Rice high-affinity potassium ion transporter encoding gene OsHAK12 and application in controlling rice potassium content and low potassium tolerance

Abstract

This invention discloses the application of the rice high-affinity potassium ion transporter gene OsHAK12. The sequence of the high-affinity potassium ion transporter gene OsHAK12 is shown in SEQ ID NO.1. This gene can be applied to control the potassium content of rice and to improve tolerance to low potassium. Through extensive experiments, this invention has discovered the biological function of the rice potassium ion transporter gene OsHAK12. Knocking out this gene leads to a decrease in the potassium content of rice grains and a significant decrease in the seedlings' tolerance to low potassium.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology and relates to the application of the rice high-affinity potassium ion transporter gene OsHAK12. Background Technology

[0002] Potassium is an essential macronutrient for plant growth and development, participating in many physiological processes in plant cells, such as enzyme activation, membrane potential maintenance, and osmotic regulation (Rozhdestvenskii et al. 1975; Wang and Wu 2013). Potassium deficiency is a common abiotic stress that significantly restricts agricultural production (Chen et al. 2008). In agricultural production, the rational application of potassium fertilizer is crucial for promoting crop growth and increasing yield. However, simply increasing the amount of potassium fertilizer applied cannot completely solve the problem, as excessive fertilizer input not only increases production costs but may also cause environmental problems. Therefore, improving the potassium use efficiency of crops has become an important strategy for reducing fertilizer costs and increasing crop yield.

[0003] Potassium use efficiency in rice is a complex quantitative trait involving multiple genes and biological processes, primarily including potassium absorption, distribution, translocation, and recycling (Rengel and Damon 2008). QTL (Quantitative Trait Loci) analysis is a method for determining the genetic basis of quantitative traits through genetic linkage and correlation analysis. By constructing genetic linkage maps, the genetic relationship between quantitative traits and molecular markers is determined. The content of most mineral nutrients in rice grains exhibits significant natural variation. Using two rice hybrids to obtain a mapping population, genetic maps are constructed and phenotypic data are collected. Further association analysis is then conducted to locate the QTLs controlling potassium accumulation in rice grains. Identifying genes controlling potassium accumulation allows for their application in rice breeding to develop rice varieties with higher or more stable potassium accumulation. These new varieties can not only improve rice yield and quality but also help farmers reduce potassium fertilizer usage, lower production costs, and contribute to environmental protection. Summary of the Invention

[0004] The purpose of this invention is to address the above-mentioned shortcomings of the prior art by providing an application of the rice high-affinity potassium ion transporter gene OsHAK12.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] The genomic nucleotide sequence of the rice high-affinity potassium ion transporter gene OsHAK12 described in this invention is shown in SEQ ID NO.1.

[0007] Furthermore, the CDS sequences of the two transcripts encoded by the rice high-affinity potassium ion transporter gene OsHAK12 are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0008] As a preferred embodiment of the present invention, knocking out the rice high-affinity potassium ion transporter gene OsHAK12 leads to a decrease in the potassium content of rice grains and a decrease in potassium absorption capacity during the seedling stage.

[0009] As a further preferred embodiment of the present invention, the knockout vector used to knock out the rice high-affinity potassium ion transporter gene OsHAK12 is a vector based on the pYLCRISPR / Cas9Pubi-H vector, in which the target sequence T1 and target sequence T2 of the high-affinity potassium ion transporter gene OsHAK12 shown in SEQ ID NO.4 and SEQ ID NO.5 are inserted between the BsaI-BsaI restriction sites of the base vector.

[0010] The application of the gene knockout vector for the rice high-affinity potassium ion transporter gene OsHAK12 in reducing the potassium content of rice grains and the potassium absorption capacity during the seedling stage.

[0011] As a preferred embodiment of the present invention, the knockout vector used for the rice high-affinity potassium ion transporter gene OsHAK12 is obtained by inserting the target sequence T1 and target sequence T2 of the high-affinity potassium ion transporter gene OsHAK12 shown in SEQ ID NO.4 and SEQ ID NO.5 between the BsaI-BsaI restriction sites of the base vector.

[0012] The beneficial effects of this invention are:

[0013] 1. Through systematic research, this invention provides for the first time a biological function of the high-affinity potassium ion transporter gene OsHAK12 in potassium absorption and transport in rice.

[0014] 2. Knocking out the OsHAK12 gene encoding the high-affinity potassium ion transporter resulted in a decrease in the plant height of rice seedlings in a low-potassium environment.

[0015] 3. Knocking out the OsHAK12 gene encoding the high-affinity potassium ion transporter reduced the ability of rice seedlings to absorb potassium.

[0016] 4. Knocking out the OsHAK12 gene encoding the high-affinity potassium ion transporter reduced the potassium content of rice grains at maturity.

[0017] The study on the high-affinity potassium ion transporter gene OsHAK12 provided by this invention effectively demonstrates the importance of OsHAK12 in potassium absorption and utilization in rice. Attached Figure Description

[0018] Figure 1 Gene mapping of OsHAK12, the gene encoding a high-affinity potassium ion transporter in rice; where:

[0019] A: QTL mapping of the plantation population in Hainan in 2020;

[0020] B: QTL mapping of the plantation population in Hunan in 2018;

[0021] Figure 2 Low potassium tolerance experiment for OsHAK12 knockout mutant, which encodes a high-affinity potassium ion transporter in rice; where: A: growth phenotypes of wild-type Lemont and OsHAK12 knockout mutant seedlings after 14 days of germination in potassium chloride solutions of different concentrations;

[0022] B: A statistical graph showing the plant height of wild-type Lemont and OsHAK12 knockout mutant seedlings.

[0023] C: A graph showing the potassium content in the aboveground parts of wild-type Lemont and OsHAK12 knockout mutant seedlings as shown in A.

[0024] Figure 3 A statistical chart showing the potassium content of grains from the OsHAK12 knockout mutant of a high-affinity potassium ion transporter gene in rice under field conditions; where:

[0025] A: Comparison of potassium content in grains between OsHAK12 knockout mutant plants and wild-type Lemont plants. Detailed Implementation

[0026] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0027] Example 1: Gene Mapping

[0028] Recombinant inbred lines were obtained by crossing TeQing and Lemont as parent rice varieties. These lines were planted in Hunan and Hainan provinces in 2018 and 2020, respectively. The elemental composition of the harvested grains was determined by inductively coupled plasma mass spectrometry (ICP-MS). The recombinant inbred lines used for resequencing were of generation F20. A total of 257 recombinant inbred lines and two parent lines, TQ and LM, were planted in Lingshui, Hainan province in the winter of 2017. Total DNA was extracted from young leaves using the CTAB method. DNA quality was assessed by electrophoresis. The DNA content of each sample was then determined using NanoDrop 2000. The DNA was broken into 500 bp fragments using an Ultrasonicator. Sequencing was then performed using an Illumina HiSeq 2500 platform. Filtered reads were aligned with the Nipponbare reference genome (Os-Nipponbare-Reference-IRGSP1.0) using BWA software. Redundant reads were removed using Picard software. The software GATK was used to identify SNPs and indels between recombinant inbred lines or between parents. To construct bin maps, SNPs within 5 bp of each other or within 5 bp of each indel were filtered out. Before constructing the bin maps, some low-quality SNPs were first filtered out. Non-homozygous SNPs, SNPs with sequencing depths below 10× between the two parents, or SNPs with sequencing depths below 4× in recombinant inbred lines were filtered out. A total of 1,262,315 high-quality SNPs were obtained. The bin map was then constructed using the windowing method.

[0029] The collected data on grain potassium content in 2018 and 2020, along with the markers used to construct the genetic map, were subjected to QTL analysis. The QTL analysis was performed using the `qtl` package in R, with a window size of 10-cM and a step size of 1-cM, using the `imp` command. In both 2018 and 2020, a QTL locus associated with grain potassium content was located on chromosome 8. Figure 1 OsHAK12 is a candidate gene for this QTL site.

[0030] Example 2

[0031] The specific process for preparing the OsHAK12 knockout mutant, which encodes a high-affinity potassium ion transporter in rice, is as follows:

[0032] 1) Knockout of OsHAK12 was performed using CRISPR / Cas9 gene editing technology. The sequences gtgccccaatagtcccaatt (SEQ ID NO.4) and agctctcagacttccctcgt (SEQ ID NO.5), which share the CDS region between the two OsHAK12 transcripts shown in SEQ ID NO.2 and SEQ ID NO.3, were selected as editing target sites T1 and T2, and primers for the knockout vector were designed and constructed.

[0033] The primer sequences are as follows:

[0034] gRT1:5'-tgccccaatagtcccaattgttttagagctagaaat-3' (SEQ ID NO.6);

[0035] OsU6aT1:5'-aattgggactattggggcacggcagccaagccagca-3' (SEQ ID NO.7);

[0036] gRT2:5'-agctctcagacttccctcgtgttttagagctagaaat-3' (SEQ ID NO.8);

[0037] OsU6bT2:5'-acgagggaagtctgagagctcaacacaagcggcagc-3'(SEQ ID NO.9)

[0038] UF: CTCCGTTTTACCTGTGGAATCG (SEQ ID NO.10);

[0039] gR-R:CGGAGGAAAATTCCATCCAC (SEQ ID NO. 11);

[0040] Pps-R: TTCAGAGGTCTCTACCGACTAGTCACGCGTATGGAATCGGCAGCAAA (SEQ ID NO. 12);

[0041] Pgs-2: AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC (SEQ ID NO. 13);

[0042] Pps-2: TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG (SEQ ID NO. 14);

[0043] Pgs-L: AGCGTGGGTCTCGCTCGACGCGTATCCATCCACTCCAAGC (SEQ ID NO. 15).

[0044] 2) Using plasmid pYLsgRNA-OsU6a as a template, the sgRNA expression cassette fragment containing T1 was amplified by PCR using primers OsU6aT1 and UF, gRT1 and gR-R; using plasmid pYLsgRNA-OsU6b as a template, the sgRNA expression cassette fragment containing T2 was amplified by PCR using primers OsU6bT2 and UF, gRT2 and gR-R.

[0045] The amplification system consisted of: 25 μl phanta mix; 1 μl plasmid; 2 μl primers each; and 20 μl ddH2O.

[0046] The amplification program is as follows: (1) 95℃, 3min; (2) 95℃, 15s; (3) 58℃, 15s; (4) 72℃, 20s; (5) 72℃, 5min; (6) 4℃, 5min; Steps (2)-(4) are repeated for 35 cycles.

[0047] 3) Using the sgRNA expression cassette fragment from 2) as a template, the complete sgRNA expression cassette OsU6a-T1-sgRNA was further amplified using primers Pps-R and Pgs-2; the complete sgRNA expression cassette OsU6b-T2-sgRNA was further amplified using primers Pps-2 and Pgs-L.

[0048] The amplification system consisted of: 25 μl phanta mix; 1 μl of each sgRNA expression cassette fragment (2) for a total of 2 μl; 2 μl of each primer; and 19 μl ddH2O.

[0049] The amplification program is as follows: (1) 95℃, 3min; (2) 95℃, 15s; (3) 58℃, 15s; (4) 72℃, 20s; (5) 72℃, 5min; (6) 4℃, 5min; Steps (2)-(4) are repeated for 35 cycles.

[0050] 4) The sgRNA expression cassettes OsU6a-T1-sgRNA and OsU6b-T2-sgRNA were assembled into the pYLCRISPR / Cas9Pubi-H knockout vector using a method of digestion and ligation with BasⅠ (NEB) endonuclease and T4 ligase.

[0051] Cut-and-ligate system: 10×CutSmart Buffer 1.5 μl; 10 mmol / L ATP 1.5 μl; pYLCRISPR / Cas9 plasmid 80 ng; OsU6a-T1-sgRNA expression cassette 15 ng; OsU6b-T2-sgRNA expression cassette 15 ng; Bsa I-HF 10 U; T4 DNA ligase 35 U; ddH2O to bring the total to 15 μl.

[0052] The cutting and connecting procedure is as follows: (1) 37℃, 5min; (2) 10℃, 5min; (3) 20℃, 5min; (4) 37℃, 5min; Steps (1)-(3) are repeated for 15 cycles.

[0053] 5) Transform the verified pYLCRISPR / Cas9Pubi-H plasmid into rice. The specific transgenic process is as follows: (1) Transform the pYLCRISPR / Cas9Pubi-H plasmid into Agrobacterium (EHA105); (2) Use Lemont rice seeds as material, peel off the husks, disinfect them and place them on the induction medium to induce callus tissue; (3) Infect the callus tissue with Agrobacterium bacterial solution, then wash it three times with sterile ddH2O and place it on the selection medium to screen out resistant callus tissue; (4) Transfer the resistant callus tissue to the differentiation medium and the rooting medium in sequence, and obtain transgenic T0 generation plants after induction differentiation and rooting.

[0054] 6) Plant T0 generation plants in the field, sequence them to identify whether the OsZIP2 coding region has mutated, select homozygous mutant plants, and harvest T1 generation seeds.

[0055] The identification primers are:

[0056] CRISPROsHAK12-F:5'-ttggatggctctttgcacct-3' (SEQ ID NO. 16);

[0057] CRISPROsHAK12-R:5'-ttgtgagtaacctgtgatgct-3' (SEQ ID NO. 17);

[0058] Upon testing, two knockout mutants, hak12-1 and hak12-2, with different editing patterns were obtained in this embodiment of the high-affinity potassium ion transporter gene OsHAK12.

[0059] Example 3:

[0060] Example 2 describes the low potassium tolerance experiment of the high-affinity potassium ion transporter gene OsHAK12 knockout mutants hak12-1 and hak12-2. The specific implementation process is as follows:

[0061] 1) After germinating wild-type Lemont seeds and T3 generation homozygous seeds of mutants hak12-1 and hak12-2 at 37℃ for 3 days, the seeds were sown on a plastic black net suspended on ddH2O. Cardboard was placed over the black net for shading to promote rooting. After 60 hours, seedlings with similar growth status were selected for the next step of treatment.

[0062] 2) Wild-type Lemont seedlings and mutants hak12-1 and hak12-2 were transferred to 6L plastic transport boxes. Two transport boxes were set up, and each transport box was used for one treatment, namely 0.4mM and 0.004mM K. + The culture was carried out in a Kimura B nutrient solution with a concentration of half. The KNO3 and KH2PO4 in the Kimura B nutrient solution were replaced with NH4H2PO4. + Add KCl. Maintain the room temperature and water temperature at approximately 28℃ and 24℃ respectively, and allow the seedlings to grow for one week under these conditions.

[0063] 3) After the treatment period, take photos to record the growth of the seedlings and measure the height of each individual plant with a ruler.

[0064] The results showed that wild-type Lemont and OsHAK12 knockout mutants were able to withstand low potassium levels (0.004 mM K) + At the concentration, the OsHAK12 knockout mutant was significantly shorter in height than the wild-type Lemont at 0.4 mM K. + In the concentration treatment, the potassium content in the aboveground parts of the OsHAK12 knockout mutant was significantly lower than that of the wild-type Lemont; this example shows that after mutation of the high-affinity potassium ion transporter gene OsHAK12, the tolerance of rice to low potassium was significantly reduced. Figure 2 ).

[0065] Example 4:

[0066] The field planting experiment of hak12-1 and hak12-2, which are high-affinity potassium ion transporter gene OsHAK12 knockout mutants prepared in Example 2, was carried out as follows:

[0067] 1) After germinating wild-type Lemont seeds and mutant hak12-1 and hak12-2 T3 generation homozygous seeds at 37℃ for 3 days, they were sown on a plastic black net suspended on tap water. Cardboard was placed over the black net for shading to promote rooting. After 3 days, the cardboard was removed and the seeds were placed under normal light to grow.

[0068] 2) Two weeks later, the wild-type Lemont seedlings and the mutants hak12-1 and hak12-2 were transferred to plastic turnover boxes containing 1 / 2 Kimura B nutrient solution and continued to grow for two weeks, during which the nutrient solution was changed every 3 days.

[0069] 3) Select wild-type Lemont seedlings with consistent growth and mutants hak12-1 and hak12-2, and transplant them into rice fields at a specific transgenic base. Transplant 6 seedlings of each line, with a spacing of 10 cm between each seedling and a row spacing of 15 cm. Water and fertilizer management and pest and disease control during the entire rice growth period are the same as in ordinary fields.

[0070] 4) Harvest the grains from wild-type Lemont and mutant hak12-1 and hak12-2 plants at the rice maturity stage.

[0071] 5) Pick the kernels from the tip of each main panicle, dry them in an oven, peel off the husks, weigh them, and then burn them in a graphite furnace. Finally, determine the potassium content using ICP-MS.

[0072] This example demonstrates that mutations in the OsHAK12 gene encoding the high-affinity potassium ion transporter significantly reduce the potassium content of rice grains at maturity under field conditions. Figure 3 ).

[0073] Plasmid sequence information:

[0074] plasmid pYLsgRNA-OsU6a:

[0075] ggatccagcgtgggtctcggttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgg

[0076] caccgagtcggtgctttttttcaagagcttggagtggatggaattttcctccgttttacctgtggaatcggcagcaaagga

[0077] ttttttcctgtagttttcccacaaccattttttaccatccgaatgataggataggaaaaatatccaagtgaacagtattcc

[0078] tataaaattcccgtaaaaagcctgcaatccgaatgagccctgaagtctgaactagccggtcacctgtacaggctatcgaga

[0079] tgccatacaagagacggtagtaggaactaggaagacgatggttgattcgtcaggcgaaatcgtcgtcctgcagtcgcatct

[0080] atgggcctggacggaataggggaaaaagttggccggataggagggaaaggcccaggtgcttacgtgcgaggtaggcctggg

[0081] ctctcagcacttcgattcgttggcaccggggtaggatgcaatagagagcaacgtttagtaccacctcgcttagctagagca

[0082] aactggactgccttatatgcgcgggtgctggcttggctgccgagagacctctgaagataacatactaagcttggcactggc

[0083] cgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccag

[0084] ctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgcg

[0085] gtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcata

[0086] gttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagaca

[0087] agctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgt

[0088] gatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcg

[0089] cggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttca

[0090] ataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcc

[0091] tgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaact

[0092] ggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgct

[0093] atgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggt

[0094] tgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgag

[0095] tgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatggggga

[0096] tcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgt

[0097] agcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggat

[0098] ggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccgg

[0099] tgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggg

[0100] gagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcaga

[0101] ccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttga

[0102] taatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttc

[0103] ttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccgga

[0104] tcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagcc

[0105] gtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgc

[0106] cagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggg

[0107] gggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgc

[0108] cacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttcc

[0109] agggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtc

[0110] aggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacat

[0111] gttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccg

[0112] aacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggcc

[0113] gattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagct

[0114] cactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttca

[0115] cacaggaaacagctatgaccatgattacgaattcgagctcggtacccgg

[0116] Plasmid pYLsgRNA-OsU6b:

[0117] ggatccagcgtgggtctcggttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtgg

[0118] caccgagtcggtgctttttttcaagagcttggagtggatggaattttcctccgttttacctgtggaatcggcagcaaagga

[0119] tgcaagaacgaactaagccggacaaaaaaaaaaggagcacatatacaaaccggttttattcatgaatggtcacgatggatg

[0120] atggggctcagacttgagctacgaggccgcaggcgagagaagcctagtgtgctctctgcttgtttgggccgtaacggagga

[0121] tacggcccacgagcgtgtactaccgcgcgggatgccgctgggcgctgcgggggccgttggatggggatcggtgggtcgcgg

[0122] gagcgttgaggggagacaggtttagtaccacctcgcctaccgaacaatgaagaacccaccttataaccccgcgcgctgccg

[0123] cttgtgttgagagacctctgaagataacatactaagcttggcactggccgtcgttttacaacgtcgtgactgggaaaaccc

[0124] tggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcg

[0125] cccttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttc

[0126] acaccgcatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgc

[0127] tgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtca

[0128] gaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgat

[0129] aataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaataca

[0130] ttcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattca

[0131] acatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagt

[0132] aaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttt

[0133] tcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgg

[0134] gcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttac

[0135] ggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaac

[0136] gatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccgga

[0137] gctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaac

[0138] tggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcg

[0139] ctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcact

[0140] ggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagaca

[0141] gatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgattt

[0142] aaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagtt

[0143] ttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctg

[0144] cttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaac

[0145] tggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagc

[0146] accgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttgga

[0147] ctcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaac

[0148] gacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggta

[0149] tccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgt

[0150] cgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaa

[0151] cgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtgga

[0152] taaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgagga

[0153] agcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcc

[0154] cgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttat

[0155] gcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgaatt

[0156] cgagctcggtacccgg

[0157] pYLCRISPR / Cas9Pubi-H Vector:

[0158] tggcaggatatattgtggtgtaaacaaattgacgcttagacaacttaataacacattgcggacgtttttaatgtactgaat

[0159] taacgccgaattaattcgggggatctggattttagtactggattttggttttaggaattagaaattttattgatagaagta

[0160] ttttacaaatacaaatacatactaagggtttcttatatgctcaacacatgagcgaaaccctataggaaccctaattccctt

[0161] atctgggaactactcacacattattatggagaaactcgagcttgtcgatcgacagatccggtcggcatctactctatttct

[0162] ttgccctcggacgagtgctggggcgtcggtttccactatcggcgagtacttctacacagccatcggtccagacggccgcgc

[0163] ttctgcgggcgatttgtgtacgcccgacagtcccggctccggatcggacgattgcgtcgcatcgaccctgcgcccaagctg

[0164] catcatcgaaattgccgtcaaccaagctctgatagagttggtcaagaccaatgcggagcatatacgcccggagtcgtggcg

[0165] atcctgcaagctccggatgcctccgctcgaagtagcgcgtctgctgctccatacaagccaaccacggcctccagaagaaga

[0166] tgttggcgacctcgtattgggaatccccgaacatcgcctcgctccagtcaatgaccgctgttatgcggccattgtccgtca

[0167] ggacattgttggagccgaaatccgcgtgcacgaggtgccggacttcggggcagtcctcggcccaaagcatcagctcatcga

[0168] gagcctgcgcgacggacgcactgacggtgtcgtccatcacagtttgccagtgatacacatggggatcagcaatcgcgcata

[0169] tgaaatcacgccatgtagtgtattgaccgattccttgcggtccgaatgggccgaacccgctcgtctggctaagatcggccg

[0170] cagcgatcgcatccatagcctccgcgaccggttgtagaacagcgggcagttcggtttcaggcaggtcttgcaacgtgacac

[0171] cctgtgcacggcgggagatgcaataggtcaggctctcgctaaactccccaatgtcaagcacttccggaatcgggagcgcgg

[0172] ccgatgcaaagtgccgataaacataacgatctttgtagaaaccatcggcgcagctatttacccgcaggacatatccacgcc

[0173] ctcctacatcgaagctgaaagcacgagattcttcgccctccgagagctgcatcaggtcggagacgctgtcgaacttttcga

[0174] tcagaaacttctcgacagacgtcgcggtgagttcaggctttttcatatctcattgccccccgggatctgcgaaagctcgag

[0175] agagatagatttgtagagagagactggtgatttcagcgtgtcctctccaaatgaaatgaacttccttatatagaggaaggt

[0176] cttgcgaaggatagtgggattgtgcgtcatcccttacgtcagtggagatatcacatcaatccacttgctttgaagacgtgg

[0177] ttggaacgtcttctttttccacgatgctcctcgtgggtgggggtccatctttgggaccactgtcggcagaggcatcttgaa

[0178] cgatagcctttcctttatcgcaatgatggcatttgtaggtgccaccttccttttctactgtccttttgatgaagtgacaga

[0179] tagctgggcaatggaatccgaggaggtttcccgatattaccctttgttgaaaagtctcaatagccctttggtcttctgaga

[0180] ctgtatctttgatattcttggagtagacgagagtgtcgtgctccaccatgttatcacatcaatccacttgctttgaagacg

[0181] tggttggaacgtcttctttttccacgatgctcctcgtgggtgggggtccatctttgggaccactgtcggcagaggcatctt

[0182] gaacgatagcctttcctttatcgcaatgatggcatttgtaggtgccaccttccttttctactgtccttttgatgaagtgac

[0183] agatagctgggcaatggaatccgaggaggtttcccgatattaccctttgttgaaaagtctcaatagccctttggtcttctg

[0184] agactgtatctttgatattcttggagtagacgagagtgtcgtgctccaccatgttggcaagctgctctagccaatacgcaa

[0185] accgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg

[0186] caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtgg

[0187] aattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgaattcgagctcggtaccaactataacgg

[0188] tcctaaggtagcgaaggatccgctcgctaccttaagagaggatatccctccatcctataatgtaggctataggaactaggg

[0189] caaggccggccatgcggccgcaagctgggtgcagcgtgacccggtcgtgcccctctctagagataatgagcattgcatgtc

[0190] taagttataaaaaattaccacatatttttttgtcacacttgtttgaagtgcagtttatctatctttatacatatatttaa

[0191] actttactctacgaataataatcttatagtactacaataatatcagtgtttttagagaatcatataaatgaacgttagac

[0192] atggtctaaaggacaattgagtattttgacaacaggactctacagttttatctttttagtgtgcatgtgttctcctttttt

[0193] tttgcaaatagcttcacctatataatacttcatccattttattagtacatccatttagggtttagggttaatggtttttat

[0194] agactaattttttagtacatctattttattctattttagcctctaaattaagaaaactaaaaactctattttagtttttt

[0195] atttaataatttagatataaaaatagataaaaaaagtgactaaaaaaaaaaaaataccctttaagaaattaaaaaaaact

[0196] agaaacatttttcttgtttcgagtagataatgccagcctgttaaacgccgtcgacgagtctaacggacaccaaccagcg

[0197] aaccagcagcgtcgcgtcgggccaagcgaagcagacggcacggcatctctgtcgctgcctctggacccctctcgagagttc

[0198] cgctccaccgttggacttgctccgctgtcggcatccagaaattgcgtggcggagcggcagacgtgagccggcacggcaggc

[0199] ggcctcctcctcctctcacggcacggcagctacgggggattcctttcccaccgctccttcgctttcccttcctcgcccgcc

[0200] gtaataaatagacaccccctccacaccctctttccccaacctcgtgttgttcggagcgcacacacacacaaccagatctcc

[0201] cccaaatccacccgtcggcacctccgcttcaaggtacgccgctcgtcctccccccccccccctctctaccttctctagatc

[0202] ggcgttccggtccatggttagggcccggtagttctacttctgttcatgtttgtgttagatccgtgtttgtgttagatccgt

[0203] gctgctagcgttcgtacacggatgcgacctgtacgtcagacacgttctgattgctaacttgccagtgtttctctttgggga

[0204] atcctgggatggctctagccgttccgcagacgggatcgatttcatgattttttttgtttcgttgcatagggtttggtttgc

[0205] ccttttcctttatttcaatatatgccgtgcacttgtttgtcgggtcatcttttcatgcttttttttgtcttggttgtgatg

[0206] atgtggtctggttgggcggtcgttctagatcggagtagaattctgtttcaaactacctggtggatttattaattttggatc

[0207] tgtatgtgtgtgccatacatattcatagttacgaattgaagatgatggatggaaatatcgatctaggataggtatacatgt

[0208] tgatgcgggttttactgatgcatatacagagatgctttttgttcgcttggttgtgatgatgtggtgtggttgggcggtcgt

[0209] tcattcgttctagatcggagtagaatactgtttcaaactacctggtgtatttattaattttggaactgtatgtgtgtgtca

[0210] tacatcttcatagttacgagtttaagatggatggaaatatcgatctaggataggtatacatgttgatgtgggttttactga

[0211] tgcatatacatgatggcatatgcagcatctattcatatgctctaaccttgagtacctatctattataataaacaagtatgt

[0212] tttataattattttgatcttgatatacttggatgatggcatatgcagcagctatatgtggatttttttagccctgccttca

[0213] tacgctatttatttgcttggtactgtttcttttgtcgatgctcaccctgttgtttggtgttacttctgcagatggctccta

[0214] agaagaagcggaaggttggtattcacggggtgcctgcggctgacaagaagtactccatcggcctcgacatcggcaccaaca

[0215] gcgtcggctgggcggtgatcaccgacgagtacaaggtcccgtccaagaagttcaaggtcctgggcaacaccgaccgccact

[0216] ccatcaagaagaacctcatcggcgccctcctcttcgactccggcgagacggcggaggcgacccgcctcaagcgcaccgccc

[0217] gccgccgctacacccgccgcaagaaccgcatctgctacctccaggagatcttctccaacgagatggcgaaggtcgacgact

[0218] ccttcttccaccgcctcgaggagtccttcctcgtggaggaggacaagaagcacgagcgccaccccatcttcggcaacatcg

[0219] tcgacgaggtcgcctaccacgagaagtaccccactatctaccaccttcgtaagaagcttgttgactctactgataaggctg

[0220] atcttcgtctcatctaccttgctctcgctcacatgatcaagttccgtggtcacttccttatcgagggtgaccttaaccctg

[0221] ataactccgacgtggacaagctcttcatccagctcgtccagacctacaaccagctcttcgaggagaaccctatcaacgctt

[0222] ccggtgtcgacgctaaggcgatcctttccgctaggctctccaagtccaggcgtctcgagaacctcatcgcccagctccctg

[0223] gtgagaagaagaacggtcttttcggtaacctcatcgctctctccctcggtctgacccctaacttcaagtccaacttcgacc

[0224] tcgctgaggacgctaagcttcagctctccaaggatacctacgacgatgatctcgacaacctcctcgctcagattggagatc

[0225] agtacgctgatctcttccttgctgctaagaacctctccgatgctatcctcctttcggatatccttagggttaacactgaga

[0226] tcactaaggctcctctttctgcttccatgatcaagcgctacgacgagcaccaccaggacctcaccctcctcaaggctcttg

[0227] ttcgtcagcagctccccgagaagtacaaggagatcttcttcgaccagtccaagaacggctacgccggttacattgacggtg

[0228] gagctagccaggaggagttctacaagttcatcaagccaatccttgagaagatggatggtactgaggagcttctcgttaagc

[0229] ttaaccgtgaggacctccttaggaagcagaggactttcgataacggctctatccctcaccagatccaccttggtgagcttc

[0230] acgccatccttcgtaggcaggaggacttctaccctttcctcaaggacaaccgtgagaagatcgagaagatccttactttcc

[0231] gtattccttactacgttggtcctcttgctcgtggtaactcccgtttcgcttggatgactaggaagtccgaggagactatca

[0232] ccccttggaacttcgaggaggttgttgacaagggtgcttccgcccagtccttcatcgagcgcatgaccaacttcgacaaga

[0233] acctccccaacgagaaggtcctccccaagcactccctcctctacgagtacttcacggtctacaacgagctcaccaaggtca

[0234] agtacgtcaccgagggtatgcgcaagcctgccttcctctccggcgagcagaagaaggctatcgttgacctcctcttcaaga

[0235] ccaaccgcaaggtcaccgtcaagcagctcaaggaggactacttcaagaagatcgagtgcttcgactccgtcgagatcagcg

[0236] gcgttgaggaccgtttcaacgcttctctcggtacctaccacgatctcctcaagatcatcaaggacaaggacttcctcgaca

[0237] acgaggagaacgaggacatcctcgaggacatcgtcctcactcttactctcttcgaggatagggagatgatcgaggagaggc

[0238] tcaagacttacgctcatctcttcgatgacaaggttatgaagcagctcaagcgtcgccgttacaccggttggggtaggctct

[0239] cccgcaagctcatcaacggtatcagggataagcagagcggcaagactatcctcgacttcctcaagtctgatggtttcgcta

[0240] acaggaacttcatgcagctcatccacgatgactctcttaccttcaaggaggatattcagaaggctcaggtgtccggtcagg

[0241] gcgactctctccacgagcacattgctaaccttgctggttcccctgctatcaagaagggcatccttcagactgttaaggttg

[0242] tcgatgagcttgtcaaggttatgggtcgtcacaagcctgagaacatcgtcatcgagatggctcgtgagaaccagactaccc

[0243] agaagggtcagaagaactcgagggagcgcatgaagaggattgaggagggtatcaaggagcttggttctcagatccttaagg

[0244] agcaccctgtcgagaacacccagctccagaacgagaagctctacctctactacctccagaacggtagggatatgtacgttg

[0245] accaggagctcgacatcaacaggctttctgactacgacgtcgaccacattgttcctcagtctttccttaaggatgactcca

[0246] tcgacaacaaggtcctcacgaggtccgacaagaacaggggtaagtcggacaacgtcccttccgaggaggttgtcaagaaga

[0247] tgaagaactactggaggcagcttctcaacgctaagctcattacccagaggaagttcgacaacctcacgaaggctgagaggg

[0248] gtggcctttccgagcttgacaaggctggtttcatcaagaggcagcttgttgagacgaggcagattaccaagcacgttgctc

[0249] agatcctcgattctaggatgaacaccaagtacgacgagaacgacaagctcatccgcgaggtcaaggtgatcaccctcaagt

[0250] ccaagctcgtctccgacttccgcaaggacttccagttctacaaggtccgcgagatcaacaactaccaccacgctcacgatg

[0251] cttaccttaacgctgtcgttggtaccgctcttatcaagaagtaccctaagcttgagtccgagttcgtctacggtgactaca

[0252] aggtctacgacgttcgtaagatgatcgccaagtccgagcaggagatcggcaaggccaccgccaagtacttcttctactcca

[0253] acatcatgaacttcttcaagaccgagatcaccctcgccaacggcgagatccgcaagcgccctcttatcgagacgaacggtg

[0254] agactggtgagatcgtttgggacaagggtcgcgacttcgctactgttcgcaaggtcctttctatgcctcaggttaacatcg

[0255] tcaagaagaccgaggtccagaccggtggcttctccaaggagtctatccttccaaagagaaactcggacaagctcatcgcta

[0256] ggaagaaggattgggaccctaagaagtacggtggtttcgactcccctactgtcgcctactccgtcctcgtggtcgccaagg

[0257] tggagaagggtaagtcgaagaagctcaagtccgtcaaggagctcctcggcatcaccatcatggagcgctcctccttcgaga

[0258] agaacccgatcgacttcctcgaggccaagggctacaaggaggtcaagaaggacctcatcatcaagctccccaagtactctc

[0259] ttttcgagctcgagaacggtcgtaagaggatgctggcttccgctggtgagctccagaagggtaacgagcttgctcttcctt

[0260] ccaagtacgtgaacttcctctacctcgcctcccactacgagaagctcaagggttcccctgaggataacgagcagaagcagc

[0261] tcttcgtggagcagcacaagcactacctcgacgagatcatcgagcagatctccgagttctccaagcgcgtcatcctcgctg

[0262] acgctaacctcgacaaggtcctctccgcctacaacaagcaccgcgacaagcccatccgcgagcaggccgagaacatcatcc

[0263] acctcttcacgctcacgaacctcggcgcccctgctgctttcaagtacttcgacaccaccatcgacaggaagcgttacacgt

[0264] ccaccaaggaggttctcgacgctactctcatccaccagtccatcaccggtctttacgagactcgtatcgacctttcccagc

[0265] ttggtggtgataagcgtcctgctgccaccaaaaaggccggacaggctaagaaaaagaagtaggatcctcccgatcgttcaa

[0266] acatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacg

[0267] ttaagcatgtaataattaacatgtaatgcatgacgttatttatgaggtgggtttttatgattagagtcccgcaattataca

[0268] tttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatc

[0269] gggagcaccggtaaggcgcgccgtagtgctcgagagacctctgaagtggccgattcattaatgcagctggcacgacaggtt

[0270] tcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactt

[0271] tatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaagaaacagctatgaccatgattacgc

[0272] caagctatttaggtgacactatagaatactcaagctatgcatcaagctcaatgggtctagtctgtagatacccatcacact

[0273] ggcgaccgctcgaacatcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtg

[0274] atattattgacacgccggggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaac

[0275] tttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgcctgtctccgttatcg

[0276] gggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataaatgt

[0277] caggcctgaatggcgaatggacgcgccctgtagcggcgcattaagcgcggcgggtgagcgtgggtctcgcggtatcattgg

[0278] cgcgcctctcgagctagcggccgcatgcatcgatctcctacatcgtataaattagcctatacgaagttattgcatctatgt

[0279] cgggtgcggagaaagaggtaatgaaatggcagtattagatctgataacttcgtataatgtatgctatacgaagttatgact

[0280] gcaggtcgacacccataatagctgtttgccaagcttggcactggccgtcgtttttacaacgtcgtgactgggaaaaccctg

[0281] gcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcc

[0282] cttcccaacagttgcgcagcctgaatggcgaatgctagagcagcttgagcttggatcagattgtcgtttcccgccttcagt

[0283] ttaaactatcagtgtttgacaggatatattggcgggtaaacctaagagaaaagagcgtttattagaataacggatatttaa

[0284] aagggcgtgaaaaggtttatccgttcgtccatttgtatgtgcatgccaaccacagggttcccctcgggatcaaagtacttt

[0285] gatccaacccctccgctgctatagtgcagtcggcttctgacgttcagtgcagccgtcttctgaaaacgacatgtcgcacaa

[0286] gtcctaagttacgcgacaggctgccgccctgcccttttcctggcgttttcttgtcgcgtgttttagtcgcataaagtagaa

[0287] tacttgcgactagaaccggagacattacgccatgaacaagagcgccgccgctggcctgctgggctatgcccgcgtcagcac

[0288] cgacgaccaggacttgaccaaccaacgggccgaactgcacgcggccggctgcaccaagctgttttccgagaagatcaccgg

[0289] caccaggcgcgaccgcccggagctggccaggatgcttgaccacctacgccctggcgacgttgtgacagtgaccaggctaga

[0290] ccgcctggcccgcagcacccgcgacctactggacattgccgagcgcatccaggaggccggcgcgggcctgcgtagcctggc

[0291] agagccgtgggccgacaccaccacgccggccggccgcatggtgttgaccgtgttcgccggcattgccgagttcgagcgttc

[0292] cctaatcatcgaccgcacccggagcgggcgcgaggccgccaaggcccgaggcgtgaagtttggcccccgccctaccctcac

[0293] cccggcacagatcgcgcacgcccgcgagctgatcgaccaggaaggccgcaccgtgaaagaggcggctgcactgcttggcgt

[0294] gcatcgctcgaccctgtaccgcgcacttgagcgcagcgaggaagtgacgcccaccgaggccaggcggcgcggtgccttccg

[0295] tgaggacgcattgaccgaggccgacgccctggcggccgccgagaatgaacgccaagaggaacaagcatgaaaccgcaccag

[0296] gacggccaggacgaaccgtttttcattaccgaagagatcgaggcggagatgatcgcggccgggtacgtgttcgagccgccc

[0297] gcgcacgtctcaaccgtgcggctgcatgaaatcctggccggtttgtctgatgccaagctggcggcctggccggccagcttg

[0298] gccgctgaagaaaccgagcgccgccgtctaaaaaggtgatgtgtatttgagtaaaacagcttgcgtcatgcggtcgctgcg

[0299] tatatgatgcgatgagtaaataaacaaatacgcaaggggaacgcatgaaggttatcgctgtacttaaccagaaaggcgggt

[0300] caggcaagacgaccatcgcaacccatctagcccgcgccctgcaactcgccggggccgatgttctgttagtcgattccgatc

[0301] cccagggcagtgcccgcgattgggcggccgtgcgggaagatcaaccgctaaccgttgtcggcatcgaccgcccgacgattg

[0302] accgcgacgtgaaggccatcggccggcgcgacttcgtagtgatcgacggagcgccccaggcggcggacttggctgtgtccg

[0303] cgatcaaggcagccgacttcgtgctgattccggtgcagccaagcccttacgacatatgggccaccgccgacctggtggagc

[0304] tggttaagcagcgcattgaggtcacggatggaaggctacaagcggcctttgtcgtgtcgcgggcgatcaaaggcacgcgca

[0305] tcggcggtgaggttgccgaggcgctggccgggtacgagctgcccattcttgagtcccgtatcacgcagcgcgtgagctacc

[0306] caggcactgccgccgccggcacaaccgttcttgaatcagaacccgagggcgacgctgcccgcgaggtccaggcgctggccg

[0307] ctgaaattaaatcaaaactcatttgagttaatgaggtaaagagaaaatgagcaaaagcacaaacacgctaagtgccggccg

[0308] tccgagcgcacgcagcagcaaggctgcaacgttggccagcctggcagacacgccagccatgaagcgggtcaactttcagtt

[0309] gccggcggaggatcacaccaagctgaagatgtacgcggtacgccaaggcaagaccattaccgagctgctatctgaatacat

[0310] cgcgcagctaccagagtaaatgagcaaatgaataaatgagtagatgaattttagcggctaaaggaggcggcatggaaaatc

[0311] aagaacaaccaggcaccgacgccgtggaatgccccatgtgtggaggaacgggcggttggccaggcgtaagcggctgggttg

[0312] tctgccggccctgcaatggcactggaacccccaagcccgaggaatcggcgtgacggtcgcaaaccatccggcccggtacaa

[0313] atcggcgcggcgctgggtgatgacctggtggagaagttgaaggccgcgcaggccgcccagcggcaacgcatcgaggcagaa

[0314] gcacgccccggtgaatcgtggcaagcggccgctgatcgaatccgcaaagaatcccggcaaccgccggcagccggtgcgccg

[0315] tcgattaggaagccgcccaagggcgacgagcaaccagattttttcgttccgatgctctatgacgtgggcacccgcgatagt

[0316] cgcagcatcatggacgtggccgttttccgtctgtcgaagcgtgaccgacgagctggcgaggtgatccgctacgagcttcca

[0317] gacgggcacgtagaggtttccgcagggccggccggcatggccagtgtgtgggattacgacctggtactgatggcggtttcc

[0318] catctaaccgaatccatgaaccgataccgggaagggaagggagacaagcccggccgcgtgttccgtccacacgttgcggac

[0319] gtactcaagttctgccggcgagccgatggcggaaagcagaaagacgacctggtagaaacctgcattcggttaaacaccacg

[0320] cacgttgccatgcagcgtacgaagaaggccaagaacggccgcctggtgacggtatccgagggtgaagccttgattagccgc

[0321] tacaagatcgtaaagagcgaaaccgggcggccggagtacatcgagatcgagctagctgattggatgtaccgcgagatcaca

[0322] gaaggcaagaacccggacgtgctgacggttcaccccgattactttttgatcgatcccggcatcggccgttttctctaccgc

[0323] ctggcacgccgcgccgcaggcaaggcagaagccagatggttgttcaagacgatctacgaacgcagtggcagcgccggagag

[0324] ttcaagaagttctgtttcaccgtgcgcaagctgatcgggtcaaatgacctgccggagtacgatttgaaggaggaggcgggg

[0325] caggctggcccgatcctagtcatgcgctaccgcaacctgatcgagggcgaagcatccgccggttcctaatgtacggagcag

[0326] atgctagggcaaattgccctagcaggggaaaaaggtcgaaaaggtctgtttcctgtggatagcacgtacattgggaaccca

[0327] aagccgtacattgggaaccggaacccgtacattgggaacccaaagccgtacattgggaaccggtcacacatgtaagtgact

[0328] gatataaaagagaaaaaaggcgatttttccgcctaaaactctttaaaacttattaaaactcttaaaacccgcctggcctgt

[0329] gcataactgtctggccagcgcacagccgaagagctgcaaaaagcgcctacccttcggtcgctgcgctccctacgccccgcc

[0330] gcttcgcgtcggcctatcgcggccgctggccgctcaaaaatggctggcctacggccaggcaatctaccagggcgcggacaa

[0331] gccgcgccgtcgccactcgaccgccggcgcccacatcaaggcaccctgcctcgcgcgtttcggtgatgacggtgaaaacct

[0332] ctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtc

[0333] agcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtatactggcttaactatgcg

[0334] gcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatc

[0335] aggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaag

[0336] gcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaac

[0337] cgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcag

[0338] aggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgacc

[0339] ctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctc

[0340] agttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggt

[0341] aactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcg

[0342] aggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgc

[0343] gctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggt

[0344] ttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgac

[0345] gctcagtggaacgaaaactcacgttaagggattttggtcatgcattctaggtactaaaacaattcatccagtaaaatataa

[0346] tattttattttctcccaatcaggcttgatccccagtaagtcaaaaaatagctcgacatactgttcttccccgatatcctcc

[0347] ctgatcgaccggacgcagaaggcaatgtcataccacttgtccgccctgccgcttctcccaagatcaataaagccacttact

[0348] ttgccatctttcacaaagatgttgctgtctcccaggtcgccgtgggaaaagacaagttcctcttcgggcttttccgtcttt

[0349] aaaaaatcatacagctcgcgcggatctttaaatggagtgtcttcttcccagttttcgcaatccacatcggccagatcgtta

[0350] ttcagtaagtaatccaattcggctaagcggctgtctaagctattcgtatagggacaatccgatatgtcgatggagtgaaag

[0351] agcctgatgcactccgcatacagctcgataatcttttcagggctttgttcatcttcatactcttccgagcaaaggacgcca

[0352] tcggcctcactcatgagcagattgctccagccatcatgccgttcaaagtgcaggacctttggaacaggcagctttccttcc

[0353] agccatagcatcatgtccttttcccgttccacatcataggtggtccctttataccggctgtccgtcatttttaaatatagg

[0354] ttttcattttctcccaccagcttatataccttagcaggagacattccttccgtatcttttacgcagcggtatttttcgatc

[0355] agttttttcaattccggtgatattctcattttagccatttattatttccttcctcttttctacagtatttaaagatacccc

[0356] aagaagctaattataacaagacgaactccaattcactgttccttgcattctaaaaccttaaataccagaaaacagcttttt

[0357] caaagttgttttcaaagttggcgtataacatagtatcgacggagccgattttgaaaccgcggtgatcacaggcagcaacgc

[0358] tctgtcatcgttacaatcaacatgctaccctccgcgagatcatccgtgtttcaaacccggcagcttagttgccgttcttcc

[0359] gaatagcatcggtaacatgagcaaagtctgccgccttacaacggctctcccgctgacgccgtcccggactgatgggctgcc

[0360] tgtatcgagtggtgattttgtgccgagctgccggtcggggagctgttggctggctgg

[0361] References:

[0362] Chen, Y.F., Wang, Y., and Wu, W.H. (2008), 'Membrane transporters for nitrogen,

[0363] phosphate and potassium uptake in plants', J Integr Plant Biol, 50(7), 835 - 48. Rengel, Z. and Damon, P.M. (2008), 'Crops and genotypes differ in efficiency of potassium uptake and use', Physiol Plant, 133(4), 624 - 36.

[0364] Rozhdestvenskii, V.I., et al. (1975), '[Control of mineral nutrition of higher plants in biological life support systems]', Kosm Biol Aviakosm Med, 9(6), 30 - 5. Wang, Y. and Wu, W.H. (2013), 'Potassium transport and signaling in higher plants', Annu Rev Plant Biol, 64, 451 - 76.

Claims

1. Knockout of the gene encoding a high-affinity potassium ion transporter in rice OsHAK12 Its application in reducing potassium content in rice grains and potassium absorption capacity during the seedling stage is characterized by... The high-affinity potassium ion transporter protein encoding gene OsHAK12 The genomic nucleotide sequence is shown in SEQ ID NO.

1. The rice variety is Lemont, and the gene encoding the high-affinity potassium ion transporter in rice was knocked out. OsHAK12 The knockout vector used was based on the vector in SEQ ID NO.20, and contained the genes encoding the high-affinity potassium ion transporter shown in SEQ ID NO.4 and SEQ ID NO.

5. OsHAK12 The target sequence T1 and target sequence T2 are obtained by inserting them between the BsaI-BsaI restriction sites in the basic vector.

2. The application according to claim 1, characterized in that, The gene encoding the high-affinity potassium ion transporter in rice. OsHAK12 The CDS sequence is shown in SEQ ID NO.2 or SEQ ID NO.

3.

3. The rice high-affinity potassium ion transporter encoding gene as described in claim 1 OsHAK12 The application of gene knockout vectors in reducing potassium content in rice grains and potassium uptake capacity during seedling stage is characterized by, The rice variety mentioned is Lemont, and the gene encoding the high-affinity potassium ion transporter protein in the rice is described. OsHAK12 The knockout vector used was based on the vector in SEQ ID NO.20, and contained the genes encoding the high-affinity potassium ion transporter shown in SEQ ID NO.4 and SEQ ID NO.

5. OsHAK12 The target sequence T1 and target sequence T2 are obtained by inserting them between the BsaI-BsaI restriction sites in the basic vector.

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