule culture solution for 4 times;

2) Carefully place the ovules into a 50ml Erlenmeyer flask filled with ovule culture medium with tweezers and a scalpel burned by an alcohol lamp, and add 5 μM UDP-galacturonic acid;

3) Put the Erlenmeyer flask containing the ovules into an incubator for dark cultivation, and be careful not to shake the Erlenmeyer flask.

No addition of UDP-galacturonic acid was used as a control.

The results are shown in Figure 3, 1 represents the control, and 2 represents the addition of 5 μM UDP-galacturonic acid; indicating that UDP-galacturonic acid can significantly promote the elongation of fibers.

5. The effect of ethylene treatment on the expression level of GhGAE1 gene

Cotton ovules of wild-type upland cotton variety Xuzhou 142 were cultured in ovule culture medium, added 0.1 μM ethylene and sealed, and cultured at a constant temperature of 37 degrees. The ovules cultured in the ovule culture medium without adding ethylene were used as the control. The ovules treated with ethylene and the control were collected at 3, 6, and 12 hours respectively, total RNA was extracted, and real-time quantitative PCR was performed according to the method in (2).

The results are shown in Figure 4, indicating that ethylene can significantly promote the expression of GhGAE1 gene.

6. Determination of Enzyme Activity of GhGAE1

1) Prokaryotic expression vector construction.

Because GhGAE1 is a Golgi-localized protein, considering that the signal peptide may affect the prokaryotic expression of the protein, the signal peptide part of the N-terminal 21 amino acids was removed from the designed protein. Using the wild-type upland cotton variety Xuzhou 142 cDNA as a template, the following primers were used for PCR amplification:

GhGAE1-5'primer: 5'-GGATCCCCACAATATGAACCGTCAATTCCA-3';

GhGAE1-3'primer: 5'-CTCGAGTTTCCCGTTACCTTCTATTTCATTC-3'.

A fragment of about 1316bp was amplified by PCR, and after recovery, it was connected to the pGEM-T Easy vector to construct a recombinant plasmid and transform it into Escherichia coli DH5α, and extract the plasmid of the positive clone identified by enzyme digestion for sequencing. The sequencing results showed that the cloned fragment The nucleotide sequence is as shown in the 233-1536 position of the 5' end of the sequence 1 in the sequence listing, and the encoded amino acid sequence is as shown in the 22-431 position of the sequence 2 in the sequence listing.

The PCR amplification product was inserted between the Xhol and BamH I sites of pET28a (Invitrogen) to obtain pET28a-GhGAE1.

2) Induced expression of GhGAE1

Transform pET28a-GhGAE1 into Escherichia coli expression strain BL21(DE3)pLysS, add 0.8mM IPTG to the positive strain when the OD ₆₀₀ is 0.6-0.8, induce for 4 hours, and collect the bacterial liquid. Resuspend the bacterial solution in sodium phosphate buffer (pH=7.6), break the bacterial solution by ultrasonic, centrifuge at 10,000 g for 30 minutes at 4°C, and dialyze the supernatant overnight at 4°C (the molecular weight cut-off of the dialysis bag is 1.2-1.4kD) To remove small molecular substances, the bacterial fluid after dialysis was used for enzyme activity analysis. At the same time, the supernatant of the bacteria transfected with pET28a empty vector was obtained as a negative control for enzyme activity analysis according to the above steps.

As shown in Figure 5, 1 represents the bacterial supernatant of the IPTG-induced pET28a empty vector transfection, 2 represents the IPTG-induced bacterial supernatant of the pET28a-GhGAE1 transfection, and the arrow points to the expressed GhGAE1-specific protein band.

3) Activity determination

3mM UDP-GlcA or 3mM UDP-GalA (UDP-GlcA was purchased from Sigma, UDP-GalA was purchased from Georgia State University Carbohydrate Research Center (CarboSource Services, University of Georgia)), 25 μL of protein bacteria solution expressing GhGAE1 was dissolved in 500 μL of 100 mM In potassium phosphate buffer (pH 7.6), react at 30°C for 60 min; add 500 μL of chloroform to terminate the reaction. The supernatant of empty vector bacteria was used as a negative control. Centrifuge at 16,000 g for 5 min; collect the aqueous phase. The aqueous phase was analyzed with ZORBAX Eclipse XDB-C18 column (0.46×15cm; Agilent Technologies), column temperature: 40°C; injection volume: 30 μL. HPLC program: 100% (volume percentage) buffer A (100mM potassium phosphate buffer contains 8mM tetrabutylammonium bisulfate, pH 6.5) for 5min, within 27min the gradient increases buffer B [70% (volume percentage) buffer A, 30% (volume percentage) methanol, pH 6.5] until the buffer B of 77% (volume percentage), 77% (volume percentage) buffer B continues 5min, flow rate is 1mL/min, 254nm detection product.

As shown in Figure 6, A is the reaction product of UDP-GlcA detected by HPLC and the crude extract expressing the empty vector; B is the reaction product of UDP-GlcA and the crude extract of GhGAE1 protein detected by HPLC. It can be seen that the crude extract expressing GhGAE1 protein can convert UDP-GlcA to UDP-GalA, but the empty vector bacterial liquid cannot complete this transformation. C is the reaction product of UDP-GalA detected by HPLC and the crude extract expressing the empty vector; D is the reaction product of UDP-GalA and the crude extract of GhGAE1 protein detected by HPLC. It can be seen that the crude bacterial solution expressing GhGAE1 protein can also convert UDP-GalA to UDP-GlcA, but the empty vector bacterial solution cannot complete this conversion. Thus it was proved that GhGAE1 has the activity of uridine diphosphate glucuronate isomerase.

sequence listing

<110> Peking University

<120>Uridine diphosphate glucuronate isomerase and its coding gene and application

<130>CGGNARL82108

<160>2

<210>1

<211>1584

<212>DNA

<213> Upland cotton (Gossypium hirsutum)

<400>1

aataaaaact cccctccctt tttttctgca tctgctttta tttctttaca gctcaaaatt 60

aataattata aatcaaatca agtctctttc ttccttatcc tttcgatctg ctaatcaagt 120

catttcaaga ttttttttct gaacaagaaa aagaaaaata gtaggacaat gccgtccttg 180

gaggatgagc tgtttccgtc gacgccaggg aagttcaagg tcgacagagc acacaatatg 240

aaccgtcaat tccatcgttg cttcgcttca accagcacca tgtttttatg ggctcttttc 300

ttgatcgcat tgacggcgtc gtatttgcgc ttccagagtt tcgttgattc tggtagccga 360

tatttcagtg cttcatgggg tggtatccaa tgggaaaaac aagtccgtaa ctccgctcag 420

atccatcgtt ccggaggcat gtccgttctg gtgactggag cggctggttt cgtcggtaca 480

cacgtttccc ttgccctcaa aaaacgcgga gatggagtcg ttgggcttga caatttcaac 540

aactattacg acccttcgtt gaaaaaggcg aggaaatccc tgcttaacag ccacggcatt 600

ttggtggttg aaggcgattt gaacgacgcc aagctgttgg ctaagctttt cgacgtggtg 660

gcttttactc acgtgatgca tttggcggct caagctggag tcaggtacgc catggaaaac 720

cccaactctt atgttcacag caacatcgcc ggtctcgtca cgcttctcga aatttgcaaa 780

tccgctaatc cccagccagc cgttgtctgg gcctcctcca gttccgttta cggtctcaac 840

gaaaaggttc ctttctctga ggccgacagg accgaccagc cggctagttt gtatgccgcc 900

accaagaagg caggcgaaga aataacccac acttataatc atatctacgg tctttcaatt 960

actggattaa gatttttcac cgtgtacggt ccatggggaa ggcctgatat ggcgtatttt 1020

tcgtttacga gaaatattct gcagggaaag cccatcacca tttatcgggg aaagaatcgg 1080

gttgatttgg cccgagattt tacctacatt gacgatatcg tgaaaggctg tttgggatcg 1140

ttggatacat ccgggaaaag caccggttct ggtgggaaga aaaaggggaa cgctccatat 1200

aggattttta atttggggaa tacgtcgccg gtcaaggtgc cggagctggt gaacatcctg 1260

gaaagacatt tgaaggtgaa agccaaaagg aatatcgtag atatgcctgg aaacggtgac 1320

gttccattca ctcatgcgaa tatcagtttg gcccaaagag aattcgggta caagccctca 1380

accgatttgc aaaccgggtt gaagaagttt gttagatggt atttatctta ttatggttat 1440

aacaaccgca aaggtctaca ataaaattta tttttggttt taaatgtaag gattttcttg 1500

gtttcccatt agaatgaaat agaaggtaac gggaaaaccg aaggaaccgc aagttaaaaa 1560

caagagaaag acgaggattc cgta 1584

<210>2

<211>431

<212>PRT

<213> Upland cotton (Gossypium hirsutum)

<400>2

Met Pro Ser Leu Glu Asp Glu Leu Phe Pro Ser Thr Pro Gly Lys Phe

1 5 10 15

Lys Val Asp Arg Ala His Asn Met Asn Arg Gln Phe His Arg Cys Phe

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Ala Ser Thr Ser Thr Met Phe Leu Trp Ala Leu Phe Leu Ile Ala Leu

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Thr Ala Ser Tyr Leu Arg Phe Gln Ser Phe Val Asp Ser Gly Ser Arg

50 55 60

Tyr Phe Ser Ala Ser Trp Gly Gly Ile Gln Trp Glu Lys Gln Val Arg

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Asn Ser Ala Gln Ile His Arg Ser Gly Gly Met Ser Val Leu Val Thr

85 90 95

Gly Ala Ala Gly Phe Val Gly Thr His Val Ser Leu Ala Leu Lys Lys

100 105 110

Arg Gly Asp Gly Val Val Gly Leu Asp Asn Phe Asn Asn Tyr Tyr Asp

115 120 125

Pro Ser Leu Lys Lys Ala Arg Lys Ser Leu Leu Asn Ser His Gly Ile

130 135 140

Leu Val Val Glu Gly Asp Leu Asn Asp Ala Lys Leu Leu Ala Lys Leu

145 150 155 160

Phe Asp Val Val Ala Phe Thr His Val Met His Leu Ala Ala Gln Ala

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Gly Val Arg Tyr Ala Met Glu Asn Pro Asn Ser Tyr Val His Ser Asn

180 185 190

Ile Ala Gly Leu Val Thr Leu Leu Glu Ile Cys Lys Ser Ala Asn Pro

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Gln Pro Ala Val Val Trp Ala Ser Ser Ser Ser Val Tyr Gly Leu Asn

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Glu Lys Val Pro Phe Ser Glu Ala Asp Arg Thr Asp Gln Pro Ala Ser

225 230 235 240

Leu Tyr Ala Ala Thr Lys Lys Ala Gly Glu Glu Ile Thr His Thr Tyr

245 250 255

Asn His Ile Tyr Gly Leu Ser Ile Thr Gly Leu Arg Phe Phe Thr Val

260 265 270

Tyr Gly Pro Trp Gly Arg Pro Asp Met Ala Tyr Phe Ser Phe Thr Arg

275 280 285

Asn Ile Leu Gln Gly Lys Pro Ile Thr Ile Tyr Arg Gly Lys Asn Arg

290 295 300

Val Asp Leu Ala Arg Asp Phe Thr Tyr Ile Asp Asp Ile Val Lys Gly

305 310 315 320

Cys Leu Gly Ser Leu Asp Thr Ser Gly Lys Ser Thr Gly Ser Gly Gly

325 330 335

Lys Lys Lys Gly Asn Ala Pro Tyr Arg Ile Phe Asn Leu Gly Asn Thr

340 345 350

Ser Pro Val Lys Val Pro Glu Leu Val Asn Ile Leu Glu Arg His Leu

355 360 365

Lys Val Lys Ala Lys Arg Asn Ile Val Asp Met Pro Gly Asn Gly Asp

370 375 380

Val Pro Phe Thr His Ala Asn Ile Ser Leu Ala Gln Arg Glu Phe Gly

385 390 395 400

Tyr Lys Pro Ser Thr Asp Leu Gln Thr Gly Leu Lys Lys Phe Val Arg

405 410 415

Trp Tyr Leu Ser Tyr Tyr Gly Tyr Asn Asn Arg Lys Gly Leu Gln

420 425 430

Claims (16)

1. A protein, which is the protein of a) or b) as follows: a) a protein consisting of the amino acid sequence shown in Sequence 2 in the sequence listing; b) A protein consisting of the 22-431 amino acid sequence of Sequence 2 in the Sequence Listing. 2. the coding gene of protein described in claim 1. 3. The gene according to claim 2, characterized in that: the gene is the following 1) or 2) or 3) gene: 1) Its nucleotide sequence is sequence 1 in the sequence listing; 2) Its coding sequence is the 169th-1464th position from the 5' end of sequence 1 in the sequence listing; 3) Its nucleotide sequence is the 233rd-1536th position from the 5' end of the sequence 1 in the sequence listing. 4. A recombinant vector containing the gene of claim 2 or 3. 5. A transgenic cell line containing the gene of claim 2 or 3. 6. The recombinant bacterium containing the gene of claim 2 or 3. 7. The protein according to claim 1 is used as uridine diphosphate glucuronate isomerase. 8. the albumen described in claim 1 is in the application of converting uridine diphosphate glucuronic acid into uridine diphosphate galacturonic acid or in converting uridine diphosphate galacturonic acid into uridine diphosphate glucuronic acid Applications. 9. The application of the gene described in claim 2 or 3 in converting uridine diphosphate glucuronic acid into uridine diphosphate galacturonic acid or converting uridine diphosphate galacturonic acid into uridine diphosphate glucuronic acid Acid application. 10. A method for cultivating transgenic plants with high uridine diphosphate galacturonic acid content, which is to introduce the gene described in claim 2 or 3 into plant cells to obtain a transgenic plant with increased uridine diphosphate galacturonic acid content . 11. The method of claim 10, wherein the plant is cotton. 12. The application of the protein described in claim 1 in cultivating cotton with increased fiber length. 13. The application of the gene according to claim 2 or 3 in cultivating cotton with increased fiber length. 14. The use of the recombinant vector according to claim 4 in cultivating cotton with increased fiber length. 15. The use of the transgenic cell line of claim 5 in cultivating cotton with increased fiber length. 16. The application of the recombinant bacterium according to claim 6 in cultivating cotton with increased fiber length.

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