Fusion gene for improving stress resistance of plant, construction method therefor, and use thereof

Abstract

The present invention relates to the technical field of genetic engineering, and in particular to a fusion gene for improving stress resistance of a plant, a construction method therefor, and a use thereof. The fusion gene is named AR1, and the nucleotide sequence thereof is as shown in SEQ NO. 1. The provided fusion gene for improving the stress resistance of a plant can significantly promote the growth of the plant while greatly improving the resistance of the plant to a high temperature and saline alkali stress, can be widely used in fields such as cultivation of new crop varieties having multi-stress resistance, and has important application value. The provided construction method for the fusion gene can successfully construct the fusion gene for improving the stress resistance of the plant. The construction method involves simple operation and easily controllable conditions, facilitating widespread promotion and use.

Description

A fusion gene for improving plant stress resistance, a construction method and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to a fusion gene for improving plant stress resistance, a construction method and application thereof. BACKGROUND

[0002] Plant stress resistance is the ability of plants to resist adverse external factors, such as drought resistance, saline-alkali resistance, waterlogging resistance, wind resistance, frost resistance, disease and pest resistance, etc.

[0003] Under natural conditions, due to different geographical locations and climate conditions, human activities and other reasons, various adverse environments are caused, which exceed the range that plants can tolerate in normal growth and development, and thus plants are damaged or even die. These environments that cause damage to plants are called stress or stress, and the adaptability and resistance of plants to adverse environments are stress resistance or stress resistance.

[0004] With global climate change and environmental degradation, plants are threatened by high temperature, salinity and other stresses during growth. These stresses seriously affect the yield and quality of plants, and bring great challenges to agricultural production. Therefore, improving the multiple stress resistance of plants through molecular breeding is of great significance for guaranteeing food security, optimizing agricultural industry structure and promoting the development of agriculture towards green, efficient and sustainable direction. SUMMARY

[0005] The present application aims to at least solve one of the technical problems in the related art. To this end, the first object of the present application is to provide a fusion gene for improving plant stress resistance, the second object of the present application is to provide a construction method of the fusion gene, and the third object of the present application is to provide an application of the fusion gene.

[0006] In order to achieve the first object, the technical solution adopted by the present application is as follows: a fusion gene for improving plant stress resistance, the nucleotide sequence of the fusion gene is shown as SEQ NO. 1, and the nucleotide sequence of the fusion gene from 5' end to 3' end is as follows: Further, the stress resistance includes saline-alkali resistance and high temperature resistance.

[0007] To achieve the second object, the technical scheme adopted by the present application is: a construction method of a fusion gene, the fusion gene being the fusion gene for improving plant stress resistance, the fusion gene being named AR1, comprising cloning and fusion construction of AR1-1 and fragment AR1-2, comprising the following steps: S100, using His-R10 as a template, amplifying fragment AR1-1-1, and connecting the amplification product to pMD18-T vector to obtain TA-AR1-1-1-HA; S200, using AR1-1-F2 and AR1-1-R2 as primers to amplify fragment AR1-1 from TA-AR1-1-1-HA plasmid, using Arabidopsis genome as a template, AR1-2-F and AR1-2-R as primers, introducing BamHI and PstI enzyme cutting sites at the upstream and downstream respectively, and cloning fragment AR1-2 by using the method of PCR amplification; S300, using BamHI and EcoO65I (BstEII) to cut pCAMBIA3301 vector, and using the seamless cloning technology to homologously recombine the amplified fragments AR1-1, fragment AR1-2 and the cut pCAMBIA3301 vector to obtain fusion gene AR1, and connecting the fusion gene AR1 on the pCAMBIA3301 vector skeleton to obtain AR1-p3301.

[0008] Further, the primers used in step S100 are AR1-1-F and AR1-1-R, respectively, the nucleotide sequence of AR1-1-F from 5' end to 3' end is shown in SEQ NO. 2, and the nucleotide sequence of AR1-1-R from 5' end to 3' end is shown in SEQ NO. 3; SEQ NO. 2 is shown as follows: SEQ NO. 3 is shown as follows:

[0009] To achieve the third object, the technical scheme adopted by the present application is: an application of a fusion gene, the fusion gene being the fusion gene for improving plant stress resistance, the application comprising plant transformation or breeding for improving plant stress resistance by using the construction method of the fusion gene.

[0010] Further, the transformation comprises the following steps: S400, using AR1-p3301 to transform GV1301 Agrobacterium to obtain transformed Agrobacterium carrying fusion gene AR1; wherein the breeding is breeding by using the transformed Agrobacterium, comprising the following steps: S500, obtaining transgenic plants by using the method of using the transformed Agrobacterium to infect receptor plant explants or inflorescences to obtain T0 generation seeds carrying fusion gene AR1.

[0011] Further, in step S400, the obtained transformed Agrobacterium is cultured in a selection medium containing antibiotics to screen the transformed Agrobacterium successfully introducing the fusion gene AR1, wherein the medium is not limited, for example, the medium can be an LB plate medium containing kanamycin, gentamicin and rifampicin.

[0012] Further, the method further comprises the following step: S600, the T0 generation seeds are subjected to resistance screening with antibiotics or herbicides to obtain T1 generation seeds successfully introducing the fusion gene AR1, wherein the resistance screening is not limited, for example, Basta resistance screening can be performed.

[0013] Further, the breeding comprises breeding of crops and horticultural crops.

[0014] Further, the crops comprise at least one of rice, wheat, corn, cotton, soybean, rape, potato, fruits and vegetables, and the horticultural crops comprise at least one of forest trees and flowers.

[0015] The one or more technical solutions in the embodiments of the present application have at least one of the following technical effects: the fusion gene AR1 for improving plant stress resistance provided by the present application can significantly promote plant growth while greatly improving the resistance of plants to high temperature and saline-alkali, and can be widely applied in the field of new variety breeding of crops with multiple stress resistance, and has important application value. The present application also provides a construction method of the fusion gene, according to which the fusion gene AR1 for improving plant stress resistance can be successfully constructed, and the method is simple to operate, the conditions are easy to control, and is conducive to wide promotion and use.

[0016] Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following and / or can be learned by practice of the application. BRIEF DESCRIPTION OF DRAWINGS

[0017] Fig. 1 is a graph of AR1 protein level detection results in the fusion gene AR1 transgenic line provided by the embodiment 1 of the present application.

[0018] Fig. 2 is an image of the growth status of rosette leaves of different plants provided by the experimental example 1 of the present application.

[0019] Fig. 3 is an image of the height growth status of different plants provided by the experimental example 1 of the present application.

[0020] Fig. 4 is an image of the growth status of WT and AR1-4 plants after high temperature treatment provided by the experimental example 2 of the present application.

[0021] Fig. 5 is an image of the growth status of different plants under saline-alkali conditions provided by the experimental example 3 of the present application.

[0022] Figure 6 is a growth condition image of the root length of different plants at the age of 18 days under saline-alkali conditions according to Example 3 of the present application. DETAILED DESCRIPTION

[0023] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below in combination with the drawings in the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application. The following embodiments are used to illustrate the present application, but cannot be used to limit the scope of the present application.

[0024] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0025] Example 1 Construction of fusion gene AR1.

[0026] (I) Fusion gene AR1 (as shown in SEQ NO. 1), the nucleotide sequence of SEQ NO. 1 from 5' end to 3' end is as follows:

[0027] (II) Amplification of fusion gene AR1.

[0028] AR1-1-1, the upstream and downstream were introduced PstI and EcoO65I (BstEII) enzyme cutting sites respectively, the primers used were AR1-1-F (as shown in SEQ NO. 2) and AR1-1-R (as shown in SEQ NO. 3), the amplification product was connected to pMD18-T vector to obtain TA-AR1-1-1-HA. The fragment AR1-1 was amplified from the TA-AR1-1-1-HA plasmid with AR1-1-F2 (as shown in SEQ NO. 4) and AR1-1-R2 (as shown in SEQ NO. 5) as primers; the fragment AR1-2 was cloned from the Arabidopsis genome with AR1-2-F (as shown in SEQ NO. 6) and AR1-2-R (as shown in SEQ NO. 7) as primers, and BamHI and PstI enzyme cutting sites were introduced at the upstream and downstream respectively. The pCAMBIA3301 vector was cut with BamHI and EcoO65I (BstEII), the PCR amplified fragment AR1-1, the fragment AR1-2 and the cut pCAMBIA3301 vector were homologously recombined by seamless cloning technology to obtain the fusion gene AR1 connected to the pCAMBIA3301 vector skeleton (AR1-p3301), the bacterial screening resistance was Kan, and the plant screening resistance was Basta. When used, the primers AR1-F (as shown in SEQ NO. 8) and AR1-R (as shown in SEQ NO. 9) can be used to amplify the target gene.

[0029] The nucleotide sequence of SEQ NO. 2 from 5' end to 3' end is as follows: The nucleotide sequence of SEQ NO. 3 from 5' end to 3' end is as follows: The nucleotide sequence of SEQ NO. 4 from 5' end to 3' end is as follows: The nucleotide sequence of SEQ NO. 5 from 5' end to 3' end is as follows: The nucleotide sequence of SEQ NO. 6 from 5' end to 3' end is as follows: The nucleotide sequence of SEQ NO. 7 from 5' end to 3' end is as follows: The nucleotide sequence of SEQ NO. 8 from 5' end to 3' end is as follows: The nucleotide sequence of SEQ NO. 9 from 5' end to 3' end is as follows: (Three) Screening and identification of transgenic Arabidopsis.

[0030] AR1-p3301 vector was transformed into GV3101 Agrobacterium, and the specific process was as follows: 1 μg of DNA was added to 200 μL of Agrobacterium competent cells, and then the mixture was placed on ice for 30 min, frozen with liquid nitrogen for 1 min, and then thawed at 37 °C. 1 mL of YEP liquid medium was added to the tube, and the mixture was incubated at 28 °C for 4 h. The bacterial cells were collected by centrifugation and then spread on LB plates containing 50 μg / mL of kanamycin, 25 μg / mL of gentamicin, and 25 μg / mL of rifampicin, and then the plates were placed in a 28 °C incubator and incubated upside down. Single colonies were picked and identified by PCR using primers AR1-F2 (as shown in SEQ NO. 10) and AR1-R2 (as shown in SEQ NO. 11). The positive clones identified by PCR were shaken to an OD 600 1.0, and then used to infect Arabidopsis inflorescences.

[0031] SEQ NO. 10 is as follows from 5' end to 3' end: SEQ NO. 11 is as follows from 5' end to 3' end:

[0032] The wild-type Col was transformed by the method of Agrobacterium-mediated Arabidopsis inflorescence infection, and the specific process of the inflorescence infection was as follows: the bacterial solution was centrifuged, the precipitate was resuspended with the prepared transformation solution (10 mM 1 / 2MS, 2.5 mM MES, 5% sucrose, 1 mg / mL 6-BA), and then Silwet L-77 (50 mL / L) was added before the Arabidopsis inflorescences were infected. The Arabidopsis inflorescences were fully contacted with the bacterial solution, and then the infection was completed. After that, the mixture was cultured in the dark for 24 h, and then cultured under normal conditions until the end of the growth cycle. The seeds were harvested and then screened with Basta-resistant screening medium. The T1 generation seeds were identified by genomic PCR, and the positive plants obtained by soil screening were used to collect T2 generation seeds for Basta-resistant screening. According to Mendelian genetic law, the single copy lines were identified according to the segregation ratio, and the T3 generation single copy homozygous lines were obtained.

[0033] The specific method of genomic PCR was as follows: sampling, extraction of genomic DNA, and PCR amplification with 1301-1-F and Nos-1-R primers.

[0034] The PCR amplification system is shown in Table 1.

[0035] Table 1. PCR amplification system

[0036] The PCR amplification program was as follows: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 1 min 30 s, 31 cycles, extension at 72 °C for 10 min, and storage at 4 °C.

[0037] SEQ ID NO. 12 nucleotide sequence from 5' end to 3' end as follows: SEQ ID NO. 13 nucleotide sequence from 5' end to 3' end as follows:

[0038] The protein level detection method of T3 generation single copy homozygous lines is as follows: total plant protein of different single copy homozygous lines is extracted by protein extraction buffer, AR1 level of fusion gene is detected by SDS-PAGE electrophoresis combined with Western blot immunoblotting method using HA antibody (Abeam ab9110), and actin antibody (Abmart: M20009L) is used as loading control. The AR1 protein accumulation level of each single copy homozygous line of T3 generation is identified, and the identification result is shown in Figure 1, wherein the actin protein is used as the loading control, the wild type (wild type, referred to as WT) is the negative control without transgene, the exposure time of α-HA is 5s, and the exposure time of α-actin is 5s.

[0039] The composition of the protein extraction buffer is as follows: 50mM Tris-HCl (pH 8.0), 150mM NaCl, 0.5M EDTA (pH 8.0), 1% Triton X-100, EDTA-free protease inhibitor.

[0040] Experimental Example 1: Growth and development phenotype observation experiment.

[0041] The seeds of 3 fusion gene AR1 transgenic lines (4, 6, 25 lines) of Arabidopsis thaliana and wild type Col are soaked in 1% (v / v) NaClO for 2-3 min, then washed with sterile distilled water for 6-8 times, and then sowed on normal 1 / 2MS medium. After two days of dark treatment, they are placed in an Arabidopsis thaliana incubator with a culture condition of 20-22℃, long day (16h light, 8h dark), relative humidity of 60%-70%, and light intensity of 90-110μmol m -2 s -1 After 12 days of growth, they are moved to the soil for further culture with a culture condition of 20-22℃, long day (16h light, 8h dark). Phenotype observation shows that the rosette leaves and plant height of AR1 transgenic plants are significantly higher than that of WT, as shown in Figures 2-3, wherein L4 is the 4th plant line, L6 is the 6th plant line, and L25 is the 25th plant line. The results show that the fusion gene AR1 can significantly promote plant growth.

[0042] Experimental Example 2: High temperature stress phenotype observation experiment.

[0043] AR1-4) were soaked in 1% (v / v) NaClO for 2-3 min, washed with sterile distilled water for 6-8 times, and then placed in a refrigerator at 4°C for two days in the dark. The seeds were then placed on normal 1 / 2MS medium in an Arabidopsis incubator under the following conditions: 20-22°C, long day (16h light, 8h dark), relative humidity of 60%-70%, and light intensity of 90-110 μmol m -2 s -1 After 11 days of growth, the treated groups were placed in a water bath at 44°C for 18 min, and then placed in an Arabidopsis incubator under the following conditions: 20-22°C, long day (16h light, 8h dark), relative humidity of 60%-70%, and light intensity of 90-110 μmol m -2 s -1 for 5 days. The results showed that there was no significant difference in the growth of WT and AR1-4 under normal culture conditions, but the survival rate of WT was significantly reduced after high temperature treatment, and the survival rate of the Arabidopsis overexpressing the fusion gene AR1 was almost not inhibited, which was significantly higher than that of WT, showing obvious high temperature tolerance, as shown in Figure 4.

[0044] Experimental Example 3: Salt-alkali stress phenotype observation.

[0045] (I) Salt-alkali stress survival rate experiment.

[0046] Three independent transgenic lines (4, 6, and 25 lines) of the fusion gene AR1 and wild type Col seeds were soaked in 1% (v / v) NaClO for 2-3 min, washed with sterile distilled water for 6-8 times, and then placed on normal 1 / 2MS medium and 1 / 2MS medium containing 10 mM NaHCO3. After two days of dark treatment, the seeds were placed in an Arabidopsis incubator under the following conditions: 20-22°C, long day (16h light, 8h dark), relative humidity of 60%-70%, and light intensity of 90-110 μmol m -2 s -1 After 14 days of growth, the survival rates of WT and Arabidopsis overexpressing the AR1 fusion gene were counted. The results showed that the survival rates of WT and Arabidopsis overexpressing the AR1 fusion gene were both 100% under normal culture conditions, but the survival rate of WT was only 21% under salt-alkali stress, and the survival rate of Arabidopsis overexpressing the AR1 fusion gene was more than 80%, which was significantly higher than that of WT, showing obvious salt-alkali resistance, as shown in Figure 5.

[0047] (II) Salt-alkali stress root length experiment.

[0048] The seeds of three independent transgenic lines (4, 6, 25 lines) of AR1 fusion gene Arabidopsis and wild type Col were soaked with 1% (v / v) NaClO for 2-3 min, then washed with sterile distilled water for 6-8 times, and then the seeds were placed on normal 1 / 2MS medium and 1 / 2MS medium containing 5mM NaHCO3, and then placed in the dark for two days, and then placed in the Arabidopsis incubator, and the culture conditions were 20-22℃, long day (16h light, 8h dark), relative humidity 60%-70%, and light intensity 90-110μmol m -2 s -1 The results are shown in Figure 6. Under normal culture conditions, there was no significant difference in root length between WT and AR1 fusion gene overexpression Arabidopsis, but under salt stress, the root length growth of WT was inhibited, and the root length growth of AR1 fusion gene overexpression Arabidopsis was almost not inhibited, and was significantly higher than that of WT, showing obvious salt and alkali resistance.

[0049] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and 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: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; 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 fusion gene for enhancing plant stress resistance, characterized in that, The nucleotide sequence of the fusion gene is shown as SEQ NO.

1.

2. The fusion gene of claim 1, wherein the stress tolerance of a plant is improved. The stress resistance includes saline-alkali resistance and high temperature resistance.

3. A method for constructing a fusion gene, characterized by, The fusion gene for improving plant stress resistance according to claim 1 or 2 is named AR1, and the cloning and fusion construction of fragment AR1-1 and fragment AR1-2 includes the following steps: S100, AR1-1-1 is amplified by taking His-R10 as a template, and the amplified product is connected to pMD18-T vector to obtain TA-AR1-1-1-HA; S200, AR1-1 is amplified from the TA-AR1-1-1-HA plasmid by taking AR1-1-F2 and AR1-1-R2 as primers, and AR1-2-F and AR1-2-R are taken as primers for amplifying AR1-2 from the Arabidopsis genome, and a BamHI and PstI enzyme cutting site is introduced at the upstream and downstream, respectively, and fragment AR1-2 is cloned by using the method of PCR amplification; S300, the pCAMBIA3301 vector is cut by BamHI and EcoO65I, the amplified fragments AR1-1 and AR1-2 and the cut pCAMBIA3301 vector are homologously recombined by using the seamless cloning technology to obtain the fusion gene AR1, and the fusion gene AR1 is connected to the pCAMBIA3301 vector skeleton to obtain AR1-p3301.

4. The method for constructing a fusion gene according to claim 3, wherein The primers used in step S100 are AR1-1-F and AR1-1-R, respectively, the nucleotide sequence of AR1-1-F from 5' end to 3' end is shown as SEQ NO. 2, and the nucleotide sequence of AR1-1-R from 5' end to 3' end is shown as SEQ NO.

3.

5. Use of a fusion gene, characterized in that The application of the fusion gene for improving plant stress resistance according to claim 1 or 2 includes plant transformation or breeding for improving plant stress resistance by using the construction method of the fusion gene according to claim 3 or 4.

6. The application of the fusion gene as described in claim 5, characterized in that, The transformation includes the following steps: S400, AR1-p3301 is used to transform GV1301 agrobacterium to obtain transformed agrobacterium carrying the fusion gene AR1; The breeding is performed by using the transformed agrobacterium, including the following steps: S500, the method of using the transformed agrobacterium to infect the receptor plant explant or inflorescence is used to obtain a transgenic plant, and T0 generation seeds carrying the fusion gene AR1 are obtained.

7. The application of the fusion gene as described in claim 6, characterized in that, In step S400, the obtained transformed agrobacterium is cultured in a culture medium containing antibiotics to screen out the transformed agrobacterium successfully introducing the fusion gene AR1, and the culture medium includes LB plate culture medium containing kanamycin, gentamicin and rifampicin.

8. The use of a fusion gene according to claim 6, wherein The breeding includes the following steps: S600, the T0 generation seeds are subjected to resistance screening by using antibiotics or herbicides to obtain T1 generation seeds successfully introducing the fusion gene AR1.

9. The use of a fusion gene according to claim 5, wherein The breeding includes the breeding of crops and horticultural crops.

10. Use of a fusion gene according to claim 9, wherein the fusion gene is a fusion gene according to claim 1. The crops include at least one of rice, wheat, corn, cotton, soybean, rape, potato, fruits and vegetables, and the horticultural crops include at least one of forest trees and flowers.

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