CN100532560C - Expression vector of high specific activity xylanase - Google Patents

Abstract

The invention provides an expression vector of high specific activity xylanase. Specifically, the present invention provides expression vectors pBinxynB' and pBinSPxynB' containing high specific activity xylanase genes and their application in transgenic plant bioreactors. The high-specific activity xylanase gene is inserted into a high-efficiency plant expression vector, the plant is transformed by transgenic technology, and regenerated plants are obtained, and the transgenic plant expressing the high specific activity xylanase is obtained after screening. The expression amount of xylanase in the transgenic plant of the present invention can reach up to 170 IU/g fresh leaves. The transxylanase plant in the present invention can be directly applied in the feed industry.

Description

Expression vector of high specific activity xylanase

technical field

The invention relates to the field of plant genetic engineering. Specifically, the present invention relates to expression vectors pBinxynB' and pBinSPxynB' comprising high specific activity xylanase genes and their application in transgenic plant bioreactors.

Background technique

Xylan (xylan) is a hybrid five-carbon sugar, the main chain is connected by multiple xylopyranosyl groups through xylosidic bonds. A variety of short substituents of different sizes are attached to the side chains. Xylan mainly exists in the secondary wall of plant cells and acts as a link between lignin and other polysaccharides. Xylan is an important component of plant hemicellulose, which accounts for one-third of the total plant carbohydrates, and is the second most abundant renewable material resource after cellulose in nature. In some annual plants such as wheat, sugarcane, and cottonseed hulls, the content of xylan is very high, generally reaching more than 30%. (Gregory A C E et al. Biotech. and Gentic Engi. Rev., 15: 439-455, 1998)

Now it is generally believed that xylan may affect the nutritional value of feed and the digestion and utilization of feed by animals in various ways, and is an important anti-nutritional factor in feed. In actual production, xylan in the diet cannot be effectively degraded, which will significantly reduce the digestibility of nutrients, reduce feed intake, and affect the production performance of livestock and poultry. Excretion of viscous feces brings difficulties to sanitation control and increases the incidence of livestock and poultry. (Morgan A J et al. Proc Aust Poult Sym, 7:109-115, 1995)

Xylanases are a class of hydrolytic enzymes capable of degrading xylan. The research results show that if xylanase is added to the feed, the anti-nutritional effect of xylan can be significantly reduced or eliminated. Improve animal feed intake, weight gain and feed conversion.

The research on xylanase has started as early as the 1960s, mainly focusing on xylanase suitable for food industry, pulp and paper industry, energy industry, etc., and a large number of xylanases have been isolated from microorganisms from different sources Different types of xylanases with different functions. At present, xylanase is mainly produced by microbial fermentation, and the cultivation of transgenic plants will be a further development direction.

Plants are diverse, low-cost and renewable resources. Plant cells transformed with exogenous genes can regenerate complete plants with stable inheritance. So far, a variety of monocotyledonous and dicotyledonous plants have been successfully transformed, such as tobacco, potato, tomato, corn, soybean and rice. There are many reports on Agrobacterium-mediated plant transformation methods, among which the leaf disc transformation method can efficiently carry out gene transformation, plant regeneration and selection of dicotyledonous plants. Biolistic methods are more effective in the transformation of monocots.

Advances in the field of genetic engineering research have made it possible to use plants to express xylanase for the feed industry. On the one hand, transgenic plants can be used as bioreactors to provide an economical, efficient and safe xylanase production method to produce xylanase preparations; on the other hand, xylanase can be expressed in feed materials and directly fed , so that the process of enzyme production and addition is completely omitted, and the current method of producing enzyme preparations through microbial fermentation is replaced.

In recent years, studies on the expression of xylanase by transgenic plants have been carried out abroad. In 1995, Herbers et al. (Bio/Technology, 1995, 13:63-66) first carried out the protein expression research of xylanase. The heat-resistant xylanase derived from Clostridium thermocellum is transformed into tobacco, and the transgenic plant expresses xylanase, and its relative content is about 4% of the total protein in the leaf extract. The enzyme has an activity of 1.5 IU per mg of total protein and remains thermostable. Their research group (Molecular Breeding, 1996, 2: 81-87) also studied the expression of another microbial source (Ruminococcus flavefaciens) xylanase XYND-A. The enzyme is extracellularly expressed in transgenic tobacco plants, and the activity of hydrolyzing xylan is 40-170 μmol/min.m ² leaves, which is about 12 IU per mg of total protein. In 1997, Sun et al. (Journal of fermentation and bioengineering, 1997, 84: 219-223) used tobacco suspension cells to transiently express xylanase XYNB derived from Clostridium stercorarium. In the highly expressed transformant B1-11, the enzyme activity can reach 30 units per gram of wet weight cells, and the relative content of the enzyme is about 5% of the total soluble protein. The suspension cells were co-incubated with the cell wall extract of barley stems , found that recombinant enzymes can degrade complex polymers in barley cell wall tissue. In 2000, Cheng et al. (United States Patent, 6137032, 2000) formed a fusion gene from the xylanase gene xynC derived from the aquatic fungus Neocallimastix patriciarum and the oil body protein gene oleosin, and transformed rapeseed. The transgenic rapeseed expressed the fusion protein, and the enzyme of the transgenic plant The highest activity is 0.03IU/mg total protein. The enzyme can be located on the oil body membrane of rapeseed, and has high stability. In the same year, Patel et al. (Molecular breeding, 2000, 6: 113-123) expressed xylanase XYNA derived from the rumen fungus Neocallimastix patriciarum using monocot barley. Xylanase is specifically expressed in the seed endosperm and exhibits activity. In the most active plants, the enzyme activity was equivalent to 48 IU per gram of dry grain. Exogenous proteins accumulate with grain development, and xylanase remains stable during grain ripening, drying, and harvesting and storage. In 2003, Kimura et al. (Appl. Microbiol. Biotechol. 2003, 62: 374-9) constitutively expressed xylanase derived from the fungus Clostridium thermocellum in rice. The protein can be stably expressed in rice straw and seeds. The expressed xylanase activity was 0.5 IU/mg total protein.

These findings demonstrate that xylanase expression in transgenic plants does not affect plant growth, development, and reproduction. It is feasible to use transgenic plants to express xylanase. However, the problem existing in the research is that the activity of transxylanase plants is low, and it is difficult to adapt to the needs of the feed industry. transgenic plants.

The xylanase XYNB from Streptomyces olivaceoviridis has excellent enzymatic properties (Zhang HL, Yao B, Chinese Science Bulletin, 48:761-765, 2003; He Yongzhi, Yao Bin et al. Acta Microbiology, 44:340- 344, 2004). It is the xylanase with the highest specific activity isolated so far, it has the ability to resist pepsin and trypsin, and metal ions and surfactants have no obvious effect on the enzymatic reaction of XYNB, and it also has good pH stability . But its heat resistance is average. Improving its thermal stability through molecular modification can make it more suitable for practical application in feed.

Contents of the invention

The present invention is proposed and completed based on solving the above problems.

One of the purposes of the present invention is to provide a constitutive vector pBIxynB' containing an improved xylanase gene with high specific activity.

Another object of the present invention is to provide a specific carrier pBISPxynB' containing an improved xylanase gene with high specific activity.

Another object of the present invention is to provide an expression vector containing an improved xylanase gene with high specific activity.

Another object of the present invention is to provide a method for cultivating transgenic plants expressing high specific activity xylanase.

Another object of the present invention is to transform cells with the above expression vectors to obtain suspension cell lines.

First, a xylanase gene (XYNB') is provided by genetic engineering mutagenesis technology. Homologous modeling of XYNB is done at http://www.expasy.org/swissmod/SWISS-MODEL.html. The deduced high-order structure of xylanase XYNB consists of two reversed β-sheets and a short a-helix, and the whole enzyme molecule is in a right-handed structure. Thr11 and Tyr16 are marked in Figure 1 . They are located on the nitrogen-terminal β-sheet strands B1 and B2, respectively. From the structural analysis of the four thermophilic xylanases belonging to the 11th family, it was found that there are hydrophobic interactions of aromatic amino acids on the nitrogen-terminal β-sheet strands B1 and B2 (Table 1), and this interaction It may play a role in stabilizing the structure of the enzyme and improving the thermal stability of the enzyme. The similar position in the XYNB structure is T11-Y16, so if Thr11 is mutated to Tyr11, it is possible to form similar hydrophobic interactions.

Mutation of the xylanase gene can be accomplished by gene mutation techniques known in the art. Gene mutation methods that can be used include site-directed mutation, PCR error-causing mutation, and DNA shuffling. The invention applies site-directed mutagenesis technique to mutate xylanase gene. Mutant molecules are obtained by designing mutation primers and amplifying by PCR. In order to realize the T11Y mutation, the designed PCR primers Z9 and Z10 are as follows:

Z9:

G

CCACGGTCATCACCACCAACCAGACCGGCTACA5'-TA

G

CCACGGTCATCACCACCAACCAGACCGGC TA CA

ACAACGGGTTC-3' (contains restriction sites for EcoR I and Nco I)

Z10:

TCAGCCGCTGACCGTGATGTT-3’(含Kpn I的酶切位点)5'-TA

TCAGCCGCTGACCGTGATGTT-3' (contains Kpn I restriction site)

Wherein Z9 is a mutation primer, and the underlined part is the mutated nucleotide. Using the gene xynB as a template, carry out PCR amplification, and perform site-directed mutation by PCR to obtain the mutant gene xynB', which has the nucleotide sequence shown in Figure 1.

Further, the constitutive vector pBIxynB' containing the above-mentioned improved xylanase gene and the specific vector pBISPxynB' were constructed using molecular biology test techniques.

The present invention provides an expression vector of a xylanase gene, specifically, expression pBinxynB' and pBinSPxynB' are provided, and the vector includes an improved xylanase gene having a nucleotide sequence as shown in FIG. 1 .

The invention provides a method for cultivating a transgenic plant expressing a high specific activity xylanase, the method comprising inserting a high specific activity xylanase gene having a nucleotide sequence as shown in Figure 1 into a high-efficiency plant expression vector 1. Transforming plant receptors by transgenic technology to obtain regenerated plants, and obtaining transgenic plants expressing high specific activity xylanase through screening. The plant recipient is one or more plants selected from the group including tobacco, corn, rice, rape, wheat, potato, cabbage, soybean, and alfalfa.

The present invention also provides the cell line transformed by using the above expression vector.

The expression vector of the present invention can express high specific activity xylanase in plants. The expression level of xylanase can reach up to 170IU/g fresh leaves. The transgenic plants transformed with the vector can be directly applied to the feed industry. The utilization efficiency of nutritional factors in the feed is improved, and the cost of feed production is reduced.

Description of drawings

The present invention clones the xylanase gene from Streptomyces olivaceoviridis, and Streptomyces olivaceoviridis is classified as Streptomyces olivaceoviridis (Streptomyces olivaceoviridis), which has been preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee on April 11, 2005. (CGMCC, address, No. 13, North Zhongguancun, Haidian District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100080), and the preservation number is CGMCC No.1348.

Fig. 1 is the nucleotide sequence of the gene of the improved xylanase xynB'.

Fig. 2 is the structure of xylanase plant expression vector

Figure 3 is the PCR analysis of transgenic tobacco plants

1-6 are pBIXy-transformed plants, 7-8 are pBISPXy-transformed plants, and 9 are non-transgenic control plants

Figure 4 is the SDS-PAGE analysis of transgenic tobacco expressing xylanase

1 is the standard protein molecular weight; 2-4 are transgenic lines 26, 55 and 60 respectively; 5, 6 are non-transgenic plants

Figure 5 is the Western Blot analysis of transgenic tobacco expressing xylanase

1-3 are transgenic lines 26, 55 and 60 respectively; 4 are non-transgenic plants; 5 is the standard molecular weight of protein

Figure 6 is an analysis of the activity of transgenic plants expressing xylanase

Figure 7 is the stability of transgenic tobacco expressing xylanase

Figure 8 is the thermal stability of transgenic tobacco expressing xylanase

Figure 9 is an analysis of the activity of xylanase expressed by transgenic F1 generation plants

Figure 10 is the PCR analysis of transgenic F1 generation plants

Detailed ways

Experimental conditions

1. Bacterial strain, carrier and gene Xylanase gene xynB (EMBL accession number is: AJ292317) from Streptomyces olivaceoviridis (preservation number: CGMCCNo. : CGMCC, address of depository unit: No. 13, North Yiyi Road, Zhongguancun, Haidian District, Beijing, Institute of Microbiology, Chinese Academy of Sciences 100080, date of deposit: April 11, 2005).

Plant expression vectors pBI525 and pBinplus, Agrobacterium tumefacience LBA4404, and shuttle plasmid pRK2013 were purchased from Invitrgon.

2. Enzymes and other biochemical reagents: endonucleases were purchased from Takara Company, and ligases were purchased from Invitrgon Company. Xylose and Birchwood xylan, MS medium basic salt were purchased from Sigma. Others are domestic reagents.

3. Antiserum and immunoassay reagents: The xylanase expressed by Pichia pastoris fermentation was purified, dissolved in PBS buffer, quantified by Bradford method, and 4 mg protein was obtained from the Animal Research Center of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. Immunization test was carried out to obtain rabbit anti-xylanase serum. The titer was determined to be 500-1000× by ELISA method. Alkaline phosphatase-labeled goat anti-rabbit IgG and BCIP/NBT detection kit were purchased from Huamei Company.

4. Culture medium: Escherichia coli medium is LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH7.0); Agrobacterium medium YEB (5g/l tryptone, 0.5% yeast extract, 5g /l sucrose, 0.5g/l MgSO4 7H2O, pH7.2-7.5); tobacco transformation medium MS (containing in 1 liter: 6.2mg boric acid, 22.3mg manganese sulfate, 8.6mg zinc sulfate, 0.25mg sodium molybdate, 0.025mg copper sulfate, 0.025mg cobalt chloride, 100mg inositol, 1mg niacin, 1mg pyridoxine hydrochloride, 10mg thiamine hydrochloride, 1.65g ammonium nitrate, 1.9g potassium nitrate, 0.37g magnesium sulfate, 0.17g diphosphate potassium hydrogen, 0.04g Fe-330, 0.4g calcium chloride, 0.83g potassium iodide).

Experiment 1 Acquisition of mutant genes

Design PCR primers Z9 and Z10 as follows:

Z9: '-TAGAATTCGCCATGGCCACGGTCATCACCACCAACCAGACCGGCTACAACAACGGGTTC-3' (including restriction sites of EcoR I and Nco I)

Z10: 5'-TAGGTACCTCAGCCGCTGACCGTGATGTT-3' (contains Kpn I restriction site)

Wherein Z9 is a mutation primer, and the underlined part is the mutated nucleotide. Using the gene xynB as a template, PCR amplification was performed, and site-directed mutation was performed by the PCR method to obtain the mutant gene xynB'. The xynB' was cloned into the pUC19 vector through the Kpn I and EcoR I double restriction sites, and the recombinant colonies were screened out by electrotransformation of Escherichia coli JM109 with blue and white spots, and the recombinant plasmid pUC19-xynB' was extracted for sequence determination. It was confirmed that the designed site was mutated correctly, and its nucleotide sequence is shown in FIG. 1 .

Construction of experiment 2 plant expression vector

The above-mentioned improved xylanase gene xynB' was connected with the vector pBI525 to obtain the constitutive vector pBIxynB'; primers were designed according to the published signal peptide sequence of Potatinase II (EMBOJ., 1990, 9: 3033-3044), 5 'Primer (adding BamHI cutting point): 5'-aaa ggatcc aatggatgttcacaaggaag-3', 3'primer (adding NcoI cutting point): 5'-aac ccatgg tgcaagccttcgcatcaacatgc-3', amplified signal from genomic DNA of potato variety Berolina Peptide sequence, cloned into the vector pBluescriptSK-. Digested with pUCXyNcoI+KpnI, ligated with the signal peptide gene/BamHI+NcoI fragment, and cloned into pBI525 to obtain the extracellular expression-specific vector pBISPxynB'. The structures of the two vectors are shown in Figure 2. The obtained plasmid was digested with NcoI+KpnI to obtain a 600bp fragment, which proved that the gene was cloned correctly. Digest pBIXy and pBISPXy with HindIII+EcoRI to obtain expression cassettes, which are connected to the pBinplus vector that was also digested to obtain plant binary vectors pBinxynB' and pBinSPxynN' for transformation.

Experiment 3 Plant Genetic Transformation

The vectors pBinxynB' and pBinSPxynB' containing high specific activity xylanase genes were transferred to Agrobacterium strain LBA4404 by triparental mating.

Tobacco was transformed by the leaf disc method, callus began to form after 7 days of infection, and adventitious buds appeared after a period of time, but the untransformed leaf disc could not form callus and differentiated buds.

The differentiated adventitious buds were transferred to the rooting medium (MS+3% sucrose+0.7% agar+100mg/l Kanamycin+500mg/l Cb), and the root system could be seen in about 10 days. The obtained transgenic plants were used for further testing.

Experiment 4 PCR Analysis of Xylanase Gene in Transgenic Tobacco

Take about 0.1g of tobacco leaves, use the CTAB method (Maniatis T., et al. Molecular cloning. New York: Cold Spring harbor laboratory, 1982) to extract genomic DNA, and use primers Z9 and Z10 at both ends of the xylanase gene for PCR Amplification (Takara LA Taq with GC Buffer), Figure 3 shows the PCR results of some regenerated plants.

Experiment 5 ELISA screening of transgenic plants

Take 100 mg of resistant plant leaves, add 400 μl of grinding buffer (10 mM Tris-Cl, 0.02 NaN ₃ , 0.001% PMSF, pH 8.0) and grind into a homogenate in an ice bath, centrifuge at 15,000 r/min at 4°C for 5 min, and take the supernatant Coat the ELISA reaction plate with 200 μl of solution, overnight at 4°C, add 500-fold dilution of the prepared rabbit antiserum, react for 2 hours at 37°C, wash the plate 3 times with PBS+0.1% Tween20, each time for 2 minutes, and then add 1000-fold diluted alkaline Phosphatase-labeled goat anti-rabbit IgG, reacted at 37°C for 1 hour, washed the plate, added substrate NPP to develop color, and measured the absorbance at 495 nm with a microplate reader. Intracellular constitutive expression vector pBinxynB' and extracellular secreted expression vector pBinSPxynB' were transformed to obtain resistance-positive plants. The crude extracts of plant leaves were tested and screened by ELISA, and 26 and 7 plants with higher reaction values were obtained for further analysis.

Experiment 6 SDS-PAGE and Western blot detection of xylanase in transgenic plants

The leaf protein crude extract of transgenic tobacco with high ELISA reaction value (intracellular expression construction strain 26, 55 and extracellular expression construction strain 60) was extracted, and the method was the same as that in experiment 5. Take 10 μl of crude supernatant protein extract for SDS-PAGE analysis. Figure 4 illustrates the results of electrophoresis. 2-4 are the transgenic lines 26, 55 and 60 respectively, and the two constructed transgenic plants all have specific bands.

Further Western Blot experiments proved that it was a xylanase XYNB' protein with normal immune activity. Figure 5 shows the experimental results of Western Blot, 1-3 are transgenic lines 26, 55 and 60, respectively.

Using the analysis software BioID++, it can be known that the molecular weight of the xylanase expressed inside and outside the cell is the same, about 20.8kD, which is similar to the theoretical molecular weight, and its content accounts for 6% of the total protein content (strains 26 and 55) and 6.8% (strain 60), indicating that the protein expression efficiency is relatively high.

Experiment 7 Analysis of Xylanase Activity in Transgenic Plants

Take 100 mg of resistant plant leaves, add 400 μl of citric acid-disodium hydrogen phosphate buffer solution (pH5.2), grind it into a homogenate with a homogenizer, centrifuge at 15,000 r/min at 4°C for 5 min, and take the supernatant for enzyme Activity assay. The enzymatic determination adopts the internationally accepted Somogyi-NelSon method, 0.25ml 0.5% soluble 4-O-Me-D-glucurono-D-xylan (Sigma company, From birchwood) solution is mixed with 0.2ml citric acid-disodium hydrogen phosphate The buffer solution was added to the test tube, and placed in a 55°C water bath to preheat for 3 minutes. Then add 0.05ml of the diluted enzyme solution into the test tube, continue to react in a 37°C water bath for 10 minutes, add 0.5ml of Somogyi reagent (basic copper reagent) to the test tube to terminate the reaction, heat the test tube in boiling water for 15 minutes, immediately Cool to room temperature with running water, add 0.5ml NelSon reagent (arsenomolybdate reagent) to the test tube for color development, stir vigorously on the Voltex mixer, place at room temperature for 10min, add 1ml distilled water, and centrifuge at 10000rpm for 5 minutes to remove flocs. Absorbance was measured at 500nm. As a control, add 0.05ml of enzyme solution to 0.2ml of citric acid-disodium hydrogen phosphate buffer solution, boil it in boiling water at 100°C for 20 minutes to inactivate it, and then add the same volume of substrate to keep it warm. Definition of xylanase activity unit: Under certain conditions, the amount of enzyme required to decompose xylan to generate 1 μmol xylose per minute is 1 activity unit (IU).

Mature leaves of plants with higher ELISA reaction values were picked, and the enzyme solution was crudely extracted for enzyme activity determination, and the total protein content was determined by the Bradford method. Figure 6 illustrates the assay results of xylanase activity in transgenic plants. Among the PbinxynB' transformed plants, the No. 26 plant had the highest enzyme activity, and its activity was about 170IU/g fresh leaves, calculated as 23IU/mg total protein based on the total protein extracted from the leaves. In the construction of pBinSPxynB', the No. 60 plant showed the highest enzyme activity, reaching 150IU/g fresh leaves, which was calculated as 20IU/mg total protein based on the total protein extracted from the leaves. higher than previously reported levels.

Extraction of secreted recombinant enzyme and determination of enzyme activity: Take fresh leaves of pBISPxynB' transformed plants, wash the surface with distilled water, add citric acid-disodium hydrogen phosphate buffer (pH5.2), vacuum extract, and extract the leaves Centrifuge at 350g for 15min to obtain the intercellular extract, and use the Somogyi-Nelson method to measure the enzyme activity. The results are shown in Table 1. The results proved that the signal peptide of Potatin II can guide xylanase to be mainly secreted to the intercellular space for expression, and its expression activity is not much different from that of the intracellularly expressed enzyme.

Table 1 Xylanase activity of intercellular extracts of transgenic plants

Experiment 8 Stability of xylanase in transgenic plants

The leaf extracts of highly active transgenic tobacco lines (intracellular expression constructs 26, 55 and extracellular expression construct 60) were incubated at 25°C and 37°C without any protective agent. 1, 4, 24 and 48 hours, placed in 55 ° C, 60 ° C water bath incubation for 1, 4 hours, and then the enzyme activity was measured. And compared with untreated enzyme activity, calculate its relative activity (%). The results showed that the activity of the enzyme was maintained above 80% at 25°C and 37°C for 1 to 4 hours, and after 48 hours, the extract still maintained about 60% of the activity (Fig. 7); After 4 hours and 60°C for 1 hour, the recombinase can maintain about 50% activity (Fig. 8). It shows that the enzyme expressed in transgenic tobacco has a certain resistance to heat denaturation. The results also imply that the transgenic plants used for direct feeding or expressing xylanase can maintain certain stability during further processing.

Experiment 9 Genetic Stability of Transgenic Tobacco Expressing Xylanase

The transgenic tobacco can reproduce normally, and the F0 generation seeds of the transgenic plants constitutively expressing xylanase are harvested. The seeds were sterilized and placed in MS selection medium containing 100 mg/ml Kan. Enzyme activity analysis of the resistant offspring revealed that the segregation ratio of active plants to inactive plants was close to 3:1 (14:4). The xylanase activity of progeny plants was similar to that of their parents (Fig. 9). Genomic DNA was extracted and subjected to PCR amplification, and the results confirmed that the gene was genetically stable ( FIG. 10 ).

sequence list20050518090808

<110>applicant: Institute of Feed, Chinese Academy of Agricultural Sciences

<120>Title: Improved high specific activity xylanase and its gene, expression vector including the gene, recombinant yeast cell and expression method

sequence

________

<210>1

<211>576

<212> DNA

<213> Xylanase gene, Streptomyces olivaceoviridis

<400>1:

gccacggtca tcaccaccaa ccagaccggc tacaacaacg ggttctacta ctccttctgg 60

accgacggcg gcggttcggt ctcgatgacc ctgaactccg gcggcaacta cagcacctcg 120

tggacgaact gcgggaactt cgccgccggc aagggctgga gcaacggcgg acgcaggaac 180

gtgcagtact cgggcagctt ctacccgtcc ggcaacggct acctggcgct gtacgggtgg 240

acctcgaacc cgctcgtcga gtactacatc gtcgacaact ggggcaacta ccggcccacc 300

ggaacgtaca agggcacggt caccagcggc ggcggcacgt acgacgtcta ccagacgacg 360

cggtacaacg ccccctccgt ggaaggcacc aagaccttca accagtactg gagcgtccgg 420

cagtccaagc ggaccggcgg caccatcacc accggcaacc acttcgacgc ctgggcccgc 480

tacggcatgc aactgggcag cttcagctac tacatgatcc tcgccaccga gggctaccag 540

agcagcggct cctccaacat cacggtcagc ggctga 576

Claims (7)

1. A constitutive vector pBIxynB' comprising a xylanase gene, characterized in that the structure of the vector is as shown in Figure 2A, including a nucleotide sequence such as the improved xylanase gene shown in SEQ ID No: 1 . 2. A specific vector pBISPxynB' comprising a xylanase gene, characterized in that the structure of the vector is as shown in Figure 2B, including an improved xylanase gene with a nucleotide sequence as shown in SEQ ID No: 1 . 3. An expression vector of a xylanase gene, characterized in that the vector includes an improved xylanase gene with a nucleotide sequence as shown in SEQ ID No: 1. 4. The expression vector according to claim 3, which is the expression vector pBinxynB', characterized in that, the vector pBIxynB' is digested with restriction enzymes, and the resulting expression cassette is cloned into the pBinplus vector to obtain the expression vector pBinxynB'. 5. The expression vector according to claim 3, which is the expression vector pBinSPxynB', characterized in that the expression vector pBinSPxynB' is obtained by digesting the vector pBISPxynB' and cloning the obtained expression cassette into the pBinplus vector. 6. A method for cultivating a transgenic plant expressing a high specific activity xylanase, comprising inserting a nucleotide sequence such as a high specific activity xylanase gene shown in SEQ ID No: 1 into a high-efficiency plant expression The step of vector, transforming plant acceptor by transgenic technology to obtain regenerated plant, and obtaining transgenic plant expressing high specific activity xylanase through screening. 7. The method according to claim 6, wherein the plant recipient is one or more plants selected from the group consisting of tobacco, corn, rice, rapeseed, wheat, potato, cabbage, soybean, and alfalfa .

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