CN110157715B - Experimental method for knocking out ZmPAD1 gene to improve yield of straight-chain corn - Google Patents
Experimental method for knocking out ZmPAD1 gene to improve yield of straight-chain corn Download PDFInfo
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Abstract
The invention discloses an experimental method for knocking out ZmPAD1 gene and improving yield of straight-chain corn, which belongs to the field of genetic engineering and mainly comprises the following steps: constructing a ZmPAD1 gene knockout CRISPR/Cas9 vector to obtain a ZmPAD1 gene knockout high-amylose corn plant. The method utilizes the interaction of ZmPAD1 protein on corn starch branching enzyme SBEIIb, and improves the average weight of corn kernels by adjusting the modification effect of ZmPAD1 on the starch synthesis process in which a main gene ZmSBEIII participates. The realization of the genetic engineering method has important practical significance for improving the yield of the special corn by utilizing the starch synthesis modified gene.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to an experimental method for knocking out ZmPAD1 gene and improving the yield of straight-chain corn.
Background
Amylose contained in high-amylose corn has unique physical and chemical properties, and is widely applied to more than 30 fields of food, medical treatment, textile, paper making, packaging, petroleum, environmental protection, optical fibers, high-precision printed circuit boards, electronic chips and the like. The high amylose corn starch as resistant starch (resistant starch) slowly releases glucose in human body, has low insulin reaction, can control blood sugar balance of diabetic patients, binds lipid in intestinal tract, reduces lipid absorption of human body, and can reduce risk of colon cancer. In the United states, native corn starch is sold at a price of about 20 cents/pound, while high amylose corn starch can be sold at a price of up to $ 2.5/pound, which is seen to be of considerable economic benefit. 80. In the early years, the American Casstein Seed company (Custom Farm Seed) utilizes ae (ZmSBEIIb) and a modifier gene thereof, a recurrent selection method is adopted to culture 7-grade (70% -80% of amylose) high-amylose corn hybrid, and the modifier gene is accumulated and selected through the recurrent selection method for more than 10 years to obtain a high-amylose corn variety with the amylose content of more than 85%. However, unlike common dent corn, the high amylose corn seeds have a lower germination rate, poorer soil arching ability and poor plant type development, and the direct effect of pollen produced by non-amylose corn pollen can also reduce the quality of the high amylose corn, the yield is not only extremely unstable, but also reduced by more than 20% compared with dent corn and waxy corn hybrids, and even in the united states, the planting area of the high amylose corn is not more than 2 ten thousand hectares, and most of the high amylose corn is signed planting. Therefore, the upward research on the yield of the high amylose corn is widely regarded and becomes a latest research hotspot and an urgent problem in the corn production science at home and abroad.
At present, the formation of corn high amylose starch is mainly based on the mutation of the ae gene and is premised on the adjustment of a high amylose metabolic modification gene. In recent years, the development of protein interaction research technology is not only beneficial to quickly searching for modifying genes involved in regulation and control of genetic phenotype of plants, but also can deepen the research of plant growth and development mechanism on cellular and protein molecular level. The research on the regulation and control mechanism of starch metabolism modifying genes on endosperm starch synthesis is one of effective ways for detecting the low yield of high amylose corn. After the ZmSBEIIb gene affecting starch branching in corn endosperm is inhibited by antisense RNA, the amylose content of the ZmSBEIIb gene is greatly improved (International patent W094/04693A 2), but the research on the regulation molecular mechanism of starch metabolism by a modifier gene related to the expression degree of the ZmSBEIIb main gene is blank, which is probably also a theoretical bottleneck that the high amylose corn yield cannot break through upwards in practice. On the basis that ZmPAD1 is identified to be an effective interaction regulatory factor of SBEIIb by a series of candidate experimental technologies, zmPAD1 genes of high amylose corn are knocked out by using a CRISPR/Cas9 system, so that the average weight of corn grains can be effectively improved.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an experimental method for knocking out ZmPAD1 gene and improving the yield of straight-chain corn.
The invention is realized by the following technical scheme:
the invention provides an experimental method for knocking out ZmPAD1 gene to improve yield of straight-chain corn, which comprises the following steps:
constructing a ZmPAD1 gene knockout CRISPR/Cas9 vector to obtain a ZmPAD1 gene knockout high amylose corn plant; the average weight of corn grains of the ZmPAD1 gene knockout high amylose corn plant is higher than that of a non-transformed plant; the corresponding nucleotide sequence of sgRNA in the ZmPAD1 gene knockout CRISPR/Cas9 vector is SEQ ID NO.1, wherein a sgRNA promoter is OsU3, a promoter of Cas9 protein is an enhanced corn Ubi promoter, and a promoter of a kanamycin resistance gene is CaMV 35S.
Further, the ZmPAD1 gene in the special corn of interest is knocked out by adopting a gene editing method, and the method comprises the following steps:
determining a sgRNA targeting sequence (SEQ ID NO. 1) according to the ZmPAD1 gene sequence;
based on the polynucleotide of the sgRNA targeting sequence, adding A to the 5 'of the sense strand and adding T to the 5' of the antisense strand;
annealing sense and antisense oligonucleotide double strands to form a dimer, and then connecting the dimer with a modified CRISPR/Cas9 vector to construct a eukaryotic expression recombinant plasmid;
amplifying the constructed CRISPR/Cas9 plasmid in bacteria and then transferring the amplified CRISPR/Cas9 plasmid into agrobacterium;
and (3) introducing CRISPR/Cas9 plasmids into high amylose corn by using agrobacterium, and screening to obtain a transgenic plant with ZmPAD1 gene silencing.
According to the invention, on the basis of confirming that ZmPAD1 is a modifying gene for regulating starch anabolism, zmPAD1 gene of high amylose corn is knocked out by using a genetic engineering technology, so that the average weight of corn grains can be effectively increased. The research on the possible mechanism that ZmPAD1 influences the corn endosperm starch metabolism is expected to lead the research on the problem of low yield of the high amylose corn to be developed in a breakthrough way.
Drawings
FIG. 1 is a graph of the identification of co-immunoprecipitation by interaction of ZmPAD1 with ZmSBEII.
FIG. 2 is a CRISPR/Cas9 vector map.
FIG. 3 is a PCR identification diagram of the ZmPAD1 gene of transgenic corn.
FIG. 4 is a histogram of the weight of corn kernels and the amylose content ratio of transgenic and control groups.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1.
The embodiment provides an experimental method for improving yield of straight-chain corn by knocking out ZmPAD1 gene, which comprises the following steps.
(1) The co-immunoprecipitation (co-IP) technique verifies the interaction of ZmPAD1 with ZmSBEIIb.
The interaction relationship between ZmSBEIIb and ZmPAD1 is detected by carrying out co-immunoprecipitation by using anti-ZmSBEIIb and anti-ZmPAD 1 antiserum owned by the laboratory. 5 g of 20-DAP Jdan 264 endosperm material was ground to powder in liquid nitrogen and ground again after addition of 10 ml protein extraction buffer (0.05M NaAc, 0.02M DTT, pH 6.0). After centrifugation (6000 g, 10 min, 4 ℃ C.), the supernatant was retained (-80 ℃ C.) for further use. Mu.l of anti-ZmSBEIIb antiserum or preimmune serum was added to 1 ml of the soluble protein extract, incubated at 0 ℃ for 2 hr, then 50. Mu.l of protein A/G-sepharose was added, the precipitate was separated after centrifugation, and the precipitate was washed 3 times with buffer (50 mM Tris-HCl and 150 mM NaCl, pH 7). Finally, the coprecipitated protein is separated by 12% SDS-PAGE, transferred to a 12% SDS-PAGE membrane, and the coprecipitated product is detected by antiserum against ZmPAD 1. The results of this experiment indicate that ZmSBEIIb and ZmPAD interact in vivo (FIG. 1).
(2) ZmPAD1 SgRNA targeting sequence design.
Downloading a ZmPAD1 (GenBank accession number: EF 406364) gene sequence, comparing the gene sequence with a genome sequence of a PE0075 corn strain in a MaizeGDB database, and determining that a first exon sequence is 1-223 amino acid residues in a ZmPAD1 transcript. The SgRNA sequences were obtained using an online SgRNA targeting sequence design tool (https:// zlab. Bio/guide-design-resources) as follows: GGGAGAAACGGTGATGGCG. When synthesizing DNA double-chain, adding BstN I (CC ^ AGG) enzyme cutting site in two sections of the sequence.
(3) And (5) constructing CRISPR/Cas9 plasmid.
Separately synthesizing Forward oligo: AGGGAGAAAACGGTGATGGCG and Reverse oligo: TCGCCATCACCGTTTTCCC. Mu.l of each of 100. Mu.M oligo was added to a 10. Mu.l system, heated to 95 ℃ for 5 min using a PCR instrument, slowly cooled to room temperature for 1 hr, and the dimer was diluted to 1.
Mu.g of CRISPR/Cas9 plasmid (FIG. 2) was digested with 10. Mu.l system (NEBuffer. RTM. 2.1) according to the BstN I digestion instructions and incubated at 60 ℃ for 1 hr. The plasmid fragments were recovered from the gel and stored at-20 ℃ for further use. Mu.l plasmid and 6. Mu.l dimer were ligated overnight at 16 ℃ using T4 DNA ligase, 2. Mu.l ligation product was taken, competent DH 5. Alpha. Was transformed, cultured at 37 ℃ for 16 hr, monoclonal colonies were picked, plasmid was extracted by alkaline lysis miniprep and PCR was performed. After the identification is correct, the sequence is sent to a company for sequencing. Single colonies of CRISPR/Cas9 plasmid containing the correct insert were stored at-80 ℃.
(4) The CRISPR/Cas9 plasmid was transformed to agrobacterium LBA4404.
Taking out the agrobacterium LBA4404 strain from a refrigerator at the temperature of-80 ℃,streaking at 28 deg.C (TYNG/Rif, TYNG: 10 g/L Bacto-tryptone,5 g/L Yeast extract,5 g/L NaCl, 0.2 g/L MgSO 4 pH 7.5, final Rif concentration 50. Mu.g/ml), after forming a single clone, a single colony was picked up and cultured overnight at 28 ℃ in 5ml TYNG/Rif/Kan (Kan, 50. Mu.g/ml) medium. The next day, 0.5 ml of the culture was inoculated to 60 ml of TYNG/Rif/kan and cultured overnight (225-250 rpm) at 26.5 ℃ with shaking. Precooling centrifugal tube and sterile CaCl on ice at noon of the next day 2 Solution (20 mM), and precooled centrifuge, the culture was placed on ice for 10 min, centrifuged at 5200 rpm at 4 ℃ for 6 min, the supernatant discarded, and then 1 ml of precooled CaCl was used 2 Rinsing the solution, centrifuging, discarding the supernatant, and adding 1 ml CaCl 2 The solution was resuspended, split-charged and stored frozen at-70 ℃. Mu.l of plasmid (40 ng/. Mu.l, 3. Mu.g total) was added to 150. Mu.l of ice-thawed competent LBA4404, gently flicked to mix well, placed in liquid nitrogen for 5 min, left at room temperature for 5-10 min, added with 1 ml of TYNG (without antibiotics), incubated overnight at 200 rpm (28 ℃), and the culture poured into plates (TYNG/Rif/Kan, 25. Mu.g/ml each of Rif and Kan) and incubated at 28 ℃.
(5) Agrobacterium-mediated transformation of maize callus.
The agrobacterium-positive single colony is picked up for overnight culture (LB/Rif/Kan, 3 ml), and is added into 50 ml of culture medium (without resistance) for amplification culture until OD is 0.5. And taking the well-grown corn callus 14-20 days after three successive generations, clamping the corn callus into small blocks with the size of two soybeans by using forceps, soaking the small blocks into the bacterial liquid for 20 min, taking out the small blocks, sucking the redundant bacterial liquid by using sterile filter paper, and culturing the small blocks on an MS agar plate without resistance for two days. The calli were washed 4 times with sterile water, then soaked in sterile water containing Amp 500. Mu.g/ml for 60 minutes, the calli were removed and blotted dry with sterile filter paper and placed on MS agar plates containing double antibody (Amp 500. Mu.g/ml and Kan 25. Mu.g/ml) until new calli grew out (about 2-3 weeks). The new calli were transferred to plates containing the resistance of the desired gene (Kan 25. Mu.g/ml) and cultured for further generations every four weeks.
(6) And (5) performing differentiation culture.
The differentiation culture medium consists of an MS culture medium, inositol, sucrose, 6-BA and plant gel, wherein the concentration of the inositol is 0.15 g/L, the concentration of the sucrose is 30 g/L, the concentration of the 6-BA is 0.5 mu g/ml, and the concentration of the plant gel is 8 g/L. The differentiation culture conditions were 28 deg.C, and the culture was performed under light irradiation for 16 days at 18 hr per day. After rooting and seedling strengthening, transplanting into a field.
(7) And (5) identifying the transgenic plant.
The F0 generation corn leaf tissue is put into a 1.5 ml centrifuge tube, then 750 mul of DNA extraction buffer solution is added, and the mixture is fully mashed by a gun head and then mixed evenly. Placing the centrifuge tube in 65 deg.C water bath for 1-2 hr, and gently mixing several times during the water bath process. The centrifuge tubes were removed, and an equal volume of 600 to 700. Mu.l phenol/chloroform (V/V = 1) solution was added to each tube, and after mixing, centrifugation was performed at 10000 rpm for 10 min. Transferring the supernatant into another centrifuge tube, adding equal volume of chloroform, mixing uniformly at 10000 rpm for 6 min. Transferring the supernatant to another centrifuge tube, adding 0.6 times volume of isopropanol, mixing, centrifuging at 10000 rpm for 10 min, washing with 70% ethanol for 2 times, drying, dissolving in 500 μ l TE, adding 3 μ l RNase solution, and keeping the temperature at 37 deg.C for 1 hr. Adding equal volume of phenol/chloroform solution, mixing, and centrifuging at 10000 rpm for 6 min. And taking the supernatant, adding chloroform with the same volume, gently mixing uniformly, and centrifuging at 10000 rpm for 6 min. Taking the supernatant, adding 1/10 volume of 3M sodium acetate, mixing uniformly, and adding 2 times volume of cold absolute ethyl alcohol. 10000 Centrifuging at rpm for 6 min, washing with 70% ethanol for 2 times, drying, and dissolving in 50 μ l TE solution at-80 deg.C. The ZmPAD1 detection primers are as follows: (5 'AAGCAAGCTCAAACCCTAA) -3', 5 'ACTGTCGCTGTCCAGATGA-3'). Denaturation at 94 ℃ for 1min, annealing at 58 ℃ for 1min, and elongation at 72 ℃ for 1min for 25 cycles. The amplification products were detected on a 1.0% agarose gel, and the results are shown in FIG. 3. Sequencing verification of the PCR product shows that the ZmPAD1 gene is knocked out.
(8) Starch quality and amylose content determination.
Random selection of T for 3 transgenic event lines 1 And taking 60 corns, drying the corns at 50 ℃ overnight, and weighing the corns. After repeating the steps for 3 times, measuring the corresponding amylose content by using a GB/T15683-2008 standard method. As can be seen from FIG. 4, in the high amylose line, the quality of corn grains is obviously improved by knocking out ZmPAD1 geneThe height range is 18.8 to 22.4 percent. The composition ratio of amylose and amylopectin did not change significantly.
And (4) conclusion: the invention obtains the ZmPAD1 gene knockout high amylose corn strain by using a genetic engineering technology. T is 1 The average quality of the corn seeds is obviously improved, the improvement range is 18.8 to 22.4 percent, but the composition ratio of amylose and amylopectin is not obviously changed, which shows that ZmPDA1 has the modification effect of starch anabolism. The interaction relationship of ZmPDA1 and SBEIIb shows that the ZmPDA is possibly an effective regulator of SBEIIb, and provides new experimental evidence for perfecting understanding of starch metabolism.
The above is a detailed embodiment and a specific operation process of the present invention, which are implemented on the premise of the technical solution of the present invention, but the protection scope of the present invention is not limited to the above-mentioned examples.
Sequence listing
<110> chenchao
Long Kai
Zhang Jiaxi
By over-white
Huang Ruipeng
Li Zhipeng
<120> experimental method for knocking out ZmPAD1 gene to improve yield of straight-chain corn
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 20
<212> DNA
<213> corn (Zea mays)
<400> 2
Claims (2)
1. A method for knocking out ZmPAD1 gene to improve yield of high linear chain corn is characterized by comprising the following steps: constructing a ZmPAD1 gene knockout CRISPR/Cas9 vector to obtain a ZmPAD1 gene knockout high amylose corn plant; the average weight of corn grains of the ZmPAD1 gene knockout high amylose corn plant is higher than that of a non-transformed plant; the corresponding nucleotide sequence of SgRNA in the ZmPAD1 gene knockout CRISPR/Cas9 vector is SEQ ID No.1, wherein the SgRNA promoter is OsU3, the promoter of Cas9 protein is a reinforced maize Ubi promoter, the promoter of kanamycin resistance gene is CaMV 35S, and the accession number of the ZmPAD1 gene in GenBank is EF406364.
2. The method for improving the yield of the high-amylose corn by knocking out the ZmPAD1 gene according to claim 1, wherein the ZmPAD1 gene in the special corn of interest is knocked out by adopting a gene editing method, and the method comprises the following steps of: determining the SgRNA targeting sequence as follows according to the ZmPAD1 gene sequence: SEQ ID No.1; based on the polynucleotide of the SgRNA targeting sequence, adding A to the 5 'of the sense strand and adding T to the 5' of the antisense strand; annealing sense and antisense oligonucleotide double strands to form a dimer, and then connecting the dimer with a modified CRISPR/Cas9 vector to construct a eukaryotic expression recombinant plasmid; amplifying the constructed CRISPR/Cas9 plasmid in bacteria and then transferring the amplified CRISPR/Cas9 plasmid into agrobacterium; and introducing the CRISPR/Cas9 plasmid into the high amylose corn by using agrobacterium, and screening to obtain a ZmPAD1 gene knockout transgenic plant.
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