CN116949054A - Method for improving crape myrtle leaf callus regeneration rate by over-expressing LiMYB75 gene - Google Patents

Method for improving crape myrtle leaf callus regeneration rate by over-expressing LiMYB75 gene Download PDF

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CN116949054A
CN116949054A CN202310297821.5A CN202310297821A CN116949054A CN 116949054 A CN116949054 A CN 116949054A CN 202310297821 A CN202310297821 A CN 202310297821A CN 116949054 A CN116949054 A CN 116949054A
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limyb75
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crape myrtle
callus
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CN116949054B (en
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吕芬妮
王鹏
姜聖姬
高露璐
王淑安
杨如同
李亚
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Institute of Botany of CAS
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Abstract

The invention relates to the technical field of genetic engineering application, and provides an overexpressionLiMYB75A method for improving the regeneration rate of crape myrtle leaf callus by using genes.LiMYB75The nucleotide sequence of the gene is shown as Seq 1, and the coding amino acid sequence is shown as Seq 2. The invention relates to a key gene for improving the callus regeneration rate of crape myrtle leavesLiMYB75Is a clone of (2); overexpression by Agrobacterium-mediated genetic transformationLiMYB75The gene obviously improves the regeneration rate of the crape myrtle leaf callus and the regeneration plant number of each callus, the regeneration rate is improved by 1.4 times compared with the wild type, and the regeneration plant number of each callus is increased to 4 times of the wild type. By means of over-expression of endogenous genes, to a certain extent, by improvement of the traditional methodsThe method for improving the plant regeneration rate under the exogenous tissue culture condition provides a new research thought and has important application value for crape myrtle molecular breeding.

Description

Method for improving crape myrtle leaf callus regeneration rate by over-expressing LiMYB75 gene
Technical Field
The invention belongs to the technical field of genetic engineering application, and particularly relates to a method for improving the callus regeneration rate of crape myrtle leaves by over-expressing LiMYB75 genes.
Background
Lagerstroemia (Lagerstroemia indica) is Lagerstroemia (Lagerstroemia) Lagerstroemia (Lythraceae) Lagerstroemia deciduous shrubs or small arbor, the tree is also called as a bark-free tree, is red in full, is native in China, and has a cultivation history of more than 1600 years. Banaba starts to bloom in succession in 6 months, and flowers in Xia Qiushao season are just about half a year in flowering period, so the banaba is called "century red".
The crape myrtle has rich color, long flowering phase and good tolerance, and can grow well no matter calcareous soil or acid soil; the tree is beautiful in pose, and the trunk is smooth and clean; the flower is a bonsai fine wood with flowers, stems and roots, and the praise of the flower is 'green in midsummer and eye-shading'; root, bark, leaf and flower can be used as herbs. With the development of modern gardens, crape myrtle also becomes an indispensable important landscape tree species for urban and rural public greenhouses, roads and living areas.
Like most woody plants, crape myrtle also suffers from relatively slow development of tissue culture techniques, often with low regeneration rates, browning during growth, vitrification, and the like. Due to the lack of effective callus induction and regeneration technology, the application of crape myrtle transgenic breeding, gene editing and other technologies is seriously hindered. The plant regeneration system is established by using the over-expression endogenous genes and combining with the tissue culture technology, the regeneration efficiency is improved, and the improvement of woody ornamental plant varieties and the development of molecular breeding can be greatly promoted.
Disclosure of Invention
The invention provides a method for improving the regeneration rate of crape myrtle leaf callus by over-expressing LiMYB75 gene. By using Lagerstroemia indica leaves as explants and through an agrobacterium-mediated genetic transformation method, the Lagerstroemia indica endogenous gene LiMYB75 is overexpressed, the regeneration rate of leaf callus and the number of regenerated plants of each callus are improved, and the method can greatly promote the improvement of woody ornamental plant varieties and the development of molecular breeding.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention provides a gene LiMYB75 for improving the callus regeneration rate of crape myrtle leaves, and the nucleotide sequence of the gene LiMYB75 is shown as Seq 1.
The invention also provides a coding protein of the gene LiMYB75 for improving the callus regeneration rate of the crape myrtle leaves, and the amino acid sequence of the coding protein is shown as Seq 2.
A plant expression vector pBI121-LiMYB75 containing a key gene LiMYB75 for improving the leaf callus regeneration rate of crape myrtle, wherein the LiMYB75 gene is started by a double CaMV35S promoter, and a green fluorescent protein eGFP reporter gene is fused at the 3' end of the LiMYB75 gene. The invention provides application of an endogenous gene LiMYB75 in improving the callus regeneration rate of crape myrtle leaves.
The specific application comprises the following steps: cloning to obtain LiMYB75 gene; recombining the gene LiMYB75 into a plant expression vector by using Gateway technology to obtain a plant over-expression vector of the LiMYB75 gene; the over-expression vector of the gene LiMYB75 is transformed into crape myrtle leaves by adopting an agrobacterium-mediated method, so that the leaf callus regeneration rate and the number of each callus regeneration plant are improved.
Compared with herbaceous plants, the tissue culture work of the woody plants is relatively slow, the invention improves the callus regeneration capacity of the banaba by a molecular means, and is an important technical means for promoting the variety improvement and molecular breeding development of woody ornamental plants.
The expression of the gene LiMYB75 is related to the regeneration capacity of the crape myrtle, and the callus regeneration capacity of the crape myrtle is obviously enhanced after the gene is overexpressed, and the specific expression is that the callus regeneration rate and the number of each callus regeneration plant are obviously improved; by applying the gene, a new research idea can be provided for improving the crape myrtle callus regeneration rate.
Drawings
FIG. 1 cloning of the Lagerstroemia speciosa LiMYB75 gene.
FIG. 2. Fluorescence detection of LiMYB75 transgenic regenerated plants and PCR detection of the gene of interest. a, eGFP fluorescence detection; b and c, target gene PCR detection. WT, receptor control; m, marker; p, plasmid positive control; 1-14, randomly selected transgenic regenerated plants.
FIG. 3 shows that the over-expression of LiMYB75 gene effectively improves the leaf callus regeneration rate of crape myrtle. a, transferring the callus induction condition of no-load; b, callus induction of over-expression LiMYB75 gene; c, a and b; d, transferring the no-load callus regeneration condition; e, callus regeneration condition of over-expression LiMYB75 gene; f, d and e; the number of regenerated plants per callus piece in g, d and e was counted.
Detailed Description
The invention is further illustrated below in conjunction with specific embodiments. It should be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The molecular biology-related kits and the like used in the embodiments are commercially available. The method provided by the embodiment of the invention is conventional operation in experiments.
Example 1 cloning of the Lagerstroemia speciosa LiMYB75 Gene
1. Extraction of Lagerstroemia indica petal total RNA and cDNA inversion
Fresh petals of crape myrtle are collected, after quick freezing by liquid nitrogen, total RNA is extracted by using an RNA extraction kit FastPure Plant Total RNA Isolation Kit (Nuo Wei Zan, RC 401), and the operation steps are carried out according to the instruction book of the kit. Total RNA was quantified using One Drop instrument and integrity detected by 1% agarose gel electrophoresis.
The cDNA was synthesized according to the instructions of the kit HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wind) (Northenan, R312) and the reverse transcription product was stored at-20 ℃.
LiMYB75 gene clone and sequencing
Primers were designed on the 5 'and 3' non-coding regions of the LiMYB75 gene according to the genomic information of Lagerstroemia indica (MYB 75-F:5'-TGGAAGGCTGGATGAGAG-3'; MYB75-R: 5'-TGGGATACGGAACAGGGAC-3'), and the LiMYB75 gene was cloned by PCR.
PCR amplification was performed using high fidelity enzymes (Norflu, P505).
PCR reaction system: 2X Phanta Max Buffer, 25. Mu.L; dNTP Mix (10 mM each), 1. Mu.L; 100ng/L cDNA, 2. Mu.L; f (10. Mu.M), 2. Mu.L; r (10. Mu.M), 2. Mu.L; phanta Max Super-Fidelity DNA Polymerase,1 μl; ddH 2 O，up to 50μL。
PCR amplification procedure: pre-denaturation at 95℃for 3min, denaturation at 95℃for 15s, annealing at 56℃for 15s, extension at 72℃for 1min,35 cycles, and complete extension at 72℃for 10min.
After completion of the reaction, the target fragment was excised and the PCR product was recovered by agarose gel DNA recovery kit according to the instructions after detection by 1% agarose gel electrophoresis. Was ligated into the pMD19-T vector (Takara, D102A) and the procedure was performed according to the instructions. The ligation product was transferred into DH 5. Alpha. E.coli competent cells.
E.coli transformation:
(a) Adding 5 mu L of plasmid into 100 mu L of competent cells of Escherichia coli, ice-bathing for 25min, performing water bath heat shock for 45s at 42 ℃, and rapidly cooling on ice for 1-2min;
(b) 800. Mu.L of blank LB liquid medium is added, and resuscitated at 37 ℃ and 200rpm for 1h;
(c) Centrifuging at 5000rpm for 1min, discarding 700 μl of supernatant, mixing the rest culture medium and thallus, sucking 100 μl of the mixture into LB solid culture medium containing ampicillin Amp (50 mg/L), and culturing at 37deg.C in an inverted manner until colony is grown.
(d) Single colonies were picked from the plates and incubated overnight on a shaker at 37℃in 1mL of liquid LB plus antibiotics.
PCR detection of E.coli transformants:
and (3) performing PCR amplification by taking the cultured escherichia coli as a template. The primer is as follows: MYB75-F/R.
The specific operation is as follows:
PCR reaction system: 2 XTaq PCR Mix, 10. Mu.L; f (10. Mu.M), 1. Mu.L; r (10. Mu.M), 1. Mu.L; bacterial liquid, 1 mu L; ddH 2 O，up to 20μL。
PCR amplification procedure: pre-denaturation at 94℃for 3min, denaturation at 94℃for 30s, annealing at 56℃for 1min, extension at 72℃for 1min,35 cycles, total extension at 72℃for 10min.
Clones positive to PCR amplification were sequenced (Nanjing Sipu gold Biotechnology Co., ltd.) and the correct sequence of the monoclonal bacteria were shaken and then mixed with 50% glycerol 7:3 and stored in a-80℃freezer for further use. Sequencing analysis shows that the sequence of the coding region of the gene is shown as Seq 1, the ORF is 1962bp (comprising a stop codon), the coding amino acid sequence is shown as Seq 2, and 653 amino acids are coded.
Example 2 construction of plant overexpression vector of Lagerstroemia indica LiMYB75 Gene
According to the sequence information of LiMYB75 gene and vector pBI121-eGFP, a primer 5 was used to design a specific cloning primer with cleavage site (MYB 75-eGFP-F:5'-ATTTACGAACGATAGGGTACCATGAAAGATGGTGTTTTAAA-3'; MYB75-eGFP-R:
5'-GCCCTTGCTCACCATGGATCCGTCTATCTCAGGCAGGAAAT-3') and amplifying the target fragment by PCR using the pMD19-T-LiMYB75 vector plasmid which is successfully sequenced as a template. The target gene fragment is inserted into Kpn I and BamH I sites of pBI121-eGFP vector by utilizing recombination method to obtain 2X 35S: liMYB75: eGFP vector.
The plant expression vector containing the target gene is introduced into agrobacterium competent EHA105 by freeze thawing. The method comprises the following specific steps: mu.L of 2X 35S:: liMYB75: eGFP vector plasmid was added to 100. Mu.L of Agrobacterium EHA105 competent cells; quick-freezing with liquid nitrogen for 5min, and water-bathing at 37 ℃ for 5min; adding 800 mu L of LB liquid medium into an ultra-clean workbench, and shaking by a shaking table at 28 ℃ and 200rpm for 4 hours; after centrifugation at 5000rpm for 1min at room temperature, 700. Mu.L of the supernatant was removed, and the remaining cells and supernatant were resuspended by pipetting and then spread evenly on LB solid medium containing kanamycin Kana (100 mg/L) +rifampicin Rif (50 mg/L), and the culture was inverted at 28℃for 48-72 h until monoclonal colonies were grown. Selecting monoclonal, shaking, mixing with 50% glycerol at a ratio of 1:1, and storing in a refrigerator at-80deg.C.
Embodiment 3 overexpression of LiMYB75 Gene improves regeneration Rate of Lagerstroemia indica callus
Genetic transformation of the limyb75 gene:
the plant expression vector constructed in example 2 was used for subsequent genetic transformation of crape myrtle leaves.
(a) Preparation of an infectious microbe liquid: the preserved agro-pole containing LiMYB75 plant over-expression vectorThe strain was streaked on LB plate containing Kana (100 mg/L) +Rif (50 mg/L), and was selected and cultured by shaking in 1mL of LB liquid medium containing Kana (100 mg/L) +Rif (50 mg/L) at 28℃for 24 hours with shaking. The agrobacteria solution was then inoculated into 100mL of LB liquid medium containing Kana (100 mg/L) +Rif (50 mg/L) +acetosyringone AS (200. Mu. Mol/L) at a ratio of 1:100 and cultured overnight. Detecting the OD of the bacterial liquid cultured overnight by using an ultraviolet spectrophotometer 600 Value of (d), OD 600 Between 0.8 and 1.0. And collecting thalli, re-suspending the thalli, and placing the thalli in a dark place for 2-3 hours.
And (3) heavy suspension: 10mmol/L ethanesulfonic acid (MES) +10mmol/L MgCl 2 +200μmol/L AS，pH＝6.0。
(b) Genetic transformation of crape myrtle leaves by agrobacterium-mediated method: the LiMYB75 gene is introduced into the genome of the lagerstroemia indica by using the inverted 3-inverted 4 leaves of the tissue-cultured lagerstroemia indica seedling close to the terminal buds as explants through an agrobacterium-mediated method.
(c) Obtaining and screening transgenic plants: after the transformed crape myrtle regenerated plant is obtained, performing eGFP positive preliminary screening on leaves of the transformed plant by using a fluorescence imaging instrument, extracting DNA of the leaves of the transformed plant by using a kit, detecting a reporter gene eGFP by using PCR (polymerase chain reaction), detecting eGFP specific primers (eGFP-F: 5'-AGTGCTTCAGCCGCTACCCC-3' and eGFP-R: 5'-CCATGCCGAGAGTGATCCCG-3'), and detecting a target band of the PCR product by agarose gel electrophoresis, thus obtaining the success of transformation.
PCR reaction system: 2 XTaq PCR Mix, 10. Mu.L; f (10. Mu.M), 0.5. Mu.L; r (10. Mu.M), 0.5. Mu.L; DNA, 1. Mu.L; ddH 2 O，up to 20μL。
PCR amplification procedure: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 2min,35 cycles, total extension at 72℃for 10min.
As shown in FIG. 2, the combination of the fluorescence detection result and the PCR detection result proves that LiMYB75-eGFP is successfully integrated into the genome of the crape myrtle, and the transgenic positive plant 90 is obtained after detection.
Phenotypic characterization of LiMYB75 transgenic Lagerstroemia speciosa
As shown in fig. 3, there was no significant statistical difference between the transgenic crape myrtle leaf callus induction rate of the no-load control and the transgenic crape myrtle leaf callus induction rate of the LiMYB75 gene overexpression; the number of regenerated plants of each callus of the banaba leaves without load is 1-2, and after the LiMYB75 gene is over-expressed, the number of regenerated plants of each callus reaches 8-9, the regeneration rate is improved by 1.4 times compared with that of the wild type, and the number of regenerated plants of each callus is increased by 4 times compared with that of the wild type. Experimental results show that the regeneration capacity of the crape myrtle leaf callus is obviously improved by over-expressing the LiMYB75 gene.
In conclusion, the invention overexpresses the endogenous gene LiMYB75 gene in the crape myrtle, improves the regeneration capacity of the callus, and can effectively promote the improvement of woody ornamental plant varieties and the development of molecular breeding.
The foregoing embodiments are merely preferred embodiments of the present invention, which are intended to be illustrative of the present invention and not to limit the scope of the present invention, and other embodiments can be easily made by those skilled in the art based on the technical disclosure in the present specification by substitution or modification, so that all changes and modifications made in the principles of the present invention shall be included in the scope of the present invention.

Claims (4)

1. Over-expressionLiMYB75A method for improving the regeneration rate of crape myrtle leaf callus by using genes is characterized in that,LiMYB75the nucleotide sequence of the gene is shown as Seq 1.
2. An overexpression according to claim 1LiMYB75A method for improving the regeneration rate of crape myrtle leaf callus by using genes is characterized in that,LiMYB75the gene coding amino acid sequence is shown as Seq 2.
3. An overexpression according to claim 1 or 2LiMYB75The method for improving the regeneration rate of the crape myrtle leaf callus by the gene is characterized by being used for improving the regeneration rate of the crape myrtle leaf callus.
4. An overexpression according to claim 1 or 2LiMYB75A method for improving the regeneration rate of crape myrtle leaf callus by gene is characterized by usingAnd improving the regeneration plant number of the crape myrtle leaf callus blocks.
CN202310297821.5A 2023-03-24 2023-03-24 Method for improving crape myrtle leaf callus regeneration rate by over-expressing LiMYB gene Active CN116949054B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110305894A (en) * 2019-06-29 2019-10-08 江苏省中国科学院植物研究所 A kind of Chinese catalpa genetic transforming method rapidly and efficiently
CN112493126A (en) * 2020-11-23 2021-03-16 河北科技师范学院 Method for induction of lagerstroemia indica somatic embryo and plant regeneration

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110305894A (en) * 2019-06-29 2019-10-08 江苏省中国科学院植物研究所 A kind of Chinese catalpa genetic transforming method rapidly and efficiently
CN112493126A (en) * 2020-11-23 2021-03-16 河北科技师范学院 Method for induction of lagerstroemia indica somatic embryo and plant regeneration

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