CN111171121B - Transcription factor and application thereof in activating expression of banana MaSBE2.3 - Google Patents
Transcription factor and application thereof in activating expression of banana MaSBE2.3 Download PDFInfo
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Abstract
The invention provides a transcription factor MaARF2, which is a protein encoded by a gene with a nucleotide sequence shown as SEQ ID NO. 1. The invention also provides a coding gene of the transcription factor MaARF2 and application thereof. The transcription factor MaARF2 can be combined with a promoter region of banana fruit starch branching enzyme gene MaSBE2.3, can interact with the MaSBE2.3 gene and regulate and control the up-regulated expression of the gene, for example, the transcription factor MaARF2 gene and the promoter of the banana fruit starch branching enzyme gene MaSBE2.3 are introduced into banana fruit slices, so that the up-regulated expression of the banana fruit starch branching enzyme gene MaSBE2.3 can be obviously activated, and the transcription factor MaARF2 has important significance for regulating and controlling the amylopectin content or improving the crop yield, improving the starch quality and cultivating a new variety of high-quality bananas, and provides candidate transcription factor resources for improving the quality of bananas or other fruit amylopectin.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a transcription factor and application thereof in activating expression of banana MaSBE2.3.
Background
Amylopectin content directly affects plant yield, quality and processability, while Starch Branching Enzyme (SBE) gene is a key enzyme gene affecting amylopectin synthesis. It cleaves the alpha-1, 4-glycosidic bond of the alpha-1, 4-D-glucan linear chain donor (linear region of amylose or amylopectin) and simultaneously catalyzes the formation of an alpha-1, 6-glycosidic bond between the cleaved short chain and the acceptor chain (original or other chain), thereby creating a branch.
In maize and rice, the transcription factors ZmNAC36 and OsbZIP8 are reported to be capable of binding to SBE gene promoter and regulating its up-regulated expression (Zhang et al, 2014; Wang et al, 2018). Auxin Response Factors (ARFs) as a class of transcription factors for regulating plant growth and signal transduction have important functions in the aspects of lateral root generation, apical dominance, tropism, cell elongation, cell division, cell differentiation and the like (Hagen et al, 2002). Meanwhile, ARF transcription factors can activate or inhibit the expression of target genes Aux/IAA, SAUR and GH3 which are rapidly induced by auxin (Liscum et al, 2002; Hagen et al, 2002). However, whether or not the ARF transcription factor can interact with the SBE gene and regulate the expression thereof has not been reported yet. Therefore, the research of the banana transcription factor MaARF2 and MaSBE2.3 genes is of great significance for regulating and controlling amylopectin synthesis or improving crop yield, improving starch quality and cultivating high-quality new banana varieties.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a transcription factor MaARF2 capable of activating the expression of banana fruit starch branching enzyme gene SBE2.3, and a coding gene and application thereof.
The first aspect of the invention provides a transcription factor MaARF2, which is a protein coded by a gene with a nucleotide sequence shown as SEQ ID NO. 1.
The second aspect of the invention provides a transcription factor MaARF2 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.
In a third aspect of the present invention, there is provided a recombinant vector comprising the original vector and the transcription factor MaARF2 gene according to the second aspect of the present invention.
As the original vector, there can be used a vector commonly used in the field of gene recombination, such as a virus, a plasmid, etc. The invention is not limited in this regard. In one embodiment of the present invention, the original vector is pGreenII 62SK vector plasmid, but it is understood that other plasmids, viruses, etc. can be used.
Preferably, the original vector is pGreenII 62SK vector plasmid, and the nucleotide sequence shown in SEQ ID NO 1 is positioned between Sac I and EcoR I restriction enzyme sites of the pGreenII 62SK vector plasmid.
In a fourth aspect, the invention provides the use of the transcription factor MaARF2 according to the first aspect, or the transcription factor MaARF2 gene according to the second aspect, or the recombinant vector according to the third aspect, for activating the expression of the banana fruit starch branching enzyme gene masbe 2.3.
The fifth aspect of the invention provides a promoter of banana fruit starch branching enzyme gene MaSBE2.3, the nucleotide sequence of which is shown as SEQ ID NO. 2.
The sixth aspect of the present invention provides another recombinant vector comprising the original vector and the promoter of banana fruit starch branching enzyme gene MaSBE2.3 according to the fifth aspect of the present invention.
As the original vector, there can be used a vector commonly used in the field of gene recombination, such as a virus, a plasmid, etc. The invention is not limited in this regard. In one embodiment of the present invention, the original vector is pGreenII 0800 vector plasmid, but it is understood that other plasmids, viruses, etc. may be used.
Preferably, the original vector is pGreenII 0800 vector plasmid, and the nucleotide sequence shown in SEQ ID NO:2 is positioned between two restriction enzyme sites of Xho I and Sma I of pGreenII 0800 vector plasmid.
The seventh aspect of the invention provides the use of the banana fruit starch branching enzyme gene MaSBE2.3 promoter according to the fifth aspect of the invention, or the recombinant vector according to the sixth aspect of the invention, for activating the expression of the banana fruit starch branching enzyme gene MaSBE2.3.
Wherein the banana fruit starch branching enzyme gene MaSBE2.3 promoter of the fifth aspect interacts with the transcription factor MaARF2 of the first aspect of the invention, thereby activating the expression of the banana fruit starch branching enzyme gene MaSBE2.3.
The eighth aspect of the invention provides a primer pair for amplifying the transcription factor MaARF2 gene, the nucleotide sequence of which is shown as SEQ ID NO. 3 and SEQ ID NO. 4.
The ninth aspect of the invention provides a primer pair for amplifying a promoter of a banana fruit starch branching enzyme gene MaSBE2.3, and the nucleotide sequences of the primer pair are shown as SEQ ID NO. 5 and SEQ ID NO. 6.
The transcription factor MaARF2 can be combined with a promoter region of banana fruit starch branching enzyme gene MaSBE2.3, can interact with the MaSBE2.3 gene and regulate and control the up-regulated expression of the gene, for example, the transcription factor MaARF2 gene and the promoter of the banana fruit starch branching enzyme gene MaSBE2.3 are introduced into banana fruits, so that the up-regulated expression of the banana fruit starch branching enzyme gene MaSBE2.3 can be obviously activated, and the transcription factor MaARF2 has important significance for regulating and controlling the amylopectin content or improving the crop yield, improving the starch quality and cultivating a new variety of high-quality bananas, and provides a candidate transcription factor resource for improving the quality of bananas or other fruit amylopectin.
Drawings
FIG. 1 shows the results of verification of self-activation and yeast single-hybrid of the MaSBE2.3 gene promoter and verification of yeast single-hybrid interaction between MaARF2 and the MaSBE2.3 gene promoter, A: self-activation of a MaSBE2.3 gene promoter and yeast single hybridization verification; b: MaARF2 was verified by single cross interaction with the MaSBE2.3 gene promoter yeast. p53 promoter: a positive control promoter; MaSBE2.3 promoter: the MaSBE2.3 gene promoter; AD-Rec-p53+ p53 promoter: a positive control combination; AD + Empty-MaSBE2.3 promoter: a negative control combination; MaARF2+ masbe2.3 promoter: the interaction between the transcription factor MaARF2 and the promoter of the MaSBE2.3 gene is verified.
FIG. 2 is a diagram of the result of the two-restriction enzyme-digestion-verified electrophoresis of pGreenII 0800-MaSBE2.3 promoter, M: DL2000 DNA Marker; lane 1: is the result of double enzyme digestion of pGreenII 0800-MaSBE2.3 promoter recombinant plasmid.
FIG. 3 is a diagram of the result of double-restriction enzyme-catalyzed check electrophoresis of pGreenII 62SK-ARF2, M: DL2000 DNA Marker; lane 1: the result is the double digestion result of pGreenII 62SK-ARF2 recombinant plasmid.
FIG. 4 is a construction pattern diagram of pGreen-MaSBE2.3 promoter and pGreenII 62SK-MaARF2 vectors and LUC activity assay, A: pGreenII 0800-MaSBE2.3 promoter and pGreenII 62SK-MaARF2 vector construction pattern diagram; b: detecting LUC activity; SK: pGreenII 62SK vector; SK + MaARF 2: pGreenII 62SK-MaARF2 vector.
Detailed Description
In order that the objects and advantages of the invention will be more clearly understood, the invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
First, obtaining a Gene
1. Acquisition of Banana transcription factor MaARF2 Gene
Taking Brazil banana fruit cDNA as a template, and
5’-ATGGGGATCGATTTGAACACGATAG-3’
5’-CTAAATTGGAGCTGCACGCAAAGCA-3’
the nucleotide sequence of the primer obtained by PCR amplification is 2,118bp, and the sequence is shown in SEQ ID No. 1.
2. Obtaining of banana fruit starch branching enzyme gene MaSBE2.3 promoter
Using Brazil banana fruit DNA as template, using
5’-AGGTTCTATATACTTAATTACTC-3’
5’-TCGTATGGAGCTGAGGCGGGTGATC-3’
Used as a primer, a primer obtained by PCR amplification contains a base sequence of 1,824bp, and the sequence is shown as SEQ ID No.2
Two, one yeast hybrid
(1) Construction and screening of Yeast Single hybrid libraries
Total RNA in 0d, 20d and 80d Brazil banana flesh after bud extraction was extracted using RNA extraction kit (TIANGEN Biotech), and then cDNA library was constructed by Mathchmaker TM yeast single-hybrid kit (Clontech, Mountain View, Calif.) and loaded into the game vector pGADT7-Rec (Clontech). The MaSBE2.3 promoter plasmid is constructed in a bait vector pAbAi (Clontech) by carrying out double digestion, recovery and ligation by Sac I and Xho I. The pGADT7-Rec prey vector carrying the cDNA library and the pAbAi decoy vector carrying the MaSBE2.3 promoter sequence were co-transformed into the Y1 HGold yeast strain (Clontech) at SD/-Leu + AbA200The selection medium was incubated at 30 ℃ for 3 d. Candidate transcription factor MaARF2 was obtained by PCR detection, sequencing analysis and alignment annotation of banana a genome database.
(2) Self-activation experiment of MaSBE2.3 promoter and interaction verification of the experiment and transcription factor MaARF2
To further verify that the MaSBE2.3 promoter interacts with MaARF2, the self-activating activity of the MaSBE2.3 promoter was in pAbAi SD/-Ura + AbA200Verified on the medium, see FIG. 1A, in selection medium SD/-Ura + AbA200Above, the pAbAi-MaSBE2.3 promoter strain was unable to grow, indicating that the MaSBE2.3 promoter has no self-activating activity.
By constructing the transcription factor MaARF2 gene in the prey vector pGADT7 AD, the MaSBE2.3 promoter in the decoy vector pAbAi, and then co-transforming the yeast strain Y1 HGold (Clontech) at SD/-Leu + AbA200The growth was normal in the selection medium, SD/-Leu + AbA, see FIG. 1B, 3d at 30 ℃200In the above, pGADT7 AD-MaARF2+ pAbAi-MaSBE2.3 promoter strain was able to grow normally, indicating that MaARF2 was able to interact with the MaSBE2.3 promoter.
Construction of recombinant vector
1. pGreenII 0800-MaSBE2.3 promoter vector
And (2) carrying out double enzyme digestion on the target fragment and pGreenII 0800 vector plasmid by using the nucleotide sequence of the banana fruit starch branching enzyme gene MaSBE2.3 promoter by using two restriction endonucleases of Xho I and Sma I, and recovering, connecting, transforming and sequencing the enzyme-digested target fragment and pGreenII 0800 vector fragment to verify the correctness, thereby obtaining the banana fruit starch branching enzyme gene MaSBE2.3 promoter vector pGreenII 0800-MaSBE2.3 promoter (figure 2).
2. pGreenII 62SK-MaARF2 vector
And (2) carrying out double enzyme digestion on the target fragment and pGreenII 62SK vector plasmid by using the nucleotide sequence of the banana transcription factor MaARF2 gene by using Sac I and EcoR I restriction enzymes respectively, and recovering, connecting, transforming and sequencing the enzyme digested target fragment and pGreenII 62SK vector fragment to verify the correctness, so as to obtain the pGreenII 62SK-MaARF2 vector of the banana transcription factor MaARF2 gene (figure 3).
Fourth, recombinant vector is transformed into banana fruit slice and dual-luciferase activity detection method
(1) Agrobacterium transformation of pGreenII 0800-MaSBE2.3 promoter vector
Adding 2 mug of pGreenII 0800-MaSBE2.3 promoter recombinant plasmid into 200 mug of agrobacterium tumefaciens GV3101 competent cells melted on ice bath, gently mixing, and placing in ice bath for 30 min; freezing in liquid nitrogen for 3min, and rapidly incubating in 37 deg.C water bath for 5 min; adding 800 mu L YEP liquid culture medium, and pre-culturing at 28 ℃ and 250rpm for 4-5 h; sucking 300 mu L of bacterial liquid to YEP solid selection culture medium containing 25mg/L Rif and 50mg/L Kan, and uniformly coating the bacterial liquid on the whole plate; and (3) placing the plate at 28 ℃ until the liquid is absorbed, inverting the plate, culturing for 23d at 28 ℃, selecting a single colony, verifying and detecting, and using the correctly transformed agrobacterium liquid for the next experiment.
(2) Agrobacterium transformation of pGreenII 62SK-MaARF2 vector
Adding 2 mu g of pGreenII 62SK-MaARF2 recombinant plasmid into 200 mu L of Agrobacterium tumefaciens GV3101 competent cells melted on ice bath, gently mixing, and placing in ice bath for 30 min; freezing in liquid nitrogen for 3min, and rapidly incubating in 37 deg.C water bath for 5 min; adding 800 mu L YEP liquid culture medium, pre-culturing at 28 ℃ and 250rpm for 4-5 h; sucking 300 mu L of bacterial liquid to YEP solid selection culture medium containing 25mg/L Rif and 50mg/L Kan, and uniformly coating the bacterial liquid on the whole plate; and (3) placing the flat plate at 28 ℃ until the liquid is absorbed, inverting the flat plate, culturing for 2-3 d at 28 ℃, selecting a single colony, verifying and detecting, and using the correctly transformed agrobacterium liquid for the next experiment.
(3) Agrobacterium tumefaciens mediated banana fruit slice genetic transformation
Soaking banana fruit slices (thickness 1mm) in 5mL of 75% ethanol sterile centrifuge tube on a superclean bench for 1min, fully shaking during soaking, and washing with sterile water for 3 times; soaking in 20% sodium hypochlorite solution for 15min, shaking, and washing with sterile water for 3 times. Inoculating the agrobacterium tumefaciens GV3101 bacterial liquid transformed with pGreenII 0800-MaSBE2.3 promoter recombinant vector and the agrobacterium tumefaciens GV3101 bacterial liquid transformed with pGreenII 62SK-MaARF2 recombinant vector to 10mL of YEP liquid culture medium containing 50mg/L Kan according to the volume ratio of 1:7 for overnight activation culture; sucking 1mL of activated culture liquid into a new 50mL of YEP liquid culture medium containing 50mg/L Kan, and culturing until OD600 is 0.6; transferring the bacterial liquid with the required concentration to a 50mL sterile centrifuge tube on an ultra-clean workbench, centrifuging at 4 ℃ and 6000rpm for 5min, removing supernatant, and adding an MS liquid culture medium with the same volume (the volume of the bacterial liquid before centrifugation) to resuspend the thalli; transferring the bacterial liquid into a 100mL sterile triangular flask, adding 0.1% by volume (the volume of the resuspended bacterial liquid) of Acetosyringone (AS), fully mixing uniformly, transferring the banana fruit slices into the agrobacterium liquid, soaking for 15min, and shaking the bacterial liquid to make the fruit slices fully contact with the bacterial liquid; taking out the slices, placing the slices on sterile filter paper, sucking off redundant bacterial liquid on the surfaces of the slices, transferring the slices to an MS culture medium containing acetosyringone, and culturing for 3d at 25 ℃; the co-cultured banana fruit slices were used for dual luciferase activity assay.
(4) Dual-luciferase activity detection method
Renilla luciferase-firefly luciferase (REN-LUC) activity in co-cultured 3d banana fruit slices was detected using the Dual-LUC Reporter assay system and the experiment was repeated three times. Firstly, the banana fruit slices are ground on ice to be pulp, the cell lysate is added in equal volume, and the mixture is fully and uniformly mixed. Incubate on ice for 5min and lyse the cells thoroughly. Centrifuging at 13000rpm for 1min, and collecting the supernatant. Add 20. mu.L of cell lysate to black microplate, set 3 wells repeatedly. LUC reaction solution and REN reaction solution are prepared, namely LUC substrate (50X) and REN substrate (50X) are respectively diluted to 1 time of working solution by corresponding buffer solution. And incubated to room temperature. Adding 100 μ L LUC reaction solution, shaking plate, mixing, and detecting LUC activity within 30 min. Adding 100 μ L REN reaction solution, shaking the plate, mixing, and detecting REN activity within 30 min. The LUC/REN ratio was calculated to obtain the relative LUC activity. As a result, as shown in FIG. 4B, in the presence of both MaARF2 and MaSBE2.3 promoter, the LUC activity was increased by about 3.5-fold compared to the control (in the presence of only MaSBE2.3 promoter), indicating that MaARF2 activates the upregulation of the MaSBE2.3 gene.
The invention discovers that the transcription factor MaARF2 can be combined with a promoter region of a banana fruit starch branching enzyme gene MaSBE2.3 based on a yeast single hybridization experiment. By constructing pGreenII 0800-MaSBE2.3 promoter and pGreenII 62SK-MaARF2 vectors, co-expressing ARF2-MaSBE2.3 promoter in banana fruit slices, and detecting dual-luciferase activity, the MaARF2 can extremely obviously activate the up-regulation expression of the MaSBE2.3 gene (p < 0.01). These results show that MaARF2 is able to interact with the masbe2.3 gene and regulate its up-regulated expression, providing a candidate transcription factor resource for improving amylopectin quality in banana or other plants.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Sequence listing
<110> research institute of tropical biotechnology of Chinese tropical academy of agricultural sciences
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catatgaggt gtttaactag ctgtaaccat tgagctcgag gaataaactt cagtgataaa 240
atttaagggt tcagaaattc atatattaaa tgcagtaaat acaacttatt tgattccagt 300
tggtgctgat gttgatgaaa aaaagattga gcttctcttg tcccaagtcc atggtaatga 360
cttgacggaa cttattgcta ctgggagaga caagattgcc tcggtgcctt gtggtggtgg 420
cggtgctgtt gcagttgctg caattgctgg aggtggtggt ggtgcttctg cagcccctgc 480
agtttctgaa ccaaagaaag aggaaaaggt agaagagaag gaagaatcag atgatgtaag 540
ttacttttta tgatttaact gcagagtttc taatgttcag tccttttggc tggaaacacc 600
ttggctatag atgcagtaat cgacttatct ctggatgata cctttgaaaa aaaagacaaa 660
cctttcctgt ccagccttat tatttgattg actgattgtg ctctaatcgc atttgtgcag 720
gacatgggct ttagcttgtt cgactgattg gtctactgta gtccagcaga agctgctgtt 780
tttggtgcag taattaggga aaaatcaatc tgcattgaag cttcagcttt gatgtttgtt 840
gtttattttc cctttttttt tccatcatga tctctaattt tgttcctcaa catattgctc 900
ggttatattt ctgcttcact caagtcgaca aacttgttag gagtttgatt gagatttaac 960
ttcattgaag acatttgtgt tttgtatgcc gggtaggcgg tccaaggtaa ccacatttct 1020
taacggagat ttcattcatg tagttgtgcc tcgcttctct agtttctgtt gatacgcaaa 1080
gccatgtcga tagatatcag tggtcctatc tttgtttacg cagtttacgt tttagcatgc 1140
ctggtgccta gtgataatga ttgttgcaga tggagacgaa tacatcaacg tcggacgatc 1200
atatggtgac cacttgtcgg atgcagatca atataccacc ttacctcact gattggcaac 1260
taggatggag ggttcagtca cacatcgcaa cttcgatgga gggtcgtcaa ccatccacca 1320
aagtaagaat ctcaaagcac gaacggacgg ccttcaacga tttcggttca cacagattga 1380
ggtgcgattg ggcccatggg cgatcgatga acggtcgaga tgagacgaat cgaccagctt 1440
ttttgttcat gagctacgtg cggtattcga acggaccaaa aggccccacc aaaaaaaaaa 1500
gtgtttttac tcttcaattt ttatttattt aatatttgat ataatgagag attttgcata 1560
agaaaatata tagaatgtga cattaacgta ttttcgaaaa taaataaata aaatgagaaa 1620
aaagtgaacg gcctttacag tgtggggcag ctgaatctgg agaaagcaaa gcaaaaccgt 1680
tcattcctca ccccctgcct tcgccagttt cgctgacgcg gcctcgttcc ccacctgtat 1740
taaacgacac gtccgctggg gcccagttcc catctccatg cacctctccg ccccacgcgg 1800
atcacccgcc tcagctccat acga 1824
<210> 3
<211> 25
<212> DNA
<213> Artificial
<400> 3
atggggatcg atttgaacac gatag 25
<210> 4
<211> 25
<212> DNA
<213> Artificial
<400> 4
ctaaattgga gctgcacgca aagca 25
<210> 5
<211> 23
<212> DNA
<213> Artificial
<400> 5
aggttctata tacttaatta ctc 23
<210> 6
<211> 25
<212> DNA
<213> Artificial
<400> 6
tcgtatggag ctgaggcggg tgatc 25
Claims (6)
1. The application of the transcription factor MaARF2, or the transcription factor MaARF2 gene, or the recombinant vector in activating the expression of the banana fruit starch branching enzyme gene MaSBE2.3 is disclosed, wherein the transcription factor MaARF2 is protein coded by a gene with a nucleotide sequence shown as SEQ ID NO.1, the transcription factor MaARF2 gene with a nucleotide sequence shown as SEQ ID NO.1, and the recombinant vector comprises an original vector and the transcription factor MaARF2 gene.
2. A promoter of banana fruit starch branching enzyme gene MaSBE2.3 is characterized in that the nucleotide sequence is shown as SEQ ID NO. 2.
3. A recombinant vector comprising the original vector and the promoter of banana fruit starch branching enzyme gene masbe2.3 according to claim 2.
4. Use of the banana fruit starch branching enzyme gene MaSBE2.3 promoter according to claim 2 or the recombinant vector according to claim 3 for activating expression of banana fruit starch branching enzyme gene MaSBE2.3.
5. The use according to claim 4, characterized in that the banana fruit starch branching enzyme gene MaSBE2.3 promoter of claim 2 interacts with the transcription factor MaARF2, the transcription factor MaARF2 being a protein encoded by the gene having the nucleotide sequence shown in SEQ ID NO. 1.
6. A primer pair for amplifying a promoter of a banana fruit starch branching enzyme gene MaSBE2.3 is characterized in that the nucleotide sequence is shown as SEQ ID NO. 5 and SEQ ID NO. 6.
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CN105837670A (en) * | 2016-05-06 | 2016-08-10 | 上海交通大学 | African agapanthus auxin response factor ApARF2 and encoding gene and probe thereof |
CN110106187A (en) * | 2019-05-28 | 2019-08-09 | 中国热带农业科学院橡胶研究所 | The ARF gene and its coding albumen of a kind of rubber tree and application |
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GB9623095D0 (en) * | 1996-11-05 | 1997-01-08 | Nat Starch Chem Invest | Improvements in or relating to starch content of plants |
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CN105837670A (en) * | 2016-05-06 | 2016-08-10 | 上海交通大学 | African agapanthus auxin response factor ApARF2 and encoding gene and probe thereof |
CN110106187A (en) * | 2019-05-28 | 2019-08-09 | 中国热带农业科学院橡胶研究所 | The ARF gene and its coding albumen of a kind of rubber tree and application |
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M0TKL9_MUSAM;None;《EMBL-EBI》;20130403;TR:M0TKL9 * |
PREDICTED: Musa acuminata subsp. malaccensis auxin response factor 3-like (LOC103992811), mRNA;Automated computational analysis;《GenBank Database》;20161025;Accession NO:XM_009412664.2 * |
The auxin response factor gene family in banana: genome-wide identification and expression analyses during development, ripening, and abiotic stress;Hu 等;《frontiers in Plant Science》;20150915;第6卷;第1-16页 * |
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