Application of BnaMATE43b gene in improving plant antioxidant activity and / or improving plant aluminum toxicity tolerance

Abstract

The present invention relates to the field of genetic engineering technology, and in particular to the use of the BnaMATE43b gene in improving plant antioxidant properties and / or improving plant aluminum toxicity resistance. The present invention introduces the BnaMATE43b gene cloned from aluminum-resistant rapeseed FDH188 into rapeseed westar, and the antioxidant properties and aluminum toxicity resistance of the rapeseed overexpressing the BnaMATE43b gene are significantly higher than those of the wild-type rapeseed westar. It can be seen that the rapeseed BnaMATE43b gene of the present invention can improve the antioxidant properties and aluminum toxicity resistance of rapeseed.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to the application of the BnaMATE43b gene in improving plant antioxidant properties and / or improving plant aluminum toxicity resistance. Background Art

[0002] Due to irregular fertilizer application and large amounts of acid deposition, soil acidity increases. When the soil pH is below 5.5, exchangeable aluminum (mainly A1 3+ 、Al(OH) 2+ and Al(OH) 2 + ) will be released from silicates or oxides, causing aluminum toxicity stress to plant roots, affecting the roots' absorption of water and nutrients in the soil, and ultimately restricting the growth of the entire plant, causing crop yields to decrease by more than 20%.

[0003] Plants' tolerance to aluminum toxicity can be divided into two types: external rejection and internal tolerance. The internal tolerance mechanism is mainly that aluminum chelates with organic acids, phenols and other compounds in the plasma membrane to form a complex, isolating aluminum ions in the vacuole or other storage organelles to reduce aluminum toxicity. The external rejection mechanism is mainly that citric acid, malic acid, oxalic acid and other transport proteins are transported to the outside of the cell to chelate the active Al outside the plasma membrane. 3+ , reducing the active Al 3+ concentration, thereby alleviating the damage of aluminum toxicity to the root system. In rapeseed, there are few reports on the function of the BnaMATE43b gene and its encoded protein. Summary of the invention

[0004] In order to solve the above problems, the present invention provides the use of BnaMATE43b gene in improving plant antioxidant activity and / or improving plant aluminum toxicity resistance. The present invention finds that BnaMATE43b gene and its encoded protein can improve plant antioxidant activity and aluminum toxicity resistance.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides the use of the BnaMATE43b gene in improving plant antioxidant properties and / or improving plant aluminum toxicity resistance. The amino acid sequence of the protein encoded by the BnaMATE43b gene is shown in SEQ ID NO.1.

[0007] Preferably, the nucleotide sequence of the BnaMATE43b gene includes the sequence shown in SEQ ID NO.2.

[0008] Preferably, the plants include dicotyledonous plants and / or monocotyledonous plants.

[0009] Preferably, the dicotyledonous plants include cruciferous plants; and the monocotyledonous plants include grass plants.

[0010] Preferably, the cruciferous plants include rapeseed and / or Arabidopsis thaliana; and the grass plants include rice and / or corn.

[0011] The present invention provides an expression vector of a BnaMATE43b gene, the expression vector comprising the BnaMATE43b gene and a basic vector; the amino acid sequence of the protein encoded by the BnaMATE43b gene is shown in SEQ ID NO.1.

[0012] Preferably, the basic vector comprises a plant expression vector.

[0013] The present invention provides a recombinant bacterium expressing the BnaMATE43b gene, wherein the recombinant bacterium comprises the expression vector described in the above technical solution.

[0014] The present invention provides the use of the expression vector described in the above technical solution or the recombinant bacteria described in the above technical solution in improving plant antioxidant properties and / or improving plant aluminum toxicity resistance.

[0015] The present invention provides the use of the BnaMATE43b gene, the expression vector described in the above technical solution or the recombinant bacteria described in the above technical solution in cultivating transgenic plants. The amino acid sequence of the protein encoded by the BnaMATE43b gene is shown in SEQ ID NO.1.

[0016] Beneficial effects:

[0017] The present invention provides an application of a BnaMATE43b gene in improving plant antioxidant properties and / or improving plant aluminum toxicity resistance, and the amino acid sequence of the protein encoded by the BnaMATE43b gene is shown in SEQ ID NO.1. The present invention introduces the BnaMATE43b gene cloned from aluminum-resistant rapeseed FDH188 into rapeseed westar, and the antioxidant properties and aluminum toxicity resistance of rapeseed overexpressing the BnaMATE43b gene are significantly higher than those of wild-type rapeseed westar. It can be seen that the rapeseed BnaMATE43b gene of the present invention can improve the antioxidant properties and aluminum toxicity resistance of rapeseed. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0019] Figure 1 This is the agarose gel electrophoresis diagram of the PCR of BnaMATE43b gene of rapeseed;

[0020] Figure 2 This is a schematic diagram of the BnaMATE43b gene overexpression vector;

[0021] Figure 3 This is the molecular detection diagram of the T0 generation of transgenic rapeseed; lanes 1 to 21 are the T0 generation transgenic rapeseed plants, and M is Maker;

[0022] Figure 4 The results of identification of BnaMATE43b gene expression in wild type and three transgenic positive lines;

[0023] Figure 5 The phenotypes of the wild type and three transgenic positive lines under aluminum toxicity stress and control treatments;

[0024] Figure 6 The bar graph shows the aluminum toxicity resistance index of the wild type and three transgenic positive lines; * means p < 0.05, ** means p < 0.01;

[0025] Figure 7 The bar graphs show the physiological indexes of roots of wild type and three transgenic plants under aluminum toxicity stress and control treatments. * indicates p < 0.05 and ** indicates p < 0.01. DETAILED DESCRIPTION

[0026] The present invention provides an application of a BnaMATE43b gene in improving plant antioxidant properties and / or improving plant resistance to aluminum toxicity. The amino acid sequence of a protein encoded by the BnaMATE43b gene is shown in SEQ ID NO.1, which is specifically as follows: MTETGDLTSVPASVVTQIPFLVIFNELTRVFSKDAIGREILG IAFPAALALAADPIASLIDTAYIGRLGAVELAAVGVSIAIFNQASRITIFPIVSVTTSFVAEENTMEKIKEEEEEAKKNNRVHANTLAVQDSLEKGIASPTSNNMNQPQQTPASDTKSSSGNKANKKGKKNIKSASTAMIIGLILGLVQAIFLIFSSKVLLGVMGVKPNSAVLSPAHKYLTIRSLGAPALLLSLSMQGIFRGFKDTKTPLYATIVADVINIILDPIFIFVLHLGI SGAAIAHVISQYFMTLILFTRLASKVNLMPPNFAALQFGKFLKNGGLLLARTIAVTFCQTLAAAMAARLGATPMAAFQICLQVWLTTSLLADGLAVAGQAILACSFTEKDYNKVSSCA SRVLQMGFVLGLGLSVFVGLGLYFGAGIFTKDPAVIHLIAIGIPFVAATQPINSLAFVLDGVNFGASDFAYTAYSMVGVAAISIAAIIYMAKFNGFLGIWIALTIYMGLRAITGIAR.

[0027]

[0028] In the present invention, the plants preferably include dicotyledonous plants and / or monocotyledonous plants; the dicotyledonous plants preferably include cruciferous plants; the cruciferous plants preferably include rapeseed and / or Arabidopsis thaliana; the monocotyledonous plants preferably include grass plants; the grass plants preferably include rice and / or corn.

[0029] In the embodiment of the present invention, the BnaMATE43b gene cloned in aluminum-resistant rapeseed FDH188 is introduced into rapeseed westar to obtain transgenic plants; after the transgenic plants and wild-type plants are treated with aluminum poisoning for 7 days, the relative elongation of the main root of the wild-type is 57.27%; the relative elongation of the main root of the transgenic plants T2-15, T2-16, and T2-18 is 66.57%, 67.25%, and 65.74%, respectively, which is significantly higher than that of the wild-type; the relative total root length of the wild-type is 69.97%; the relative total root length of the transgenic plants T2-15, T2-16, and T2-18 is 77.07%, 77.71%, and 76.13%, respectively, which is significantly higher than that of the wild-type. After 48 hours of treatment, the content of CAT, MDA, and soluble protein in the root system of the transgenic strain is significantly higher than that of the wild-type plant, the activity of the SOD enzyme is significantly higher than that of the wild-type, the POD enzyme activity of T2-15 is significantly higher than that of the wild-type, and there is no significant difference in PRO content. The above results indicate that overexpression of BnaMATE43b increased the antioxidant properties and aluminum toxicity resistance of the transgenic lines, that is, the BnaMATE43b gene and its encoded protein can improve plant antioxidant properties and aluminum toxicity resistance.

[0030] The present invention also provides an expression vector of the BnaMATE43b gene, which comprises the BnaMATE43b gene and a basic vector; the amino acid sequence of the protein encoded by the BnaMATE43b gene is shown in SEQ ID NO.1.

[0031] In the present invention, the basic vector preferably includes a plant expression vector; the plant expression vector preferably includes a pCAMBIA1301 vector; and the BnaMATE43b gene is preferably located between the BglⅡ and BstEⅡ restriction sites of the pCAMBIA1301 vector.

[0032] The present invention also provides a recombinant bacterium expressing the BnaMATE43b gene, wherein the recombinant bacterium comprises the expression vector described in the above technical solution. In the present invention, the original bacterium of the recombinant bacterium preferably comprises an Agrobacterium strain, more preferably Agrobacterium tumefaciens EHA105.

[0033] The present invention also provides the use of the expression vector described in the above technical solution or the recombinant bacteria described in the above technical solution in improving plant antioxidant properties and / or improving plant aluminum toxicity resistance.

[0034] In the present invention, the plants preferably include dicotyledonous plants and / or monocotyledonous plants; the dicotyledonous plants preferably include cruciferous plants; the cruciferous plants preferably include rapeseed and / or Arabidopsis thaliana; the monocotyledonous plants preferably include grass plants; the grass plants preferably include rice and / or corn.

[0035] The present invention also provides the use of the BnaMATE43b gene, the expression vector described in the above technical solution or the recombinant bacteria described in the above technical solution in cultivating transgenic plants. The amino acid sequence of the protein encoded by the BnaMATE43b gene is shown in SEQ ID NO.1.

[0036] In the present invention, the wild-type plants for cultivating transgenic plants preferably include dicotyledonous plants and / or monocotyledonous plants; the dicotyledonous plants preferably include cruciferous plants; the cruciferous plants preferably include rapeseed and / or Arabidopsis thaliana; the monocotyledonous plants preferably include grass plants; the grass plants preferably include rice and / or corn.

[0037] The present invention utilizes the expression vector described in the above technical scheme or the recombinant bacteria described in the above technical scheme to introduce the BnaMATE43b gene into plants, thereby obtaining transgenic plants with higher antioxidant properties and aluminum toxicity resistance than the wild type, providing technical support for molecular breeding related to plant aluminum toxicity resistance.

[0038] To further illustrate the present invention, the application of the BnaMATE43b gene provided by the present invention in improving plant antioxidant properties and / or improving plant aluminum toxicity resistance is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] 1. Full-length cloning of CDS encoding BnaMATE43b

[0041] A plant total RNA extraction kit (purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd.) was used to extract total RNA from the roots of aluminum-tolerant rapeseed FDH188 plants, and a reverse transcription kit was used to reverse transcribe it into cDNA (purchased from Baoriyi Biotechnology (Beijing) Co., Ltd.); the aluminum-tolerant rapeseed FDH188 was preserved in the Key Laboratory of Crop Physiology, Ecology and Genetic Breeding of the Ministry of Education, Jiangxi Agricultural University, and was published in [Zhou HW, Xiao XJ, Ali Asjad, et al. Integration of GWAS and transcriptome analyses to identify SNPs and candidate genes for aluminum tolerance inrapeseed (Brassica napus L.) [J]. BMC Plant Biology. 2022, 22: 130.].

[0042] The cDNA is used as a template, and PCR amplification is performed using a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is: 5'-ATGACGGAAACTGGTGATC-3' (as shown in SEQ ID NO.3); the nucleotide sequence of the reverse primer is: 5'-TTACCTGGCAATTCCAGTAA-3' (as shown in SEQ ID NO.4); the PCR amplification system is 25 μl, specifically: 1 μl forward primer (primer concentration is 10 μM), 1 μl reverse primer (primer concentration is 10 μM), 2 μl template (template concentration is 150 ng / μl), 0.5 μl high-fidelity enzyme, 2 μl dNTPs, 2.5 μl 10×Buffer and 18 μl sterile water ddH 2 O; the PCR amplification program is: 94°C pre-denaturation for 3 min; 94°C denaturation for 45 s, 59°C annealing for 45 s, 72°C extension for 2 min, 30 cycles; 72°C extension for 10 min; 12°C storage.

[0043] The product obtained by PCR amplification is 1539 bp in total. The specific cloning results are as follows: Figure 1 The PCR product was sent for sequencing, and the nucleotide sequence of the PCR product was shown in SEQ ID NO.2. The gene was named BnaMATE43b; the protein encoded by the gene was BnaMATE43b, and the amino acid sequence of the protein was shown in SEQ ID NO.1.

[0044] 2. Construction of BnaMATE43b overexpression vector and genetic transformation

[0045] The specific construction process of the recombinant vector pCAMBIA1301-BnaMATE43b overexpressing the BnaMATE43b gene is as follows:

[0046] The nucleotide sequence of the BnaMATE43b gene shown in SEQ ID NO.2 was inserted between the BglⅡ and BstEⅡ restriction sites of the plant expression vector pCAMBIA1301, that is, the 5' end of the first position of SEQ ID NO.2 was adjacent to the BglⅡ restriction site, and the 3' end of the last position of SEQ ID NO.2 was adjacent to the BstEⅡ restriction site, to obtain the recombinant vector pCAMBIA1301-BnaMATE43b. The recombinant vector is as shown in Figure 2 shown.

[0047] The recombinant vector pCAMBIA1301-BnaMATE43b was introduced into Agrobacterium EHA105 by freeze-thaw method to obtain recombinant Agrobacterium EHA105 / pCAMBIA1301-BnaMATE43b.

[0048] The recombinant Agrobacterium EHA105 / pCAMBIA1301-BnaMATE43b was genetically transformed into wild-type rapeseed westar by the cotyledon node method. After the transgenic plants grew roots, the lid of the test tube containing the transgenic strain was opened, the culture medium was cleaned, and sterile water was added to culture at room temperature for 2 to 3 days. Transplanted and cultured in a flower pot, after it grew normally, moved it outdoors for potted planting. Cultivation management was carried out according to conventional field management methods.

[0049] The genomic DNA of the transgenic T0 generation transgenic rapeseed was extracted by the CTAB method, and PCR amplification was performed using a vector primer and a target gene primer to detect positive plants. The nucleotide sequence of the vector primer is shown in SEQ ID NO.5: 5'-GACGTAAGGGATGACGCACA-3', and the nucleotide sequence of the target gene primer is shown in SEQ ID NO.6: 5'-TGCACAAGGCCCAGGATTAG-3'; the amplification system was 15 μl, specifically: 1 μl vector primer (primer concentration was 10 μM), 1 μl target gene primer (primer concentration was 10 μM), 1 μl genomic DNA template, 7.5 μl 2×PCR Mix and 4.5 μl sterile water ddH 2 O; the PCR amplification program is: 94℃ pre-denaturation for 3min; 94℃ denaturation for 45s, 59℃ annealing for 45s, 72℃ extension for 1min, 28 cycles; 72℃ extension for 5min; 12℃ storage. Figure 3 shown.

[0050] After harvesting the T0 generation positive transgenic lines, T1 and T2 generation positive plants were obtained through continuous self-pollination, and the gene expression level of BnaMATE43b in T2-15, T2-16, and T2-18 was detected by fluorescence quantitative PCR technology;

[0051] The detection steps of the fluorescent quantitative PCR are as follows: the cDNA of the transgenic rapeseed plant and the wild-type rapeseed plant are used as templates for fluorescent quantitative PCR amplification; the amplification primers of the BnaMATE43b gene are composed of the forward primer shown in SEQ ID NO.7 and the reverse primer shown in SEQ ID NO.8; the internal reference gene is ACT6, and the amplification primers of the internal reference gene ACT6 are composed of the forward primer shown in SEQ ID NO.9 and the reverse primer shown in SEQ ID NO.10, and the specific primer nucleotide sequences are as follows:

[0052] SEQ ID NO.7: 5'-CTGGGCCTTGTGCAAGCTAT-3';

[0053] SEQ ID NO.8: 5'-TTGGTGTCCTTGAAGCCACG-3';

[0054] SEQ ID NO.9: 5'-CCTCTCAACCCGAAAGCCAA-3';

[0055] SEQ ID NO.10: 5'-CATCACCAGAGTCGAGCACA-3';

[0056] The fluorescence quantitative PCR system was 20 μl, and the fluorescence quantitative reagent was purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd., specifically: 0.8 μl forward primer (primer concentration was 10 μM), 0.8 μl reverse primer (primer concentration was 10 μM), 10 μl 2×qPCR Mix, 2 μl template and 6.4 μl RNase free ddH 2 O;

[0057] The program of the fluorescent quantitative PCR amplification is: 95°C for 5 min; 95°C for 15 s, 60°C for 30 s, 40 cycles; the melting curve setting temperature is 60-95°C; the gene expression level detection results are as follows Figure 4 As shown, the expression of BnaMATE43b in leaves and roots of transgenic lines T2-15, T2-16, and T2-18 was upregulated 5.09 to 10.76 times compared with that of wild-type plants.

[0058] 3. Verification of aluminum toxicity resistance of positive transgenic rapeseed lines

[0059] The wild type (denoted as WT) and positive transgenic rapeseed lines T2-15, T2-16, and T2-18 were treated with aluminum poisoning for 48 h, and three parallel experiments were conducted for each line; the aluminum poisoning treatment was as follows: seeds with full and uniform grains were selected and sown on gauze in a square plastic basin filled with pure water. After 6 days, when the cotyledons were fully expanded but the lateral roots had not yet differentiated from the main root, seedlings with basically the same growth were selected from each material and transferred to a square basin filled with 1 / 4 improved Hoagland nutrient solution (Beijing Coolaibo Technology Co., Ltd.), and the seedlings were slowed down for 3 days, and then transferred to 1 / 2 improved Hoagland nutrient solution respectively. After culturing for 3 days, aluminum stress test was carried out; the plants were taken out of the 1 / 2 nutrient solution and transferred to 0.5 mmol / L CaCl 2 solution (pH 4.3) for 24 h, and then transferred into 60 μmol / L AlCl 3 and 0.5mmol / L CaCl 2 1 / 2 modified Hoagland nutrient solution (pH 4.3) for 7 days without adding AlCl 3 The taproot lengths of wild-type and positive transgenic rapeseed lines T2-15, T2-16, and T2-18 were measured before and after treatment. The total root lengths of wild-type and positive transgenic rapeseed lines T2-15, T2-16, and T2-18 were analyzed after treatment. The relative elongation of the taproot and the relative total root length were calculated to identify the aluminum toxicity resistance of transgenic plants. The wild type was used as the control. The phenotypic results are shown in Table 1. Figure 5 and Figure 6 shown.

[0060] The phenotypic identification results were Figure 5 and Figure 6 The results showed that the relative elongation rate of the wild type taproot was 57.27%; the relative elongation rates of the taproots of transgenic plants T2-15, T2-16, and T2-18 were 66.57%, 67.25%, and 65.74%, respectively, which were significantly higher than those of the wild type; the relative total root length of the wild type was 69.97%; the relative total root length of transgenic plants T2-15, T2-16, and T2-18 were 77.07%, 77.71%, and 76.13%, respectively, which were significantly higher than those of the wild type.

[0061] Physiological indices of plant roots were determined 48 h after aluminum poisoning, specifically catalase (CAT), malondialdehyde (MDA), peroxidase (POD), superoxide dismutase (SOD), soluble protein (Protein) and proline (PRO) according to the instructions of the kit (the kit was purchased from Suzhou Grace Biotechnology Co., Ltd.). The results of physiological activity tests are shown in the figure. Figure 7 , as shown in Table 1 and Table 2.

[0062] Table 1 Results of physiological indexes of wild-type and transgenic plants T2-15 at different times

[0063]

[0064] Table 2 Physiological indexes of transgenic plants T2-16 and T2-18 at different times

[0065]

[0066]

[0067] Depend on Figure 7 As shown in Table 1 and Table 2, after 48 hours of aluminum toxicity treatment, the CAT, MDA, and soluble protein content in the roots of the transgenic lines were significantly higher than those of the wild-type plants, the SOD enzyme activity was significantly higher than that of the wild-type, the POD enzyme activity of T2-15 was significantly higher than that of the wild-type, and there was no significant difference in PRO content. The results showed that overexpression of BnaMATE43b increased the antioxidant properties of the transgenic lines, thereby improving the aluminum toxicity resistance of the transgenic lines.

[0068] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of the BnaMATE43b gene in improving the antioxidant capacity of rapeseed, the amino acid sequence of the protein encoded by the BnaMATE43b gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the BnaMATE43b gene includes the sequence shown in SEQ ID NO.2.

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