Rice high-affinity potassium ion transporter coding gene OsHAK12 and application thereof in controlling potassium content of rice and tolerating low potassium

Abstract

The invention discloses an application of a rice high-affinity potassium ion transport protein coding gene OsHAK12. The invention relates to a high-affinity potassium ion transporter coding gene OsHAK12, and the sequence of the high-affinity potassium ion transporter coding gene OsHAK12 is as shown in SEQ ID The gene can be applied to the aspects of controlling the potassium content of rice, tolerating low potassium and the like. The biological function of the potassium ion transporter coding gene OsHAK12 in rice is found through a large number of experiments, after the gene is knocked out, the potassium content in rice grains is reduced, and the tolerance of seedlings to low potassium is remarkably reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering and relates to the application of rice high-affinity potassium ion transporter encoding gene OsHAK12. Background Art

[0002] Potassium is a macronutrient essential for plant growth and development, and is involved in many physiological processes of 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 the development of agricultural production (Chen et al. 2008). In agricultural production, the rational application of potassium fertilizer is crucial to promoting crop growth and increasing yield. However, simply increasing the application of potassium fertilizer cannot completely solve the problem, because excessive fertilizer input not only increases production costs, but may also cause environmental problems. Therefore, improving the utilization efficiency of crop potassium has become an important strategy to reduce fertilizer cost input and increase crop yield.

[0003] Potassium use efficiency in rice is a complex quantitative trait involving multiple genes and multiple biological processes, which mainly include potassium absorption, distribution, transport and recycling (Rengel and Damon 2008). QTL (Quantitative Trait Loci) analysis and positioning is a method to determine the genetic basis of quantitative traits through genetic linkage and correlation analysis. The genetic relationship between quantitative traits and molecular markers is determined by constructing a genetic linkage map. The content of mineral nutrients in rice grains mostly has a large natural variation. Using the positioning population obtained by hybridizing two rice plants, a genetic map is constructed and phenotypic data are collected, and further association analysis is performed to locate the QTL that controls the accumulation of potassium in rice grains. The genes that control potassium accumulation can be identified and applied to rice breeding to cultivate 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 the use of potassium fertilizers, reduce production costs, and contribute to environmental protection. Summary of the invention

[0004] The purpose of the present invention is to provide an application of the rice high-affinity potassium ion transporter encoding gene OsHAK12 in view of the above-mentioned deficiencies in the prior art.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] The genomic nucleotide sequence of the rice high-affinity potassium ion transporter encoding gene OsHAK12 of the present 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 encoding gene OsHAK12 are shown as 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 encoding gene OsHAK12 will lead to a decrease in the potassium content of rice grains and a decrease in the potassium absorption capacity of the seedling stage.

[0009] As a further preference of the present invention, the knockout vector used for knocking out the rice high-affinity potassium ion transporter encoding gene OsHAK12 is a pYLCRISPR / Cas9Pubi-H vector as a basic vector, and the target sequence T1 and target sequence T2 of the high-affinity potassium ion transporter encoding gene OsHAK12 shown in SEQ ID NO.4 and SEQ ID NO.5 are inserted between the BsaI-BsaI restriction sites of the basic vector.

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

[0011] As a preferred embodiment of the present invention, the knockout vector used for the rice high-affinity potassium ion transporter encoding gene OsHAK12 is a pYLCRISPR / Cas9Pubi-H vector as a basic vector, and the target sequence T1 and target sequence T2 of the high-affinity potassium ion transporter encoding gene OsHAK12 shown in SEQ ID NO.4 and SEQ ID NO.5 are inserted between the BsaI-BsaI restriction sites of the basic vector.

[0012] Beneficial effects of the present invention:

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

[0014] 2. After knocking out the high-affinity potassium ion transporter encoding gene OsHAK12, the plant height of rice seedlings decreased in a low potassium environment.

[0015] 3. After knocking out the high-affinity potassium ion transporter encoding gene OsHAK12, the rice seedlings' ability to absorb potassium decreased.

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

[0017] Through the research on the high-affinity potassium ion transporter encoding gene OsHAK12 provided by the present invention, the importance of OsHAK12 in rice potassium absorption and utilization is effectively demonstrated. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the location map of the rice high-affinity potassium ion transporter encoding gene OsHAK12; among them:

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

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

[0021] Figure 2 This is an experiment on the low potassium tolerance of the rice high-affinity potassium ion transporter encoding gene OsHAK12 knockout mutant; A: The growth phenotype of the seedlings of the wild-type Lemont and the OsHAK12 knockout mutant germinated for 14 days in potassium chloride solutions of different concentrations;

[0022] B: Plant height statistics of wild-type Lemont and OsHAK12 knockout mutant seedlings shown in A.

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

[0024] Figure 3 This is a statistical chart of the potassium content in the grains of the rice high-affinity potassium ion transporter encoding gene OsHAK12 knockout mutant under field conditions; among which:

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

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

[0027] Example 1: Gene localization

[0028] Recombinant inbred lines were obtained by hybridizing TeQing and Lemont as parental rice. They were planted in Hunan and Hainan in 2018 and 2020, respectively. The harvested grains were measured for elemental content using inductively coupled plasma mass spectrometry. The recombinant inbred lines used for resequencing were F20 generation. A total of 257 recombinant inbred lines and two parents, TQ and LM, were planted in Lingshui, Hainan in the winter of 2017. Total DNA was extracted from young leaves using the CTAB method. DNA quality was identified by electrophoresis. The DNA content of each sample was then determined using NanoDrop 2000. The DNA was fragmented into 500 bp fragments using Ultrasonicator. Then, sequencing was performed using the Illumina HiSeq2500 platform. The filtered reads were aligned with the Nipponbare reference genome (Os-Nipponbare-Reference-IRGSP1.0) using BWA software. Redundant reads were removed using the software Picard. The software GATK was used to identify SNPs and Indels between recombinant inbred lines or between parents. In order to construct the bin map, SNPs within 5 bp or within 5 bp of Indel were filtered out. Before constructing the bin map, some low-quality SNPs were first filtered out. Non-homozygous SNPs or SNPs with a sequencing depth of less than 10× for both parents or less than 4× for the 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 window method.

[0029] The collected grain potassium content and genetic map markers in 2018 and 2020 were used for QTL analysis. The QTL analysis was performed using the qtl package in R language, with a window of 10-cM, a step size of 1-cM, and the method used was "imp". In both 2018 and 2020, a QTL locus related to grain potassium content was located on chromosome 8 ( Figure 1 ), OsHAK12 is the candidate gene of this QTL locus.

[0030] Example 2

[0031] The preparation of the knockout mutant of rice high-affinity potassium ion transporter encoding gene OsHAK12 is specifically implemented as follows:

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

[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, primers OsU6aT1 and UF, gRT1 and gR-R were used to amplify the sgRNA expression cassette fragment containing T1 by PCR; using plasmid pYLsgRNA-OsU6b as a template, primers OsU6bT2 and UF, gRT2 and gR-R were used to amplify the sgRNA expression cassette fragment containing T2 by PCR.

[0045] The amplification system is: 25 μl phanta Mix; 1 μl plasmid; 2 μl of each primer; 20 μl ddH2O;

[0046] The amplification program was as follows: (1) 95°C, 3 min; (2) 95°C, 15 s; (3) 58°C, 15 s; (4) 72°C, 20 s; (5) 72°C, 5 min; (6) 4°C, 5 min; steps (2)-(4) were repeated for 35 cycles.

[0047] 3) Using the sgRNA expression cassette fragment in 2) as a template, primers Pps-R and Pgs-2 were further used to amplify the complete sgRNA expression cassette OsU6a-T1-sgRNA; primers Pps-2 and Pgs-L were used to further amplify the complete sgRNA expression cassette OsU6b-T2-sgRNA.

[0048] The amplification system is: 25 μl phanta Mix; 1 μl of each sgRNA expression cassette fragment in 2) for a total of 2 μl; 2 μl of each primer; 19 μl ddH2O;

[0049] The amplification program was as follows: (1) 95°C, 3 min; (2) 95°C, 15 s; (3) 58°C, 15 s; (4) 72°C, 20 s; (5) 72°C, 5 min; (6) 4°C, 5 min; steps (2)-(4) were 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 by using BasⅠ (NEB) endonuclease and T4 ligase for cutting and ligation.

[0051] Cutting and ligating system: 10×CutSmart Buffer 1.5μl; 10mmol / LATP 1.5μl; pYLCRISPR / Cas9 plasmid 80ng; OsU6a-T1-sgRNA expression cassette 15ng; OsU6b-T2-sgRNA expression cassette 15ng; Bsa I-HF 10U; T4 DNA ligase 35U; ddH2O is added to 15μl.

[0052] The cutting and ligation procedure was as follows: (1) 37°C, 5 min; (2) 10°C, 5 min; (3) 20°C, 5 min; (4) 37°C, 5 min; steps (1)-(3) were repeated for 15 cycles.

[0053] 5) The verified correct pYLCRISPR / Cas9Pubi-H plasmid was transformed into rice. The specific transgenic process is as follows: (1) The pYLCRISPR / Cas9Pubi-H plasmid was transformed into Agrobacterium (EHA105); (2) The rice variety Lemont seeds were used as materials, shelled, sterilized and placed on an induction medium to induce callus tissue; (3) The callus tissue was infected with Agrobacterium bacterial liquid, then washed three times with sterile ddH2O and placed on a selection medium to screen out resistant callus tissue; (4) The resistant callus tissue was transferred to a differentiation medium and a rooting medium in turn, and transgenic T0 generation plants were obtained after inducing differentiation and rooting.

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

[0055] The primers for identification are:

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

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

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

[0059] Embodiment 3:

[0060] The low potassium tolerance experiment of the high affinity potassium ion transporter encoding gene OsHAK12 knockout mutants hak12-1 and hak12-2 prepared in Example 2 was specifically carried out as follows:

[0061] 1) Wild-type Lemont seeds and T3 homozygous seeds of mutants hak12-1 and hak12-2 were germinated at 37°C for 3 days and then sown on a plastic black net suspended in ddH2O. Cardboard was covered over the black net to shade the seeds and promote rooting. After 60 hours, seedlings with similar growth status were selected for the next step.

[0062] 2) The seedlings of wild-type Lemont and mutants hak12-1 and hak12-2 were transferred to 6-L plastic boxes. Two boxes were set up in total, with one treatment in each box, 0.4 mM and 0.004 mM K + The culture was carried out in Kimura B nutrient solution with a concentration of half. KNO3 and KH2PO4 in Kimura B nutrient solution were replaced with NH4H2PO4. + The room temperature and water temperature were kept at about 28°C and 24°C respectively, and the seedlings were grown under this condition for one week.

[0063] 3) After the treatment period, take photos to record the growth status 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 could be activated under low potassium (0.004 mM K) + The plant height of OsHAK12 knockout mutant was significantly shorter than that of wild-type Lemont at 0.4 mM K. + In the concentration treatment, the potassium content of the aboveground part of the OsHAK12 knockout mutant was significantly lower than that of the wild type Lemont; this example shows that after the high-affinity potassium ion transporter encoding gene OsHAK12 mutated, the tolerance of rice to low potassium was significantly reduced ( Figure 2 ).

[0065] Embodiment 4:

[0066] The field planting test of the high-affinity potassium ion transporter encoding gene OsHAK12 knockout mutants hak12-1 and hak12-2 prepared in Example 2 was specifically carried out as follows:

[0067] 1) T3 homozygous seeds of wild-type Lemont and mutants hak12-1 and hak12-2 were germinated at 37°C for 3 days and then sown on a plastic black net suspended in tap water. Cardboard was covered over the black net to shade the seeds and promote rooting. After 3 days, the cardboard was removed and the seeds were grown under normal light.

[0068] 2) Two weeks later, the seedlings of wild-type Lemont and 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 replaced every 3 days.

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

[0070] 4) At the maturity stage of rice, grains of wild-type Lemont and mutants hak12-1 and hak12-2 were harvested.

[0071] 5) The kernels on the tip of the main ear of each individual plant were picked, dried in an oven, and then the husks were removed. The kernels were weighed and boiled in a graphite furnace, and then the potassium content was determined using ICP-MS.

[0072] This example shows that after the high-affinity potassium ion transporter encoding gene OsHAK12 mutated, the potassium content of mature rice grains under field conditions decreased significantly ( 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 Qiaochu 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. Application of rice high-affinity potassium ion transporter encoding gene OsHAK12 in rice potassium absorption and low potassium tolerance, the genomic nucleotide sequence of the high-affinity potassium ion transporter encoding gene OsHAK12 is shown in SEQ ID NO.

1.

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

3.

3. The use according to claim 1, characterized in that Knocking out the rice high-affinity potassium ion transporter encoding gene OsHAK12 will lead to a decrease in the potassium content of rice grains and a decrease in the potassium absorption capacity during the seedling stage.

4. The use according to claim 3, characterized in that: The knockout vector used for knocking out the rice high-affinity potassium ion transporter encoding gene OsHAK12 is a basic vector based on the SEQ ID NO.20 vector, in which the target sequence T1 and target sequence T2 of the high-affinity potassium ion transporter encoding gene OsHAK12 shown in SEQ ID NO.4 and SEQ ID NO.5 are inserted between the BsaI-BsaI restriction sites of the basic vector.

5. Use of the gene knockout vector of the rice high-affinity potassium ion transporter encoding gene OsHAK12 as claimed in claim 1 in reducing the potassium content of rice grains and the potassium absorption capacity at the seedling stage.

6. The use according to claim 5, characterized in that The knockout vector used for the rice high-affinity potassium ion transporter encoding gene OsHAK12 is obtained by using the SEQ ID NO.20 vector as the basic vector, and inserting the target sequence T1 and target sequence T2 of the high-affinity potassium ion transporter encoding gene OsHAK12 shown in SEQ ID NO.4 and SEQ ID NO.5 between the BsaI-BsaI restriction sites of the basic vector.

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