Dangshan pear aux / iaa gene family gene pbraux1 and application thereof

Abstract

The application 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 has a nucleotide sequence as shown in SEQ ID NO.1 and has an amino acid sequence as shown in SEQ ID NO.2.The application has the beneficial effect that a low-temperature-resistant gene of Dangshan pear is obtained, a super-expression recombinant vector is constructed by using the gene, and the function of the gene in resisting low temperature is proved by using molecular biology and transgenic technology.The PbrAux1 improves the low-temperature-resistant capability of transgenic Arabidopsis thaliana, and is reflected in that, compared with WT, the survival rate of the Arabidopsis thaliana after low-temperature freezing injury, the activities of POD, SOD and CAT are improved, and the MDA accumulation amount and the relative electrolyte permeability are reduced.Therefore, the PbrAux1 can be introduced into plants as a target gene to promote the low-temperature-resistant capability of the plants.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological genetic engineering, and particularly relates to a PbrAux1 gene of Aux / IAA gene family of Pyrus bretschneideri and application thereof. BACKGROUND

[0002] Aux / IAA is a key transcription factor protein family inducing the regulation of auxin response, and the Aux / IAA gene family affects the early response of auxin by expressing nuclear localization proteins. The Aux / IAA protein includes four highly conserved domains (Domain I, II, III, IV): Domain I exists in the transcriptional repressor domain related to ethylene response factors, and the transcriptional repressor inhibits the expression of genes downstream of the auxin signal through the domain; Domain II has a highly conserved characteristic and is controlled by TIR1, and can directly bind to SCFTIR1 to control the degradation of Aux / IAA protein; Domain III and IV are the binding sites of ARF, and have the homologous domain of CTDs of ARFs protein, which is responsible for the dimerization and multimerization of other Aux / IAA proteins, and further regulates the auxin signal.

[0003] The Aux / IAA family genes have large differences in length and are unevenly distributed on eight chromosomes; the genes are located in the nucleus, and the encoded proteins contain 189-363 amino acids and have large differences in acid-base, and are unstable proteins and hydrophilic proteins. The Aux / IAA gene has been cloned and the regulation mechanism has been studied in plants such as Arabidopsis, tomato, cucumber and rice (Wang et al., 2010; Piya et al., 2014; Ambreetha et al., 2018). Researches have confirmed that Aux / IAA is involved in regulating the growth and development process of plants, for example, knocking out IAA3 / SHY2 of Arabidopsis affects the balance of auxin and lateral root formation. TIR1 / AFB2 can form a special sensing complex with AtIAA2, AtIAA6, AtIAA9 or AtIAA17, and can regulate the balance of jasmonic acid and the initial development of adventitious roots after auxin treatment. Aux / IAA gene is also involved in the response to various stress responses, for example, OsIAA9 and OsIAA20 of rice are induced to express under drought and salt stress, and the transcription level of GmIAA47 and GmIAA49 of soybean in leaf is significantly increased at the reproductive stage under drought conditions. The above is the research of Aux / IAA gene family in regulating the growth and development of plants, and the research of the gene family in regulating the plant resistance to low temperature in Pyrus bretschneideri has not been reported. SUMMARY

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

[0005] The present application solves the above technical problems by the following technical means:

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

[0007] The second aspect of the present application provides a protein encoded by the above gene PbrAux1, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

[0008] The third aspect of the present application provides a biological material containing the above gene PbrAux1, and the biological material includes but is not limited to a recombinant DNA, an expression vector, a host bacterium 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 includes Arabidopsis thaliana.

[0012] The fourth aspect of the present application provides an application of the above gene PbrAux1 or the biological material containing the gene PbrAux1 in any of the following cases:

[0013] 1) for improving the resistance of plants to low temperature stress;

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

[0015] 3) for preparing transgenic plants;

[0016] 4) for plant breeding.

[0017] Preferably, the plant is Arabidopsis thaliana.

[0018] The fifth aspect of the present application provides a method for regulating the low temperature resistance of Arabidopsis thaliana, and the method comprises:

[0019] (1) making the plant contain the gene PbrAux1 of claim 1 or

[0020] (2) making the plant overexpress the gene PbrAux1 of claim 1.

[0021] Preferably, the method includes but is not limited to cloning the PbrAux1 sequence, constructing the PbrAux1 gene sequence into an overexpression vector, transforming a strain with a recombinant vector, transgenesis, and propagation of transgenic materials.

[0022] The sixth aspect of the present application provides an application of the protein encoded by the PbrAux1 gene of the Aux / IAA gene family of Dangshan pear in regulating the low-temperature resistance of plants.

[0023] The present application has the following advantages:

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

[0025] Figure 1 Fig. 3 is a result diagram of genomic PCR identification of PbrAux1 transgenic Arabidopsis thaliana obtained by using a floral infection method mediated by Agrobacterium in Example 3 of the present application. 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 Fig. 4 is a comparison diagram of freeze injury phenotypes and mortality of PbrAux1 transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana after being cultured on a culture medium for 2 weeks and then being treated at-2℃ for 1.5h in Example 3 of the present application;

[0027] Figure 3 Fig. 5 is a comparison diagram of relative electrolyte permeability of PbrAux1 transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana before and after being treated at-2℃ for 2h in Example 3 of the present application;

[0028] Figure 4 Fig. 6 is a comparison diagram of malondialdehyde (MDA) content of PbrAux1 transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana after being treated at-2℃ for 2h in Example 3 of the present application;

[0029] Figure 5 Fig. 7 is a comparison diagram of peroxidase (POD) activity of PbrAux1 transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana after being treated at-2℃ for 2h in Example 3 of the present application;

[0030] Figure 6is a comparison chart of different Arabidopsis thaliana superoxide dismutase (SOD) activities after PbrAux1 transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana in Example 3 of the present application are subjected to low temperature treatment at-2℃ for 2h;

[0031] Figure 7 is a comparison chart of different Arabidopsis thaliana catalase (CAT) activities after PbrAux1 transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana in Example 3 of the present application are subjected to low temperature treatment at-2℃ for 2h. DETAILED DESCRIPTION

[0032] To make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described below in a clear and complete manner in combination with the embodiments of the present application. Obviously, the described embodiments are only some, but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0033] In the following examples, the test materials and reagents used, etc. can be obtained from commercial channels unless otherwise specified.

[0034] If no specific technology or condition is specified in the examples, the technology or condition described in the literature in the art or according to the product manual can be used.

[0035] Example 1: Obtaining of PbrAux1 gene

[0036] A number of plant leaves were collected from Pyrus bretschneideri Rehd. trees in Dangshan pear production areas in Hefei, Anhui, and total RNA of the P. bretschneideri leaf samples was extracted using an RNA extraction kit, and cDNA was obtained by RNA reverse transcription.

[0037] According to the transcriptome sequencing result analysis of the P. bretschneideri 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 on the upstream and downstream of the CDS of the gene by using Primer Premier 5 software;

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

[0039] P2: TCAAGCTTTGTGAGGGGGG. (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 the overexpression vector of PbrAuxl gene

[0050] It is known that the enzyme cutting sites of pCAMBIA-1300 vector plasmid are Xba I and BamH I. The above enzyme cutting sites were double-cut by using the restriction endonuclease produced by Sanying Biotechnology Co., Ltd. After adding sample according to the system requirements, the reaction was carried out at 37°C for 2 h, and then the double-cut target band was obtained by agarose gel electrophoresis. After cutting and recovering, the linearized vector of pCAMBIA-1300 vector plasmid was obtained.

[0051] Xba I and BamH I two enzyme cutting sites 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] Using the target gene cDNA with correct sequencing results as a template, PCR amplification was performed using Novizan high-fidelity enzyme. After amplification, 10 μL of 5× Loading Buffer was added to the PCR product, mixed well, and then detected by agarose gel electrophoresis. The target band was obtained after gel extraction and storage at -20℃.

[0055] Finally, we used the technology provided by TOLOBIO. Universal CloneMix performs a recombination reaction between the insert fragment and the linearized vector. The reaction product is then transformed into *E. coli*, plated, and incubated at 37°C for 12 hours. Single colonies are picked and cultured in liquid LB medium containing 100 mg / L kanamycin sulfate for 3 hours. Detection is then performed using the vector and primers. After successful sequencing alignment, 50% glycerol is added at a 1:1 volume ratio, and the mixture is stored at -80°C.

[0056] Example 3: Arabidopsis thaliana transformation and screening

[0057] PbrAuxl transgenic Arabidopsis was obtained by Agrobacterium-mediated inflorescence infection. Wild-type Arabidopsis was planted, and when the wild-type Arabidopsis plants reached the full bloom stage, the flower buds were left and the rest of the flowers and pods were removed. The Agrobacterium carrying the PbrAuxl overexpression vector was taken out from the -80°C refrigerator, activated, and then 30 ml of bacterial solution was placed in a 28°C shaker for 10-12 hours until the bacterial solution was turbid. Before infection, the infection solution was prepared by adding sucrose at a final concentration of 30 g / L and Silwet L-77 at a final concentration of 200 l / L to the MS liquid medium. The Agrobacterium bacterial solution was centrifuged at 5000 rpm for 8 minutes, the supernatant was discarded, and then the Agrobacterium precipitate was resuspended with the infection solution. The OD600 of the infection solution was measured to be 0.8-1.0 using a UV spectrophotometer. The wild-type Arabidopsis inflorescences with only flower buds were completely immersed in the infection solution for 120 seconds, and after the infection was completed, the inflorescences were completely wrapped with a film and cultured in the dark for 1 day. After the film was removed, the plants were normally cultured until the seeds matured. After the seeds were harvested, they were washed with 75% alcohol and 95% alcohol in a clean bench, and after drying, they were evenly sown on MS selection medium containing hygromycin in a 4°C environment for 1 day, and then vertically placed in a light incubator for culture. After the seedlings grew to 2-3 true leaves, the seedlings were transplanted to the substrate and normally cultured for 1 week. Two healthy leaves were selected from each Arabidopsis plant for DNA extraction and identification, and the positive plants were T0 generation transgenic plants (as shown in Figure 1 ). The T0 generation transgenic plants were normally cultured until the seeds matured, and the seeds were harvested from each plant to obtain T1 generation transgenic Arabidopsis seeds.

[0058] (1) Freezing resistance identification (MS medium method): PbrAuxl overexpression and wild-type Arabidopsis were sown on MS medium, and the phenotypic differences were observed. During the entire growth process, the MS plates were placed horizontally.

[0059] After 2 weeks of growth on the medium, the plants were treated at -2°C for 1.5 hours. Then the medium was removed and cultured at normal temperature. After 2 days, the Arabidopsis plants showed freezing injury phenotypes. The results showed that the survival rate of PbrAuxl overexpression Arabidopsis was significantly higher than that of wild-type plants. Through observation and analysis, it was found that the survival rate of PbrAuxl overexpression Arabidopsis after low temperature was significantly higher than that of WT wild-type Arabidopsis (as shown in Figure 2 ).

[0060] (2) Low temperature physiological index determination: 4-week-old transgenic Arabidopsis and wild-type Arabidopsis under normal growth conditions were treated at -2℃ for 2h, and the relative electrolyte permeability of each Arabidopsis before and after low temperature treatment was measured. It can be found that there is no difference in the relative electrolyte permeability content between each Arabidopsis under normal growth conditions, and after low temperature treatment, the transgenic Arabidopsis shows lower relative electrolyte permeability compared with the wild-type Arabidopsis, indicating that the damage degree of the transgenic Arabidopsis leaf is significantly lower than that of the wild-type Arabidopsis (as shown in Figure 3 ).

[0061] The MDA content of each Arabidopsis before and after -2℃ treatment was detected. It can be found that the MDA content in each Arabidopsis under normal conditions is less, and there is no difference in MDA content between each genotype Arabidopsis; and after low temperature treatment, it can be found that the MDA content of each Arabidopsis shows an upward trend, and the MDA content of the transgenic Arabidopsis is significantly lower than that of the wild-type Arabidopsis, indicating that the transgenic Arabidopsis is less damaged (as shown in Figure 4 ). It is shown that the freeze resistance of the transgenic Arabidopsis is higher than that of the 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 obvious difference in the POD, SOD and CAT activities of each Arabidopsis before low temperature treatment; and after low temperature treatment, the POD, SOD and CAT activities of PbrAux1 overexpression Arabidopsis were significantly higher than those of the wild-type Arabidopsis (as shown in Figure 5 , Figure 6 and Figure 7 ), indicating that PbrAux1 enhances the freeze resistance of Arabidopsis plants.

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

[0064] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A gene family of Aux / IAA from Dangshan crisp pear PbrAux1 Its characteristics are, Its nucleotide sequence is shown in SEQ ID NO.

1.

2. A gene family of Aux / IAA from Dangshan pear as described in claim 1. PbrAux1 The encoded protein is characterized by, Its amino acid sequence is shown in SEQ ID NO.

2.

3. A gene containing the Aux / IAA gene family of Dangshan pear as described in claim 1. PbrAux1 The biomaterial is characterized by, The biological materials are recombinant DNA, expression vector, and host bacteria.

4. The biomaterial according to claim 3, characterized in that, The biomaterial is an expression vector.

5. The biomaterial according to claim 4, characterized in that, The base vector for the expression vector is the pCAMBIA-1300 vector plasmid.

6. The biomaterial according to claim 3, characterized in that, The host bacterium is Agrobacterium GV3101.

7. A gene as described in claim 1 PbrAux1 Or the use of the biomaterial of claim 3 in any of the following situations: 1) Used to improve the resistance of Arabidopsis thaliana to low-temperature stress; 2) Used for the preparation of transgenic Arabidopsis thaliana; 3) Used for breeding low-temperature resistant Arabidopsis thaliana.

8. A method for enhancing the low-temperature resistance of Arabidopsis thaliana, characterized in that, The method includes: (1) To make Arabidopsis thaliana contain the gene of claim 1 PbrAux1 or (2) Overexpressing the gene of claim 1 in Arabidopsis thaliana. PbrAux1 .

9. The method according to claim 8, characterized in that, The method is cloning. PbrAux1 sequence, PbrAux1 Gene sequence construction to overexpression vector, recombinant vector transformation of strains, transgenic, and transgenic material propagation.

10. The Aux / IAA gene family gene of Dangshan crisp pear as described in claim 2 PbrAux1 Application of the encoded protein in upregulating the freeze resistance of Arabidopsis thaliana.

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