Brassica napus e3 ubiquitin ligase encoding gene bnaa02.sip1 and application thereof

Abstract

This invention provides a gene encoding the E3 ubiquitin ligase BnaA02.SIP1 from Brassica napus and its application, belonging to the field of bioengineering technology. The nucleotide sequence of the BnaA02.SIP1 gene described in this invention is shown in SEQ ID NO:1. Specific primers were designed based on the coding region sequence of the BnaA02.SIP1 gene from Brassica napus, and a plant genetic transformation vector overexpressing the BnaA02.SIP1 gene was constructed using the constitutive expression promoter CaMV35S. The gene was then introduced into wild-type Arabidopsis thaliana and Brassica napus using Agrobacterium-mediated transformation, resulting in transgenic Arabidopsis thaliana and Brassica napus plants. The invention also verified that this gene plays a role in regulating salt tolerance in seedlings. This invention provides a theoretical basis and gene source for cultivating new salt-tolerant rapeseed varieties.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to a gene encoding E3 ubiquitin ligase BnaA02.SIP1 in Brassica napus and its application. Background Technology

[0002] Soil salinization is one of the major abiotic stresses in agricultural production, and its adverse effects on crop growth and yield are becoming increasingly serious. Currently, nearly 20% to 50% of farmland is adversely affected by soil salinity to varying degrees. This escalating problem poses a significant challenge to the sustainable development of agriculture. Cultivating salt-tolerant crops to restore these salinized lands, improve the ecological environment, and increase crop yields is the most direct, economical, and effective way.

[0003] Rapeseed (Brassica napus L.) is an important oilseed crop, with rapeseed oil accounting for approximately 45% of edible vegetable oil production, ranking first among oilseed crops. Rapeseed is also a major oilseed crop planted on saline-alkali land, playing a vital role in the restoration, transformation, and utilization of saline-alkali land. Currently, the self-sufficiency rate of edible vegetable oil is only 40%, a very serious situation. Expanding the rapeseed planting area and increasing yield on saline-alkali land is the primary issue facing rapeseed production. Discovering key genes for salt-alkali tolerance in rapeseed and analyzing their functions for the innovation of salt-alkali tolerant rapeseed germplasm resources and the breeding of new varieties is of great significance for increasing the rapeseed planting area on marginal saline-alkali land and ensuring high and stable rapeseed yields on saline-alkali land. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a Brassica napus E3 ubiquitin ligase encoding gene BnaA02.SIP1 and its application. This invention discovers that overexpression of the Brassica napus E3 ubiquitin ligase encoding gene BnaA02.SIP1 can significantly improve the crop's salt tolerance.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a gene encoding E3 ubiquitin ligase BnaA02.SIP1 from Brassica napus, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0007] This invention provides a Brassica napus E3 ubiquitin ligase, the amino acid sequence of which is shown in SEQ ID NO:2.

[0008] This invention provides a recombinant expression vector, which includes the BnaA02.SIP1 gene and a primary expression vector;

[0009] The initial expression vector was pMDC83;

[0010] The BnaA02.SIP1 gene was cloned between the Spe I and Kpn I restriction sites of the primary expression vector.

[0011] This invention provides a method for constructing the recombinant expression vector, comprising the following steps:

[0012] (1) Total RNA was extracted from rapeseed and cDNA was obtained by reverse transcription;

[0013] (2) Based on the BnaA02.SIP1 gene, specific primers were designed to amplify the cDNA by PCR to obtain the BnaA02.SIP1 gene.

[0014] (3) The BnaA02.SIP1 gene was cloned into the pMDC83 expression vector to obtain the recombinant expression vector.

[0015] Preferably, the specific primers are as shown in SEQ ID NO:3 and SEQ ID NO:4.

[0016] The present invention also provides a transformant, wherein the BnaA02.SIP1 gene, the recombinant expression vector, or the recombinant expression vector obtained according to the construction method is transformed into Agrobacterium competent cells;

[0017] The Agrobacterium competent cells were Agrobacterium GV3101 competent cells.

[0018] This invention also provides the application of the Brassica napus E3 ubiquitin ligase encoding gene BnaA02.SIP1, the Brassica napus E3 ubiquitin ligase, the recombinant expression vector, the recombinant expression vector obtained according to the construction method, or the transformant in improving plant salt tolerance.

[0019] As a preferred method, overexpression of the BnaA02.SIP1 gene in plants can improve the salt tolerance of plants.

[0020] Preferably, the plant is Arabidopsis thaliana or rapeseed.

[0021] This invention also provides the application of the Brassica napus E3 ubiquitin ligase encoding gene BnaA02.SIP1 in plant-assisted breeding, in which plants overexpressing the BnaA02.SIP1 gene are screened during the breeding process.

[0022] Compared with existing technologies, this invention has the following beneficial effects: Through cloning the BnaA02.SIP1 gene sequence from the Shuang 11 seedling of Brassica napus L., this invention discovered that the BnaA02.SIP1 gene participates in the regulation of salt tolerance in plants. Based on the coding region sequence of the BnaA02.SIP1 gene in Brassica napus L., this invention designed specific primers and constructed a plant genetic transformation vector overexpressing the BnaA02.SIP1 gene using the constitutive expression promoter CaMV35S. Using Agrobacterium-mediated transformation, this gene was introduced into wild-type Arabidopsis thaliana and Brassica napus, resulting in transgenic Arabidopsis thaliana and Brassica napus plants. The study verified that this gene plays a role in regulating plant salt tolerance during the seedling stage. This indicates that the BnaA02.SIP1 gene plays a positive regulatory role in plant salt stress response, revealing that overexpression of the BnaA02.SIP1 gene can significantly improve plant salt tolerance. This invention provides a theoretical basis and gene source for breeding new salt-tolerant rapeseed varieties. Attached Figure Description

[0023] Figure 1 Electrophoresis results of RT-PCR products from T3 generation transgenic Arabidopsis thaliana plants;

[0024] Figure 2 The main root growth phenotype of T3 generation transgenic Arabidopsis thaliana plants after 7 days of treatment with 150 mM NaCl stress;

[0025] Figure 3 The results show the relative taproot length of T3 generation transgenic Arabidopsis thaliana plants after 7 days of treatment with 100mM NaCl.

[0026] Figure 4 Electrophoresis results of RT-PCR products from T3 generation transgenic rapeseed plants;

[0027] Figure 5 The growth phenotype of T3 generation transgenic rapeseed plants after 12 days of treatment with 100mM NaCl stress;

[0028] Figure 6 The results show the statistical results of the aboveground fresh weight of T3 generation transgenic Arabidopsis plants after 12 days of treatment with 100mM NaCl. Detailed Implementation

[0029] This invention provides a gene encoding the E3 ubiquitin ligase BnaA02.SIP1 from Brassica napus, the nucleotide sequence of which is shown in SEQ ID NO:1, and is detailed below:

[0030] ATGGGAGATCATTTCGTGCTCCTGGTGGATCGGTTGATAACAGAAGCGACGATTGAGGAAGCCATTCAGAGCAGGAACCGTATGTTGCAAGGCAACGCACCCGTCGAGGAGGAATGTAGAATCCTGGACGAGAAAACACTTGAGAAGCTGCGTAATGGAGATTTGAAGATGGTGCAGTGTAGGATTTGTCACGACGAGGATTTGGATTGTAACATGGAGACTCCTTGCTCTTGCAGCGGTAGCTTGAAGTATGCACATAGGAGGTGCGTGCAGAGATGGTGTAACGAGAAGGGAGACACCACCTGCGAGATTTGCCATCAGGAGTTTAAGCCGGGCTATACAGCACCATCTCCATTGTTAGAGTTAGGTCATGTTCCTCTTCACTTCAGGGGAAACTGGGGAGTGTCTCAACGTGAGCATCGTTTCATC ACAGTTGTACCTGCAGATCCCACTTTCCTCGACGACCATCATCAGTATCCTCTTTCTTCCTCCACAAGCTTCATCTGTTGCCGTTCCCTTGTTCTAATCTTCATGGCTCTTCTGATTCTCCGACACACGCTCCCCTTAGTCCTCACCGGTTCAAACCTCCATGTTTTCCCTTTGTTCACGTTGTTGTTTCTGAGAATACTCGGGATCATGCTACCAATCTATATCGTCACAAAAGCAGTAGCCACCTGTCGCCGCCATTCTCAGACCTTAGAAACCTCTGAGTCTGAAGATTCATCTGACGAAGAAGCAGAGTTATGGCGCTTCCCTCGAACGCAATCTTACATTATAGGAGTGCCA。

[0031] The present invention provides a Brassica napus E3 ubiquitin ligase, and the amino acid sequence of the Brassica napus E3 ubiquitin ligase is as shown in SEQ ID NO:2, specifically as follows:

[0032] MGDHFVLLVDRLITEATIEEAIQSRNRMLQGNAPVEEECRILDEKTLEKLRNGDLKMVQCRICHDEDLDCNMETPCSCSGSLKYAHRRCVQRWCNEKGDTTCEICHQEFKPGYTAPSPLLELGHVPLHFRG NWGVSQREHRFITVVPADPTFLDDHHQYPLSSSTSFICCRSLVLIFMALLILRHTLPLVLTGSNLHVFPLFTLLFLRILGIMLPIYIVTKAVATCRRHSQTLETSESEDSSDEEAELWRFPRTQSYIIGVP.

[0033] This invention provides a recombinant expression vector, the vector comprising the BnaA02.SIP1 gene and a primary expression vector;

[0034] The initial expression vector was pMDC83;

[0035] The BnaA02.SIP1 gene was cloned between the Spe I and Kpn I restriction sites of the primary expression vector.

[0036] This invention provides a method for constructing the recombinant expression vector, comprising the following steps:

[0037] (1) Total RNA was extracted from rapeseed and cDNA was obtained by reverse transcription;

[0038] (2) Based on the BnaA02.SIP1 gene, specific primers were designed to amplify the cDNA by PCR to obtain the BnaA02.SIP1 gene.

[0039] (3) The BnaA02.SIP1 gene was cloned into the pMDC83 expression vector to obtain the recombinant expression vector.

[0040] In this invention, total RNA was extracted from rapeseed and reverse transcribed to obtain cDNA. Two-week-old seedlings of the Brassica napus variety Shuang 11 were used, and total RNA was extracted from the seedlings using the Simply P Total RNA Extraction Kit. 2 μg of the total RNA was then reverse transcribed using the TIANScript cDNA First-Strand Synthesis Kit to obtain single-stranded cDNA.

[0041] In this invention, specific primers were designed based on the BnaA02.SIP1 gene to amplify cDNA by PCR, thereby obtaining the BnaA02.SIP1 gene. Using the single-stranded cDNA obtained by reverse transcription as a template, specific primer pairs were designed based on the BnaA02.SIP1 nucleotide sequence for PCR amplification, thereby obtaining the BnaA02.SIP1 gene. The specific primers are shown in SEQ ID NO:3 and SEQ ID NO:4, specifically: SEQ ID NO:3: GACCTCGACTCTAGAACTAGTATGGGAGATCATT TCGTGCTCC; SEQ ID NO:4: CTCATTTTTTCTACCGGTACCGGTGGCACT CCTATAATGTAAGATTGC; The polymerase used for PCR amplification is TopTaq high-efficiency hot-start DNA polymerase, and the PCR amplification reaction system is prepared in 50 μL according to the TopTaq high-efficiency hot-start DNA polymerase instructions; The PCR amplification program is as follows: 94℃ for 5 min; 94℃ for 30 sec, 60℃ for 30 sec, 72℃ for 45 sec, 30 cycles; 72℃ for 10 min.

[0042] In this invention, the BnaA02.SIP1 gene was cloned into the pMDC83 expression vector to obtain a recombinant expression vector. The Seamless Cloning and Assembly Kit was used to perform homologous recombination of the obtained BnaA02.SIP1 gene fragment with the plant expression vector pMDC83, which had been digested with Spe I and Kpn I, to obtain the recombinant expression vector. The recombination reaction was performed in 10 μL volumes according to... The instructions for the Seamless Cloning and Assembly Kit state that the reaction solution should be prepared and the reaction conditions should be: 50°C for 15 min.

[0043] This invention also includes the step of scaling up the obtained recombinant expression vector to obtain a plant expression vector. The obtained recombinant expression vector is transformed into Trans5α chemocompetent cells. The transformed competent cells are then added to LB agar medium containing kanamycin and spread evenly. The plates are incubated at 37°C until the liquid is absorbed, then inverted and incubated overnight at 37°C. Ten different single colonies from the overnight kanamycin-containing LB agar medium are picked and subjected to PCR amplification to obtain PCR products. The PCR products are then detected. Positive amplification products are inoculated into 5 mL of LB liquid medium containing kanamycin for scaling up. The Plasmid MiniPrep Kit was used to extract plasmids according to the instructions. The extracted plasmids were sequenced, and the sequenced plasmids accurately identified the obtained plant expression vector pMDC83-BnaA02.SIP1. The primers used for PCR amplification were GAGCCCAATCCCACTATCC (SEQ ID NO:5) and GGTAACGGGAGA AGCACTG (SEQ ID NO:6); the enzyme used for PCR amplification was polymerase, specifically… Taq DNA Polymerase; the PCR amplification reaction system is in 20 μL increments, according to... Prepare Taq DNA Polymerase according to the instructions; the PCR amplification reaction program is as follows: 94℃ for 3 min; 94℃ for 5 sec, 58℃ for 15 sec, 72℃ for 60 sec, 31 cycles; 72℃ for 5 min.

[0044] The present invention also provides a transformant, wherein the BnaA02.SIP1 gene, the recombinant expression vector, or the recombinant expression vector obtained according to the construction method is transformed into Agrobacterium competent cells;

[0045] The Agrobacterium competent cells were Agrobacterium GV3101 competent cells.

[0046] In this invention, the obtained recombinant expression vector is transformed into Agrobacterium GV3101 competent cells by electroporation, plated onto LB agar medium containing kanamycin, rifamycin, and gentamicin, inverted, and cultured overnight at 28°C to obtain the transformant.

[0047] This invention also provides the application of the Brassica napus E3 ubiquitin ligase encoding gene BnaA02.SIP1, the Brassica napus E3 ubiquitin ligase, the recombinant expression vector, the recombinant expression vector obtained according to the construction method, or the transformant in improving plant salt tolerance.

[0048] In this invention, overexpression of the BnaA02.SIP1 gene in plants can improve the salt tolerance of plants.

[0049] In this invention, the plant is Arabidopsis thaliana or rapeseed.

[0050] This invention also provides the application of the Brassica napus E3 ubiquitin ligase encoding gene BnaA02.SIP1 in plant-assisted breeding, in which plants overexpressing the BnaA02.SIP1 gene are screened during the breeding process.

[0051] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0052] Example 1: Cloning and Overexpression Vector Construction of BnaA02.SIP1 Gene

[0053] Two-week-old seedlings of the Brassica napus variety Shuang 11 were collected. Total RNA was extracted from the seedlings using the Simply P Total RNA Extraction Kit (Hangzhou Bori Technology Co., Ltd., catalog number: BSC52M1). The nucleic acid concentration was determined using a UV spectrophotometer. 2 μg of total RNA was used to perform reverse transcription to obtain single-stranded cDNA using the TIANScript cDNA First-Strand Synthesis Kit (Tiangen Biotech (Beijing) Co., Ltd., catalog number: KR107-01).

[0054] Using single-stranded cDNA obtained by reverse transcription as a template, primer pairs F1 / R1 (F1: GACCTCGACTCTAGAACTAGTATGGGAGATCATTTCG TGCTCC, SEQ ID NO:3 and R1: CTCATTTTTTCTACCGGTACCGGTGGCACT CCTATAATGTAAGATTGC, SEQ ID NO:4) were designed based on the BnaA02.SIP1 nucleotide sequence for PCR amplification. The F1 and R1 primers were synthesized by Yangling Zhongke Yutong Biotechnology Co., Ltd., and the polymerase used for amplification was TopTaq high-efficiency hot-start DNA polymerase (containing 2.5 mM dNTPs) (Transgen, catalog number: AP151-11). The PCR reaction volume was 50 μL, prepared according to the TopTaq high-efficiency hot-start DNA polymerase instructions. The PCR reaction conditions were: 94℃ for 5 min; 94℃ for 30 sec, 60℃ for 30 sec, 72℃ for 45 sec, 30 cycles; 72℃ for 10 min. A fragment of approximately 786 bp was obtained. Using 1% agarose gel electrophoresis, the target fragment was excised and placed into a 2 mL centrifuge tube. The PCR Purification Kit (Transgen, catalog number: EP101-01) recovers the target fragment.

[0055] use The Seamless Cloning and Assembly Kit (Transgen, catalog number: CU201-02) allows for homologous recombination of the gel-recovered target fragment with the plant expression vector pMDC83, which has been double-digested with Spe I and Kpn I. The cloning reaction volume is 10 μL, prepared according to... Prepare the reaction solution according to the Seamless Cloning and Assembly Kit instructions. The reaction conditions are: 50°C for 15 min. Afterwards, cool the centrifuge tubes on ice for 30 s. Then, use the recombinant product directly for transformation. Transform Trans5α chemocompetent cells (Transgen, catalog number: CD201) using the above reaction solution. Add the transformed competent cells to LB agar medium containing carbamycin and spread the cells evenly. Incubate the plates at 37°C until the liquid is absorbed, then invert the plates and incubate overnight at 37°C.

[0056] Ten different single colonies were selected from LB agar medium containing kanamycin that had been cultured overnight. Colonies were amplified by PCR using primers F2: GAGCACAATCCCACTATCC (SEQ ID NO:5) and R2: GGTAACGGGAGA AGCACTG (SEQ ID NO:6) synthesized by Yangling Zhongke Yutong Biotechnology Co., Ltd. The polymerase used for amplification was [missing information - likely a specific polymerase or enzyme]. Taq DNA Polymerase (Transgen, catalog number: AP101), PCR reaction volume is 20 μL, according to... The reaction solution was prepared according to the Taq DNA Polymerase instructions, and the reaction conditions were: 94℃ for 3 min; 94℃ for 5 sec, 58℃ for 15 sec, 72℃ for 60 sec, 31 cycles; 72℃ for 5 min. After electrophoresis on a 1% agarose gel, positive clones were inoculated into 5 mL of LB liquid medium containing kanamycin for expansion culture.

[0057] use Plasmid MiniPrep Kit (Transgen, catalog number: EM101-02) was used to extract plasmids according to the instructions. 20 μL of plasmid with a concentration greater than 50 ng / μL was sent to Yangling Tianrun Aoke Biotechnology Co., Ltd. for sequencing verification. After accurate sequencing, the plant expression vector pMDC83-BnaA02.SIP1 was obtained.

[0058] Example 2: Obtaining Transgenic Arabidopsis

[0059] The plant expression recombinant vector pMDC83-BnaA02.SIP1 obtained in Example 1 was transformed into Agrobacterium GV3101 competent cells (WEIDI, catalog number: EM101-02) by electroporation. The cells were then plated onto LB agar medium containing kanamycin (50 μg / mL), rifamycin (40 μg / mL), and gentamicin (50 μg / mL). The plates were inverted and cultured overnight at 28°C to obtain recombinant Agrobacterium GV3101: pMDC83-BnaA02.SIP1.

[0060] A single clone of recombinant Agrobacterium GV3101:pMDC83-BnaA02.SIP1 was inoculated into 100 mL LB liquid medium containing 50 mg / L kanamycin, 50 mg / L rifamycin, and 50 mg / L gentamicin. The medium was cultured at 28°C with shaking at 180 rpm for 2 days until the OD600 reached 2.0. The bacterial culture was centrifuged at 3000 rpm for 10 min to obtain Agrobacterium precipitate. The Agrobacterium precipitate was resuspended in 100 mL of infection solution containing 2.36 g / L MS, 25 g / L sucrose, and 100 μL / L Silwet L-77. The solution was incubated in the dark at 28°C for 2 h. The solution was then used to infect wild-type Arabidopsis thaliana Col-0 inflorescences. The culture was carried out normally until the T1 generation of transgenic Arabidopsis thaliana seeds were harvested.

[0061] T1 generation transgenic Arabidopsis seeds were screened for germination on 1 / 2 MS solid medium containing 300 mg / L Tim (termethin), 35 mg / L Hygromycin (hygromycin), 2% sucrose, and 0.75% agar powder. Normally growing T1 generation seedlings were transplanted into nutrient soil and cultured normally until T2 generation seeds were harvested. The harvested T2 generation transgenic Arabidopsis seeds were then subjected to the same screening process to obtain homozygous T3 generation transgenic Arabidopsis seeds.

[0062] Using specific primers F2: GACGCCACAATCCCACTATCC (SEQ ID NO:5) and R2: GGTAACGGGAGAAGCACTG (SEQ ID NO:6), and internal reference ACTIN2 primers (F3: TGTGCCAATCTACGAGGGTTT, SEQ ID NO:7 and R3: TTTCCCGCTCTGCT GTTGT, SEQ ID NO:8), The expression level of BnaA02.SIP1 in homozygous T3 generation transgenic Arabidopsis thaliana plants was detected using Taq DNA Polymerase and RT-PCR. The specific steps were as follows: Wild-type Arabidopsis thaliana Col-0 and homozygous T3 generation transgenic Arabidopsis thaliana seedlings carrying the BnaA02.SIP1 gene were collected. Total RNA was extracted using the SimplyP Total RNA Extraction Kit, and the nucleic acid concentration was determined. 2 μg of total RNA was used to perform reverse transcription using the TIANScript cDNA First-Strand Synthesis Kit to obtain single-stranded cDNA. Using the obtained cDNA as a template, specific primer pair F2 / R2, internal control primer pair F3 / R3, and... Taq DNA Polymerase was used for PCR amplification. The PCR reaction volume was 20 μL, and the reaction was performed according to... The reaction solution was prepared according to the Taq DNA Polymerase instructions. The reaction conditions were: 94℃ for 3 min; 94℃ for 5 sec, 58℃ for 15 sec, 72℃ for 60 sec, 31 cycles; 72℃ for 5 min. The expression level of the BnaA02.SIP1 gene in the overexpressing transgenic Arabidopsis thaliana lines was determined by 1% agarose gel electrophoresis. The results are shown below. Figure 1 As shown.

[0063] Depend on Figure 1 It was found that the expression level of the target gene BnaA02.SIP1 in the T3 generation Arabidopsis lines OE#1, OE#2, and OE#3, which were transgenic with the BnaA02.SIP1 gene, was significantly higher than that in the wild-type Col-0. Subsequent salt tolerance tests were conducted on the OE#1 and OE#2 transgenic lines.

[0064] Example 3: Overexpression of the BnaA02.SIP1 gene improves salt tolerance in Arabidopsis thaliana.

[0065] Wild-type Arabidopsis thaliana Col-0 seeds and BnaA02.SIP1 transgenic Arabidopsis thaliana seeds (OE#1 and OE#2) obtained in Example 2 were sterilized in a 1% sodium hypochlorite solution for 15 min, followed by washing four times with sterile deionized water for 5 min each time in a sterile laminar flow hood. The surface-sterilized seeds were placed in a 4°C refrigerator for 2 days, then seeded onto 1 / 2 MS solid medium and placed in a light incubator (experimental conditions: 22°C, 16 hours light / 8 hours dark) for 2 days. Seeds with consistent germination were transferred to 1 / 2 MS solid medium (1% sucrose) containing 0 or 150 mM NaCl. These culture dishes were then vertically placed in a light incubator (experimental conditions: 22°C, 16 hours light / 8 hours dark) for 7 days. Observations and photographs were taken, and the taproot length was measured. The relative root lengths of wild-type Arabidopsis thaliana Col-0 and BnaA02.SIP1 transgenic Arabidopsis thaliana under NaCl stress were statistically analyzed. Results are as follows: Figure 2 and Figure 3 As shown.

[0066] Relative root length = (Length of taproot after 150mM NaCl stress / Length of taproot in the unstressed group) × 100%.

[0067] Depend on Figure 2 and Figure 3 It can be seen that under 150mM NaCl treatment, the taproot length of Arabidopsis thaliana transgenic BnaA02.SIP1 is significantly greater than that of wild-type Arabidopsis thaliana. Therefore, the salt tolerance of Arabidopsis thaliana transgenic BnaA02.SIP1 (OE#1 and OE#2) is significantly higher than that of wild-type Arabidopsis thaliana.

[0068] Example 4: Obtaining Transgenic Brassica napus

[0069] The recombinant Agrobacterium GV3101: pMDC83-BnaA02.SIP1 obtained in Example 2 was inoculated into 100 mL of LB liquid medium containing 50 mg / L kanamycin, 50 mg / L rifamycin, and 50 mg / L gentamicin. The culture was incubated overnight at 28°C and 200 rpm until the OD value reached 0.6–0.8. The Agrobacterium cell pellet was collected and resuspended in 100 mL of infection solution containing 4.42 g MS powder, 30 g sucrose, and 100 mmol / L LAS (acetylsyleugenol). This solution was then used to inoculate hypocotyls of Brassica napus K407 grown in the dark on 1 / 2 MS medium for 7 days. T0 generation transgenic positive plants were obtained by screening on medium containing 20 mg / L Hyg antibiotics and 300 mg / L Titim antibiotics.

[0070] Normally rooted T0 generation transgenic seedlings were transferred to nutrient soil and cultured normally until T1 generation transgenic seeds were harvested. T1 generation transgenic seeds were then placed on filter paper soaked in 150 mg / L Hyg antibiotic to screen for T1 transgenic resistant seedlings. The selected normally rooted T1 generation transgenic seedlings were then transferred to nutrient soil and cultured normally until T2 generation transgenic seeds were harvested. T2 generation transgenic seeds were then placed on filter paper soaked in 150 mg / L Hyg antibiotic to screen for T2 transgenic resistant seedlings. The selected normally rooted T2 generation transgenic seedlings were then transferred to nutrient soil and cultured normally until homozygous T3 generation transgenic seeds were harvested.

[0071] Using specific primers F2: GACGCCACAATCCCACTATCC (SEQ ID NO:5) and R2: GGTAACGGGAGAAGCACTG (SEQ ID NO:6), and internal reference primers BnACTIN7 (F4: GCTGACCGTATGAGCAAAG, SEQ ID NO:9 and R4: AAGATGATGGATGGA CCCGAC, SEQ ID NO:10), The expression level of BnaA02.SIP1 in homozygous T3 generation transgenic rapeseed plants was detected using Taq DNA Polymerase and RT-PCR. The PCR reaction system and conditions were the same as in Example 2. Lines with high expression levels were selected from the screened positive transgenic rapeseed plants, and the detection results are as follows: Figure 4 As shown.

[0072] Depend on Figure 4 It was found that the expression level of the target gene BnaA02.SIP1 in the T3 generation Brassica napus lines OE#1, OE#2, and OE#3, which were transgenic with the BnaA02.SIP1 gene, was significantly higher than that in the control plant K407. These three transgenic rapeseed lines (OE#1, OE#2, and OE#3) were selected for subsequent salt tolerance tests.

[0073] Example 5: Overexpression of the BnaA02.SIP1 gene improves salt tolerance in Brassica napus.

[0074] Seeds from the T3 generation homozygous transgenic BnaA02.SIP1 rapeseed lines OE#1, OE#2, and OE#3 obtained in Example 4, and the wild-type Brassica napus inbred line K407, were sown in small flowerpots containing 200g of nutrient soil substrate. After 10 days of growth, seedlings with uniform growth were selected and watered with an equal volume of solution containing 100mmol / L NaCl. Stress treatment was applied every 3 days for 12 days. The aboveground fresh weight of control and transgenic plants was observed, photographed, and measured. The results are as follows: Figure 5 and Figure 6 As shown.

[0075] Depend on Figure 5 and Figure 6 It can be seen that under 100mM NaCl treatment, the fresh weight of the aboveground parts of the BnaA02.SIP1 transgenic rapeseed was significantly higher than that of the control K407. Therefore, the salt tolerance of the BnaA02.SIP1 transgenic rapeseed (OE#1, OE#2 and OE#3) was significantly better than that of wild-type rapeseed K407.

[0076] In summary, BnaA02.SIP1 is a gene that plays a positive regulatory role in the salt stress response of Arabidopsis thaliana and rapeseed, revealing that BnaA02.SIP1 participates in the salt tolerance regulation process of Arabidopsis thaliana and rapeseed. This invention provides a theoretical basis and gene source for breeding new salt-tolerant rapeseed varieties.

[0077] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Gene encoding E3 ubiquitin ligase in Brassica napus BnaA02.SIP1 Its application in improving plant salt tolerance is characterized by, Overexpression in plants BnaA02.SIP1 Genes can enhance a plant's salt tolerance; The nucleotide sequence of the gene is shown in SEQ ID NO:1; The plant in question is either Arabidopsis thaliana or Brassica napus.

2. The application of E3 ubiquitin ligase in improving salt tolerance in Brassica napus, characterized in that, The Brassica napus E3 ubiquitin ligase was overexpressed in plants. BnaA02.SIP1 Genes can enhance a plant's salt tolerance; The amino acid sequence of the Brassica napus E3 ubiquitin ligase is shown in SEQ ID NO:2; The plant in question is either Arabidopsis thaliana or Brassica napus.

3. Gene encoding E3 ubiquitin ligase in Brassica napus BnaA02.SIP1 Its application in assisted plant breeding is characterized by, During the breeding process, expression was screened. BnaA02.SIP1 Genetically modified plants; The plant in question is Arabidopsis thaliana or Brassica napus; The nucleotide sequence of the gene is shown in SEQ ID NO:1.

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