NL2029636B1 - Soybean seed-specific promoter gmp34p and use thereof - Google Patents
Soybean seed-specific promoter gmp34p and use thereof Download PDFInfo
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Abstract
The present disclosure relates to the field of plant genetic engineering. The present disclosure provides a seed—specific promoter GmP34P, which is a novel effective seed—specific promoter that drives the expression of downstream genes to be seed—specific and is highly expressed in seeds. According to the invention, the seed—specific promoter has a nucleotide sequence shown in SEQ ID No. l.
Description
P791/NLpd SOYBEAN SEED-SPECIFIC PROMOTER GMP34P AND USE THEREOF
TECHNICAL FIELD The present disclosure relates to the field of plant genetic engineering, and in particular to a soybean seed-specific promoter GmP34P and use thereof.
BACKGROUND ART In crop guality breeding, an appropriate promoter should be selected to construct upstream of a target gene. A seed-specific promoter regulates the specific expression of exogenous genes in a seed, which can increase the content of some substances in seeds as desired, achieving the objective of seed quality improvement. Sometimes, use of non-parent plant promoters may lead to a change in tissue specificity or a decrease in expression intensi- ty, but parent plant promoters tend to be needed during crop seed quality improvement. Studies of the soybean seed-specific promot- ers have been reported, such as promoters of soybean oleosin gene and soybean B-conglycinin o-subunit, but in general, they are sel- dom used in soybean molecular breeding at present. Therefore, a novel soybean seed-specific promoter is still needed.
SUMMARY To solve the above problems, the present disclosure provides a soybean seed-specific promoter GmP34P and use thereof. The seed- specific promoter drives the expression of downstream genes to be seed-specific and is highly expressed in seeds, providing an ef- fective promoter for molecular breeding for soybean seed quality improvement. To achieve the above objective, the present disclosure pro- vides the following technical solutions: The present disclosure provides a seed-specific promoter GmP34P, where the seed-specific promoter has a nucleotide sequence shown in SEQ ID No. 1. The present disclosure further provides a primer pair for identifying the above seed-specific promoter GmP34P, where an up- stream primer of the primer pair has a nucleotide sequence shown in SEQ ID No. 2; and a downstream primer of the primer pair has a nucleotide sequence shown in SEQ ID No. 3. The present disclosure further provides a recombinant expres- sion vector including the above seed-specific promoter GmP34P.
Preferably, a basic plasmid of the recombinant expression vector may include pCAMBIA1301. The present disclosure further provides use of the above seed-specific promoter GmP34P, the above primer pair, or the above recombinant expression vector in seed quality improvement of a transgenic plant.
Preferably, the plant may include a leguminous plant.
Preferably, the leguminous plant may include soybean.
Beneficial effects: The present disclosure provides a seed-specific promoter GmP34P, where the seed-specific promoter has a nucleotide sequence shown in SEQ ID No. 1. The seed-specific promoter GmP34P provided by the present disclosure is a novel effective seed-specific pro- moter suitable as a seed-specific promoter in research on molecu- lar breeding for soybean seed quality improvement.
In the examples of the present disclosure, expression of a GmP34P promoter-driven GUS gene in transgenic tobacco seeds is significantly higher than that in roots, stems, leaves and flow- ers; relative expression of the GmP34P promoter-driven GUS gene in seeds is 122.36 times the expression thereof in roots; relative expression of a CaMV35S promoter-driven GUS gene in seeds is 95.61 times the expression thereof in roots.
Visibly, the GmP34P promot- er drives the expression of downstream genes in seeds more active- ly than the CaMV353 promoter.
BRIEF DESCRIPTION OF THE DRAWINGS: FIG. 1 illustrates the expression of soybean P34 gene in dif- ferent soybean organs detected by real-time fluorescent quantita- tive PCR assay in Example 1; FIG. 2 illustrates the PCR amplification result of GmP34P promoter, where M represents DL2000 Marker, and 1 represents PCR-
amplified bands; FIG. 3 illustrates a double digestion of a recombinant plas- mid pMD18-T-GmP34P, where M represents DL2000 Marker, and 1 repre- sents a double digestion result of the plasmid; FIG. 4 illustrates a double digestion of a plant expression vector pCAM-GmP34P, where M represents DL2000 Marker, and 1 repre- sents a double digestion result of the plasmid; FIG. 5 illustrates the identification result of PCR for Ty positive transgenic tobacco plants with pCAM-GmP34P, where M rep- resents DL2000 Marker, 1 to 6 represent amplified bands of T; posi- tive transgenic tobacco plants, “+” represents a positive control of PCAM-GmP34P plasmid, and “-" represents a negative control of wild-type tobacco; FIG. 6 illustrates the identification result of RT-PCR for T, positive transgenic tobacco plants with pCAM-GmP34P, where M rep- resents DL2000 Marker, 1 to 6 represent amplified bands of T, posi- tive transgenic tobacco plants, “+” represents a positive control of pCAM-GmP34P plasmid, and “-" represents a negative control of wild-type tobacco; FIG. 7 illustrates the result of the expression of a GUS gene in different organs of transgenic tobacco detected by real-time fluorescent quantitative PCR, where root, stem, leaf, flower, and seed represent the relative expression of the GUS gene in the root, stem, leaf, flower, and seed of a positive pCAM-GmP34P- transfected tobacco plant, respectively, and 353 represents the relative expression of the GUS gene in the seed of a positive PCAMBIA1301-transfected tobacco plant; FIG. 8 illustrates the detection of GUS activity by a fluo- rescence spectrophotometer, where WT represents the GUS activity in the seeds of wild-type tobacco, GmP34P represents the GUS ac- tivity in the seeds of positive pCAM-GmP34P-transfected tobacco, and CaMV35S represents the GUS activity in the seeds of positive PCAMBIA1301-transfected tobacco.
DETAILED DESCRIPTION OF THE EMBODIMENTS The present disclosure provides a seed-specific promoter GmP34P, where the seed-specific promoter has a nucleotide sequence shown in SEQ ID No. 1, specifically: TGAGTTGTTTCAGGTTCCATTGCCTTATTGCTAAAACTCCAACTAAAATAACAAA- TAGCACATGCAGGTGCAAACAACACGTTACTCTGATGAAGGTGATGTGCCTCTAGCAG- TCTAGCTTATGAGGCTCGCTGCTTATCAACGATTCATCATTCCCCAAGACGTGTACGCAGAT- TAAACAATGGACAAAACTTCAATCGATTATAGAATAATAATTTTAACAG- TGCCGACTTTTTTCTGTAAACAAAAGGCCAGAATCATATCGCACATCATCTTGAATGCAG- TGTCGAGTTTGGACCATTTGAGTACAAAGCCAATATTGAATGATTTTTCGAT- TTTACATGTGTGAATCAGACAAAAGTGCATGCAATCACTTGCAAGTAAATTAAGGATACT- AATCTATTCCTTTCATTTTATATGCTCCACTTTTATATAAAAAAATATACATTATTATA- TATGCATTATTAATTATTGCAGTATTATGCTATTGGTTTTATGGCCCTGCTAAATAAC- CTAAATGAGTCTAACTATTGCATATGAATCAAATGAAGGAAGAATCATGATCTAAACCTGAG- TACCCAATGCAATAAAATGCGTCCTATTACCTAAACTTCAAACACACATTGCCATCGGAC- GTATAAATTAATGCATATAGATTATTTTGAGAAAAGAAAACATCAAAA- GCTCTAAAACTTCTTTTAACTTTGAAATAAGCTGATAAAAATAC- GCTTTAAATCAACTGTGTGCTGTATATAAGCTGCAATTTCACATTTTACCAAACCGAAACAA- GAATGGTAACAGTGAGGCAAAAATTTGAAAAATGTCC- TACTTCACATTCACATCAAATTAATTACAACTAAATAAATAAACATCGTGATTCAAGCAG- TAATGAAAGTCGAAATCAGATAGAATATACACGTTTAACATCAATT- GAATTTTTTTTTAAATGGATATATACAAGTTTACTATTTTATATATAATGAAAATTCATTTT- GTGTTAGCACAAAACTTACAGAAAGAGATAAATTTTAAATAAAGAGAATTA- TATCCAATTTTATAATCCAAAATAATCAAATTAAAGAATATTGGCTAGATAGAC- CGGCTTTTTCACTGCCCCTGCTGGATAATGAAAATTCAT- ATCAAAACAATACAGAAGTTCTAGTTTAATAATAAAAAAGTTGG- CAAACTGTCATTCCCTGTTGGTTTTTAAGCCAAATCACAATTCAATTAC- GTATCAGAAATTAATTTAAACCAAATATATAGCTACGAGGGAACTTCTTCAGTCATTACTAGC- TAGCTCACTAATCACTATATATACGACATGCTACAAGTGAAGTGAC- CATATCTTAATTTCAAATCATAAAATTCTTCCACCAAGTT. In the present disclo- sure, the GmP34P promoter may preferably be obtained by amplifica- tion of ATG upstream sequence of a soybean P34 protein sequence (NM 001251290.2).
The present disclosure further provides a primer pair for identifying the above seed-specific promoter GmP34P, where an up- stream primer of the primer pair has a nucleotide sequence shown in SEQ ID No. 2, specifically: 5'-TGAGTTGTTTCAGGTTCC-3'; and a downstream primer of the primer pair has a nucleotide se- quence shown in SEQ ID No. 3, specifically: 5'- AACTTGGTGGAAGAATTTTATG-3".
Preferably, the present disclosure further provides a primer pair for PCR amplification of the above seed-specific promoter GmP34P, where an upstream primer of the primer pair has a nucleo- tide sequence shown in SEQ ID No. 2, specifically: 5'- 5 TGAGTTGTTTCAGGTTCC-3'; and a downstream primer of the primer pair has a nucleotide se- quence shown in SEQ ID No. 4, specifically: 5'- GGGCCATGGAACTTGGTGGAAGAATTTTATG-3" {the underline is an Ncol re- striction site).
In the present disclosure, reaction conditions of the PCR am- plification may preferably include: initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 95°C for 30 s, annealing at 53.7°C for 40 s, and extension at 72°C for 1 min; extension at 72°C for 8 min.
The present disclosure further provides a recombinant expres- sion vector, where the recombinant expression vector includes the above seed-specific promoter GmP34P. In the present disclosure, a basic plasmid of the recombinant expression vector may preferably include pCAMBIA1301; the recombinant expression vector may prefer- ably be constructed by: constructing the GmP34P promoter upstream of a GUS gene of a pCAMBIA1301 vector, and substituting a CaMV35S promoter.
The present disclosure further provides use of the above seed-specific promoter GmP34P, the above primer pair, or the above recombinant expression vector in seed quality improvement of a transgenic plant. In the present disclosure, the plant may prefer- ably include a leguminous plant, and the leguminous plant may preferably include soybean. The seed-specific promoter GmP34P pro- vided by the present disclosure is a novel effective seed-specific promoter that drives the expression of downstream genes to be seed-specific and is highly expressed in seeds. The seed-specific promoter GmP34P is suitable as a seed-specific promoter in re- search on molecular breeding for soybean seed quality improvement.
In order to further describe the present disclosure, the seed-specific promoter GmP34P provided by the present disclosure will be described in detail below with reference to the accompany- ing drawings and examples, but they should not be construed as limiting the protection scope of the present disclosure.
Example 1 Detection of the expression of soybean P34 gene in different soybean organs by real-time fluorescent quantitative PCR assay Total RNA was extracted from the roots, stems, leaves, flow- ers, and seeds of soybean by using RNAiso Plus (Takara); a first strand of cDNA was separately synthesized as a template for real- time fluorescent quantitative PCR according to the steps of re- verse transcription. Soybean housekeeping gene B-tubulin (GMU12286) was used as an internal reference; primers were Sl: 5'- GGAAGGCTTTCTTGCATTGGTA-3! (SEQ ID No. 5) and S2: 5H'- AGTGGCATCCTGGTACTGC-3!' (SEQ ID No. 6). Detection primers were de- signed as Tl: 5'-TGTGACCATCCACCTGCATCATG-3" (SEQ ID No. 7) and T2: 5'-CAACCGCTTCCACAGCCC -3' (SEQ ID No. 8) according to the nucleo- tide sequence (NM 001251290.2) of the soybean P34 gene. The reac- tion system included 10 pL of SYBR Primer Ex Taq (Takara), 0.2 uL of ROX Reference Dye II, 2 pL of cDNA, and 0.4 pL each of upstream and downstream primers, making up the water to a total volume of 20 pL. The reaction program of the real-time quantitative PCR sys- tem was set as: 95°C for 10 s, 59°C for 20 s, and 72°C for 30 s. Three biological replicates and three technical replicates were set at the sampling point. The result of the expression of the soybean P34 gene in different soybean organs is shown in FIG. 1: when the expression of this gene in roots is 1, its relative ex- pression in the stems, leaves, flowers, and seeds is 6.38, 47.06,
3.89, and 1331.41, respectively. Visibly, the expression of this gene in soybean seeds is significantly higher than that in roots, stems, leaves, and flowers, indicating that the soybean P34 gene is a seed-specifically expressed gene.
Example 2 Cloning and cis-acting element analysis of soybean GmP34P promoter sequence The 5' upstream sequence of the P34 gene was searched from the soybean genome sequence (http://www.Phytozome.net/soybean). Soybean genomic DNA (Tiangen kit) was extracted as a template, and upstream and downstream primers were designed as Fl: 5'-
TGAGTTGTTTCAG GTTCC -3' (SEQ ID No. 2) and F2: 5'-GGGCCATGGAACTTGGTGG AA- GAATTTTATG-3' (SEQ ID No. 4) (the underline is an Ncol restriction site), respectively. The PCR conditions were: initial denaturation at 95°C for 5 min; 30 cycles of 95°C for 30 s, 53.7°C for 40 s, and 72°C for 1 min; extension at 72°C for 8 min. A seguence with a length of 1,380 bp was obtained by PCR amplification, and named GmP34P (FIG. 2). The PCR-amplified fragment was ligated to a pMD18-T cloning vector and transformed into Escherichia coli DH5a competent cells (Tiangen Biotech (Beijing) Co., Ltd.) to obtain a recombinant plasmid pMD18-T-GmP34P; the recombinant plasmid was double digested with EcoRI and NcoI (both restriction endonucleas- es were purchased from Takara) for identification (FIG. 3). Se- quencing results were subjected to homology alignment using DNAMAN software, and the sequence (SEQ ID No. 1) was correct.
Bioinformatics prediction and analysis indicated that the GmP34P promoter contained a plurality of cis-acting elements com- monly present in seed-specific promoters, including: one RY-repeat element (CATGCA) playing an important role in high-level expres- sion of seed-specifically expressed genes; 10 embryo-specifically expressed cis-acting elements SEF4 (RTTTTTR, where R is A or G); 9 E-box elements (CANNTG, where N is A, T, C, or G) commonly present in promoters that participate in triacylglycerol synthesis and plant seed-specific expression of genes; 6 seed-specifically ex- pressed cis-acting elements Skn-1 (RTCAT, where R is A or G); 1 napin element related to seed-specific regulation (TACACAT); 3 seed-specifically expressed cis-acting elements AATAAA; 5 seed- specifically expressed cis-acting elements ACGT; and 1 seed- specifically expressed cis-acting element AACACA. Soybean P34 gene was specifically highly expressed in the soybean seed and closely related to the above cis-acting elements present in the GmP34P promoter.
Example 3 Construction of a plant expression vector with GmP34P-driven GUS gene and tobacco transformation
A pMD18-T-GmP34P vector and a pPCAMBIA1301 plant expression vector were double digested with restriction endonucleases EcoRI and NcoI, respectively; digested fragments were extracted from the gel and ligated by using DNA Ligation Kit Ver.2.0 (Takara), in or- der to substitute the GmP34P promoter with the CaMV35S promoter to construct upstream of the GUS gene in the pCAMBIA1301 vector; the ligated fragment was transformed into E. coli DHSx and a plasmid was extracted; the plasmid was double digested (FIG. 4) to obtain an expression vector pCAM-GmP34P. The pCAM-GmP34P and pCAM- BIA1301vector plasmids were transformed into Agrobacterium tumefa- ciens EHA105 (Biovector) by the freeze-thaw method, respectively.
Screening and molecular identification of transgenic tobacco Through the leaf disc method, leaves of aseptic tobacco (NC89) seedlings were infected with the successfully constructed A. tumefaciens strains transformed with plant expression vector PCAM-GmP34P and empty vector PCAMBIA-1301, respectively; the leaves were placed on the Murashige and Skoog (MS) medium, cul- tured in the dark for three days, sterilized, and transferred into the differential medium (supplemented with 8 mg/L hygromycin); af- ter 2-3 subcultures, green resistant buds grew around the leaves; after the resistant buds grew to around 1-3 cm, they were cut off and transferred to the rooting medium (supplemented with 5 mg/L hygromycin), and they were transplanted into the soil after their roots were developed.
Positive transgenic tobacco plants were identified by the PCR assay and the RT-PCR assay. In the PCR assay, the genomic DNA of a transgenic tobacco plant was used as a template, the detection primers of the pCAM-GmP34P-transfected tobacco plant were Fl: 5'- TGAGTTGTTTCAGGTTCC-3" (SEQ ID No. 2) and F3: 57'- AACTTGGTGGAAGAATTTTATG -3' (SEQ ID No. 3), the wild-type tobacco was used as a negative control, and the plasmid pCAM-GmP34P was used as a positive control. Correctly identified tobacco plants were subjected to the RT-PCR assay; total RNA was extracted from the transgenic tobacco plants and reverse transcribed to cDNA as a template; upstream and downstream primers were designed according to the sequence of the GUS gene, specifically NF: 5'- GTAGAAACCCCAACCCGTGAA-3’ (SEQ ID No. 9) and NR: 5'-
TGAGCGTCGCAGRACATTACAT-3' (SEQ ID No. 10), respectively; the wild- type tobacco was used as a negative control, and the plasmid pCAM- GmP34P was used as a positive control. Positive transgenic tobacco seeds were harvested, namely T; seeds; the T,; seeds were sown in the soil to obtain T; tobacco seedlings, and they were subjected to subsequent PCR and RT-PCR assays. FIG. 5 illustrates the identifi- cation result of PCR for T, transgenic tobacco plants with pCAM- GmP34P, and FIG. 6 illustrates the identification result of RT-PCR for T, transgenic tobacco plants with pCAM-GmP34P correctly identi- fied by PCR. The identified T: positive transgenic tobacco plants will be subjected to subseguent experiments.
Example 4 Detection of the expression of soybean GUS gene in different organs of transgenic tobacco by real-time fluorescent quantitative PCR assay Total RNA was extracted from the roots, stems, leaves, flow- ers, and seeds of the positive pCAM-GmP34P-transfected tobacco plant and the seeds of the positive pCAMBIA1301-transfected tobac- co plant; the first strand of the cDNA was synthesized according to the steps of reverse transcription, respectively.
The reference gene was tobacco housekeeping gene a-tubulin; reference primers were KF: 5'-ATGAGAGAGTGCATATCGAT-3' (SEQ ID No. 11) and KR: 5'-TTCACTGAAGAAGGTGTTGAA-3' (SEQ ID No. 12). The de- tection primers of the GUS gene were SF: 5'-GATCGCGAAAACTGTGGAAT- 3' (SEQ ID No. 13) and SR: 5'-TAATGAGTGACCGCATCGAA-3' (SEQ ID No. 14). The real-time fluorescent quantitative PCR was the same as the detection of the expression of the P34 gene in different soy- bean organs. Results (FIG. 7) showed that: in the pCAM-GmP34P- transfected tobacco plant, when the expression of the GUS gene in roots was 1, the relative expression of this gene in stems, leaves, flowers, and seeds was 5.35, 11.92, 2.04, and 122.36, re- spectively, indicating that GmP34P promoter-driven expression of downstream GUS gene was seed-specific; in addition, when the ex- pression of the GUS gene in the roots of the pCAM-GmP34P- transfected tobacco plant was 1, the relative expression of the GUS gene in the seeds of the pCAMBIA1301-transfected tobacco plant was 95.61. Visibly, the GmP34P promoter drove the expression of downstream genes in seeds more actively than the CaMV35S promoter. Example 5 GUS activity was analyzed by using a fluorescence spectropho- tometer. The sample was ground into a powder with liquid nitrogen, mixed with an extraction buffer (100 mL, supplemented with 50 mL of 50 mmol/L pH7.0 sodium phosphate buffer solution, 100 pL of f£- mercaptoethanol, 2 mL of 0.5 mmol/L EDTA, 1 mL of 10% SDS, and 100 pL of TritenX 100), and centrifuged at 12,000 rpm for 10 min at 4°C; a supernatant was collected to obtain a crude GUS soluble protein solution. A part of the crude GUS soluble protein solution was mixed with 1 mmol/L GUS reaction substrate 4-MUG, and held at 37°C for 20 min; 0.2 mmol/L Na,CO; stop solution was added to stop the reaction. The content of soluble protein was determined by the Bradford assay. The amount of enzymes required by hydrolysis of 4- MUG into 1 nmol/L 4-MU within 1 min was considered as an activity unit, the enzyme activity per milligram of protein represented the GUS activity, and the above experiment was repeated thrice. Re- sults (FIG. 8) showed that: the GUS activity in the negative con- trol wild-type (WT) tobacco seeds was 0.67 nmol 4-MU mint ‘mg: pro- tein, with very low activity, and that in the pCAMBIA1301 {CaMV353: GUS)-transfected tobacco seeds was 82.7 nmol 4-MU min” ''mg™* protein, indicating that the GUS reporter gene in the vector was activated and expressed by the CaMV35S constitutive promoter. The GUS activity in the pCAM-GmP34P (GmP34P: GUS) -transfected to- bacco seeds was 102.5 nmol 4-MU min” mg” protein, indicating that the GmP34P promoter drove the expression of downstream GUS gene in plant seeds more actively; its GUS activity was relatively higher than that in the pCAMBIA1301 (CaMV35S: GUS) -transfected tobacco seeds, indicating that the GmP34P promoter drove the expression of downstream genes in seeds more actively than the CaMV35S promoter.
In conclusion, the GmP34P promoter provided by the present disclosure is an effective seed-specific promoter suitable as a candidate promoter in molecular breeding for soybean seed quality improvement.
Although the present disclosure has been disclosed as above in preferred examples, it is not intended to limit the present disclosure.
Those skilled in the art can make various variations and modifications without departing from the spirit and scope of the present disclosure.
Therefore, the protection scope of the present disclosure should be subject to that defined by the claims.
NL2029636 sequence listing
SEQUENCE LISTING <110> Qiqihar University <120> SOYBEAN SEED-SPECIFIC PROMOTER GmP34P AND USE THEREOF <130> HKJP202199542 <160> 14 <170> PatentIn version 3.5 <210> 1 <211> 1380 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of the seed-specific promoter <400> 1 tgagttgttt caggttccat tgccttattg ctaaaactcc aactaaaata acaaatagca 60 catgcaggtg caaacaacac gttactctga tgaaggtgat gtgcctctag cagtctagct 120 tatgaggctc gctgcttatc aacgattcat cattccccaa gacgtgtacg cagattaaac 180 aatggacaaa acttcaatcg attatagaat aataatttta acagtgccga cttttttctg 240 taaacaaaag gccagaatca tatcgcacat catcttgaat gcagtgtcga gtttggacca 300 tttgagtaca aagccaatat tgaatgattt ttcgatttta catgtgtgaa tcagacaaaa 360 gtgcatgcaa tcacttgcaa gtaaattaag gatactaatc tattcctttc attttatatg 420 ctccactttt atataaaaaa atatacatta ttatatatgc attattaatt attgcagtat 480 tatgctattg gttttatggc cctgctaaat aacctaaatg agtctaacta ttgcatatga 540 atcaaatgaa ggaagaatca tgatctaaac ctgagtaccc aatgcaataa aatgcgtcct 600 attacctaaa cttcaaacac acattgccat cggacgtata aattaatgca tatagattat 660 tttgagaaaa gaaaacatca aaagctctaa aacttctttt aactttgaaa taagctgata 720 aaaatacgct ttaaatcaac tgtgtgctgt atataagctg caatttcaca ttttaccaaa 780 ccgaaacaag aatggtaaca gtgaggcaaa aatttgaaaa atgtcctact tcacattcac 840 atcaaattaa ttacaactaa ataaataaac atcgtgattc aagcagtaat gaaagtcgaa 900 atcagataga atatacacgt ttaacatcaa ttgaattttt ttttaaatgg atatatacaa 960 Pagina 1
NL2029636 sequence listing gtttactatt ttatatataa tgaaaattca ttttgtgtta gcacaaaact tacagaaaga 1020 gataaatttt aaataaagag aattatatcc aattttataa tccaaaataa tcaaattaaa 1080 gaatattggc tagatagacc ggctttttca ctgcccctgc tggataatga aaattcatat 1140 caaaacaata cagaagttct agtttaataa taaaaaagtt ggcaaactgt cattccctgt 1200 tggtttttaa gccaaatcac aattcaatta cgtatcagaa attaatttaa accaaatata 1260 tagctacgag ggaacttctt cagtcattac tagctagctc actaatcact atatatacga 1320 catgctacaa gtgaagtgac catatcttaa tttcaaatca taaaattctt ccaccaagtt 1380 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Upstream primer for amplifying GmP34P <400> 2 tgagttgttt caggttcc 18 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Downstream primer for amplifying GmP34P <400> 3 aacttggtgg aagaatttta tg 22 <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Downstream primer for amplifying GmP34P <400> 4 gggccatgga acttggtgga agaattttat g 31 Pagina 2
NL2029636 sequence listing <210>5 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer S1 <400> 5 ggaaggcttt cttgcattgg ta 22 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer S2 <400> 6 agtggcatcc tggtactgc 19 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer T1 <400> 7 tgtgaccatc cacctgcatc atg 23 <2105 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer T2 <400> 8 caaccgcttc cacagccc 18 <216> 9 <211> 21 <212> DNA <213> Artificial Sequence
Pagina 3
NL2029636 sequence listing <220> <223> Primer NF <400> 9 gtagaaaccc caacccgtga a 21 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer NR <400> 10 tgagcgtcgc agaacattac at 22 <21e> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer KF <400> 11 atgagagagt gcatatcgat 20 <21e> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer KR <400> 12 ttcactgaag aaggtgttga a 21 <2105 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer SF
Pagina 4
NL2029636 sequence listing <400> 13 gatcgcgaaa actgtggaat 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer SR <400> 14 taatgagtga ccgcatcgaa 20 Pagina 5
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