Alfalfa zinc finger protein-like gene al3 for enhancing plant resistance to heavy metal cadmium and application thereof

Abstract

The present application relates to a kind of enhanced plant anti-heavy metal cadmium alfalfa zinc finger protein analog protein gene AL3 and its application.The alfalfa zinc finger protein analog protein gene AL3 is Chinese cabbage homologous gene BrAL3, nucleotide sequence is as shown in SEQ ID NO.1;Or is arabidopsis homologous gene AtAL3, nucleotide sequence is as shown in SEQ ID NO.2.The application of alfalfa zinc finger protein analog protein gene AL3 in enhancing the application of plant anti-heavy metal cadmium is disclosed for the first time, alfalfa zinc finger protein analog protein gene AL3 is connected to expression vector, and plant is transformed using agrobacterium infection method, and the construction of strong plant anti-heavy metal cadmium plant strain is completed.The root length of the plant strain of alfalfa zinc finger protein analog protein gene AL3 is significantly greater than wild type, which indicates that the plant strain of alfalfa zinc finger protein analog protein gene AL3 has good anti-cadmium stress, significantly enhances the ability of plant anti-heavy metal cadmium, reduces plant cadmium accumulation and cadmium absorption.

Description

Technical Field

[0001] This invention relates to an alfalfa zinc finger protein-like protein gene AL3 that enhances plant resistance to heavy metal cadmium and its application, belonging to the field of plant genetic engineering. Background Technology

[0002] Currently, using molecular biology techniques to improve plants is an effective way to address cadmium pollution in plants. In the past decade or so, botanists and crop scientists have screened many cadmium transport proteins in different plants, such as the ABC protein family, the HMA protein family, and the NRAMP protein family, and applied them to plant and crop improvement processes. However, most of these proteins merely transport cadmium ions to the vacuoles or a specific part of the plant, simply isolating the cadmium ions, which poses a risk of cadmium pollution to the food chain. For example, the cadmium ion efflux transporter AtPDR8, a typical ABC (ATP-binding cassette) transporter, requires a large amount of energy and has a relatively limited ability to efflux cadmium ions. In the molecular breeding of low-cadmium crops, researchers often use gene editing technology to knock out cadmium ion transporter genes (such as NRAMP5 and HMA3) to cultivate low-cadmium crops.

[0003] Therefore, there is an urgent need for further research to discover new genes that enhance plants' resistance to the heavy metal cadmium. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides an alfalfa zinc finger protein-like protein gene AL3 that enhances plant resistance to the heavy metal cadmium and its applications.

[0005] The technical solution of the present invention is as follows:

[0006] A zinc finger protein-like protein gene AL3 of alfalfa that enhances plant resistance to heavy metal cadmium, wherein the alfalfa zinc finger protein-like protein gene AL3 is a homolog of Chinese cabbage BrAL3, with a nucleotide sequence as shown in SEQ ID NO.1; or a homolog of Arabidopsis thaliana AtAL3, with a nucleotide sequence as shown in SEQ ID NO.2.

[0007] An expression vector containing the alfalfa zinc finger protein-like protein gene AL3.

[0008] According to a preferred embodiment of the present invention, the method for constructing the expression vector includes the following steps:

[0009] (1) RNA was extracted from Chinese cabbage, and then the RNA was reverse transcribed to obtain cDNA;

[0010] (2) PCR amplification was performed using cDNA as a template to obtain the AL3 sequence of the alfalfa zinc finger protein-like protein gene. The PCR primer sequences are as follows:

[0011] Forward primer: 5'-cgggggactctagagATGGAAGGAGGAGCTGGTCTT-3',

[0012] Reverse primer:

[0013] 5'-gaccacccggggatcTTCCTCCTCCTCCTCCTCCTTAAGGTCGTGCTCTTTTGTT-3';

[0014] (3) The alfalfa zinc finger protein-like protein gene AL3 sequence and the cauliflower mosaic virus (CaMV) 35S promoter p35S were ligated into the pCambia3300 plasmid to obtain an expression vector containing the alfalfa zinc finger protein-like protein gene AL3.

[0015] The application of the alfalfa zinc finger protein-similar gene AL3 in enhancing plant resistance to heavy metal cadmium.

[0016] According to a preferred embodiment of the present invention, the application method includes: transforming an expression vector containing the alfalfa zinc finger protein-like protein gene AL3 into plants.

[0017] According to a preferred embodiment of the invention, the plant includes crops.

[0018] More preferably, the crops include any one or more of the following: corn, rice, tomato, potato, peanut, soybean, cotton, tobacco, cucumber, melon, watermelon, cabbage, rapeseed, bok choy, spinach, and radish.

[0019] Beneficial effects:

[0020] 1. This invention discloses for the first time the application of the alfalfa zinc finger protein analog gene AL3 in enhancing plant resistance to the heavy metal cadmium. The alfalfa zinc finger protein analog gene AL3 is linked to an expression vector, and plants are transformed using Agrobacterium-mediated transformation to construct plants with strong resistance to cadmium. The root length of the plants transformed with the alfalfa zinc finger protein analog gene AL3 is significantly greater than that of the wild type, indicating that the plants transformed with the alfalfa zinc finger protein analog gene AL3 have excellent resistance to cadmium stress, significantly enhancing the plant's ability to resist the heavy metal cadmium and reducing cadmium accumulation and absorption.

[0021] 2. The alfalfa zinc finger protein-like protein gene AL3, which enhances plant resistance to heavy metal cadmium, discovered in this invention can be used for gene editing molecular breeding, providing a basis for breeding plant varieties resistant to cadmium stress and offering a new approach for germplasm innovation of cadmium-resistant crops. Attached image description:

[0022] Figure 1 Phenotypic images of Chinese cabbage and wild-type Chinese cabbage after being transferred to 0.5×MS hydroponic medium containing 50 μM CdCl2 and grown for 7 days.

[0023] Figure 2 Figure 1 shows the cadmium content of Chinese cabbage and wild-type Chinese cabbage after 7 days of growth, transformed with the alfalfa zinc finger protein similar protein gene BrAL3. Error bars represent the mean SD of three independent replicates, ***P<0.001.

[0024] Figure 3 Phenotypic images of Arabidopsis mutant atal3-1 and wild-type Arabidopsis after 7 days of growth on 0.5×MS hydroponic medium containing 50 μM CdCl2.

[0025] Figure 4 Cadmium content was determined in Arabidopsis thaliana mutant atal3-1 and wild-type Arabidopsis thaliana after 7 days of growth. Error bars represent the mean SD of three independent replicates, ***P<0.001. Detailed Implementation

[0026] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof. The present invention provides a general and / or specific description of the materials and test methods used in the experiments. Although many materials and operating methods used to achieve the objectives of the present invention are known in the art, the present invention is still described in as much detail as possible herein.

[0027] Example 1

[0028] 1. RNA was extracted from Chinese cabbage using the following method:

[0029] Pre-cool the mortar with liquid nitrogen, weigh 2g of leaves of "Guangdong Early" Chinese cabbage, add 10mL of liquid nitrogen and grind quickly, transfer to a 1.5mL centrifuge tube, add 1mL of Trizol and mix well, then incubate at 25℃ for 5min to allow for complete lysis; centrifuge at 12000rpm at 4℃ for 5min, discard the precipitate and collect the supernatant; add 200μL of chloroform to the supernatant, vortex and mix well, then incubate at 25℃ for 15min; centrifuge at 12000rpm at 4℃ for 5min, aspirate the upper aqueous phase to another centrifuge tube, add 500μL of isopropanol, incubate at 25℃ for 10min; centrifuge at 12000rpm at 4℃ for 10min, discard the supernatant, and the total RNA settles at the bottom of the tube. Add 1 mL of 75% ethanol to the precipitate and wash gently by inverting. Centrifuge at 8000 rpm for 5 min at 4 °C, discard the supernatant, and air-dry the precipitate at 25 °C. Dissolve the total RNA sample in 50 μL of DEPC-treated ddH2O. Store the prepared total RNA sample in an ultra-low temperature freezer at -80 °C for later use.

[0030] 2. Reverse transcription PCR

[0031] The total RNA sample was used as a template for reverse transcription PCR amplification (two-step method). The specific reverse transcription PCR amplification system and conditions are as follows:

[0032] Step 1:

[0033]

[0034] Reaction conditions: Incubate at 42℃ for 2 minutes, then store at 4℃.

[0035] Step 2:

[0036]

[0037] Reaction conditions: 37℃ for 15 min, 85℃ for 5 s.

[0038] Total cDNA from Chinese cabbage was obtained using the above method. The reagents in Step 1 and Step 2 were from PrimeScript, a product of TAKALA. TM II 1st Strand cDNASynthesis Kit.

[0039] Example 2

[0040] 1. Construct an expression vector containing the AL3 sequence of an alfalfa zinc finger protein-like protein gene. The specific method is as follows:

[0041] (1) Using total cDNA from Chinese cabbage as a template, PCR amplification was performed to obtain the BrAL3 sequence (SEQ ID NO.1), a similar protein to alfalfa zinc finger protein. The primers for the PCR amplification are as follows:

[0042] Forward primer: 5'-cgggggactctagagATGGAAGGAGGAGCTGGTCTT-3',

[0043] Reverse primer:

[0044] 5'-gaccacccggggatcTTCCTCCTCCTCCTCCTCCTTAAGGTCGTGCTCTTTTGTT-3';

[0045] The PCR system is as follows:

[0046]

[0047] The PCR conditions were as follows: pre-denaturation, 98℃, 30s; denaturation, 98℃, 10s; annealing, 60℃, 30s; extension, 72℃, 1min (35 cycles); termination extension, 72℃, 10min; and final incubation at 4℃.

[0048] (2) The pCambia3300 plasmid was digested with BamH1 and ligated into the pCambia3300 plasmid with the sequence of the alfalfa zinc finger protein similar protein gene BrAL3 and the cauliflower mosaic virus (CaMV) 35S promoter p35S to obtain the expression vector p35:BrAL3 containing the alfalfa zinc finger protein similar protein gene AL3.

[0049] The connection system is as follows:

[0050]

[0051] Ligation reaction conditions: 50℃, 15 min; store at 4℃.

[0052] 2. Preparation of transgenic plants

[0053] Remove Agrobacterium tumefaciens GV3101 competent cells and freeze-thaw them on ice. Add 2 μL of the expression vector p35:BrAL3 containing the alfalfa zinc finger protein-like protein gene AL3 to 100 μL of Agrobacterium tumefaciens GV3101 competent cells and gently mix with a pipette tip. Transfer the mixture of cells and plasmids to an electroporation cuvette, pre-cool it, and electroporate it at 2500V. Remove the electroporation cuvette, add 800 μL of pre-cooled LB liquid medium, gently mix by pipetting, and transfer the bacterial solution to a 1.5 mL centrifuge tube. Incubate at 28℃ and 200 rpm for 5 h with shaking. Spread 30-40 μL of the bacterial solution onto LB solid medium containing the corresponding antibiotics and incubate upside down at 28℃ for 1.5-2 days. Then screen Agrobacterium tumefaciens resistant to rifampicin and kanamycin for colony PCR. Select positive colonies for shaking culture, expand the culture, and use them for Agrobacterium infection.

[0054] Agrobacterium tumefaciens containing the alfalfa zinc finger protein analog gene AL3 expression vector p35:BrAL3 was selected and Chinese cabbage 'Guangdong Early' was infected using the floret infection method. After resistance selection, Chinese cabbage transgenic with the alfalfa zinc finger protein analog gene AL3 was obtained.

[0055] Example 3

[0056] Seedlings of Chinese cabbage (BrAL3) and wild-type Chinese cabbage 'Guangdong Early' (WT) prepared in Example 2, which were transgenic with the alfalfa zinc finger protein-similar gene AL3, were first grown on 0.5×MS hydroponic medium for 3 days. Seedlings with similar growth were then selected and transferred to 0.5×MS hydroponic medium containing 50 μM CdCl2 for another 7 days. The plant phenotypes of both were as follows: Figure 1 As shown, the cadmium content determination results are as follows: Figure 2 As shown.

[0057] Depend on Figure 1 It can be seen that the root length of Chinese cabbage transgenic with alfalfa zinc finger protein analogue AL3 was significantly greater than that of wild-type Chinese cabbage after growing under cadmium stress. This indicates that the growth of Chinese cabbage transgenic with alfalfa zinc finger protein analogue AL3 under cadmium stress was significantly better than that of wild-type Chinese cabbage, that is, alfalfa zinc finger protein analogue AL3 can enhance the plant's cadmium resistance.

[0058] Depend on Figure 2 It can be seen that the cadmium content of Chinese cabbage grown under cadmium stress was significantly lower than that of wild-type Chinese cabbage, indicating that the alfalfa zinc finger protein analog gene AL3 can reduce cadmium accumulation and absorption in plants.

[0059] Example 4

[0060] The applicant purchased atal3-1 (SALK_139843C, Tair), a mutant of Arabidopsis thaliana with decreased expression of the alfalfa zinc finger protein homolog AtAL3 (SEQ ID NO.2), from AraShare for resistance testing and cadmium content testing.

[0061] Then, wild-type Arabidopsis thaliana (WT) and the Arabidopsis thaliana mutant atal3-1 (atal3-1) with decreased AL3 expression were first grown on 0.5×MS hydroponic medium for 3 days. Seedlings with similar growth were then transferred to 0.5×MS hydroponic medium containing 50 μM CdCl2 for another 7 days. The plant phenotypes of both were as follows: Figure 3 As shown, the cadmium content determination results are as follows: Figure 4 As shown.

[0062] Depend on Figure 3 It can be seen that the root length of wild-type Arabidopsis thaliana after growing under cadmium stress is significantly greater than that of the Arabidopsis thaliana mutant atal3-1, indicating that the growth of the mutant atal3-1 with decreased AL3 expression is significantly worse than that of wild-type Arabidopsis thaliana. That is, decreased expression of the alfalfa zinc finger protein-like protein gene AL3 will reduce the plant's resistance to cadmium.

[0063] Depend on Figure 4 It can be seen that the cadmium content of wild-type Arabidopsis thaliana after growth under cadmium stress is significantly lower than that of the Arabidopsis thaliana mutant atal3-1, indicating that the decreased expression of the alfalfa zinc finger protein-similar gene AL3 will increase cadmium accumulation and cadmium absorption in plants.

[0064] In summary, this invention has discovered that the alfalfa zinc finger protein analog gene AL3 can enhance the plant's resistance to heavy metal cadmium. Plants transfected with the alfalfa zinc finger protein analog gene AL3 have excellent resistance to cadmium stress, significantly enhance the plant's ability to resist heavy metal cadmium, and reduce cadmium accumulation and absorption in plants.

Claims

1. The application of the alfalfa zinc finger protein-like protein gene BrAL3 in enhancing the resistance of Chinese cabbage to heavy metal cadmium, characterized in that, The nucleotide sequence of the alfalfa zinc finger protein-like protein gene BrAL3 is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The application method includes: transforming an expression vector containing the alfalfa zinc finger protein-like protein gene BrAL3 into Chinese cabbage.

3. The application as described in claim 2, characterized in that, The method for constructing the expression vector includes the following steps: (1) RNA was extracted from Chinese cabbage, and then cDNA was obtained by reverse transcription of the RNA; (2) PCR amplification was performed using cDNA as a template to obtain the BrAL3 sequence of alfalfa zinc finger protein-like protein. The PCR primer sequences are as follows: Forward primer: 5'-cgggggactctagagATGGAAGGAGGAGCTGGTCTT-3', Reverse primer: 5'-gaccacccggggatcTTCCTCCTCCTCCTCCTCCTTAAGGTCGTGCTCTTTTGTT-3'; (3) The alfalfa zinc finger protein-similar protein gene BrAL3 sequence and the cauliflower mosaic virus 35S promoter p35S were ligated to the pCambia3300 plasmid to obtain an expression vector containing the alfalfa zinc finger protein-similar protein gene BrAL3.

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