NL2027897A - Use of gmaap protein and gmaap gene in breeding soybeans - Google Patents
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Abstract
The present disclosure belongs to the field of plant genetic engineering, and particularly relates to use of a GmAAP protein and a GmAAP gene in breeding soybeans. The present 5 disclosure provides use of a GmAAP protein in breeding soybeans and the GmAAP protein has an amino acid sequence shown in SEQ ID NO: 1. The present disclosure significantly increases the number of branches of soybeans and enhances the soybean yield by using a genetic engineering technology to reduce expression of the GmAAP gene. It can be specifically seen from examples that the present disclosure can effectively lO increase the number of branches of the soybeans, increase the number of pods per plant, and thus increase the soybean yield.
Description
TECHNICAL FIELD The present disclosure belongs to the field of plant genetic engineering, and particularly relates to use of a GmAAP protein and a GmAAP gene in breeding soybeans.
BACKGROUND Nitrogen is one of macroelements essential for growth of plants and a lack of the nitrogen directly affects yield and quality of crops. In order to ensure the yield and quality of crops, a large amount of nitrogen fertilizers are applied to the soil every year. However, due to extremely low absorption and utilization of the nitrogen fertilizers, excessive application of the nitrogen fertilizers makes a limited contribution to the yield of crops and also leads to waste of resources, environmental pollution and the like. Therefore, improving the ability of plants to use the nitrogen is of great significance to sustainable development of agricultural production. The genetic engineering technology is an effective way to solve the above problems by improving the abilities of crops to absorb, utilize and transport the nitrogen in the soil and cultivating new varieties of nitrogen- efficient crops.
Roots of plants can absorb nitrate ions, ammonium ions or amino acids from the soil to obtain the nitrogen. Absorption and transport of the nitrogen mainly rely on ammonium transporter (AMT), nitrate transporter (NRT), amino acid transporter (AAT), peptide transporter (PTR) and other transporters. Absorption and transmembrane transport of amino acids produced by degrading macromolecules in source organs are mainly completed by the AAT. In higher plants, the AAT is a type of transmembrane protein that transports amino acids from outsides of cells to insides of the cells. At the same time, the AAT also plays important roles in amino acid input, pathogenic response, abiotic stress, and the like during processes of long-distance transport of the amino acids to a growing "library", phloem loading of the amino acids in the "sources" organs in remobilization of nitrogen and seed development.
An AAT gene has two superfamilies: an amino acid, polyamine and choline transport (APC) superfamily and an amino acid/auxin permease (AAAP) superfamily. The AAAP superfamily has six sub-families: an amino acid permease (AAP) family, a lysine and histidine transporter (LHTs) family, a proline transporter (ProTs) family, a y-amino acid butyrate transporter (GATS) family, an auxin transporter (AUXs) family and an aromatic and neutral amino acid transporter (ANTs) family. Studies showed that regulating expressions of genes encoding the AAT can regulate growth and development and yield- related traits of transgenic plants. For example, T-DNA insertion mutants of OsAATS, OsAAT7, OsAAT24, OsAAT49, and OsAAT60 have decreased rice yield and plant dry weight, proving that the AAT plays an important role in accumulation of nitrogen and distribution of carbon and nitrogen in rice. An overexpression of AfLHT! in arabidopsis thaliana can enhance absorption of multiple amino acids by transgenic plants and at the same time, biomass accumulation of the transgenic plants increased (Hinier et al., 2006), expressing an A4P/ gene of kidney beans in peas significantly improved content of total nitrogen and proteins in transgenic pea seeds and weight of the seeds increased (Rol Ietschek et al., 2005); and an overexpression of S/CAT9 in tomato fruits can improve an amino acid composition of the fruits (Snowden et al., 2015). Therefore, it can be seen that regulation of an expression level of the AAT gene is of great significance to improving crop yield and quality. However, there is currently no report on an effect of AAT on plant types of soybeans.
SUMMARY In order to solve the problems, the present disclosure provides use of a GmAAP protein and a GmAAP gene in breeding soybeans. The present disclosure explores an effect of an amino acid transfer protein on plant types of the soybeans. The GmAAP gene plays an extremely important role in branching of the soybeans and can be applied to improve plant types to increase soybean yield. To achieve the above objective, the present disclosure provides the following technical solution. The present disclosure provides use of a GmAAP protein in breeding soybeans, and the GmAAP protein has an amino acid sequence shown in SEQ ID NO: 1. The present disclosure also provides use of a GmAAP gene in breeding soybeans, and the GmAAP gene has a cDNA sequence shown in SEQ ID NO: 2. The present disclosure also provides use of regulation of an expression of a GmAAP gene in improving plant types of soybeans. The present disclosure also provides use of GmAAP gene silencing in increasing the number of branches of soybeans.
The present disclosure also provides use of GmAAP gene silencing in increasing the number of pods per plant of soybeans. The present disclosure also provides use of GmAAP gene silencing in improving soybean yield.
The present disclosure provides use of a GmAAP protein in breeding soybeans. The GmAAP protein has an amino acid sequence shown in SEQ ID NO: 1. A genetic engineering technology is used to silence an expression of a GmAAP gene to significantly increase the number of branches of soybeans and thus increase the soybean yield. It can be seen from examples that the use can effectively increase the number of branches of soybeans and increase the number of pods per plant.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a structural diagram of main functional elements of a gene editing vector, where, 33S, atU6 and Ubi are promoters; Bar is a marker gene and gRNA is a target sequence; and dpCas9 is a Cas? gene; FIG. 2 is a recombinant plant expression vector D7S6001-GmAAP, FIG. 3 is a phenogram of whole plants of soybean wild-type Jack (OE-CK) and soybean wild-type variety Williams82 (KO-CK), 3 lines (OE-1, OE-2 and OE-3) of GmAAP gene-overexpressing plants and 3 lines (KO-1, KO-2 and KO-3) of GmAAP gene- silenced plants; FIG. 4 is a statistical diagram of the number of branches per plant of each plant; FIG. 5 is a statistical diagram of the number of pods per plant of each plant; FIG. 6 is relative expression of a GmAAP gene in each plant.
DETAILED DESCRIPTION The present disclosure provides use of a GmAAP protein in breeding soybeans, and the GmAAP protein has an amino acid sequence shown in SEQ ID NO:
1. >Glyma.04G209100.1 487aa
SYNOQNVFSLVWRTVFVIITTVISMLLPFFNDILGVIGALGFWPLTVYFPVEMYIL QKRIPKWSMRWISLELLSVVCLIVTIAAGLGSMVGVLLDLQKYKPFSSDY*, SEQ ID NO: 1. The method in breeding soybeans preferably includes reducing an expression of GmAAP protein in plants. In the present disclosure, under a premise of not affecting an activity of the GmAAP protein (that is, not in an active center of the protein), those skilled in the art can make various substitutions, additions and/or deletions of one or more amino acids of the amino acid sequence shown in the SEQ ID IO NO. 1 to obtain an amino acid sequence with the same function. Therefore, the GmAAP protein also includes a protein with the same activity obtained by substituting, replacing and/or adding one or more amino acids to the amino acid sequence shown in the SEQ ID NO. 1.
The present disclosure also provides use of a GmAAP gene in breeding soybeans, and the GmAAP gene has a cDNA sequence shown in SEQ ID NO:
2. >Glyma.04G209100.1 CDS 1464bp
TATGCTCAACCCCTCTTTGCATTTGTCGAGAAGGAGGCAGCAAAAAGATGG 5 CCCAAAATTGACAAGGAATTCCAAATTTCAATTCCCGGTTTGCAATCCTACA
GTGTGGTGTGCCTCATAGTAACAATTGCGGCTGGTCTTGGCTCAATGGTTGG TGTCTTGCTTGACCTCCAGAAATACAAACCATTCAGTTCAGATTATTAA, SEQ ID NO: 2. The method in breeding soybeans preferably includes silencing the GmAAP gene to obtain GmAAP gene-silenced transgenic plants. In the present disclosure, considering degeneracy of codons and preference of the codons of different species, those skilled in the art can use the codons suitable for expression of specific species as needed.
The present disclosure also provides use of regulation of expression of a GmAAP gene in improving plant types of soybeans. The use can effectively improve the plant types of the soybeans, so that the plant types of genetically modified soybeans are shorter and stronger and have more branches and larger number of pods per plant compared to plant types of unmodified soybeans.
The present disclosure also provides use of GmAAP gene silencing in increasing the number of branches of soybeans. The present disclosure also provides use of GmAAP gene silencing in increasing the number of pods per plant of soybeans.
In the use of the GmA4AP gene silencing in increasing the number of branches of soybeans or increasing the number of pods per plant of the soybeans, a method of the GmAAP gene silencing preferably includes: an amino acid transporter gene GmAAP of soybeans is used as an object, a cDNA sequence of the GmAAP gene is cloned from a soybean wild-type variety Williams82, a GmAAP gene-silenced expression vector is constructed by a Cas9 gene editing technology, the Cas9-edited expression vector is introduced into plants of a normal soybean wild-type variety Williams82 by a genetic transformation method mediated by agrobacterium tumefaciens EHA105 to obtain
GmAAP gene-silenced transgenic plants, the number of branches of soybeans of the GmAAP gene-silenced transgenic plants can increase by 50% compared with that of the soybean wild-type variety Williams82, and the number of pods per plant of the GmAAP gene-silenced transgenic plants increases by 57.5% compared with that of the soybean wild-type variety Williams82. The present disclosure also provides use of GAAP gene silencing in improving soybean yield. The use can effectively increase the number of branches and the number of pods per plant of soybeans, thereby increasing soybean yield. The present disclosure also constructs a GmAAP gene over-expressing vector, the over-expression vector is introduced into a soybean wild-type Jack to obtain GmAAP gene over-expressing transgenic plants whose number of branches does not change significantly compared with the soybean wild-type Jack. The results show that the number of branches of soybeans can be increased by reducing an expression of the GmAAP gene, which further increases the number of pods per plant of the soybeans and the soybean yield.
The present disclosure also clarifies a molecular mechanism of plant yield increase and has a great promoting effect to clarify the molecular mechanism of the plant yield increase.
In order to further describe the present disclosure, the use of the GmAAP protein and the GmAAP gene in breeding soybeans 1s described in detail below with reference to the accompanying drawings and examples, but the accompanying drawings and examples should not be understood as limiting the protection scope of the present disclosure. Embodiment Construction of GmAAP Gene-Silenced Plants GmAAP gene information in a soybean reference genome in a soybean genome database (https://www.soybase.org) was referred and an online website (http://crispr.hzau.edu.cn /CRISPR2/) was used to design targets of the gene.
Principles for designing the targets were as follows: 1) knockout sites were located in a coding (CDS) region and as far as possible in the front of a protein or in an important functional domain region; 2) a higher proportion of transcripts was covered as much as possible; 3) no off-targets existed or the off-targets were located in an intergenic region; 4) the targets with higher editing efficiency were optimized; and
5) a sequence had a relatively balanced GC content and was not easy to form a secondary structure. The target sequence was shown in SEQ ID NO: 3: gRNA: 5'-ATGTCATATTCCAAGTATCC-3', SEQ ID NO: 3.
Astructural diagram of main functional elements of a gene editing vector was shown in FIG. 1, where, 355, atU6 and Ubi were promoters; Bar was a marker gene and gRNA was the target sequence; and dp(Cas9 was a Cas? gene.
According to an instruction in a CRISPR/Cas9 rapid construction kit VK005-04 (purchased from Beijing Viewsolid Biotech Co.Ltd), the gRNA shown in the SEQ ID NO: 3 was inserted into the gene editing vector to construct a CRISPR/Cas9 vector containing the GmAAP target sequence. The gene-silencing vector was introduced into a normal soybean wild-type variety Williams82 to obtain transgenic seedlings by using a genetic transformation method mediated by agrobacterium tumefaciens EHA 105. Processes for obtaining edited plants were as follows: All the obtained transgenic seedlings were transplanted in baskets with the soil and watered and fertilized regularly; when the transgenic seedlings grew to about 10 cm in height, the transgenic seedlings were planted in the field; after the transgenic seedlings grew up, LibertyLink® Bar test strips (EnviroLogix Inc., USA) were used to detect Bar protein. A specific method was as follows: (1) leaves of the grown transgenic seedlings were taken as samples, the sample leaf tissues were placed between lids and tube bodies of disposable tissue extraction tubes, the lids were quickly covered to obtain round leaf tissues, and the leaves were placed at bottom parts of the extraction tubes by using a pestle; (2) the pestle was inserted into the tube and rotated to crush the leaves, and pressing was kept for 20-30 seconds; 0.5 mL of an extraction buffer was added, (3) the crushing was repeated to enable the samples and the buffer to be in full contact and mixed, and the pestle was removed; and (4) reaction tubes were kept upright, test paper was inserted into the reaction tubes, a sample solution would rise along the test paper, and a result was read after 10 minutes of reaction; Genomic DNA was extracted, Bar in the transgenic plants was detected by PCR and detection primer pairs were shown in SEQ ID NO: 4 and SEQ ID NO: 5: F3: 5-TGCACCATCGTCAACCACTACAT-3', SEQ ID NO: 4; and
R3: 5'-AGAAACCCACGTCATGCCAGT-3', SEQ ID NO:5. If an amplified 480bp fragment was detected, the transgenic plants may be positive plants. Then seeds of single plants were harvested, the seeds were planted, samples were taken, DNA was extracted, sequencing primers were used to sequence near the target sequence; and if there were changes of insertion or deletion of a base sequence in sequencing double peaks or near the target sequence, the transgenic plants may be positive plants. The sequencing primer pairs were shown in SEQ ID NO: 6 and SEQ ID NO: 7: F4: S'-TACACAATTGCGGCTTCCCT-3', SEQ ID NO: 6; and R4: 5'-TGCAGGGGTTATGTGCCAAT-3', SEQ ID NO: 7. Homozygous transgenic plants were identified in a T2 generation, that is, the GmAAP gene-edited plants were obtained, three subsequent lines were selected and respectively marked as KO-1, KO-2 and KO-3 and the soybean wild-type variety Williams82 without GmAAP gene editing was marked as KO-CK.
Construction of GmAAP Gene-overexpressing Plants RNA of a soybean wild-type variety Jack was extracted and reverse-transcribed into cDNA, and primer pairs as shown in SEQ ID NO: 8 and SEQ ID NO: 9 were used: F5: S'-TTTGGAGAGAACACGTATGGCTGAGCTTCACTACCA AC-3', SEQ ID NO: 8:and R5: 5'-TCGGGGAAATTCGGGGTTAATAATCTGAACTGAATG- 3', SEQ ID NO: 9. A GmAAP gene was forwardly introduced into a soybean expression vector PCAMBIA3300 by a seamless cloning technology to construct a recombinant plant expression vector DTS6001-GmAAP (as shown in FIG. 2 and the FIG. 2 was the recombinant plant expression vector DTS6001-GmAAP), the vector contained a selectable marker gene 5-enolpyruvvl-shikimate-3-phosphate synthase (EPSPS) in a T- DNA region, and the encoded 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) which can block an interference of glyphosate to a biosynthetic pathway and thus was not killed by the glyphosate. The overexpressing vector was introduced into a normal soybean wild-type variety Jack to obtain transgenic seedlings of the soybean wild-type variety Jack by using a genetic transformation method mediated by agrobacterium tumefaciens EHA 105. All the obtained transgenic seedlings of the soybean wild-type variety Jack were transplanted in baskets with the soil and watered and fertilized regularly, when the seedlings grew to about 10 cm in height, the seedlings were planted in the field, after the seedlings grew up, genomic DNA was extracted, the transgenic seedlings were detected by PCR and detection primer pairs were as shown in SEQ ID NO: 10 and SEQ ID NO: 11: F3: S-GACGCACAATCCCACTATCC-3', SEQ ID NO: 10; and R3: S'-TTAATAATCTGAACTGAATGGT-3', SEQ ID NO: 11. If a fragment of 1,656bp is amplified, the transgenic plants were positive plants.
Seeds of the single positive plants were harvested, the seeds were planted, homozygous transgenic plants were identified until a T2 generation, that is, the GmAAP gene- overexpressing plants were obtained, three subsequent lines were selected and respectively marked as OE-1, OE-2 and OE-3 and the soybean wild-type variety Jack without GmAAP gene editing was marked as OE-CK.
Application Example 1 The soybean wild-type variety Jack (OE-CK), the 3 lines (OE-1, OE-2 and OE-3) of the GmAAP gene over-expressing plants, the soybean wild-type variety Williams82 (KO- CK), and the 3 lines (KO-1, KO-2 and KO-3) of the GmAAP gene-silenced plants provided by the embodiment were cultivated, cultivation results were shown in FIG. 3, FIG. 4 and FIG. 5 and Table 1 and Table 2, FIG. 3 was a phenogram of whole plants of the soybean wild-type Jack (OE-CK) and the soybean wild-type Williams82 (KO-CK), the 3 lines (OE-1, OE-2 and OE-3) of the GmAAP gene-overexpressing plants and the 3 lines (KO-1, KO-2 and KO-3) of the GmAAP gene-silenced plants, FIG. 4 was a statistical diagram of the number of branches per plant of each plant, and FIG. 5 was a statistical diagram of the number of pods per plant of each plant.
Table 1 Statistical results of the number of branches per plant of each plant oe pe It can be seen from FIG. 3, FIG. 4 and Table 1, the number of branches of the GmAAP gene-edited plants (KO-1, KO-2 and KO-3) was significantly greater than that of the soybean wild-type variety Williams82 plant.
Compared with the soybean wild-type variety Jack, the number of branches of the GmAAP gene-overexpressing plants was not significantly reduced, indicating that GmAAP gene silencing can effectively increase the number of branches of soybeans.
Table 2. Statistical results of the number of pods per plant of each plant It can be seen from FIG. 5 and Table 2, the number of pods of the GmAAP gene-edited plants (KO-1, KO-2 and KO-3) was significantly greater than that of the soybean wild- type variety Williams82 plant.
Compared with the soybean wild-type variety Jack, the number of branches of the GmAAP gene-overexpressing plants was not significantly different, indicating the GmAAP gene silencing can effectively increase the number of pods per plant of soybeans.
Application Example 2 Leaves of GmAAP gene-edited plants and GmAAP gene-overexpressing plants were taken, RNA was extracted and reverse-transcribed into cDNA, and expression of a GmAAP gene in the edited plants and overexpressing plants was detected by real-time fluorescent quantitative PCR.
Detection results were shown in FIG. 6 and Table 3 and the FIG. 6 was relative expression of the GmAAP gene in each plant.
The used primer pairs by the real-time fluorescent quantitative PCR were shown in SEQ ID NO: 12 and SEQ ID NO: 13: FS: 5-TGGCCTCTAACGGTGTACTT-3', SEQ ID NO: 12; and R5: 5'-AGACACCAACCATTGAGCCA-3', SEQ ID NO: 13. Table 3 Relative expression of a GmAAP gene in each plant It can be seen from FIG. 6 and Table 3, the expression of the GmAAP gene in the gene- edited plants (KO-1, KO-2 and KO-3) was significantly lower than that of a soybean wild-type variety Williams82 plant without gene editing.
The expression of the GmAAP gene in the overexpressing plants was much higher than that of a soybean wild-type variety Jack.
The results showed that reducing the expression of the GmAAP gene can improve the number of branches of soybeans and the number of pods per plant of the soybeans, and finally affects soybean yield.
Although the present disclosure has been disclosed with exemplary examples, the content of this specification shall not be construed as a limitation to the present disclosure.
Any person skilled in the art can make changes and variations without departing from the spirit and scope of the present disclosure.
The present disclosure shall fall within the protection scope defined in the following claims.
SEQLTXT Sequence Listing <120> USE OF GMAAP PROTEIN AND GMAAP GENE IN BREEDING SOYBEANS <160> 13 <170> SIPOSequenceListing 1.0 <210> 1 <211> 487 <212> PRT <213> Artificial Sequence <400> 1 Met Val Glu Tyr Ala Ser Arg Thr Asn Leu Ser Tyr Cys Arg Asp Tyr 1 5 10 15 Asp Ile Glu Glu Asp Ser Met Asp Gly Met Pro Leu Lys Ser Asp Pro
Glu Cys Tyr Asp Asp Asp Gly Arg Leu Lys Arg Thr Gly Thr Ile Trp 40 45 Thr Thr Ser Ser His Ile Ile Thr Ala Val Val Gly Ser Gly Val Leu 50 55 60 Ser Leu Ala Trp Ala Ile Ala Gln Met Gly Trp Ile Ala Gly Pro Ala 65 70 75 80 Val Met Ile Leu Phe Ser Ile Val Thr Leu Tyr Thr Ser Ser Phe Leu 85 90 95 Ala Asp Cys Tyr Arg Thr Gly Asp Pro Ile Phe Gly Lys Arg Asn Tyr 100 105 110 Thr Phe Met Asp Ala Val Ser Thr Ile Leu Gly Gly Tyr Ser Val Thr 115 120 125 Phe Cys Gly Ile Val Gln Tyr Leu Asn Leu Phe Gly Ser Ala Ile Gly 130 135 140 Tyr Thr Ile Ala Ala Ser Leu Ser Met Lys Ala Ile Gln Arg Ser His 145 150 155 160 Cys Ile Ile Gln Phe Ser Asp Gly Glu Asn Gln Cys His Ile Pro Ser 165 170 175 Ile Pro Tyr Met Ile Gly Phe Gly Ala Val Gln Ile Phe Phe Ser Gln 180 185 190 Ile Pro Asp Phe His Asn Met Trp Trp Leu Ser Ile Val Ala Ser Val 195 200 205 Met Ser Phe Thr Tyr Ser Ile Ile Gly Leu Val Leu Gly Val Thr Lys 210 215 220 Ile Ala Glu Thr Gly Thr Phe Lys Gly Ser Leu Thr Gly Ile Ser Ile 225 230 235 240 Gly Thr Val Thr Glu Ala Gln Lys Val Trp Gly Val Phe Gln Ala Leu 245 250 255 Gly Asn Ile Ala Phe Ala Tyr Ser Tyr Ser Phe Val Leu Leu Glu Ile 260 265 270 Gln Asp Thr Ile Lys Ser Pro Pro Ser Glu Val Lys Thr Met Lys Lys 275 280 285 Ala Ala Lys Leu Ser Ile Ala Val Thr Thr Thr Phe Tyr Met Leu Cys 290 295 300 Pagina 1
Gly Cys Val Gly Tyr Ala Ala Phe Gly Asp Ser Ala Pro Gly Asn Leu 305 310 315 320 Leu Ala Gly Phe Gly Phe His Lys Leu Tyr Trp Leu Ile Asp Ile Ala
325 330 335 Asn Ala Ala Ile Val Ile His Leu Val Gly Ala Tyr Gln Val Tyr Ala
340 345 350 Gln Pro Leu Phe Ala Phe Val Glu Lys Glu Ala Ala Lys Arg Trp Pro 355 360 365 Lys Ile Asp Lys Glu Phe Gln Ile Ser Ile Pro Gly Leu Gln Ser Tyr 370 375 380
Asn Gln Asn Val Phe Ser Leu Val Trp Arg Thr Val Phe Val Ile Ile 385 390 395 400 Thr Thr Val Ile Ser Met Leu Leu Pro Phe Phe Asn Asp Ile Leu Gly
405 410 415 Val Ile Gly Ala Leu Gly Phe Trp Pro Leu Thr Val Tyr Phe Pro Val
420 425 430 Glu Met Tyr Ile Leu Gln Lys Arg Ile Pro Lys Trp Ser Met Arg Trp 435 440 445 Ile Ser Leu Glu Leu Leu Ser Val Val Cys Leu Ile Val Thr Ile Ala 450 455 460
Ala Gly Leu Gly Ser Met Val Gly Val Leu Leu Asp Leu Gln Lys Tyr 465 470 475 480 Lys Pro Phe Ser Ser Asp Tyr
485 <210> 2 <211> 1464 <212> DNA <213> Artificial Sequence <400> 2 atggtagaat atgcttcgag aacaaacctt agctactgtc gagattatga cattgaggag 60 gactccatgg atggcatgcc tttaaaaagt gatcctgaat gctatgacga tgatggccgt 120 cttaaacgaa cagggaccat ttggactaca agctcccaca taataacagc tgtggtagga 180 tctggggtgc tctccttagc ctgggcaata gctcagatgg gttggattgc tggtcctgca 240 gtgatgatct tattcagcat agtcactttg tatacttcat catttctagc tgattgttat 300 cgtactggtg accccatatt cgggaagaga aattatactt tcatggatgc agttagcacc 360 attctaggcg ggtacagtgt tacgttctgt gggatagttc agtacttaaa tcttttcgga 420 agtgcgatag gatacacaat tgcggcttcc cttagcatga aggcaatcca aaggtctcac 480 tgtatcatcc aattctctga tggagaaaac caatgtcata ttccaagtat cccatacatg 540 atcggttttg gtgcagtgca aattttcttt tctcaaattc cagattttca taacatgtgg 600 tggctctcaa tagttgcttc agtcatgtct ttcacctatt ccataattgg tctcgttctt 660 ggagttacca aaattgcaga aacgggaact ttcaagggta gcctcactgg aataagcatt 720 ggaactgtga cagaggccca aaaagtatgg ggtgttttcc aagctcttgg taacatagcc 780 ttcgcctatt catattcttt cgttctcctt gaaattcagg ataccatcaa atctccacca 840 tctgaagtaa aaacaatgaa gaaggctgca aaattaagta ttgcagtgac cacaacattt 900 tatatgcttt gtggctgcgt aggctatgct gcttttgggg attcagcacc tgggaacctg 960 cttgctggat ttggtttcca taaactatat tggcttatag atattgctaa tgctgctatt 1020 gtaattcacc ttgtgggggc ataccaagtg tatgctcaac ccctctttgc atttgtcgag 1080 aaggaggcag caaaaagatg gcccaaaatt gacaaggaat tccaaatttc aattcccggt 1140 ttgcaatcct acaatcagaa cgtatttagc ctagtttgga ggacagtgtt tgtgatcata 1200 accactgtta tatcaatgtt gcttccattc ttcaatgata ttttgggagt gattggagca 1260
Pagina 2
SEQLTXT ttggggtttt ggcctctaac ggtgtacttt cctgtggaga tgtatatctt gcaaaagagg 1320 atcccaaaat ggagtatgag atggatttct ctggaattgc tgagtgtggt gtgcctcata 1380 gtaacaattg cggctggtct tggctcaatg gttggtgtct tgcttgacct ccagaaatac 1440 aaaccattca gttcagatta ttaa 1464 <210> 3 <211> 13 <212> DNA <213> Artificial Sequence <400> 3 agcaaccaag acc 13 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <400> 4 tgcaccatcg tcaaccacta cat 23 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <400> 5 agaaacccac gtcatgccag t 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 tacacaattg cggcttccct 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 tgcaggggtt atgtgccaat 20 <210> 8 <211> 38 <212> DNA <213> Artificial Sequence <400> 8 tttggagaga acacgtatgg ctgagcttca ctaccaac 38 Pagina 3
<210> 9
<211> 36
<212> DNA
<213> Artificial Sequence
<400> 9 tcggggaaat tcggggttaa taatctgaac tgaatg 36
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 10 gacgcacaat cccactatcc 20
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 11 ttaataatct gaactgaatg gt 22
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 12 tggcctctaa cggtgtactt 20
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 13 agacaccaac cattgagcca 20 Pagina 4
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001012801A2 (en) * | 1999-08-18 | 2001-02-22 | Pioneer Hi-Bred International, Inc. | Defense-related signaling genes and methods of use |
| WO2003095654A2 (en) * | 2002-05-13 | 2003-11-20 | Wolf-Bernd Frommer | Method for producing a transgenic plant having modified transport of substances |
| CN106518993A (en) * | 2016-10-25 | 2017-03-22 | 武汉生物工程学院 | Application of amino acid transporter gene OsAAP3 in rice seed selection |
| CN108103087A (en) * | 2017-12-18 | 2018-06-01 | 南开大学 | The canaline transport protein fusion and its application that one insertion introne is modified |
| CN108220305A (en) * | 2017-12-15 | 2018-06-29 | 中国烟草总公司郑州烟草研究院 | Tobacco amino acid permease NtAAP2 genes and its application |
| CN111440808A (en) * | 2020-03-30 | 2020-07-24 | 中国农业大学 | Plant amino acid permease and application of coding gene thereof in regulating and controlling high temperature resistance of plants |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2687635A1 (en) * | 2007-05-22 | 2008-11-27 | Basf Plant Science Gmbh | Plant cells and plants with increased tolerance and/or resistance to environmental stress and increased biomass production-ko |
| CN106591354B (en) * | 2016-12-02 | 2019-08-23 | 武汉生物工程学院 | Application of the amino acid transport gene OsAAP5 in rice breeding |
| CN106929522B (en) * | 2017-02-23 | 2019-11-15 | 武汉生物工程学院 | Amino acid transport gene OsAAP1 promotes the application of paddy growth under low nitrogen |
-
2020
- 2020-11-27 CN CN202011354096.3A patent/CN112410309B/en active Active
-
2021
- 2021-04-01 NL NL2027897A patent/NL2027897B1/en active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001012801A2 (en) * | 1999-08-18 | 2001-02-22 | Pioneer Hi-Bred International, Inc. | Defense-related signaling genes and methods of use |
| WO2003095654A2 (en) * | 2002-05-13 | 2003-11-20 | Wolf-Bernd Frommer | Method for producing a transgenic plant having modified transport of substances |
| CN106518993A (en) * | 2016-10-25 | 2017-03-22 | 武汉生物工程学院 | Application of amino acid transporter gene OsAAP3 in rice seed selection |
| CN108220305A (en) * | 2017-12-15 | 2018-06-29 | 中国烟草总公司郑州烟草研究院 | Tobacco amino acid permease NtAAP2 genes and its application |
| CN108103087A (en) * | 2017-12-18 | 2018-06-01 | 南开大学 | The canaline transport protein fusion and its application that one insertion introne is modified |
| CN111440808A (en) * | 2020-03-30 | 2020-07-24 | 中国农业大学 | Plant amino acid permease and application of coding gene thereof in regulating and controlling high temperature resistance of plants |
Non-Patent Citations (4)
| Title |
|---|
| ANONYMOUS: "D0Y65_010371 - Amino acid permease 2 - Glycine soja (Wild soybean) - D0Y65_010371 gene & protein", 8 May 2019 (2019-05-08), XP055849959, Retrieved from the Internet <URL:https://www.uniprot.org/uniprot/A0A445L343> [retrieved on 20211011] * |
| KOCH W ET AL: "Reduced amino acid content in transgenic potato tubers due to antisense inhibition of the leaf H+/amino acid symporter StAAP1", THE PLANT JOURNAL, BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD, GB, vol. 33, no. 2, 1 January 2003 (2003-01-01), pages 211 - 220, XP002266693, ISSN: 0960-7412, DOI: 10.1046/J.1365-313X.2003.01618.X * |
| MOLLY PERCHLIK ET AL: "Improving Plant Nitrogen Use Efficiency through Alteration of Amino Acid Transport Processes", PLANT PHYSIOLOGY, VOL. 175, N. 1, 21 July 2017 (2017-07-21), pages 235 - 247, XP055659935, Retrieved from the Internet <URL:http://www.plantphysiol.org/content/plantphysiol/175/1/235.full.pdf> [retrieved on 20200121], DOI: 10.1104/pp.17.00608 * |
| PERCHLIK MOLLY ET AL: "Leaf Amino Acid Supply Affects Photosynthetic and Plant Nitrogen Use Efficiency under Nitrogen Stress", PLANT PHYSIOLOGY, vol. 178, no. 1, 1 September 2018 (2018-09-01), Rockville, Md, USA, pages 174 - 188, XP055849974, ISSN: 0032-0889, Retrieved from the Internet <URL:https://academic.oup.com/plphys/article-pdf/178/1/174/36066881/plphys_v178_1_174.pdf> DOI: 10.1104/pp.18.00597 * |
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| CN112410309B (en) | 2022-02-08 |
| NL2027897B1 (en) | 2021-11-09 |
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