Aux / IAA gene family gene PbrAux1 of Dangshan pears and application of gene PbrAux1

Abstract

The invention discloses a Dangshan pear Aux / IAA gene family gene PbrAux1 and application thereof, and belongs to the technical field of biological gene engineering. The PbrAux1 gene disclosed by the invention has a nucleotide sequence as shown in SEQ ID NO. 1 and an amino acid sequence as shown in SEQ ID NO. 2. The Dangshan pear low-temperature-resistant gene has the beneficial effects that the Dangshan pear low-temperature-resistant gene is obtained, an overexpression recombinant vector is constructed by utilizing the gene, and the function of the gene in the aspect of low temperature resistance is proved by utilizing molecular biology and transgenic technologies. The PbrAux1 improves the low-temperature resistance of the transgenic arabidopsis thaliana, which is shown in that compared with WT, the PbrAux1 improves the survival rate of the arabidopsis thaliana after low-temperature freeze injury and the activity of POD, SOD and CAT, and reduces the MDA accumulation amount and the relative electrolyte permeability. Therefore, the PbrAux1 can be introduced into the plant as a target gene to promote the low-temperature resistance of the plant.

Description

Technical Field

[0001] The invention relates to the technical field of biological genetic engineering, and in particular to a Dangshan pear Aux / IAA gene family gene PbrAux1 and an application thereof. Background Art

[0002] Aux / IAA is a key transcription factor protein family that induces auxin response regulation. The Aux / IAA gene family affects the early response of auxin by expressing nuclear localization proteins. Aux / IAA proteins include four highly conserved domains (DomainⅠ, Ⅱ, Ⅲ, Ⅳ): DomianⅠ has a transcriptional repressor domain related to ethylene response factor, and transcriptional repressor factors inhibit the expression of downstream genes of auxin signals through this domain; DomianⅡ has highly conserved characteristics and is regulated by TIR1. It can directly bind to SCFTIR1 to control the degradation of Aux / IAA proteins; DomianⅢ and Ⅳ are the sites that bind to ARFs, and have homologous domains of CTDs of ARFs proteins, which are responsible for dimerization and multimerization with other Aux / IAA proteins, thereby regulating auxin signals.

[0003] The length of Aux / IAA family genes varies greatly and is unevenly distributed on 8 chromosomes. The genes are located in the cell nucleus and the proteins they encode contain 189 to 363 amino acids with large differences in acidity and alkalinity. They are unstable and hydrophilic proteins. Aux / IAA genes have been cloned and their regulatory mechanisms studied in plants such as Arabidopsis, tomato, cucumber, and rice (Wang Lei et al., 2010; Piya et al., 2014; Ambreetha et al., 2018). Studies have confirmed that Aux / IAA is involved in regulating the growth and development of plants. For example, knocking out Arabidopsis IAA3 / SHY2 affects auxin balance and lateral root formation. TIR1 / AFB2 can form a special sensor complex with AtIAA2, AtIAA6, AtIAA9 or AtIAA17, which can regulate the balance of jasmonic acid and the initiation of adventitious root development in plants after auxin treatment. Aux / IAA genes are also involved in a variety of stress responses, such as the induction of rice OsIAA9 and OsIAA20 expression under drought and salt stress, and the transcription levels of soybean GmIAA47 and GmIAA49 in the reproductive stage significantly increased under drought conditions. The above is the research on the Aux / IAA gene family in regulating plant growth and development, but the research on this gene family in Dangshan pear in regulating plant resistance to low temperature has not been reported. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to provide an application of a pear PbrAux1 gene in regulating the low temperature resistance of plants.

[0005] The present invention solves the above technical problems through the following technical means:

[0006] The first aspect of the present invention provides a Dangshan pear Aux / IAA gene family gene PbrAux1, whose nucleotide sequence is shown as SEQ ID NO.1.

[0007] The second aspect of the present invention provides a protein encoded by the above gene PbrAux11, whose amino acid sequence is shown in SEQ ID NO.2.

[0008] The third aspect of the present invention provides a biological material containing the gene PbrAux1, wherein the biological material includes but is not limited to recombinant DNA, an expression vector, a host bacteria or a plant material.

[0009] Preferably, the expression vector is a pCAMBIA-1300 vector plasmid.

[0010] Preferably, the host bacterium is Agrobacterium GV3101.

[0011] Preferably, the plant material comprises Arabidopsis thaliana.

[0012] The fourth aspect of the present invention proposes the use of the gene PbrAux1 or a biological material containing the gene PbrAux1 in any of the following situations:

[0013] 1) Used to improve the resistance of plants to low temperature stress;

[0014] 2) Used to increase the activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) in plants under low temperature stress;

[0015] 3) Used for preparing transgenic plants;

[0016] 4) Used in plant breeding.

[0017] Preferably, the plant is Arabidopsis thaliana.

[0018] A fifth aspect of the present invention provides a method for regulating the low temperature resistance of Arabidopsis thaliana, the method comprising:

[0019] (1) Making a plant contain the gene PbrAux1 or

[0020] (2) Overexpressing the gene PbrAux1 according to claim 1 in a plant.

[0021] Preferably, the method includes but is not limited to cloning the PbrAux1 sequence, constructing the PbrAux1 gene sequence into an overexpression vector, transforming the strain with the recombinant vector, genetic modification, and propagating the genetically modified material.

[0022] The sixth aspect of the present invention proposes the use of the protein encoded by the Dangshan pear Aux / IAA gene family gene PbrAux1 in regulating the low temperature resistance of plants.

[0023] The beneficial effects of the present invention are:

[0024] The present invention discloses a gene PbrAux1 of the Dangshan pear Aux / IAA gene family and a protein encoded by it. This gene is reported in pear for the first time. Functional verification of transgenic Arabidopsis thaliana shows that overexpression of this gene significantly promotes the tolerance of Arabidopsis thaliana plants to low temperature environments. Therefore, it is expected to be introduced into plants as a target gene to improve the low temperature resistance of plants, thereby improving plant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The figure is a result of genomic PCR identification of PbrAux1 transgenic Arabidopsis thaliana obtained by Agrobacterium-mediated inflorescence infection method using specific primers in Example 3 of the present invention. WT: wild-type Arabidopsis thaliana, as a negative control. P: PbrAux1-1300 plasmid, as a negative control. PbrAux1-OE-1 and PbrAux1-OE-2 are different strains of PbrAux1 transgenic Arabidopsis thaliana.

[0026] Figure 2 The PbrAux1 transgenic Arabidopsis and wild-type Arabidopsis in Example 3 of the present invention were grown on a culture medium for 2 weeks and then placed under -2°C for 1.5 hours. The culture medium was then removed and cultured at normal temperature for 2 days, and the Arabidopsis plants were observed to show a freezing injury phenotype and a mortality comparison chart;

[0027] Figure 3 The graph is a comparison of the relative electrolyte permeability of the PbrAux1 transgenic Arabidopsis thaliana and the wild-type Arabidopsis thaliana that are grown normally for 4 weeks and are subjected to -2°C low temperature treatment for 2 hours in Example 3 of the present invention, and before and after the low temperature treatment;

[0028] Figure 4 This is a comparison diagram of malondialdehyde (MDA) contents of different Arabidopsis thaliana after PbrAux1 transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana were subjected to -2°C low temperature treatment for 2 hours in Example 3 of the present invention;

[0029] Figure 5 This is a comparison of the peroxidase (POD) activities of different Arabidopsis thaliana after the PbrAux1 transgenic Arabidopsis thaliana and the wild-type Arabidopsis thaliana were subjected to -2°C low temperature treatment for 2 hours in Example 3 of the present invention;

[0030] Figure 6This is a comparison diagram of superoxide dismutase (SOD) activities of different Arabidopsis thaliana after PbrAux1 transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana were subjected to -2°C low temperature treatment for 2 hours in Example 3 of the present invention;

[0031] Figure 7 This is a comparison of catalase (CAT) activities of different Arabidopsis thaliana after the PbrAux1 transgenic Arabidopsis thaliana and the wild-type Arabidopsis thaliana were subjected to -2°C low temperature treatment for 2 hours in Example 3 of the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0034] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0035] Example 1: Acquisition of PbrAux1 gene

[0036] Several plant leaves were collected from Dangshan pear trees in the pear producing area of ​​Hefei City, Anhui Province. Total RNA of the Dangshan pear leaf samples was extracted using an RNA extraction kit, and cDNA was obtained by RNA reverse transcription.

[0037] According to the transcriptome sequencing analysis of Dangshan pear samples before and after low temperature treatment, the PbrAux1 sequence with complete ORF and differential expression was selected, and primers P1 and P2 were designed upstream and downstream of the CDS of the gene using Primer Premier 5 software;

[0038] P1: ATGGCTTCCGAGAAGGTTGA; (SEQ ID NO: 3)

[0039] P2: TCAAGCTTGTGAGGGGGG. (SEQ ID NO: 4)

[0040]

[0041] MASEKVETVIAGNYVEMERESDVKTSAKSKLSTFLWHGGSAYDAWFSSASNQVAQV

[0042] LLTLPYSFSQLGLLSGILFQLFYGLMGSWTAYLISLLYVEYRTRKEREKVDFRNHVIQW

[0043] FEVLDGLFGKHCRNVGLFFNCTFLLFGSVIQLIACASNIYYINDNLDKRTWTYIFGACC

[0044] ATTVFIPSFHNYRIWSFLGLIMTTYTAWYLTIASLIHGQVEGVKHSGPSTMVLYFTGAT

[0045] NILYTFGGHAVTVEIMHAMWKPQKFKLIYLMATLYVLTLTLPSASAVYWAFGDNLLTH

[0046] SNALAMLPKTRFRDTAVVLMLIHQFITFGFACTPLYFVWEKFIQMHETKSMVKRALA

[0047] RLPVVIPIWFLATIFPFFGPINSTVGSLLVSFTVYIIPALAHMVTFASASARENAVEKPPSF

[0048] LGGWAGSYTMNIFVVVWVLIVGFGFGGWASMLNFINQVNTFGLFTNRHQCPPHKA (SEQ ID NO: 2)

[0049] Example 2: Construction of overexpression vector of PbrAux1 gene

[0050] According to the information, the restriction sites of the pCAMBIA-1300 vector plasmid are Xba I and BamH I. The restriction endonucleases produced by Saningsi Biotechnology Co., Ltd. are used to perform double restriction digestion on the above restriction sites. After adding the sample according to the system requirements, the reaction is carried out at 37°C for 2 hours, and then the double restriction target band is obtained by agarose gel electrophoresis. After gel cutting and recovery, the linearized vector of the pCAMBIA-1300 vector plasmid is obtained.

[0051] Two restriction sites, Xba I and BamH I, were selected, and primers P3 and P4 were designed using CE Design V1.04 software;

[0052] P3: TATGACCATGATTACGAATTCATGGCTTCCGAGAAGGTTGAG; (SEQ ID NO: 5)

[0053] P4: ACGGGGGACTCTAGAGGATCCTCAAGCTTTTGTGAGGGGGG. (SEQ ID NO: 6)

[0054] The target gene cDNA with the correct sequencing result was used as a template for PCR amplification using the Novozyme high-fidelity enzyme. 10 μL 5× Loading Buffer was added to the amplified PCR product and mixed well, and then detected by agarose gel electrophoresis. The target band was obtained after gel excision and recovery and stored at -20°C.

[0055] Finally, we used the one provided by TOLOBIO Universal CloneMix will insert the fragment and linearized vector for recombination reaction. The reaction product is then transformed into E. coli, plated and placed in a 37°C incubator for 12 hours, single clone colonies are picked, shaken and cultured in liquid LB medium containing 100 mg / L kanamycin sulfate (Kan) for 3 hours, and then tested with vectors and primers. After the sequencing is correct, 50% glycerol is added in a volume ratio of 1:1 and stored in a -80°C refrigerator.

[0056] Example 3: Arabidopsis transformation and screening

[0057] The PbrAux1 transgenic Arabidopsis was obtained by Agrobacterium-mediated inflorescence infection. Wild-type Arabidopsis was planted, and when the wild-type Arabidopsis plants reached the flowering stage, the flower buds were left and the remaining flower pods were removed. The Agrobacterium transferred with the PbrAux1 overexpression vector was taken out from the -80°C refrigerator. After activation, 30 ml of the bacterial solution was placed in a 28°C shaker for 10-12 hours until the bacterial solution became turbid. The infection solution was prepared before infection, and sucrose with a final concentration of 30 g / L and SilwetL-77 with a final concentration of 200 l / L were added to the MS liquid culture medium. The Agrobacterium bacterial solution was centrifuged at 5000 rpm for 8 minutes, the supernatant was discarded, and the Agrobacterium precipitate was resuspended with the infection solution. The infection solution OD600 was measured by a UV spectrophotometer to be 0.8-1.0. The wild-type Arabidopsis inflorescence with only the flower buds left was completely immersed in the infection solution for 120 seconds. After the infection, the inflorescence was completely wrapped with a film and cultured in the dark for 1 day. After the film was removed, it was cultured normally until the seeds matured. After harvesting the seeds, they were cleaned with 75% alcohol and 95% alcohol in a clean bench. After washing and air-drying, they were evenly sown on the MS screening medium containing hygromycin using a toothpick. They were cold-treated at 4°C for 1 day, and then placed vertically in a light incubator for culture. After the seedlings grew to 2-3 true leaves, they were transplanted into the substrate and cultured normally for 1 week. Two healthy leaves were selected from each Arabidopsis plant to extract leaf DNA for identification. The positive plants were T0 generation transgenic plants (such as Figure 1 As shown). After the T0 generation transgenic plants are cultured normally until the seeds mature, the seeds of each plant are harvested to obtain T1 generation transgenic Arabidopsis seeds.

[0058] (1) Identification of freezing resistance (MS medium method): PbrAux1 overexpression and wild-type Arabidopsis were sown on MS medium to observe phenotypic differences. During the entire growth process, the MS plate was placed horizontally.

[0059] After growing on the culture medium for 2 weeks, it was placed at -2℃ for 1.5h. Then the culture medium was taken out and cultured at normal temperature. After 2 days, it was observed that the Arabidopsis plants showed a frost injury phenotype. The results showed that the survival rate of PbrAux1 overexpressing Arabidopsis was significantly higher than that of wild-type plants. Through observation and analysis, it can be found that the survival rate of PbrAux1 overexpressing Arabidopsis after low temperature is significantly higher than that of WT wild-type Arabidopsis. (such as Figure 2 shown).

[0060] (2) Determination of low temperature physiological indicators: Transgenic Arabidopsis and wild-type Arabidopsis that were 4 weeks old were treated with -2℃ low temperature for 2 hours, and the relative electrolyte permeability of each Arabidopsis was measured before and after low temperature treatment. It can be found that there is no difference in the relative electrolyte permeability content of each Arabidopsis strain under normal growth conditions, but after low temperature treatment, the transgenic Arabidopsis showed a lower relative electrolyte permeability than the wild-type Arabidopsis, indicating that the degree of damage to the leaves of the transgenic Arabidopsis is significantly lower than that of the wild-type Arabidopsis. (e.g. Figure 3 shown).

[0061] The MDA content of each Arabidopsis before and after -2℃ treatment was detected. It can be found that under normal circumstances, the MDA content of each Arabidopsis is low, and there is no difference in the MDA content between Arabidopsis genotypes; after low temperature treatment, it can be found that the MDA content of each Arabidopsis is on the rise, and the MDA content of transgenic Arabidopsis is significantly lower than that of wild-type Arabidopsis, indicating that the transgenic Arabidopsis is less damaged (such as Figure 4 ). This indicates that the freezing resistance of transgenic Arabidopsis is higher than that of wild-type Arabidopsis.

[0062] At the same time, the POD, SOD and CAT activities of PbrAux1 overexpression and wild-type Arabidopsis before and after low temperature treatment were also detected. The results showed that there was no significant difference in the POD, SOD and CAT activities of each strain of Arabidopsis before low temperature treatment; however, after low temperature treatment, the POD, SOD and CAT activities of PbrAux1 overexpression Arabidopsis were significantly higher than those of wild-type Arabidopsis (such as Figure 5 , Figure 6 and Figure 7 ), indicating that PbrAux1 enhances the freezing tolerance of Arabidopsis plants.

[0063] In summary, it can be found that PbrAux1 can improve the freezing resistance of Arabidopsis by increasing the activities of POD, SOD and CAT in Arabidopsis while reducing the MDA content in Arabidopsis.

[0064] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Dangshan pear Aux / IAA gene family gene PbrAux1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A protein encoded by the Dangshan pear Aux / IAA gene family gene PbrAux1 according to claim 1, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

3. A biological material containing the Dangshan pear Aux / IAA gene family gene PbrAux1 according to claim 1, characterized in that: The biological material is recombinant DNA, expression vector, host bacteria or plant material.

4. The biomaterial according to claim 3, characterized in that The expression vector is pCAMBIA-1300 vector plasmid.

5. The biomaterial according to claim 3, characterized in that The host bacteria is Agrobacterium GV3101; the plant material is Arabidopsis thaliana.

6. Use of the gene PbrAux1 according to claim 1 or the biomaterial according to claim 3 in any of the following situations: 1) Used to improve the resistance of plants to low temperature stress; 2) Used to increase the activity of peroxidase, superoxide dismutase and catalase in plants under low temperature stress; 3) Used for preparing transgenic plants; 4) Used in plant breeding.

7. The use according to claim 6, characterized in that: The plant is Arabidopsis thaliana.

8. A method for regulating the low temperature resistance of Arabidopsis thaliana, characterized in that: The method comprises: (1) Making a plant contain the gene PbrAux1 or (2) Overexpressing the gene PbrAux1 according to claim 1 in a plant.

9. The method according to claim 8, characterized in that The method comprises cloning a PbrAux1 sequence, constructing a PbrAux1 gene sequence into an overexpression vector, transforming a strain with a recombinant vector, and multiplying a transgenic gene or a transgenic material.

10. Use of the protein encoded by the Dangshan pear Aux / IAA gene family gene PbrAux1 according to claim 2 in regulating plant frost resistance.

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