CN108410846A - Improve wheat plant types GAP-associated protein GAP TaAAA-ATPase1 and its gene and application - Google Patents
Improve wheat plant types GAP-associated protein GAP TaAAA-ATPase1 and its gene and application Download PDFInfo
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- CN108410846A CN108410846A CN201810447198.6A CN201810447198A CN108410846A CN 108410846 A CN108410846 A CN 108410846A CN 201810447198 A CN201810447198 A CN 201810447198A CN 108410846 A CN108410846 A CN 108410846A
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Abstract
The present invention relates to genetic engineering fields, and in particular to improvement wheat plant types GAP-associated protein GAP TaAAA ATPase1 and its gene and application.The amino acid sequence of the albumen TaAAA ATPase1 is as shown in SEQ ID NO.1.It will be imported in wheat cell containing the recombinant expression carrier of excessive TaAAA ATPase1 genes, transgenic over expression TaAAA ATPase1 can improve the tillering number of wheat, big fringe appropriate, thick bar and mass of 1000 kernel increase, wheat yield is caused to increase up to 16%, this will produce huge impetus and economic benefit to crop breeding and agricultural production.
Description
Technical field
The present invention relates to genetic engineering fields, and in particular to improvement wheat plant types GAP-associated protein GAP TaAAA-ATPase1 and its
Gene and application.
Background technology
Currently, traditional breeding method, which improves yield of wheat, has been in bottleneck stage, unit yield is further increased, profit
It is combined with traditional breeding method and transgenic technology, becomes one of modern SOYBEAN IN HIGH-YIELD BREEDING main means.
Wheat plant types are to determine one of the principal element of wheat yield, and determine the Other Main Agronomic Characters of lodging resistance, small
Moulding for wheat ideotype is the important channel for improving wheat yield.Wheat big fringe, appropriate tiller and the thick stalk appropriate of being formed resists
The on the contrary key of ideotype.Obtaining has the factors such as example best tillering number of best ideotype, big fringe appropriate and thick bar
Wheat breed be product that China's wheat breeding main target and existing market are badly in need of exploitation.
AAA-ATPase gene families (ATPase associated with various cellular
Activities it) is widely present in eukaryon and prokaryotes.To the AAA-ATPase results of study hair of people, mouse and yeast etc.
Existing, which participates in film fusion, cell division cycle regulating, the transport of intracellular protein and regulation and control and Apoptosis etc. a system
Row physiological activity.It is relatively fewer for the function and Study on mechanism of plant AAA-ATPase, and about wheat AAA-
The research that ATPase gene of MP family member participates in reason plant type has not been reported.
AAA-ATPase gene family albumen has conservative Walker A and Walker B structures domain, and specifically same
The secondary region in source (second region of homology, SRH).Participate in protein degradation, protein folding, film transport, cell bone
Frame adjusts and a variety of physiological activities such as expression regulation.The skin of junket containing face albumen (valosin-containing protein, VCP) is
First certified 174-263 tPA is a kind of film combination glycoprotein being widely present, mainly as the effect of similar molecular chaperones
It plays an important role in the relevant protein degradation of endoplasmic reticulum and cell cycle regulating.The p97/VCP and yeast of mammal
P97/Cdc48 is the widest AAA-ATPase gene families member of research, and the gene also has height in other biological
Conservative.P97/VCP is one of molecular chaperones AAA-ATPase gene family members, is exercised in intracellular associated with endoplasmic reticulum
The critical functions such as degradation, film fusion, transcription factor activation, and the enforcement of these functions is all related with ubiquitination.
Research shows that arabidopsis AAA-ATPase family members RPT2a is a subunit of 26S proteasomes, the gene work(
It can lose, reduce the activity of 26S proteasomes, the stalk cell so as to cause arabidopsis increases and cell quantity is reduced.However,
In unifacial leaf model plant, the research of AAA-ATPase genes is very limited.
Invention content
The object of the present invention is to provide improvement wheat ideotype GAP-associated protein GAP TaAAA-ATPase1.
Another object of the present invention is to provide the above-mentioned improvement wheat ideotype GAP-associated protein GAP TaAAA-ATPase1's of coding
Gene.
It is a further object of the present invention to provide the recombinant vectors comprising said gene.
Another object of the present invention provide above-mentioned plant improvement wheat ideotype related gene TaAAA-ATPase1 and its
The application of gene.
The relevant gene TaAAA-ATPase1 of improvement wheat ideotype provided by the present invention derives from the big grain of strain
The big fringe more tiller wheat S301 of thick bar, amino acid are 500, and amino acid sequence is as shown in SEQ ID NO.1.
SEQ ID NO.1
MEMILDWRSVGSLIATVMVFRTAMRDFIPPEAEQWLRRLLARLAAAFRAPTATILIDEADGASSGATND
LYDAAQLYLGSRCLAAAPAVRLYKPRQSERAVASLPDSHTADDTFQGVRVKWTSTARPVERGAGHNPYNVFGSRGGG
AGGDHRSLELQFPRQHRDFVHDTYIPHIIAEATRMRLKSRERRLYTNRAAAPGDDHHRLWTSHTFSHPSTFDTLAVD
PALREEIRADLLRFAARREHYARVGRAWKRGYLLHGPPGTGKTSLVAAIANLLEFDVYDLELTTVPTNSHLRRLLVS
TTPKSVVVVEDIDCSLDLSDRKKNSGGADEDNAQLAMLSPAAAAAMAAIGRESISLSGVLNFVDGLWSSCVGERLMI
FTTNHPERLDPALLRPGRMDRKIELGYCTPAALRVLAKNYIGVGEDPMDEPDTVVDGLMAEAEGLLAADVRITPADI
DEVFMGCDGAGASAALRRLVGELRGRRDTPAVDTVQPGALTEEKME
According to the above-mentioned albumen of TaAAA-ATPase1 gene codes of the present invention, there can be the nucleotide as shown in SEQ ID NO.2
Sequence, TaAAA-ATPase1 open reading frame (ORF) length are 1500bp.
SEQ ID NO.2
ATGGAGATGATACTGGACTGGCGCTCGGTGGGCTCGCTGATCGCCACGGTCATGGTGTTCCGAACGGCC
ATGCGGGACTTCATCCCGCCGGAGGCCGAGCAGTGGCTGCGGCGCCTGCTCGCGCGCCTCGCCGCCGCGTTCCGGGC
GCCCACGGCCACCATCCTCATCGACGAGGCCGACGGCGCCAGCTCCGGCGCCACCAACGACCTCTACGACGCCGCGC
AGCTCTACCTCGGCTCGCGCTGCCTCGCCGCCGCCCCCGCCGTGCGCCTCTACAAGCCGCGCCAGTCGGAGCGCGCC
GTCGCCTCGCTCCCGGACTCGCACACCGCGGACGACACCTTCCAGGGCGTCCGTGTCAAGTGGACCTCCACCGCGCG
CCCCGTCGAGCGCGGGGCCGGGCACAACCCCTACAACGTGTTCGGCAGCCGCGGCGGCGGTGCCGGCGGCGACCACC
GCAGCCTCGAGCTCCAGTTCCCGCGCCAGCACCGCGACTTCGTGCACGACACCTACATCCCCCACATCATCGCCGAG
GCCACCCGGATGCGGCTCAAGTCGCGGGAGCGCAGGCTCTACACCAACCGCGCCGCCGCGCCCGGCGACGACCACCA
CCGCCTCTGGACCTCCCACACCTTCTCCCACCCCTCCACCTTCGACACGCTCGCTGTCGACCCGGCGCTCCGCGAGG
AGATCCGCGCCGACCTACTCCGCTTCGCCGCCCGCCGGGAGCACTACGCGCGCGTCGGCCGCGCCTGGAAGCGCGGG
TACCTGCTCCACGGCCCGCCCGGCACCGGCAAGACCAGCCTCGTGGCCGCCATCGCCAACCTCCTCGAGTTCGACGT
GTACGACCTGGAGCTCACCACGGTGCCCACCAACTCCCACCTCCGCCGCCTGCTCGTCTCCACCACGCCCAAGTCCG
TCGTCGTCGTCGAGGACATCGACTGCTCCCTCGACCTCTCCGACCGCAAGAAGAACTCCGGCGGCGCCGACGAGGAC
AACGCGCAGCTGGCGATGCTCTCCCCGGCCGCCGCGGCGGCCATGGCGGCGATCGGGCGCGAGTCCATCAGCCTCTC
CGGCGTCCTCAACTTCGTGGACGGGCTCTGGTCGTCCTGCGTGGGCGAGCGCCTGATGATCTTCACCACCAACCATC
CGGAGCGGCTGGACCCGGCGCTGCTCCGCCCGGGCCGCATGGACCGCAAGATCGAGCTGGGCTACTGCACGCCCGCG
GCGCTCCGCGTGCTCGCCAAGAACTACATCGGCGTCGGCGAGGACCCCATGGACGAGCCCGACACTGTGGTGGACGG
CCTCATGGCCGAGGCCGAGGGCCTGCTCGCCGCCGACGTGCGCATCACGCCGGCAGACATCGACGAGGTGTTCATGG
GTTGCGACGGCGCGGGCGCCTCCGCCGCGCTGAGGAGGCTTGTCGGGGAGCTGCGCGGCAGGAGAGACACGCCGGCC
GTCGACACGGTGCAGCCCGGTGCATTGACAGAGGAGAAGATGGAGTGA
The present invention also provides the recombinant expression carriers for including said gene TaAAA-ATPase1.
The present invention also provides the recombinant cells for including said gene TaAAA-ATPase1.
In addition, using the characteristic of wheat different tissues, expression of the analysis wheat TaAAA-ATPase1 in different tissues is poor
Different analysis finds that TaAAA-ATPase1 is expressed in various degree in root, stem, leaf, immature seed etc., illustrates wheat TaAAA-
ATPase1 plays an important role in Wheat Development process.
The present invention also provides above-mentioned improvement wheat ideotype GAP-associated protein GAP TaAAA-ATPase1 and its encoding genes
Using.
The present invention also provides a kind of methods for cultivating ideal wheat plant types, include the following steps：Any of the above-described kind is contained
There is the recombinant expression carrier of excessive TaAAA-ATPase1 genes to import in wheat cell, transgenic over expression TaAAA-
ATPase1 can improve the tillering number of wheat, big fringe appropriate, and thick bar and mass of 1000 kernel increase, and wheat yield increase is caused to reach
16%, this will produce huge impetus and economic benefit to crop breeding and agricultural production.
As in crop breeding with high-quality candidate gene, TaAAA-ATPase1 gene-correlations albumen of the invention and its volume
Code gene pairs crop breeding and raising yield, acceleration molecular breeding process and world food have safely highly important
Theoretical and practical significance.
Description of the drawings
Fig. 1 shows TaAAA-ATPase1 in different tissues expression pattern analysis result.
Fig. 2 shows the character variation of overexpression TaAAA-ATPase1 (OE-JING18) transfer-gen plant.
Specific implementation mode
Embodiment 1, the clone of TaAAA-ATPase1 genes and sequence motif analysis
Wheat S301 kinds are planted in Beijing field, take 10 days roots of Post flowering (root) respectively, stem (stem), leaf (leaf),
Seed (seed) and fringe (spike) sampling, are immediately placed in liquid nitrogen frozen, -80 DEG C save backup.According to RNA extracts kits (north
Capital Tiangeng biochemical technology Co., Ltd) complete plant leaf blade tissue Total RNAs extraction, later using the total serum IgE of extraction as template, with
TaAAA-ATPase1-F/R sequences are primer, carry out reverse transcription with reverse transcriptase (M-MLV), obtain for subsequent experimental
CDNA templates.Design pair of primers：
TaAAA-ATPase1-F1ACAAACCGACGCGCACCAAAGC
TaAAA-ATPase1-R1GTCTACGGTGGATGGTGACCAGTT
The present invention obtains Gene A AA-ATPase, AAA-ATPase open reading frame (ORF) length 1500bp, coding
500 amino acid, sequence analysis determines that the gene is a coding AAA-ATPase gene family, to be cloned in wheat for the first time
One new AAA-ATPase gene.
Embodiment 2：TaAAA-ATPase1 different tissues expression characteristics are analyzed.
10 days roots of Post flowering (root), stem (stem), leaf (leaf), seed (seed) and fringe are taken respectively to wheat S301
(spike) the RNA reverse transcriptions of above-mentioned various Stress treatment materials are cDNA by sampling sampling, as template, utilize RT-PCR points
Analyse the expression pattern of different tissues.As shown in Figure 1, TaAAA-ATPase1 different tissues express, 10 days roots of Post flowering, stem,
The high efficient expression of TaAAA-ATPase1 genes is detected in leaf, seed and fringe.
Embodiment 3：The small Function Identification of TaAAA-ATPase1 transgenosis
TaAAA-ATPase1 is building up in overexpression pCAMBIA3300 carriers, using agrobacterium-mediated transformation, by this
In genetic transformation to capital winter 18, T2 has been obtained at present for transgenic line (OE-18:OE-2, OE-3), by Molecular Detection,
Positive transgenic plant is obtained, the strains such as TaAAA-ATPase1 gene plant type wheats OE-2, OE-3,3 repetitions, cell face are turned
Product is 0.02 mu, as shown in Fig. 2, carrying out Yield comparison experiment finds that overexpression TaAAA-ATPase1 (OE-JING18) turns
Gene plant improves tillering ability than control, and stalk is thicker, increases spike length and mass of 1000 kernel, hence it is evident that improves wheat yield, wherein
In Fig. 2, A is that overexpression OE-JING18 transfer-gen plants become with growing state, transgenic line tillering ability, stalk is compareed
Slightly, increase spike length, yield significantly improves；B be overexpression OE-JING18 transfer-gen plants with compare tassel, transgenic line
Spike length significantly improves；Compared with the control for overexpression OE-JING18 transfer-gen plants, seed significantly increases C；D was scale
Compared with the control up to OE-JING18 transfer-gen plants, TaAAA-ATPase1 expression is remarkably reinforced；E is overexpression OE-
Compared with the control, transgenic line is remarkably reinforced JING18 transfer-gen plants；F is overexpression OE-JING18 transfer-gen plants
With compare tassel, transgenic line spike length significantly improves, G. overexpression OE-JING18 transfer-gen plants with compare tassel, turn
Genetic material output increased reaches 16% or more.
Therefore, TaAAA-ATPase1 is used as in crop breeding with high yield and high quality candidate gene.The TaAAA- of the present invention
ATPase1 gene-correlations albumen and its encoding gene to crop breeding and improve yield, acceleration molecular breeding process, Yi Jishi
Boundary's grain security has highly important theoretical and practical significance.
Sequence table
<110>Beijing City Agriculture and Forestry Institute
<120>Improve wheat plant types GAP-associated protein GAP TaAAA-ATPase1 and its gene and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 500
<212> PRT
<213> Triticum aestivuml.
<400> 1
Met Glu Met Ile Leu Asp Trp Arg Ser Val Gly Ser Leu Ile Ala Thr
1 5 10 15
Val Met Val Phe Arg Thr Ala Met Arg Asp Phe Ile Pro Pro Glu Ala
20 25 30
Glu Gln Trp Leu Arg Arg Leu Leu Ala Arg Leu Ala Ala Ala Phe Arg
35 40 45
Ala Pro Thr Ala Thr Ile Leu Ile Asp Glu Ala Asp Gly Ala Ser Ser
50 55 60
Gly Ala Thr Asn Asp Leu Tyr Asp Ala Ala Gln Leu Tyr Leu Gly Ser
65 70 75 80
Arg Cys Leu Ala Ala Ala Pro Ala Val Arg Leu Tyr Lys Pro Arg Gln
85 90 95
Ser Glu Arg Ala Val Ala Ser Leu Pro Asp Ser His Thr Ala Asp Asp
100 105 110
Thr Phe Gln Gly Val Arg Val Lys Trp Thr Ser Thr Ala Arg Pro Val
115 120 125
Glu Arg Gly Ala Gly His Asn Pro Tyr Asn Val Phe Gly Ser Arg Gly
130 135 140
Gly Gly Ala Gly Gly Asp His Arg Ser Leu Glu Leu Gln Phe Pro Arg
145 150 155 160
Gln His Arg Asp Phe Val His Asp Thr Tyr Ile Pro His Ile Ile Ala
165 170 175
Glu Ala Thr Arg Met Arg Leu Lys Ser Arg Glu Arg Arg Leu Tyr Thr
180 185 190
Asn Arg Ala Ala Ala Pro Gly Asp Asp His His Arg Leu Trp Thr Ser
195 200 205
His Thr Phe Ser His Pro Ser Thr Phe Asp Thr Leu Ala Val Asp Pro
210 215 220
Ala Leu Arg Glu Glu Ile Arg Ala Asp Leu Leu Arg Phe Ala Ala Arg
225 230 235 240
Arg Glu His Tyr Ala Arg Val Gly Arg Ala Trp Lys Arg Gly Tyr Leu
245 250 255
Leu His Gly Pro Pro Gly Thr Gly Lys Thr Ser Leu Val Ala Ala Ile
260 265 270
Ala Asn Leu Leu Glu Phe Asp Val Tyr Asp Leu Glu Leu Thr Thr Val
275 280 285
Pro Thr Asn Ser His Leu Arg Arg Leu Leu Val Ser Thr Thr Pro Lys
290 295 300
Ser Val Val Val Val Glu Asp Ile Asp Cys Ser Leu Asp Leu Ser Asp
305 310 315 320
Arg Lys Lys Asn Ser Gly Gly Ala Asp Glu Asp Asn Ala Gln Leu Ala
325 330 335
Met Leu Ser Pro Ala Ala Ala Ala Ala Met Ala Ala Ile Gly Arg Glu
340 345 350
Ser Ile Ser Leu Ser Gly Val Leu Asn Phe Val Asp Gly Leu Trp Ser
355 360 365
Ser Cys Val Gly Glu Arg Leu Met Ile Phe Thr Thr Asn His Pro Glu
370 375 380
Arg Leu Asp Pro Ala Leu Leu Arg Pro Gly Arg Met Asp Arg Lys Ile
385 390 395 400
Glu Leu Gly Tyr Cys Thr Pro Ala Ala Leu Arg Val Leu Ala Lys Asn
405 410 415
Tyr Ile Gly Val Gly Glu Asp Pro Met Asp Glu Pro Asp Thr Val Val
420 425 430
Asp Gly Leu Met Ala Glu Ala Glu Gly Leu Leu Ala Ala Asp Val Arg
435 440 445
Ile Thr Pro Ala Asp Ile Asp Glu Val Phe Met Gly Cys Asp Gly Ala
450 455 460
Gly Ala Ser Ala Ala Leu Arg Arg Leu Val Gly Glu Leu Arg Gly Arg
465 470 475 480
Arg Asp Thr Pro Ala Val Asp Thr Val Gln Pro Gly Ala Leu Thr Glu
485 490 495
Glu Lys Met Glu
500
<210> 2
<211> 1503
<212> DNA
<213> Triticum aestivuml.
<400> 2
atggagatga tactggactg gcgctcggtg ggctcgctga tcgccacggt catggtgttc 60
cgaacggcca tgcgggactt catcccgccg gaggccgagc agtggctgcg gcgcctgctc 120
gcgcgcctcg ccgccgcgtt ccgggcgccc acggccacca tcctcatcga cgaggccgac 180
ggcgccagct ccggcgccac caacgacctc tacgacgccg cgcagctcta cctcggctcg 240
cgctgcctcg ccgccgcccc cgccgtgcgc ctctacaagc cgcgccagtc ggagcgcgcc 300
gtcgcctcgc tcccggactc gcacaccgcg gacgacacct tccagggcgt ccgtgtcaag 360
tggacctcca ccgcgcgccc cgtcgagcgc ggggccgggc acaaccccta caacgtgttc 420
ggcagccgcg gcggcggtgc cggcggcgac caccgcagcc tcgagctcca gttcccgcgc 480
cagcaccgcg acttcgtgca cgacacctac atcccccaca tcatcgccga ggccacccgg 540
atgcggctca agtcgcggga gcgcaggctc tacaccaacc gcgccgccgc gcccggcgac 600
gaccaccacc gcctctggac ctcccacacc ttctcccacc cctccacctt cgacacgctc 660
gctgtcgacc cggcgctccg cgaggagatc cgcgccgacc tactccgctt cgccgcccgc 720
cgggagcact acgcgcgcgt cggccgcgcc tggaagcgcg ggtacctgct ccacggcccg 780
cccggcaccg gcaagaccag cctcgtggcc gccatcgcca acctcctcga gttcgacgtg 840
tacgacctgg agctcaccac ggtgcccacc aactcccacc tccgccgcct gctcgtctcc 900
accacgccca agtccgtcgt cgtcgtcgag gacatcgact gctccctcga cctctccgac 960
cgcaagaaga actccggcgg cgccgacgag gacaacgcgc agctggcgat gctctccccg 1020
gccgccgcgg cggccatggc ggcgatcggg cgcgagtcca tcagcctctc cggcgtcctc 1080
aacttcgtgg acgggctctg gtcgtcctgc gtgggcgagc gcctgatgat cttcaccacc 1140
aaccatccgg agcggctgga cccggcgctg ctccgcccgg gccgcatgga ccgcaagatc 1200
gagctgggct actgcacgcc cgcggcgctc cgcgtgctcg ccaagaacta catcggcgtc 1260
ggcgaggacc ccatggacga gcccgacact gtggtggacg gcctcatggc cgaggccgag 1320
ggcctgctcg ccgccgacgt gcgcatcacg ccggcagaca tcgacgaggt gttcatgggt 1380
tgcgacggcg cgggcgcctc cgccgcgctg aggaggcttg tcggggagct gcgcggcagg 1440
agagacacgc cggccgtcga cacggtgcag cccggtgcat tgacagagga gaagatggag 1500
tga 1503
Claims (10)
1. improveing wheat plant types GAP-associated protein GAP TaAAA-ATPase1, which is characterized in that the ammonia of the albumen TaAAA-ATPase1
Base acid sequence is as shown in SEQ ID NO.1.
2. the gene of coding improvement wheat plant types GAP-associated protein GAP TaAAA-ATPase1 described in claim 1.
3. gene according to claim 2, which is characterized in that the nucleotide sequence of shown gene such as SEQ ID NO.2 institutes
Show.
4. including the recombinant vector of gene described in claim 2.
5. including the recombinant cell of gene described in claim 2.
6. improveing the application that wheat plant types GAP-associated protein GAP TaAAA-ATPase1 is used to improve wheat plant types described in claim 1.
7. gene described in claim 2 is used to improve the application of wheat plant types.
8. application according to claim 7, which is characterized in that the wheat plant types include tillering number, fringe size, thick bar
And mass of 1000 kernel.
9. the gene described in claim 2 is used to improve the application of wheat yield.
10. a kind of method of improvement wheat plant types, which is characterized in that the method includes being overexpressed claim 2 in wheat
The step of gene.
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| CN107254455A (en) * | 2016-12-14 | 2017-10-17 | 南京农业大学 | One kind miniaturization cucumber plant GAP-associated protein GAP and its encoding gene and application |
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