Nanguo pear lncRNA-EIF2 gene, overexpression vector and application

Abstract

The invention belongs to the technical field of molecular biology gene engineering, and particularly discloses a Nanguo pear lncRNA-EIF2 gene, an overexpression vector and application. In order to solve the problem that respiratory climacteric pears are poor in storage property, the invention provides a Nanguo pear lncRNA-EIF2 gene, and the nucleotide sequence of the Nanguo pear lncRNA-EIF2 gene is as shown in SEQ ID NO.1. The invention further constructs an overexpression vector comprising the Nanguo pear lncRNA-EIF2 gene, the vector is transferred into Nanguo pear fruits through an agrobacterium-mediated transformation method, and identification proves that the Nanguo pear lncRNA-EIF2 gene can inhibit the generation of Nanguo pear ethylene and improve the storage property of Nanguo pears. The invention provides a theoretical basis and a technical means for inhibiting the generation of ethylene so as to improve the storage property of fruits, and has a very high application value.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology gene engineering, and in particular relates to a Nanguo pear long-chain non-coding RNA lncRNA-EIF2 gene, an overexpression vector and an application thereof. Background Art

[0002] Pear (Scientific name: Pyrus spp), a plant of the genus Pyrus in the Rosaceae family, is usually a deciduous tree or shrub, with a very small number of evergreen species. The leaves are mostly oval, and the size varies depending on the variety.

[0003] There are two types of pears, the climacteric type and the non-climacteric type. Among them, the Nanguo pear (Pyrusussuriensis Maxim.) is a climacteric type fruit. The climacteric type fruit will suddenly release a large amount of ethylene during the ripening process, causing a sudden increase in the respiration intensity. The fruit matures quickly, and then the fruit quality will decrease and it will be intolerant to storage, resulting in a large waste of resources.

[0004] The present invention found that lncRNA-EIF2 is a long non-coding RNA. Overexpression of this gene in respiratory climacteric pear fruit can significantly inhibit the release of ethylene from pear fruit and prolong the storage period of the fruit. The lncRNA-EIF2 gene is differentially expressed as the plant grows and develops. Functional studies on this gene clarify its mechanism of ethylene production, which can provide a theoretical basis for the molecular level regulation of ethylene production in Nanguo pear fruit. There is no report in the prior art on the application of lncRNA-EIF2 in the regulation of ethylene production and ripening of fruit. Summary of the invention

[0005] In order to solve the problem that the prior art fails to apply long non-coding RNA lncRNA-EIF2 to respiratory climacteric pear fruits to inhibit ethylene production and prolong the storage period, the present invention aims to provide a Nanguo pear lncRNA-EIF2 gene, an overexpression vector and an application.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] A Nanguo pear lncRNA-EIF2 gene, the nucleic acid sequence of the Nanguo pear lncRNA-EIF2 gene is shown as SEQ ID NO.1.

[0008] A Nanguo pear lncRNA-EIF2 gene overexpression vector, the overexpression vector comprising the Nanguo pear lncRNA-EIF2 gene.

[0009] Furthermore, the overexpression vector is constructed by inserting the Nanguo pear lncRNA-EIF2 gene into the plant expression vector pRI101-CaMV35S.

[0010] Furthermore, the method for constructing the overexpression vector comprises the following steps:

[0011] Step 1, using the total RNA of Nanguo pear as a template for reverse transcription to obtain cDNA, and then using the obtained cDNA as a template, using the lncRNA-EIF2 primer set for PCR amplification to obtain the Nanguo pear lncRNA-EIF2 gene;

[0012] Step 2: Connect the Nanguo pear lncRNA-EIF2 gene obtained in step 1 to the plant expression vector pRI101-CaMV35S to obtain the overexpression vector pPI1 0 1-lncRNA-EIF2.

[0013] Further, the lncRNA-EIF2 primer set includes primer lncRNA-EIF2-F and primer lncRNA-EIF2-R;

[0014] The nucleotide sequence of the primer lncRNA-EIF2-F is: 5′-CCAAGACAAGAAGTCGTACG-3′;

[0015] The nucleotide sequence of the primer lncRNA-EIF2-R is: 5′-TCACATTCACGTCCACACAT-3′.

[0016] The present invention also protects the use of the above-mentioned Nanguo pear lncRNA-EIF2 gene in inhibiting fruit ethylene production and prolonging the storage period of the fruit, and the fruit is a respiratory climacteric pear or apple.

[0017] In addition, the functions of the overexpression vector containing the above gene, the transgenic plant line and the host cell containing the vector in inhibiting the production of ethylene in fruits and prolonging the storage period of fruits also fall within the protection scope of the present invention.

[0018] Furthermore, the overexpression vector pRI101-lncRNA-EIF2 carrying the Nanguo pear lncRNA-EIF2 gene was transferred into Agrobacterium, and the Nanguo pear fruit was transiently infected using the Agrobacterium-mediated method.

[0019] Furthermore, the Agrobacterium-mediated transient infection is to inject Agrobacterium containing the overexpression vector pRI101-lncRNA-EIF2 into the Nanguo pear fruit.

[0020] The present invention also discloses a method for reducing fruit ethylene production and improving fruit storability, by promoting the expression of lncRNA-EIF2 gene in target plant to obtain a plant with reduced fruit ethylene production and higher storability than the target plant. Preferably, the target plant is Nanguo pear.

[0021] The method of promoting the expression of lncRNA-EIF2 gene is overexpression or overexpression, wherein the overexpression method can be transient overexpression mediated by Agrobacterium.

[0022] The above-mentioned method of reducing fruit ethylene production and improving fruit storage properties is also applicable to other recipient plants having homologous genes with Nanguo pear. There is no special limitation on the recipient plants suitable for the present invention, which include not only Nanguo pear, but also respiratory burst-type pear or apple, and other plants with high homology, as long as they are suitable for gene transformation operations.

[0023] The "plant" mentioned in the present invention includes the whole plant, its parent and progeny plants and different parts of the plant, including seeds, fruits, stems, buds, leaves, roots, flowers, tissues and organs, and these different parts all have our target gene or nucleic acid. The "plant" mentioned here also includes plant cells, suspension cultures, callus, embryos, meristem areas and pollen, and similarly, each of the aforementioned objects contains the target gene / nucleic acid.

[0024] The present invention includes any plant cell, or any plant obtained or obtainable by the method therein, and all plant parts and propagules thereof. This patent also includes transfected cells, tissues, organs or whole plants obtained by any of the aforementioned methods. The only requirement is that the progeny exhibit the same genotypic or phenotypic characteristics, and the progeny obtained using the method of this patent have the same characteristics.

[0025] The present invention also extends to the harvestable parts of the plants as described above, but not limited to seeds, fruits, peels. It also further relates to other derivatives of the plants after harvest, such as organic acids, sugars, lycopene, tomatine, volatile substances, vitamins, minerals and proteins. The present invention also relates to foods or food additives obtained from the relevant plants.

[0026] Advantages of the present invention:

[0027] The invention utilizes plant genetic engineering technology, and through gene expression analysis, gene cloning and sequence analysis technology, separates and identifies the sequence information of lncRNA-EIF2 gene related to the ripening of Nanguo pear from Nanguo pear, and constructs an overexpression vector of the lncRNA-EIF2 gene of Nanguo pear. The overexpression vector containing the lncRNA-EIF2 gene of Nanguo pear is transferred into Nanguo pear fruit through Agrobacterium-mediated transformation. It is proved through identification that the lncRNA-EIF2 gene of Nanguo pear can inhibit the generation of ethylene in Nanguo pear fruit and prolong the storage period of Nanguo pear, thus providing a theoretical basis and technical means for enhancing the storage property of respiratory climacteric pear, and having high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 This is a photo of the amplification electrophoresis detection result of the lncRNA-EIF2 gene cDNA sequence in Example 1 of the present invention, where M is DL2000;

[0030] Figure 2 This is a picture of the electrophoresis detection result of the overexpression vector pRI101-lncRNA-EIF2 in Example 2 of the present invention;

[0031] Figure 3 This is a photo of the electrophoresis detection result of Escherichia coli pRI101-lncRNA-EIF2 in Example 3 of the present invention;

[0032] Figure 4 This is a comparative diagram of phenotypic analysis in Nanguo pear fruit after transient transformation in Example 1 and Example 3;

[0033] Figure 5 This is a comparison chart of the expression levels of lncRNA-EIF2 gene in Nanguo pear fruit after transient transformation in Example 1 and Example 3;

[0034] Figure 6 This is a comparison chart of the ethylene production in Nanguo pear fruit after instantaneous transformation in Example 1 and Example 3. DETAILED DESCRIPTION

[0035] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0036] Example 1 Cloning of lncRNA-EIF2 gene

[0037] This example provides a method for cloning the lncRNA-EIF2 gene related to ethylene synthesis in Nanguoli.

[0038] The fruits of Pyrus ussuriensis Maxim. were used as experimental materials for RNA extraction.

[0039] The total RNA from the fruit of Nanguo pear was extracted by CTAB method (Li et al., 2013). The total RNA was used as template and reverse transcription kit (PrimeScript TM cDNA was obtained by reverse transcription using RT reagent Kit with gDNA Eraser.

[0040] The cDNA obtained by reverse transcription was used as a template and PCR amplification was performed using the lncRNA-EIF2 primer set.

[0041] The lncRNA-EIF2 primer set includes primer lncRNA-EIF2-F and primer lncRNA-EIF2-R;

[0042] The nucleotide sequence of the primer lncRNA-EIF2-F is: 5′-CCAAGACAAGAAGTCGTACG-3′ (shown in SEQ ID NO. 2);

[0043] The nucleotide sequence of the primer lncRNA-EIF2-R is: 5′-TCACATTCACGTCCACACAT-3′ (shown in SEQ ID NO. 3).

[0044] The reaction system for PCR amplification in this embodiment is: 2×ExTaq TM Mix 25.0μL, lncRNA-EIF2-F 2.5μL, lncRNA-EIF2-R 2.5μL, cDNA 1μL, ddH2O 19μL, a total of 50.0μL. PCR amplification program: 95℃, 3min; 95℃, 30s; 55℃, 30s; 72℃, 1min20s, return to the second step (95℃, 30s) for 35 cycles; 72℃, 5min, and store at 4℃.

[0045] The PCR products were detected by agarose gel electrophoresis. Figure 1 As shown, the specific band product produced is the target fragment, that is, the lncRNA-EIF2 gene related to ethylene synthesis in Nanguoli. The PCR product was recovered by gel and sequenced. The results showed that the amplified gene was 684 bp in length, and its DNA nucleotide sequence was shown in SEQ ID NO.1:

[0046]

[0047] Example 2 Construction of overexpression vector of Nanguo pear lncRNA-EIF2 gene

[0048] Any expression vector for introducing exogenous genes into plants can be used to construct an overexpression vector of the Nanguo pear lncRNA-EIF2 gene. The preferred plant expression vector in this embodiment is pRI101-CaMV35S, and the restriction endonucleases NdeI and SacI are selected, which are purchased from Bioengineering (Dalian) Co., Ltd.

[0049] pEASY-T1 (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) containing the target gene lncRNA-EIF2 was amplified; the pRI101-CaMV35S plasmid was double-digested, the vector large fragment and the target gene small fragment were recovered, and connected with a seamless cloning kit (Seamless Cloning Kit) to obtain the overexpression vector PRI101-lncRNA-EIF2. The PCR amplification electrophoresis detection results of the overexpression vector pRI101-lncRNA-EIF2 are shown in Figure 2 shown.

[0050] The obtained overexpression vector pRI101-lncRNA-EIF2 was used to transform the competent E. coli Transl-T1 (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and the bacterial solution was preserved by shaking after amplification and identification to obtain E. coli carrying the expression vector pRI101-lncRNA-EIF2. The PCR electrophoresis detection results are as follows: Figure 3 The bacterial solution with the correct amplification verification was selected and sent to Shanghai Biotech Co., Ltd. for sequencing.

[0051] The Escherichia coli plasmid carrying the overexpression vector pRI101-lncRNA-EIF2 was used to transform Agrobacterium EHA105 competent cells, and the obtained Agrobacterium containing the overexpression vector pRI101-lncRNA-EIF2 was used to infect Nanguo pear fruits.

[0052] Example 3 Functional verification of lncRNA-EIF2 gene

[0053] In this example, the Agrobacterium containing the overexpression vector pRI101-lncRNA-EIF2 obtained in Example 2 was used to infect Nanguo pear to verify the function of the Nanguo pear lncRNA-EIF2 gene.

[0054] In this example, Nanguo pear fruits 135 days after the full flowering period were used as test materials, and the Nanguo pear fruits were instantly infected by Agrobacterium-mediated method. The specific method was as follows:

[0055] Pick the positive monoclonal Agrobacterium colony containing the target gene Nanguoli lncRNA-EIF2 gene and inoculate it into 5mL YEP liquid medium. The YEP liquid medium contains 5μL 100mg / mL Rif and 5μL 50mg / mL Kana, mix well and place in a 28℃ incubator for 12h. Take 200μL of the cultured bacterial solution and add it to 300mL YEP liquid medium, mix well and place in a 28℃ incubator, and culture at 180rpm until OD600=0.4.

[0056] Transfer the bacterial solution to a 50mL centrifuge tube, place it in a centrifuge, and centrifuge at 10000×g for 5min. Add an appropriate amount of sterile water to wash the bacteria and then centrifuge. Collect the Agrobacterium cells and add a suspension to suspend the Agrobacterium cells. The suspension contains 1mL 1MMES-KOH, 1mL 1M MgCl2, and 100μL 100mM acetosyringone (ACE); add water to make up to 100mL. After the Agrobacterium cells and the suspension are fully mixed, adjust to OD600=0.5, stand at room temperature and avoid light for 2-3h, and then push it into the flesh of the Nanguo pear with a syringe. The single injection volume is 0.3mL.

[0057] Comparative Example 1

[0058] In this comparative example, Agrobacterium containing only the expression vector pRI101-CaMV35S was used for transformation of Nanguo pear.

[0059] The expression vector pRI101-CaMV35S was used to transform the competent Escherichia coli Transl-T1 (purchased from Beijing Quanshijin Biotechnology Co., Ltd.). After amplification and identification, the bacterial liquid was shaken to obtain Escherichia coli carrying the empty vector pRI101. The Escherichia coli carrying the empty vector pRI101 was used to transform the competent Agrobacterium EHA105 cells. The obtained Agrobacterium containing the empty vector pRI101 was used to transform Nanguo pear fruit.

[0060] This comparative example also uses Nanguo pear fruits 135 days after the full flowering period as test materials, and adopts Agrobacterium-mediated method to transiently infect Nanguo pear fruits. The specific method is the same as that of Example 3.

[0061] Figure 4This is a comparison chart of phenotypic analysis in Nanguo pear fruit after transient transformation in Comparative Example 1 and Example 3; Figure 4 It can be seen that the maturity of Nanguo pear overexpressing lncRNA-EIF2 gene is lower than that of the control group.

[0062] The ethylene content in the Nanguo pear fruit after instantaneous transformation of Example 3 and Comparative Example 1 was determined by the following method:

[0063] The Nanguo pear fruits transiently infected by the Agrobacterium-mediated method in Example 3 and Comparative Example 1 were stored at room temperature (25±1° C.) for 9 days, and the expression level of the lncRNA-EIF2 gene and the amount of ethylene produced in the Nanguo pear fruits after transient transformation were measured.

[0064] Figure 5 This is a comparative chart of the expression analysis of lncRNA-EIF2 gene in Nanguo pear fruit after transient transformation in Comparative Example 1 and Example 3; Figure 6 The figure is a comparison chart of ethylene production in Nanguo pear fruit after instantaneous transformation in Comparative Example 1 and Example 3. Figure 5 and Figure 6 It can be seen that under the same storage conditions, compared with Comparative Example 1, the expression of the lncRNA-EIF2 gene in the Nanguo pear fruit in Example 3 was significantly increased, the ethylene production was significantly reduced, and the fruit ripening was inhibited. It can be confirmed that the Nanguo pear lncRNA-EIF2 gene can inhibit the production of ethylene components in Nanguo pear fruit, prolong the storage time of the fruit, and provide a theoretical basis and technical means for improving the storage of the fruit, which has a high application value.

Claims

1. A Nanguo pear lncRNA-EIF2 gene, characterized in that The nucleotide sequence of the Nanguo pear lncRNA-EIF2 gene is shown in SEQ ID NO.

1.

2. An overexpression vector of the lncRNA-EIF2 gene of Nanguo Pear, characterized in that: The overexpression vector comprises the Nanguo pear lncRNA-EIF2 gene according to claim 1.

3. According to claim 2, an overexpression vector of the Nanguo pear lncRNA-EIF2 gene, characterized in that: The overexpression vector is constructed by inserting the Nanguoli lncRNA-EIF2 gene described in claim 1 into the plant expression vector pRI101-CaMV35S.

4. According to claim 3, an overexpression vector of the Nanguo pear lncRNA-EIF2 gene, characterized in that: The method for constructing the overexpression vector comprises the following steps: Step 1, using the total RNA of Nanguo pear as a template for reverse transcription to obtain cDNA, and then using the obtained cDNA as a template, using the lncRNA-EIF2 primer set for PCR amplification to obtain the Nanguo pear lncRNA-EIF2 gene; Step 2: Connect the Nanguo pear lncRNA-EIF2 gene obtained in step 1 to the plant expression vector pRI101-CaMV35S to obtain the overexpression vector pRI101-lncRNA-EIF2.

5. According to claim 4, an overexpression vector of the Nanguo pear lncRNA-EIF2 gene, characterized in that: The lncRNA-EIF2 primer set includes primer lncRNA-EIF2-F and primer lncRNA-EIF2-R; the primer sequences are: lncRNA-EIF2-F: 5′-CCAAGACAAGAAGTCGTACG-3′; lncRNA-EIF2-R: 5′-TCACATTCACGTCCACACAT-3′.

6. Use of the Nanguoli lncRNA-EIF2 gene according to claim 1 or the overexpression vector of the Nanguoli lncRNA-EIF2 gene according to any one of claims 2 to 5 in inhibiting ethylene production and improving fruit storage properties.

7. The use of the overexpression vector of the Nanguo Pear lncRNA-EIF2 gene in inhibiting ethylene production and improving fruit storage properties according to claim 6, characterized in that: The overexpression vector pRI101-lncRNA-EIF2 carrying the Nanguo pear lncRNA-EIF2 gene was transferred into Agrobacterium, and the Nanguo pear fruit was transiently infected by Agrobacterium-mediated method.

8. A method for reducing ethylene production in fruits and improving the storage properties of fruits, characterized in that: By promoting the expression of the lncRNA-EIF2 gene in the target plant, a plant with reduced fruit ethylene production and higher storage capacity than the target plant is obtained, the nucleotide sequence of the lncRNA-EIF2 gene is shown in SEQ ID NO.1, and the target plant is a respiratory climacteric pear or apple.

9. The method according to claim 8, characterized in that The way to promote lncRNA-EIF2 gene expression is overexpression or overexpression.

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