CN115896137A - Nanguo pear PuAAT gene, overexpression vector and application thereof - Google Patents
Nanguo pear PuAAT gene, overexpression vector and application thereof Download PDFInfo
- Publication number
- CN115896137A CN115896137A CN202211538503.5A CN202211538503A CN115896137A CN 115896137 A CN115896137 A CN 115896137A CN 202211538503 A CN202211538503 A CN 202211538503A CN 115896137 A CN115896137 A CN 115896137A
- Authority
- CN
- China
- Prior art keywords
- puaat
- gene
- nanguo pear
- pear
- overexpression vector
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 235000014443 Pyrus communis Nutrition 0.000 title claims abstract description 78
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 53
- 239000013598 vector Substances 0.000 title claims abstract description 37
- 230000002018 overexpression Effects 0.000 title claims abstract description 31
- 235000013399 edible fruits Nutrition 0.000 claims abstract description 50
- 241000589158 Agrobacterium Species 0.000 claims abstract description 21
- 239000003205 fragrance Substances 0.000 claims abstract description 14
- 239000002773 nucleotide Substances 0.000 claims abstract description 10
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 10
- 230000001404 mediated effect Effects 0.000 claims abstract description 8
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 4
- 239000013604 expression vector Substances 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- 239000002299 complementary DNA Substances 0.000 claims description 8
- 230000001052 transient effect Effects 0.000 claims description 7
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 238000010839 reverse transcription Methods 0.000 claims description 4
- 238000010276 construction Methods 0.000 claims description 3
- 208000015181 infectious disease Diseases 0.000 claims description 3
- 238000003208 gene overexpression Methods 0.000 claims 3
- 230000002708 enhancing effect Effects 0.000 claims 1
- 150000002148 esters Chemical class 0.000 abstract description 9
- 239000000126 substance Substances 0.000 abstract description 6
- 230000015572 biosynthetic process Effects 0.000 abstract description 5
- 238000003786 synthesis reaction Methods 0.000 abstract description 4
- OPCRGEVPIBLWAY-QNRZBPGKSA-N ethyl (2E,4Z)-deca-2,4-dienoate Chemical compound CCCCC\C=C/C=C/C(=O)OCC OPCRGEVPIBLWAY-QNRZBPGKSA-N 0.000 abstract description 3
- 230000006872 improvement Effects 0.000 abstract description 3
- 230000001105 regulatory effect Effects 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 101150038499 ATT gene Proteins 0.000 abstract description 2
- 230000001276 controlling effect Effects 0.000 abstract description 2
- 238000011426 transformation method Methods 0.000 abstract description 2
- 238000010835 comparative analysis Methods 0.000 abstract 1
- 230000001737 promoting effect Effects 0.000 abstract 1
- 230000009261 transgenic effect Effects 0.000 abstract 1
- 241000220324 Pyrus Species 0.000 description 55
- 230000000052 comparative effect Effects 0.000 description 9
- 108010083294 ethanol acyltransferase Proteins 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 230000014509 gene expression Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 230000009466 transformation Effects 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 4
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 description 4
- SHZIWNPUGXLXDT-UHFFFAOYSA-N ethyl hexanoate Chemical compound CCCCCC(=O)OCC SHZIWNPUGXLXDT-UHFFFAOYSA-N 0.000 description 4
- AOGQPLXWSUTHQB-UHFFFAOYSA-N hexyl acetate Chemical compound CCCCCCOC(C)=O AOGQPLXWSUTHQB-UHFFFAOYSA-N 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 235000021017 pears Nutrition 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 230000001131 transforming effect Effects 0.000 description 4
- 240000000220 Panda oleosa Species 0.000 description 3
- 235000016496 Panda oleosa Nutrition 0.000 description 3
- 101150037054 aat gene Proteins 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000009630 liquid culture Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- HNAGHMKIPMKKBB-UHFFFAOYSA-N 1-benzylpyrrolidine-3-carboxamide Chemical compound C1C(C(=O)N)CCN1CC1=CC=CC=C1 HNAGHMKIPMKKBB-UHFFFAOYSA-N 0.000 description 2
- 244000173166 Pyrus ussuriensis Species 0.000 description 2
- 235000011572 Pyrus ussuriensis Nutrition 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- OBNCKNCVKJNDBV-UHFFFAOYSA-N butanoic acid ethyl ester Natural products CCCC(=O)OCC OBNCKNCVKJNDBV-UHFFFAOYSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108700001094 Plant Genes Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 235000004789 Rosa xanthina Nutrition 0.000 description 1
- 241000220222 Rosaceae Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to a PuAAT gene of Nanguo pear, an overexpression vector and application thereof, belonging to the technical field of molecular biological gene engineering. In order to solve the problem that the prior art still fails to apply the south fruit pear ester fragrance related ATT gene to the improvement of fruit fragrance content, the invention provides the south fruit pear PuAAT gene, the nucleotide sequence of which is shown as SEQ ID No.1, and the amino acid sequence of which is shown as SEQ ID No. 2. The invention also provides an overexpression vector comprising the Nanguo pear PuAAT gene, the overexpression vector containing the Nanguo pear PuAAT gene is transferred into Nanguo pear fruits by an agrobacterium-mediated transformation method, research shows that the gene has the function of promoting ester fragrance synthesis of the Nanguo pear, and comparative analysis proves that the ester fragrance substance content of the transgenic fruits is obviously improved. The invention provides theoretical basis and technical means for regulating and controlling the fruit aroma and has great application value.
Description
Technical Field
The invention belongs to the technical field of molecular biological gene engineering, and particularly relates to a Nanguo pear PuAAT gene, an overexpression vector and application thereof.
Background
The Nanguo pear (Pyrus ussuriensis) is a plant of Rosaceae and Pyritum, belongs to typical breath transition type fruit, is ripe from the fruit which is then ripe for about 10-15 days to the best edible period at normal temperature after being picked, has strong and pleasant fragrance, and is a scarce test material for researching the fragrance of the pear fruit. The aroma is one of important quality indexes of the fruits and directly influences the economic value of the fruits, and the ester aroma substances are important sources of the aroma. Earlier researches find that the content of ethyl hexanoate, hexyl acetate and ethyl butyrate in the nanguo pear ester fragrance substances is highest.
Alcohol Acyltransferase (AAT) is a key enzyme in the very end of the anabolic pathway for ester fragrances and plays an important role in ester synthesis. AAT is a member of the BAHD family, and the amino acid sequence of AAT contains two highly conserved motifs, HXXXD and FGWG. The alcohol acyltransferase AAT gene is subjected to functional research, the fragrance regulation mechanism of the alcohol acyltransferase AAT gene is clarified, and a theoretical basis can be provided for regulating the fragrance synthesis mechanism of Nanguo pears at a molecular level. However, the prior art still fails to apply the AAT gene related to the aroma of Nanguo pears to the improvement of the aroma content of fruits.
Disclosure of Invention
In order to solve the problem that the prior art still fails to apply the south fruit pear ester fragrance related ATT gene to fruit fragrance content improvement, the invention provides a south fruit pear PuAAT gene, an overexpression vector and application thereof.
The technical scheme of the invention is as follows:
a Nanguo pear PuAAT gene, the nucleotide sequence of which is shown in SEQ ID No. 1.
Furthermore, the amino acid sequence of the PuAAT gene code of Nanguo pear is shown in SEQ ID No. 2.
An overexpression vector of a Nanguo pear PuAAT gene comprises the Nanguo pear PuAAT gene.
Furthermore, the overexpression vector is constructed by inserting the Nanguo pear PuAAT gene into a plant expression vector pRI101-CaMV 35S.
Further, the construction method of the overexpression vector comprises the following steps:
step 1, reverse transcription is carried out by taking the total RNA of the Nanguo pear as a template to obtain cDNA, and then the obtained cDNA is taken as the template, and PCR amplification is carried out by utilizing a PuAAT primer group to obtain the PuAAT gene of the Nanguo pear;
and 2, connecting the PuAAT gene of the Nanguo pear obtained in the step 1 with a plant expression vector pRI101-CaMV35S to obtain an overexpression vector pRI101-CaMV35S-PuAAT.
Further, the PuAAT primer group comprises a primer PuAAT-F and a primer PuAAT-R;
the nucleotide sequence of the primer PuAAT-F is as follows: 5 '-ATGATGCCACTCTCAGTACT-3';
the nucleotide sequence of the primer PuAAT-R is as follows: 5 '-TTACATCATTGACATGATCC-3'.
An application of a Nanguo pear PuAAT gene in improving fruit aroma is disclosed, wherein the fruit is Nanguo pear fruit, american pear or golden-crown apple.
An application of an overexpression vector of a Nanguo pear PuAAT gene in improving fruit aroma is disclosed, wherein the fruit is Nanguo pear fruit, american pear or golden-cap apple.
Furthermore, an overexpression vector pRI101-CaMV35S-PuAAT carrying the PuAAT gene of Nanguo pear is transferred into agrobacterium and the Nanguo pear fruit is transiently infected by adopting an agrobacterium-mediated method.
Further, the agrobacterium-mediated transient infection is to inject agrobacterium containing an overexpression vector pRI101-CaMV35S-PuAAT into Nanguo pear fruits.
The invention has the beneficial effects that:
the invention utilizes plant gene engineering technology, separates and identifies the sequence information of the ester fragrance synthesis related gene PuAAT of Nanguo pear from Nanguo pear through gene expression analysis, gene cloning and sequence analysis technology, constructs an overexpression vector of the PuAAT gene of Nanguo pear, and transfers the overexpression vector containing the PuAAT gene of Nanguo pear into Nanguo pear fruits through an agrobacterium-mediated transformation method. The invention provides theoretical basis and technical means for regulating and controlling the fruit aroma by utilizing the genetic engineering technology, and has great application value.
Drawings
FIG. 1 is a photograph showing the result of amplification of a cDNA sequence of the PuAAT gene in example 1, wherein M is DL2000;
FIG. 2 is a graph comparing the expression levels of the PuAAT gene in Nanguo pear fruits after transient transformation in comparative example 1 and example 3;
FIG. 3 is a graph comparing the content of ester-type aroma substances in Nanguo pear fruits after transient transformation in comparative example 1 and example 3.
Detailed Description
The technical solutions of the present invention are further described below with reference to the embodiments, but the present invention is not limited thereto, and any modifications or equivalent substitutions made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention should be covered in the protection scope of the present invention. The process equipment or apparatus not specifically mentioned in the following examples are conventional in the art, and if not specifically mentioned, the raw materials and the like used in the examples of the present invention are commercially available; unless otherwise specified, the technical means used in the examples of the present invention are conventional means well known to those skilled in the art.
Example 1
The embodiment provides a cloning method of a PuAAT gene related to aroma of Nanguo pears.
RNA extraction was performed using Nanguo pear (Pyrus ussuriensis Maxim.) fruit as a test material.
Total RNA in the nanguo pear fruit was extracted using the CTAB method (Li et al, 2013). The total RNA was used as a template and reverse transcribed with a reverse transcription kit (PrimeScriptTM RTreagentkit with gDNAeraser) to obtain cDNA.
And carrying out PCR amplification by using the cDNA obtained by reverse transcription as a template and a PuAAT primer group.
The PuAAT primer group comprises a primer PuAAT-F and a primer PuAAT-R;
the nucleotide sequence of the primer PuAAT-F is as follows: 5 '-ATGATGCCACTCTCAGTACT-3';
the nucleotide sequence of the primer PuAAT-R is as follows: 5 '-TTACATCATTGACATGATCC-3'.
The reaction system for PCR amplification in this example is: 2 XExTaq TM Mix 25.0μL、PuAAT-F 2.5μL、PuAAT-R 2.5μL、cDNA 1μL、ddH 2 O19. Mu.L, 50.0. Mu.L total.
The PCR amplification procedure was: at 95 ℃ for 3min; at 95 ℃ for 30s; 30s at 55 ℃; returning to the second step (95 ℃,30 s) for 35 cycles at 72 ℃,1min and 20 s; storing at 72 deg.C for 5min and 4 deg.C.
The PCR product is subjected to agarose gel electrophoresis detection, and the result is shown in figure 1, and the generated specific strip product is a target fragment, namely the PuAAT gene related to the aroma of Nanguo pear. After the PCR product is recovered by gel, the gel is connected to a T1-Simple (purchased from Beijing all-style gold biotechnology Co., ltd.) carrier, and sequencing is carried out after screening positive clone, and the result shows that the full length of the amplified gene is 1380bp, and the nucleotide sequence of the amplified gene is shown as SEQ ID No. 1.
Example 2
The embodiment provides an overexpression vector of a PuAAT gene of Nanguo pear and a construction method thereof.
Any expression vector for introducing a foreign gene into a plant can be used to construct an overexpression vector for the PuAAT gene of Nanguo pear, and the preferred plant expression vector in this example is pRI101-CaMV35S, available from TaKaRa. The pRI101-CaMV35S is digested by restriction enzymes NdeI and SacI.
pRI101-CaMV35S, restriction enzymes NdeI and SacI used in this example were purchased from TaKaRa.
The target gene Nanguo pear PuAAT gene is connected to the pRI101-CaMV35S vector after the enzyme digestion is successful by using a Seamless Cloning Kit (Seamless Cloning Kit), and an over-expression vector pRI101-CaMV35S-PuAAT is obtained.
The obtained overexpression vector pRI101-CaMV35S-PuAAT was used to transform E.coli competent DH5a (purchased from Beijing Quanjin Biotechnology Co., ltd.), and positive clones were amplified and identified. And selecting the recombinant plasmid which is verified to be correct, and sending the recombinant plasmid to Shanghai bioengineering GmbH for sequencing. And then, transforming agrobacterium EHA105 competent cells by using an overexpression vector pRI101-CaMV35S-PuAAT to obtain agrobacterium containing an overexpression vector pRI101-CaMV35S-PuAAT recombinant plasmid for infecting Nanguo pear fruits.
Example 3
In the embodiment, the agrobacterium containing the overexpression vector pRI101-CaMV35S-PuAAT recombinant plasmid obtained in the embodiment 2 is used for transient infection of Nanguo pear to verify the function of the PuAAT gene of the Nanguo pear.
In the embodiment, 135d of Nanguo pear fruits after full-bloom stage are used as test materials, and the Nanguo pear fruits are instantly infected by adopting an agrobacterium-mediated method, wherein the specific method comprises the following steps:
selecting positive monoclonal Agrobacterium colony containing target gene Nanguo pear PuAAT gene, inoculating in 5mL YEP liquid culture medium containing kanamycin (Kana) and rifampicin (Rif) antibiotics. YEP liquid culture medium contains 5 mu L of 100mg/mL Rif and 5 mu L of 50mg/mL Kana, and is uniformly mixed and placed in an incubator at 28 ℃ for overnight culture at 180 rpm; sucking 1mL of the cultured bacterial liquid, inoculating the bacterial liquid into 50mL of YEP liquid culture medium containing Kana and Rif antibiotics, and culturing at 28 ℃ and 180rpm until the OD of the bacterial liquid 600 The value is about 0.5.
Transferring the bacterial liquid into a 50mL centrifuge tube, placing the centrifuge tube in a centrifuge, and centrifuging the centrifuge tube for 5min at 10000 g. Adding a proper amount of sterile water to wash the bacteria and then centrifuging. Collecting Agrobacterium cells, and adding a suspension comprising 1mL of 1MMES-KOH, 1mL of 1M MgCl 2 100 μ L of 100mM Acetosyringone (ACE, acetosyringone); adding water to a constant volume of 100mL. The Agrobacterium cells and the suspension are mixed well and then adjusted to OD 600 And (4) =0.5, standing at room temperature in a dark place for 2-3h, pushing into the pulp of the Nanguo pear by using an injector, and injecting 6 holes into each fruit, wherein the injection amount of each hole is 0.2ml.
Comparative example 1
This comparative example uses Agrobacterium containing only the expression vector pRI101-CaMV35S for transformation of Nanguo pears.
Transforming Escherichia coli competent DH5a (purchased from Beijing Quanjin biotechnology limited) by using expression vector pRI101-CaMV35S, performing amplification and identification, shaking and storing bacterial liquid to obtain Escherichia coli carrying empty vector pRI101, transforming Agrobacterium EHA105 competent cells by using the Escherichia coli carrying empty vector pRI101, and transforming the obtained Agrobacterium containing empty vector pRI101 into Nanguo pear fruits.
In the comparative example, the Nanguo pear fruit 135 days after full-bloom stage is also used as a test material, and the Nanguo pear fruit is instantly infected by adopting an agrobacterium-mediated method, and the specific method is the same as that in example 3.
The PuAAT gene expression amount of the injected fruit is measured by sampling every 3d, FIG. 2 is a comparison graph of PuAAT gene expression amount analysis in the Nanguo pear fruit after transient transformation of comparative example 1 and example 3, and as can be seen from FIG. 2, the 9d gene expression amount after injection is obviously higher than that in the comparative example, which indicates the successful injection. And then, detecting aroma components of the 9d fruit sample by using an Agilent 7890-5975GC-MS1 gas chromatography-mass spectrometer, performing qualitative analysis by retrieving a NIST/WILEY standard spectrum library, and performing internal standard methodQuantitative analysis is carried out, fig. 3 is a graph comparing the content of ester aroma substances in the Nanguo pear fruits after the transient transformation of comparative example 1 and example 3, and as can be seen from fig. 3, the content of the ester aroma substances of the Nanguo pear fruits in example 3, namely ethyl caproate, hexyl acetate and ethyl butyrate, is 0.2480 mug.g respectively under the same storage condition -1 、0.2344μg·g -1 And 0.0416. Mu.g.g -1 All higher than in comparative example 1.
Therefore, the PuAAT gene of the Nanguo pear can promote the accumulation of ester aroma components in the Nanguo pear fruit, improve the fruit aroma of the Nanguo pear, provide theoretical basis and technical means for increasing the aroma content of the fruit, and have great application value.
Claims (10)
1. A Nanguo pear PuAAT gene is characterized in that the nucleotide sequence of the Nanguo pear PuAAT gene is shown in SEQ ID No. 1.
2. The Nanguo pear PuAAT gene according to claim 1, wherein the encoded amino acid sequence of the Nanguo pear PuAAT gene is shown as SEQ ID No. 2.
3. An overexpression vector of the Nanguo pear PuAAT gene, which is characterized by comprising the Nanguo pear PuAAT gene of claim 1.
4. The Nanguo pear PuAAT gene overexpression vector of claim 3, which is constructed by inserting the Nanguo pear PuAAT gene of claim 1 into a plant expression vector pRI101-CaMV 35S.
5. The Nanguo pear PuAAT gene overexpression vector according to claim 4, wherein the construction method of the overexpression vector comprises the following steps:
step 1, reverse transcription is carried out by taking the total RNA of the Nanguo pear as a template to obtain cDNA, and then the obtained cDNA is taken as the template, and PCR amplification is carried out by utilizing a PuAAT primer group to obtain the PuAAT gene of the Nanguo pear;
and 2, connecting the PuAAT gene of the Nanguo pear obtained in the step 1 with a plant expression vector pRI101-CaMV35S to obtain an overexpression vector pRI101-CaMV35S-PuAAT.
6. The Nanguo pear PuAAT gene overexpression vector of claim 5, wherein the PuAAT primer group comprises a primer PuAAT-F and a primer PuAAT-R;
the nucleotide sequence of the primer PuAAT-F is as follows: 5 '-ATGATGCCACTCTCAGTACT-3';
the nucleotide sequence of the primer PuAAT-R is as follows: 5 '-TTACATCATTGACATGATCC-3'.
7. Use of the Nanguo pear PuAAT gene of claim 1 or 2 for enhancing fruit aroma.
8. Use of the overexpression vector of the PuAAT gene of Nanguo pear according to any one of claims 3 to 6 for improving fruit aroma.
9. The application of the overexpression vector of the Nanguo pear PuAAT gene in improving the fragrance of fruits according to claim 8, wherein the overexpression vector pRI101-CaMV35S-PuAAT carrying the Nanguo pear PuAAT gene is transferred into agrobacterium and the Nanguo pear fruits are transiently infected by an agrobacterium-mediated method.
10. The application of the overexpression vector of the Nanguo pear PuAAT gene in improving fruit aroma according to claim 9, wherein the agrobacterium-mediated transient infection is injection of agrobacterium containing the overexpression vector pRI101-CaMV35S-PuAAT into Nanguo pear fruit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211538503.5A CN115896137A (en) | 2022-12-01 | 2022-12-01 | Nanguo pear PuAAT gene, overexpression vector and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211538503.5A CN115896137A (en) | 2022-12-01 | 2022-12-01 | Nanguo pear PuAAT gene, overexpression vector and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115896137A true CN115896137A (en) | 2023-04-04 |
Family
ID=86489366
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211538503.5A Pending CN115896137A (en) | 2022-12-01 | 2022-12-01 | Nanguo pear PuAAT gene, overexpression vector and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115896137A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116554291A (en) * | 2023-04-28 | 2023-08-08 | 南京农业大学 | Pear bZIP transcription factor PubZIP914 and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000032789A1 (en) * | 1998-12-02 | 2000-06-08 | Centrum Voor Plantenveredelings- En Reproduktieonderzoek (Cpro-Dlo) | Fruit flavour related genes and use thereof |
| CN106256907A (en) * | 2016-06-17 | 2016-12-28 | 山东省果树研究所 | Pears PuADH1 gene, separating clone and the method for expression analysis, Subcellular Localization method and application thereof |
-
2022
- 2022-12-01 CN CN202211538503.5A patent/CN115896137A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000032789A1 (en) * | 1998-12-02 | 2000-06-08 | Centrum Voor Plantenveredelings- En Reproduktieonderzoek (Cpro-Dlo) | Fruit flavour related genes and use thereof |
| CN106256907A (en) * | 2016-06-17 | 2016-12-28 | 山东省果树研究所 | Pears PuADH1 gene, separating clone and the method for expression analysis, Subcellular Localization method and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| LUO等: "Salicylic acid treatment alleviates diminished ester production in cold-stored ‘Nanguo’ pear by promoting the transcription of PuAAT", POSTHARVEST BIOLOGY AND TECHNOLOGY, vol. 187, 20 January 2022 (2022-01-20), pages 1 - 11, XP086973947, DOI: 10.1016/j.postharvbio.2022.111849 * |
| ZHOU等: "Pyrus ussuriensis cultivar Nanguoli alcohol acyltransferase (aat) mRNA, complete cds, KJ775788.1", pages 1 - 2, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/nucleotide/KJ775788.1?report=genbank&log$=nuclalign&blast_rank=1&RID=2GWHMVWD016> * |
| 周鑫等: "间歇升温诱导南果梨冷藏转常温酯类香气的变化和相关基因表达", 食品科学, vol. 36, no. 14, 31 December 2015 (2015-12-31), pages 206 - 211 * |
| 纪淑娟;董玲;周鑫;李蕊;程顺昌;: "水杨酸对1-MCP处理南果梨冷藏后酯类香气的影响及作用机理", 中国食品学报, no. 06, 30 June 2016 (2016-06-30), pages 1670173 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116554291A (en) * | 2023-04-28 | 2023-08-08 | 南京农业大学 | Pear bZIP transcription factor PubZIP914 and application thereof |
| CN116554291B (en) * | 2023-04-28 | 2024-02-09 | 南京农业大学 | Pear bZIP transcription factor PubZIP914 and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109943586B (en) | Plant circRNA overexpression vector and construction method thereof | |
| CN111548399B (en) | MYB transcription factor for regulating and controlling accumulation of tobacco cembratriene diol, coding gene and application | |
| CN111088237A (en) | Kenaf HcPDS gene VIGS silencing system | |
| CN115896137A (en) | Nanguo pear PuAAT gene, overexpression vector and application thereof | |
| CN114634939B (en) | PgJMT1 gene for regulating synthesis of methyl jasmonate in ginseng and application thereof | |
| CN113430180B (en) | Sweet potato flavonol synthase IbFLS1, and coding gene and application thereof | |
| CN114561369B (en) | Glycosyltransferase for biosynthesis of paris polyphylla saponin, encoding gene and application thereof | |
| CN116254243A (en) | Anthocyanin glycosyltransferase and acyltransferase, and encoding genes and application thereof | |
| CN108473972B (en) | Drimenol synthase III | |
| CN104894150B (en) | Saussurea involucrate cell phenylalanine ammonia lyase gene SiPAL and encoding product and application thereof | |
| CN109735523B (en) | Cedarol synthase Lc-CedS coding gene and application thereof | |
| CN115927406B (en) | Nanguo pear PuAOX1a gene, over-expression vector and application | |
| CN114507657B (en) | Chrysanthemum brain monoterpene synthase gene CnTPS2 and application thereof | |
| CN114350634B (en) | Glucoside glycosyl transferase for synthesizing epimedin and coding gene and application thereof | |
| CN110760519B (en) | Gene for regulating and controlling synthesis of chlorogenic acid and encoding protein and application thereof | |
| CN114369582B (en) | Brucella bifidus source ester synthetase JG536_25355, coding gene and application | |
| CN115819537B (en) | AP2/ERF transcription factor, and coding gene and application thereof | |
| CN117778425B (en) | Use and method of methyltransferase in enhancing nitrogen fixation capacity of nitrogen fixation microorganism | |
| CN117025631A (en) | Transcription factor NtGATA8 related to synthesis of tobacco terpenoid, encoding gene, vector and application thereof | |
| CN114717247B (en) | Cassava transcription factors MebHLH72 and MebHLH114 and application thereof in inhibiting synthesis of linolenyl bitter | |
| CN116042663A (en) | Dendrobium candidum beta-ionone synthesis key enzyme gene DoCCD1 and cloning method and application thereof | |
| CN115976068B (en) | SiHQT gene for improving chlorogenic acid content of saussurea involucrata as well as encoding product and application thereof | |
| CN112322564B (en) | Escherichia coli engineering bacterium and construction method and application thereof | |
| CN110317823B (en) | Function identification and application of gossypol biosynthetic pathway enzyme gene 2-ODD-1 | |
| CN117230029A (en) | Biological enzyme for catalyzing hydroxylation of C9 position in taxane compounds, nucleic acid molecule, biological material and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |