Cultivation method of insect-resistant transgenic small black poplar

Abstract

The invention relates to a method for cultivating insect-resistant transgenic Xiaoblack poplar, in particular to a method for cultivating insect-resistant transgenic Xiaoblack poplar. The invention aims to solve the problem of low insect resistance of the populus similis. The cultivation method comprises the following steps: 1, synthesizing a Cry1Ac gene, wherein the nucleotide sequence of the gene is shown as Seq ID No: 1 in a sequence table; 2, a plant expression vector pBI121-Cry1Ac expressed by the Cry1Ac gene is constructed, and the plant expression vector pBI121-Cry1Ac is constructed; 3, preparing an infection solution; 4, cultivating the Cry1Ac-transgenic small black poplar by using an agrobacterium-mediated leaf disc transformation method, the Cry1Ac-transgenic poplar is created, the average concentration of leaf Cry1Ac protein is more than 200ng / g, the insect resistance of the transgenic poplar can be improved, all fall webworms die after being fed for 5 days, and the corrected death rate reaches 100%. The method is applied to the field of molecular breeding.

Description

Technical Field

[0001] The present invention relates to an insect-resistant transgenic Populus simonii×P. nigra and a cultivation method thereof, and particularly relates to a transgenic Populus simonii×P. nigra transformed with the Cry1Ac gene, which expresses the exogenous Bt gene Cry1Ac and is resistant to Hyphantria cunea. Background Art

[0002] Populus simonii×P. nigra is widely used in afforestation, industrial timber, pulp and paper making and other fields, and is one of the important greening tree species and economic tree species. Among the many pests of poplars, Hyphantria cunea is an alien invasive species. Due to its fast reproduction speed and strong diffusion ability, it has caused great damage to forest trees and crops. Hyphantria cunea has been rampant in China in recent years. At present, the main measures for controlling this pest are the combination of chemical control and biological control. In order to reduce the losses caused by pests to forestry, it is particularly important to improve the insect resistance of poplars by means of genetic engineering. Summary of the Invention

[0003] The purpose of the present invention is to solve the problem of low insect resistance of Populus simonii×P. nigra. The Cry1Ac gene is introduced into the genome of Populus simonii×P. nigra to cultivate a transgenic Populus simonii×P. nigra expressing the Cry1Ac gene, thereby endowing it with insect resistance.

[0004] The cultivation method of an insect-resistant transgenic Populus simonii×P. nigra of the present invention is carried out according to the following steps:

[0005] First, synthesize the Cry1Ac gene, and the nucleotide sequence of the gene is as shown in Sequence Listing Seq ID No: 1;

[0006] Second, construct a plant expression vector pBI121-Cry1Ac for driving the expression of the Cry1Ac gene downstream by 35S;

[0007] Third, transfer PBI121-Cry1Ac into Agrobacterium tumefaciens EHA105, and then prepare an infection solution;

[0008] Fourth, cultivate transgenic Populus simonii×P. nigra transformed with the Cry1Ac gene by the Agrobacterium-mediated leaf disc transformation method to obtain insect-resistant transgenic Populus simonii×P. nigra.

[0009] The beneficial effects of the present invention are as follows:

[0010] The present invention has created a transgenic poplar transformed with the Cry1Ac gene. The exogenous gene Cry1Ac is integrated into the poplar genome and correctly expressed. The average concentration of Cry1Ac protein in the leaves is above 200 ng / g, which can improve the insect resistance of transgenic poplars. All Hyphantria cunea died after being fed for 5 days, and the corrected mortality rate reached 100%. Brief Description of the Drawings

[0011] Figure 1 It is the map of the pBI121-Cry1Ac plant expression vector;

[0012] Figure 2 This is the electrophoresis diagram for DNA-level detection of transgenic poplars using PCR in the present invention, where M: DL2000 Marker; 1-3: transgenic plants; 4: wild type of Populus simonii × P. nigra; 5: positive plasmid;

[0013] Figure 3 This is the electrophoresis diagram for RNA-level detection of transgenic poplars using RT-PCR in the present invention, where M: DL2000 Marker; 1-3: transgenic plants; 4: wild type of Populus simonii × P. nigra; 5: positive plasmid;

[0014] Figure 4 This is for determining the content of Cry1Ac protein in the leaves of transgenic Populus simonii × P. nigra using an ELISA detection kit, where WT: wild type poplar; H1-H3: transgenic poplars;

[0015] Figure 5 This shows the changes in leaves after feeding transgenic Populus simonii × P. nigra and wild type leaves to Hyphantria cunea for 24 hours, where WT: wild type poplar; H1-H3: transgenic poplars;

[0016] Figure 6 This is the corrected mortality rate of feeding transgenic Populus simonii × P. nigra leaves to Hyphantria cunea, where WT: wild type poplar; H1-H3: transgenic poplars. Detailed implementation manners

[0017] Detailed implementation manner 1: The cultivation method of an insect-resistant transgenic Populus simonii × P. nigra in this implementation manner is carried out according to the following steps:

[0018] 1. Synthesize the Cry1Ac gene, and the nucleotide sequence of the gene is as shown in Sequence Listing Seq ID No: 1;

[0019] 2. Construct a plant expression vector pBI121-Cry1Ac for expressing the Cry1Ac gene driven by 35S downstream;

[0020] 3. Transfer PBI121-Cry1Ac into Agrobacterium tumefaciens EHA105, and then prepare an infection solution;

[0021] 4. Cultivate transgenic Populus simonii × P. nigra with Cry1Ac gene by Agrobacterium-mediated leaf disc transformation method to obtain insect-resistant transgenic Populus simonii × P. nigra.

[0022] Detailed implementation manner 2: The difference between this implementation manner and detailed implementation manner 1 is that the construction method of the pBI121-Cry1Ac vector is:

[0023] The gene was synthesized according to the cDNA sequence of the Cry1Ac gene (Gene ID: KF630361.1) in the database. Specific primers were designed, and the restriction enzyme sites BamH I and EcoR I were introduced at the 5' and 3' ends. The KOD high-fidelity enzyme was used for PCR amplification. Then, the PCR product and the pBI121 plasmid were double-digested with EcoRⅠ and BamHⅠ, and the products were recovered separately and ligated using T4 ligase to construct the vector pBI121-Cry1Ac. Others are the same as in the first specific embodiment.

[0024] Specific Embodiment 3: The difference between this embodiment and the first or second specific embodiment is as follows: The method for preparing the infection solution is as follows: The vector pBI121-Cry1Ac was transferred into Agrobacterium tumefaciens EHA105. A single colony of Agrobacterium tumefaciens containing the pBI121-Cry1Ac vector was picked and inoculated into an LB liquid medium containing antibiotics and cultured overnight at 28°C. The next day, 1 mL of the Agrobacterium tumefaciens solution was taken and added to 50 mL of the LB liquid medium containing antibiotics, and cultured with shaking at 28°C until the OD600 of the bacterial solution was 0.6 - 0.8. Then, the cells were collected by centrifugation at 4000 rpm for 10 min, and the cells were resuspended in 1 / 2MS + 50 μL AS liquid medium to an OD600 of 0.1, which was used as the infection solution for the genetic transformation of Populus simonii×P. nigra. Others are the same as in the first or second specific embodiment.

[0025] Specific Embodiment 4: The difference between this embodiment and any one of the first to third specific embodiments is as follows: The LB liquid medium containing antibiotics is the LB liquid medium containing 50 mg / L of kanamycin and 50 mg / L of rifampicin. Others are the same as any one of the first to third specific embodiments.

[0026] Specific Embodiment 5: The difference between this embodiment and any one of the first to fourth specific embodiments is as follows: The insect resistance mentioned refers to resistance to Hyphantria cunea. Others are the same as any one of the first to fourth specific embodiments.

[0027] The effects of the present invention were verified through the following experiments:

[0028] Example 1:

[0029] Cloning of the Cry1Ac Gene

[0030] Using the cDNA sequence of the Cry1Ac gene (Gene ID: KF630361.1) in the database as a template, specific primers shown in Seq ID No: 3 and Seq ID No: 4 were designed using Primer5 to amplify the Cry1Ac gene fragment.

[0031] The PCR reaction system is as follows:

[0032]

[0033] The reaction procedure was as follows: pre-denaturation at 94°C for 2 min; denaturation at 98°C for 10 sec; annealing at 53°C for 30 sec, extension at 68°C for 1 min, for 35 cycles; after the cycles ended, extension at 68°C for 7 min, and finally preservation at 4°C. The Cry1Ac gene sequence shown in Seq ID No: 1 was finally obtained. In this example, the Cry1Ac gene with a length of 1848 bp was synthesized, which is a structural gene encoding the Bt toxin protein and encodes a protein with the amino acid sequence of SeqID No: 2 in the sequence listing.

[0034] Example 2: Construction of the pBI121-Cry1Ac vector

[0035] A gene was synthesized according to the cDNA sequence of the Cry1Ac gene (Gene ID: KF630361.1) in the database. Specific primers Cry1Ac-F and Cry1Ac-R were used, and restriction enzyme sites BamH I and EcoR I were introduced at the 5' and 3' ends. A PCR amplification reaction was carried out using KOD high-fidelity enzyme. Then, the Cry1Ac gene fragment and the pBI121 plasmid were double-digested with EcoRⅠ and BamHⅠ. The pBI121 digested product and the Cry1Ac gene fragment were ligated using T4 DNA ligase to construct the vector pBI121-Cry1Ac. The map of the pBI121-Cry1Ac plant expression vector is as Figure 1 shown. The PBI121-Cry1Ac plasmid was transferred into Agrobacterium tumefaciens EHA105 for cultivating transgenic poplar trees expressing Cry1Ac.

[0036] The primer sequence information is as follows:

[0037] Cry1Ac-F: 5'-CGGGATCCCGGTTGTAAAACGACGGCCAGATGGACAACAACCCAAACATC-3';

[0038] Cry1Ac-R: 5'-GGAATTCCAGGAAACAGCTATGACTCATTCAGCCTCGAGTGTTGC-3'.

[0039] Example 3: Cultivation of transgenic Populus simonii × P. nigra and analysis of plant insect resistance

[0040] Transgenic Populus simonii × P. nigra plants were cultivated by the Agrobacterium-mediated leaf disc transformation method. The specific steps were as follows:

[0041] I. Two days before preparing the infection solution, pick a single colony of Agrobacterium containing the pBI121-Cry1Ac vector and inoculate it into LB liquid medium containing antibiotics (50 mg / L kanamycin and 50 mg / L rifampicin) and culture overnight at 28 °C. The next day, pipette 1 mL of the Agrobacterium solution into 50 mL of liquid LB (containing 50 mg / L Kan and 50 mg / L Rif) medium and culture with shaking at 28 °C until the OD 600 of the bacterial solution reaches 0.6 - 0.8. Centrifuge at 4000 rpm for 10 min to collect the bacteria, and then resuspend the bacteria in 1 / 2MS + 50 μL AS liquid medium to an OD 600 of 0.1, which is used as the infection solution for Populus simonii × P. nigra genetic transformation.

[0042] II. Agrobacterium-mediated genetic transformation:

[0043] Use the leaf disc method mediated by Agrobacterium tumefaciens for genetic transformation. The specific transformation steps are as follows:

[0044] (1) Pre-culture: Cut the leaves of the sterile seedlings into pieces of 1 cm × 1 cm and inoculate them onto the differentiation medium for pre-culture for 1 day.

[0045] (2) Co-culture: Immerse the explants in the Agrobacterium solution (OD 600 = 0.1), gently shake for infection for 10 min, and then blot off the excess bacterial solution with sterile filter paper. Then, transfer the explants to the differentiation medium (MH + 0.5 mg / L 6-BA + 0.05 mg / L NAA + 200 mg / L cefotaxime) and culture in the dark at 28 °C for 2 days.

[0046] (3) Selection culture: Transfer the co-cultured explants to the differentiation selection medium with selective pressure (MH + 0.5 mg / L 6-BA + 0.05 mg / L NAA + 50 mg / L Kan + 200 mg / L cefotaxime) and culture at 25 °C. Replace the medium once a week until resistant buds grow.

[0047] (4) Rooting culture: Cut the resistant buds and insert them onto the rooting medium with selective pressure (1 / 2MS + 0.2 mg / L IBA + 10 mg / L kanamycin + 100 mg / L cefotaxime) for rooting culture.

[0048] (5) Pick the Kan-resistant plants that grow normally in the rooting medium and extract DNA and RNA for molecular identification.

[0049] PCR detection proves that the exogenous Cry1Ac gene has been integrated into the poplar genome, and TR-PCR detection proves that the exogenous Cry1Ac gene is correctly expressed in poplar cells. The average concentration of Cry1Ac protein in the transgenic Populus simonii × P. nigra leaves is above 200 ng / g, specifically asFigure 2 , Figure 3 and Figure 4 as shown in Figure 2 , Figure 3 and Figure 4 , transgenic Populus simonii×P. nigra was obtained.

[0050] The insects used for insect resistance identification were the larvae of Hyphantria cunea. The eggs and feed were purchased from the Insect Virus R & D Center of the Chinese Academy of Forestry. The egg masses were soaked in 10% formaldehyde solution for 15 min and then washed thoroughly with water. The culture conditions were (25 ± 1) °C, the photoperiod was 16 h, the dark period was 8 h, and the relative humidity was (70 ± 5)%. The newly hatched larvae were cultured to the 3rd instar, starved for 24 h and then used for the insect resistance identification of poplar.

[0051] Using wild-type poplar as the negative control, the resistance of Cry1Ac gene-transformed poplar to Hyphantria cunea was identified. The 2nd - 4th fresh leaves from different poplar lines were used to feed the larvae of Hyphantria cunea in the culture box, with 3 replicates, 10 larvae in each replicate. The leaves were replaced every 24 h, and the number of dead larvae was recorded to calculate the corrected mortality rate. It was found that after 24 h of feeding, the damaged area of the transgenic leaves was significantly smaller than that of the wild type. Mortality began to occur 2 d after feeding the larvae of Hyphantria cunea with the transgenic poplar lines, and all died after 5 d of feeding, and the corrected mortality rates all reached 100%, as specifically shown in Figure 5 and Figure 6 as shown.

[0052] In summary, it was determined that the exogenous gene Cry1Ac was integrated into the poplar genome and correctly expressed, which could significantly improve the insect resistance of plants and had good application prospects in the field of forest tree insect resistance breeding.

Claims

1. A method for cultivating insect-resistant transgenic Populus simonii×P. nigra, characterized in that The method proceeds as follows:

1. Synthesize Cry1Ac gene, the nucleotide sequence of which is shown in Seq ID No: 1; Second, construct the plant expression vector pBI121-Cry1Ac that drives the expression of the downstream Cry1Ac gene driven by 35S; 3. Transform PBI121-Cry1Ac into EHA105 Agrobacterium, and then prepare the infection solution; 4. Use Agrobacterium-mediated leaf disc transformation method to cultivate Cry1Ac gene-transgenic Populus xiaohei to obtain insect-resistant transgenic Populus xiaohei.

2. The cultivation method of an insect-resistant transgenic Populus simonii×P. nigra according to claim 1, characterized in that, The construction method of pBI121-Cry1Ac vector is as follows: The gene was synthesized according to the cDNA sequence of the Cry1Ac gene in the database, specific primers were designed, restriction sites BamH I and EcoR I were introduced at the 5' and 3' ends, and PCR amplification reaction was carried out using KOD high-fidelity enzyme; then the PCR product and pBI121 plasmid were double-digested with EcoRⅠ and BamHⅠ, the products were recovered separately and connected using T4 ligase to construct the vector pBI121-Cry1Ac.

3. The cultivation method of an insect-resistant transgenic Populus simonii×P. nigra according to claim 1, characterized in that, The method for preparing the infection solution is as follows: the vector pBI121-Cry1Ac is transferred into EHA105 Agrobacterium, a single colony of Agrobacterium containing the pBI121-Cry1Ac vector is picked, and inoculated into LB liquid culture medium containing antibiotics and cultured overnight at 28°C. The next day, 1 mL of Agrobacterium solution is added to 50 mL of LB liquid culture medium containing antibiotics, and cultured at 28°C with shaking until the OD600 of the bacterial solution is 0.6-0.8, and then the bacteria are collected by centrifugation at 4000 rpm for 10 min, and then the bacteria are resuspended in 1 / 2MS+50μLAS liquid culture medium to an OD600 of 0.1, and used as the infection solution for genetic transformation of Populus xiaohei.

4. The cultivation method of an insect-resistant transgenic Populus simonii×P. nigra according to claim 1, characterized in that, The insect resistance refers to resistance to the gypsy moth.

Images