CN102250908B - Rice seed 26kD globulin Glb-1 gene terminator and application thereof - Google Patents

Abstract

The invention discloses a rice seed 26kD globulin Glb-1 gene terminator and application thereof. The terminator is a DNA molecule, which is a molecule of the following 1) or 2) or 3): 1) a DNA molecule consisting of the nucleotide sequence shown in Sequence 1 in the sequence listing; 2) a DNA defined by 1) The sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% % homology; 3) Molecules that hybridize under stringent conditions to the DNA sequence defined by 1) or 2). Using the terminator of the present invention to cooperate with different promoters can increase the expression and accumulation level of foreign genes in target plants, laying a foundation for the use of biotechnology to improve seed quality, molecular medicine farms, etc., and has great applications prospect.

Description

Rice seed 26kD globulin Glb-1 gene terminator and its application

technical field

The invention relates to a rice seed 26kD globulin Glb-1 gene terminator and application thereof.

Background technique

The latest development of plant biotechnology not only realizes the improvement of traditional agronomic traits (such as increasing crop yield, enhancing disease resistance, insect resistance, herbicide resistance, improving quality, etc.), but also makes plants become bioreactors for biomedicine and industrial products . Most gramineous crops have the characteristics of high yield, low production cost, storage resistance, easy control of production scale, direct consumption, etc., and they have the ability of post-translational modification in vivo, so they become ideal carriers for the second generation of transgenic products. In recent years, the use of rice seeds to produce exogenous proteins and edible vaccines with health effects has developed rapidly, and has achieved great success. Such as vitamin A, glycinin which can lower blood sugar, soybean ferritin which can prevent and treat iron deficiency anemia and improve autoimmune function, GLP which can stimulate insulin secretion and prevent diabetes, hepatitis B vaccine and pollen allergy vaccine were successfully expressed and accumulated in high levels in rice. However, to further achieve high-efficiency expression of exogenous genes must increase gene expression at both transcriptional and post-transcriptional levels. Promoters mainly regulate gene expression at the transcriptional level, while terminators regulate both transcriptional and post-transcriptional levels.

Although currently widely used terminators, such as Nos and Ocs, combined with heterologous promoters can initiate high expression of reporter genes in plants, they can meet the research needs of biochemistry, physiology and cell localization. However, there are few studies on other terminators that can improve gene expression. Since some important agronomic traits and plant secondary metabolites are controlled by multiple genes, increasing the expression of a single gene has no obvious effect on improving related traits. It is time-consuming and labor-intensive to achieve multigene transformation through traditional transformation methods, such as repeated transformation or hybridization. The multi-gene transformation system developed in recent years can simultaneously insert multiple genes into an expression vector. In order to avoid transgene silencing caused by high homology of transgenes, these genes need to be driven by different promoters and terminated by different terminators. transcription.

Contents of the invention

One object of the present invention is to provide a terminator named tGlb-1, which can increase the expression level of foreign genes compared with the nos terminator.

The terminator provided by the present invention is the following 1) or 2) or 3) DNA molecule:

1) A DNA molecule composed of the nucleotide sequence shown in Sequence 1 in the sequence listing;

2) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% of the DNA sequence defined in 1), Molecules with at least 98% or at least 99% homology;

3) A molecule that hybridizes under stringent conditions to the DNA sequence defined in 1) or 2).

The stringent conditions may be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na ₃ PO ₄ and 1 mM EDTA, at 50°C, 2×SSC, 0.1% SDS Rinse in medium; can also be: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M Na ₃ PO ₄ and 1mM EDTA, rinse in 50°C, 1×SSC, 0.1% SDS; can also be: 50°C , hybridize in a mixed solution of 7% SDS, 0.5M Na ₃ PO ₄ and 1mM EDTA, rinse at 50°C, 0.5×SSC, 0.1% SDS; also: 50°C, in 7% SDS, 0.5M Na ₃ Hybridize in a mixed solution of PO ₄ and 1mM EDTA, rinse at 50°C in 0.1×SSC, 0.1% SDS; also: 50°C, in a mixed solution of 7% SDS, 0.5M Na ₃ PO ₄ and 1mM EDTA Hybridization at 65°C, rinsing in 0.1×SSC, 0.1% SDS; alternatively: in a solution of 6×SSC, 0.5% SDS, hybridization at 65°C, and then with 2×SSC, 0.1% SDS and 1 ×SSC and 0.1% SDS were used to wash the membrane once.

Wherein, sequence 1 in the sequence listing consists of 506 nucleotides.

Recombinant vectors, expression cassettes, transgenic cell lines, recombinant bacteria or recombinant viruses containing said DNA molecules also belong to the protection scope of the present invention.

An existing plant expression vector can be used to construct a recombinant expression vector containing the DNA molecule. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment and the like. Such as pROKII, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Company), etc. When using the DNA molecule to construct a recombinant plant expression vector, any enhanced promoter (such as the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin promoter of maize) can be added before the transcription initiation nucleotide of the foreign gene. promoter (Ubiquitin)), constitutive promoter or tissue-specific expression promoter (such as the promoter of seed-specific expression), they can be used alone or in combination with other plant promoters; in addition, using the DNA molecule of the present invention to construct plant When expressing vectors, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence, to Ensure correct translation of the entire foreign gene sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes (GUS gene, luciferase gene, etc.) genes, etc.), antibiotic marker genes (such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, and the hph gene that confers resistance to the antibiotic hygromycin , and the dhfr gene that confers resistance to metharexate, the EPSPS gene that confers resistance to glyphosate) or the marker gene for resistance to chemical agents (such as the herbicide resistance gene), the mannose-6- that provides the ability to metabolize mannose Phosphate isomerase gene.

Specifically, the recombinant vector can be pGluB-3-tGlb-1. The pGluB-3-tGlb-1 is a recombinant vector obtained by replacing the nos terminator in pGluB-3-nos with the tGlb-1 terminator shown in Sequence 1 in the sequence listing.

The expression cassette refers to a promoter, a gene of interest transcribed by the promoter, and the DNA molecule located downstream of the gene of interest. Wherein, the promoter can be a constitutive promoter or a tissue-specific expression promoter (such as an endosperm-specific promoter). The target gene can be a protein-coding gene and/or a non-protein-coding gene; the protein-coding gene is preferably a quality-improving gene; and the non-protein-coding gene is a sense RNA gene and/or an antisense RNA gene.

Another object of the present invention is to provide a method for breeding transgenic plants.

The method for cultivating transgenic plants provided by the present invention is to introduce the expression cassette containing the DNA molecule into the target plant to obtain a transgenic plant whose expression level of the target gene is higher than that of introducing the following expression cassette into the target plant: the The expression cassette obtained by replacing the DNA molecule in the above expression cassette with the nos terminator of nopaline synthase.

The target plant can specifically be a monocotyledonous plant or a dicotyledonous plant.

Specifically, the monocotyledon can be rice, wheat, corn, sorghum or barley, and the dicot can be soybean, rapeseed, cotton, tobacco, potato, sweet potato or tung tree.

The transgenic plant is understood to include not only the first-generation transgenic plant obtained by transforming the target plant with the expression cassette containing the DNA molecule, but also its progeny. For transgenic plants, the expression cassette containing the DNA molecule can be propagated in that species or transferred into other varieties of the same species, particularly including commercial varieties, using conventional breeding techniques.

The use of said DNA molecule in breeding transgenic plants or as a terminator is also within the protection scope of the present invention.

Using the terminator of the present invention to cooperate with different promoters can increase the expression and accumulation level of foreign genes in target plants, improve seed quality, and introduce physiologically active proteins or short peptides into seeds to create new health-care products. Varieties, using seeds as bioreactors to produce useful exogenous proteins or edible vaccines, increasing the added value of agricultural products technology, etc. The terminator of the present invention and its application have laid a foundation for the use of biotechnology to improve the quality of seeds, the research of molecular medicine farms, etc., and have great application prospects.

Description of drawings

Fig. 1 is the electrophoretic pattern of PCR amplification of 26kD globulin Glb-1 gene terminator (tGlb-1) from rice genomic DNA. Wherein 1 is a DNA molecular weight standard, and 2 is a fragment of tGlb-1. Among them, the standard molecular weight sizes from bottom to top are: 0.1kb, 0.2kb, 0.3kb, 0.4kb, 0.5kb, 0.6kb, 0.7kb, 0.8kb, 0.9kb, 1.0kb, 1.2kb, 1.5kb, 2.0kb, 3.0kb, 4.0kb, 5.0kb, 6.0kb, 8.0kb, 10.0kb.

Figure 2 is the identification map of vector pMD-tGlb-1 by Sac I and EcoR I double enzyme digestion. Among them, 1 is the molecular weight standard of DNA, and 2 is the digested fragment of pMD-tGlb-1. Among them, the standard molecular weight sizes from bottom to top are: 0.1kb, 0.2kb, 0.3kb, 0.4kb, 0.5kb, 0.6kb, 0.7kb, 0.8kb, 0.9kb, 1.0kb, 1.2kb, 1.5kb, 2.0kb, 3.0kb, 4.0kb, 5.0kb, 6.0kb, 8.0kb, 10.0kb, the enzyme-digested fragments from bottom to top are: tGlb-1 fragment, 506bp; vector frame fragment, 2700bp.

Fig. 3 is a schematic diagram of the structure of the vector pGluB-3-tGlb-1. Wherein, Glb-1T is tGlb-1.

Figure 4 is the identification map of vector pGluB-3-tGlb-1 after Sac I and EcoR I double digestion. Among them, 1 is the molecular weight standard of DNA, and 2 is the digested fragment of pGluB-3-tGlb-1. Among them, the standard molecular weight sizes from bottom to top are: 0.1kb, 0.2kb, 0.3kb, 0.4kb, 0.5kb, 0.6kb, 0.7kb, 0.8kb, 0.9kb, 1.0kb, 1.2kb, 1.5kb, 2.0kb, 3.0kb, 4.0kb, 5.0kb, 6.0kb, 8.0kb, 10.0kb, the enzyme-digested fragments from bottom to top are: tGlb-1 fragment, 506bp; vector frame fragment, 16.6kb.

Fig. 5 is the electrophoresis pattern of PCR amplification of chimeric primers of rice plants of T ₀ generation transduced with pGluB-3-tGlb-1 vector. 1 is a DNA molecular weight standard, 2 is a positive control using pGluB-3-tGlb-1 as a template, 3 is a negative control using an untransformed strain as a template, 4-11 are plant expression vectors transformed with pGluB-3-tGlb-1 regenerated plants.

Figure 6 shows the GUS staining results of non-transgenic wild-type rice Kitaake plants. Among them, 1, 2, 3, and 4 are the staining results of the roots, stems, leaves, and seeds of the non-transgenic wild-type rice Kitaake plants, respectively.

Fig. 7 shows the GUS staining results of the transgenic rice Kitaake/pGluB-3-nos plants of the T ₀ generation. Among them, 1, 2, 3, and 4 are the staining results of the roots, stems, leaves, and seeds of pGluB-3-nos-transformed plants, respectively.

Figure 8 shows the GUS staining results of the transgenic rice Kitaake/pGluB-3-tGlb-1 plants of the T ₀ generation. Among them, 1, 2, 3, and 4 are the staining results of the roots, stems, leaves, and seeds of the pGluB-3-tGlb-1-transformed plants, respectively.

Fig. 9 is the result of measuring GUS fluorescence activity of seeds produced by T ₀ transgenic rice Kitaake/pGluB-3-tGlb-1.

Fig. 10 is the result of measuring the GUS fluorescence activity of the seeds of _T1 transgenic rice Kitaake/pGluB-3-tGlb-1.

Detailed ways

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

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, the acquisition of rice seed 26kD globulin Glb-1 gene terminator (tGlb-1)

According to the cDNA sequence of rice 26kD globulin Glb-1 gene (GenBank number is AY427575), search the genome DNA sequence of 26kD globulin Glb-1 gene from GenBank, the sequence of 506bp after the stop codon is rice seed 26kD of the present invention The terminator (tGlb-1) of the globulin Glb-1 gene was designed to amplify tGlb-1. To facilitate vector construction, restriction sites (underlined) were added to the primers respectively. The forward primer of tGlb-1 is tGlb-1 SacF: 5′-AA GAGCTC TAGGCTTAGCTGGCTTGCCT-3′ (Sac I), and the reverse primer is tGlb-1 EcoR: 5′-A GAATTC AAGGACGGATTTGAATTGAG-3′ (EcoR I).

A small amount of genomic DNA was extracted from the leaves of wild-type rice Kitaake (Qu et al., J. Exp. Bot. 2008, 59: 2417-2424) by CTAB method, using it as a template and using tGlb-1SacF and tGlb-1EcoR as primers , PCR reaction program: 94°C pre-denaturation for 5min, then 30 cycles at 94°C for 30sec, 55°C for 30sec, 72°C for 1min, and finally 72°C for 10min. The target band of 506 bp was obtained by PCR amplification, as shown in FIG. 1 . The amplified product was recovered, directly connected to the pMD18-T vector (purchased from TaKaRa Company), and sequenced. The results showed that the amplified tGlb-1 sequence was 506bp in size and had the nucleotide sequence of sequence 1 in the sequence table. The recombinant vector containing the tGlb-1 fragment was named as pMD-tGlb-1 by sequencing detection. The identification map of pMD-tGlb-1 by Sac I and EcoRI double enzyme digestion is shown in Figure 2.

Embodiment 2, the stable expression vector construction of rice seed 26kD globulin Glb-1 gene terminator (tGlb-1)

1. Construction of plant stable expression vector fused with GUS gene of tGlb-1

Digest the pMD-tGlb-1 plasmid with Sac I and EcoR I, recover the 506bp restriction fragment of tGlb-1, and insert the fragment into the SacI and EcoR I double enzymes of pGluB-3-nos containing the GluB-3 promoter The stable transformation vector pGluB-3-tGlb-1 was constructed between the cut recognition sites (the schematic diagram of which is shown in Figure 3). pGluB-3-nos was constructed according to the method described in the literature Qu et al., J. Exp. Bot. 2008, 59: 2417-2424: the genomic DNA of rice Taichung 65 was used as a template, and

GluB-3 Forward Primer 5'-CCC AAGCTT ATTTTACTTGTACTGTTTAACC-3'(HIndIII) and

GluB-3 reverse primer 5'AAA CCCGGG AGCTTTCTGTATATGCTAATG-3'(SmaI) is a primer,

The GluB-3 promoter was amplified by PCR, and the GluB-3 promoter was double-digested with HindIII and SmaI, and inserted into

The HindIII and SmaI sites upstream of the GUS of pGPTV-35S-HPT (Qu et al., J. Exp. Bot. 2008, 59: 2417-2424), the resulting recombinant vector is pGluB-3-nos. pGPTV-35S-HPT uses GUS as the exogenous gene and nos as the terminator.

The resulting recombinant vector pGluB-3-tGlb-1 was identified by Sac I and EcoR I on the basis of PCR identification to carry out double enzyme digestion identification, and a fragment containing 506bp tGlb-1 was obtained, as shown in Figure 4.

2. Obtaining of transgenic pGluB-3-tGlb-1 rice and pGluB-3-nos regenerated rice plants

After the construction of the completed pGluB-3-tGlb-1 expression vector was verified by enzyme digestion and sequencing, it was respectively introduced into Agrobacterium EHA105 by freeze-thawing method. In the cells, lightly flick and mix them and freeze them in liquid nitrogen for 7 minutes, then transfer them to a 37°C water bath and let them stand for 3 minutes, then add 800ulYEB liquid culture and restore the culture based on 28°C for 3h to 5h, take 400μl of the bacterial solution and spread it on On the YEB resistance plate (kanamycin 50 mg/L, rifampicin Rif 50 mg/L), culture upside down at 28°C for 2 to 3 days. Pick a few single colonies of Agrobacterium for colony PCR detection of the target gene, and then streak the positive colonies on the YEB resistance plate for multiplication and culture, use the Agrobacterium infection method to transform the callus of rice variety kitaake, and screen with hygromycin Resistant calluses were further cultured and differentiated into seedlings, and then transplanted to the greenhouse.

Using the hygromycin screening method (performed according to the method described in the literature Hiei et al., Plant J. 1994, 6: 271-282), 8 T ₀ generation transgenic pGluB-3-tGlb-1 rice plants were obtained. Utilize the PCR method to carry out PCR molecular detection to the partial pGluB-3-tGlb-1 rice that above-mentioned screening obtains, and PCR primer is the chimeric primer of tGlb-1 and GUS gene sequence, namely: tGlb-1 sequence reverse primer tGlb-1EcoR :

5'-A GAATTC AAGGACGGATTTGAATTGAG-3' and forward primer GUSF185 inside the GUS sequence: 5'-TCGTCGGTGAACAGGTATGG-3'. The PCR reaction program was: pre-denaturation at 94°C for 5 minutes, followed by 30 cycles at 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min, and finally 72°C for 10 min. The target band of 0.7 kb obtained by PCR amplification is positive for detection. The results showed that the eight pGluB-3-tGlb-1 transformed plants (expressed as Kitaake/pGluB-3-tGlb-1) detected by PCR were all positive (as shown in FIG. 5 ).

According to the above method, pGluB-3-nos was transformed into wild-type rice Kitaake, and 9 positive pGluB-3-nos plants transformed with pGluB-3-nos (expressed as Kitaake/pGluB-3-nos) were obtained. PCR detection method for pGluB-3-nos-transformed rice plants: use tnos sequence reverse primer tnosR: 5′-GATCTAGTAACATAGATGAC-3′ and GUS sequence internal forward primer GUSF185: 5′-TCGTCGGTGAACAGGTATGG-3′ as primers to amplify The chimeric sequence of tnos and GUS genes, the target band of 500bp is a positive transformant.

The seeds produced by the transgenic plants of the T ₀ generation and the plants grown from the seeds are the T ₁ generation, and so on, T ₂ and T ₃ represent the second and third generation of the transgenic plants, respectively.

Example 3, Functional verification of rice seed 26kD globulin Glb-1 gene terminator (tGlb-1)

1. GUS histochemical detection of T ₀ transgenic rice Kitaake/pGluB-3-nos and Kitaake/pGluB-3-tGlb-1

Histochemical staining was performed on T ₀ plants of transgenic rice Kitaake/pGluB-3-nos and Kitaake/pGluB-3-tGlb-1 that were positive by PCR, and wild-type rice Kitaake plants that were not transgenic were used as controls. The specific steps are: cutting the T ₀ generation plants of transgenic rice Kitaake/pGluB-3-nos and Kitaake/pGluB-3-tGlb-1 and part of the leaves, roots and stem tissues of non-transgenic wild type rice Kitaake plants into small pieces block; the seeds at the filling stage 17 days after flowering were cut longitudinally from the middle with a scalpel. Soak the treated sample in GUS staining reaction solution (0.1M sodium phosphate buffer (pH 7.0), 10mM Na ₂ -EDTA (pH 7.0), 5mM potassium ferricyanide, 5mM potassium ferrocyanide, 1.0mM X -Gluc, 0.1% Triton X-100), react at 37°C for 0.5-4 hours. Store and observe in 70% ethanol. Observe and take pictures under the stereoscope. Result As shown in Figure 6, Figure 7, and Figure 8, GUS expression was not observed in the aleurone layer and sub-aleurone layer of the roots, leaves, stems, and seeds of the non-transgenic wild-type rice Kitaake plants 17 days after flowering; GUS expression was not observed in the roots, leaves, and stems of T ₀ transgenic rice Kitaake/pGluB-3-nos and Kitaake/pGluB-3-tGlb-1 plants, and T ₀ transgenic rice Kitaake/pGluB-3-nos flowered The seeds of the last 17 days were only expressed in the aleurone layer and sub-aleurone layer, while the blue color of the aleurone layer, sub-aleurone layer and the whole endosperm of T ₀ transgenic rice Kitaake/pGluB-3-tGlb-1 was clear Visible expression. The results showed that tGlb-1 enhanced the expression intensity of β-glucuronidase (GUS) reporter gene in rice seed endosperm compared with nos terminator, but did not change the specific expression of GUS reporter gene in endosperm.

2. Determination of GUS fluorescence activity of T ₀ and T ₁ transgenic rice Kitaake/pGluB-3-nos and Kitaake/pGluB-3-tGlb-1

Quantitative GUS analysis was performed on the seeds of the T ₀ and T ₁ generation plants of the transgenic rice Kitaake/pGluB-3-nos and Kitaake/pGluB-3-tGlb-1 that were positive in PCR detection 17 days after flowering. The method was carried out according to the fluorescence detection method of Jefferson et al. (Jefferson, Plant Mo1. Bio1. Report, 1987, 5: 387-405). Specifically: take 3 seeds from each plant, place them in 3 1.5ml eppendorf tubes, crush the seeds with a pestle, add 200 μl of extract (50mM sodium phosphate buffer solution (pH7.0), 10mM β-mercaptoethanol , 10mM Na2EDTA (pH 8.0), 0.1% SDS, 0.1% Triton X-100), shake and mix. Centrifuge at 13,000 rpm at 4°C for 5 min, and take the supernatant into a new tube. 10 μl of supernatant was added to 90 μl of reaction solution (1 mM 4-MUG dissolved in GUS extract), reacted at 37°C for 60 minutes, added 900 μl of stop solution (0.2M Na ₂ CO ₃ ), and terminated at room temperature. 4-MU was used as a standard curve, and the relative 4-MU content was detected with an F-4500 (Hitachi) fluorescence spectrophotometer at an excitation wavelength of 360nm and an absorption wavelength of 460nm. Using bovine serum albumin as a control, the protein content was determined using BIO-Rad Protein Assay Kit II (purchased from Bio-Rad, USA, No. GPR6611).

The results of GUS fluorescence activity measurement on the seeds of the transgenic rice line T ₀ generation are shown in FIG. 9 . Among the 8 T ₀ strains of Kitaake/pGluB-3-tGlb-1, the highest value of GUS activity was 46.04pmol 4-MU min ^-1 μg ^-1 protein, and the lowest value was 9.65pmol 4-MU min ^-1 μg ^{- 1} protein, with an average of 23.9±12.7 pmol 4-MU min ^-1 μg ^-1 protein. In the 9 control vector lines transfected with Kitaake/pGluB-3-nos, the highest value of GUS activity was 17.38pmol 4-MU min ^-1 μg ^-1 protein, and the lowest value was 2.39pmol 4-MU min ^-1 μg ^-1 Protein, with an average of 11.2±5.0 pmol 4-MU min ^-1 μg ^-1 protein. One of the 8 T ₀ strains of Kitaake/pGluB-3-tGlb-1 had a GUS expression level less than 1, and the rest were greater than or equal to the average value of the T ₀ strains of the control Kitaake/pGluB-3-nos, and the former had the maximum value of the latter 2.64 times of the maximum value, 19.23 times of the minimum value, and 2.13 times of the average value of the control.

The GUS activity was measured on the seeds produced by some _T1 generations of the transgenic rice lines (shown in FIG. 10 ). The highest GUS activity of the six Kitaake/pGluB-3-tGlb-1 strains was 40.72pmol 4-MU min ^-1 μg ^-1 protein, the lowest was 14.4pmol 4-MU min ^-1 μg ^-1 protein, the average It is 25.4±9.1pmol 4-MU min ^-1 μg ^-1 protein. In contrast, among the 7 T ₁ strains of Kitaake/pGluB-3-nos, the highest value of GUS activity was 21.52pmol4-MU min ^-1 μg ^-1 protein, and the lowest value was 2.82pmol 4-MU min ^-1 μg ^{- 1} protein, with an average of 9.9±5.8pmol4-MU min ^-1 μg ^-1 protein. The average value of the former is 2.55 times that of the control.

The results showed that tGlb-1 enhanced the expression level of exogenous genes in rice endosperm compared with the nos terminator.

Claims (12)

1. A DNA molecule, characterized in that: the DNA molecule is a DNA molecule composed of the nucleotide sequence shown in Sequence 1 in the Sequence Listing. 2. A recombinant vector containing the DNA molecule of claim 1. 3. An expression cassette comprising the DNA molecule of claim 1. 4. A transgenic cell line containing the DNA molecule of claim 1. 5. A recombinant bacterium containing the DNA molecule of claim 1. 6. A recombinant virus comprising the DNA molecule of claim 1. 7. The expression cassette according to claim 3, characterized in that: the expression cassette consists of a promoter, a gene of interest for transcription initiated by the promoter, and the DNA molecule according to claim 1 positioned at the downstream of the gene of interest composition. 8. The expression cassette according to claim 7, wherein the promoter is a constitutive promoter or a tissue-specific expression promoter, and the tissue-specific expression promoter is an endosperm-specific expression promoter. 9. The expression cassette according to claim 7 or 8, characterized in that: the target gene is a protein-coding gene and/or a non-protein-coding gene; the protein-coding gene is a quality-improving gene; the non-protein-coding gene Because of the sense RNA gene and/or the antisense RNA gene. 10. A method for cultivating transgenic rice, which is to introduce the expression cassette described in any one of claims 7-9 into the rice of interest, and obtain the expression level of the gene of interest higher than that of the transgene that introduces the following expression cassette into the rice of interest Rice: an expression cassette obtained by replacing the DNA molecule of claim 1 in the expression cassette of claim 7 with a nos terminator. 11. The application of the DNA molecule according to claim 1 in cultivating transgenic rice. 12. Use of the DNA molecule of claim 1 as a terminator.

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