CN102277355A - Rice seed glutelin GluA-2 gene terminator and application thereof - Google Patents

Abstract

The invention discloses a rice seed glutelin GluA-2 gene terminator and application thereof. The terminator is a DNA (deoxyribonucleic acid) molecule, which is a molecule shown as 1), 2) or 3): 1) a DNA molecule consisting of nucleotide sequences shown as a sequence 1 in a sequence table; 2) a molecule with at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99 percent of homology with the DNA sequence limited by 1); and 3) a molecule hybridized with the DNA sequence limited by 1) or 2) under a strict condition. The terminator disclosed by the invention is utilized to cooperate with different promoters, the expression and accumulation levels of an exogenous gene in a target plant can be improved, and foundation is established for researches in improving seed quality, molecular medicine farm and the like by utilizing biotechnology. Therefore the terminator has great application prospect.

Description

Rice Seed Glutenin GluA-2 Gene Terminator and Its Application

technical field

The invention relates to a rice seed glutelin GluA-2 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 tGluA-2, 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 728 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 any of the following vectors: pGluB-3-tGluA-2, pGluC-tGluA-2, p35S-tGluA-2, pUbi-tGluA-2.

The pGluB-3-tGluA-2 is a recombinant vector obtained by replacing the nos terminator in pGluB-3-nos with the tGluA-2 terminator shown in Sequence 1 in the sequence listing, and the pGluC-tGluA-2 is a recombinant vector obtained by replacing The nos terminator in pGluC-nos is replaced by the tGluA-2 terminator shown in sequence 1 in the sequence listing. The recombinant vector obtained by the tGluA-2 terminator shown in the pUbi-tGluA-2 is a recombinant vector obtained by replacing the nos terminator in pUbi-221 with the tGluA-2 terminator shown in Sequence 1 in the sequence listing, The pUbi-221 is a recombinant vector obtained by replacing the 35S promoter in pBI221 with the Ubiquitin promoter.

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 an electrophoretic pattern of PCR amplification of glutelin GluA-2 gene terminator (tGluA-2) from rice genomic DNA. Among them, 1 is a DNA molecular weight standard, and 2 is a fragment of tGluA-2. Among them, the standard molecular weight sizes from bottom to top are: 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp, 5000bp.

Figure 2 is the identification map of vector pMD-tGluA-2 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-tGluA-2. Among them, the standard molecular weight sizes from bottom to top are: 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp, 5000bp, and the restriction enzyme fragments from bottom to top are: tGluA-2, 728bp; pMD-18T vector fragment, 2700bp.

Fig. 3 is a schematic diagram of the structure of the vector containing tGluA-2. Among them, Figure A is a schematic diagram of the structure of pGluB-3-tGluA-2, Figure B is a schematic diagram of the structure of pGluC-tGluA-2, Figure C is a schematic diagram of the structure of p35S-tGluA-2, Figure D is a schematic diagram of the structure of pUbi-tGluA-2, GluA- 2T stands for tGluA-2.

Figure 4 is the identification map of the vector containing tGluA-2 after Sac I and EcoR I double digestion. Among them, A-D diagrams are the vectors pGluB-3-tGluA-2, pGluC-tGluA-2, p35S-tGluA-2, pUbi-tGluA-2 respectively; 1 is the DNA molecular weight standard, and 2 is the digested fragment of each vector. Among them, the standard molecular weight sizes in Figure A 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, 2.0 kb, 3.0kb, 4.0kb, 5.0kb, 6.0kb, 8.0kb, 10.0kb, the restriction fragments from bottom to top are: tGluA-2, 728bp; pGluB-3-tGluA-2 vector frame fragment, 16.6kb. Figure 4B-4D standard molecular weight sizes from bottom to top are: 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp, 5000bp. Figure 4B enzyme-digested fragments from bottom to top are: tGluA-2, 728bp; 2 vector framework fragments, 5.2kb. Figure 4D restriction fragments from bottom to top are: tGluA-2, 728bp; GUS and part of the Ubiquitin promoter fusion fragment 2.5kb (of which part of the Ubiquitin promoter size is 0.6kb), pUbi-tGluA-2 fragment after removing the above two parts 3.9kb.

Fig. 5 is the PCR amplification electrophoresis pattern of the chimeric primers of some rice plants of the T ₀ generation transfected with pGluB-3-tGluA-2 and pGluC-tGluA-2. 1 is the DNA molecular weight standard, 2 is the positive control using pGluB-3-tGluA-2 as the template, 3 is the negative control using the untransformed strain as the template, 4-13 are the regeneration of partially transformed pGluB-3-tGluA-2 Rice plants, 14-23 are regenerated rice plants partially transfected with pGluC-tGluA-2.

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-tGluA-2 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-tGluA-2-transformed plants, respectively.

Fig. 9 is the measurement result of GUS fluorescence activity of seeds produced by T ₀ transgenic rice Kitaake/pGluB-3-tGluA-2.

Fig. 10 is the measurement result of GUS fluorescence activity of seeds produced by _T1 transgenic rice Kitaake/pGluB-3-tGluA-2.

Fig. 11 shows the results of measuring GUS fluorescence activity of seeds produced by T ₀ transgenic rice Kitaake/pGluC-tGluA-2.

Figure 12 shows the activity determination of GUS extracted from protoplasts of rice transfected with p35S-tGluA-2 and pBI221 incubated for 14 hours. Among them, 1 is the average value of three repetitions of pBI221 transformation, and 2, 3, and 4 are the three measured values of p35S-tGluA-2 transformation respectively.

Figure 13 shows the activity determination of GUS extracted from protoplasts of rice transfected with pUbi-tGluA-2 and pUbi-221 after incubation for 14 hours. Among them, 1 is the average value of three replicates transformed into pUbi-221, and 2, 3, and 4 are the measured values of three times transformed into pUbi-tGluA-2, respectively.

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 glutelin GluA-2 gene terminator (tGluA-2)

According to the cDNA sequence of rice glutelin GluA-2 gene (GenBank number is AK107314), search the genomic DNA sequence of glutelin GluA-2 gene from GenBank, the sequence of 728bp behind the stop codon is the rice seed glutelin GluA of the present invention -2 gene terminator (tGluA-2), design primers to amplify tGluA-2. To facilitate vector construction, restriction sites (underlined) were added to the primers respectively. The forward primer of tGluA-2 is tGluA-2SacF: 5′-G GAGCTC GTTGGCAATGCGGATAAAG-3′ (Sac I), and the reverse primer is tGluA-2EcoR: 5′-A GAATTC GAATTCGGAAGCTCACTT-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, tGluA-2 SacF and tGluA-2 EcoR As a primer, the tGluA-2 sequence was amplified by PCR. 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 728bp was amplified by PCR, as shown in Figure 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 tGluA-2 sequence was 728bp in size and had the nucleotide sequence of sequence 1 in the sequence table. The recombinant vector containing the tGluA-2 fragment was named pMD-tGluA-2 according to the sequencing detection. The identification map of pMD-tGluA-2 by Sac I and EcoR I double enzyme digestion is shown in Figure 2.

Example 2, Vector Construction and Transformation of Rice Seed Glutenin GluA-2 Gene Terminator (tGluA-2)

1. Construction of tGluA-2 fusion GUS gene plant expression vector

Digest the pMD-tGluA-2 plasmid with Sac I and EcoR I, recover the 728bp fragment of tGluA-2, and insert the fragment into pGluB-3-nos containing GluB-3 promoter and pGluC of GluC promoter respectively The stable transformation vectors pGluB-3-tGluA-2 (the schematic diagram of which is shown in Figure 3A) and pGluC-tGluA-2 (the schematic diagram of which is shown in Figure 3B) were constructed between the SacI and EcoR I double restriction recognition sites of -nos Show).

pGluB-3-nos and pGluC-nos were 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

The GluB-3 reverse primer 5'AAA CCCGGG AGCTTTCTGTATATGCTAATG-3'(Sma I) was used as a primer, and the GluB-3 promoter was amplified by PCR. The GluB-3 promoter was double digested with HindIII and Sma I, and inserted into pGPTV- 35S-HPT (Qu et al., J.Exp.Bot.2008, 59:2417-2424) upstream of the GUS HindIII and SmaI sites, the resulting recombinant vector is pGluB-3-nos; rice Taichung 65 GluC forward primer 5'-GGG AAGCTT GTTCAAGATTTATTTTTGG-3'(HindIII); GluC reverse primer 5'-ACGC GTCGAC AGTTATTCACTTAGTTTCCC-3'(Sal I) was used as primer, and the GluC primer was amplified by PCR The GluC promoter was double digested with HindIII and Sal I, and inserted into the HindIII and Sal I sites upstream of the GUS of pGPTV-35S-HPT, and the resulting recombinant vector was pGluC-nos. pGPTV-35S-HPT uses GUS as the exogenous gene and nos as the terminator.

tGluA-2 was constructed between pBI221 (Chen et al., Mol. Breed.2003, 11: 287-293) and pUbi-221 between the Sac I and EcoR I double restriction recognition sites to construct the transient expression vector p35S- tGluA-2 (the schematic diagram of which is shown in FIG. 3C ) and pUbi-tGluA-2 (the schematic diagram of which is shown in FIG. 3D ). Among them, pUbi-221 is a recombinant vector obtained by replacing the 35S promoter of pBI221 with the Ubiquitin promoter.

The four recombinant vectors obtained above were identified by double enzyme digestion with Sac I and EcoRI on the basis of PCR identification, and a tGluA-2 fragment containing 728bp was obtained, as shown in Figure 4.

2. Obtainment of regenerated rice plants transformed with pGluB-3-tGluA-2, pGluC-tGluA-2, pGluB-3-nos and pGluC-nos

After the constructed pGluB-3-tGluA-2 and pGluC-tGluA-2 expression vectors were digested and sequenced to verify their correctness, they were respectively introduced into Agrobacterium EHA105 by freeze-thawing method. In 100μl EHA105 Agrobacterium competent cells, lightly flick and mix well, freeze in liquid nitrogen for 7min, then transfer to 37℃ water bath and let stand for 3min, then add 800ulYEB liquid culture based on 28℃ for 3-5 hours to recover and cultivate. Take 400 μl of the bacterial liquid and spread it on the YEB resistance plate (Kanamycin 50 mg/L, Rifampicin Rif 50 mg/L), and culture it 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 (according to the method described in the literature Hiei et al., Plant J.1994, 6: 271-282), 14 T ₀ generation transgenic pGluB-3-tGluA-2 rice plants and 17 T _{The 0th} generation was transformed into pGluC-tGluA-2 rice plants. Use the PCR method to carry out PCR molecular detection on the transpGluB-3-tGluA-2 rice and transpGluC-tGluA-2 rice obtained by the above screening, and the PCR primer is a chimeric primer of tGluA-2 and GUS gene sequence, namely: tGluA-2 Sequence reverse primer tGluA-2 EcoR: 5'-A GAATTC GAATTCGGAAGCTCACTT-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. A target band of 0.9 kb obtained through PCR amplification is positive for detection (as shown in FIG. 5 ).

The results showed that 14 pGluB-3-tGluA-2 plants (expressed as Kitaake/pGluB-3-tGluA-2) were all positive in PCR detection, and 17 plants transformed with pGluC-tGluA-2 (expressed as Kitaake/pGluC-tGluA- 2) PCR tests were all positive.

According to the above method, pGluB-3-nos and pGluC-nos were respectively transformed into wild-type rice Kitaake to obtain 9 positive plants transformed with pGluB-3-nos (expressed as Kitaake/pGluB-3-nos), and 10 plants transformed with pGluC -nos positive plants (indicated as Kitaake/pGluC-nos). PCR detection method of rice plants transfected with pGluB-3-nos and pGluC-nos: tnos sequence reverse primer tnosR: 5′-GATCTAGTAACATAGATGAC-3′ and GUS sequence internal forward primer GUSF185: 5′-TCGTCGGTGAACAGGTATGG-3′ As primers, amplify the chimeric sequences of tnos and GUS genes, and obtain the target band of 500bp, which is the 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.

4. Obtaining of p35S-tGluA-2, pUbi-tGluA-2, pBI221 and pUbi-221 protoplasts

By PEG-mediated method, p35S-tGluA-2, pUbi-tGluA-2, pBI221 and pUbi-221 were respectively transferred into the protoplasts made from the etiolated seedlings of wild-type rice Kitaake (according to the literature Chen et al, Mol PlantPathol. 2006, 7: 417-427), in triplicate for each vector, incubated at 28°C for 14-16 hours.

Example 3, Functional Verification of Rice Seed Glutenin GluA-2 Gene Terminator (tGluA-2)

1. GUS histochemical detection of _T0 transgenic rice

Histochemical staining was performed on T ₀ plants of transgenic rice Kitaake/pGluB-3-nos and Kitaake/pGluB-3-tGluA-2 that were positive in PCR detection, 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-tGluA-2 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. The results are 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 17 days after flowering of the non-transgenic wild-type rice Kitaake plants ; GUS expression was not observed in the roots, leaves, and stems of T ₀ transgenic rice Kitaake/pGluB-3-nos and Kitaake/pGluB-3-tGluA-2 plants, and T ₀ transgenic rice Kitaake/pGluB-3-nos Seeds 17 days after flowering were only expressed in the aleurone layer and sub-aleurone layer, while the aleurone layer, sub-aleurone layer and the whole endosperm of T ₀ transgenic rice Kitaake/pGluB-3-tGluA-2 were blue. clearly visible expression. The results showed that tGluA-2 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

Transgenic rice Kitaake/pGluB-3-nos and Kitaake/pGluB-3-tGluA-2 transgenic rice Kitaake/pGluB-3-nos and Kitaake/pGluB-3-tGluA-2 T ₀ generation plants 17 days after flowering Seeds at filling stage, Kitaake/pGluC-nos and Kitaake/pGluC-tGluA GUS quantitative analysis of the seeds of the T ₀ generation plants of -2 at the filling stage 17 days after flowering and the seeds of the T ₁ generation plants of Kitaake/pGluB-3-nos and Kitaake/pGluB-3-tGluA-2 at the filling stage 17 days after flowering . The method was carried out according to the fluorescence detection method of Jefferson et al. (Jefferson, Plant Mol. Biol. 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 Na ₂ EDTA (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).

Figure 9 and Figure 11 show the results of the GUS activity assay on the seeds of the transgenic line T ₀ generation. Among the 14 T ₀ strains of Kitaake/pGluB-3-tGluA-2 (Figure 9), the highest value of GUS activity was 53.46pmol 4-MUmin ^-1 μg ^-1 protein, and the lowest value was 18.24pmol 4-MU min ^{- 1} μg ^-1 protein, the average value is 27.5±9.2 pmol4-MU min ^-1 μg ^-1 protein. In the 9 control Kitaake/pGluB-3-nos T ₀ strains, 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. The expression levels of GUS in the 14 lines transfected with pGluB-3-tGluA-2 vector were all greater than the maximum value of the pGluB-3-nos transfected plants, the former maximum value was 3.1 times the latter maximum value, and the latter was 22.33 times the minimum value. The value is 2.5 times of the average value of the control.

Among the 17 T ₀ lines of Kitaake/pGluC-tGluA-2 (Figure 11), the highest value of GUS activity was 76.12pmol 4-MU min ^-1 μg ^-1 protein, and the lowest value was 31.74pmol 4-MU min ^-1 μg ^-1 protein with an average of 52.9±12.4 pmol 4-MU min ^-1 μg ^-1 protein. In the 10 control Kitaake/pGluC-nos T ₀ strains, the highest value of GUS activity was 43.55pmol 4-MU min ^-1 μg ^-1 protein, the lowest value was 6.46pmol 4-MU min ^-1 μg ^-1 protein, The mean value was 20.38±10.64 pmol 4-MU min ^-1 μg ^-1 protein. The maximum value of the former is 1.75 times of the maximum value of the latter, 11.8 times of its minimum value, and the average value is 2.6 times of the control average value.

Figure 10 shows the results of the GUS activity assay on the seeds produced by the _T1 generation of the transgenic lines. The highest GUS activity of 8 Kitaake/pGluB-3-tGluA-2 T ₁ strains was 55.45pmol 4-MU min ^-1 μg ^-1 protein, the lowest was 16.75pmol 4-MU min ^-1 μg ^-1 protein, The mean value was 31.6±12.8 pmol 4-MU min ⁻¹ μg ⁻¹ protein. In the 7 control Kitaake/pGluB-3-nos T ₁ strains, 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, the average value was 9.9±5.8pmol4-MU min ^-1 μg ^-1 protein. The average value of the former is 3.17 times that of the control.

The results showed that tGluA-2 enhanced the expression level of exogenous genes in rice endosperm compared with the nos terminator.

3. Determination of GUS fluorescence activity of rice protoplasts

The activity of GUS extracted from wild-type rice Kitaake protoplasts transformed with p35S-tGluA-2 and pBI221 and incubated for 14 hours was measured (shown in FIG. 12 ). 1 is the average value of 0.51pmol 4-MU min ^-1 μg ^-1 protein of three repetitions of pBI221 transfection, while 2, 3 and 4 are the three measured values of 1.48, 1.28 and 1.08pmol 4-MUmin of p35S-tGluA-2 respectively ^-1 μg ^-1 protein, respectively 2.90, 2.51 and 2.12 times of the control.

The activity of GUS extracted from wild-type rice Kitaake protoplasts incubated with pUbi-tGluA-2 and pUbi-221 for 14 hours was determined (shown in FIG. 13 ). 1 is the average value of 9.22pmol 4-MUmin ^-1 μg ^-1 protein of three repetitions of pUbi-221 transfection, while 2, 3 and 4 are the three measured values of 24.0, 21.86 and 20.82pmol 4-MUmin -1 of pUbi-tGluA-2 respectively MU min ^-1 μg ^-1 protein was 2.60, 2.37 and 2.25 times that of the control, respectively.

The results showed that tGluA-2 enhanced the expression level of exogenous genes in rice compared with the nos terminator.

Claims (10)

1. DNA molecule, is characterized in that: described DNA molecule is following 1) or 2) or 3) 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). 2. A recombinant vector, an expression cassette, a transgenic cell line, a recombinant bacterium or a recombinant virus containing the DNA molecule of claim 1. 3. The expression cassette according to claim 2, 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. 4. The expression cassette according to claim 3, wherein the promoter is a constitutive expression promoter or a tissue-specific expression promoter, and the tissue-specific expression promoter is an endosperm-specific expression promoter. 5. The expression cassette according to claim 3 or 4, characterized in that: the target gene is a protein-coding gene and/or a non-protein-coding gene; the protein-coding gene is preferably a quality-improving gene; the non-protein-coding gene The gene is a sense RNA gene and/or an antisense RNA gene. 6. A method for cultivating transgenic plants, which is to introduce the expression cassette described in any one of claims 3-5 into the plant 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 plant of interest Plant: an expression cassette obtained by replacing the DNA molecule of claim 1 in the expression cassette of claim 3 with a nos terminator. 7. The method according to claim 6, characterized in that: the target plant is a monocot or a dicot. 8. The method according to claim 7, characterized in that: the monocot is rice, wheat, corn, sorghum or barley, and the dicotyledon is soybean, rape, cotton, tobacco, potato, sweet potato or oil Tong. 9. The application of the DNA molecule according to claim 1 in cultivating transgenic plants. 10. Use of the DNA molecule of claim 1 as a terminator.

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