Method for improving inductivity and genetic transformation efficiency of hairy roots of toona ciliata

Abstract

The invention discloses a method for improving the inductivity and genetic transformation efficiency of hairy roots of toona ciliata. According to the method, agrobacterium rhizogenes carrying RUBY-TcGRF8-GIF2 chimeric protein are injected into stems and petioles of aseptic seedlings of the toona ciliata, and the TcGRF8-GIF2 chimeric gene is over-expressed, so that the induction efficiency and the genetic transformation efficiency of the toona ciliata hairy roots are remarkably improved, and the transformation period of the toona ciliata hairy roots is remarkably shortened. Specifically, after overexpression of the TcGRF8-GIF2 gene, the induction time of the hairy roots is shortened from 70 days of a RUBY-XbaI no-load plasmid control group to 25 days, the induction rate and conversion rate of the hairy roots mediated by agrobacterium tumefaciens are improved, the operation efficiency of genetic transformation of the toona ciliata is effectively improved, and the method has important significance for promoting genetic improvement of the toona ciliata.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a method for improving the induction rate and genetic transformation efficiency of hairy roots of Toona sinensis. Background Technology

[0002] It belongs to the genus Toona sinensis in the family Meliaceae. Toona Red Toona sinensis ( ) Toona ciliata Toona sinensis (red cedar) is a high-quality, fast-growing timber species in my country with significant economic and ecological value. Currently, its natural resources are relatively scarce, its natural regeneration and reproductive capacity are limited, and it is severely affected by human interference and destruction. Due to over-logging and habitat destruction, Toona sinensis resources are dwindling and on the verge of extinction. If effective protection and management measures are not implemented in a timely manner, the natural population of Toona sinensis will face an extremely severe survival crisis, and may even face extinction. Currently, Toona sinensis is an endangered species, listed as a Class II protected wild plant in China, and included in the "Reference List of Major Cultivated Precious Tree Species in China." Therefore, genetic improvement of Toona sinensis using genetic engineering technology is crucial for cultivating varieties with superior traits and promoting its large-scale application and sustainable development.

[0003] However, as a woody plant, Toona sinensis has inherent biological limitations such as a long growth cycle and poor internal regeneration capacity. In the process of in vitro tissue culture, it is generally constrained by problems such as endophytic bacteria contamination and explant browning, which makes it difficult to establish its genetic transformation system and the transformation efficiency extremely low. This seriously hinders the gene function research and molecular breeding process of Toona sinensis.

[0004] The GRF (Growth-Regulating Factor) protein binds to its transcriptional cofactor GIF (GRF-Interacting Factor) to form a functional transcriptional complex that endows cells with the potential to proliferate and form meristems during organ development. The activity of GRF-GIF requires regulation by the chromatin remodeling complex SWI / SNF (Switch / Sucrosene Onfermenting), which enables cell redifferentiation during cell development. Summary of the Invention

[0005] Based on the shortcomings and deficiencies of existing technologies, this invention aims to provide a method for improving the induction rate and genetic transformation efficiency of hairy roots in Toona sinensis. Specifically, by constructing a TcGRF-GIF chimeric protein complex, its application in the genetic transformation system of Toona sinensis is explored. By promoting cell proliferation and inducing stem cells to differentiate into transitional amplified cells, the overall efficiency of the genetic transformation system of Toona sinensis is improved. The fundamental goal is to establish an efficient and stable genetic transformation technology platform for Toona sinensis, directly serving gene function research and precise gene editing breeding of this tree species.

[0006] The first object of the present invention is to provide TcGRF8-GIF2 The chimeric gene has the nucleotide sequence shown in SEQ ID NO. 17.

[0007] The second object of the present invention is to provide the aforementioned TcGRF8-GIF2 Application of chimeric genes in improving the efficiency of the genetic transformation system of hairy roots of Toona sinensis.

[0008] Preferably, the application is overexpression. TcGRF8-GIF2 Application of chimeric genes in improving the induction rate and genetic transformation efficiency of hairy roots of Toona sinensis.

[0009] The third object of the present invention is to provide the aforementioned TcGRF8-GIF2 Application of chimeric genes in the preparation of products that improve the efficiency of the genetic transformation system for hairy roots of Toona sinensis.

[0010] The fourth object of the present invention is to provide a method for utilizing the aforementioned TcGRF8-GIF2 Products prepared by chimeric gene synthesis that improve the efficiency of the genetic transformation system for hairy roots of Toona sinensis, wherein the products are one or more of the following (1) to (2): (1) Containing the aforementioned TcGRF8-GIF2 Recombinant expression vectors for chimeric genes; (2) Contains the aforementioned TcGRF8-GIF2 Genetically engineered bacteria with chimeric genes.

[0011] The fifth objective of this invention is to provide a method for improving the induction rate and genetic transformation rate of hairy roots of Toona sinensis, including overexpression of the aforementioned method. TcGRF8-GIF2 The steps involved in chimeric gene synthesis.

[0012] Preferably, the method includes the following steps: TcGRF8-GIF2 Chimeric genes were cloned into overexpression vectors to achieve overexpression. TcGRF8-GIF2 Recombinant vectors of chimeric genes will overexpress... TcGRF8-GIF2 After the chimeric gene recombinant vector was transferred into competent Agrobacterium rhizogenes cells, it was transfected into the stem segments or petioles of Toona sinensis seedlings.

[0013] Preferably, the overexpression vector is the RUBY-XbaⅠ vector, which is obtained by linearizing the 35S:RUBY vector through HindⅢ restriction site and then adding a 35S promoter-XbaⅠ-NOS terminator expression cassette.

[0014] Preferably, the Agrobacterium rhizogenes is Ar Qual.

[0015] Preferably, the *Toona sinensis* seedlings are 3-month-old sterile *Toona sinensis* seedlings.

[0016] The beneficial effects of this invention are: This invention will TcGRF8 as well as TcGIF2 The nucleotide sequence of the vector was inserted into the RUBY-XbaⅠ vector to construct the RUBY-TcGRF8-GIF2 vector. Through fusion expression of these two vectors and their interaction, the genetic transformation efficiency of *Toona sinensis* hairy roots was improved, and the transformation cycle was significantly shortened, making the genetic transformation system of *Toona sinensis* more efficient. Specifically, the TcGRF8-GIF2 chimeric protein significantly shortened the time for hairy root emergence in *Toona sinensis*. RUBY-XbaⅠ induced hairy root production in approximately 70 days, while overexpression of TcGRF8-GIF2 reduced the induction time to approximately 25 days. This indicates that the TcGRF8-GIF2 chimeric protein has a significant effect on the genetic transformation efficiency of *Toona sinensis* and is of great importance for promoting genetic improvement of *Toona sinensis*. Attached Figure Description

[0017] Figure 1 It relates to the functions of Arabidopsis thaliana (At), wheat (Ta), and poplar (Pt). GRF And red camellia (Tc) GRF The phylogenetic tree of a family.

[0018] Figure 2 It relates to the functions of Arabidopsis thaliana (At), wheat (Ta), and poplar (Pt). GIF And red camellia (Tc) GIF The phylogenetic tree of a family.

[0019] Figure 3 This is a schematic diagram of the RUBY-TcGRF8-GIF2 recombinant plasmid.

[0020] Figure 4 It is the empty vector plasmid RUBY-XbaⅠ and the overexpression of RUBY- TcGRF8-GIF2 A comparison of the induction rate and positive rate of hairy roots of Toona sinensis var. mongolica induced by recombinant vector plasmid.

[0021] Figure 5 This is an example diagram of positive hairy roots. Detailed Implementation

[0022] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0023] Example 1: TcGRF-GIF Construction of expression vectors (1) Primer design and synthesis according to Figure 1 and Figure 2 Phylogenetic trees, selecting those with high homology TcGRF8 , TcGRF9 and TcGIF1, TcGIF2 Combine them in pairs to construct different TcGRF-GIF Expressive vehicle.

[0024] Based on the target gene downloaded from the database TcGRF8 , TcGRF9 and TcGIF1 , TcGIF2 The nucleotide sequences are shown in SEQ ID NO.1-SEQ ID NO.4, respectively, encoding proteins with amino acid sequences shown in SEQ ID NO.5-SEQ ID NO.8. Cloning primers were designed using Snapgene software, and after adding the required fragment ligation or the homologous arms required for vector ligation, they were sent to a branch of Qingke Biotechnology Co., Ltd. for synthesis. The obtained primers are shown in Table 1. Gene sequences with homologous arms of the RUBY-XbaⅠ vector were cloned based on the primers, and the sequences were then removed. TcGRF8 and TcGRF9 The stop codon is inserted, and a sequence of four alanine residues (5'-GCGGCCGCTGCC-3') is inserted as a linker to form a chimeric sequence. TcGRF8-GIF1 , TcGRF8-GIF2 , TcGRF9-GIF1 , TcGRF9-GIF2 .

[0025] Table 1 Cloning Primer Information (2) Amplification TcGRF-GIF Chimeric genes. The reaction system shown in Table 2 was prepared in sterilized PCR reaction tubes using 2×XKL-Super mix (purchased from Guangzhou Xinkailai Enzyme Biotechnology Co., Ltd., catalog number XKL0201).

[0026] Table 2 PCR reaction system After preparing the reaction system as shown in Table 2, the reaction tubes were briefly centrifuged to concentrate the reaction solution at the bottom. Then, the reaction tubes were placed in a PCR instrument to carry out the amplification reaction, following the PCR reaction procedure outlined in Table 3.

[0027] Table 3 PCR reaction procedure After the PCR reaction is complete, a small amount of the PCR product is taken for electrophoresis to detect the amplification results. If the band size after electrophoresis is consistent with the expected size, the amplification is successful.

[0028] The PCR products were purified and recovered using the universal OMEGA purification and recovery kit (D250002000D28W013). The recovered products can be stored at -20°C for use when needed.

[0029] (3) Carrier modification After digesting the HindIII restriction site of the commercial plasmid 35S:RUBY, linearizing it, and then adding the 35S promoter-XbaⅠ (restriction site)-NOS terminator expression cassette to modify it into plasmid RUBY-XbaⅠ.

[0030] (4) Carrier linearization and linkage reaction Based on the XbaI restriction site selected during primer design, the vector RUBY-XbaI was linearized by XbaI enzyme digestion. Homologous recombination was then used to recombine different... TcGRF-GIF Chimeric genes were ligated into the vector RUBY-XbaⅠ, enabling 35S to drive different... TcGRF-GIF Chimeric genes were combined to obtain different recombinant vectors, which were named according to their combinations. RUBY- TcGRF8-GIF1 , RUBY-TcGRF8-GIF2 , RUBY-TcGRF9-GIF1 and RUBY-TcGRF9-GIF2 .

[0031] Will TcGRF-GIF The specific steps for ligating the chimeric gene into the vector RUBY-XbaⅠ are as follows: Prepare the enzyme digestion system according to Table 4 and incubate it in a 37℃ metal bath for 15 min. Detect the digestion effect by electrophoresis. If the electrophoresis results are as expected, perform gel extraction and recover the digested products. Then, use a homologous recombinase (specifically, XKL-Gib seamless cloning enzyme Super Fusion Cloning Mix 2×, purchased from Guangzhou Xinkailai Enzyme Biotechnology Co., Ltd., catalog number XKL0612) to digest the obtained homologous arms. TcGRF-GIF The chimeric gene was ligated into the linearized RUBY-XbaⅠ vector, and the enzyme digestion system was prepared according to Table 5. The mixture was then placed in a 50℃ water bath for 45 min. After processing, different recombinant vectors were obtained. RUBY- TcGRF-GIF .

[0032] Table 4 Enzyme digestion reaction system Table 5 Homologous recombination reaction system The obtained recombinant vector was transformed into competent Escherichia coli DH5α cells using a heat shock method. Different cells with correct sequences were obtained through bacterial culture PCR and plasmid extraction sequencing.RUBY-TcGRF-GIF Recombinant vector: RUBY-TcGRF8-GIF1 , RUBY-TcGRF8-GIF2 , RUBY-TcGRF9-GIF1 , RUBY-TcGRF9-GIF2 .

[0033] Example 2: Agrobacterium-mediated induction and transformation of hairy roots of Toona sinensis 1. Obtain expression RUBY-TcGRF-GIF Agrobacterium strains with recombinant vectors will contain RUBY-TcGRF-GIF Recombinant vector ( RUBY-TcGRF8-GIF1 , RUBY-TcGRF8-GIF2 , RUBY- TcGRF9-GIF1 , RUBY-TcGRF9-GIF2 Agrobacterium tumefaciens ArQual was transformed with the empty vector RUBY-XbaⅠ using the heat shock method. After incubation at 28°C for 2 days, positive clones were obtained by colony PCR identification. The positive clones were inoculated into 3 mL of LB liquid medium (containing 100 mg / L spectinomycin) and incubated at 28°C for 1 day. The bacterial culture was then aliquoted into 1.5 mL centrifuge tubes and mixed with 50% glycerol at a volume ratio of 1:1, and stored in an ultra-low temperature (-80°C) freezer.

[0034] 2. Procedures for Agrobacterium rhizogenes-mediated genetic transformation (1) One day before infection, 20 μL of Ar Qual glycerol bacteria containing different recombinant vectors RUBY-TcGRF-GIF were added to 3 mL of LB liquid medium containing 100 mg / L spectinomycin and cultured overnight at 28°C with shaking speed of 220 rpm until the bacterial culture reached OD. 600 =0.6, the obtained bacterial solution was centrifuged at 8000 rpm for 3 min, the supernatant was discarded, and the enriched bacterial cells were resuspended in 3 mL of 1 / 2 MS liquid medium containing 100 μM acetylsyl syringone. The culture was carried out at 28℃ in the dark and at a shaking speed of 220 rpm for 2 h to prepare the infection solution.

[0035] (2) Prepare 3-month-old aseptic seedlings of Toona sinensis and a 1 mL syringe. Inject the aseptic seedlings of Toona sinensis into the sterile solution in a clean bench. Use a 1 mL syringe to draw 300 μL of the infection solution and inject it into the petiole and stem segments of the seedlings. One aseptic seedling of Toona sinensis can be injected 8 to 10 times.

[0036] (3) After the infected Toona sinensis seedlings were cultured in the dark for 3 days, they were transferred to a temperature of 25±2℃, a light intensity of 2500 lx, and a light / dark time of 16 h / 8 h. After 20 days, the number of explants that induced hairy roots was counted. The positive hairy roots of the target gene were red. Figure 5The total number of explants with positive hairy roots induced was counted to calculate the hairy root induction rate and the hairy root positivity rate.

[0037] The calculation formula is as follows: Hairy root plant induction rate (%) = (Number of hairy root plants / Total number of infected plants) × 100; Positive rate of hairy-rooted plants (%) = (Number of positive hairy-rooted plants / Number of plants with hairy roots) × 100.

[0038] Based on the calculation results, the effects of different TcGRF-GIF chimeric proteins on the conversion efficiency of Toona sinensis were initially obtained.

[0039] Table 6. Initial screening results of GRF-GIF chimeric protein combinations from *Toona sinensis*. As shown in Table 6, the chimeric protein composed of TcGRF8 and TcGIF2 induced more hairy roots, with an induction rate as high as 24.44% and a positive rate of 27.2%, significantly better than the other three combinations. Therefore, the TcGRF8-GIF2 chimeric protein was selected for constructing the genetic transformation system of Toona sinensis. RUBY-TcGRF8-GIF2 carrier RUBY-TcGRF8-GIF2 The structure of the carrier is as follows Figure 3 As shown, where TcGRF8-GIF2 The chimeric nucleotide sequence is shown in SEQ ID NO. 17.

[0040] Example 3: Validation of the *Toona sinensis* genetic transformation system constructed using the TcGRF8-GIF2 chimeric protein Referring to "2. Steps for Agrobacterium rhizogenes-mediated genetic transformation" in Example 2, using... RUBY-TcGRF8- GIF2 The genetic transformation system of *Agrobacterium rhizogenes* transformed by the recombinant vector was used to verify the genetic transformation of *Toona sinensis* constructed using the TcGRF8-GIF2 chimeric protein, with *Agrobacterium rhizogenes* transformed by the RUBY-XbaⅠ vector without the addition of TcGRF8-GIF2 as a control.

[0041] Table 7. Transformation of hairy roots of *Toona sinensis* in the RUBY-XbaⅠ vector without the addition of TcGRF8-GIF2. Note: Different serial numbers indicate different experimental replicates.

[0042] Table 8 RUBY-TcGRF8-GIF2 Transformation of the hairy roots of the vector *Toona sinensis* Note: Different serial numbers indicate different experimental replicates.

[0043] In overexpressionTcGRF8-GIF2 (i.e., utilizing) RUBY-TcGRF8-GIF2 When Agrobacterium rhizogenes transformed with the recombinant vector infected Toona sinensis, the chimeric gene was found to significantly shorten the time for hairy root emergence in Toona sinensis and simultaneously increase the hairy root induction rate. Figure 4 Compared with the results in Table 7, where Agrobacterium rhizogenes transformed with the RUBY-XbaⅠ vector induced hairy root production in approximately 70 days, with a hairy root induction rate of 11.2% and a hairy root positivity rate of 8.1%, the transformation... RUBY-TcGRF8-GIF2 Agrobacterium rhizogenes overexpression TcGRF8-GIF2 The time for inducing hairy roots was shortened to about 25 days, the hairy root induction rate increased to 28.56%, and the hairy root positive rate increased to 25.16% (Table 8). This indicates that the TcGRF8-GIF2 chimeric protein has a significant effect on improving the genetic transformation efficiency of Toona sinensis.

Claims

1. TcGRF8-GIF2 Chimeric genes, characterized by, Its nucleotide sequence is shown in SEQ ID NO.

17.

2. The claim 1 TcGRF8-GIF2 Application of chimeric genes in improving the efficiency of the genetic transformation system of hairy roots of Toona sinensis.

3. The application according to claim 2, characterized in that, For overexpression TcGRF8-GIF2 Application of chimeric genes in improving the induction rate and genetic transformation efficiency of hairy roots of Toona sinensis.

4. The claim 1 TcGRF8-GIF2 Application of chimeric genes in the preparation of products that improve the efficiency of the genetic transformation system for hairy roots of Toona sinensis.

5. Using the method described in claim 1 TcGRF8-GIF2 A product prepared using chimeric genes to improve the efficiency of the genetic transformation system for the hairy roots of *Toona sinensis* is characterized by, The product is one or more of the following (1) to (2): (1) Containing the aforementioned TcGRF8-GIF2 Recombinant expression vectors for chimeric genes; (2) Contains the aforementioned TcGRF8-GIF2 Genetically engineered bacteria with chimeric genes.

6. A method for improving the induction rate and genetic transformation rate of hairy roots of Toona sinensis, characterized in that, Including the overexpression described TcGRF8-GIF2 The steps involved in chimeric gene synthesis.

7. The method according to claim 6, characterized in that, Includes the following steps: Will TcGRF8-GIF2 Chimeric genes were cloned into overexpression vectors to achieve overexpression. TcGRF8-GIF2 Recombinant vectors of chimeric genes will overexpress... TcGRF8-GIF2 After the chimeric gene recombinant vector was transferred into competent Agrobacterium rhizogenes cells, it was transfected into the stem segments or petioles of Toona sinensis seedlings.

8. The method according to claim 7, characterized in that, The overexpression vector is the RUBY-XbaⅠ vector, which is obtained by linearizing the 35S:RUBY vector through HindⅢ restriction site and then adding the 35S promoter-XbaⅠ-NOS terminator expression cassette.

9. The method according to claim 7, characterized in that, The Agrobacterium rhizogenes mentioned is Ar Qual.

10. The method according to claim 7, characterized in that, The aforementioned Toona sinensis seedlings are 3-month-old sterile Toona sinensis seedlings.

Images