CN109576288A - A kind of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene and its application - Google Patents
A kind of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene and its application Download PDFInfo
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Abstract
The invention discloses a kind of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene and its applications, first according to the CDS sequence design PCR amplification primer pair of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene；Using Fragariavesca strawberry achene cDNA as template, using PCR amplification primer pair, FveCYP707A4a gene complete sequence is obtained by PCR amplification, obtains the gene of control strawberry fruit size；Again by the way that FveCYP707A4a gene complete sequence is constructed silent carrier or building over-express vector, strawberry is infected after carrier is mixed with Agrobacterium, so that strawberry fruit expands the ABA content decline that is suppressed and dissociates in strawberry fruit, and then adjust the development and maturation of strawberry fruit.
Description
Technical field
The present invention relates to plant biotechnology field, especially a kind of strawberry ABA degradation pathway key enzyme
FveCYP707A4a gene and its application.
Background technique
Strawberry (FragariaX ananassa) belongs to rosaceae strawberry plants, is important one of fresh food fruit crop,
It is also the mode species of the important functional gene research of rosaceous plant, the relevant heredity of fruit development and molecular biology are ground
Study carefully that more and more attention has been paid to Giovannoni James J..2004.Wherein, the regulation of plant hormone is for fruit development and product
Matter forms most crucial (Zhong Xiaohong, 2004).
Abscisic Acid (A B A) can not only promote absorption of the strawberry fruit to sugar, and in strawberry fruit maturation
It plays an important role in regulation process.A B A also plays central role in other non-respiratory transition type fruits, such as in cherry
In Peach fruits maturation, A B A takes part in the growth and development and maturation of fruit；A large amount of A is had accumulated in citrus maturation
B A；And more and more research also indicates that, A B A participate in fruit development and maturation regulation (KanoY, 1981,
John O A1994, Jiang Y M 2003, Zhong Xiaohong, 2004).
The rapid catabolism of ABA is an important factor for influencing plant tissue ABA content.ABA Oxidative inactivation is high plant
The major way that object endogenous ABA is decomposed mainly is completed by 8 ' position methylhydroxy approach of ABA；8 '-hydroxylase (8-hydoxy
Lase) be the oxidative pathway key enzyme, which belongs to cytochrome P 450 monooxygenases (Krochko etc., 1998), in strawberry
In the growth course of fruit, the change of the expression of the gene affects the content of ABA hormone in sporocarp, and then adjusts grass
The development and maturation of certain kind of berries fruit.Gene Silencing (virus-induced gene silencing, VIGS) is a kind of
Easy to operate, quickly and easily Functional identification of genes method.Viral vectors with target gene fragment is infected into plant, plant
Cell can spontaneous identification intrusive viruses threat, then resisted using the defense mechanism of itself and destroy virus and virus carry
Target gene on body occurs degradation in post-transcriptional level so as to cause target gene and even eliminates (Lange etc. 2013；
Purkayastha and Dasgupta2009) there is in various plants VIGS gene silencing system for studying corresponding gene function
Reported success, especially targetedly research plant propagation organ in specific gene, to understand the hair of fruit
It educates and quality responses mechanism.Widely with VIGS silent carrier be diplornavirus Tobacco rattle virus (TRV) (Liu et al.
2002) (Jia etc. 2011), is applied successfully in strawberry.
The strawberry production in China deserves to be called " strawberry big country " but beautiful with " world strawberry power " on the gross area and total output
Compared to there are still certain gaps, the ratio that the high-quality large fruited strawberry kind of self-fertile accounts for world's strawberry breeding is very low for state, Spain etc., and
Average per unit area yield is significantly lower.Strawberry fruit size control molecular mechanism is disclosed, the correlation functions such as fruit size, weight are marked
Exploitation and assist-breeding are suitble to the large fruited strawberry new germ plasm of China's planting environment and consumption habit, are of great significance；Market needs
Such problems can be solved to the method that strawberry fruit size is regulated and controled, the present invention by wanting a kind of.
Summary of the invention
To solve the deficiencies in the prior art, the purpose of the present invention is to provide a kind of strawberry ABA degradation pathway key enzymes
FveCYP707A4a gene and its application, the present invention is by constructing silent carrier or structure for FveCYP707A4a gene complete sequence
Over-express vector is built, infects strawberry after carrier is mixed with Agrobacterium, so that strawberry fruit expands suppressed and strawberry fruit
In dissociate ABA content decline, and then adjust strawberry fruit development and maturation.
In order to achieve the above objectives, the present invention adopts the following technical scheme that:
A kind of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene,
FveCYP707A4a gene complete sequence is as follows:
SEQ ID No.1:FveCYP707A4a
ATGAAGAAGAGCAAGGGAGGAGGAGAAGAAGACCCCCATGATGATCATGGTCATAAGAATAGGGCGGC
TCAGCTCCCTCCAGGCTCATTTGGTTGGCCTTATATCGGTGAGACCCTTCAGCTCTATTCTCAGGACCCAAACACT
TTCTTCTCTTCCAGACAGAAAAGGTATGGGAAAATTTTTAAGACACATATACTTGGGAGTCCATGTGTGATGCTGG
CGAGCCCGGAGGCTGCAAAGTTTGTATTGGTCACTCAAGCTCACTTGTTCAAGCCCACCTATCCCAAAAGCAAAGA
GGCTCTGATTGGTCCCTCCGCATTATTTTTCCACCATGGAGATTACCATTTCAGACTGAGGAAGCTTGTTCAGCGA
TCTCTCAGTCCTGATGCTATTCGGAATTTGGTGCCCCATATCGACGCCACAGCTGCCTCTGTGACCTCGGAATGGT
GGGGCACCGGGAAAGTCATCAACACCTTCCATGAGATGAAGAAGTTTTCTTTCGAAGTTGGTGTACTAGTAATTTT
TGGCCAATTGGAGACCCGCTACAAAGAAGAACTGAGGAAAAACTATATGGCAGTGAACAAAGGCTACAATTCATTT
CCCATAAACATTCCTGGAACGCCATACAAAAAGGCTTTGTTGGCGAGGGAGAGGCTGAGGCACATTATCGGTGACA
TTATCCATGAGAGAAAGGAGAAGAGGTTACCTGAAAAGGATCTGTTGGGTTGTTTGCTGAGATCAATAAACGAAGG
AGGGGAAGTTTTGAGTGATGACCAAATCGCAGACAACATAATAGGTGTTCTCTTTGCTGCTCAAGACACCACAGCC
AGTGTCATGACCTGGATTTTCAAGTACCTCCATGACGAACCAAAAATCCTAGAAGCTGTTAAGGCCGAACAAAATG
CAATTCGCCTATCAAATGAACAAGCAGGTAACCAACCATTGAGTTGGGCAGACACCAGAAACATGCCAATTAGTTA
CAAGGTTGTGTTGGAGAGTTTGAGACTGTCAAGCATTATATCATTCCTTTTTAGAGAAGCTGTGGTTGATGTGGAG
TACAAAGGTTACTTGATTCCAAAAGGTTGGAAGGTGATGCCTTTGTTCAGGAACATTCATCATAATCCTGAATTCT
TCACCGACCCTCAGAAATTCGATCTTTCTAGATTCGAGGTTGCACCAAAGCCAAATACATTTATGCCATTTGGCAG
TGGAGTCCATGCTTGTCCAGGAAACGAGCTTGCTAAGCTGGAATTACTGATCATGATCCACCATTTAGTCACCAAT
TTCAGGTGGGAAATTGAGGGATCCCAAAGCGGGACCGAGTATAGTCCATTTCCTGTACCTCTGAATGGACTTCCGG
TCAAACTTTGGAAATTAGAATAG；
Amino acid sequence is as follows:
SEQ ID No.2:
MKKSKGGGEEDPHDDHGHKNRAAQLPPGSFGWPYIGETLQLYSQDPNTFFSSRQKRYGKIFKTHILGS
PCVMLASPEAAKFVLVTQAHLFKPTYPKSKEALIGPSALFFHHGDYHFRLRKLVQRSLSPDAIRNLVPHIDATAAS
VTSEWWGTGKVINTFHEMKKFSFEVGVLVIFGQLETRYKEELRKNYMAVNKGYNSFPINIPGTPYKKALLARERLR
HIIGDIIHERKEKRLPEKDLLGCLLRSINEGGEVLSDDQIADNIIGVLFAAQDTTASVMTWIFKYLHDEPKILEAV
KAEQNAIRLSNEQAGNQPLSWADTRNMPISYKVVLESLRLSSIISFLFREAVVDVEYKGYLIPKGWKVMPLFRNIH
HNPEFFTDPQKFDLSRFEVAPKPNTFMPFGSGVHACPGNELAKLELLIMIHHLVTNFRWEIEGSQSGTEYSPFPVP
LNGLPVKLWKLE。
A kind of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene above-mentioned, sequencing approach include following step
It is rapid:
Step 1, according to the CDS sequence design PCR amplification of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene
Primer pair；
Step 2, using PCR amplification primer pair, is expanded using Fragaria vesca strawberry achene cDNA as template by PCR
Increase and obtains FveCYP707A4a gene complete sequence.
A kind of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene above-mentioned, PCR amplification program are as follows: 94 DEG C,
4min carries out initial denaturation；94 DEG C, 40s denaturation；Anneal 30s under 60 DEG C of items；Extend 1rnin at 72 DEG C；30 cyclic amplifications, 72 DEG C
Re-extending 10min, reaction was completed.
A kind of application of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene, comprising: by FveCYP707A4a base
After silencing, it is able to suppress strawberry fruit and expands, ABA content of dissociating in strawberry fruit decline.
The application of a kind of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene above-mentioned, by FveCYP707A4a
The method of gene silencing are as follows: using the method for Gene Silencing, the Tobacco rattle virus for constructing FveCYP707A4a is heavy
Silent carrier establishes virus induction after silent carrier is transferred to Agrobacterium, then with the young green fruit of microinjection method infection strawberry
The Transgenic Strawberry strain of FveCYP707A4a gene silencing.
A kind of application of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene above-mentioned, virus induction gene are heavy
Silent method includes:
FveCYP707A4a gene complete sequence is cloned into expression vector with the method for infusion by step 1
PCAMBIA1305, sequencing are correct；
FveCYP707A4a gene complete sequence is as follows:
SEQ ID No.1:FveCYP707A4a
ATGAAGAAGAGCAAGGGAGGAGGAGAAGAAGACCCCCATGATGATCATGGTCATAAGAATAGGGCGGC
TCAGCTCCCTCCAGGCTCATTTGGTTGGCCTTATATCGGTGAGACCCTTCAGCTCTATTCTCAGGACCCAAACACT
TTCTTCTCTTCCAGACAGAAAAGGTATGGGAAAATTTTTAAGACACATATACTTGGGAGTCCATGTGTGATGCTGG
CGAGCCCGGAGGCTGCAAAGTTTGTATTGGTCACTCAAGCTCACTTGTTCAAGCCCACCTATCCCAAAAGCAAAGA
GGCTCTGATTGGTCCCTCCGCATTATTTTTCCACCATGGAGATTACCATTTCAGACTGAGGAAGCTTGTTCAGCGA
TCTCTCAGTCCTGATGCTATTCGGAATTTGGTGCCCCATATCGACGCCACAGCTGCCTCTGTGACCTCGGAATGGT
GGGGCACCGGGAAAGTCATCAACACCTTCCATGAGATGAAGAAGTTTTCTTTCGAAGTTGGTGTACTAGTAATTTT
TGGCCAATTGGAGACCCGCTACAAAGAAGAACTGAGGAAAAACTATATGGCAGTGAACAAAGGCTACAATTCATTT
CCCATAAACATTCCTGGAACGCCATACAAAAAGGCTTTGTTGGCGAGGGAGAGGCTGAGGCACATTATCGGTGACA
TTATCCATGAGAGAAAGGAGAAGAGGTTACCTGAAAAGGATCTGTTGGGTTGTTTGCTGAGATCAATAAACGAAGG
AGGGGAAGTTTTGAGTGATGACCAAATCGCAGACAACATAATAGGTGTTCTCTTTGCTGCTCAAGACACCACAGCC
AGTGTCATGACCTGGATTTTCAAGTACCTCCATGACGAACCAAAAATCCTAGAAGCTGTTAAGGCCGAACAAAATG
CAATTCGCCTATCAAATGAACAAGCAGGTAACCAACCATTGAGTTGGGCAGACACCAGAAACATGCCAATTAGTTA
CAAGGTTGTGTTGGAGAGTTTGAGACTGTCAAGCATTATATCATTCCTTTTTAGAGAAGCTGTGGTTGATGTGGAG
TACAAAGGTTACTTGATTCCAAAAGGTTGGAAGGTGATGCCTTTGTTCAGGAACATTCATCATAATCCTGAATTCT
TCACCGACCCTCAGAAATTCGATCTTTCTAGATTCGAGGTTGCACCAAAGCCAAATACATTTATGCCATTTGGCAG
TGGAGTCCATGCTTGTCCAGGAAACGAGCTTGCTAAGCTGGAATTACTGATCATGATCCACCATTTAGTCACCAAT
TTCAGGTGGGAAATTGAGGGATCCCAAAGCGGGACCGAGTATAGTCCATTTCCTGTACCTCTGAATGGACTTCCGG
TCAAACTTTGGAAATTAGAATAG；
Amino acid sequence is as follows:
SEQ ID No.2:
MKKSKGGGEEDPHDDHGHKNRAAQLPPGSFGWPYIGETLQLYSQDPNTFFSSRQKRYGKIFKTHILGS
PCVMLASPEAAKFVLVTQAHLFKPTYPKSKEALIGPSALFFHHGDYHFRLRKLVQRSLSPDAIRNLVPHIDATAAS
VTSEWWGTGKVINTFHEMKKFSFEVGVLVIFGQLETRYKEELRKNYMAVNKGYNSFPINIPGTPYKKALLARERLR
HIIGDIIHERKEKRLPEKDLLGCLLRSINEGGEVLSDDQIADNIIGVLFAAQDTTASVMTWIFKYLHDEPKILEAV
KAEQNAIRLSNEQAGNQPLSWADTRNMPISYKVVLESLRLSSIISFLFREAVVDVEYKGYLIPKGWKVMPLFRNIH
HNPEFFTDPQKFDLSRFEVAPKPNTFMPFGSGVHACPGNELAKLELLIMIHHLVTNFRWEIEGSQSGTEYSPFPVP
LNGLPVKLWKLE；
Step 2, using pMD19-T-FveCYP707A4a plasmid as template, 326bp is left in design special primer amplification CDS
Right distinguished sequence, EcoRI with BamHI double digestion connect the downstream of the 2X35S promoter of pTRV2 carrier, form introne
(intron) the two-way hairpin structure separated.
A kind of application of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene, comprising: by FveCYP707A4a base
After being overexpressed, it is able to suppress strawberry fruit and expands, ABA content of dissociating in strawberry fruit decline.
The application of a kind of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene above-mentioned, by FveCYP707A4a
The method of gene overexpression includes the following steps:
Step 1 constructs the seamless cloning vector of In-Fusion；
The seamless cloning vector of In-Fusion is carried out qRT-PCR and quantified by step 2.
A kind of application of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene above-mentioned, step 1 construct In-
Fusion is seamless cloning vector,
Carrier framework: pCAMBIA1305；
Restriction enzyme site: upstream primer Mlu I, downstream primer Sal I；
Reaction system:
*<0.5kb:10–50ng,0.5to 10kb:50–100ng,>10kb:50–200ng；
**<10kb:50–100ng,>10kb:50–200ng。
A kind of application of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene above-mentioned, step 2, by In-
Fusion is seamless, and cloning vector progress qRT-PCR is quantitative,
QRT-PCR quantitative reaction system is as follows:
Response procedures:
One: 95 DEG C, 30s；Two: 95 DEG C, 5s；Three: 58 DEG C, 15s；Four: 72 DEG C, 10s；Five: solubility curve；
Circulation from two to three totally 40 circulation.
The invention has the beneficial effects that:
The present invention is according to the CDS sequence design pcr amplification primer of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene
Object pair；Using Fragaria vesca strawberry achene cDNA as template, using PCR amplification primer pair, obtained by PCR amplification
FveCYP707A4a gene complete sequence obtains the gene of control strawberry fruit size；
The present invention will be carried by the way that FveCYP707A4a gene complete sequence is constructed silent carrier or building over-express vector
Body infects strawberry after mixing with Agrobacterium, so that strawberry fruit expands the ABA content decline that is suppressed and dissociates in strawberry fruit,
And then adjust the development and maturation of strawberry fruit.
Detailed description of the invention
Fig. 1 is virus induction silent carrier (pTRV2-FveCYP707A4a carrier) figure of present invention building target gene
Spectrum；
Fig. 2 is the over-express vector pCAMBIA1305-FveCYP707A4a Vector map of present invention building target gene；
When Fig. 3 is using gene silencing methods, in control group, in empty plasmid control group and gene silencing group fruit at
The expression schematic diagram of the ripe critical period S5 and FveCYP707A4a in RS1 period；
When Fig. 4 is using gene silencing methods, in control group, fruit in empty plasmid control group and overexpression processing group
FveCYP707A4a expression schematic diagram；
When Fig. 5 is FveCYP707A4a virus induction expression silencing of the invention, in fruit maturation critical period S5 and RS1
The ABA changes of contents situation schematic diagram in period；
When Fig. 6 is that the present invention uses overexpression FveCYP707A4a carrier, the situation of change signal of ABA content in fruit
Figure；
When Fig. 7 is that the present invention uses overexpression FveCYP707A4a carrier, strawberry fruit phenotypic analysis result；
When Fig. 8 is that the present invention uses FveCYP707A4a silent carrier, strawberry fruit phenotypic analysis result.
Specific embodiment
Specific introduce is made to the present invention below in conjunction with the drawings and specific embodiments.
A kind of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene,
FveCYP707A4a gene complete sequence is as follows:
SEQ ID No.1:FveCYP707A4a
ATGAAGAAGAGCAAGGGAGGAGGAGAAGAAGACCCCCATGATGATCATGGTCATAAGAATAGGGCGGC
TCAGCTCCCTCCAGGCTCATTTGGTTGGCCTTATATCGGTGAGACCCTTCAGCTCTATTCTCAGGACCCAAACACT
TTCTTCTCTTCCAGACAGAAAAGGTATGGGAAAATTTTTAAGACACATATACTTGGGAGTCCATGTGTGATGCTGG
CGAGCCCGGAGGCTGCAAAGTTTGTATTGGTCACTCAAGCTCACTTGTTCAAGCCCACCTATCCCAAAAGCAAAGA
GGCTCTGATTGGTCCCTCCGCATTATTTTTCCACCATGGAGATTACCATTTCAGACTGAGGAAGCTTGTTCAGCGA
TCTCTCAGTCCTGATGCTATTCGGAATTTGGTGCCCCATATCGACGCCACAGCTGCCTCTGTGACCTCGGAATGGT
GGGGCACCGGGAAAGTCATCAACACCTTCCATGAGATGAAGAAGTTTTCTTTCGAAGTTGGTGTACTAGTAATTTT
TGGCCAATTGGAGACCCGCTACAAAGAAGAACTGAGGAAAAACTATATGGCAGTGAACAAAGGCTACAATTCATTT
CCCATAAACATTCCTGGAACGCCATACAAAAAGGCTTTGTTGGCGAGGGAGAGGCTGAGGCACATTATCGGTGACA
TTATCCATGAGAGAAAGGAGAAGAGGTTACCTGAAAAGGATCTGTTGGGTTGTTTGCTGAGATCAATAAACGAAGG
AGGGGAAGTTTTGAGTGATGACCAAATCGCAGACAACATAATAGGTGTTCTCTTTGCTGCTCAAGACACCACAGCC
AGTGTCATGACCTGGATTTTCAAGTACCTCCATGACGAACCAAAAATCCTAGAAGCTGTTAAGGCCGAACAAAATG
CAATTCGCCTATCAAATGAACAAGCAGGTAACCAACCATTGAGTTGGGCAGACACCAGAAACATGCCAATTAGTTA
CAAGGTTGTGTTGGAGAGTTTGAGACTGTCAAGCATTATATCATTCCTTTTTAGAGAAGCTGTGGTTGATGTGGAG
TACAAAGGTTACTTGATTCCAAAAGGTTGGAAGGTGATGCCTTTGTTCAGGAACATTCATCATAATCCTGAATTCT
TCACCGACCCTCAGAAATTCGATCTTTCTAGATTCGAGGTTGCACCAAAGCCAAATACATTTATGCCATTTGGCAG
TGGAGTCCATGCTTGTCCAGGAAACGAGCTTGCTAAGCTGGAATTACTGATCATGATCCACCATTTAGTCACCAAT
TTCAGGTGGGAAATTGAGGGATCCCAAAGCGGGACCGAGTATAGTCCATTTCCTGTACCTCTGAATGGACTTCCGG
TCAAACTTTGGAAATTAGAATAG；
Amino acid sequence is as follows:
SEQ ID No.2:
MKKSKGGGEEDPHDDHGHKNRAAQLPPGSFGWPYIGETLQLYSQDPNTFFSSRQKRYGKIFKTHILGS
PCVMLASPEAAKFVLVTQAHLFKPTYPKSKEALIGPSALFFHHGDYHFRLRKLVQRSLSPDAIRNLVPHIDATAAS
VTSEWWGTGKVINTFHEMKKFSFEVGVLVIFGQLETRYKEELRKNYMAVNKGYNSFPINIPGTPYKKALLARERLR
HIIGDIIHERKEKRLPEKDLLGCLLRSINEGGEVLSDDQIADNIIGVLFAAQDTTASVMTWIFKYLHDEPKILEAV
KAEQNAIRLSNEQAGNQPLSWADTRNMPISYKVVLESLRLSSIISFLFREAVVDVEYKGYLIPKGWKVMPLFRNIH
HNPEFFTDPQKFDLSRFEVAPKPNTFMPFGSGVHACPGNELAKLELLIMIHHLVTNFRWEIEGSQSGTEYSPFPVP
LNGLPVKLWKLE。
The sequencing approach of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene includes the following steps:
Step 1, according to the CDS sequence design PCR amplification of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene
Primer pair, as shown in SEQ ID No.3 and No.4:
SEQ ID No.3:(MluI) ACGCGTATGAAGAAGAGCAAGGGAGG
SEQ ID No.4:(SalI) GTCGACCTATTCTAATTTCCAAAGTTTGACC；
Step 2, using PCR amplification primer pair, is expanded using Fragaria vesca strawberry achene cDNA as template by PCR
Increase and obtains FveCYP707A4a gene complete sequence；
PCR amplification program are as follows: 94 DEG C, 4min carries out initial denaturation；94 DEG C, 40s denaturation；Anneal 30s under 60 DEG C of items；At 72 DEG C
Extend 1rnin；30 cyclic amplifications, 72 DEG C re-extend 10min reaction was completed.
△ as one embodiment, wrap by a kind of application of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene
It includes: after FveCYP707A4a gene silencing, being able to suppress strawberry fruit and expand, ABA content of dissociating in strawberry fruit decline.
By the method for FveCYP707A4a gene silencing are as follows: utilize the method for Gene Silencing, building
The Tobacco rattle virus silent carrier of FveCYP707A4a, after silent carrier is transferred to Agrobacterium, then with microinjection method infect
The green fruit of strawberry children, establishes the Transgenic Strawberry strain of virus induction FveCYP707A4a gene silencing.
The method of Gene Silencing includes:
FveCYP707A4a gene complete sequence is cloned into expression vector with the method for infusion by step 1
PCAMBIA1305, sequencing are correct；
FveCYP707A4a gene complete sequence is as follows:
SEQ ID No.1:FveCYP707A4a
ATGAAGAAGAGCAAGGGAGGAGGAGAAGAAGACCCCCATGATGATCATGGTCATAAGAATAGGGCGGC
TCAGCTCCCTCCAGGCTCATTTGGTTGGCCTTATATCGGTGAGACCCTTCAGCTCTATTCTCAGGACCCAAACACT
TTCTTCTCTTCCAGACAGAAAAGGTATGGGAAAATTTTTAAGACACATATACTTGGGAGTCCATGTGTGATGCTGG
CGAGCCCGGAGGCTGCAAAGTTTGTATTGGTCACTCAAGCTCACTTGTTCAAGCCCACCTATCCCAAAAGCAAAGA
GGCTCTGATTGGTCCCTCCGCATTATTTTTCCACCATGGAGATTACCATTTCAGACTGAGGAAGCTTGTTCAGCGA
TCTCTCAGTCCTGATGCTATTCGGAATTTGGTGCCCCATATCGACGCCACAGCTGCCTCTGTGACCTCGGAATGGT
GGGGCACCGGGAAAGTCATCAACACCTTCCATGAGATGAAGAAGTTTTCTTTCGAAGTTGGTGTACTAGTAATTTT
TGGCCAATTGGAGACCCGCTACAAAGAAGAACTGAGGAAAAACTATATGGCAGTGAACAAAGGCTACAATTCATTT
CCCATAAACATTCCTGGAACGCCATACAAAAAGGCTTTGTTGGCGAGGGAGAGGCTGAGGCACATTATCGGTGACA
TTATCCATGAGAGAAAGGAGAAGAGGTTACCTGAAAAGGATCTGTTGGGTTGTTTGCTGAGATCAATAAACGAAGG
AGGGGAAGTTTTGAGTGATGACCAAATCGCAGACAACATAATAGGTGTTCTCTTTGCTGCTCAAGACACCACAGCC
AGTGTCATGACCTGGATTTTCAAGTACCTCCATGACGAACCAAAAATCCTAGAAGCTGTTAAGGCCGAACAAAATG
CAATTCGCCTATCAAATGAACAAGCAGGTAACCAACCATTGAGTTGGGCAGACACCAGAAACATGCCAATTAGTTA
CAAGGTTGTGTTGGAGAGTTTGAGACTGTCAAGCATTATATCATTCCTTTTTAGAGAAGCTGTGGTTGATGTGGAG
TACAAAGGTTACTTGATTCCAAAAGGTTGGAAGGTGATGCCTTTGTTCAGGAACATTCATCATAATCCTGAATTCT
TCACCGACCCTCAGAAATTCGATCTTTCTAGATTCGAGGTTGCACCAAAGCCAAATACATTTATGCCATTTGGCAG
TGGAGTCCATGCTTGTCCAGGAAACGAGCTTGCTAAGCTGGAATTACTGATCATGATCCACCATTTAGTCACCAAT
TTCAGGTGGGAAATTGAGGGATCCCAAAGCGGGACCGAGTATAGTCCATTTCCTGTACCTCTGAATGGACTTCCGG
TCAAACTTTGGAAATTAGAATAG；
Amino acid sequence is as follows:
SEQ ID No.2:
MKKSKGGGEEDPHDDHGHKNRAAQLPPGSFGWPYIGETLQLYSQDPNTFFSSRQKRYGKIFKTHILGS
PCVMLASPEAAKFVLVTQAHLFKPTYPKSKEALIGPSALFFHHGDYHFRLRKLVQRSLSPDAIRNLVPHIDATAAS
VTSEWWGTGKVINTFHEMKKFSFEVGVLVIFGQLETRYKEELRKNYMAVNKGYNSFPINIPGTPYKKALLARERLR
HIIGDIIHERKEKRLPEKDLLGCLLRSINEGGEVLSDDQIADNIIGVLFAAQDTTASVMTWIFKYLHDEPKILEAV
KAEQNAIRLSNEQAGNQPLSWADTRNMPISYKVVLESLRLSSIISFLFREAVVDVEYKGYLIPKGWKVMPLFRNIH
HNPEFFTDPQKFDLSRFEVAPKPNTFMPFGSGVHACPGNELAKLELLIMIHHLVTNFRWEIEGSQSGTEYSPFPVP
LNGLPVKLWKLE；
Step 2 designs special primer SEQ ID No.5 and No.6 using pMD19-T-FveCYP707A4a plasmid as template
The distinguished sequence of 326bp or so in CDS is expanded, EcoRI with BamHI double digestion connects under the 2X35S promoter of pTRV2 carrier
Trip forms the two-way hairpin structure that introne (intron) is separated.
As one embodiment, shown in SEQ ID No.5 and No.6:
SEQ ID No.5:(EcorI) GAATTCTTGGTCCCTCCGCATTATT；
SEQ ID No.6:(BamHI) GGATCCTTTGTATGGCGTTCCAGGA.
As shown in Fig. 2, FveCYP707A4a+TRV2.
The method of microinjection are as follows: select Agrobacterium single bacterium fall within 5ml LB liquid medium (rifampin containing 100mg/ml,
50mg/ml kanamycins, 40mg/ml gentamicin, 10mM MES PH5.5,20 μM of acetosyringones) in 28 DEG C of 100r/min
Cultivate 12h；Being inoculated in 50ml LB liquid medium, (containing 1 00mg/ml rifampin, 50mg/ml kanamycins, 40mg/ml celebrating are big
Mycin, 10mM MES PH5.5,20 μM of acetosyringones) in culture OD600 value to 0.8-1.0；Then 4 DEG C, 4000 × g centrifugation
5min abandons supernatant precipitating and is washed with dip dyeing buffer (10mM Mgcl2,10mM MES PH5.5,200 μM of acetosyringones) suspension
Bacterium is repeated once.It suspends and adjusts OD600To 1.0-2.0,25 DEG C, 50r/min shakes 4h.PTRVl and pTRV2 or pTRV2-
FaMYB utilizes the sterile micro syringe of 0.5ml after mixing in the ratio of 1:l, 300- is injected at base of fruit in the strawberry gingko phase
500 μ l, condition of culture are 25 DEG C/16h on daytime, 15 DEG C/8h of night, relative humidity 70-90%.It observes after a week.
△ as another embodiment, wrap by a kind of application of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene
It includes: after FveCYP707A4a gene overexpression, being able to suppress strawberry fruit and expand, ABA content of dissociating in strawberry fruit decline.
The method of FveCYP707A4a gene overexpression is included the following steps:
Step 1 constructs the seamless cloning vector of In-Fusion,
Method: In-Fusion Cloning
Reagent:HD Cloning Kit
Company: A Takara Bio Company,Laboratories,Inc.
Article No.: Cat.Nos.Many (011614)
Carrier framework: pCAMBIA1305
Restriction enzyme site: upstream primer Mlu I, downstream primer Sal I；
Primer:
Upstream primer F:(Mlu I) cgccCCTCAGCacgcgt+ATGAAGAAGAGCAAGGGAGG；
Downstream primer R:(Sal I) ATGCCTGCAGGTCGAC+CTATTCTAATTTCCAAAGTTTGACC.
The seamless cloning vector of In-Fusion is carried out qRT-PCR and quantified by step 2,
Mix is Takara companyPremix Ex TaqTM II (article No.: RR820A)；Instrument are as follows:
Bio-RAD CFX Connect Real-Time System。
QRT-PCR quantitative reaction system is as follows:
Response procedures:
One: 95 DEG C, 30s；Two: 95 DEG C, 5s；Three: 58 DEG C, 15s；Four: 72 DEG C, 10s；Five: solubility curve；Circulation from two to
Three totally 40 circulation；
The fluorescent quantitation primer of use, as follows:
Upstream region of gene primer (5 ' -3 '), downstream primer (5 ' -3 ')；
ACTIN GCCAGAAAGATGCTTATGTCGGTG, GGGGCAACACGAAGCTCAT；
FveCYP707A4a CCCGCTACAAAGAAGAACTGA, TGTCACCGATAATGTGCCTC；
Step 3 establishes virus after over-express vector is transferred to Agrobacterium, then with the green fruit of microinjection method infection strawberry children
Induce the Transgenic Strawberry strain of FveCYP707A4a gene overexpression.
The method of microinjection are as follows: select Agrobacterium single bacterium fall within 5ml LB liquid medium (rifampin containing 100mg/ml,
50mg/ml kanamycins, 40mg/ml gentamicin, 10mM MES PH5.5,20 μM of acetosyringones) in 28 DEG C of 100r/min
Cultivate 12h；Being inoculated in 50ml LB liquid medium, (containing 1 00mg/ml rifampin, 50mg/ml kanamycins, 40mg/ml celebrating are big
Mycin, 10mM MES PH5.5,20 μM of acetosyringones) in culture OD600 value to 0.8-1.0；Then 4 DEG C, 4000 × g centrifugation
5min abandons supernatant precipitating and is washed with dip dyeing buffer (10mM Mgcl2,10mM MES PH5.5,200 μM of acetosyringones) suspension
Bacterium is repeated once.It suspends and adjusts OD600To 1.0-2.0,25 DEG C, 50r/min shakes 4h.PTRVl and pTRV2 or pTRV2-
FaMYB utilizes the sterile micro syringe of 0.5ml after mixing in the ratio of 1:l, 300- is injected at base of fruit in the strawberry gingko phase
500 μ l, condition of culture are 25 DEG C/16h on daytime, 15 DEG C/8h of night, relative humidity 70-90%.It observes after a week.
Experimental verification part:
The identification experiment of Gene Silencing strawberry:
In order to detect whether the strawberry infected converts success, wild type is extracted respectively and is converted the total of strawberry fruit
RNA determines whether conversion succeeds using the expression of the method detection target gene FveCYP707A4a of QRT-PRC with this.
If the expression of FveCYP707A4a significantly reduces after gene silencing processing；And after doing overexpression processing,
The expression of FveCYP707A4a significantly increases；Show to convert successfully.
The specific method of QRT-PRC includes:
Mix is Takara companyPremix Ex TaqTM II (article No.: RR820A)；Instrument are as follows:
Bio-RAD CFX Connect Real-Time System。
QRT-PCR quantitative reaction system is as follows:
Response procedures:
One: 95 DEG C, 30s；Two: 95 DEG C, 5s；Three: 58 DEG C, 15s；Four: 72 DEG C, 10s；Five: solubility curve；Circulation from two to
Three totally 40 circulation；
Primer used in QRT-PCR:
PG:F:GGTGGTGCCATTGAACACTT, R:CAAGAAACTCAGCCAGGTGG；
PL:F:TCAACTCGTCAATGGCAGAC, R:GGTCACATCTCCAGCAGTCA；
CEL2:F:CGAGTTTGGTTGGGATAACA, R:AAGTAGGAACGAGAGCGAAGTT；
The qualification result for the strawberry that silenced gene expression carrier infects is as shown in figure 3, silencing success；
The qualification result for the strawberry that gene overexpression carrier infects is as shown in figure 4, be overexpressed successfully.
FveCYP707A4a virus induction expression silencing influences to test on ABA changes of contents；
Two experiments are done,
The raw material of experiment one is:
Used in the strawberry in S5 period fruit maturation critical period:
Group 1-1: the strawberry of conversion processing is not done as non-treated group；
Group 2-1: conversion does not carry the strawberry of the empty plasmid of any gene as empty plasmid control group；
The group 3-1 strawberry of using silencing FveCYP707A4a genophore to infect is as overexpression processing group (OE)；
Used in the strawberry in RS1 period fruit maturation critical period:
Group 1-2: the strawberry of conversion processing is not done as non-treated group；
Group 2-2: conversion does not carry the strawberry of the empty plasmid of any gene as empty plasmid control group；
The group 3-2 strawberry of using silencing FveCYP707A4a genophore to infect is as overexpression processing group (OE)；
The raw material of experiment two is:
Group 1: the strawberry of conversion processing is not done as non-treated group；
Group 2: conversion does not carry the strawberry of the empty plasmid of any gene as empty plasmid control group；
Group 3, which use, is overexpressed the strawberries infected of FveCYP707A4a genophore as overexpression processing group (OE)；
Carry out the detection of ABA content respectively according to grouping above.
Detect the experimental procedure of ABA content in fruit:
1, by each group of strawberry by liquid nitrogen grinding at powdered, grouping is proceeded as follows；
2, every group takes 100mg strawberry to be fitted into 6 centrifuge tubes respectively, and 100 μ L isotopes are added into 6 centrifuge tubes；
3,100 μ L heterotopes and 800 μ L methanol are added into 3 centrifuge tubes respectively, and add in the other three centrifuge tube
Enter 900 μ L methanol；
4, sufficiently after oscillation, -20 spend night；
5, second day oscillation sample, ultrasonic 2min, centrifugation；Supernatant is transferred in the centrifuge tube of 2ml specification；
6,0.5ml70% methanol is added into precipitating, oscillation, ultrasonic 5min；
7, centrifugation, supernatant moves in the centrifuge tube in 5 steps, is concentrated into 300 μ L；
8,1% formic acid water (about 3 hours) of 700 μ L is added into the concentrate of step 7, is centrifuged 3min；
9, prepare Water MCX column, 2ml methanol, 2ml0.1MHCl, 2ml 1%FA is added；
10, loading (sample in step 8)
11,2ml1%FA is added, cleans impurity, 2ml methanol (collects fraction3:IAA, ABA, JA, SA, GA, i-
IAA, i-ABA), 2ml methanol, 2ml2% ammonium hydroxide, in methanol (collection fraction5: containing tz, i-tz).
Result is quantitatively obtained as shown in Figure 5 by chromatograph: the FveCYP707A4a virus induction expression silencing in this experiment
When, fruit maturation closes in non-treated group (control), empty plasmid control group (empty vector) and RNAi processing group
The ABA changes of contents situation of key period S5 and RS1 period, it is therefore seen that RNAi processing group ABA changes of contents is big.
Result is quantitatively obtained by chromatograph as shown in fig. 6, in this experiment be overexpressed FveCYP707A4a gene when, nowhere
Reason group is not done conversion processing (control), empty plasmid control group, conversion does not carry the empty plasmid of any gene
In (empty vector) and (conversion with target gene) overexpression processing group (OE) in fruit ABA content variation feelings
Condition；After over-express vector infects, the content of ABA is significantly reduced.
Experiment three, from the influence of phenetic analysis FveCYP707A4a gene pairs strawberry fruit:
The influence of strawberry fruit size is as shown in fig. 7, be the overexpression FveCYP707A4a in this experiment, strawberry fruit table
Type analysis is as a result, at non-treated group (control), in empty plasmid control group (empty vector) and overexpression processing group
The situation of change of the form of fruit processing after a week.Compared with the control, it is obvious to be overexpressed FveCYP707A4a gene Fruit
It is suppressed.
As shown in figure 8, after for the FveCYP707A4a gene silencing in this experiment, strawberry fruit phenotypic analysis as a result,
Non-treated group (control), fruit processing one in empty plasmid control group (empty vector) and gene silencing processing group
The situation of change of form after week.Compared with the control, the elongation of Gene Silencing fruit is obvious suppressed, and Fruit
Enhancing.
Protovirus is infected into strawberry fruit with wild type compared with the strawberry fruit of developmental stage, discovery is containing original
There is slight depression at the top of fruit after the Agrobacterium injection of TRV virus, furthermore Fruit and grown form, which are not affected by, significantly affects.
Further phenotypic analysis discovery, the strawberry fruit of injection recombinant viral vector Agrobacterium, which expands, to be suppressed, part fruit warp
Growth in one month is crossed almost without becoming larger, the small green fruit stage is still within, in addition has inhibition suffered by the Fruit of part to want
It is slight.
The present invention is according to the CDS sequence design pcr amplification primer of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene
Object pair；Using Fragaria vesca strawberry achene cDNA as template, using PCR amplification primer pair, obtained by PCR amplification
FveCYP707A4a gene complete sequence obtains the gene of control strawberry fruit size；Again by the way that FveCYP707A4a gene is complete
Sequence construct silent carrier or building over-express vector, infect strawberry after carrier is mixed with Agrobacterium, so that strawberry fruit
The ABA content decline that is suppressed and dissociates in strawberry fruit is expanded, and then adjusts the development and maturation of strawberry fruit.
The basic principles, main features and advantages of the invention have been shown and described above.The technical staff of the industry should
Understand, the above embodiments do not limit the invention in any form, all obtained by the way of equivalent substitution or equivalent transformation
Technical solution is fallen within the scope of protection of the present invention.
Sequence table
<110>Zhejiang pentahapto Biotechnology Co., Ltd
<120>a kind of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene and its application
<141> 2018-12-05
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1383
<212> DNA
<213> Fragaria vesca
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atgaagaaga gcaagggagg aggagaagaa gacccccatg atgatcatgg tcataagaat 60
agggcggctc agctccctcc aggctcattt ggttggcctt atatcggtga gacccttcag 120
ctctattctc aggacccaaa cactttcttc tcttccagac agaaaaggta tgggaaaatt 180
tttaagacac atatacttgg gagtccatgt gtgatgctgg cgagcccgga ggctgcaaag 240
tttgtattgg tcactcaagc tcacttgttc aagcccacct atcccaaaag caaagaggct 300
ctgattggtc cctccgcatt atttttccac catggagatt accatttcag actgaggaag 360
cttgttcagc gatctctcag tcctgatgct attcggaatt tggtgcccca tatcgacgcc 420
acagctgcct ctgtgacctc ggaatggtgg ggcaccggga aagtcatcaa caccttccat 480
gagatgaaga agttttcttt cgaagttggt gtactagtaa tttttggcca attggagacc 540
cgctacaaag aagaactgag gaaaaactat atggcagtga acaaaggcta caattcattt 600
cccataaaca ttcctggaac gccatacaaa aaggctttgt tggcgaggga gaggctgagg 660
cacattatcg gtgacattat ccatgagaga aaggagaaga ggttacctga aaaggatctg 720
ttgggttgtt tgctgagatc aataaacgaa ggaggggaag ttttgagtga tgaccaaatc 780
gcagacaaca taataggtgt tctctttgct gctcaagaca ccacagccag tgtcatgacc 840
tggattttca agtacctcca tgacgaacca aaaatcctag aagctgttaa ggccgaacaa 900
aatgcaattc gcctatcaaa tgaacaagca ggtaaccaac cattgagttg ggcagacacc 960
agaaacatgc caattagtta caaggttgtg ttggagagtt tgagactgtc aagcattata 1020
tcattccttt ttagagaagc tgtggttgat gtggagtaca aaggttactt gattccaaaa 1080
ggttggaagg tgatgccttt gttcaggaac attcatcata atcctgaatt cttcaccgac 1140
cctcagaaat tcgatctttc tagattcgag gttgcaccaa agccaaatac atttatgcca 1200
tttggcagtg gagtccatgc ttgtccagga aacgagcttg ctaagctgga attactgatc 1260
atgatccacc atttagtcac caatttcagg tgggaaattg agggatccca aagcgggacc 1320
gagtatagtc catttcctgt acctctgaat ggacttccgg tcaaactttg gaaattagaa 1380
tag 1383
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<211> 460
<212> PRT
<213> Fragaria vesca
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20 25 30
Pro Tyr Ile Gly Glu Thr Leu Gln Leu Tyr Ser Gln Asp Pro Asn Thr
35 40 45
Phe Phe Ser Ser Arg Gln Lys Arg Tyr Gly Lys Ile Phe Lys Thr His
50 55 60
Ile Leu Gly Ser Pro Cys Val Met Leu Ala Ser Pro Glu Ala Ala Lys
65 70 75 80
Phe Val Leu Val Thr Gln Ala His Leu Phe Lys Pro Thr Tyr Pro Lys
85 90 95
Ser Lys Glu Ala Leu Ile Gly Pro Ser Ala Leu Phe Phe His His Gly
100 105 110
Asp Tyr His Phe Arg Leu Arg Lys Leu Val Gln Arg Ser Leu Ser Pro
115 120 125
Asp Ala Ile Arg Asn Leu Val Pro His Ile Asp Ala Thr Ala Ala Ser
130 135 140
Val Thr Ser Glu Trp Trp Gly Thr Gly Lys Val Ile Asn Thr Phe His
145 150 155 160
Glu Met Lys Lys Phe Ser Phe Glu Val Gly Val Leu Val Ile Phe Gly
165 170 175
Gln Leu Glu Thr Arg Tyr Lys Glu Glu Leu Arg Lys Asn Tyr Met Ala
180 185 190
Val Asn Lys Gly Tyr Asn Ser Phe Pro Ile Asn Ile Pro Gly Thr Pro
195 200 205
Tyr Lys Lys Ala Leu Leu Ala Arg Glu Arg Leu Arg His Ile Ile Gly
210 215 220
Asp Ile Ile His Glu Arg Lys Glu Lys Arg Leu Pro Glu Lys Asp Leu
225 230 235 240
Leu Gly Cys Leu Leu Arg Ser Ile Asn Glu Gly Gly Glu Val Leu Ser
245 250 255
Asp Asp Gln Ile Ala Asp Asn Ile Ile Gly Val Leu Phe Ala Ala Gln
260 265 270
Asp Thr Thr Ala Ser Val Met Thr Trp Ile Phe Lys Tyr Leu His Asp
275 280 285
Glu Pro Lys Ile Leu Glu Ala Val Lys Ala Glu Gln Asn Ala Ile Arg
290 295 300
Leu Ser Asn Glu Gln Ala Gly Asn Gln Pro Leu Ser Trp Ala Asp Thr
305 310 315 320
Arg Asn Met Pro Ile Ser Tyr Lys Val Val Leu Glu Ser Leu Arg Leu
325 330 335
Ser Ser Ile Ile Ser Phe Leu Phe Arg Glu Ala Val Val Asp Val Glu
340 345 350
Tyr Lys Gly Tyr Leu Ile Pro Lys Gly Trp Lys Val Met Pro Leu Phe
355 360 365
Arg Asn Ile His His Asn Pro Glu Phe Phe Thr Asp Pro Gln Lys Phe
370 375 380
Asp Leu Ser Arg Phe Glu Val Ala Pro Lys Pro Asn Thr Phe Met Pro
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<213> artificial gene
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Claims (10)
1. a kind of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene, which is characterized in that
FveCYP707A4a gene complete sequence is as follows:
SEQ ID No.1:FveCYP707A4a
ATGAAGAAGAGCAAGGGAGGAGGAGAAGAAGACCCCCATGATGATCATGGTCATAAGAATAGGGCGGCTCAG
CTCCCTCCAGGCTCATTTGGTTGGCCTTATATCGGTGAGACCCTTCAGCTCTATTCTCAGGACCCAAACACTTTCT
TCTCTTCCAGACAGAAAAGGTATGGGAAAATTTTTAAGACACATATACTTGGGAGTCCATGTGTGATGCTGGCGAG
CCCGGAGGCTGCAAAGTTTGTATTGGTCACTCAAGCTCACTTGTTCAAGCCCACCTATCCCAAAAGCAAAGAGGCT
CTGATTGGTCCCTCCGCATTATTTTTCCACCATGGAGATTACCATTTCAGACTGAGGAAGCTTGTTCAGCGATCTC
TCAGTCCTGATGCTATTCGGAATTTGGTGCCCCATATCGACGCCACAGCTGCCTCTGTGACCTCGGAATGGTGGGG
CACCGGGAAAGTCATCAACACCTTCCATGAGATGAAGAAGTTTTCTTTCGAAGTTGGTGTACTAGTAATTTTTGGC
CAATTGGAGACCCGCTACAAAGAAGAACTGAGGAAAAACTATATGGCAGTGAACAAAGGCTACAATTCATTTCCCA
TAAACATTCCTGGAACGCCATACAAAAAGGCTTTGTTGGCGAGGGAGAGGCTGAGGCACATTATCGGTGACATTAT
CCATGAGAGAAAGGAGAAGAGGTTACCTGAAAAGGATCTGTTGGGTTGTTTGCTGAGATCAATAAACGAAGGAGGG
GAAGTTTTGAGTGATGACCAAATCGCAGACAACATAATAGGTGTTCTCTTTGCTGCTCAAGACACCACAGCCAGTG
TCATGACCTGGATTTTCAAGTACCTCCATGACGAACCAAAAATCCTAGAAGCTGTTAAGGCCGAACAAAATGCAAT
TCGCCTATCAAATGAACAAGCAGGTAACCAACCATTGAGTTGGGCAGACACCAGAAACATGCCAATTAGTTACAAG
GTTGTGTTGGAGAGTTTGAGACTGTCAAGCATTATATCATTCCTTTTTAGAGAAGCTGTGGTTGATGTGGAGTACA
AAGGTTACTTGATTCCAAAAGGTTGGAAGGTGATGCCTTTGTTCAGGAACATTCATCATAATCCTGAATTCTTCAC
CGACCCTCAGAAATTCGATCTTTCTAGATTCGAGGTTGCACCAAAGCCAAATACATTTATGCCATTTGGCAGTGGA
GTCCATGCTTGTCCAGGAAACGAGCTTGCTAAGCTGGAATTACTGATCATGATCCACCATTTAGTCACCAATTTCA
GGTGGGAAATTGAGGGATCCCAAAGCGGGACCGAGTATAGTCCATTTCCTGTACCTCTGAATGGACTTCCGGTCAA
ACTTTGGAAATTAGAATAG；
Amino acid sequence is as follows:
SEQ ID No.2:
MKKSKGGGEEDPHDDHGHKNRAAQLPPGSFGWPYIGETLQLYSQDPNTFFSSRQKRYGKIFKTHILGSPCVM
LASPEAAKFVLVTQAHLFKPTYPKSKEALIGPSALFFHHGDYHFRLRKLVQRSLSPDAIRNLVPHIDATAASVTSE
WWGTGKVINTFHEMKKFSFEVGVLVIFGQLETRYKEELRKNYMAVNKGYNSFPINIPGTPYKKALLARERLRHIIG
DIIHERKEKRLPEKDLLGCLLRSINEGGEVLSDDQIADNIIGVLFAAQDTTASVMTWIFKYLHDEPKILEAVKAEQ
NAIRLSNEQAGNQPLSWADTRNMPISYKVVLESLRLSSIISFLFREAVVDVEYKGYLIPKGWKVMPLFRNIHHNPE
FFTDPQKFDLSRFEVAPKPNTFMPFGSGVHACPGNELAKLELLIMIHHLVTNFRWEIEGSQSGTEYSPFPVPLNGL
PVKLWKLE。
2. a kind of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene according to claim 1, feature exist
In sequencing approach includes the following steps:
Step 1, according to the CDS sequence design PCR amplification primer of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene
It is right；
Step 2, using PCR amplification primer pair, is obtained using Fragaria vesca strawberry achene cDNA as template by PCR amplification
Obtain FveCYP707A4a gene complete sequence.
3. a kind of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene according to claim 2, feature exist
In PCR amplification program are as follows: 94 DEG C, 4min carries out initial denaturation；94 DEG C, 40s denaturation；Anneal 30s under 60 DEG C of items；Extend at 72 DEG C
1rnin；30 cyclic amplifications, 72 DEG C re-extend 10min reaction was completed.
4. a kind of application of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene characterized by comprising will
After FveCYP707A4a gene silencing, it is able to suppress strawberry fruit and expands, ABA content of dissociating in strawberry fruit decline.
5. a kind of application of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene according to claim 4, special
Sign is, by the method for FveCYP707A4a gene silencing are as follows: utilizes the method for Gene Silencing, building
The Tobacco rattle virus silent carrier of FveCYP707A4a, after silent carrier is transferred to Agrobacterium, then with microinjection method infect
The green fruit of strawberry children, establishes the Transgenic Strawberry strain of virus induction FveCYP707A4a gene silencing.
6. a kind of application of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene according to claim 5, special
Sign is that the method for the Gene Silencing includes:
FveCYP707A4a gene complete sequence is cloned into expression vector pCAMBIA1305 with the method for infusion by step 1,
Sequencing is correct；
FveCYP707A4a gene complete sequence is as follows:
SEQ ID No.1:FveCYP707A4a
ATGAAGAAGAGCAAGGGAGGAGGAGAAGAAGACCCCCATGATGATCATGGTCATAAGAATAGGGCGGCTCAG
CTCCCTCCAGGCTCATTTGGTTGGCCTTATATCGGTGAGACCCTTCAGCTCTATTCTCAGGACCCAAACACTTTCT
TCTCTTCCAGACAGAAAAGGTATGGGAAAATTTTTAAGACACATATACTTGGGAGTCCATGTGTGATGCTGGCGAG
CCCGGAGGCTGCAAAGTTTGTATTGGTCACTCAAGCTCACTTGTTCAAGCCCACCTATCCCAAAAGCAAAGAGGCT
CTGATTGGTCCCTCCGCATTATTTTTCCACCATGGAGATTACCATTTCAGACTGAGGAAGCTTGTTCAGCGATCTC
TCAGTCCTGATGCTATTCGGAATTTGGTGCCCCATATCGACGCCACAGCTGCCTCTGTGACCTCGGAATGGTGGGG
CACCGGGAAAGTCATCAACACCTTCCATGAGATGAAGAAGTTTTCTTTCGAAGTTGGTGTACTAGTAATTTTTGGC
CAATTGGAGACCCGCTACAAAGAAGAACTGAGGAAAAACTATATGGCAGTGAACAAAGGCTACAATTCATTTCCCA
TAAACATTCCTGGAACGCCATACAAAAAGGCTTTGTTGGCGAGGGAGAGGCTGAGGCACATTATCGGTGACATTAT
CCATGAGAGAAAGGAGAAGAGGTTACCTGAAAAGGATCTGTTGGGTTGTTTGCTGAGATCAATAAACGAAGGAGGG
GAAGTTTTGAGTGATGACCAAATCGCAGACAACATAATAGGTGTTCTCTTTGCTGCTCAAGACACCACAGCCAGTG
TCATGACCTGGATTTTCAAGTACCTCCATGACGAACCAAAAATCCTAGAAGCTGTTAAGGCCGAACAAAATGCAAT
TCGCCTATCAAATGAACAAGCAGGTAACCAACCATTGAGTTGGGCAGACACCAGAAACATGCCAATTAGTTACAAG
GTTGTGTTGGAGAGTTTGAGACTGTCAAGCATTATATCATTCCTTTTTAGAGAAGCTGTGGTTGATGTGGAGTACA
AAGGTTACTTGATTCCAAAAGGTTGGAAGGTGATGCCTTTGTTCAGGAACATTCATCATAATCCTGAATTCTTCAC
CGACCCTCAGAAATTCGATCTTTCTAGATTCGAGGTTGCACCAAAGCCAAATACATTTATGCCATTTGGCAGTGGA
GTCCATGCTTGTCCAGGAAACGAGCTTGCTAAGCTGGAATTACTGATCATGATCCACCATTTAGTCACCAATTTCA
GGTGGGAAATTGAGGGATCCCAAAGCGGGACCGAGTATAGTCCATTTCCTGTACCTCTGAATGGACTTCCGGTCAA
ACTTTGGAAATTAGAATAG；
Amino acid sequence is as follows:
SEQ ID No.2:
MKKSKGGGEEDPHDDHGHKNRAAQLPPGSFGWPYIGETLQLYSQDPNTFFSSRQKRYGKIFKTHILGSPCVM
LASPEAAKFVLVTQAHLFKPTYPKSKEALIGPSALFFHHGDYHFRLRKLVQRSLSPDAIRNLVPHIDATAASVTSE
WWGTGKVINTFHEMKKFSFEVGVLVIFGQLETRYKEELRKNYMAVNKGYNSFPINIPGTPYKKALLARERLRHIIG
DIIHERKEKRLPEKDLLGCLLRSINEGGEVLSDDQIADNIIGVLFAAQDTTASVMTWIFKYLHDEPKILEAVKAEQ
NAIRLSNEQAGNQPLSWADTRNMPISYKVVLESLRLSSIISFLFREAVVDVEYKGYLIPKGWKVMPLFRNIHHNPE
FFTDPQKFDLSRFEVAPKPNTFMPFGSGVHACPGNELAKLELLIMIHHLVTNFRWEIEGSQSGTEYSPFPVPLNGL
PVKLWKLE；
Step 2, using pMD19-T-FveCYP707A4a plasmid as template, 326bp or so in design special primer amplification CDS
Distinguished sequence, EcoRI with BamHI double digestion connect the downstream of the 2X35S promoter of pTRV2 carrier, form introne
(intron) the two-way hairpin structure separated.
7. a kind of application of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene characterized by comprising will
After FveCYP707A4a gene overexpression, it is able to suppress strawberry fruit and expands, ABA content of dissociating in strawberry fruit decline.
8. a kind of application of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene according to claim 7, special
Sign is, the method for FveCYP707A4a gene overexpression is included the following steps:
Step 1 constructs the seamless cloning vector of In-Fusion；
The seamless cloning vector of In-Fusion is carried out qRT-PCR and quantified, obtains over-express vector by step 2；
Step 3 establishes virus induction after over-express vector is transferred to Agrobacterium, then with the green fruit of microinjection method infection strawberry children
The Transgenic Strawberry strain of FveCYP707A4a gene overexpression.
9. a kind of application of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene according to claim 7, special
Sign is that step 1 constructs the seamless cloning vector of In-Fusion,
Carrier framework: pCAMBIA1305；
Restriction enzyme site: upstream primer Mlu I, downstream primer Sal I；
Reaction system:
*<0.5kb:10–50ng,0.5to 10kb:50–100ng,>10kb:50–200ng；
**<10kb:50–100ng,>10kb:50–200ng。
10. a kind of application of strawberry ABA degradation pathway key enzyme FveCYP707A4a gene according to claim 7,
It is characterized in that, step 2, the seamless cloning vector of In-Fusion is subjected to qRT-PCR and is quantified,
QRT-PCR quantitative reaction system is as follows:
Response procedures:
One: 95 DEG C, 30s；Two: 95 DEG C, 5s；Three: 58 DEG C, 15s；Four: 72 DEG C, 10s；Five: solubility curve；Circulation is total to from two to three
40 circulations.
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| CN110396519A (en) * | 2019-06-27 | 2019-11-01 | 西北农林科技大学 | - 3 fruit maturation gene FvTCP9 of fraises des bois Heilungkiang and its application |
| CN111019911A (en) * | 2019-11-01 | 2020-04-17 | 浙江万里学院 | Protein of 8' -hydroxylase CYP707A protein critical to abscisic acid degradation pathway, coding gene and application thereof |
| CN114395019A (en) * | 2021-12-15 | 2022-04-26 | 山东农业大学 | Strawberry FvMYB79 gene and application thereof |
| CN114438102A (en) * | 2022-03-15 | 2022-05-06 | 扬州大学 | Strawberry ethylene response FaERF13 gene and application thereof in changing strawberry fruit mature period |
| CN114540407A (en) * | 2022-01-13 | 2022-05-27 | 安庆市长三角未来产业研究院 | Application of SlCYP707A gene as negative regulatory factor in promoting tomato resistance at sub-low temperature |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110396519A (en) * | 2019-06-27 | 2019-11-01 | 西北农林科技大学 | - 3 fruit maturation gene FvTCP9 of fraises des bois Heilungkiang and its application |
| CN111019911A (en) * | 2019-11-01 | 2020-04-17 | 浙江万里学院 | Protein of 8' -hydroxylase CYP707A protein critical to abscisic acid degradation pathway, coding gene and application thereof |
| CN114395019A (en) * | 2021-12-15 | 2022-04-26 | 山东农业大学 | Strawberry FvMYB79 gene and application thereof |
| CN114395019B (en) * | 2021-12-15 | 2023-06-16 | 山东农业大学 | Strawberry FvMYB79 gene and application thereof |
| CN114540407A (en) * | 2022-01-13 | 2022-05-27 | 安庆市长三角未来产业研究院 | Application of SlCYP707A gene as negative regulatory factor in promoting tomato resistance at sub-low temperature |
| CN114540407B (en) * | 2022-01-13 | 2023-11-28 | 安庆市长三角未来产业研究院 | Application of SlCYP707A gene as negative regulation factor in promotion of sub-low temperature resistance of tomatoes |
| CN114438102A (en) * | 2022-03-15 | 2022-05-06 | 扬州大学 | Strawberry ethylene response FaERF13 gene and application thereof in changing strawberry fruit mature period |
| CN114438102B (en) * | 2022-03-15 | 2023-06-16 | 扬州大学 | Strawberry ethylene response FaERF13 gene and application thereof in changing strawberry fruit maturity |
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