A method for improving regeneration and genetic transformation efficiency of peanut plants by ectopic expression of AhLEC1a gene

Abstract

The application belongs to the technical field of plant genetic engineering and genetic breeding, and particularly relates to a method for improving regeneration and genetic transformation efficiency of peanut plants by ectopic expression of a gene AhLEC1a The application discloses a method for improving regeneration efficiency and genetic transformation success rate of peanut plants by ectopic expression of a gene AhLEC1a The method is characterized in that a beta-estradiol inducible expression vector is constructed AhLEC1a The transgenic plants are obtained by using somatic embryo regeneration or a direct organ regeneration approach. AhLEC1a The ectopic expression of the gene can significantly improve somatic embryo induction rate (up to 100%) and the number of cells of a single explant (2.13 times of that of a control group); the explant bud induction efficiency of the direct organ regeneration approach is improved by 22% compared with that of the control group; and the transgenic positive rates of the two methods are improved by 27.81% and 6.15% respectively compared with those of the control group. The application breaks through the difficulties of low regeneration efficiency and low transformation success rate in peanut genetic transformation, and provides important technical support for peanut gene function research and molecular breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and genetic breeding technology, specifically relating to a method of expressing genetic material in ectopic locations. AhLEC1a A method for improving peanut plant regeneration and genetic transformation efficiency through gene modification. This invention is applicable to peanut gene function research, molecular breeding, and the creation of new transgenic peanut varieties. Background Technology

[0002] peanut( Arachis hypogaea Peanuts, as a globally important oilseed and economic crop, occupy a vital position in agricultural production. However, the development of its genetic transformation technology is relatively lagging, severely restricting the progress of molecular breeding. Currently, the main technical bottlenecks include low transformation efficiency (usually less than 5%), strong genotype dependence (transformation efficiency differences between different varieties can reach more than 5 times), and an imperfect regeneration system (long cycle, many chimeras). Traditional transformation methods, such as Agrobacterium-mediated transformation, are easily limited by genotype, gene gun methods are costly, and pollen tube pathway methods have poor stability. Although some improvements have been made, such as optimizing culture media and adding antioxidants, the effects are limited, with transformation efficiency only increasing by 2-3 percentage points, which is insufficient to meet the needs of modern molecular breeding. Especially against the backdrop of the rapid development of gene editing technology, inefficient transformation systems have become a major obstacle to peanut functional genomics research and molecular design breeding. Taking the improvement of important traits such as aflatoxin resistance and high oleic acid as examples, the low transformation efficiency significantly prolongs the cycle from gene identification to variety breeding. Therefore, developing efficient, stable, and universally applicable peanut genetic transformation technologies and breaking through existing technological bottlenecks is of great significance for promoting peanut molecular breeding and industrial upgrading.

[0003] LEC1 ( Leafy Cotyledon 1 ) is an NF-YB family transcription factor that encodes a homolog of the CCAAT-box binding transcription factor HAP3 subunit and is a key transcription factor regulating plant embryonic development. In Arabidopsis thaliana (… Arabidopsis thaliana Ectopic expression in (L.) Heynh. LEC1 This can lead to abnormal seedling growth, exhibiting embryonic characteristics such as undeveloped cotyledons, impaired root elongation, and the appearance of embryo-like structures in the shoot apical meristem. This indicates... LEC1 It can induce somatic cells to transform into embryoid cells. Besides Arabidopsis, it has been reported that... LEC Genes play a role in embryogenesis in many other plant species, including alfalfa (Brachys punctata). Medicago truncatula Gaertn., corn ( Zea maize L.), carrots ( Carrot L . ), alfalfa ( M. sativa L.) and cocoa ( Theobroma cacaoL.). Domoki et al. (Domoki M, J Györgyey, J Bíró, et al. Identification and characterization of genes associated with the induction of embryogenic competence in leaf-protoplast-derived alfalfa cells.[J]. BBA - Gene Structure and Expression, 2006, 1759 (11): 543-551. DOI:10.1016 / j.bbaexp.2006.11.005) treated alfalfa with 2,4-D and analyzed the relative expression levels of specific genes during leaf somatic cell embryo induction, finding that including MsLEC1 Ten genes, including [list of genes], exhibited different expression patterns during the induction and differentiation stages of somatic embryogenesis, suggesting that the expression of these genes may be closely related to developmental transitions such as differentiation and dedifferentiation during somatic embryogenesis. (Zhang et al. Similarity of expression patterns of [data missing]) knotted1 and ZmLEC1 during somatic andzygotic embryogenesis in maize ( Zea mays L.)[J]. Planta, 2002, 215(2): 191-194. DOI: 10.1007 / s00425-002-0735-3) The in situ hybridization technique was used to study the process of somatic embryogenesis of maize Hi-II genotype. ZmLEC1 The expression pattern was discovered. ZmLEC1 It is highly expressed throughout the entire somatic embryo, and its expression pattern is similar to that during the development of Arabidopsis thaliana zygote embryos. LEC1 Their expression patterns are similar, indicating that... ZmLEC1It may play an important regulatory role in maize somatic embryogenesis. Yazawa et al. (Yazawa K, Takahata K, Kamada H. Isolation of the gene encoding Carrot leafy cotyledon1 and expression analysis during somatic and zygotic embryogenesis[J]. Plant Physiology and Biochemistry, 2004, 42(3): 215-223. DOI: 10.1016 / j.plaphy.2003.12.003) identified and isolated it from carrot. LEC1 The homologous gene was named C-LEC1 This gene is expressed in embryogenic cells, somatic embryos, and early globular embryos in immature seeds, and can be complementary in Arabidopsis thaliana. lec1-1 The phenotype of the mutant. Within the regulatory network of somatic embryogenesis (SE) in upland cotton, GhLEC1 It has been identified as a direct upstream negative regulator. It works by binding... GhCKI Cis-elements (CTTTTC) in the promoter region negatively regulate the casein kinase I gene. GhCKI The expression of [something] affects the transition of SE cells from proliferation to differentiation and the formation of somatic embryos.

[0004] In peanuts, current research has identified... LEC1 The homologous genes were identified and their expression patterns were preliminarily analyzed; however, no studies have yet been conducted that... AhLEC1 Reports on its application in tissue culture and genetic transformation. AhLEC1 The study investigated the effects of plant regeneration and genetic transformation efficiency, and found that improving peanut genetic transformation efficiency through ectopic expression is of great significance for constructing an efficient peanut transformation system. Summary of the Invention

[0005] The purpose of this invention is to provide a method for expressing through ectopic sites. AhLEC1a The technology aims to improve the regeneration efficiency and genetic transformation success rate of peanut plants through gene enhancement, thereby addressing existing problems such as weak regeneration capacity of peanut plants, low efficiency of genetic transformation systems, and genotype limitations.

[0006] This invention is achieved through the following technical solution: A method of expression via ectopic sites AhLEC1a Methods to improve peanut plant regeneration and genetic transformation efficiency through gene therapy include the following steps: (1) Constructing a system containing AhLEC1a β-estradiol-induced expression vector for the gene; (2) The constructed inducible expression vector was transformed into Agrobacterium and then transformed into explants using the Agrobacterium-mediated transformation method; (3) Culture the transformed explants and induce tissue regeneration; (4) Select transgenic plants.

[0007] Furthermore, the steps described in step (1) AhLEC1a The gene sequence is shown in SEQ ID NO:1.

[0008] Further, the expression vector mentioned in step (1) is pBWA(V)BVE, induced by β-estradiol. XVE Promoter-driven expression.

[0009] Furthermore, the explant mentioned in step (3) is a peanut embryo or mesocotyl.

[0010] Furthermore, the culture described in step (3) adopts either the somatic embryo regeneration pathway or the direct organ regeneration pathway.

[0011] An expression vector for improving peanut regeneration efficiency, the expression vector comprising... AhLEC1a Genes, say AhLEC1a Genes are XVE Promoter-driven expression.

[0012] A transgenic peanut plant obtained by the above method.

[0013] The present invention has the following advantages over the prior art: This invention discovers ectopic expression AhLEC1a The gene significantly improved the somatic embryo induction rate, shoot induction rate, and genetic transformation efficiency of peanuts. In the somatic embryo induction experiment, ectopic expression... AhLEC1a The somatic embryo induction rate was significantly higher than that of the control group, with the number of somatic embryos induced from a single explant reaching 2.13 times that of the control group; the transgenic positivity rate was 27.81% higher than that of the control. In the direct organ regeneration pathway, AhLEC1a Ectopic gene expression significantly improved the bud induction efficiency of mesocotyls by 22% compared to the control group; the resistant bud screening rate increased by 6.15% compared to the control. Attached Figure Description

[0014] Figure 1 The results of phylogenetic analysis and protein sequence alignment of AhLEC1; Figure 2 This refers to the induction of somatic embryos; Figure 3 This describes the induction of mesocotyl buds. Detailed Implementation

[0015] To further explain the present invention, the following specific embodiments are described.

[0016] Example 1: Construction of Inducible Expression Vector 1.1 Extraction of total RNA from peanuts and synthesis of cDNA first strand Fresh, developing peanut seeds were quickly placed in liquid nitrogen and ground into powder using a plant tissue homogenizer. Total RNA was extracted according to the instructions of the RNAsimple Total RNA Extraction Kit, followed by agarose gel electrophoresis and concentration determination. Based on the determined RNA concentration in different tissues, appropriate amounts of RNA were taken for reverse transcription experiments according to the instructions of the reverse transcription kit.

[0017] 1.2 Gene amplification and vector construction Obtained from the genome of "Fuhua" AhLEC1a The full-length CDS sequence was obtained, and primers were designed for gene fragment amplification. Using peanut seed cDNA as a template, the gene fragment was amplified using the high-fidelity enzyme KOD FXne. The reaction program was 94℃ pre-denaturation for 2 min, followed by 98℃ denaturation for 10 s and 68℃ extension for 1 min, repeated 40 times. Detection was then performed by agarose gel electrophoresis. The target band was excised using a blue light scanning gel for purification and sent to the company for sequencing. If the alignment results were correct, the concentration was determined and stored at -20℃ for later use. AhLEC1a The gene CDS sequence (678 bp) is shown in SEQ ID NO: 1. The overexpression vector pBWA(V)BVE was digested with restriction endonucleases BasI / Eco31I according to the manufacturer's instructions. Then, the digested vector and the target gene fragment were recombined using 2 × EasyClone Mix. The resulting mixture was transformed into *E. coli* DH5α. Positive clones were selected for sequencing. Successful sequencing confirmed the vector was successfully constructed. The constructed overexpression vector was transformed into *Agrobacterium* GV3101, and colony PCR was performed for verification. After successful verification, the bacterial culture was mixed with 50% glycerol at a 1:1 ratio and stored at -80℃ for later use.

[0018] Table 1 shows the primer design table.

[0019] SEQ ID NO. 1: 5'--3' This invention obtained the full-length LEC1 protein sequences from species such as Arabidopsis thaliana, soybean, rice, and maize from public databases such as NCBI, and identified the LEC1 protein in the peanut genome through homology alignment. LEC1 Homologous genes were used, and phylogenetic analysis was performed with 1000 bootstrap iterations. The results showed that ( Figure 1 The peanut genome contains two LEC1 Homologous genes: AhLEC1a ( Ahy_A01G032050 )and AhLEC1b ( Ahy_B01G029970 The two groups converged with a high support rate of 98%, and joined forces with species such as Arabidopsis thaliana and soybean. LEC1 Genes clearly diverged, forming independent evolutionary branches. This indicates that... AhLEC1This is a species-specific sequence formed during the evolution of peanuts. Protein sequence alignment shows that AhLEC1a and AhLEC1b share a sequence similarity of up to 95.13%.

[0020] Example 2: Genetic transformation of peanuts 2.1 Preparation of culture medium Pre-medium (EM): (1) For somatic embryo regeneration pathway: 4.33 g of MS plant salt mixture (without vitamins; Phytotech M524), 1 mL of 1000 × B5 vitamin solution, 30 g of sucrose, 900 mL of ddH2O, adjust the pH to 5.6-5.8, add 3 g of Gellan gum, bring the volume to 1 L, and then place it in an autoclave and sterilize at 121℃ for 30 min. When the temperature drops to about 50℃, add 0.1% PPM (plant tissue culture antibacterial agent) in a clean bench, then unplate and store at 4℃ for later use. (2) For direct organogenesis pathway: 4.33 g of MS plant salt mixture (without vitamins), 1 mL of 1000× B5 vitamin solution, 30 g of sucrose, 900 mL of ddH2O, adjust the pH to 5.6-5.8, add 3 g of Gellan gum, bring the volume to 1 L, and then place it in an autoclave and sterilize at 121℃ for 30 min. When the temperature drops to about 50℃, add 0.1% PPM, 100 mg / L Tim (termethin) and 3 mg / L 6-BA (6-benzyladenine) in a laminar flow hood, then plate it and store it in a refrigerator at 4℃ for later use.

[0021] Liquid co-culture medium (LCCM): (1) For somatic embryo regeneration: 2.165 g of MS plant salt mixture (without vitamins), 500 μL of 1000 × B5 vitamin solution, 15 g of sucrose, add ddH2O to make up to 500 mL, adjust the pH to 5.6-5.8, then put it in an autoclave and sterilize at 121℃ for 30 min. When the temperature drops to about 50℃, add 100 μmol / L AS (acetylsalicylic acid) and 1 mmol / L DTT (dithiothreitol) in a clean bench and store at 4℃ for later use. (2) For direct organogenesis pathway: 2.165 g of MS plant salt mixture (without vitamins), 500 μL of 1000 × B5 vitamin solution, 15 g of sucrose, add ddH2O to make up to 500 mL, adjust the pH to 5.6-5.8, make up to 500 mL, then put it in an autoclave and sterilize at 121℃ for 30 min. When the temperature drops to about 50℃, add 3 mg / L 6-BA and 100 mMAS in a clean bench and store in a refrigerator at 4℃ for later use.

[0022] Co-culture medium (CCM): (1) For somatic embryo regeneration pathway: 4.33 g MS plant salt mixture (without vitamins), 1 mL 1000 × B5 vitamin solution, 30 g sucrose, 900 mL ddH2O, adjust pH to 5.6-5.8, add 3 g Gellan gum, bring volume to 1 L, then place in an autoclave, sterilize at 121℃ for 30 min, when the temperature drops to about 50℃, add 3 mg / L PIC (picostatin), 100 μmol / L AS and 1 mmol / L DTT in a clean bench, then unplate and store at 4℃ for later use. (2) For direct organogenesis pathway: 4.33 g of MS plant salt mixture (without vitamins), 1 mL of 1000× B5 vitamin solution, 30 g of sucrose, 900 mL of ddH2O, adjust the pH to 5.6-5.8, add 3 g of Gellangum, bring the volume to 1 L, and then place it in an autoclave and sterilize at 121℃ for 30 min. When the temperature drops to about 50℃, add 3 mg / L 6-BA and 100 mM AS in a clean bench, then turn the plate over and store it at 4℃ for later use.

[0023] Somatic embryo induction medium (SEIM): 4.33 g MS plant salt mixture (without vitamins), 1 mL 1000 × B5 vitamin solution, 30 g sucrose, 900 mL ddH2O added, pH adjusted to 5.6-5.8, 3 g Gellangum added, volume adjusted to 1 L, then placed in an autoclave and sterilized at 121℃ for 30 min. When the temperature dropped to about 50℃, 3 mg / L PIC, 2 mg / L Gln (glutamine), 2 mg / L AgNO3, 100 mg / L Tim and 200 mg / L Cef (cefotaxime) were added in a clean bench. The plates were then inverted and stored at 4℃ for later use.

[0024] Somatic embryo regeneration medium (SERM): 4.33 g MS plant salt mixture (vitamin-free), 1 mL 1000 × B5 vitamin solution, 30 g sucrose, 900 mL ddH2O added, pH adjusted to 5.6-5.8, 3 g Gellan gum added, volume adjusted to 1 L, then placed in an autoclave and sterilized at 121℃ for 30 min. When the temperature dropped to about 50℃, 3 mg / L 6-BA, 0.5 mg / L NAA, 100 mg / L Tim and 200 mg / L Cef were added in a clean bench, then the plates were inverted and stored at 4℃ for later use.

[0025] Rooting medium (RM): 2.165 g MS plant salt mixture (vitamin-free), 500 μL 1000× B5 vitamin solution, 15 g sucrose, 400 mL ddH2O added, pH adjusted to 5.6-5.8, 1.5 g Gellan gum added, volume adjusted to 500 mL, then placed in an autoclave and sterilized at 121℃ for 30 min. When the temperature dropped to about 50℃, 0.8 mg / L NAA, 100 mg / L Tim and 200 mg / L Cef were added in a clean bench, then the plates were inverted and stored at 4℃ for later use.

[0026] Shoot induction medium (SIM): 4.33 g MS plant salt mixture (vitamin-free), 1 mL 1000 × B5 vitamin solution, 30 g sucrose, 900 mL ddH2O added, pH adjusted to 5.6-5.8, 3 g Gellan gum added, volume adjusted to 1 L, then placed in an autoclave and sterilized at 121℃ for 30 min. When the temperature dropped to about 50℃, 3 mg / L 6-BA, 100 mg / L Tim and 200 mg / L Cef were added in a clean bench, then the mixture was inverted and stored at 4℃ for later use.

[0027] Elongation medium (ECM): 4.33 g MS plant salt mixture (vitamin-free), 1 mL 1000 × B5 vitamin solution, 30 g sucrose, 900 mL ddH2O added, pH adjusted to 5.6-5.8, 3 g Gellan gum added, volume adjusted to 1 L, then placed in an autoclave and sterilized at 121℃ for 30 min. When the temperature dropped to about 50℃, 0.5 mg / L 6-BA, 0.1 mg / L IBA, 0.5 mg / L GA3 (Gibberellic Acid), 100 mg / L Tim and 200 mg / L Cef were added in a clean bench. The plates were then inverted and stored at 4℃ for later use.

[0028] Screening medium (SM): 4.33 g MS plant salt mixture (vitamin-free), 1 mL 1000 × B5 vitamin solution, 30 g sucrose, 900 mL ddH2O added, pH adjusted to 5.6-5.8, 3 g Gellan gum added, volume adjusted to 1 L, then placed in an autoclave and sterilized at 121℃ for 30 min. When the temperature dropped to about 50℃, 0.5 mg / L 6-BA, 0.1 mg / L IBA, 0.5 mg / L GA3, 3 mg / L Basta (glufosinate), 100 mg / L Tim and 200 mg / L Cef were added in a clean bench. The plates were then unplated and stored at 4℃ for later use.

[0029] 2.2 Genetic transformation via somatic embryo regeneration pathway (1) Preparation of experimental materials: After sterilization, the embryos of mature peanut seeds were taken and pre-cultured on EM medium for 0.5-1 day.

[0030] (2) Activation of bacterial strain: The Agrobacterium tumefaciens culture of the constructed vector was taken out from -80℃ and added to LB liquid medium containing Kanamycin and Rifampicin antibiotics. The culture was carried out at 28℃ and 200 rpm for 24-48 h with shaking until the bacterial culture OD was reached. 600 It reaches 0.6-0.8.

[0031] (3) Agrobacterium infection: After centrifuging the bacterial culture, add 15 mL of LCCM and mix well to resuspend the bacteria. Then add the germ, sonicate for 1 min, and place on a shaker for 30 min. Discard the infection solution, spread the germ flat on sterile filter paper to air dry, and then place it on CCM medium and incubate in the dark at 25℃ for 2 days.

[0032] (4) Subculture and regeneration: After co-culturing for 2 days, the embryos were transferred to SEIM medium and cultured in the dark at 25°C. The embryos were changed to new SEIM medium every half month. The induced somatic embryos were subcultured for 4 months and then transferred to SERM medium. They were cultured in alternating light and dark (16 h / 8 h) for 2-3 months to induce buds. After buds were induced, they were transferred to SM medium for screening.

[0033] (5) Data statistics: The number of somatic embryos was counted one month after being transferred to SEIM medium, and the induction rate of callus was observed. The number of buds induced and the screening rate were also counted.

[0034] 2.3 Genetic transformation through the direct organ regeneration pathway (1) Preparation of experimental materials: After sterilization, the middle segment of the embryo of mature peanut seeds was taken and pre-cultured on EM medium for 0.5-1 day.

[0035] (2) Strain activation: The strain activation steps are the same as those in the somatic embryo regeneration pathway.

[0036] (3) Agrobacterium infection: the Agrobacterium infection step in the same somatic embryo regeneration pathway.

[0037] (4) Bud induction and elongation: After co-culturing for 2 days, the mid-embryo was transferred to SIM medium and cultured in the dark at 25°C for about one week. Then, it was transferred to light and dark (16 h / 8 h) alternating culture for about half a month to induce buds. After bud induction, it was transferred to ECM medium for elongation, and then transferred to SM medium for selection. Two months after selection, it was transferred to RM medium for rooting.

[0038] (5) Data statistics: The germination rate was statistically analyzed half a month after being transferred to SIM medium, and then the screening rate was statistically analyzed.

[0039] Example 3: Effect Verification 3.1 Verification of the transformation effect of somatic embryo regeneration pathway (1) Somatic embryo induction efficiency: After 30 days of somatic embryo induction culture, observation and statistical analysis using a stereomicroscope showed that... AhLEC1a The somatic embryo induction rate in the gene-induced expression treatment group was significantly higher than that in the control group, with an average induction rate approaching 100%. Figure 2 B). This indicates that AhLEC1a Gene overexpression can improve the induction efficiency of peanut somatic embryos to some extent.

[0040] (2) Average number of somatic embryogenesis: By statistically analyzing the number of somatic cells induced by a single explant, it was found that... AhLEC1a The number of somatic cells in a single explant expressing ectopic expression was approximately 2.13 times that of the control group. p < 0.0001), and the number of somatic cells that can be induced by a single explant is 6.68 ( Figure 2 C). This further confirms... AhLEC1a The role of genes in promoting somatic embryogenesis.

[0041] (3) Somatic embryo morphology: The induced somatic embryos mainly exhibited typical torpedo-shaped and spherical morphological characteristics, consistent with the morphology of normally developing somatic embryos, indicating that AhLEC1a The expression of this did not have an adverse effect on the morphology of somatic embryos.

[0042] (4) Transgenic positivity rate: The induced somatic embryos were transferred to selection medium supplemented with Basta for selection culture. GFP fluorescence signals were observed at an excitation wavelength of 488 nm, and the transgenic positivity rate was calculated. The results showed that induced expression... AhLEC1a The positive rate of somatic embryos was 53.29%, significantly higher than the 27.81% in the control group without β-estradiol supplementation. Figure 2 DE). This indicates... AhLEC1a The expression of this substance can significantly improve the efficiency of genetic transformation in peanut somatic embryos.

[0043] Figure 2In the diagram: (A) is a schematic diagram of the β-estradiol-induced expression vector; (B) and (C) show the somatic embryo induction rate and the average number of somatic embryos per explant; (D) shows the frequency statistics of transgenic positive somatic embryos; (E) shows the GFP fluorescence verification results, with white arrows indicating transgenic positive somatic embryos with GFP fluorescence signals. Data are expressed as mean ± SD (n = 3 biological replicates) and were analyzed using one-way ANOVA with GraphPad Prism 9.0 software. Significant differences were marked as follows: **** indicates... P < 0.0001 (highly significant); ** indicates 0.001 < P < 0.01 (highly significant).

[0044] 3.2 Verification of the conversion effect of the direct organ regeneration pathway Bud induction rate and number of buds per explant: After culturing in SIM medium supplemented with 3.0 mg / L 6-BA for 28 days, the hypocotyl bud induction rate and the number of buds induced per explant were statistically analyzed. The experimental results showed that... AhLEC1a The induced expression of [the substance] significantly improved the shoot induction rate, increasing it by 22% compared to the control group. p < 0.01)( Figure 3 A). Furthermore, the number of adventitious shoots induced by a single explant was significantly higher than that in the control group ( p < 0.01), the average number of buds induced per explant in the treatment group reached 6.56 ( Figure 3 B) further confirms AhLEC1a Its important role in promoting the regeneration of peanut adventitious shoots.

[0045] Figure 3 In Chinese: (A) bud induction rate; (B) number of shoot clusters induced per unit explant. Data are expressed as mean ± SD (n = 3 biological replicates) and were analyzed using one-way ANOVA with GraphPad Prism 9.0 software. Significant differences were marked as follows: ** indicates 0.001 < P < 0.01 (highly significant).

[0046] Transgenic resistant shoot selection rate: The induced clustered shoots were transferred to a selection medium supplemented with Basta for selection culture. After 60 days of selection culture, the seedling survival rate was counted and the resistant shoot selection rate was calculated. The results showed that the selection efficiency of the control group and the treatment group was low, at 25.86% and 32.01%, respectively (Table 2). The selected plants were verified by green fluorescent lamp, and it was found that the green fluorescence was mostly concentrated on the hypocotyl, and all positive plants were chimeras.

[0047] The above results indicate that ectopic expression AhLEC1a It can promote direct regeneration of peanut organs and improve genetic transformation efficiency by 6.15%.

[0048] Table 2. Screening results of positive plants using mesocotyls as explants for the direct organ regeneration pathway.

[0049] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method of expression via ectopic delivery AhLEC1a A method for improving peanut plant regeneration and genetic transformation efficiency through gene therapy, characterized in that... Includes the following steps: (1) Constructing a system containing AhLEC1a β-estradiol-induced expression vector for the gene; (2) The constructed inducible expression vector was transformed into Agrobacterium and then transformed into explants using the Agrobacterium-mediated transformation method; (3) Culture the transformed explants and induce tissue regeneration; (4) Select transgenic plants.

2. The method according to claim 1, characterized in that, The steps described in step (1) AhLEC1a The gene sequence is shown in SEQ ID NO:

1.

3. The method according to claim 1, characterized in that, The expression vector mentioned in step (1) is pBWA(V)BVE, induced by β-estradiol. XVE Promoter-driven expression.

4. The method according to claim 1, characterized in that, The explants mentioned in step (3) are peanut embryos or mesocotyls.

5. The method according to claim 1, characterized in that, The culture described in step (3) uses either the somatic cell embryo regeneration pathway or the direct organ regeneration pathway.

6. An expression vector for improving peanut regeneration efficiency, characterized in that, The expression vector contains AhLEC1a Genes, AhLEC1a Genes are XVE Promoter-driven expression.

7. A transgenic peanut plant obtained by the method of any one of claims 1-5.

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