CN117965606A - Editing target point of simultaneously mutating rubber tree HbMLO gene and HbMLO gene, gene editing vector and application - Google Patents
Editing target point of simultaneously mutating rubber tree HbMLO gene and HbMLO gene, gene editing vector and application Download PDFInfo
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Abstract
The invention provides an editing target point, a gene editing vector and application of simultaneously mutating genes of rubber trees HbMLO and HbMLO. Meanwhile, the editing targets of the rubber tree HbMLO and HbMLO genes are mutated, and the nucleotide sequence of the editing targets is shown as SEQ ID No. 1. By comparing sequences of 2 candidate powdery mildew genes HbMLO and HbMLO in rubber trees, sgRNA targets are designed at the positions where sequences of coding regions of the two genes are consistent, so that two genes HbMLO and HbMLO can be mutated simultaneously, and compared with one candidate powdery mildew gene, the mutant 2 genes are expected to show stronger and wider-spectrum resistance to powdery mildew.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to an editing target point, a gene editing vector and application of simultaneously mutating genes of rubber trees HbMLO, hbMLO and HbMLO.
Background
Powdery mildew of rubber trees is the first disease faced by rubber production in China, the disease can cause yield loss of more than 45%, and a large amount of manpower, material resources and financial resources are needed to be input for powdery mildew control every year, so that the cultivation of new varieties with high yield and disease resistance is one of important targets of rubber tree breeding, and the MLO gene for disease-sensitive in mutant plants is a general method for cultivating powdery mildew-resistant varieties. In rubber trees HbMLO, 23 has been demonstrated as a disease-causing MLO gene, while HbMLO is a candidate disease-causing gene, and mutation of these two genes is expected to create powdery mildew-resistant varieties of rubber trees.
The research of the gene of the rubber tree HbMLO is still in the identification, cloning, expression, heterologous transformation or transient transformation identification stage and cannot be carried out by technical means such as knockout or editing, so that the function of the HbMLO gene cannot be accurately identified and a new disease-resistant germplasm cannot be created by losing the function of HbMLO.
HbMLO is used as powdery mildew-sensing gene, and the loss of function mutation of the powdery mildew-sensing gene is a necessary way for identifying the functions and cultivating disease-resistant varieties. In 2023, the team defend the country of Hainan university carries out RNAi interference on mRNA of HbMLO23 by a method of transient transformation of leaves, and the in vivo function identification of HbMLO genes is completed for the first time. However, this method has the following disadvantages: firstly, the interference is incomplete, and the function of HbMLO genes cannot be completely lost; secondly, hbMLO is a gene family containing a plurality of homologous genes, and the specificity of RNAi interference is not high, so that the specificity and accuracy of HbMLO gene identification are poor. Thirdly, the study did not mutate the HbMLO gene which is highly homologous to HbMLO.sup.23, so the degree of contribution of mutation HbMLO14 to disease resistance was missed.
Disclosure of Invention
In order to solve the problems, the invention provides an editing target point, a gene editing vector and application of simultaneously mutating genes of rubber trees HbMLO, hbMLO and HbMLO, and can simultaneously mutate genes of rubber trees HbMLO, hbMLO.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides an editing target of simultaneously mutating the rubber tree HbMLO gene and the HbMLO gene, wherein the nucleotide sequence of the editing target is shown as SEQ ID No. 1.
The invention also provides a gene editing vector for simultaneously mutating the rubber tree HbMLO gene and the HbMLO gene, and the preparation method of the gene editing vector comprises the following steps:
1) Annealing the MLO-both-F forward primer and the MLO-both-R reverse primer to obtain a double-chain target sequence;
the nucleotide sequence of the MLO-both-F forward primer is shown as SEQ ID No.2, and the nucleotide sequence of the MLO-both-R reverse primer is shown as SEQ ID No. 3;
2) Connecting the double-chain target sequence obtained in the step 1) into a HbU6.2-163Cas9M vector to obtain a gene editing vector;
the HbU6.2-163Cas9M carrier has a nucleotide sequence shown as SEQ ID No. 4.
Preferably, the annealing condition in the step 1) includes: the temperature was 100deg.C and the time was 5min.
Preferably, the HbU6.2-163Cas9M vector in the step 2) is connected with the double-chain target sequence after being subjected to enzyme digestion by AarI;
the enzyme digestion system comprises: 10 XAarI buffer 5. Mu.L, 50 Xoligonucleotide 1. Mu. L, aarI. Mu.L, hbU6.2-163Cas9M vector 1. Mu.g, ddH 2 O to 50. Mu.L;
the conditions for the cleavage include: the temperature was 37℃and the time was 5 hours.
Preferably, the system connected in step 2) comprises: hbU6.2-163Cas9M vector 50ng, 10×T4 Ligase buffer 1 μL, double stranded target sequence 7 μ L, T4 ligase 0.5 μL, make up ddH 2 O to 10 μL;
The conditions of the connection include: the temperature is 20-30 ℃ and the time is 1h.
The invention also provides application of the editing target spot in the technical scheme in simultaneous mutation of the rubber tree HbMLO gene and the HbMLO gene.
The invention also provides application of the gene editing vector in the technical scheme in simultaneously mutating the rubber tree HbMLO genes and the HbMLO genes.
Preferably, the application comprises: the gene editing vector is transferred into rubber tree protoplast by PEG mediated method.
Preferably, the PEG-mediated method comprises the steps of:
A. mixing the gene editing carrier with a rubber tree protoplast suspension, and standing to obtain a standing substance;
B. mixing the obtained standing substance with an equal volume of PEG solution, and standing in the dark to realize simultaneous mutation of the rubber tree HbMLO gene and the HbMLO gene.
Preferably, the ratio of the mass of the gene editing vector to the volume of the rubber tree protoplast suspension is 50 mug to 1ml; the concentration of the rubber tree protoplast suspension is 5 multiplied by 10 6 per ml; the standing time is 15min;
The mass percentage of the PEG solution is 40%; the temperature of the dark standing is 28 ℃ and the time is 15min.
The beneficial effects of the invention are as follows:
The CRISPR/Cas9 gene editing technology has the functions of high efficiency, specificity and thorough mutation of target genes, and the report of the rubber tree gene editing technology at home and abroad is limited to the team at present, and the technology is firstly applied to research HbMLO genes, and can complete the thorough loss-of-function mutation of HbMLO genes compared with the prior technology. In the invention, by comparing the sequences of 2 candidate powdery mildew genes HbMLO and HbMLO in the rubber tree, the sgRNA targets are designed at the positions where the sequences of the coding regions of the two genes are consistent, so that the two genes HbMLO and HbMLO can be mutated simultaneously, and compared with the mutation of one candidate powdery mildew gene, the mutation of 2 genes simultaneously is expected to show stronger and wider-spectrum resistance to powdery mildew.
Drawings
FIG. 1 is a HbMLO, coding region nucleotide sequence alignment HbMLO;
FIG. 2 is an alignment of the amino acid sequences of HbMLO and HbMLO proteins;
FIG. 3 is a HbU6.2-163Cas9M vector structure;
FIG. 4 shows the mutant sequence and ratio at HbMLO's 14 target
FIG. 5 shows the mutant sequence and the ratio at the HbMLO target point.
Detailed Description
The invention provides an editing target point of simultaneously mutating rubber tree HbMLO gene and HbMLO gene, wherein the nucleotide sequence of the editing target point is shown as SEQ ID No.1, and the method specifically comprises the following steps:
5'-TTTGCAAGGGATACATCATTTGG-3', the bolded part is PAM base sequence.
The invention also provides application of the editing target spot in the technical scheme in simultaneous mutation of the rubber tree HbMLO gene and the HbMLO gene.
In the invention, the full-length sequence of the rubber tree HbMLO gene is 3272bp, and contains 15 exons, and the full-length 1656bp of the coding region sequence is specifically as follows:
SEQ ID No.5: hbMLO14 Gene sequence (the bold is the exon sequence, the italic base is the sgRNA target shared by HbMLO and HbMLO, the single underlined base is the PAM motif)
In the present invention, the HbMLO gene has a full-length sequence of 3435 bp and also contains 15 exons, and the full-length 1725 bp of the coding region sequence is as follows:
SEQ ID No.6: hbMLO23 Gene sequence (the bold is the exon sequence, the italic base is the sgRNA target shared by HbMLO and HbMLO, the single underlined base is the PAM motif)
In the present invention, the coding region sequences of the rubber tree HbMLO and HbMLO genes are highly homologous (FIG. 1), the nucleotide sequences reach 87.2% homology, and the amino acid sequences also have 86.6% homology (FIG. 2).
The invention also provides a gene editing vector for simultaneously mutating the rubber tree HbMLO gene and the HbMLO gene, and the preparation method of the gene editing vector comprises the following steps:
1) Annealing the MLO-both-F forward primer and the MLO-both-R reverse primer to obtain a double-chain target sequence;
the nucleotide sequence of the MLO-both-F forward primer is shown as SEQ ID No.2, and the nucleotide sequence of the MLO-both-R reverse primer is shown as SEQ ID No. 3;
2) Connecting the double-chain target sequence obtained in the step 1) into a HbU6.2-163Cas9M vector to obtain a gene editing vector;
the HbU6.2-163Cas9M carrier has a nucleotide sequence shown as SEQ ID No. 4.
Annealing an MLO-both-F forward primer and an MLO-both-R reverse primer to obtain a double-chain target sequence; the nucleotide sequence of the MLO-both-F forward primer is shown as SEQ ID No.2, and the nucleotide sequence of the MLO-both-R reverse primer is shown as SEQ ID No. 3.
In the present invention, the annealing conditions preferably include: the temperature was 100deg.C and the time was 5min. In the invention, the nucleotide sequence of the MLO-both-F forward primer is shown as SEQ ID No.2, and the nucleotide sequence of the MLO-both-R reverse primer is shown as SEQ ID No.3, and is specifically as follows:
The thickening part is a sequence complementary to 4 sticky end bases after enzyme cutting of the carrier;
the bolded part is a sequence complementary to the 4 sticky end bases of the carrier after cleavage.
The double-stranded target sequence is connected to HbU6.2-163Cas9M carrier to obtain gene editing carrier; the HbU6.2-163Cas9M carrier has a nucleotide sequence shown as SEQ ID No. 4.
In the invention, the HbU6.2-163Cas9M vector is connected with a double-chain target sequence after being subjected to enzyme digestion by AarI. In the present invention, the cleavage system preferably comprises: 10 XAarI buffer 5. Mu.L, 50 Xoligonucleoteide 1. Mu. L, aarI. Mu.L, hbU6.2-163Cas9M vector 1. Mu.g, ddH2O to 50. Mu.L; the conditions for the cleavage include: the temperature was 37℃and the time was 5 hours. In the present invention, the linked system preferably comprises: hbU6.2-163Cas9M vector 50ng, 10×T4 ligase buffer 1 μL, double stranded target sequence 7 μ L, T4.5 μL of 4 ligase, make up ddH2O to 10 μL; the conditions of the connection preferably include: the temperature is 20-30 ℃ and the time is 1h.
In the invention, the nucleotide sequence of the HbU6.2-163Cas9M vector is shown as SEQ ID No.4, and is specifically as follows:
GAGCTCGGTACCGAGGATCCTCTAGACTCGAGGATTATGTGGAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTCAACTTGCTATGCTGTTTCCAGCATAGCTCTGAAACGTGTGCAGGTGTTGTGTTCACCTGCGAGCCAATTGCTACTGCCTATTCTTTGCTTATGAAGGCTTGCAACTATATGTAACTAAATGATGTGGGACTAAGTTCAATCCAACCAACCAGATGACTTTACAACCAGGATGGGTTAAATAATTTCATTAGGCCCACAAATGGGTTCTGAGTTGTACATCAGGCCTAAGTTGGTCAAAGCCCAATGCTAAGACACCCTACTTGCCATGCGCTAAGGGCACCTGGTGTTGGCATAAGCTTACAAGTTACAAGATACTTCCACTTCTATAACAATTATAAAGACCTTCATTTTTCACACCCTTAAAATAACCAACATTAAAGTTGGTGCTAAGCTATTCCTTTAGCTTAGATCCGTACCCCTACTCCAAAAATGTCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATTTCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCGAAAGGACAGTAGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCATTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGACATCTCCACTGACGTAAGGGATGACGCACAATCCCACCCCTACTCCAAAAATGTCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATTTCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCGAAAGGACAGTAGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCATTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGACATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACAGCCCAAGCTAGATCCATGGCCCCTAAGAAGAAGAGAAAGGTCGGTATTCACGGCGTTCCTGCGGCGATGGACAAGAAGTATAGTATTGGTCTGGACATTGGGACGAATTCCGTTGGCTGGGCCGTGATCACCGATGAGTACAAGGTCCCTTCCAAGAAGTTTAAGGTTCTGGGGAACACCGATCGGCACAGCATCAAGAAGAATCTCATTGGAGCCCTCCTGTTCGACTCAGGCGAGACCGCCGAAGCAACAAGGCTCAAGAGAACCGCAAGGAGACGGTATACAAGAAGGAAGAATAGGATCTGCTACCTGCAGGAGATTTTCAGCAACGAAATGGCGAAGGTGGACGATTCGTTCTTTCATAGATTGGAGGAGAGTTTCCTCGTCGAGGAAGATAAGAAGCACGAGAGGCATCCTATCTTTGGCAACATTGTCGACGAGGTTGCCTATCACGAAAAGTACCCCACAATCTATCATCTGCGGAAGAAGCTTGTGGACTCGACTGATAAGGCGGACCTTAGATTGATCTACCTCGCTCTGGCACACATGATTAAGTTCAGGGGCCATTTTCTGATCGAGGGGGATCTTAACCCGGACAATAGCGATGTGGACAAGTTGTTCATCCAGCTCGTCCAAACCTACAATCAGCTCTTTGAGGAAAACCCAATTAATGCTTCAGGCGTCGACGCCAAGGCGATCCTGTCTGCACGCCTTTCAAAGTCTCGCCGGCTTGAGAACTTGATCGCTCAACTCCCGGGCGAAAAGAAGAACGGCTTGTTCGGGAATCTCATTGCACTTTCGTTGGGGCTCACACCAAACTTCAAGAGTAATTTTGATCTCGCTGAGGACGCAAAGCTGCAGCTTTCCAAGGACACTTATGACGATGACCTGGATAACCTTTTGGCCCAAATCGGCGATCAGTACGCGGACTTGTTCCTCGCCGCGAAGAATTTGTCGGACGCGATCCTCCTGAGTGATATTCTCCGCGTGAACACCGAGATTACAAAGGCCCCGCTCTCGGCGAGTATGATCAAGCGCTATGACGAGCACCATCAGGATCTGACCCTTTTGAAGGCTTTGGTCCGGCAGCAACTCCCAGAGAAGTACAAGGAAATCTTCTTTGATCAATCCAAGAACGGCTACGCTGGTTATATTGACGGCGGGGCATCGCAGGAGGAATTCTACAAGTTTATCAAGCCAATTCTGGAGAAGATGGATGGCACAGAGGAACTCCTGGTGAAGCTCAATAGGGAGGACCTTTTGCGGAAGCAAAGAACTTTCGATAACGGCAGCATCCCTCACCAGATTCATCTCGGGGAGCTGCACGCCATCCTGAGAAGGCAGGAAGACTTCTACCCCTTTCTTAAGGATAACCGGGAGAAGATCGAAAAGATTCTGACGTTCAGAATTCCGTACTATGTCGGACCACTCGCCCGGGGTAATTCCAGATTTGCGTGGATGACCAGAAAGAGCGAGGAAACCATCACACCTTGGAACTTCGAGGAAGTGGTCGATAAGGGCGCTTCCGCACAGAGCTTCATTGAGCGCATGACAAATTTTGACAAGAACCTGCCTAATGAGAAGGTCCTTCCCAAGCATTCCCTCCTGTACGAGTATTTCACTGTTTATAACGAACTCACGAAGGTGAAGTATGTGACCGAGGGAATGCGCAAGCCCGCCTTCCTGAGCGGCGAGCAAAAGAAGGCGATCGTGGACCTTTTGTTTAAGACCAATCGGAAGGTCACAGTTAAGCAGCTCAAGGAGGACTACTTCAAGAAGATTGAATGCTTCGATTCCGTTGAGATCAGCGGCGTGGAAGACAGGTTTAACGCGTCACTGGGGACTTACCACGATCTCCTGAAGATCATTAAGGATAAGGACTTCTTGGACAACGAGGAAAATGAGGATATCCTCGAAGACATTGTCCTGACTCTTACGTTGTTTGAGGATAGGGAAATGATCGAGGAACGCTTGAAGACGTATGCCCATCTCTTCGATGACAAGGTTATGAAGCAGCTCAAGAGAAGAAGATACACCGGATGGGGAAGGCTGTCCCGCAAGCTTATCAATGGCATTAGAGACAAGCAATCAGGGAAGACAATCCTTGACTTTTTGAAGTCTGATGGCTTCGCGAACAGGAATTTTATGCAGCTGATTCACGATGACTCACTTACTTTCAAGGAGGATATCCAGAAGGCTCAAGTGTCGGGACAAGGTGACAGTCTGCACGAGCATATCGCCAACCTTGCGGGATCTCCTGCAATCAAGAAGGGTATTCTGCAGACAGTCAAGGTTGTGGATGAGCTTGTGAAGGTCATGGGACGGCATAAGCCCGAGAACATCGTTATTGAGATGGCCAGAGAAAATCAGACCACACAAAAGGGTCAGAAGAACTCGAGGGAGCGCATGAAGCGCATCGAGGAAGGCATTAAGGAGCTGGGGAGTCAGATCCTTAAGGAGCACCCGGTGGAAAACACGCAGTTGCAAAATGAGAAGCTCTATCTGTACTATCTGCAAAATGGCAGGGATATGTATGTGGACCAGGAGTTGGATATTAACCGCCTCTCGGATTACGACGTCGATCATATCGTTCCTCAGTCCTTCCTTAAGGATGACAGCATTGACAATAAGGTTCTCACCAGGTCCGACAAGAACCGCGGGAAGTCCGATAATGTGCCCAGCGAGGAAGTCGTTAAGAAGATGAAGAACTACTGGAGGCAACTTTTGAATGCCAAGTTGATCACACAGAGGAAGTTTGATAACCTCACTAAGGCCGAGCGCGGAGGTCTCAGCGAACTGGACAAGGCGGGCTTCATTAAGCGGCAACTGGTTGAGACTAGACAGATCACGAAGCACGTGGCGCAGATTCTCGATTCACGCATGAACACGAAGTACGATGAGAATGACAAGCTGATCCGGGAAGTGAAGGTCATCACCTTGAAGTCAAAGCTCGTTTCTGACTTCAGGAAGGATTTCCAATTTTATAAGGTGCGCGAGATCAACAATTATCACCATGCTCATGACGCATACCTCAACGCTGTGGTCGGAACAGCATTGATTAAGAAGTACCCGAAGCTCGAGTCCGAATTCGTGTACGGTGACTATAAGGTTTACGATGTGCGCAAGATGATCGCCAAGTCAGAGCAGGAAATTGGCAAGGCCACTGCGAAGTATTTCTTTTACTCTAACATTATGAATTTCTTTAAGACTGAGATCACGCTGGCTAATGGCGAAATCCGGAAGAGACCACTTATTGAGACCAACGGCGAGACAGGGGAAATCGTGTGGGACAAGGGGAGGGATTTCGCCACAGTCCGCAAGGTTCTCTCTATGCCTCAAGTGAATATTGTCAAGAAGACTGAAGTCCAGACGGGCGGGTTCTCAAAGGAATCTATTCTGCCCAAGCGGAACTCGGATAAGCTTATCGCCAGAAAGAAGGACTGGGACCCGAAGAAGTATGGAGGTTTCGACTCACCAACGGTGGCTTACTCTGTCCTGGTTGTGGCAAAGGTGGAGAAGGGAAAGTCAAAGAAGCTCAAGTCTGTCAAGGAGCTCCTGGGTATCACCATTATGGAGAGGTCCAGCTTCGAAAAGAATCCGATCGATTTTCTCGAGGCGAAGGGATATAAGGAAGTGAAGAAGGACCTGATCATTAAGCTTCCAAAGTACAGTCTTTTCGAGTTGGAAAACGGCAGGAAGCGCATGTTGGCTTCCGCAGGAGAGCTCCAGAAGGGTAACGAGCTTGCTTTGCCGTCCAAGTATGTGAACTTCCTCTATCTGGCATCCCACTACGAGAAGCTCAAGGGCAGCCCAGAGGATAACGAACAGAAGCAACTGTTTGTGGAGCAACACAAGCATTATCTTGACGAGATCATTGAACAGATTTCGGAGTTCAGTAAGCGCGTCATCCTCGCCGACGCGAATTTGGATAAGGTTCTCTCAGCCTACAACAAGCACCGGGACAAGCCTATCAGAGAGCAGGCGGAAAATATCATTCATCTCTTCACCCTGACAAACCTTGGGGCTCCCGCTGCATTCAAGTATTTTGACACTACGATTGATCGGAAGAGATACACTTCTACGAAGGAGGTGCTGGATGCAACCCTTATCCACCAATCGATTACTGGCCTCTACGAGACGCGGATCGACTTGAGTCAGCTCGGGGGGGATAAGAGACCAGCGGCAACCAAGAAGGCAGGACAAGCGAAGAAGAAGAAGTAGCAATTCGGTACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCTATTTTCTCCATAAATAATGTGTGAGTAGTTTCCCGATAAGGGAAATTAGGGTTCTTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGATCTCCTAAAGTCCCTATAGATCTTTGTCGTGAATATAAACCAGACACGAGACGACTAAACCTGGAGCCCAGACGCCGTTCGAAGCTAGAAGTACCGCTTAGGCAGGAGGCCGTTAGGGAAAAGATGCTAAGGCAGGGTTGGTTACGTTGACTCCCCCGTAGGTTTGGTTTAAATATGATGAAGTGGACGGAAGGAAGGAGGAAGACAAGGAAGGATAAGGTTGCAGGCCCTGTGCAAGGTAAGAAGATGGAAATTTGATAGAGGTACGCTACTATACTTATACTATACGCTAAGGGAATGCTTGTATTTATACCCTATACCCCCTAATAACCCCTTATCAATTTAAGAAATAATCCGCATAAGCCCCCGCTTAAAAATTGGTATCAGAGCCATGAATAGGTCTATGACCAAAACTCAAGAGGATAAAACCTCACCAAAATACGAAAGAGTTCTTAACTCTAAAGATAAAAGATCTTTCAAGATCAAAACTAGTTCCCTCACACCGGTGACGGGGATCGCATGCGATATCTCGAGATCTAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGT.
The invention also provides application of the editing target spot in the technical scheme in simultaneous mutation of the rubber tree HbMLO gene and the HbMLO gene.
The invention also provides application of the gene editing vector in the technical scheme in simultaneously mutating the rubber tree HbMLO genes and the HbMLO genes.
In the present invention, the application preferably includes: the gene editing vector is transferred into rubber tree protoplast by PEG mediated method. In the present invention, the PEG-mediated method preferably comprises the steps of:
A. mixing the gene editing carrier with a rubber tree protoplast suspension, and standing to obtain a standing substance;
B. mixing the obtained standing substance with an equal volume of PEG solution, and standing in the dark to realize simultaneous mutation of the rubber tree HbMLO gene and the HbMLO gene.
In the present invention, the ratio of the mass of the gene editing vector to the volume of the suspension of the rubber tree protoplast is preferably 50. Mu.g/1 ml; the concentration of the rubber tree protoplast suspension is 5X 106 per ml; the standing time is 15min. In the present invention, the mass percentage of the PEG solution is preferably 40%. In the present invention, the temperature of the dark rest is preferably 28℃for 15 minutes.
The present invention will be described in detail with reference to examples for further illustration of the invention, but they should not be construed as limiting the scope of the invention.
Example 1
1. SgRNA design
In the CRISPR/Cas9 gene editing system, the downstream requirement of the sgRNA target must have the PAM (Protospacer Adjacent Motif) motif with the sequence NGG. Although the HbMLO gene has up to 87.2% sequence homology with the HbMLO gene coding region, there are also a large number of SNP and In/Del differences, and In view of the specificity of CRISPR/Cas9 for target recognition, to edit two genes simultaneously, it is necessary to design a sgRNA target where the two gene coding regions are completely identical In sequence and have a PAM motif, by sequence alignment, one sgRNA target is designed In the 6 th exon of two genes, the sequence is: 5'-TTTGCAAGGGATACATCATTTGG-3' the sequence of the target region is shown below (bolded part is the target sequence, italic base is PAM motif, single underlined base is SNP of both):
2. Vector construction
The gene editing vector is constructed by taking HbU6.2-163Cas9M (structure shown in the sequence of FIG. 3 and SEQ ID No. 4) rubber tree protoplast transient transformation gene editing vector as a framework, and the specific flow is as follows:
1) Synthesizing a pair of complementary targets with joints, wherein the sequences are as follows;
The thickened bases are 4 bases added which are complementary to the viscous end of the carrier, as follows;
2) Primer annealing to double strand
The MLO-both-F forward primer and the MLO-both-R reverse primer were diluted to 100. Mu.M with ddH2O water, 20. Mu.L each was mixed in a PCR tube, and then placed in a PCR apparatus, run at 100℃for 5min, and cooled at room temperature, thereby forming a double-stranded target sequence with sticky ends.
3) Enzyme cutting of carrier
And placing the prepared enzyme digestion system at 37 ℃ for enzyme digestion for 5 hours, and purifying and recovering enzyme digestion vectors by using a Tiangen mass DNA product purification kit (DP 205).
4) Target fragment-editing vector ligation
5) Coli transformation and cloning identification
Adding 10 mu L of the connection product into 100 mu L of DH5 alpha competent cells, slightly mixing, ice-bathing for 30min, immediately placing in a metal bath at 42 ℃ for heat shock for 45sec, immediately placing on ice for standing for 2min, transferring into 500 mu L of LB liquid medium without antibiotics, standing upside down, mixing uniformly, placing on a shaking table at 180rpm for shaking culture at 37 ℃ for 1h to revive the cells, then uniformly smearing 100 mu L of bacterial liquid on a plate of LB solid medium with kana resistance, drying, and then culturing in a culture box at 37 ℃ for 16h in an inverted manner.
After bacterial plaque grows on the flat plate on the next day, the primer MLO-both-F+163-F (5'-AGGGTTATTGTCTCATGAGCGG-3') is used for colony PCR identification, and clones which can be amplified to specific 268bp are selected and sent to a company for sequencing identification.
3. Transient transformation of protoplasts
The positive clones identified above were inoculated with a plasmid, which was then extracted and transferred into rubber tree protoplasts by PEG-mediated method. The method comprises the following steps:
1) Preparation of protoplasts
About 2g of 5-7 days of yellow leaf blades were collected from 7-33-97 tissue culture seedlings of shading-treated rubber tree clones planted in a greenhouse and immediately soaked in 0.6M mannitol for 10 minutes. The main vein was then excised with a razor blade, the remaining mesophyll was cut into strips 0.5-1.0mm wide, and then placed in a 150ml flask containing 20ml of an enzyme solution [1.5% cellulase R10,0.6% pectinase R10 (Yakult Pharma., tokyo, japan), 0.6M mannitol, 20mM KCl,20mM MES,10mM CaCl 2 and 0.1% BSA, pH5.7], and shaken at 60rpm under dark conditions at 26℃for 4-5h. The digestion process was then terminated by adding equal amounts of W5 solution (154 mM NaCl, 5mM KCl, 125mM CaCl 2, 5mM glucose, 2mM MES, pH 5.7). The digestate was then filtered into a 50ml round bottom tube with a 30 μm sterilized metal mesh pre-wetted with W5 solution, and the protoplasts were washed twice with W5 solution and centrifuged at 800rpm for 3 minutes at 4 ℃. The protoplasts were then suspended with MMG solution (15 mM MgCl 2, 0.4M mannitol, 4mM MES, pH 5.7) and adjusted to a concentration of 5X 106/ml (calculated by hemocytometer). Finally, the suspended protoplasts were placed on ice for 30 minutes before transformation.
2) PEG-mediated protoplast transformation
50 Mug of gene editing plasmid is added into a 2ml round bottom centrifuge tube to 1ml protoplast suspension, the protoplast suspension is flicked and mixed uniformly, the mixture is kept stand for 15min at room temperature, 40% PEG solution is added into the equal volume for rapid mixing, the mixture is kept stand for 15min at 28 ℃ in a dark room, the equal volume of W5 solution is added into the centrifuge tube for uniform mixing to stop the reaction, the supernatant is removed after centrifugation at 800rpm/min for 3min, the equal volume of WI solution is added into the centrifuge tube for rinsing once, the supernatant is removed after centrifugation at 800rpm/min for 3min, and finally 1ml of WI solution is added for resuspension, and the mixture is transferred into a 12-hole cell culture plate for culturing for 48h at 26 ℃ in the dark room.
3) Editing detection of target sites
Extracting the genomic DNA of the transformed protoplasts with a plant genome extraction kit from Tiangen biospecimen, followed by PCR amplification of the genomic DNA comprising the target region with HbMLO gene-specific primers (MLO14-b-msF SEQ ID S No.7：5'-ggagtgagtacggtgtgcGCACAATTCCATTGTATAACATC-3';MLO14-b-msR SEQ IDS No.8：5'-gagttggatgctggatggTCCGCTACTTACAATCCAAAG-3'), and HbMLO gene-specific primer (MLO23-b-msF SEQ IDS No.9：5'-ggagtgagtacggtgtgcCCCACTGTATAGTCAGCAAT-3';MLO23-b-msR SEQ IDS No.10：5'-gagttggatgctggatggTTCTGGAGCTTACAATCCAAAT-3',, respectively, wherein the lower case letters are high throughput sequencing linker sequences), in accordance with the following amplification system and procedure:
The amplification was performed using 2X MAGIC GREEN TAQ Supermix enzyme from Tu Long Kong, the amplification system was as follows:
Forward and reverse primers (10. Mu.M) 1. Mu.L each, 2X MAGIC GREEN TAQ Supermix 10. Mu.L, DNA template 1. Mu.L, ddH 2 O7. Mu.L; the PCR procedure was as follows: pre-denaturation at 95℃for 3min; the next 35 cycles are as follows: 95℃10s,58℃10s,72℃30s; finally, the primer is extended at 72 ℃ for 5min, and the amplicon size is 268bp.
The amplified product was sent to the company for high throughput sequencing (Hi-TOM, http:// 121.40.237.174/Hi-TOM/logic. Php), and the results showed that specific mutations were generated at the target sites for both HbMLO gene and HbMLO gene, with the ratio of mutations being 20.3% (FIG. 4) and 16.81% (FIG. 5), respectively.
The results show that the invention successfully identifies an editing target point capable of simultaneously mutating HbMLO genes and HbMLO genes for the first time, and the target point can be utilized to mutate the two genes by a CRISPR/Cas9 mediated gene editing method, so that a foundation is laid for cultivating new varieties with high rubber powdery mildew resistance.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. Meanwhile, the nucleotide sequence of the editing target point of the mutant rubber tree HbMLO and HbMLO genes is shown as SEQ ID No. 1.
2. A gene editing vector for simultaneously mutating genes of rubber tree HbMLO and HbMLO, wherein the method for preparing the gene editing vector comprises the following steps:
1) Annealing the MLO-both-F forward primer and the MLO-both-R reverse primer to obtain a double-chain target sequence;
the nucleotide sequence of the MLO-both-F forward primer is shown as SEQ ID No.2, and the nucleotide sequence of the MLO-both-R reverse primer is shown as SEQ ID No. 3;
2) Connecting the double-chain target sequence obtained in the step 1) into a HbU6.2-163Cas9M vector to obtain a gene editing vector;
the HbU6.2-163Cas9M carrier has a nucleotide sequence shown as SEQ ID No. 4.
3. The gene editing vector according to claim 2, wherein the annealing conditions of step 1) include: the temperature was 100deg.C and the time was 5min.
4. The gene editing vector according to claim 2, wherein the step 2) hbu6.2-163Cas9M vector is linked to a double-stranded target sequence after AarI cleavage;
the enzyme digestion system comprises: 10 XAarI buffer 5. Mu.L, 50 Xoligonucleotide 1. Mu. L, aarI. Mu.L, hbU6.2-163Cas9M vector 1. Mu.g, ddH 2 O to 50. Mu.L;
the conditions for the cleavage include: the temperature was 37℃and the time was 5 hours.
5. The gene editing vector according to claim 2, wherein the system of step 2) ligation comprises: hbU6.2-163Cas9M vector 50ng, 10×T4 Ligase buffer 1 μL, double stranded target sequence 7 μ L, T4 ligase 0.5 μL, make up ddH 2 O to 10 μL;
The conditions of the connection include: the temperature is 20-30 ℃ and the time is 1h.
6. Use of the editing target of claim 1 for mutating the genes of rubber tree HbMLO, hbMLO and HbMLO simultaneously.
7. Use of the gene editing vector according to any one of claims 2 to 5 for simultaneously mutating genes of rubber tree HbMLO and HbMLO.
8. The application according to claim 7, characterized in that it comprises: the gene editing vector is transferred into rubber tree protoplast by PEG mediated method.
9. The use according to claim 8, wherein the PEG-mediated method comprises the steps of:
A. mixing the gene editing carrier with a rubber tree protoplast suspension, and standing to obtain a standing substance;
B. mixing the obtained standing substance with an equal volume of PEG solution, and standing in the dark to realize simultaneous mutation of the rubber tree HbMLO gene and the HbMLO gene.
10. The use according to claim 9, characterized in that the ratio of the mass of the gene editing vector to the volume of the suspension of rubber tree protoplasts is 50 μg to 1ml; the concentration of the rubber tree protoplast suspension is 5 multiplied by 10 6 per ml; the standing time is 15min;
The mass percentage of the PEG solution is 40%; the temperature of the dark standing is 28 ℃ and the time is 15min.
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