VND gene promoter with vascular specificity and application of VND gene promoter in driving expression of xylan hydrolase in plants

Abstract

The invention discloses a vascular specific VND gene promoter and application of the vascular specific VND gene promoter in driving expression of xylan hydrolase in plants. The invention provides a DNA fragment. The DNA fragment is any one of the following: 1) the nucleotide sequence of the DNA fragment is sequence 1; and 2) a nucleic acid sequence which has more than 90% of homology with the nucleic acid sequence shown in 1) and has the same function. Experiments prove that a SbNAC31 gene promoter specifically expressed by a vascular tissue is connected with CDS of a xylan hydrolase gene SbXyl, and a vector with the fusion gene is used for converting a sorghum xylan hydrolase function deletion mutant, so that the growth and development defects of the mutant are made up; meanwhile, the excellent lignocellulose quality (high feed quality) of the mutant is reserved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and relates to a vascular-specific VND gene promoter and an application thereof in driving the expression of xylan hydrolase in plants, and specifically relates to a sorghum vascular tissue-specifically expressed VND gene promoter and an application thereof in driving the specific expression of xylan hydrolase in restoring the growth and development phenotype of a sorghum xylan hydrolase mutant. Background Art

[0002] The promoter of a gene is a sequence upstream of the transcription start site of the gene, which participates in controlling the expression of the gene by recruiting transcription factors. At present, the promoter of constitutively expressed genes is often used in plant genetic engineering to drive the expression of specific genes. The promoter of such genes can drive the target gene to be continuously and efficiently expressed in various growth and development periods and various tissues, such as the ubiquitin promoter commonly used in monocotyledons and the tobacco mosaic virus 35S promoter in dicotyledons. However, the expression of many genes has tissue and spatiotemporal specificity, and the continuous high expression of certain genes will affect the growth and development of plants. Moreover, when conducting plant gene function research or transgenic breeding, it is sometimes necessary to express the target gene in a specific tissue. At this time, the promoter of the tissue-specific expression gene should be used to drive the expression of the exogenous gene.

[0003] The vascular tissue is responsible for transporting water and nutrients, and is essential for the normal growth and development of plants. In the vascular tissue, vessels, tracheids, and fiber cells all have thickened secondary cell walls, and xylan is the main hemicellulose in the secondary cell walls. Abnormal xylan metabolism can cause xylem collapse, affect the water transport function of the vessels, and ultimately hinder plant growth and development. On the other hand, mutants with defects in xylan metabolism can affect cell wall composition and structure, and improve the efficiency of plant lignocellulose utilization. Using the promoter of vascular tissue-specifically expressed genes to drive the expression of normal genes in these mutants is expected to restore the kinetic energy of water transport, compensate for growth and development defects, and retain the excellent lignocellulose quality of the mutants. Summary of the invention

[0004] The technical problem solved by the invention is to provide a vascular bundle-specifically expressed VND gene promoter and application thereof in driving the specific expression of xylan hydrolase.

[0005] In order to solve the above technical problems, the present invention provides a DNA fragment in a first aspect, which is any one of the following:

[0006] A1) the nucleotide sequence of the DNA fragment is sequence 1;

[0007] A2) is a DNA molecule that has more than 90% identity with the nucleic acid sequence shown in A1) and has the same function.

[0008] The above DNA fragment is derived from sorghum and is the promoter of the SbNAC31 gene.

[0009] In a second aspect, the present invention provides use of the DNA fragment described in the first aspect in driving the expression of a target gene in a plant.

[0010] In the above applications, the expression in plants refers to specific expression in plant tissues.

[0011] In the above application, the plant tissue is vascular tissue.

[0012] In the above, the target gene is a gene encoding a protein related to plant lignocellulose metabolism.

[0013] The protein related to plant lignocellulose metabolism is xylan hydrolase.

[0014] In a second aspect, the present invention provides a fused DNA fragment, which comprises the DNA fragment described in the first aspect and a gene encoding a functional protein located downstream thereof.

[0015] In the fused DNA fragments described above, the functional protein is a protein related to plant lignocellulose metabolism.

[0016] In the fused DNA fragments described above, the protein related to plant lignocellulose metabolism is xylan hydrolase;

[0017] The fusion DNA fragment is composed of the DNA fragment described in the first aspect and the gene encoding the xylan hydrolase in sequence;

[0018] The gene encoding the xylan hydrolase is any one of the following:

[0019] C1) The nucleotide sequence is the DNA molecule shown in Sequence 2;

[0020] C2) a DNA molecule that has 90% or more identity with the nucleotide sequence defined in C1), is derived from a plant and encodes the same protein;

[0021] C3) A DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined in C1) and encodes the same protein.

[0022] The term "identity" refers to sequence similarity to a natural nucleic acid sequence. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0023] The 90% or more identity may be at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity.

[0024] In a third aspect, the present invention provides an expression cassette, a recombinant vector or a recombinant bacterium containing the DNA fragment described in the first or second aspect.

[0025] In an embodiment of the present invention, the recombinant vector enables the target gene connected downstream of the DNA fragment in the first aspect to be specifically expressed in plant vascular tissue.

[0026] In the recombinant expression vector, the DNA fragment in the first aspect is connected to the upstream of the target gene sequence to be expressed in the plant expression vector pCAMBIA2300 to drive the expression of the target gene. The plant expression vector pCAMBIA2300 is a binary vector that can be expressed in sorghum.

[0027] In a fourth aspect, the present invention provides the use of the DNA fragment described in the first aspect in driving the specific expression of the gene encoding xylan hydrolase in plant vascular tissue;

[0028] Or the present invention provides the use of the DNA fragment described in the second aspect or the expression cassette, recombinant vector or recombinant bacteria described in the third aspect in any of the following:

[0029] B1) restoring the growth and development phenotype of plants lacking xylan hydrolase function;

[0030] B2) restoring the growth and development phenotype of plants lacking xylan hydrolase function while retaining high lignocellulose quality;

[0031] B3) cultivating plants with restored xylan hydrolase function and high lignocellulose quality;

[0032] B4) Production of high lignocellulosic quality plants.

[0033] In a fifth aspect, the present invention provides a method for preparing a plant with restored xylan hydrolase function and high lignocellulose quality, comprising the following steps: introducing the DNA fragment described in the second aspect into a plant lacking xylan hydrolase function to obtain a target plant;

[0034] The target plant recovers the growth and development phenotype and maintains the high lignocellulose quality of the plant lacking the xylan hydrolase function.

[0035] Alternatively, the present invention provides use of the method or the target plant prepared by the method in preparing animal feed.

[0036] In the above, the high lignocellulose quality refers to high feed quality, which is specifically reflected in the improvement of rumen digestibility of feed.

[0037] The above-mentioned improvement in rumen digestibility of feed means that the rumen digestibility of feed is higher than that of wild-type plants before the xylan hydrolase function is deleted.

[0038] The above-mentioned recovery of growth and development phenotype means recovery to the level of wild-type plants before the loss of xylan hydrolase function, which is specifically reflected in the absence of leaf tip dieback, or the plant height, vascular bundle area or fiber cell secondary cell wall thickness are all restored to the level of wild-type plants.

[0039] The plant lacking the function of xylan hydrolase is a sorghum E048 xylan hydrolase mutant.

[0040] The plant lacking xylan hydrolase function is specifically M19.

[0041] In the above, the plant is a monocotyledonous plant or a dicotyledonous plant.

[0042] Furthermore, the plant is a grass plant.

[0043] Furthermore, the plant is sorghum.

[0044] The experiments of the present invention prove that the promoter of the SbNAC31 gene specifically expressed in vascular tissue is connected to the CDS of the xylan hydrolase gene SbXyl, and a vector carrying the fusion gene is used to transform a sorghum xylan hydrolase function-deficient mutant, thereby compensating for the growth and development defects of the mutant and retaining the excellent lignocellulose quality (high feed quality) of the mutant. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The in situ hybridization results of sorghum SbNAC31 gene in developing stem node tissues; the left picture is the result of antisense probe (Antisense probe), and the right picture is the result of sense probe (Sense probe) as a negative control.

[0046] Figure 2 Schematic diagram of the vector for driving the expression of SbXyl gene CDS by sorghum SbNAC31 gene promoter and PCR detection diagram of positive transgenic strains; wherein, (a) is a schematic diagram of the construction of the recombinant vector SbNAC31Promoter::SbXyl; (b) is the PCR detection of positive transgenic strains, and lane M19 is a negative control using xylan hydrolase mutant DNA as a template; lanes VSC-1, VSC-2, and VSC-3 are three positive transgenic strains, respectively.

[0047] Figure 3Phenotypic observation and determination of the vascular tissue-specific complementation materials created; (a), (b), (c), (d) and (i) are wild type E048, mutant M19 and three positive vascular tissue-specific complementation lines (VSC-1, VSC-2 and VSC-3) from left to right. (a) is a plant grown for 50 days, (b) is a leaf vein vascular bundle, (c) is a stem vascular bundle, (d) is a vascular fiber cell, (e) to (h) are the determination and statistics of the leaf tip dead length, leaf vascular bundle area, stem vascular bundle area and fiber cell secondary wall thickness in (a) to (d). (i) is the plant morphology at the heading stage, and (j) is the plant height statistics at the heading stage.

[0048] Figure 4 The vascular tissue-specific complementary material created retains the excellent lignocellulose quality of the mutant; (a) shows the rumen digestibility of the wild type E048, mutant M19 and VSC strain before ensilage treatment; (b) shows the rumen digestibility of the wild type E048, mutant M19 and VSC strain after ensilage treatment. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0050] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0051] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0052] Example 1: Cloning of the Sorghum Vascular Tissue-Specifically Expressed Gene SbNAC31 and Its Promoter

[0053] Studies on the model plant Arabidopsis have shown that AtVND6 and AtVND7 are specifically expressed in xylem vessels, with AtVND6 mainly expressed in metaxylem, while AtVND7 is expressed in both the differentiating primary and metaxylem. In the monocot model plant rice, the homologous gene OsNAC31 of AtVND7 is specifically expressed in vascular tissues. Phylogenetic tree analysis identified the homologous genes of AtVND6 and AtVND7 in sorghum, which are Sobic.010G002900 and Sobic.007G003000, respectively. Transcriptome sequencing analysis of young roots, seedlings, young leaves, mature leaves, developing nodes, mature nodes, spikelets before flowering, spikelets after flowering, seeds in the filling stage, and seeds in the mature stage of sorghum E048 showed that Sobic.007G003000 was specifically and highly expressed in the developing nodes with rich vascular tissues in sorghum. The results of in situ hybridization further confirmed that Sobic.007G003000 was specifically expressed in the vascular tissue of developing stem nodes (e.g. Figure 1 As shown in the figure, Sobic.007G003000 was selected as the target gene and named as SbNAC31.

[0054] According to the genome sequence of sorghum E048 (variety protection number 20191004941) and the vector to be connected, primers were designed to amplify the SbNAC31 promoter. The primer sequences are as follows:

[0055] Forward Primer 1: 5′-GAGAGTGTCGTGCTCCACCATGGCAACAATTAAGGGATGATC-3′

[0056] Reverse Primer 1: 5'-CCATTTCGATCTCTATAGCTAGCTTCCTGCAATTGCTCGAT-3'

[0057] The genomic DNA of sorghum E048 was used as a template to amplify the promoter of SbNAC31 using forward primer 1 and reverse primer 1. The PCR amplification procedure was as follows:

[0058] Pre-denaturation at 95°C for 3 minutes; denaturation at 98°C for 10 seconds, annealing at 65°C for 30 seconds, extension at 68°C for 1 minute, for a total of 33 cycles; final extension at 68°C for 5 minutes.

[0059] The PCR product was electrophoresed using 1% agarose gel, and the target band was cut out and recovered to obtain the promoter of SbNAC31.

[0060] After sequencing, the promoter nucleotide sequence of SbNAC31 was sequence 1, and the promoter of SbNAC31 was named SbNAC31Promoter.

[0061] Example 2, Application of SbNAC31 Promoter

[0062] 1. Construction of SbNAC31Promoter::SbXyl fusion expression vector

[0063] The SbXyl gene encodes a xylan hydrolase that is mainly expressed in the developing nodes of sorghum. Its functional loss causes obstruction of water transport in sorghum, affecting normal growth and development. However, changes in cell wall components significantly improve its feed quality.

[0064] According to the gene sequence of SbXyl, using the cDNA of SbXyl (sequence 2) as a template, forward primer 2 and reverse primer 2 were designed to amplify the CDS sequence of SbXyl. The primer sequences are as follows:

[0065] Forward Primer 2: 5'-AAGCTAGCTATAGAGATCGAAATGGCGGCGATCGGCGGCGA-3'

[0066] Reverse Primer 2: 5'-CAGGTCGACTCTAGAGGATCCTCACACTTTAATGTTGAGCAC-3'

[0067] The PCR amplification procedure was the same as before to obtain the PCR product.

[0068] The PCR product was subjected to 1% agarose gel electrophoresis, and the target band was cut out and recovered to obtain the SbXyl gene CDS fragment.

[0069] 100 ng of SbNAC31Promoter obtained in Example 1 and 100 ng of the CDS fragment of the SbXyl gene obtained above were added as templates to the PCR reaction system, amplified with forward primer 1 and reverse primer 2, and the CDS sequences of SbNAC31Promoter and SbXyl genes were fused by overlapping PCR to obtain the SbNAC31Promoter::SbXyl fragment.

[0070] The PCR amplification procedure was the same as above. The PCR products obtained by overlapping PCR fusion were subjected to 1% agarose gel electrophoresis, and the target band was cut out and recovered to obtain the SbNAC31Promoter::SbXyl fragment.

[0071] Use BstXI and BamHI to digest the pCAMBIA2300 vector. The digestion system is as follows:

[0072] 10X Cutsmart buffer 5μL, plasmid vector 1μg, BstXI 1μL, BamHI 1μL, ddH2O to 50μL. Digest at 37℃ for 1 hour. After electrophoresis of the digested product using 1% agarose gel, cut out the target band and recover it to obtain the linearized pCAMBIA2300 vector.

[0073] The linearized pCAMBIA2300 vector recovered from the gel was connected with the SbNAC31Promoter::SbXyl fragment recovered from the gel by homologous recombination. The reaction system is as follows:

[0074] 5X CE buffer 4μL, linearized pCAMBIA2300 vector 5μL, gel-recovered SbNAC31Promoter::SbXyl fragment 2μL, EnaseII 2μL, ddH2O 7μL. React at 37℃ for 30 minutes.

[0075] Transform the ligation product into E. coli: take E. coli XL1-blue competent cells, thaw on ice, add the ligation product, mix gently, and place on ice for 30 minutes; heat shock at 42°C for 90 seconds, immediately place on ice for 3 minutes, add 500 μL of LB liquid culture medium without resistance, and culture at 37°C, 220rpm for 1 hour; centrifuge at 5,000rpm for 1 minute, collect the bacterial precipitate; discard the excess liquid LB culture medium and resuspend the precipitate with the remaining liquid LB, spread it on the screening plate containing kanamycin, and culture at 37°C for 16 hours; pick the single clone on the plate, use forward primer 3 and reverse primer 3 for PCR verification, and obtain 685bp as the positive clone.

[0076] Forward Primer 3: 5'-TCCAGCTGCATGCCTCTATC-3'

[0077] Reverse Primer 3: 5'-GCGCCGTACTCAAGATTCTC-3'

[0078] The PCR amplification procedure is as follows:

[0079] Pre-denaturation at 95°C for 3 minutes; denaturation at 98°C for 10 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 30 seconds, for a total of 33 cycles; final extension at 72°C for 5 minutes.

[0080] The above PCR products were subjected to agarose gel electrophoresis, and the positive clones with the target bands were sent to the company for sequencing for further verification, and the plasmids were extracted and the recombinant vectors were preserved as follows:

[0081] Inoculate the verified positive monoclonal clone into 5 mL LB liquid medium containing the corresponding antibiotics, and culture at 37°C and 220 rpm for 14 hours; collect the bacterial solution by centrifugation at 12,000 rpm for 30 seconds, and discard the supernatant; add 250 μL P1 buffer and vortex to mix the precipitate; add 250 μL P2 buffer and slowly invert and mix 4 to 6 times to make the bacterial solution transparent and clear. This step should not exceed 5 minutes; add 350 μL N3 and immediately invert and mix slowly; centrifuge at 12,000 rpm for 5 minutes, transfer the supernatant to the adsorption column, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate; add 150 μL PB, centrifuge for 30 seconds, and discard the filtrate; add 700 μL washing buffer, centrifuge for 1 minute, and discard the filtrate; empty at 12,000 rpm for 1 minute, move the adsorption column to a new 1.5 mL centrifuge tube, leave it at room temperature for 2 minutes, and add 30 μL EB after the washing buffer is thoroughly dried buffer, let stand at room temperature for about 2 minutes, centrifuge at 12,000 rpm for 2 minutes, and elute with EB buffer to obtain the plasmid, which is the recombinant vector.

[0082] The concentration was measured using NanoDrop 2000 and stored at -20°C.

[0083] The recombinant vector SbNAC31Promoter::SbXyl is a vector obtained by replacing the fragment between the BstXI and BamHI restriction sites of the pCAMBIA2300 vector with the SbNAC31Promoter::SbXyl fragment (eg Figure 2 (as shown in (a)).

[0084] The nucleotide sequence of the SbNAC31Promoter::SbXyl fragment consists of sequence 1 and sequence 2, and the last base of sequence 1 is adjacent to the first base of sequence 2.

[0085] 2. Transformation of sorghum xylan hydrolase mutants with SbNAC31Promoter::SbXyl fusion expression vector

[0086] The recombinant vector SbNAC31Promoter::SbXyl was introduced into the sorghum xylan hydrolase loss-of-function mutant using Agrobacterium-mediated genetic transformation.

[0087] 1. The recombinant vector SbNAC31Promoter::SbXyl constructed above was transformed into Agrobacterium by electroporation to obtain the recombinant bacterium EHA105 / SbNAC31Promoter::SbXyl.

[0088] Take Agrobacterium EHA105 competent cells, thaw on ice and add the plasmid to be transformed; add the competent cell and plasmid mixture to the electroporation cup, 1.8kV, electroporation 5.6ms; aspirate the competent cells in the electroporation cup to a 1.5mL centrifuge tube and add 500μL of LB liquid medium without resistance, 28℃, 200rpm for 3 hours; after the culture, take 30μL of bacterial liquid and spread it on LB solid medium containing kanamycin and rifampicin antibiotics, and invert and culture it in a 28℃ incubator for 2 days. After the colony grows, take a single colony and use forward primer 3 and reverse primer 3 for PCR detection. The amplified 685bp fragment is the positive colony EHA105 / SbNAC31Promoter::SbXyl.

[0089] 2. Agrobacterium-mediated genetic transformation of sorghum

[0090] The sorghum E048 xylan hydrolase mutant is a mutant obtained by mutating the gene encoding the xylan hydrolase in sorghum E048, and the xylan hydrolase function is reduced or lost.

[0091] Among them, the sorghum E048 xylan hydrolase mutant M19 is a mutant in which the 1789th C of the xylan hydrolase encoding gene (sequence 2) in sorghum E048 is mutated to T, and other nucleotide residues remain unchanged, resulting in a mutant with reduced or lost xylan hydrolase function; compared with the wild-type sorghum E048, the mutant phenotype has curled leaves and reduced plant height.

[0092] The above mutants can be obtained by conventional methods in the art (such as point mutation or gene editing).

[0093] Immature embryos were isolated from seeds of sorghum E048 xylan hydrolase mutant M19 that developed 12-14 days after pollen shedding. After the immature seeds were shelled, they were surface sterilized with 50% bleach containing 0.1% Tween-20 for 30 minutes, and then rinsed with high-pressure distilled water three times. The young embryos isolated under a dissecting microscope were mixed with recombinant Agrobacterium cells EHA105 / SbNAC31Promoter::SbXyl (OD600=0.2) resuspended in liquid infection medium at room temperature for 5 minutes. After removing the liquid, the infected young embryos were transferred to sterile filter paper to drain the remaining liquid, and then spread on solid infection medium.

[0094] After 3-4 days of co-cultivation, the young embryos were transferred to resting medium and cultured at 28°C in the dark for 7-10 days.

[0095] After resting, the young embryos were transferred to the selection medium (resting medium supplemented with 50 mg / L paromomycin) and selected in the dark at 28° C. Healthy calli were re-cultured on the selection medium every two weeks until fast-growing resistant calli were selected.

[0096] The resistant callus was cultured on bud induction medium at 28°C in the dark for 7-10 days to germinate, and cultured at 28°C (16 hours of light; 8 hours of darkness) after the buds appeared. The regenerated buds were transferred to root induction medium after they grew to 2-4 cm. After 1-2 weeks of root formation, the plants with healthy buds and roots were moved to pots and grown in a greenhouse to maturity to obtain T0 generation SbNAC31Promoter::SbXyl complemented sorghum.

[0097] The culture medium formula used in the above process is as follows:

[0098] Infection medium (per liter): 4.3 g MS salts, 1.0 mg nicotinamide, 1.0 mg pyridoxine hydrochloride, 5.0 mg thiamine hydrochloride, 100 mg inositol, 2.0 mg 2,4-D, 30.0 g sucrose, 10.0 g glucose, 700.0 mg L-proline, 0.5 g 2-(N-morpholino)ethanesulfonic acid (MES), 10.0 mg ascorbic acid, and 8.0 g agar, the balance being water. The medium was adjusted to pH 5.8 and autoclaved. Acetosyringone was added after autoclaving to a final concentration of 100 μM.

[0099] Resting medium (per liter): 4.3 g MS basal salt mixture, 100 mg inositol, 1.0 g casein peptone, 1.0 mg niacin, 1.0 mg pyridoxine HCl, 5.0 mg thiamine·HCl, 2.0 mg 2,4-D, 1.22 mg copper sulfate, 30 g maltose, 0.69 g L-proline, 8 g agar, 200 mg carbenicillin, balance water, pH 5.6.

[0100] Shoot induction medium (per liter): 4.3 g MS basal salt mixture, 1.0 g casein peptone, 1.0 mg niacin, 1.0 mg pyridoxine hydrochloride, 5.0 mg thiamine hydrochloride, 100 mg inositol, 1.25 mg copper sulfate, 30 g sucrose, 8.0 g agar, 2 mg BAP, 200 mg carbenicillin, balance water, pH 5.6.

[0101] Root induction medium (per liter): 2.15 g MS basal salt mix, 2.5 ml MS vitamin stock solution, 0.05 g inositol, 20 g sucrose, 3.0 g phytagel, balance water, pH 5.6.

[0102] 3. PCR detection of transgenic plants

[0103] The genomic DNA from leaves of T0 generation SbNAC31Promoter::SbXyl-complemented sorghum plants was extracted using the CTAB method. Take about 100 mg of sorghum sample and put it into a 2mL centrifuge tube together with 5mm steel beads. After quick freezing in liquid nitrogen, use a grinder to grind the sample into powder, set the parameters to 900rpm, 2min; add 650μL DNA extraction buffer and shake well, place it in a 65℃ water bath or constant temperature oven for 40min, then add 650μL chloroform / isoamyl alcohol (24:1), shake it vigorously, and centrifuge it at 4℃, 12,000rpm for 10min; take 500μL supernatant, add an equal volume of isopropanol, place it at -20℃ for more than 30min, precipitate DNA, centrifuge it at 12,000rpm for 10min, and discard the supernatant; add 1mL 70% ethanol to wash the precipitate, centrifuge it at 12,000rpm for 2min, and repeat the operation once; after centrifugation, dry the precipitate at room temperature, add 100μL sterile water, and store it at -20℃ after the DNA is completely dissolved.

[0104] The extracted DNA was used as a template, and PCR amplification was performed using forward primer 3 and reverse primer 3. The DNA of sorghum E048 xylan hydrolase mutant M19 was used as a negative control. The plants that amplified a 685 bp fragment were positive plants.

[0105] The results are as follows Figure 2 As shown in (b), it can be seen that positive T0 generation SbNAC31Promoter::SbXyl complemented sorghum VSC-1, VSC-2, and VSC-3 were obtained.

[0106] 3. Phenotypic observation and determination of positive transgenic sorghum lines

[0107] The seeds of positive T0 transgenic SbNAC31Promoter::SbXyl complemented sorghum VSC-1, VSC-2, and VSC-3 were harvested, and positive T1 transgenic SbNAC31Promoter::SbXyl complemented sorghum VSC-1, VSC-2, and VSC-3 were identified and planted to observe growth and development, observe and measure vascular bundle morphology, and measure feed rumen digestibility. Specifically, the positive T1 transgenic SbNAC31Promoter::SbXyl complemented sorghum was planted in greenhouse pots, and the sorghum xylan hydrolase mutant M19 and wild-type sorghum E048 were used as controls.

[0108] 1. Observation of growth and development:

[0109] During the entire growth and development period, plant phenotypes were observed, and plant height was counted at the heading stage (120 days after sowing).

[0110] The results are as follows Figure 3As shown in (a), (e), (i) and (j), it can be seen that at 50 days of growth (starting from sowing), compared with the wild-type sorghum, the sorghum xylan hydrolase mutant showed a leaf tip dieback phenotype and the plant height at the heading stage was significantly reduced, while the vascular tissue-specific complementation line (positive T1 generation SbNAC31Promoter::SbXyl complemented sorghum) compensated for the growth and development defects of the sorghum xylan hydrolase mutant, did not show a leaf tip dieback phenotype, and the plant height at the heading stage also recovered to the wild-type level.

[0111] 2. Observation and determination of vascular bundle morphology

[0112] The morphology of the vascular bundles in the stems of each plant grown for 50 days was observed using a scanning electron microscope.

[0113] Select samples from different materials at the same growth period and the same internode, use a new blade to cross-cut the middle of the stem, and then cut the stem into cross-section samples about 2 mm thick; place the samples in paraformaldehyde fixative and fix them for more than 24 hours; use 10%, 30%, 50%, 70% and 90% ethanol to dehydrate the fixed samples in sequence, each time for 0.5 hour, and finally place them in anhydrous ethanol; after drying the samples with a critical point dryer, spray gold on the cross-section to be observed; and observe and take pictures with a scanning electron microscope.

[0114] The results are as follows Figure 3 (b), (c), (d), (f), (g), (h), it was found that compared with wild-type sorghum, the vascular bundle area of ​​sorghum xylan hydrolase mutant was reduced and the secondary cell wall of fiber cells was thinner. After vascular tissue-specific complementation in the mutant, the above phenotypes of positive T1 generation SbNAC31Promoter::SbXyl complemented sorghum were restored to wild-type levels.

[0115] 3. Determination of rumen digestibility of feed

[0116] The feed digestibility of wild-type sorghum materials, xylan hydrolase mutant materials and vascular bundle-specific complementation materials (positive T1 generation SbNAC31Promoter::SbXyl complemented sorghum) was determined.

[0117] The materials at the heading stage were crushed and silaged or not, and the silage time of the silage materials was 30 days. After that, the materials were dried and ground into powder, and the rumen fluid of the cow was taken for in vitro digestion experiments (Effects of Lactic Acid Bacteria Isolated From Rumen Fluid and Feces of Dairy Cows on Fermentation Quality, Microbial Community, and in vitro Digestibility of Alfalfa Silage, Linna Guo, Dandan Yao, Dongxia Li, Yanli Lin, Smerjai Bureenok, Kuikui Ni and Fuyu Yang, ORIGINAL RESEARCH published: 09 January 2020, doi: 10.3389 / fmicb.2019.02998).

[0118] Digestibility formula: Dry matter digestibility (%) = (dry matter weight of sample before digestion – dry matter weight of sample after digestion) ÷ dry matter weight of sample × 100%

[0119] The results are as follows Figure 4 As shown, it can be seen that before ensilage treatment, the digestibility of the xylan hydrolase mutant M19 was 14.6% higher than that of the wild-type sorghum, and the digestibility of the three vascular tissue-specific complementation lines (positive T1 generation SbNAC31Promoter::SbXyl complemented sorghum) was increased by 7.5% to 13.2%. After ensilage treatment, the digestibility of the xylan hydrolase mutant M19 was 7.6% higher than that of the wild-type sorghum, and the digestibility of the three vascular tissue-specific complementation lines (positive T1 generation SbNAC31Promoter::SbXyl complemented sorghum) was increased by 7.5% to 7.9%.

[0120] The above results confirmed that the vascular tissue-specific complementation strains compensated for the growth and development defects of the xylan hydrolase mutants while retaining excellent feed quality.

[0121] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A DNA fragment, which is any of the following: A1) the nucleotide sequence of the DNA fragment is sequence 1; A2) is a DNA molecule that has more than 90% identity with the nucleic acid sequence shown in A1) and has the same function.

2. Use of the DNA fragment according to claim 1 in driving the expression of a target gene in a plant.

3. The use according to claim 2, characterized in that: The expression in plants refers to specific expression in plant tissues.

4. The use according to claim 3, characterized in that: The plant tissue is vascular tissue.

5. A fused DNA fragment, comprising the DNA fragment according to claim 1 and a gene encoding a functional protein located downstream thereof.

6. The fused DNA fragment according to claim 5, characterized in that: The functional protein is a protein related to plant lignocellulose metabolism.

7. The fused DNA fragment according to claim 6, characterized in that: The protein related to plant lignocellulose metabolism is xylan hydrolase; The fusion DNA fragment is composed of the DNA fragment of claim 1 and the gene encoding the xylan hydrolase in sequence; The gene encoding the xylan hydrolase is any one of the following: C1) The nucleotide sequence is the DNA molecule shown in Sequence 2; C2) a DNA molecule that has 90% or more identity with the nucleotide sequence defined in C1), is derived from a plant and encodes the same protein; C3) A DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined in C1) and encodes the same protein.

8. An expression cassette, recombinant vector or recombinant bacterium containing the DNA fragment according to any one of claims 1 or 5-7.

9. Use of the DNA fragment of claim 1 in driving the specific expression of a gene encoding a xylan hydrolase in plant vascular tissues; Or the use of the DNA fragment according to any one of claims 5 to 7 or the expression cassette, recombinant vector or recombinant bacteria according to claim 8 in any of the following: B1) restoring the growth and development phenotype of plants lacking xylan hydrolase function; B2) restoring the growth and development phenotype of plants lacking xylan hydrolase function while retaining high lignocellulose quality; B3) cultivating plants with restored xylan hydrolase function and high lignocellulose quality; B4) Production of high lignocellulosic quality plants.

10. A method for preparing a plant with restored xylan hydrolase function and high lignocellulose quality, comprising the following steps: introducing the DNA fragment according to any one of claims 5 to 7 into a plant lacking xylan hydrolase function to obtain the target plant; The target plant recovers the growth and development phenotype and maintains the high lignocellulose quality of the plant lacking xylan hydrolase function; Or, use of the method or the target plant prepared by the method in preparing animal feed.

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