Vascular-specific vnd gene promoter and its use to drive expression of xylanolytic enzymes in plants

Abstract

The present application discloses a vascular specific VND gene promoter and its application in driving xylanase to express in plants. The present application provides a DNA fragment, which is any one of the following: 1) the nucleotide sequence of the DNA fragment is sequence 1; 2) a nucleic acid sequence with more than 90% homology with the nucleic acid sequence shown in 1) and with the same function. The experiment of the present application proves that the vascular tissue specific expression SbNAC31 gene promoter is connected with the CDS of xylanase gene SbXyl, the vector with the fusion gene is used to transform the xylanase functional deficiency mutant of sorghum, the growth and development defects of the mutant are made up, and meanwhile the excellent lignocellulose quality (high forage quality) of the mutant is retained.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the application of a vascular-specific VND gene promoter and its driving xylan hydrolase expression in plants. Specifically, it relates to the application of a sorghum vascular tissue-specific VND gene promoter and its driving xylan hydrolase expression in restoring the growth and development phenotype of sorghum xylan hydrolase mutants. Background Technology

[0002] A gene promoter is a sequence upstream of the transcription start site that recruits transcription factors to control gene expression. Currently, constitutive gene promoters are commonly used in plant genetic engineering to drive the expression of specific genes. These promoters can drive the sustained and efficient expression of the target gene in various growth and development stages and 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 exhibits tissue and spatiotemporal specificity, and sustained high expression of certain genes can negatively impact plant growth and development. Moreover, in plant gene function studies or transgenic breeding, it is sometimes necessary to express the target gene in specific tissues; in such cases, tissue-specific gene promoters are used to drive the expression of exogenous genes.

[0003] Vascular tissue plays a crucial role in transporting water and nutrients, essential for normal plant growth and development. Within vascular tissue, vessels, tracheids, and fiber cells all possess thickened secondary cell walls, with xylan being the primary hemicellulose component. Abnormal xylan metabolism can cause xylem collapse, impairing the water transport function of vessels and ultimately hindering plant growth and development. Conversely, xylan metabolism-deficient mutants can affect cell wall composition and structure, improving the utilization efficiency of lignocellulose in plants. Utilizing promoters of vascular tissue-specific genes to drive the expression of normal genes in these mutants holds promise for restoring water transport capabilities, compensating for growth and developmental deficiencies, and preserving the superior lignocellulose quality of the mutants. Summary of the Invention

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

[0005] To address the aforementioned technical problems, the first aspect of the present invention provides a DNA fragment, which is any of the following:

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

[0007] DNA molecules with more than 90% identity and the same function as the nucleic acid sequences shown in A2) and A1).

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

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

[0010] In the above-described application, the expression in the plant refers to specific expression in plant tissues.

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

[0012] The target gene mentioned above is a gene encoding a protein related to plant lignocellulose metabolism.

[0013] The protein involved in plant lignocellulose metabolism is xylan hydrolase.

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

[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 coding genome of 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] The nucleotide sequences defined by C2 and C1) have 90% or more identity, are derived from plant DNA molecules and encode the same protein;

[0021] C3) hybridizes with the nucleotide sequence defined by C1) under strict conditions and encodes the same protein in a DNA molecule.

[0022] The term "identity" refers to sequence similarity to a natural nucleic acid sequence. Identity can be evaluated visually or using 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 higher identity can be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.

[0024] Thirdly, the present invention provides an expression cassette, recombinant vector, or recombinant bacteria containing the DNA fragments described in the first or second aspect.

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

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

[0027] Fourthly, the present invention provides the application of the DNA fragment described in the first aspect in the specific expression of the gene encoding xylan hydrolase in plant vascular tissues;

[0028] Alternatively, 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) restores the growth and development phenotype of plants lacking xylan hydrolase function;

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

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

[0032] B4) Prepare high-quality lignocellulose plants.

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

[0034] The target plant restores its growth and development phenotype while maintaining the high lignocellulose quality of the plant lacking xylan hydrolase function.

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

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

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

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

[0039] The above-mentioned plants lacking xylan hydrolase function are sorghum E048 xylan hydrolase mutants.

[0040] The plant with the aforementioned lack of xylan hydrolase function is specifically M19.

[0041] The plants mentioned above are either monocotyledonous or dicotyledonous plants.

[0042] Furthermore, the plant in question is a grass belonging to the Poaceae family.

[0043] Furthermore, the plant in question is sorghum.

[0044] The experiments of this invention demonstrate that linking the promoter of the SbNAC31 gene, which is specifically expressed in vascular tissue, with the CDS of the xylan hydrolase gene SbXyl, and transforming the sorghum xylan hydrolase-deficient mutant with a vector carrying this fusion gene, can compensate for the growth and development defects of the mutant while retaining the excellent lignocellulose quality (high feed quality) of the mutant. Attached Figure Description

[0045] Figure 1 The results show the in situ hybridization of the sorghum SbNAC31 gene in developing stem and node tissues; the left image shows the results of the antisense probe, and the right image shows the results of the sense probe as a negative control.

[0046] Figure 2 This diagram shows the vector for driving the CDS expression of the SbXyl gene driven by the promoter of the SbNAC31 gene in sorghum and the PCR detection diagram of the positive transgenic lines. Among them, (a) is a schematic diagram of the construction of the recombinant vector SbNAC31Promoter::SbXyl; (b) shows the positive transgenic lines detected by PCR, with lane M19 being a negative control using xylan hydrolase mutant DNA as a template; lanes VSC-1, VSC-2, and VSC-3 are three positive transgenic lines.

[0047] Figure 3Phenotypic observation and determination of the created vascular tissue-specific complementary materials; (a), (b), (c), (d), and (i) from left to right represent wild-type E048, mutant M19, and three positive vascular tissue-specific complementary lines (VSC-1, VSC-2, and VSC-3). (a) shows the plant after 50 days of growth; (b) shows the leaf vein vascular bundles; (c) shows the stem vascular bundles; (d) shows the vascular bundle fiber cells; and (e) to (h) show the determination and statistical analysis of leaf tip necrosis length, leaf vascular bundle area, stem vascular bundle area, and fiber cell secondary wall thickness from (a) to (d). (i) shows the plant morphology at the heading stage, and (j) shows the plant height at the heading stage.

[0048] Figure 4 The vascular tissue-specific complementary material created retains the superior lignocellulose quality of the mutant; (a) represents the rumen digestibility of wild-type E048, mutant M19 and VSC lines without silage treatment; (b) represents the rumen digestibility of wild-type E048, mutant M19 and VSC lines after silage treatment. Detailed Implementation

[0049] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0050] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0051] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0052] Example 1: Cloning of the sorghum vascular tissue-specific expression gene SbNAC31 and its promoter

[0053] Studies on the model plant Arabidopsis thaliana have shown that AtVND6 and AtVND7 are specifically expressed in xylem vessels, with AtVND6 mainly expressed in the metaxylem, while AtVND7 is expressed in both the differentiating primary and metaxylem. In the monocotyledonous model plant rice, the homolog of AtVND7, OsNAC31, is specifically expressed in vascular tissue. Phylogenetic analysis identified the homologs of AtVND6 and AtVND7 in sorghum as Sobic.010G002900 and Sobic.007G003000, respectively. Transcriptome sequencing analysis of young roots, seedlings, young leaves, mature leaves, developing nodes, mature nodes, pre-flowering spikelets, post-flowering spikelets, seeds at the grain-filling stage, and mature seeds of sorghum E048 revealed that Sobic.007G003000 is specifically highly expressed in the developing nodes rich in vascular tissue of sorghum. The results of in situ hybridization further confirmed the vascular tissue-specific expression of Sobic.007G003000 in developing stem nodes (e.g. Figure 1 As shown in the figure, Sobic.007G003000 was chosen as the target gene and named SbNAC31.

[0054] Based on the genome sequence of sorghum variety E048 (variety protection number 20191004941) and the vector to be ligated, 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] Using genomic DNA from sorghum E048 as a template, the promoter of SbNAC31 was amplified using forward primer 1 and reverse primer 1. The PCR amplification procedure is as follows:

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

[0059] The PCR product was subjected to 1% agarose gel electrophoresis, and the target band was excised 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 the SbNAC31 promoter

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

[0063] The SbXyl gene encodes a xylan hydrolase that is mainly expressed in the stem nodes during sorghum development. Its loss of function hinders water transport in sorghum, affecting normal growth and development. However, changes in cell wall composition significantly improve its feed quality.

[0064] Based on the SbXyl gene sequence, using the SbXyl cDNA (sequence 2) as a template, forward primer 2 and reverse primer 2 were designed to amplify the SbXyl CDS sequence. 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 is the same as before, and the PCR product is obtained.

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

[0069] 100 ng of the SbNAC31Promoter obtained in Example 1 and 100 ng of the SbXyl gene CDS fragment obtained above were added to the PCR reaction system as templates. Amplification was performed using forward primer 1 and reverse primer 2. The CDS sequences of the SbNAC31Promoter and SbXyl genes were fused by overlap 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 excised and recovered to obtain the SbNAC31Promoter::SbXyl fragment.

[0071] The pCAMBIA2300 vector was digested with BstXI and BamHI enzymes. The digestion system is as follows:

[0072] 5 μL of 10X Cutsmart buffer, 1 μg of plasmid vector, 1 μL of BstXI, 1 μL of BamHI, and ddH2O were added to a final volume of 50 μL. The mixture was digested at 37°C for 1 hour. After electrophoresis of the digested product on a 1% agarose gel, the target band was excised and recovered to obtain the linearized pCAMBIA2300 vector.

[0073] The linearized pCAMBIA2300 support after gel recovery was linked to the gel-recovered SbNAC31Promoter::SbXyl fragment using homologous recombination. The reaction system is as follows:

[0074] 4 μL of 5X CE buffer, 5 μL of linearized pCAMBIA2300 support, 2 μL of gel-recovered SbNAC31Promoter::SbXyl fragment, 2 μL of Enase II, and 7 μL of ddH2O. Incubate at 37°C for 30 minutes.

[0075] Transform E. coli with the ligation product: Take E. coli XL1-blue competent cells, thaw them on ice, add the ligation product, mix gently, and incubate on ice for 30 minutes; heat shock at 42°C for 90 seconds, immediately place on ice for 3 minutes, then add 500 μL of antibiotic-free LB liquid medium, and incubate at 37°C and 220 rpm for 1 hour; centrifuge at 5,000 rpm for 1 minute and collect the bacterial precipitate; discard the excess liquid LB medium, resuspend the precipitate with the remaining liquid LB, spread it on a selection plate containing kanamycin, and incubate at 37°C for 16 hours; pick single clones on the plate and perform PCR verification using forward primer 3 and reverse primer 3, obtaining a 685 bp 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℃ for 3 minutes; denaturation at 98℃ for 10 seconds, annealing at 56℃ for 30 seconds, extension at 72℃ for 30 seconds, for a total of 33 cycles; final extension at 72℃ for 5 minutes.

[0080] The PCR products were subjected to agarose gel electrophoresis. Positive clones with the target band were sent to the company for sequencing for further verification. The plasmid was extracted and the recombinant vector was preserved using the following method:

[0081] Validated positive monoclonal antibodies were inoculated into 5 mL of LB liquid medium containing the appropriate antibiotic and incubated at 37°C and 220 rpm for 14 hours. The bacterial culture was collected by centrifugation at 12,000 rpm for 30 seconds, and the supernatant was discarded. 250 μL of P1 buffer was added, and the precipitate was vortexed. 250 μL of P2 buffer was added, and the culture was slowly inverted 4 to 6 times until clear; this step should not exceed 5 minutes. 350 μL of N3 was added, and the culture was immediately and slowly inverted. The culture was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was transferred to an adsorption column. The column was centrifuged at 12,000 rpm for 30 seconds, and the filtrate was discarded. 150 μL of PB was added, and the culture was centrifuged for 30 seconds, and the filtrate was discarded. 700 μL of washing buffer was added, and the culture was centrifuged for 1 minute, and the filtrate was discarded. The culture was centrifuged at 12,000 rpm for 1 minute, and the adsorption column was transferred to a new 1.5 mL centrifuge tube. The tube was incubated at room temperature for 2 minutes to allow the washing buffer to dry completely before adding 30 μL of EB. After rinsing with EB buffer and letting stand at room temperature for about 2 minutes, centrifuge at 12,000 rpm for 2 minutes. The plasmid can be obtained by eluting with EB buffer, which is the recombinant vector.

[0082] 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 (e.g., ...). Figure 2 (a) is shown.

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

[0085] II. Transformation of sorghum lignan hydrolase mutant with SbNAC31Promoter::SbXyl fusion expression vector

[0086] Agrobacterium-mediated genetic transformation was used to transfer the recombinant vector SbNAC31Promoter::SbXyl into a sorghum polysaccharide hydrolase-deficient mutant.

[0087] 1. The recombinant vector SbNAC31Promoter::SbXyl constructed above was transferred into Agrobacterium using an electroporation method to obtain the recombinant strain EHA105 / SbNAC31Promoter::SbXyl.

[0088] Agrobacterium EHA105 competent cells were thawed on ice and then the plasmid to be transformed was added. The mixture of competent cells and plasmid was added to an electroporation cuvette and electroporated at 1.8 kV for 5.6 ms. The competent cells were then transferred to a 1.5 mL centrifuge tube and 500 μL of antibiotic-free LB liquid medium was added. The mixture was incubated at 28°C and 200 rpm for 3 hours. After incubation, 30 μL of the bacterial culture was spread onto LB solid medium containing kanamycin and rifampin antibiotics and incubated upside down at 28°C for 2 days. After colonies grew, single colonies were analyzed by PCR using forward primer 3 and reverse primer 3. A 685 bp fragment amplified was identified as a positive colony: EHA105 / SbNAC31Promoter::SbXyl.

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

[0090] The xylan hydrolase mutant of sorghum E048 is a mutant obtained by mutating the gene encoding xylan hydrolase in sorghum E048, resulting in a reduction or loss of xylan hydrolase function.

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

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

[0093] Immature embryos were isolated from sorghum seeds that had developed for 12-14 days after pollination of the xylan hydrolase mutant M19 (E048). After dehulling, the immature seeds were surface-sterilized with 50% bleach containing 0.1% Tween-20 for 30 minutes, followed by rinsing three times with autoclaved distilled water. Under a dissecting microscope, the isolated embryos were mixed with recombinant Agrobacterium cells EHA105 / SbNAC31 Promoter::SbXyl (OD600 = 0.2) resuspended in liquid infection medium at room temperature for 5 minutes. After removing the liquid, the infected embryos were transferred to sterile filter paper to drain excess liquid, and then spread onto solid infection medium.

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

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

[0096] Resistant callus tissue was cultured on shoot induction medium at 28°C in the dark for 7-10 days to germinate, and then cultured at 28°C after shoot emergence (16 hours light; 8 hours dark). Once the regenerated shoots reached 2-4 cm in length, they were transferred to root induction medium. After 1-2 weeks of root formation, plants with healthy shoots and roots were transplanted into pots and grown in a greenhouse until maturity, yielding T0 generation SbNAC31Promoter::SbXyl reintroduced sorghum.

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

[0098] Infection medium (per liter): 4.3 g MS salt, 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, with the remainder being water. The medium was adjusted to pH 5.8 and autoclaved. After autoclaving, acetylsuccine was added 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 nicotinic acid, 1.0 mg pyridoxine hydrochloride, 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] Sprout induction medium (per liter): 4.3 g MS basal salt mixture, 1.0 g casein peptone, 1.0 mg nicotinic acid, 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 mixture, 2.5 mL MS vitamin stock solution, 0.05 g inositol, 20 g sucrose, 3.0 g plant gel, balance water, pH 5.6.

[0102] 3. PCR detection of transgenic plants

[0103] Genomic DNA was extracted from the leaves of T0 generation SbNAC31Promoter::SbXyl supplemented sorghum plants using the CTAB method. Take approximately 100 mg of sorghum sample and place it in a 2 mL centrifuge tube along with a 5 mm steel bead. After quick-freezing in liquid nitrogen, grind the sample into powder using a grinder at 900 rpm for 2 min. Add 650 μL of DNA extraction buffer and shake well. Incubate at 65°C in a water bath or constant temperature oven for 40 min. Then add 650 μL of chloroform / isoamyl alcohol (24:1), shake vigorously, and centrifuge at 12,000 rpm for 10 min at 4°C. Take 500 μL of the supernatant, add an equal volume of isopropanol, and incubate at -20°C for at least 30 min to precipitate the DNA. Centrifuge at 12,000 rpm for 10 min and discard the supernatant. Wash the precipitate with 1 mL of 70% ethanol and centrifuge at 12,000 rpm for 2 min. Repeat this process once. After centrifugation, dry the precipitate at room temperature, add 100 μL of sterile water, and store at -20°C after the DNA has completely dissolved.

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

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

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

[0107] Seeds of positive T0 generation SbNAC31Promoter::SbXyl-transferred sorghum VSC-1, VSC-2, and VSC-3 were harvested. These seeds were then planted with positive T1 generation SbNAC31Promoter::SbXyl-transferred sorghum VSC-1, VSC-2, and VSC-3 for observation of growth and development, vascular bundle morphology, and rumen digestibility. Specifically, positive T1 generation SbNAC31Promoter::SbXyl-transferred sorghum was planted in greenhouse pots, with the sorghum xylan hydrolase mutant M19 and wild-type sorghum E048 serving as controls.

[0108] 1. Observation of growth and development:

[0109] Throughout the growth and development period, observe the plant phenotype and count the plant height 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 wild-type sorghum, the sorghum xylan hydrolase mutant exhibited a leaf tip dieback phenotype and a significantly reduced plant height at the heading stage. In contrast, the vascular tissue-specific complementary line (positive T1 generation SbNAC31Promoter::SbXyl supplemented sorghum) compensated for the growth and developmental defects of the sorghum xylan hydrolase mutant, did not exhibit the leaf tip dieback phenotype, and the plant height at the heading stage also recovered to the level of the wild type.

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

[0112] The morphology of the vascular bundles in the stems of various plants that had grown for 50 days was observed using scanning electron microscopy.

[0113] Samples from different materials at the same growth stage and internode were selected. The middle of the stem was transversely cut using a new blade, and then cut into stem transverse samples of about 2 mm thickness. The samples were placed in paraformaldehyde fixative and fixed for more than 24 hours. The fixed samples were dehydrated successively with 10%, 30%, 50%, 70%, and 90% ethanol for 0.5 hours each time, and finally placed in anhydrous ethanol. After drying the samples using a critical point desiccator, the cross-section to be observed was sputter-coated with gold. The cross-section was observed and photographed using a scanning electron microscope.

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

[0115] 3. Determination of rumen digestibility of feed

[0116] Feed digestibility was determined for wild-type sorghum, xylan hydrolase mutant, and vascular bundle-specific complementary material (positive T1 generation SbNAC31Promoter::SbXyl supplemented sorghum).

[0117] After crushing the various materials at the heading stage, they were treated with either silage or no silage. The silage time for the silage material was 30 days. The materials were then dried and ground into powder. Bovine rumen fluid was used 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, without silage treatment, the digestibility of the xylan hydrolase mutant M19 was 14.6% higher than that of wild-type sorghum, and the digestibility improvement of the three vascular tissue-specific complementary lines (positive T1 generation SbNAC31 Promoter::SbXyl supplemented sorghum) ranged from 7.5% to 13.2%. After silage treatment, the digestibility of the xylan hydrolase mutant M19 was 7.6% higher than that of wild-type sorghum, and the digestibility improvement of the three vascular tissue-specific complementary lines (positive T1 generation SbNAC31 Promoter::SbXyl supplemented sorghum) ranged from 7.5% to 7.9%.

[0120] The above results confirm that the vascular tissue-specific complementary lines not only compensate for the growth and developmental defects of the xylan hydrolase mutant, but also retain excellent feed quality.

[0121] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A DNA fragment, wherein the nucleotide sequence of the DNA fragment is sequence 1.

2. The application of the DNA fragment according to claim 1 in driving the expression of the target gene in plants; The expression in the plant is specific to plant tissues; the plant tissues are vascular tissues. The plant in question is sorghum.

3. A fused DNA fragment comprising the DNA fragment of claim 1 and a gene encoding a functional protein located downstream therefrom.

4. The fused DNA fragment according to claim 3, characterized in that: The functional protein is a protein related to plant lignocellulose metabolism.

5. The fused DNA fragment according to claim 4, characterized in that: The protein involved in plant lignocellulose metabolism is a xylan hydrolase; The fusion DNA fragment is composed of the DNA fragment described in claim 1 and the coding genome of 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; The nucleotide sequences defined by C2 and C1) have 90% or more identity, are derived from plant DNA molecules and encode the same protein; C3) hybridizes with the nucleotide sequence defined by C1) under strict conditions and encodes the same protein in a DNA molecule.

6. An expression cassette, recombinant vector, or recombinant bacteria containing any of the DNA fragments described in claims 1 or 3-5.

7. The application of the DNA fragment of claim 1 in the specific expression of the gene encoding xylan hydrolase in plant vascular tissue, wherein the plant is sorghum.

8. The DNA fragment of claim 5 or an expression cassette, recombinant vector or recombinant bacteria containing the DNA fragment of claim 5, used to cultivate plants with restored xylan hydrolase function and high lignocellulose quality, wherein the plant is sorghum.

9. A method for preparing plants with restored xylan hydrolase function and high lignocellulose quality, comprising the following steps: introducing the DNA fragment of claim 5 into plants lacking xylan hydrolase function to obtain the target plant; The target plant restores its growth and development phenotype while maintaining the high lignocellulose quality of the plant lacking xylan hydrolase function. The plant in question is sorghum.

10. The use of the method of claim 9 or the target plant prepared by the method of claim 9 in the preparation of animal feed, wherein the plant is sorghum.

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