CN116732091A - Cultivation method and detection method for corn with efficient utilization of potassium nutrition and herbicide resistance - Google Patents
Cultivation method and detection method for corn with efficient utilization of potassium nutrition and herbicide resistance Download PDFInfo
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- CN116732091A CN116732091A CN202310456152.1A CN202310456152A CN116732091A CN 116732091 A CN116732091 A CN 116732091A CN 202310456152 A CN202310456152 A CN 202310456152A CN 116732091 A CN116732091 A CN 116732091A
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Abstract
The invention discloses a cultivation method and a detection method for efficiently utilizing potassium nutrition of herbicide-resistant corn by transferring AlAKT1 gene and bar gene, and belongs to the technical field of biology. According to the invention, exogenous genes AlAKT1 and bar are introduced into a corn strain to obtain corn with high-efficiency potassium nutrition utilization and herbicide resistance, wherein AlAKT1 is a double-affinity potassium channel protein, and the potassium absorption efficiency of corn plants can be improved by over-expressing the AlAKT1 gene; the exogenous marker gene bar codes glufosinate-ammonium acetyl transferase PAT, so that the acceptor corn is endowed with the capability of resisting herbicide glufosinate-ammonium, the potassium absorption capability and the herbicide glufosinate-ammonium resistance capability can be improved, both the potassium channel protein gene AlAKT1 and the glufosinate-ammonium resistance gene bar can be stably inherited in the acceptor corn, and the efficient overexpression of the potassium channel protein gene can improve the efficient potassium nutrition utilization performance of the corn.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a cultivation method and a detection method for high-efficiency utilization of potassium nutrition of herbicide-resistant corn by transferring AlAKT1 genes and bar genes.
Background
Corn is one of three large grain crops in China, plays an important role in grain storage, feed and industrial raw material production, and the quality of the corn yield and the economic benefit directly influence the grain safety and the agricultural production development in China.
Potassium is the most abundant inorganic cation in plant cells, and is present in the plant body in the form of soluble inorganic salts or ions, accounting for about 2% -10% of the dry weight of the plant. Potassium is one of the essential nutrient elements in plants, and is involved in the processes of enzyme activation, osmotic regulation, cell expansion, membrane potential regulation, photosynthesis, maintenance of intracellular and extracellular osmotic pressure and the like, and plays a vital role in plant cells to influence the whole process of plant growth and development. The potassium deficiency of the plants can cause root system dysplasia, growth retardation, yellowing of leaves, reduced stress resistance and reduced crop yield in severe cases.
The improvement of the corn yield is not separated from the supply of potassium, the potassium supply at present mainly depends on the application of potassium fertilizer, but the self-sufficiency rate of the potassium fertilizer in China is low, the imported potassium fertilizer is high in price, the agricultural production cost is increased, and the problems of resource waste, environmental pollution and the like are caused. Not only ensures the yield, but also solves the problem of insufficient potassium fertilizer supply, and is one of the difficult problems that the agricultural development of China needs to overcome. Therefore, the cultivation of new varieties of low-potassium-resistant transgenic crops has attracted extensive attention, and becomes an important subject to be studied at present.
Disclosure of Invention
In view of the above problems in the prior art, the present invention aims to provide a cultivation method and a detection method for corn with efficient use of potassium nutrition and herbicide resistance, which are characterized in that exogenous genes AlAKT1 and bar are introduced into a corn strain to obtain corn with efficient use of potassium nutrition and herbicide resistance, wherein AlAKT1 is a double-affinity potassium channel protein, exogenous target gene AlAKT1 is from a halophyte festuca arundinacea, an open reading frame 2292bp codes a polypeptide composed of 763 amino acids, and the over-expression of AlAKT1 gene can improve the potassium absorption efficiency of corn plants; the exogenous marker gene bar, the glufosinate-resistant gene from streptomyces hygroscopicus, with the total length of 552bp, codes for glufosinate-acetyltransferase PAT, and endows the recipient corn with the herbicide glufosinate resistance.
In order to achieve the purpose of the invention, the following technical scheme is provided:
the invention provides a potassium channel protein gene AlAKT1 capable of realizing the efficient utilization of plant potassium nutrition, and the nucleotide sequence of the gene is shown as SEQ ID NO. 3.
Based on the technical scheme, the amino acid sequence of the potassium channel protein AlAKT1 is shown as SEQ ID NO.4.
In another aspect, the invention provides a recombinant expression vector containing potassium channel protein gene AlAKT1, which comprises an exogenous T-DNA gene, wherein the insertion sequence of the exogenous T-DNA gene is shown as SEQ ID NO. 5.
Based on the technical scheme, further, the exogenous T-DNA gene comprises a potassium channel protein gene AlAKT1 and a herbicide-resistant glufosinate-ammonium gene bar, the nucleotide sequence of the potassium channel protein gene AlAKT1 is shown as SEQ ID NO.3, and the nucleotide sequence of the herbicide-resistant glufosinate-ammonium gene bar is shown as SEQ ID NO. 8.
Based on the technical scheme, further, the exogenous T-DNA gene contains a potassium efficient utilization gene expression frame and a herbicide-resistant glufosinate-ammonium gene expression frame.
Based on the technical scheme, the potassium efficient utilization gene expression frame is further composed of a corn ZmUbi promoter, a potassium channel protein gene AlAKT1 and a nos terminator; wherein the ZmUbi promoter has the size of 2020bp, and the nucleotide sequence shown in SEQ ID NO.6 is a constitutive promoter, so that the target gene can be driven to express in corn; the nos terminator is 270bp in size, comes from the nopaline synthase gene terminator in the T-DNA region of the Ti plasmid of the agrobacterium tumefaciens, and stops transcription of the AlAKT1 gene, and the nucleotide sequence is shown as SEQ ID NO. 7.
Based on the technical scheme, further, the nucleotide sequence of the potassium efficient utilization gene is SEQ ID NO.3, the full length of an open reading frame is 2292bp, 763 amino acid polypeptide is encoded, the molecular weight of the protein is about 19.0kDa, the theoretical isoelectric point is 4.92, and the encoded amino acid sequence is SEQ ID NO.4.
Based on the technical scheme, the herbicide-resistant glufosinate-ammonium expression frame is composed of a CaMV35S promoter, a herbicide-resistant glufosinate-ammonium gene bar and a TVSP terminator, wherein the size of the CaMV35S promoter is 345bp, and the herbicide-resistant glufosinate-ammonium expression frame is from cauliflower mosaic virus CaMV and is responsible for starting the expression of the plant glufosinate-ammonium-resistant gene bar, and the nucleotide sequence is SEQ ID NO.10; TVSP terminator is 682bp in size, terminates bar gene transcription, and has the nucleotide sequence of SEQ ID NO.11.
Based on the technical scheme, further, the total length of the herbicide-resistant glufosinate-ammonium gene is 552bp, the nucleotide sequence is SEQ ID NO.8, PAT protein is formed by encoding, the sequence of the PAT protein is shown as SEQ ID NO.9, the protein belongs to an acetyl transferase family, the molecular weight of the protein is about 23kDa, the herbicide-resistant glufosinate-ammonium is acetylated, the activity of the glufosinate-ammonium is inhibited, and finally the effect of the glufosinate-resistant is achieved.
In another aspect, the invention provides agrobacterium strain EHA105 comprising a recombinant expression vector.
The invention also provides a method for improving the efficient utilization of corn potassium and herbicide resistance, which comprises the steps of constructing a potassium channel protein gene AlAKT1 from swertia, and a herbicide resistant glufosinate-ammonium gene bar from streptomycin hygroscopicus onto a pTF101 plant expression vector through an agrobacterium-mediated method, transferring the vector into a genome of corn KN5585, and screening to obtain transgenic corn with efficient utilization of potassium nutrition and herbicide resistance.
Based on the technical scheme, further, the exogenous T-DNA is inserted into chromosome 1 of the corn genome.
Based on the technical scheme, further, the nucleotide sequence of the left flank of the T-DNA is shown as SEQ ID NO.1, and the nucleotide sequence of the right flank of the T-DNA is shown as SEQ ID NO. 2.
Based on the above technical scheme, further, the exogenous T-DNA gene is inserted into the corn genome in a single copy form.
In another aspect, the invention provides a method for identifying transgenic corn that is highly efficient in potassium utilization and resistant to herbicides comprising:
(1) Extracting genomic DNA from a corn sample to be identified;
(2) Taking the extracted DNA sample as a template, and carrying out PCR fragment amplification of exogenous target genes AlAKT1 and marker gene bar by using the primer pair ALF-ALR and BarF-BarR;
(3) Detecting PCR amplified products, and if the length of the amplified products is consistent with the theoretical length between the sequences of the PCR primer pair on a transformation event, indicating that the sample is transgenic corn with high potassium nutrition utilization and herbicide resistance.
Based on the above technical scheme, further, the primer (AZ 2 is used for detecting whether the left side of the exogenous gene is connected with the corn genome specific site, AY2 is used for detecting whether the right side of the exogenous gene is connected with the corn genome specific site) of the method is as follows:
the PCR reaction system of the invention is as follows:
PCR reaction conditions:
the specific PCR reaction system of the invention is as follows:
specific PCR reaction conditions:
the nested PCR reaction system comprises:
nested PCR reaction conditions:
wherein the AZ2 nested primer pair detects whether the left side of the exogenous gene is connected with a corn genome specific site, and the expected amplification target fragment size is 272bp; the AY2 primer pair detects whether the right side of the exogenous gene is connected with a corn genome specific site, the size of a target fragment for expected amplification is 128bp, and whether the transgenic corn with high-efficiency potassium nutrition utilization and herbicide resistance is identified according to whether the target fragment which accords with the expected fragment size is amplified.
The invention also provides a method for cultivating the herbicide-resistant glufosinate-ammonium maize plant with high-efficiency utilization of potassium nutrition, which is characterized in that the transgenic maize plant is hybridized with maize breeding materials to obtain the filial generation with high-efficiency utilization of potassium nutrition and glufosinate resistance.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a method for cultivating corn with high-efficiency utilization of potassium nutrition of AlAKT1 gene and bar gene and herbicide tolerance, which can realize that exogenous genes are specifically introduced into corn lines, so that the receptor corn can improve the potassium absorption capacity and the herbicide tolerance glufosinate-ammonium capacity; the potassium channel protein gene AlAKT1 and the glufosinate-ammonium resistant gene bar can be stably inherited in the recipient corn; the efficient overexpression of the potassium ion channel protein gene can improve the efficient utilization of potassium nutrition of corn.
Drawings
In order to more clearly illustrate the embodiments of the present invention, the drawings to which the embodiments relate will be briefly described.
FIG. 1 is a schematic representation of a plant expression vector for maize transformation;
FIG. 2 shows the Agrobacterium-mediated transformation of maize plants (A: co-cultivation; B: screening; C: differentiation; D: regeneration of seedlings);
FIG. 3 is a PCR assay for transgenic maize plants (A: alAKT1 gene; B: bar gene), wherein M: DL2000; h: ddH 2 O; WT: a wild-type control; 1-13: transgenic AlAKT1 gene corn;
FIG. 4 shows PCR detection of the hybrid generation of AlAKT gene-transferred maize (A: alAKT1 gene, B: bar gene); wherein M: DL2000; h: ddH 2 O;1-5, transforming AlAKT1 gene corn to generate hybrid generation;
FIG. 5 shows the RT-PCR detection of the cross-bred first generation of different organs of maize transformed with AlAKT1 gene (A: RNA electrophoretogram; B: alAKT1 gene; C: bar gene), wherein M: DL2000; h: ddH 2 O; WT: a wild-type control; 1. 2, 3, 4, respectively represent roots, stems, leaves and seeds;
FIG. 6 shows bar test strip detection of AlAKT1 transgenic maize hybrid generation, wherein WT: wild type corn; 1-2: transforming AlAKT1 gene corn to generate first filial generation;
FIG. 7 is a schematic diagram of the insertion site of the T-DNA region of AlAKT1 transgenic maize;
FIG. 8 shows specific PCR detection of AlAKT1 gene-transferred maize transformants (A: AZ2; B: AY 2), wherein M: DL2000; h: ddH 2 O; WT: wild type; 1: transforming AlAKT1 gene corn to generate first filial generation;
FIG. 9 is 0.1mM K + Under the condition, alAKT1 gene corn hybrid generation and wild corn exhaustion result diagram is transferred.
Detailed Description
The invention is described in detail below with reference to specific embodiments, but not in any way limited to, but the embodiments described below are only some of the embodiments, and other similar embodiments are possible for a person skilled in the art without working inventive aspects, all falling within the scope of the invention.
Example 1 obtaining AlAKT1 Gene-transferred corn
1. Construction of maize expression vectors
The plant expression vector of the study is pTF101-AlAKT1-Bar (saved in university of Conduits), the main information elements are shown in Table 1, the map is shown in FIG. 1, and the nucleotide sequence of the T-DNA gene is shown in SEQ ID NO. 5. The sequence comprises a complete AlAKT1 gene expression frame and an anti-glufosinate expression frame which are used for high efficiency of potassium, and specifically comprises the following parts:
AlAKT1 gene expression cassette: consists of a corn ZmUbi promoter (the nucleotide sequence is shown as SEQ ID NO. 6), a potassium channel protein gene AlAKT1 (the nucleotide sequence is shown as SEQ ID NO. 3) and a nos terminator (the nucleotide sequence is shown as SEQ ID NO. 7).
Herbicide-resistant glufosinate expression cassette: consists of a CaMV35S promoter (the nucleotide sequence of which is shown as SEQ ID NO. 10), a herbicide-resistant glufosinate-ammonium gene bar (the nucleotide sequence of which is shown as SEQ ID NO. 8) and a TVSP terminator (the nucleotide sequence of which is shown as SEQ ID NO. 11). The herbicide-resistant glufosinate-ammonium expression frame is connected with the potassium efficient gene expression frame and is jointly inserted into a skeleton vector pTF101, so that pTF101-AlAKT1-Bar is constructed and obtained.
TABLE 1 information table of the major originals of pTF101-AlAKT1-Bar vector
2. Obtaining transformed plant by transforming young embryo of corn with agrobacterium
Selecting KN5585 maize immature embryo with pollination of 9-12d as an explant, infecting fresh peeled maize immature embryo with agrobacterium strain EHA105 containing pTF101-AlAKT1-bar plasmid, culturing in dark on a co-culture medium for 3d after infecting, transferring the immature embryo with enlarged volume and hardened texture to a recovery medium for culturing in dark for 6d, transferring the young embryo with good growth state to a screening medium for screening for 14d, at the moment, growing callus on partial young embryo, transferring to a fresh screening medium for culturing for 14d, at the moment, making the resistant callus in a more fluffy state, cutting the callus into pieces, transferring to a regeneration medium for culturing for 21d under illumination, transferring to a rooting medium for rooting after differentiating seedlings, and transferring the seedlings to a culture medium with components shown in Table 2. Seedlings grown to 10cm after rooting were transferred to outdoor soil culture to complete the transformation process (see fig. 2).
The method comprises the following specific steps:
(1) Recombinant plasmid extraction
Coli containing pTF101-AlAKT1-bar recombinant plasmid was cultured overnight at 37℃with 180rpm shaking table for 12 hours. The E.coli plasmid was extracted using the SanPrep column plasmid DNA miniprep kit.
(2) Preparation of Agrobacterium competent cells
(1) The agrobacterium EHA105 bacteria are kept in an ultralow temperature refrigerator at the temperature of minus 80 ℃ and taken out. Streaking on YEP plates containing 50mg/L rifampicin resistance, culturing in a constant temperature incubator at 28 ℃ for 48 hours;
(2) picking single colony and inoculating the single colony into a YEP liquid culture medium containing rifampicin, and culturing at 28 ℃ at 180rpm overnight;
(3) inoculating the shaken bacterial solution into YEP liquid culture medium without antibiotics according to the ratio of 1:100, and culturing at 28 ℃ at 180rpm until OD 600 ＝0.3-0.5；
(4) Placing the bacterial liquid in an ice-water mixture for ice bath for 30min, centrifuging at 4000rpm at 4 ℃ for 10min, and discarding the supernatant;
(5) 4mL of 20mM ice-cooled CaCl was added to the bacterial pellet 2 The solution was resuspended and centrifuged at 4000rpm at 4℃for 10min, the supernatant discarded;
(6) 1mL of 20mM ice-cooled CaCl was added to the bacterial pellet 2 The solution was resuspended, added to an equal volume of 50% glycerol and dispensed into 1.5mL centrifuge tubes with 200. Mu.L each. Quick-freezing in liquid nitrogen, and storing in a refrigerator at-80deg.C.
(3) Transformation of Agrobacterium
Transforming agrobacterium by freeze thawing method
(1) Thawing Agrobacterium competent cells on ice for 5min, adding 1 μg plasmid DNA after the competent cells are thawed, and gently mixing;
(2) ice bath for 30min, quick freezing with liquid nitrogen for 1min, water bath at 37 ℃ for 5min, and then adding YEP culture medium to make the final volume 1mL, and slowly shake culturing at 28 ℃ for 4-5h;
(3) centrifuging at 5000rpm for 2min, discarding 800 μl of supernatant, collecting bacterial heavy suspension, coating on YEP plate containing 50mg/L rifampicin and 100mg/L strong resistance, and culturing in a constant temperature incubator at 28deg.C for 2d in an inverted manner until single colony is grown;
(4) single colonies were picked and cultured overnight at 28℃at 180rpm in 10mL of YEP liquid medium containing rifampicin and resistance to observance;
(4) PCR detection of agrobacterium tumefaciens bacterial liquid
Taking 1 mu L of bacterial liquid as a PCR template, and carrying out bacterial liquid PCR detection on the ALF/ALR by using a primer pair on a target gene. And (3) picking out colonies positive in PCR detection, preparing agrobacterium according to the ratio of bacterial liquid to glycerol of 1:1, and storing in an ultralow temperature refrigerator at-80 ℃ for later use.
The detection primer pair ALF/ALR sequence of the AlAKT1 gene is as follows:
ALF:5'-AACAAGGCCATGCCTACACA-3'；
ALR:5'-TTGACCGTGGTAGGAGGACA-3'；
the size of the target fragment is 384bp.
TABLE 2 Medium for corn transformation
3. PCR detection of transformed plants
(1) Corn DNA extraction (Rapid plant genomic DNA extraction reagent purchased from Tiangen Co.)
(1) Shearing 0.1g of fresh corn leaves, placing the fresh corn leaves into a 2mL centrifuge tube, adding small steel balls sterilized at high temperature, and freezing the fresh corn leaves in liquid nitrogen for 15min;
(2) taking out the centrifuge tube, placing the centrifuge tube in an oscillator to oscillate for 1min, and ensuring that leaf tissues are fully destroyed;
(3) taking out the small steel balls, adding 400 mu L of buffer FP1 and 6 mu L of RNase A with the concentration of 10mg/mL, carrying out vortex oscillation for 1min, and standing at room temperature for 10min;
(4) adding 130 mu L buffer FP2, fully and uniformly mixing, and vortex oscillating for 1min;
(5) centrifuge at 12,000rpm for 5min and transfer the supernatant to another clean 1.5mL centrifuge tube.
(6) Repeating step (5) (this step is aimed at removing precipitated impurities from the supernatant to make the purity of the extracted genomic DNA higher);
(7) adding 0.7 times volume of isopropanol (e.g. 500 μl of supernatant plus 350 μl of isopropanol) to the supernatant, mixing thoroughly, centrifuging at 12,000rpm for 2min to obtain flocculent genomic DNA, discarding the supernatant, and retaining the precipitate;
(8) adding 600 μl of 70% ethanol, vortex oscillating for 5sec, centrifuging at 12,000rpm for 2min, discarding supernatant, and repeating the above steps once;
(9) uncovering and inverting, standing at room temperature for 30min, and thoroughly airing residual ethanol;
adding an appropriate amount of elution buffer TE, dissolving DNA in water bath at 65 ℃ for 30min, reversing and uniformly mixing for a plurality of times during the period of time to assist dissolution, and finally obtaining DNA solution.
(2) Primers used in PCR detection:
the detection primer ALF/ALR sequence of AlAKT1 gene is the same as above.
The detection primer of the Bar gene is the same as the BarF/BAR sequence.
(3) PCR reaction system:
(4) PCR reaction conditions:
the PCR amplified product was detected by agarose gel electrophoresis at 1% (10 ul of the PCR product loading).
The results showed (see FIG. 3) that the PCR products from AlAKT 1-transformed maize showed the desired band and the fragment length was identical to that expected, whereas the negative control (wild-type WT) showed no amplified band. The result shows that the exogenous target gene AlAKT1 and the marker gene bar are integrated into the corn genome, and the PCR result (see figure 4) of the first filial generation of the corn transformed with the AlAKT1 gene shows that the target gene AlAKT1 and the marker gene bar can be stably inherited.
Example 2: RT-PCR detection of AlAKT1 gene transgenic corn hybrid generation
1. RT-PCR detection of different tissues and organs of corn
(1) Total RNA extraction of different tissues and organs of corn (RNAiso Plus kit)
(1) Taking a certain amount of fresh plant tissue material, putting the fresh plant tissue material into liquid nitrogen, grinding the fresh plant tissue material into powder in the liquid nitrogen, quickly putting the powder into a precooled centrifuge tube containing 1mL of Trizol reagent, oscillating and uniformly mixing the powder, and standing the powder at room temperature for 10min to ensure that plant tissues are fully cracked;
(2) adding 200 μl of chloroform, slowly reversing and mixing thoroughly, standing at room temperature for 5min, and centrifuging at 12000rpm at 4deg.C for 15min;
(3) absorbing 500 mu L of supernatant into a new centrifuge tube, adding isopropanol with the same volume, reversing and uniformly mixing, standing at room temperature for 10min, and centrifuging at 12000rpm for 15min at 4 ℃;
(4) removing the supernatant, adding 1mL of precooled 75% ethanol solution, washing RNA precipitate with shaking, and centrifuging at 8000rpm at 4 ℃ for 10min;
(5) repeating the step (4) once;
(6) discarding the supernatant, inverting the centrifuge tube, blowing the centrifuge tube in an ultra-clean workbench until RNA is in a semitransparent state, and dissolving the centrifuge tube in 20 mu L of DEPC water;
(7) 1. Mu.L of the above solution was used for purity analysis and concentration measurement by Nanodrop2000c, and RNA was quantified to 500 ng/. Mu.L;
(8) RNA quality was detected by 1.5% agarose gel electrophoresis.
(2) Single-stranded cDNA acquisition
Reverse transcription reactions were performed according to the instructions of the PrimerScriptTM RT reagent Kit with g DNAEraser (Perfect Real Time) kit.
The reverse transcription system is as follows:
corn Total RNA 2. Mu.L
5×gDNAEraser Buffer 2μL
RNase free water Up to 10μL
Reaction conditions: 42 ℃ for 2min (this step is to degrade the genomic DNA fully)
Adding to the reaction solution:
reaction conditions:
37℃ 15min
85℃ 5s
4℃ ∞
(3) RT-PCR detection
The cDNA obtained by the reverse transcription is used as a template, and a semi-quantitative RT-PCR reaction is carried out by using the primers on the AlAKT1 and Bar genes.
Primer sequences and PCR reaction systems were as above.
PCR reaction conditions:
the results show that (see FIG. 5), the target gene AlAKT1 and the marker gene Bar can be stably and genetically expressed in different tissues organs (roots, stems, leaves and seeds) of corn.
Example 3: bar test strip detection for first-filial generation of transgenic corn
The area of the sheared leaf is about 1cm 2 Fresh corn leaves were placed at the bottom of a 1.5mL centrifuge tube, 500 μl EB2 Extraction Buffer was added to the tube, and the tube was thoroughly ground using a grinding rod, and after the corn leaves were homogenized, bar test strips were inserted in the indicated direction, and left to stand for 2min, the strips were observed. The presence of two bands (the detection band and the destination band) indicates bar protein expression; if only one detection band is present, this indicates that the bar protein is not expressed.
The results showed (see FIG. 6) that the bar gene was successfully expressed at the protein level.
Example 4: genetic stability analysis of transgenic maize
Selecting 30 seeds of a full transgenic corn hybrid generation, spraying herbicide when the corn is in a trefoil period and a heart period, spraying Basta solution with the concentration of 200mg/L twice a day, continuously spraying for 4 days, and enabling the plants without resistance to have leaf yellowing and wilting gradually until death, wherein the plants with resistance positive can grow normally. Counting the number of plants which grow normally and die, and carrying out chi-square test, wherein the result shows that the number ratio of the plants which grow normally to die is 14:16, χ 2 =0.1333, which is consistent with the mendelian split ratio, is presumed to be a single copy.
Example 5: flanking sequence analysis and transformant specific PCR of transgenic maize
1. Flanking sequence analysis
Extracting genome DNA of two-leaf and one-heart transgenic corn, comparing the flanking sequences of AlAKT1 gene insertion site with DNA as template and high-flux sequencing and relevant bioinformatics analysis, and obtaining corn genome DNA sequence of 400bp in the left flanking sequence (SEQ ID NO. 1) and corn genome DNA sequence of 390bp in the right flanking sequence (SEQ ID NO. 2). BLAST alignment of the obtained sequences in the maize database (http:// www.maizegdb.org /), revealed that the flanking sequences were located on maize chromosome 1, indicating integration of the AlAKT1 gene on maize chromosome 1.
Analysis of the flanking region of the insertion site revealed that: 107bp of deletion on the corn genome, 4bp of deletion on the left border of the T-DNA, but 12bp of unknown sequence is inserted into the site (left side of the left border of T-DNA insertion) along with the T-DNA, and 21bp of deletion on the right border of the T-DNA.
2. Transformant specific PCR detection
On the basis, a primer pair AZ1 and a nested primer pair AZ2 are designed near the left boundary of left-side corn genome sequence and T-DNA insertion, and a specific primer pair AY2 is designed on the right-side corn genome sequence and within the right boundary of right-side T-DNA insertion (see FIG. 7), so that the specific PCR detection of the transformant is performed.
TABLE 3 primers for transformant specific PCR detection
As can be seen from FIG. 8, the maize transformant ALK1 amplified the band of interest, whereas the negative control (wild-type WT) did not amplify the band of interest. The results indicate that the exogenous gene has been integrated into the maize genome and is capable of stable inheritance. Meanwhile, the primer pairs AZ2 and AY2 are used as specific primers of the corn transformant ALK1, and can be used for specifically identifying the corn transformant ALK1.
Example 6: k of transgenic maize AlAKT1 + Depletion experiment
Selecting five-leaf and one-heart wild type corn plants with consistent growth vigor and AlAKT1 gene transfer for 0.1mM K + Stress treatment. The results showed (see FIG. 9) that at 0.1mM K + The potassium absorption capacity of the AlAKT1 gene-transferred corn is better than that of the wild type after 24 hours of treatment.
Finally, it should be noted that: many possible variations and modifications may be made to the teachings of the invention, or equivalents may be made, without departing from the scope thereof. Therefore, any simple modification, equivalent variation and modification of the above embodiments according to the technical substance of the present invention shall still fall within the scope of the technical solution of the present invention.
Claims (10)
1. A recombinant expression vector containing potassium channel protein gene AlAKT1 is characterized by comprising an exogenous T-DNA gene, wherein the insertion sequence of the exogenous T-DNA gene is shown as SEQ ID NO. 5.
2. The recombinant expression vector according to claim 1, wherein the exogenous T-DNA gene comprises a potassium channel protein gene AlAKT1 and a herbicide-resistant glufosinate-ammonium gene bar, the nucleotide sequence of the potassium channel protein gene AlAKT1 is shown in SEQ ID No.3, and the nucleotide sequence of the herbicide-resistant glufosinate-ammonium gene bar is shown in SEQ ID No. 8.
3. The recombinant expression vector of claim 1, wherein the exogenous T-DNA gene comprises a potassium efficient use gene expression cassette and a herbicide-tolerant glufosinate gene expression cassette.
4. The recombinant expression vector according to claim 3, wherein the potassium efficient use gene expression cassette is composed of a maize ZmUbi promoter, a potassium channel protein gene AlAKT1 and nos terminator; wherein the nucleotide sequence of the ZmUbi promoter is shown in SEQ ID NO. 6; the nucleotide sequence of the nos terminator is shown as SEQ ID NO. 7; the herbicide-resistant glufosinate-ammonium expression frame consists of a CaMV35S promoter, a herbicide-resistant glufosinate-ammonium gene bar and a TVSP terminator, wherein the nucleotide sequence of the CaMV35S promoter is shown as SEQ ID NO.10; the nucleotide sequence of the TVSP terminator is shown as SEQ ID NO. 11; the nucleotide sequence of the herbicide-resistant glufosinate-ammonium gene bar is shown in SEQ ID NO. 8.
5. An agrobacterium strain comprising the recombinant expression vector of any of claims 1-4.
6. A method for improving the efficient utilization of potassium in corn and herbicide resistance, which is characterized in that the method is to transfer the recombinant expression vector in any one of claims 1-4 into the genome of corn KN5585 by an agrobacterium-mediated method, and screen to obtain transgenic corn with efficient utilization of potassium nutrition and herbicide resistance.
7. The method of claim 6, wherein the exogenous T-DNA is inserted into chromosome 1 of the corn genome; the exogenous T-DNA gene is inserted into the maize genome in a single copy.
8. The identification method of the herbicide-resistant transgenic corn with high-efficiency utilization of potassium nutrition is characterized by comprising the following steps of:
(1) Extracting genomic DNA from a corn sample to be identified;
(2) Using the extracted DNA sample as a template, and using a primer pair ALF-ALR and BarF-BarR to amplify PCR fragments of exogenous target genes AlAKT1 and marker genes bar;
(3) Detecting PCR amplification products, and if the length of the amplification products is consistent with the theoretical length between sequences of the PCR primer pair on a transformation event, the sample is transgenic corn with high-efficiency potassium nutrition utilization and herbicide resistance;
the nucleotide sequences of the primer pair ALF-ALR are respectively shown in SEQ ID NO. 12-13;
the nucleotide sequences of the primer pair BarF-BarR are shown in SEQ ID NO.14-15 respectively.
9. The method of claim 8, comprising detecting whether the left side of the exogenous gene is linked to a maize genome-specific site using an AZ2 nested primer pair; the AY2 primer pair detects whether the right side of the exogenous gene is connected with a corn genome specific site, and whether the transgenic corn which is high-efficiency in potassium nutrition utilization and herbicide resistant is identified according to whether a target band meeting the expected fragment size is amplified;
the nucleotide sequence of the AZ1 upstream primer is shown as SEQ ID NO.16, the nucleotide sequence of the AZ1 downstream primer is shown as SEQ ID NO.17, the nucleotide sequence of the AZ2 nest upstream primer is shown as SEQ ID NO.18, the nucleotide sequence of the AZ2 downstream primer is shown as SEQ ID NO.17, the nucleotide sequence of the AY2 upstream primer is shown as SEQ ID NO.19, and the nucleotide sequence of the AY2 downstream primer is shown as SEQ ID NO. 20.
10. A method for cultivating a maize plant with efficient utilization of potassium nutrition and herbicide resistance of glufosinate comprises the step of crossing the transgenic maize plant obtained in claim 6 with maize breeding materials to obtain a filial generation with efficient utilization of potassium nutrition and glufosinate resistance.
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