CN111876413A - Oligo DNA group of sgRNA of two site-directed knockout rice OsPLS4 genes - Google Patents
Oligo DNA group of sgRNA of two site-directed knockout rice OsPLS4 genes Download PDFInfo
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Abstract
The invention provides sgRNA for knocking out rice OsPLS4 gene. Designing a sgRNA sequence based on CRISPR/Cas9 aiming at a rice OsPLS4 gene, connecting a DNA fragment containing the sgRNA sequence to a vector carrying CRISPR/Cas9, transforming rice callus by an agrobacterium-mediated method, and realizing the knockout of the rice OsPLS4 gene through screening and identification. Wherein, the nucleotide sequence of the sgRNA action site is shown in SEQ ID NO. 1. According to the invention, the rice endogenous gene OsPLS4 is edited by a CRISPR-CAS9 technology, and an OsPLS4 knockout mutant is obtained. The sgRNA prepared by the method can efficiently, quickly and accurately target the OsPLS4 gene of rice, and has certain significance in basic research (molecular mechanism of early senescence of rice) and production practice (improvement of early senescence varieties and stress-resistant breeding of rice).
Description
Technical Field
The invention belongs to the field of plant genetic engineering. Specifically, the invention relates to two oligo DNA groups for site-directed knockout of sgRNA of rice OsPLS4 gene based on CRISPR-CAS9 technology and application thereof.
Background
The OsPLS4 gene codes 3-acyl reductase, the function of which is studied more in bacteria and animals, mainly participates in the second stage of head-on synthesis of Fatty acid, belongs to KR protease, and is an important component of Fatty acid synthase complex (FAS), while FAS enzyme is one of important complexes for Fatty acid synthesis. The fatty acid synthesis processes are respectively the de novo synthesis of saturated fatty acid, the extension of fatty acid carbon chain and the generation of unsaturated fatty acid.
In plants, epidermal waxes are the first barrier of plants against foreign and abiotic stresses, which are composed primarily of very long chain fatty acids and their derivatives, and fatty acid synthesis is therefore crucial for the formation of a waxy layer on the plant surface. In recent years, surface waxiness of plants is researched more and more, and a plurality of research results show that the accumulation of the waxiness on the surfaces of rice is reduced, so that the drought resistance and water retention capacity of the rice are reduced. Meanwhile, unsaturated fatty acids are very important for cold resistance of plants. Therefore, the research on the rice fatty acid synthesis related gene can further explore the mechanism of fatty acid regulation and control of plant surface wax synthesis, reveal the effect of fatty acid on plant stress resistance, and provide theoretical basis for grain safety production.
Recently, the CRISPR-CAS9 gene editing technology has been studied with great success on crops. Therefore, by using the technology, knocking out the fatty acid synthesis related gene OsPLS4 to obtain a mutant thereof has important effects on analyzing the function of the gene and clarifying a regulation mechanism of fatty acid in the growth, development and stress tolerance processes of rice. The invention obtains a transgenic plant by designing sgRNA to construct a gene editing vector, a transgenic technology and a CRISPR-CAS9 technology, and obtains a plant with OsPLS4 gene editing (knockout) and a sequence editing condition by sequencing analysis.
Disclosure of Invention
The invention aims to provide a method for knocking out a rice 3-acyl reductase gene OsPLS4, and provides sgRNA for knocking out rice OsPLS4 based on CRISPR-CAS9 technology and a vector for knocking out rice OsPLS4 gene. A method for targeted knockout of rice OsPLS4 by using CRISPR/Cas9 technology, which is characterized by comprising the following steps:
a) selecting two target sites of 672-690 and 766-784 nucleic acid sequences of the coding region of the OsPLS4 gene as target sequences of a CRISPR/Cas9 system (SEQ ID NO.1): TTACAGTGCTGCCAAGGCT
(SEQ ID NO.2):TGTCAGATGCAATGAAACC
Four oligo DNAs were designed based on the target sequence:
(SEQ ID NO.3)PLS4(1)F：GGCATTACAGTGCTGCCAAGGCT
(SEQ ID NO.4)PLS4(1)R：AAACAGCCTTGGCAGCACTGTAA
(SEQ ID NO.5)PLS4(2)F：GGCATGTCAGATGCAATGAAACC
(SEQ ID NO.6)PLS4(2)R：AAACGGTTTCATTGCATCTGACA
b) oligo DNA sets PLS4(1) F and PLS4(1) R, PLS4(2) F and PLS4(2) R are mixed, a dimer structure is formed through annealing reaction, and then the dimer structure is connected with a linearized pYL-Hs vector fragment to construct pYL-Hs-PLS4-cas9 plasmid containing two target sequences of the rice OsPLS4 gene.
c) The rice callus is infected by the plasmid containing pYL-PLS4-cas9, namely agrobacterium tumefaciens EHA105, and the transgenic rice plant is obtained through hygromycin screening and regeneration.
d) CX-PLS4-F (SEQ ID NO.7) and CX-PLS4-R (SEQ ID NO.8) specific primers are used for amplifying a sequence near a target sequence, sequencing an amplified genome fragment, identifying the editing condition of OsPLS4 and screening a knockout plant;
(SEQ ID NO.7)CX-PLS4-F:GCTTACAAGTTTAGTCAGGTTTC
(SEQ ID NO.8)CX-PLS4-R:TCGCGCAAATAGAAATCGTG
the invention has the advantages that after the gene editing vector pYL-Hs-PLS4-cas9 transforms rice, the rice OsPLS4 gene can be efficiently, quickly and specifically subjected to targeted editing and knockout, the gene can be directly used as a research material to discuss the functions and action mechanisms of the OsPLS4 gene, and a foundation is laid for better utilizing the OsPLS4 gene to carry out genetic improvement in production practice.
Drawings
FIG. 1 is a schematic structural view of OsPLS4 gene editing vector pYL-Hs-PLS4-cas9
FIG. 2pYL-Hs-PLS4-cas9 transgene identification map
FIG. 3 Gene editing of transgenic line No.2 OsPLS4
FIG. 4 Gene editing of transgenic line No.3 OsPLS4
FIG. 5 Gene editing of transgenic line No.8 OsPLS4
Detailed Description
The invention is further illustrated by the following specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. In the following examples, the techniques used are conventional and well known to those skilled in the art, unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 design and Synthesis of sgRNA sequence of OsPLS4 Gene of Rice
The coding region sequence of the rice OsPLS4 gene is shown in SEQ ID NO. 9.
In the embodiment, the CRISPR/Cas9 editing target sequence has the length of 19bp, and the 672-th 690 and 766-th 784 nucleic acid sequences located in the eighth and ninth exons of the conserved sequence of the coding region of the OsPLS4 gene are edited, wherein the edited target sequence is (SEQ ID NO.1): TTACAGTGCTGCCAAGGCT, (SEQ ID NO.2): TGTCAGATGCAATGAAACC, and the sequences are specific on a rice genome and have extremely low off-target probability.
Four oligo DNAs were synthesized from the target sequence:
(SEQ ID NO.3)PLS4(1)F：GGCATTACAGTGCTGCCAAGGCT
(SEQ ID NO.4)PLS4(1)R：AAACAGCCTTGGCAGCACTGTAA
(SEQ ID NO.5)PLS4(2)F：GGCATGTCAGATGCAATGAAACC
(SEQ ID NO.6)PLS4(2)R：AAACGGTTTCATTGCATCTGACA
example 2 construction of OsPLS4 editing vector pYL-Hs-PLS4-cas9
Two oligo DNA sets PLS4(1) F and PLS4(1) R, PLS4(2) F and PLS4(2) R were added ddH, respectively2Dissolving O to 10 mu M, mixing according to a reaction system, heating at 95 ℃ for 3 minutes, naturally cooling and annealing to enable PLS4(1) F/R and PLS4(2) F/R to form a dimer structure, forming the dimer structure through annealing reaction, and then respectively connecting with a vector pYL-Hs using a U3 promoter and a gRNA framework sequence fused with rice. The ligation products were transformed into E.coli by heat shock method, and single colonies were picked up and cultured in LB liquid medium (+ kan) for 12 hours, followed by sequencing. Selecting bacteria with correct sequencing to extract plasmid DNA, and obtaining an editing vector pYL-Hs-PLS4-cas9 containing a rice OsPLS4 gene target sequence, wherein a vector diagram is shown in figure 1.
Example 3, pYL-Hs-PLS4-cas9 vector Agrobacterium transformation
pYL-Hs-PLS4-cas9 plasmid is transformed into agrobacterium by a freeze-thaw method, 10 mu L plasmid DNA is added into 200 mu L agrobacterium EHA105 competence, ice bath is carried out for 30min, liquid nitrogen is used for freezing for 3min, water bath at 37 ℃ is carried out for 5min, 1mL YEB culture medium is added, and shaking culture is carried out at 28 ℃ for 3-4 h. The cells were centrifuged at 6000rpm at room temperature for 1min, the supernatant was discarded, 200. mu.L of YEB medium was added to resuspend the cells, and they were plated on YEB solid medium (+ rifampicin) and cultured at 28 ℃ for 3 days. Selecting single colony for identification.
Example 4 transformation of Rice
In the implementation, the Zhonghua 11 is used as a receptor to carry out agrobacterium transformation. Selecting about 300 seeds of medium flowers 11, removing shells, soaking for 1 minute by using 75% ethanol, pouring off the 75% ethanol, sterilizing for 30 minutes by using a sodium hypochlorite solution, washing for 6 times by using sterile water, sucking water by using sterile gauze, then planting the seeds on an NB culture medium containing 2,4-D (2mg/L) for 2 weeks in a dark place at 26 ℃, and selecting the calli which grow vigorously to be used as a transformed receptor. The rice calli were infected with an engineered bacterial solution prepared from EHA105 strain containing an editing vector (pYL-Hs-PLS4-cas9), cultured in the dark at 25 ℃ for 3 days, and then cultured on a selection medium containing 50mg/L of Hygromycin for about 14 days (light intensity 13200LX, temperature 32 ℃). Transferring the pre-differentiated callus to a differentiation medium, and culturing the pre-differentiated callus under the illumination condition (the illumination intensity is 13200LX, and the temperature is 32 ℃) for about one month to obtain a resistant transgenic plant. Using 1/2MS culture medium to take root and strengthen seedling to obtain T0 generation plant, transplanting into field to plant.
Example 5 analysis of OsPLS4 Gene editing
DNA is extracted from T0 plant leaves, primers hyg-F and hyg-R are designed according to hygromycin gene sequence, and positive transgenic plants are determined, as shown in figure 2. Then, positive transgenic plants were amplified using the primers SQ-OsPLS4-F and SQ-OsPLS4-R (FIG. 3), and the sequences near the target sites of OsPLS4 gene were analyzed by sequencing (FIGS. 4-5 are sequencing results of partially edited plants), and the statistics of the overall results are shown in Table 1. The hygromycin gene and the sequence amplification primer sequence near the target site are as follows:
SQ-OsPLS4-F(SEQ ID NO.7)：GCTTACAAGTTTAGTCAGGTTTC
SQ-OsPLS4-R(SEQ ID NO.8)：TCGCGCAAATAGAAATCGTG
TABLE 1 analysis of OsPLS4 gene coding conditions
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.
Sequence listing
<110> university of agriculture in Jiangxi
<120> oligo DNA set of sgRNA of two site-directed knockout rice OsPLS4 genes
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aaggtcctgg tgaactatgc ccgatcctca aaagaggctg aagaagtctc caaagagatc 360
gaagcatgtg gtggtcaggc tattaccttc gggggagatg tttcaaaaga agccgatgtg 420
gattctatga tgaaagcagc tcttgataaa tggggaacaa ttgatgtgct ggtaaacaat 480
gcaggaatta cccgagacac attattaatg aggatgaaga aatcacaatg gcaagacgta 540
attgacctga atcttactgg tgttttcctc tgtacacaag ctgctacaaa aataatgatg 600
aagaagaaaa agggaaaaat catcaacata gcatcagttg ttggtcttgt tggtaatatt 660
ggccaagcta attacagtgc tgccaaggct ggggttattg gtttgacgaa aacagtagct 720
agggaatatg caagcagaaa tatcaatgtg aatgcaattg cacctggttt cattgcatct 780
gacatgactg ctgaacttgg agaggatctt gagaagaaaa tcttgtcaac catcccatta 840
gggagatatg gcaaaccaga ggaggttgct ggcttggttg agtttttggc tctcaatcct 900
gcggccaact acatcacggg acaggttctt accatcgatg gagggatggt gatgtag 957
Claims (4)
1. The oligo DNA group of sgRNA of two site-directed knockout rice OsPLS4 genes is characterized in that: the target sites of two sgRNAs comprising an OsPLS4 gene have DNA sequences shown as SEQ ID No.1 and SEQ ID No.2, one oligo DNA group is PLS4(1) F and PLS4(1) R, the other oligo DNA group is PLS4(2) F and PLS4(2) R, PLS4(1) F nucleotide sequence is shown as SEQ ID No.3, PLS4(1) R nucleotide sequence is shown as SEQ ID No.4, PLS4(2) F nucleotide sequence is shown as SEQ ID No.5, and PLS4(2) R nucleotide sequence is shown as SEQ ID No. 6.
2. The use of the oligo DNA set of sgRNA of two site-directed knockout rice OsPLS4 genes according to claim 1 in rice OsPLS4 gene editing and breeding.
3. A carrier for site-directed knockout of sgRNA of a rice OsPLS4 gene is pYL-Hs-PLS4-cas9, and is characterized in that the carrier is prepared by mixing oligo DNA groups PLS4(1) F, PLS4(1) R, PLS4(2) F and PLS4(2) R, forming a dimer structure through annealing reaction, and then connecting the dimer structure with a linearized pHY-HS carrier fragment to construct an pYL-Hs-4-cas 9 plasmid containing two target sequences of the rice OsPLS4 gene, wherein the PLS4(1) F nucleotide sequence is shown as SEQ ID No.3, the PLS4(1) R nucleotide sequence is shown as SEQ ID No.4, the PLS4(2) F nucleotide sequence is shown as SEQ ID No.5, and the PLS4(2) R nucleotide sequence is shown as SEQ ID No. 6.
4. A primer pair for identifying the editing condition of an sgRNA target region of an OsPLS4 gene, which is characterized in that: the DNA sequence of the primer pair is shown as SEQ ID NO.7 and SEQ ID NO. 8.
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