Method for cultivating insect-resistant gene transformed poplar

Abstract

The invention relates to a method for cultivating an insect-resistant gene transgenic poplar, which belongs to the field of transgenosis, and comprises the following steps: obtaining PHOT2-LRE and BtCry1Ac + API, and carrying out Golden Gate assembly to obtain a pCAMBIA1305-PHOT2-CTP-Bt-API recombinant plasmid; transferring into young leaves of sterile tissue culture seedlings of poplars, and screening and culturing to obtain insect-resistant gene transformed poplars. Through photosensitive protein regulation and chloroplast polygene transformation, the insect-resistant efficiency and safety are remarkably improved, and a new direction is provided for insect-resistant poplar breeding.

Description

Technical Field

[0001] The invention belongs to the field of poplars, and in particular relates to a method for cultivating poplars with insect-resistant genes. Background Art

[0002] Poplar (Populus) is one of the fast-growing timber species with the highest wood yield and the largest planting area in the mid-latitude plains. It has the characteristics of fast growth, early maturity and high yield. With the continuous expansion of the scale of poplar plantations and concentrated planting, the

[0003] A variety of forest pests, mainly Lymantria dispar, Malacosoma neustria and Coleoptera, have begun to spread and wreak havoc, causing heavy losses to the forestry economy and ecosystem. Poplar pest control is imminent. In the past, people mostly used chemical control methods to kill insects. Although it temporarily eased the contradiction, the side effects on the ecology, such as environmental pollution, insect resistance, and the rampant increase of pests, have attracted people's attention. Breeding poplar varieties with broad-spectrum insect resistance has become an ideal and most promising method for controlling forest pests. In the past decade, with the widespread application of genetic engineering technology, a fast and effective way has been provided for insect-resistant breeding of poplars. Insect-resistant genes are introduced into forest cells to stably inherit and express them in cells, thereby breeding new insect-resistant varieties of forest trees. With the increasing maturity of poplar tissue culture technology and the development of molecular biology, scientific researchers have achieved the goal of improving varieties in a short period of time through genetic engineering, and bred new varieties that are resistant to pests and diseases, resistant to stress, and high-quality. Insect-resistant transgenic technology started in the 1980s and has formed three major technical systems with Bt toxin protein genes, protease inhibitor genes, and exogenous lectin genes as the core. Bt genetic engineering has become the mainstream direction due to its high efficiency and targeting. More than 75% of the world's transgenic insect-resistant crops rely on Bt protein expression systems. The Biotechnology Center of the Chinese Academy of Agricultural Sciences constructed a highly active BtCry1Ac expression cassette through whole genome synthesis technology and developed a chloroplast localization vector pLD-ctv, which increased the efficiency of poplar resistance to gypsy moth to 95%. The fusion gene vector designed by Zhejiang University (such as Cry1Aa-Cry9Aa) increases the insecticidal activity by 3.8 times through domain recombination. However, there are still deficiencies in multi-gene coordinated regulation and high-throughput vector assembly technology. The expression of Bt insect-resistant genes is not high, and there are problems such as interference with host metabolism and low biosafety. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a method for cultivating poplars with insect-resistant genes.

[0005] The present invention provides a method for cultivating poplars with insect-resistant genes, wherein the method is as follows:

[0006] 1) Using Arabidopsis thaliana genomic DNA as a template, perform two-step annealing PCR reactions with PHOT2 primers and Gbox primers to obtain the PHOT2-LRE product, and use it after purification;

[0007] 2) Take Populus alba × Populus glandulosa leaves, extract genomic DNA, perform PCR with BtCry1Ac primers and API primers, and amplify to obtain the BtCry1Ac+API fragment, and use it after purification;

[0008] 3) Assemble the purified PHOT2-LRE, synthetic CTP, BtCry1Ac+API and the pCAMBIA1305 vector by Golden Gate to obtain the pCAMBIA1305-PHOT2-CTP-Bt-API recombinant plasmid;

[0009] 4) Coat the pCAMBIA1305-PHOT2-CTP-Bt-API recombinant plasmid DNA with gold particles and introduce it into the young leaves of Populus alba × Populus glandulosa sterile tissue culture seedlings by gene gun transformation method. After screening with spectinomycin and blue light induction, obtain multiple resistant buds. After testing as positive resistant buds and regeneration, obtain insect-resistant Populus alba × Populus glandulosa plants transformed with the pCAMBIA1305-PHOT2-CTP-Bt-API gene.

[0010] Furthermore, the PHOT2 primers are as follows:

[0011] PHOT2-F: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3';

[0012] PHOT2-R: 5'-CACGTGCACGTGCACGTG 3';

[0013] The Gbox primers are as follows:

[0014] Gbox-F: 5'-CACGTGCACGTGCACGTG-3';

[0015] Gbox-R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3';

[0016] The PCR reaction system is 20 μL as follows:

[0017]

[0018] The PCR reaction program:

[0019] Initial denaturation: 98°C for 2 min; amplification stage: 98°C for 10 s, 65°C for 15 s, 72°C for 30 s; 25 cycles; fusion annealing: 98°C for 10 s, 55°C for 30 s, 72°C for 1 min; extension: 72°C for 5 min.

[0020] Further, the BtCry1Ac primers are as follows:

[0021] Bt-F: 5'-GGTCTCACGTATGGAACAACCCAAAC-3'

[0022] Bt-R: 5'-GAGACCGAGACCTTAGGCGTAACCTTG-3'

[0023] API-F: 5'-GCTGAATTCGACCATGGCGGCCTCCAACGCT-3'

[0024] API-R: 5'-CGATGCCCAGCAAGGTTTTT-3'

[0025] The PCR reaction system for amplifying the BtCry1Ac+API fragment is as follows:

[0026]

[0027] PCR reaction program: 98°C for 2 min, 98°C for 10 s, 55°C for 30 s, 72°C for 45 s, 35 cycles, 72°C for 5 min.

[0028] Further, the Golden Gate assembly reaction system is as follows:

[0029]

[0030] Reaction program: 37°C for 1 h, 50°C for 5 min.

[0031] Further, the poplar trees are 741 poplar, 84K poplar, Chinese white poplar, European-American poplar or eastern cottonwood.

[0032] Further, the insect resistance is against Lymantria dispar, Clostera anachoreta, Hyphantria cunea, Anoplophora glabripennis or Plagiodera versicolora.

[0033] The present invention has the following beneficial effects:

[0034] The present invention achieves light control-chloroplast dual-effect expression: the PHOT2 promoter is fused with the Prrn promoter to realize "high expression + light specificity" regulation. The Bt protein content is increased by 3 times compared with nuclear transformation, and the mortality rate is increased by 22.2%. The PHOT2-LRE promoter: initiates the expression of insect-resistant genes under blue light induction, and the leakage rate is <0.5% under dark conditions, reducing non-target environmental exposure. CTP (chloroplast transit peptide): targets the Bt protein to the chloroplast stroma, utilizes the multi-copy characteristics of the chloroplast genome to increase the expression level, and at the same time reduces the risk of gene drift (<0.1%) through maternal inheritance. The present invention significantly improves the insect resistance efficiency and safety through photosensitive protein regulation and chloroplast multi-gene transformation, providing a new direction for insect-resistant poplar breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is the agarose gel electrophoresis pattern of the PHOT2-LRE fragment;

[0036] Figure 2 It is the agarose gel electrophoresis pattern of the BtCry1Ac + API fragment. DETAILED DESCRIPTION OF THE INVENTION

[0037] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the spirit of the content disclosed by the present invention will be described in detail below. After any person skilled in the art understands the embodiments of the content of the present invention, they can make changes and modifications based on the technology taught by the content of the present invention, which do not depart from the spirit and scope of the content of the present invention.

[0038] The illustrative embodiments of the present invention and their descriptions are used to explain the present invention, but are not intended to limit the present invention.

[0039] Example 1

[0040] The insect-resistant transgenic poplar in this example is obtained as follows:

[0041] 1) Using Arabidopsis genomic DNA as a template, perform two-step annealing PCR reactions with PHOT2 primers and Gbox primers to obtain PHOT2-LRE products, and purify them for later use;

[0042] The PHOT2 primers are as follows:

[0043] PHOT2-F: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3';

[0044] PHOT2-R: 5'-CACGTGCACGTGCACGTG 3';

[0045] The Gbox primers are as follows:

[0046] Gbox-F: 5'-CACGTGCACGTGCACGTG-3';

[0047] Gbox-R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3';

[0048] The PCR reaction system of 20 μL is as follows:

[0049]

[0050]

[0051] The PCR reaction procedure:

[0052]

[0053] . The PCR results are shown in Figure 1 , Figure 1 . The band at around 1.2 kb is the PHOT2-LRE product.

[0054] 2) Take the leaves of the highly resistant strain PB29 (germplasm bank of Hebei Agricultural University) of Populus deltoides cv. 'Jilin 741', extract genomic DNA, perform PCR using BtCry1Ac primers and API primers, amplify to obtain the BtCry1Ac + API fragment, and purify the target band using a gel extraction kit (such as Qiagen QIAquick) for standby;

[0055] The BtCry1Ac primers are as follows:

[0056] Bt-F: 5'-GGTCTCACGTATGGAACAACCCAAAC-3'

[0057] Bt-R: 5'-GAGACCGAGACCTTAGGCGTAACCTTG-3'

[0058] API-F: 5'-GCTGAATTCGACCATGGCGGCCTCCAACGCT-3'

[0059] API-R: 5'-CGATGCCCAGCAAGGTTTTT-3'

[0060] The PCR reaction system for amplifying the BtCry1Ac + API fragment is as follows:

[0061]

[0062] PCR reaction procedure: 98°C for 2 min, 98°C for 10 s, 55°C for 30 s, 72°C for 45 s, 35 cycles, 72°C for 5 min. The PCR results are shown in Figure 2 , Figure 2 as shown, the band at around 1.2 kb is the BtCry1Ac + API product.

[0063] 3) Purified PHOT2-LRE, synthetic CTP, BtCry1Ac + API and pCAMBIA1305 vector were assembled by Golden Gate to obtain the pCAMBIA1305-PHOT2-CTP-Bt-API recombinant plasmid; the pCAMBIA1305-PHOT2-CTP-Bt-API recombinant plasmid was verified by sequencing. PHOT2-CTP-Bt-API was ligated to the pCAMBIA1305 vector for the next step; the synthetic CTP (chloroplast transit peptide) was synthesized by a biological company from the Arabidopsis thaliana RBCS1A gene (GenBank: NM_001203311.1).

[0064] 4) The pCAMBIA1305-PHOT2-CTP-Bt-API recombinant plasmid DNA (1 μg / mg gold powder) was coated with gold particles and introduced into the young leaves of sterile tissue-cultured poplar seedlings by gene gun transformation. After induction and screening with spectinomycin and blue light (50 μmol / m 2 / s, 16 h photoperiod), multiple resistant buds were obtained. After testing as positive resistant buds, they were induced to regenerate buds with MS + 1.0 mg / L BA + 0.1 mg / L NAA, and cultured with 1 / 2 MS + 0.3 mg / L IBA for 3 weeks to obtain insect-resistant poplar plants transformed with the pCAMBIA1305-PHOT2-CTP-Bt-API gene.

[0065] Example 2

[0066] I. Experimental verification of insect resistance

[0067] 1. Experimental conditions

[0068] 1.1 Test insects:

[0069] Lepidoptera: 3rd instar larvae of Lymantria dispar.

[0070] Coleoptera: larvae of Anoplophora glabripennis.

[0071] 1.2 Groups

[0072] Experimental group: PHOT2-CTP-Bt-API chloroplast-transformed Populus alba × Populus glandulosa '741' plants.

[0073] Control 1: BtCry1Ac + API 741 poplar plants (without CTP).

[0074] Control 2: Wild-type 741 poplar.

[0075] 2. Experimental methods

[0076] 2.1 Indoor insect feeding experiment:

[0077] Leaf treatment: Take leaves with 12 hours of light (peak Bt protein expression), and cut them into 2×2 cm fragments.

[0078] Insect feeding conditions: 30 larvae in each group, at 25 °C, 70% humidity, and 16 h light.

[0079] Data recording: Count the mortality rate, weight inhibition rate, and leaf feeding area every 24 hours.

[0080] 2.2 Monitoring of resistance evolution:

[0081] qPCR detection: Calculate the frequency of the Cry1Ac resistance allele (Vip3A-R) in the pest population.

[0082] Field experiment: Plant continuously for 3 years and count the proportion of resistant individuals.

[0083] 2.3 Experimental results

[0084]

[0085] As can be seen from the above table, the co-expression of the two genes reduces the resistance evolution rate by 4 times, and the inhibition rate against Coleoptera pests (Anoplophora glabripennis) increases by 50%; the mortality rate of the experimental group is 22.2% higher than that of Control 1. The resistance frequency of the experimental group is 7.2%, which is significantly lower than that of Control 1. API delays larval development by inhibiting the activity of insect digestive enzymes and reduces the resistance evolution rate (the resistance allele frequency is only 7.2% in 3 years).

[0086] 2.4 Indoor insect feeding experiment:

[0087] Select Lepidoptera pests (gypsy moth larvae), 30 in each group, and feed them to the experimental group (light group / dark group), Control 1, and Control 2 respectively. Record the mortality rate and larval weight inhibition rate at 72 hours.

[0088]

[0089]

[0090] The experimental group significantly improved the insecticidal effect through light.

[0091] II. Photocontrol kinetic analysis

[0092] Light intensity gradient experiment: Set the blue light intensity to 0 (dark), 20, 50, 100 μmol / m 2 / s, and measure the Bt protein content (ELISA method) after continuous irradiation for 6 hours. The results are shown in the following table.

[0093] <![CDATA[Light intensity (μmol / m 2 / s)]]> Bt protein (μg / g FW) API protein (μg / g FW) 0 0.002 0.001 50 0.125 0.098 100 0.130 0.103

[0094] The light control module increased the BtCry1Ac expression level by 3 times compared to nuclear transformation (0.15% vs. 0.05% TSP); the leakage rate under dark conditions was only 2.67% of that induced by blue light (<0.5%), which was significantly better than the traditional induction system (such as the ethanol induction leakage rate >5%).

[0095] III. Biosafety assessment

[0096] 3.1 Gene flow detection

[0097] Pollen transmission experiment: Plant the experimental group and wild-type plants in a 1:5 ratio in an isolated test area, collect the seeds of wild plants within 1 km; use BtCry1Ac specific primers (F: 5′-CTGACGTAAGGATGACGCAC-3′, R: 5′-ACTATTGATAGTCGCGGCATC-3′) to detect the offspring plants; the results show that the gene flow rate: <0.1%.

[0098] 3.2 Light control expression leakage rate

[0099] Dark induction experiment: Place the experimental group plants in a completely dark environment for 7 days, and take samples to detect the Bt protein content;

[0100] Blue light induction: Synchronously detect after irradiating with 450 - 480 nm blue light (50 μmol / m 2 / s) for 12 hours.

[0101] Light condition BtCry1Ac expression level (% TSP) Blue light induction 0.15±0.03 Dark treatment 0.004±0.001

[0102] Leakage rate: The expression level under dark conditions was only 2.67% of that induced by blue light (<0.5%).

[0103] IV. Effects of chloroplast localization on host metabolism

[0104] Metabolomics analysis: Use LC-MS to detect the differences in metabolites in the chloroplast stroma and cytoplasm between the experimental group and control 1;

[0105] Photosynthetic parameters: Use a LI-6400XT photosynthesis system to measure the net photosynthetic rate (Pn) and photosystem II efficiency (Fv / Fm).

[0106] Parameter Experimental group Control 1 <![CDATA[Pn (μmol / m 2 / s)]]> 18.7±1.2 15.3±1.0 Fv / Fm 0.82±0.03 0.75±0.04 Cytoplasmic Bt accumulation Not detected 0.03% TSP

[0107] Chloroplast localization significantly reduces the interference of Bt protein on cytoplasmic metabolism, and the photosynthetic efficiency is increased by 22%.

Claims

1. A method for cultivating transgenic insect-resistant poplar trees, characterized in that The method is as follows: 1) Using Arabidopsis genomic DNA as a template, perform two-step annealing PCR reactions with PHOT2 primers and Gbox primers to obtain PHOT2-LRE products, and use them after purification; 2) Take Populus × euramericana cv. 741 leaves, extract genomic DNA, perform PCR with BtCry1Ac primers and API primers to amplify the BtCry1Ac+API fragment, and use it after purification; 3) Assemble the purified PHOT2-LRE, synthetic CTP, BtCry1Ac+API and pCAMBIA1305 vector by GoldenGate to obtain the pCAMBIA1305-PHOT2-CTP-Bt-API recombinant plasmid; 4) Wrap the pCAMBIA1305-PHOT2-CTP-Bt-API recombinant plasmid DNA with gold particles and introduce it into the young leaves of Populus sterile tissue culture seedlings by gene gun transformation method. After screening with spectinomycin and blue light induction, obtain multiple resistant buds. After testing as positive resistant buds and regeneration, obtain insect-resistant Populus plants transformed with the pCAMBIA1305-PHOT2-CTP-Bt-API gene.

2. The method for cultivating transgenic insect-resistant poplar according to claim 1, characterized in that The PHOT2 primers are as follows: PHOT2-F: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3'; PHOT2-R: 5'-CACGTGCACGTGCACGTG-3'; The Gbox primers are as follows: Gbox-F: 5'-CACGTGCACGTGCACGTG-3'; Gbox-R: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3'; The PCR reaction system is 20 μL as follows: The PCR reaction program: Initial denaturation: 98°C for 2 min; Amplification stage: 98°C for 10 s, 65°C for 15 s, 72°C for 30 s; 25 cycles; Fusion annealing: 98°C for 10 s, 55°C for 30 s, 72°C for 1 min; Extension: 72°C for 5 min.

3. A method for cultivating transgenic insect-resistant poplar according to claim 1, characterized in that The BtCry1Ac primers: Bt-F: 5'-GGTCTCACGTATGGAACAACCCAAAC-3' Bt-R: 5'-GAGACCGAGACCTTAGGCGTAACCTTG-3' API-F: 5'-GCTGAATTCGACCATGGCGGCCTCCAACGCT-3' API-R: 5'-CGATGCCCAGCAAGGTTTTT-3' The PCR reaction system for amplifying the BtCry1Ac+API fragment: PCR reaction program: 98°C for 2 min, 98°C for 10 s, 55°C for 30 s, 72°C for 45 s, 35 cycles, 72°C for 5 min.

4. A method for cultivating transgenic insect-resistant poplar according to claim 1, characterized in that The GoldenGate assembly reaction system: Reaction program: 37°C for 1 h, 50°C for 5 min.

5. A method for cultivating transgenic insect-resistant poplar according to claim 1, characterized in that The Populus is Populus × euramericana cv. 741, Populus alba × Populus glandulosa, Populus tomentosa, Populus × euramericana or Populus deltoides.

6. The method for cultivating transgenic insect-resistant poplar according to claim 1, characterized in that The insect resistance is against Lymantria dispar, Clostera anachoreta, Hyphantria cunea, Anoplophora glabripennis or Plagiodera versicolora.

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