CN116949054B - Method for improving crape myrtle leaf callus regeneration rate by over-expressing LiMYB gene - Google Patents

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

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CN116949054B
CN116949054B CN202310297821.5A CN202310297821A CN116949054B CN 116949054 B CN116949054 B CN 116949054B CN 202310297821 A CN202310297821 A CN 202310297821A CN 116949054 B CN116949054 B CN 116949054B
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CN116949054A (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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Abstract

The invention relates to the technical field of genetic engineering application, and provides a method for improving the regeneration rate of crape myrtle leaf callus by over-expressing LiMYB genes. The nucleotide sequence of LiMYB gene is as described in Seq 1 and the coding amino acid sequence is as described in Seq 2. The invention relates to cloning of a key gene LiMYB for improving the callus regeneration rate of crape myrtle leaves; through agrobacterium-mediated genetic transformation method over-expression LiMYB genes, the regeneration rate of crape myrtle leaf calli and the regeneration plant number of each calli are obviously improved, the regeneration rate is improved by 1.4 times compared with that of a wild type, and the regeneration plant number of each calli is increased to 4 times of that of the wild type. By means of over-expression of endogenous genes, a new research thought is provided for the traditional method for improving plant regeneration rate by improving exogenous tissue culture conditions to a certain extent, and the method has important application value for crape myrtle molecular breeding.

Description

Method for improving crape myrtle leaf callus regeneration rate by over-expressing LiMYB gene
Technical Field
The invention belongs to the technical field of genetic engineering application, and particularly relates to a method for improving the regeneration rate of crape myrtle leaf callus by over-expressing LiMYB genes.
Background
Lagerstroemia speciosa (Lagerstroemia indica) is Lagerstroemia (LYTHRACEAE) Lagerstroemia (Lagerstroemia) Lagerstroemia of Lythraceae, also called bark-less tree, full-red, native to China, and has been cultivated for over 1600 years. Crape myrtle starts to bloom in succession in 6 months, flowers in Xia Qiushao season are right, and the flowering period is about half a year, so the crape myrtle is called as '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; also has ' midsummer green and eye shade ', praise of the flower is full of the hall ', is a bonsai fine wood for viewing flowers, stems and roots; 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 LiMYB genes. By using crape myrtle leaves as explants and through an agrobacterium-mediated genetic transformation method, the crape myrtle endogenous gene LiMYB 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 LiMYB for improving the callus regeneration rate of crape myrtle leaves, and the nucleotide sequence of the gene LiMYB is shown as Seq 1.
The invention also provides a coding protein of the gene LiMYB 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.
The gene pBI121-LiMYB, liMYB of the plant expression vector containing a key gene LiMYB for improving the regeneration rate of the leaf callus of the crape myrtle 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 LiMYB in improving the callus regeneration rate of crape myrtle leaves.
The specific application comprises the following steps: cloning to obtain LiMYB gene; recombining the gene LiMYB into a plant expression vector by using Gateway technology to obtain a plant over-expression vector of LiMYB75 gene; the over-expression vector of the gene LiMYB is transformed into the 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 LiMYB disclosed by the invention 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.
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FIG. 1 cloning of the Banaba LiMYB 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 gene LiMYB is over expressed to effectively improve the regeneration rate of the crape myrtle leaf callus. a, transferring the callus induction condition of no-load; b, callus induction of over-expression LiMYB genes; c, a and b; d, transferring the no-load callus regeneration condition; e, callus regeneration condition of over-expression LiMYB 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 Banaba LiMYB 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 kit HISCRIPT III, 1, st Strand cDNA Synthesis Kit (+ GDNA WIPER) (Northenan, R312), and the reverse transcription product was stored at-20 ℃.
LiMYB75 gene clone and sequencing
Primers were designed in the 5 'and 3' non-coding regions of the LiMYB gene according to the genomic information of Lagerstroemia indica (MYB 75-F:5'-TGGAAGGCTGGATGAGAG-3'; MYB75-R: 5'-TGGGATACGGAACAGGGAC-3'), and the LiMYB gene was cloned by PCR.
PCR amplification was performed using high fidelity enzymes (Norflu, P505).
PCR reaction system ：2×Phanta Max Buffer,25μL;dNTP Mix(10mM each),1μL;100ng/L cDNA,2μL;F(10μM),2μL;R(10μM),2μL;Phanta Max Super-Fidelity DNA Polymerase,1μL;ddH2O,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 20. Mu.L.
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 crape myrtle LiMYB Gene
Based on the LiMYB gene and the sequence information of vector pBI121-eGFP, specific cloning primers with cleavage sites were designed using primer 5 (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 was 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 LiMYB 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 LiMYB Gene improves the regeneration Rate of crape myrtle 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 stored agrobacterium containing LiMYB plant over-expression vector is streaked on LB plate containing Kana (100 mg/L) +Rif (50 mg/L), and then monoclonal cultured in 1mL LB liquid medium containing Kana (100 mg/L) +Rif (50 mg/L) by shaking at 28 ℃ for 24h. 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. The value of the bacterial liquid OD 600 cultured overnight was measured by an ultraviolet spectrophotometer, and OD 600 was 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 +200. Mu. Mol/L AS, pH=6.0.
(B) Genetic transformation of crape myrtle leaves by agrobacterium-mediated method: the method comprises the steps of taking the inverted 3-inverted 4-leaf of the tissue culture crape myrtle seedling close to the terminal bud as an explant, and introducing LiMYB genes into the crape myrtle genome by 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, 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 20. Mu.L.
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, by combining the fluorescence detection result and the PCR detection result, liMYB-eGFP is proved to be successfully integrated into the genome of the crape myrtle, and the transgenic positive plant 90 strains are 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 that of the LiMYB gene over-expressed transgenic crape myrtle leaf callus induction rate; the number of regenerated plants of each callus of the crape myrtle leaves without load is 1-2, and the number of regenerated plants of each callus reaches 8-9 after the LiMYB genes are over-expressed, so that 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 LiMYB genes.
In conclusion, the invention overexpresses the endogenous gene LiMYB gene in the crape myrtle, improves the regeneration capability 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. A method for improving the callus regeneration rate of crape myrtle leaves by over-expressing LiMYB genes is characterized in that the nucleotide sequence of LiMYB gene is shown as Seq ID No. 1.
2. The method for improving the callus regeneration rate of crape myrtle leaves by over-expression LiMYB gene according to claim 1, wherein LiMYB gene encodes an amino acid sequence shown in Seq ID No. 2.
3. A method for increasing the rate of regeneration of crape myrtle leaf callus by over-expression of LiMYB gene according to claim 1 or 2, for increasing the rate of regeneration of crape myrtle leaf callus.
4. The method for improving the regeneration rate of crape myrtle leaf callus by over-expression LiMYB gene according to claim 1 or 2, which is used for improving the regeneration plant number of crape myrtle leaf callus blocks.
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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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