CN111549037A - Corn ZmRLK7 gene editing target and application thereof - Google Patents
Corn ZmRLK7 gene editing target and application thereof Download PDFInfo
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Abstract
The invention discloses a corn ZmRLK7 gene editing target spot and application thereof. The invention provides two editing targets for editing a maize leucine-rich repetitive receptor-like kinase gene ZmRLK7, wherein the two editing targets can be used for editing a ZmRLK7 gene to obtain editing types such as SNP, Indel, large fragment deletion and the like, so that the function of the gene can be deeply analyzed. Provides a new way for utilizing the ZmRLK7 gene of the corn and creates germplasm resources for further improving the yield of the corn. Provides a new way for constructing the endogenous knockout or site-specific knock-in exogenous gene of the ZmRLK7 gene of the corn.
Description
Technical Field
The invention relates to a corn ZmRLK7 gene editing target spot and application thereof, belonging to the technical field of genes.
Background
Corn is not only an important grain crop, but also an important feed and industrial raw material, so that the cultivation of an excellent corn variety and the improvement of the unit yield of the corn play a significant role in the development of national economy. Corn yield depends on individual plant yield and planting density, and individual plant yield can be further subdivided into grain number per ear and grain weight per grain. Compared with other complex agronomic traits, the seed trait has high hereditary force and easy measurement, and is not only a mode trait of genetic research, but also an important target trait of genetic improvement. The corn genetic resources are rich, however, the rich natural variation existing in the corn is not completely excavated so far, the genetic basis for controlling important agronomic traits is not deeply clarified, the key genes influencing the grain traits are excavated by adopting a linkage analysis and whole genome association analysis method, the functions and the regulation and control mechanisms of the key genes are analyzed, and the related regulation and control network is constructed and has important practical and theoretical significance when being applied to breeding research
All organisms sense and transmit signals through cell surface receptors. Many of these signal transduction in plants are mediated by cell surface receptor-like protein kinases (RLKs), of which Leucine-Rich repeat receptor-like protein kinases (LRR-RLKs) are the largest family of RLKs. LRR-RLKs are a class of membrane proteins localized to the plasma membrane, comprising an extracellular domain that senses signals, a transmembrane domain that anchors the protein to the membrane, and an intracellular kinase domain that transmits the signal down by autophosphorylation and by phosphorylation of specific substrates (Shiu and Bleecker, 2001; Gou et al, 2010). The LRR-RLKs ectodomain part comprises different numbers of LRR repeats, and the diversity of LRR repeats endows the LRR-RLK with perception of different receptors comprising small molecules, peptides and proteins; the kinase domain portion typically comprises 12 conserved subdomains that fold to form a three-dimensional catalytic core with two double-lobed structures (Hanks et al, 1988; Hanks and Hunter, 1995). As shown in FIG. 3(Modified from Shiu and B leecker,2001), LRR-RLKs in plants act as receptor protein kinases, participate in signal transduction processes such as growth and development of plant tissues and organs, plant hormone signal response, biotic and abiotic stress response, and play important roles in plant life activities.
Liu et al (2015) performed eQTL analysis by using existing RNA-seq data, identified a trans-regulatory factor, which they considered to be a BAK1-like gene, located in the seventh chromosome, named ZmRLK7. they located a major QTL affecting grain width and hundred grain weight by using a recombinant inbred line population, and found that ZmRLK7 co-located with the QTL-10ZmRLK7 controls the expression of 67 genes, and thus the gene may be a key node gene.
Although Liu et al (2015) have some preliminary predictions and analyses, the function, mechanism of action and involved regulatory network of the gene are still poorly understood, and especially the genetic improvement potential of the gene in maize or other crops is lacking in effective evaluation, which is worthy of intensive study. In modern molecular biology, the identification of the function of an unknown gene by using a forward or reverse genetics method is a common means, and the mutant of the gene is obtained by using methods such as EMS mutagenesis and the like, so that the time is long and the efficiency is low; at present, the mutation of a target gene by using a genome editing means can be doubled with half the effort, but a key ring of a gene editing technology is the design and editing target.
Disclosure of Invention
The invention overcomes the defects of the prior art and provides a corn ZmRLK7 gene editing target spot and application thereof. The invention provides two editing targets for editing a maize leucine-rich repetitive receptor-like kinase gene ZmRLK7, wherein the two editing targets can be used for editing a ZmRLK7 gene to obtain editing types such as SNP, Indel, large fragment deletion and the like, so that the function of the gene can be deeply analyzed.
The invention provides two gene editing targets capable of effectively editing corn ZmRLK7, wherein the corn ZmRLK7 gene editing targets comprise Target1 and Target2, the Target1 is positioned at 2462-2471bp of a ZmRLK7 cDNA sequence, and the sequence of the Target is shown as SEQ ID NO. 1; the Target2 is positioned at the 2737-2746bp position of the ZmRLK7 cDNA sequence, and the sequence is shown as SEQ ID NO. 2; the ZmRLK7 cDNA sequence is shown in SEQ ID NO. 7.
Further, the method for editing the target gene ZmRLK7 by using the maize ZmRLK7 gene editing target point specifically comprises the following steps:
(1) constructing a gene editing vector containing editing Target points of Target1 and Target 2;
(2) carrying out corn genetic transformation on the gene editing vector obtained in the step (1) to obtain a transgenic corn event;
(3) taking the tissue material of the transgenic corn event obtained in the step (2), extracting DNA for PCR amplification, sequencing an amplification product, and verifying the gene editing condition.
And (3) comparing the sequencing result in the step (3) with the sequence of the target gene ZmRLK7, wherein if the sequence is the same, the target gene is not successfully edited, and if the sequencing result shows SNP, Indel or large fragment deletion and the like, the gene editing is successful.
Further, the method for constructing the gene editing vector in the step (1) specifically comprises the following steps:
s1 the sequence of ZmRLK7 gene of corn is called out from gene bank and input into websitehttp:// crispr.hzau.edu.cn/CRISPR2/Selecting a sequence of target sites for gene editing;
s2 designing and synthesizing corresponding primer sequence according to the sequence of the target site;
s3, using CPB-ZmUbi-hspCas9 as a template, using the primer sequence in the step S2 as an amplification primer, carrying out PCR amplification by using high-fidelity enzyme, and amplifying a homology arm and a target region in the primer sequence in the step S2 to obtain a PCR amplification product;
s4, performing restriction enzyme digestion on CPB-ZmUbi-hspCas9 to form linearity, and obtaining a linearized vector;
s5, connecting the PCR amplification product obtained in the step S3 and the linearized vector obtained in the step S4 by using a recombinase to obtain a recombinant;
s6, sequencing the target point sequence of the recon obtained in the step S5, wherein the recon with correct sequencing is the gene editing vector.
Further, the primer sequence in the above step S2 includes T1-F, T1-R, T2-F, T2-R;
the sequence of T1-F is shown as SEQ ID NO.3, the sequence of T1-R is shown as SEQ ID NO.4, the sequence of T2-F is shown as SEQ ID NO.5, and the sequence of T2-R is shown as SEQ ID NO. 6.
Further, the reaction system for PCR amplification described in the above step S3 is 25 μ l, and includes the following components: 2 x High-fidelity Master Mix 12.5 u l, upstream and downstream primers (concentration 10 u M) each 1 u l, plasmid 2 u l, adding sterile water 8.5 u l;
the PCR amplification procedure is as follows: pre-denaturation at 98 ℃ for 1 min; denaturation at 98 ℃ for 10s, annealing at 68 ℃ for 15s, extension at 72 ℃ for 45s, and circulating for 35 times; overextension was carried out for 5min at 72 ℃.
The application of the maize ZmRLK7 gene editing target point in editing ZmRLK7 gene.
Has the advantages that:
(1) the maize ZmRLK7 gene can be edited by using the editing target point to generate abundant editing types, such as SNP, Indel, large fragment deletion and other editing types.
(2) The corn ZmRLK7 gene edited by the editing target point can obtain the corn gene editing event, is favorable for deeply analyzing the function of the receptor kinase gene and knowing the action mechanism of the receptor kinase gene.
(3) Provides a new way for utilizing the ZmRLK7 gene of the corn and creates germplasm resources for further improving the yield of the corn. Provides a new way for constructing the endogenous knockout or site-specific knock-in exogenous gene of the ZmRLK7 gene of the corn.
Drawings
FIG. 1: and (3) PCR detection of the transgenic plant.
FIG. 2: editing the Target1 site of the Target gene.
FIG. 3: editing the Target2 site of the Target gene.
FIG. 4: editing results of deletion of large fragments obtained after the ZmRLK7 gene editing.
FIG. 5: and (3) a corn genetic transformation process.
Detailed Description
In order to make the technical solutions in the present application better understood, the present invention is further described below with reference to examples, which are only a part of examples of the present application, but not all examples, and the present invention is not limited by the following examples.
Example 1 target screening and vector construction
Calling out a corn ZmRLK7 gene sequence from a gene library, inputting the sequence into a website http:// criprp.hzau.edu.cn/CRISPR 2/, setting a U6 promoter screening condition, selecting a sequence with lower off-target score, and selecting a target site sequence for gene editing according to the function of target gene coding protein; the gene encodes a receptor kinase and has a kinase domain, so two targets we have chosen are in the kinase region. According to the screening conditions, two targets of Target1 and Target2 are obtained, so we select them to design primer T1-F, T1-R, T2-F, T2-R.
The plant transformation binary vector basic vector used in the experiment is modified from pCAMBIA1300, the vector CPB-ZmUbi-hspCas9 is from the subject group of the Xiaoming teacher of the academy of agriculture department of China, and the plasmid carries glufosinate-ammonium resistance gene Bar. The target sequence is connected into a vector, namely a ProZmUbi vector, which is composed of hspCs 9-ProU6, T1T2-P35S and bar editing vector. The specific operation is as follows: CPB-ZmUbi-hspCas9 as template, T1-F: 5-GCAGCTCAGGGGCCGTGTACAATTCGGTGCTTGCGG-3' (SED. ID. NO3) and T1-R: 5-GTACACGGCCCCTGAGCTGCGTTTTAGAGCTAGAAATAGC-3′(SED.ID.NO4)、T2-F: 5′-AGCGCGATCTCGTAGCGCGCAATTCGGTGCTTGCGG-3′(SEDID. NO5) and T2-R: 5-GCGCGCTACGAGATCGCGCTGTTTTAGAGCTAGAAATAGC-3' (SED. ID. NO6) as primers, and PCR amplification of homology arms and target regions using high fidelity enzymes, the underlined parts being the vector backbone homology arms and the tandem target region homology arms, respectively.
The reaction system for PCR amplification is 25. mu.l, and comprises the following components: 2 x High-fidelity Master Mix 12.5 u l, upstream and downstream primers (concentration 10 u M) each 1 u l, plasmid 2 u l, adding sterile water 8.5 u l. The PCR procedure was: pre-denaturation at 98 ℃ for 1 min; denaturation at 98 ℃ for 10s, annealing at 68 ℃ for 15s, extension at 72 ℃ for 45s, and circulating for 35 times; overextension was carried out for 5min at 72 ℃.
Cutting the vector CPB-ZmUbi-hspCas9 into linearity by using a restriction enzyme Hind III, connecting the PCR product with a linearization vector by using a recombinase, and finally identifying a target sequence by sequencing, wherein a recon with correct sequencing is an editing vector.
Example 2 genetic transformation of maize
2.1 Agrobacterium transformation
the recombinant plasmid is transformed into AGL1 Agrobacterium tumefaciens competent cell, the method refers to the competent specification, melting Agrobacterium tumefaciens competent cell preserved at-70 deg.C in ice bath, adding 1 μ g recombinant plasmid into 100 μ L competent cell, mixing, ice-cooling for 30min, quickly freezing with liquid nitrogen for 5min, incubating in 37 deg.C water bath for 5min, ice-cooling for 2min, adding 800 μ L YEP (10g ⊙ L)-1Yeast extract; 10 g.L-1Tryptone; 5 g.L-1Sodium chloride; pH 7.0), and shake culturing on a shaker at 28 deg.C for 2-3 h. After the cells were collected, they were spread on YEP plates containing kanamycin and rifampicin, and they were cultured in an inverted state at 28 ℃ for 48 to 72 hours.
2.2 Agrobacterium-mediated genetic transformation of maize embryos
Transfer Agrobacterium containing recombinant plasmid to 200ml YEP liquid medium, wait OD600When the average molecular weight is 0.8 to 1.2, 5000 r.min-1And centrifuging for 10min to collect the thallus. HiII maize embryos of 1.5-2mm after pollination are used as receptors for genetic transformation. Transformation time is 5min, then the cells are placed in induction culture and co-culture, and after 7 days, the cells are placed on screening medium until antibodies grow outAnd (3) sexual callus. Transferring the resistant callus into a regeneration culture medium, transplanting the regeneration plant, and selfing to obtain fruit. The transformation process is shown in FIG. 5, starting from the left panel of the first row and ending in the right panel of the third row, which is the transformed maize immature embryo plant obtained.
Example 3 molecular detection of transgenic Material
3.1 detection of transgenic plants
DNA was extracted from transgenic and T0 generation non-transgenic leaf tissue, and DNA expression was performed using Bar gene primers: 5'-ATGAGCCCAGAACGACGCCCG-3' (SED.ID.NO8), 5'-TCAGATCTCGGTGACGGGCAG-3' (SED.ID.NO9) amplifying transgenic positive plants; positive plants were amplified with the following primers for the target fragment, P1: 5'-GGCGTCATCTCGTCATTCAAG-3' (sed. id. no10) and P2: 5'-AGCAGCACGTTCATGGACTT-3' (SED.ID.NO11), the amplified product was sequenced and identified.
The PCR detection result of the transgenic plant is shown in FIG. 1, and the Bar gene target fragment amplified by the transgenic plant, but not the target fragment in the transgenic control (WT), can prove that the constructed vector is successfully transferred into the plant.
3.2 comparison of PCR sequencing results
And comparing the sequencing result with the target gene sequence by using BioEdit software, wherein the event different from the target region in the obtained sequencing result is the gene editing event.
FIGS. 2-4 represent 3 editing types, FIG. 2 represents Indel, FIG. 3 represents SNP, and FIG. 4 represents deletion of a large fragment between two target sites. Obtaining these edit types in the sequencing results can prove the effectiveness of the two target sites.
SEQUENCE LISTING
<110> corn institute of agriculture academy of sciences (Shandong province corn engineering research center)
<120> maize ZmRLK7 gene editing target spot and application thereof
<130>2020
<160>11
<170>PatentIn version 3.3
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agcgcgatct cgtagcgcgc aattcggtgc ttgcgg 36
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tcctgcgctt tccctgtagt acaaacaatt catggggtct ccccctctac aatcccaatg 60
cttgcgagag gcgccacctc acagagccag ccatccaccg cattgccgcc cccctcgcct 120
ctcctcacgt cacccgcccg tctcagcctc accaccgagg cgcgcgctgc caatgccgcc 180
acggtcccgc gccgcggcac cgaggctcgc gtttctcgtg ccgctcgcct tcgccttcgc 240
cttgctgctg gtgccgccgt gccactgcgt caacgagcag ggccaggcgc tgctgcgatg 300
gaaggacacc ctgcggccgg cgggcggcgc gctggcgtcg tggcgcgccg gggacgcgag 360
tccgtgccgg tggaccggcg tgtcgtgcaa cgcgcgcggc gacgtcgtcg ggctgagcat 420
cacctcggtc gatctgcagg gccctctccc ggccaacctg cagccgcttg cagcgtcgct 480
gaagacgctg gagctctctg gcacgaacct caccggcgcg atacccaagg agatcggcga 540
gtacggcgag ctgaccaccc tcgaccttag caagaaccag ctcaccggcg cggtccccgc 600
cgagctgtgc cggctggcca agcttgagtc gctcgcgctc aactccaact ccctgcgtgg 660
agccatcccg gacgacatcg gcaacctcac cagcctgacg tatctgacgc tctacgacaa 720
tgagctcagt gggccgatcc cgcccagcat cggcaacctg aagaagctgc aggtgctccg 780
cgccggcggg aaccagggga tgaagggtcc cctgccgcag gagatcggcg gatgcactga 840
cctcaccatg ctcgggctcg cggagaccgg cgtctcaggg agcctcccgg agacgatcgg 900
gcagctcaag aagatccaga ccattgccat ctacaccact ctgctctccg gccggatccc 960
ggagtccatc ggcaactgca cccagctcac cagcctgtac ctgtaccaga attctctctc 1020
cgggccgata cctccgcagc tcggctacct caagaagctc cagactctgc ttctatggca 1080
gaaccagctc gtcggcgcaa ttcccccgga actcggacag tgcaaggagc tcacgctcat 1140
tgacctgtcg ctgaattcgc ttaccgggag catcccggcg agtttgggcg ggctacctaa 1200
tctccagcag ctgcagctga gcacgaacca gctcactggc accataccgc cggagctctc 1260
caactgcacg tcgctgacgg acatcgaggt cgacaacaac ttgctgtccg gggcgatcag 1320
catcgacttc ccgagactgc gcaacctcac cctgttctac gcgtggaaga accggctcac 1380
tggcggcgtg ccgacgagcc tcgccgaggc cccgagcttg caggcggttg acctgtcata 1440
caacaacctc accggtccca tccccaaggc gctgttcggg ctccagaact tgaccaagct 1500
gctgcttctc aacaacgagc tgaccgggct cataccgtcg gagatcggta actgcaccaa 1560
cctgtacaga ctccggctca acggcaacag gctgtccggc gcgattcccg ccgagatcgg 1620
caacctcaag aacctaaact tcctcgacat gagtgagaac cacctcgtcg gcccggtgcc 1680
cgcggccata tcggggtgcg ccagcctcga gttcctcgac ctgcactcca atgctctgtc 1740
cggcgcattg ccggacacgt tgccgcgcag tctccagctc attgacgtct ccgacaacca 1800
gctcaccggg ccgttgagct ccagcatcgg gtcattgccg gagctgacga agctgtacat 1860
gggaaacaac cggctgaccg gtggcatccc gcccgagctc ggttcgtgtg agaagctcca 1920
gctgctggac ctcggcggca acgcgttctc cggtggcatc ccgtcggagc tcgggatgct 1980
accgtcattg gagatctcgc ttaacctcag ctgcaaccgg ctttcagggg agataccgtc 2040
gcagttcgcc ggccttgaca agctcggcag cctcgacctg tcgcacaacg agctctccgg 2100
gagccttgag ccgctcgcgg cgctgcagaa cctcgtcacg ttgaacatat cctacaatac 2160
cttctctggg gagctcccga acactccctt cttccagaag ctgcccctca gcgacctagc 2220
cggcaaccgc catctcgtcg tcagcgacgg ctccgacgag tcctcccggc gtggcgtcat 2280
ctcgtcattc aagatagcca tatccatcct cgccgcagcc agcgcgctgc tcctggttgc 2340
cgccgcctat atgctcgccc gcacgcaccg ccgcggcggc ggccgcatca tccacggcga 2400
gggctcgtgg gaggtgacac tgtaccagaa gctcgacatc accatggacg acgtgctccg 2460
agggctgacg tccgcgaaca tgatcggcac cggcagctca ggggccgtgt acaaggtgga 2520
cacccccaac ggctacaccc tcgccgtgaa gaagatgtgg tcgtcggacg aggtgacgtc 2580
ggcggcgttc cgcagcgaga tcgcggcgct gggctccatc cgccaccgca acatcgtgcg 2640
cctcctcggg tgggccgcga acggcggcac gaggctgctc ttctacagct acctccccaa 2700
cggcagcctg agcggcctcc tgcacggcgg ccgcgccgcc aagggctcgc ccgcggacga 2760
gtggggcgcg cgctacgaga tcgcgctcgg cgtcgcccac gccgtggcgt acctgcacca 2820
cgactgcgtg ccggccatcc tgcacggcga cgtcaagtcc atgaacgtgc tgctcggcgc 2880
gtcctacgag ccgtacctcg ctgacttcgg cctcgcccgc gtcctggccg ccgcgagctc 2940
catgctcgac accggcaagc agccccgcat cgccggctcg tacggctaca tggcaccaga 3000
gtacgcgtcg atgcagcgga tcagcgagaa gagcgacgtg tacagcttcg gcgtcgtgtt 3060
gctggagatc ttgacggggc ggcacccgct ggacccgacg ctgtccggcg gcgcgcacct 3120
ggtgcagtgg ctgcgcgagc acgtgcaggc gaagcgtgac gcgtccgagc tactggacgc 3180
gcggctccgg gccagggcgg gcgaggcgga cgtgcacgag atgcggcagg tgctgtccgt 3240
ggccacgctg tgcgtgtcgc gccgcgcgga cgaccggccc gccatgaagg acgtggtggc 3300
gctgctcaag gagatccggc gccccgcggc ggtggacgac gcgaagcagc ggccacccac 3360
ggccgccgcg ccggtgtcgc cggtgagcgc gcactcgagg ggccaatcgt cgagctgctc 3420
cttcgccgtg tcggagtact ctgcctgacg ggacgcgaat ttttgcgagt gtagtgaacc 3480
tagtaaatta ctactaacat cttagcgagc cgtaaacagt gttgattagc caaagatgta 3540
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Claims (7)
1. The corn ZmRLK7 gene editing Target point is characterized in that the corn ZmRLK7 gene editing Target point comprises Target1 and Target2, the Target1 is positioned at 2462-2471bp of ZmRLK7 cDNA sequence, and the sequence is shown as SEQ ID NO. 1; the Target2 is positioned at the position 2737-2746bp of a ZmRLK7 cDNA sequence, and the sequence is shown as SEQ ID NO. 2; the ZmRLK7 cDNA sequence is shown in SEQ ID NO. 7.
2. The use of the maize ZmRLK7 gene editing target of claim 1 in editing the ZmRLK7 gene.
3. The method for editing the target gene ZmRLK7 by using the maize ZmRLK7 gene editing target spot as claimed in claim 1, which comprises the following steps:
(1) constructing a gene editing vector containing editing Target points of Target1 and Target 2;
(2) carrying out corn genetic transformation on the gene editing vector obtained in the step (1) to obtain a transgenic corn event;
(3) taking the tissue material of the transgenic corn event obtained in the step (2), extracting DNA for PCR amplification, sequencing an amplification product, and verifying the gene editing condition.
4. The method according to claim 3, wherein the method for constructing the gene editing vector in the step (1) specifically comprises the steps of:
s1, calling out the sequence of the maize ZmRLK7 gene in a gene bank, and selecting a target site sequence for gene editing;
s2 designing and synthesizing corresponding primer sequence according to the sequence of the target site;
s3, using CPB-ZmUbi-hspCas9 as a template, using the primer sequence in the step S2 as an amplification primer, carrying out PCR amplification by using high-fidelity enzyme, and amplifying a homology arm and a target region in the primer sequence in the step S2 to obtain a PCR amplification product;
s4, performing restriction enzyme digestion on CPB-ZmUbi-hspCas9 to form linearity, and obtaining a linearized vector;
s5, connecting the PCR amplification product obtained in the step S3 and the linearized vector obtained in the step S4 by using a recombinase to obtain a recombinant;
s6, sequencing the target point sequence of the recon obtained in the step S5, wherein the recon with correct sequencing is the gene editing vector.
5. The method of claim 4, wherein the primer sequences in step S2 include T1-F, T1-R, T2-F, T2-R;
the sequence of T1-F is shown as SEQ ID NO.3, the sequence of T1-R is shown as SEQ ID NO.4, the sequence of T2-F is shown as SEQ ID NO.5, and the sequence of T2-R is shown as SEQ ID NO. 6.
6. The method of claim 4, wherein the reaction system of the PCR amplification in step S3 is 25 μ l, and comprises the following components: 2 x High-fidelity Master Mix 12.5 u l, upstream and downstream primers 1 u l, plasmid 2 u l, adding sterile water 8.5 u l.
7. The method of claim 4, wherein the PCR amplification process of step S3 is: pre-denaturation at 98 ℃ for 1 min; denaturation at 98 ℃ for 10s, annealing at 68 ℃ for 15s, extension at 72 ℃ for 45s, and circulating for 35 times; overextension was carried out for 5min at 72 ℃.
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| CN109735512A (en) * | 2019-03-18 | 2019-05-10 | 华中农业大学 | Corn gene ZmACO2 is improving the application in corn yield |
| CN110066774A (en) * | 2019-04-30 | 2019-07-30 | 山东省农业科学院玉米研究所 | Corn receptoroid kinase gene ZmRLK7 and its application |
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| CN109735512A (en) * | 2019-03-18 | 2019-05-10 | 华中农业大学 | Corn gene ZmACO2 is improving the application in corn yield |
| CN110066774A (en) * | 2019-04-30 | 2019-07-30 | 山东省农业科学院玉米研究所 | Corn receptoroid kinase gene ZmRLK7 and its application |
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