CN113073102B - Application of autophagy gene ATG9 in rice breeding and/or rice grain type mechanism research - Google Patents

Abstract

The invention provides application of an autophagy gene ATG9 in rice breeding and/or rice grain type mechanism research, belonging to the field of botany and biotechnology. The nucleotide sequence of the autophagy gene ATG9 is shown as SEQ ID NO 1 in the sequence table; the amino acid sequence of the coding protein of the autophagy gene ATG9 is shown in SEQ ID NO. 2. The autophagy gene ATG9 has remarkable significance for improving the yield and the quality of rice, and can be used as a new target for creating rice grain quality improvement. The plant height and the ear length of the over-expressed ATG9 plant are obviously improved, the grain width, the grain length and the hundred grain weight are also obviously increased, and the ATG9 positively controls the size change of various tissues of rice such as grains and the like.

Description

Application of autophagy gene ATG9 in rice breeding and/or rice grain type mechanism research

Technical Field

The invention belongs to the technical field of botany and biology, and particularly relates to application of an autophagy gene ATG9 in rice breeding and/or rice grain type mechanism research.

Background

Rice is one of the most extensive and important food crops in the world, is also one of the model organisms for plant genomics research, and provides food and energy for more than 30 hundred million people in the world. With the increasing population, global climate change, environmental stress and the like, it is important to cultivate new high-yield and high-quality rice varieties. The grain size of rice is one of the most important factors affecting the yield and quality of rice. The development of new genes and molecular mechanisms for regulating and controlling the grain size can provide important theoretical basis for genetic improvement of rice yield and quality in the future.

Autophagy is a highly conserved degradation mechanism in the biological evolution process, and mainly transports components in cells, such as damaged organelles, denatured or unfolded proteins and the like, to vacuoles or lysosomes for degradation, so that nutrients are promoted to be recycled, and the aims of assisting plants in survival, growth and development in adverse environments are fulfilled. The existing research shows that the cell autophagy is widely involved in the regulation and control of various aspects of plants, including pollen fertility, rice tillering, nitrogen utilization, various stresses and the like. It is worth mentioning that autophagy has not been found to be directly involved in the regulation of yield traits, such as grain size. The research progress on autophagy is now essentially about the anti-aging, stress resistance and plant nitrogen cycle promoting functions of autophagy genes. However, no report is found about the regulation of the autophagy gene ATG9 on rice grain type.

Disclosure of Invention

The invention aims to provide application of an autophagy gene ATG9, in particular application of the autophagy gene ATG9 in rice breeding and/or rice grain type mechanism research.

The above object of the present invention is achieved by the following technical solutions:

the autophagy gene ATG9 is applied to rice breeding and/or rice grain type mechanism research.

The nucleotide sequence of the autophagy gene ATG9 is shown as SEQ ID NO. 1 in the sequence table.

The amino acid sequence of the coding protein of the autophagy gene ATG9 is shown in SEQ ID NO. 2.

The rice breeding preferably comprises regulation and control of rice grain type improvement.

The regulation is positive regulation.

The positive regulation refers to the overexpression of the autophagy gene ATG9 in rice, the plant height and the panicle length of a rice ATG9 overexpression plant are obviously improved, and the grain width, the grain length and the hundred-grain weight are obviously increased. The autophagy gene ATG9 positively regulates the size change of various rice tissues such as seeds, so that the overexpression of the autophagy gene ATG9 has important significance for increasing the size of the seeds, and important gene resources are provided for improving the yield of the rice.

The rice is preferably japonica rice.

The japonica rice is preferably medium flower 11.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the invention clones an autophagy gene ATG9 (the corresponding locus number corresponds to LOC _ Os10g07994 published in Rice Genome alteration Project) from Rice by using a PCR method. The autophagy gene ATG9 has remarkable significance for improving the yield and the quality of rice, and can be used as a new target for improving the quality of rice grains.

(2) ATG9 is knocked out by CRISPR/Cas9 to obtain ATG9 mutant plants. The phenotype of atg9 mutant plants was analyzed in detail. The results show that the plant height of atg9 mutant plants is significantly reduced, the ear length is shortened, the kernel is reduced and the grain weight is reduced compared with the Wild Type (WT). Through intensive research, an ATG9 overexpression plant is constructed, and compared with WT, the ATG9 overexpression plant is found to be remarkably improved in plant height and spike length, grain width, grain length and hundred grain weight, and ATG9 positively regulates and controls the size change of various tissues of rice such as grains.

(3) According to the invention, the expression regulation of the ATG9 gene can be explored through deep research, and by combining a gene editing technology, an excellent ATG9 allele is expected to be created, for example, a silencer of the gene is knocked out, so that the expression level is improved, and finally, a phenotype (the grain is enlarged and the yield is improved) similar to the ATG9 overexpression is generated. In addition, the ultra-long grain high-yield and high-quality rice can be further cultivated by molecular manipulation of grain length genes and ATG9 (such as gene polymerization). In view of previous research, it is possible to improve stress resistance of plants and to improve the adaptability of rice to the environment, particularly to adverse environments. In conclusion, the ATG9 gene is considered as a potential new candidate target point for rice yield and stress resistance synergy.

Drawings

FIG. 1 is the gene editing of ATG 9: sequence analysis of Wild Type (WT) and mutant (atg 9); wherein the sequence analysis map is checked and aligned by chromas software, GAGTGGAAGCGCTCGAGTC represents a target site, PAM represents a contiguous motif of the crRNA complementary sequence, an arrow indicates a mutation site, and a font T indicated by the arrow indicates an inserted base T.

FIG. 2 is a graph showing the results of phenotypic analysis of ATG 9; wherein, from left to right, graph A shows the plant height results of Wild Type (WT), mutant (ATG9) and ATG9 overexpression plants, respectively, with the scale equal to 15 cm; FIG. B is a graph of panicle results from left to right for Wild Type (WT), mutant (ATG9) and ATG9 overexpressing plants, respectively, with scale equal to 2 cm; panel C shows, from top to bottom, the grain width results for Wild Type (WT), mutant (ATG9) and ATG9 overexpressing plants, respectively, on a scale equal to 0.5 cm; panel D shows, from top to bottom, the grain length results for Wild Type (WT), mutant (ATG9) and ATG9 overexpressing plants, respectively, on a scale equal to 0.5 cm; panel E shows, from left to right, the results of the weight of the grains of Wild Type (WT), mutant (ATG9) and ATG9 overexpressing plants, respectively, on a scale equal to 1 cm.

FIG. 3 is a graph of statistics of WT, ATG9 and ATG9 overexpression phenotype data, respectively; wherein, the graph A is a plant height result graph of Wild Type (WT), mutant (ATG9) and ATG9 Overexpression (OE) plants; FIG. B is a graph of spike length results for Wild Type (WT), mutant (ATG9) and ATG9 Overexpressing (OE) plants; FIG. C is a plot of grain width results for Wild Type (WT), mutant (ATG9) and ATG9 Overexpressing (OE) plants; FIG. D is a graph of grain length results for Wild Type (WT), mutant (ATG9) and ATG9 Overexpressing (OE) plants; panel E is a plot of the results for the weight of the grains of Wild Type (WT), mutant (ATG9) and ATG9 Overexpressing (OE) plants. The data in the figure are mean values ± standard deviation, and a, b and c respectively represent significant differences from each other.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto. The experimental procedures used in the examples are conventional, unless otherwise indicated.

Plant material: the test material in this study was japonica rice background (Oryza sativa L.). The Wild Type (WT) was Zhonghua 11(ZH11) (publicly used rice variety, commercially available). The atg9 mutant plant was created by the laboratory in conjunction with the King Kanji project group of the Chinese Rice institute using the CRISPR/Cas9 method (see the references Wang C, Shen L, Fu Y, et al. A simple CRISPR/Cas9 system for multiplex genome editing in rice [ J ]. Journal of Genetics and Genomics 2015,42(12): 703-706.). ATG9 overexpression plant OE Rice was constructed by Baige Gene technology (Jiangsu) Inc. Strains and plasmids: the strain used for the experiment, Escherichia coli DH5 alpha, was purchased from Guangzhou spring mud Co., Ltd; the plant overexpression Vector pRHVcGFP was provided by the Wang Beam project group of Chinese academy of agricultural sciences (the plant overexpression Vector pRHVcGFP is disclosed in supplementary data of He F, Zhang F, Sun W, et al. A Versatile Vector Toolkit for Functional Analysis of Rice Genes [ J ]. Rice,2018,11(1): 27.); the pC1300-Cas9 and SK-gRNA vectors are provided by the Wangkangjian subject group of Chinese Rice institute (the pC1300-Cas9 vector and the SK-gRNA vector are disclosed in the invention patent with the application number of 2015104855732 and the name of construction and application of plant polygene knockout vector).

Chemical reagents: restriction endonucleases KpnI, NotI, BamHI, BglII and T4DNA ligase were purchased from New England Biolabs; restriction endonuclease AarI was purchased from Ferent; the high fidelity enzyme KOD FX was purchased from TOYOBO; abm reverse transcription kit is purchased from Aibimeng biotechnology limited; the gel recovery kit and the plasmid extraction kit are purchased from Jiufen Biotechnology (Beijing) Co., Ltd; 2 × Taq PCR StarMix was purchased from Biotech, Inc., of Convergence, Beijing.

Example 1: acquisition of the Rice atg9 mutant

The atg9 mutant was constructed according to the CRISPR/Cas9 technique described in Wang C, Shen L, Fu Y, et al.A. simple CRISPR/Cas9 system for multiplex genome editing in rice [ J ]. Journal of Genetics and Genomics,2015,42(12):703-706.

1. Target sequence selection and primer design

A wild-type ATG9 gene is used for taking 19bp GAGTGGAAGCGCTCGAGTC as a target site, and primers are designed according to the target site, as shown in Table 1.

Table 1: target site primer

2. Intermediate vector construction

ATG9-2-g + + and ATG9-2-g- -primers were diluted with water to a concentration of 100. mu.M, 20. mu.L each of the front and rear primers were mixed together, the primers were mixed and then denatured and annealed, denatured at 100 ℃ for 5 minutes, and cooled at room temperature to form fragments with sticky ends. Performing AarI enzyme digestion on the intermediate vector SK-gRNA, performing enzyme digestion at 37 ℃ for 3-6h, purifying the enzyme digestion product by using the kit to form a vector with a sticky end, wherein an enzyme digestion system is shown in Table 2.

Table 2: AarI enzyme cutting SK-gRNA vector system

The vector with the cohesive end and the fragment with the cohesive end were ligated (molar concentration 1:3-10) to obtain recombinant plasmid 1, which was named SK-gRNA-ATG9 recombinant vector. After the system preparation is completed as shown in Table 3, the components are gently pipetted and mixed uniformly, the reaction solution is collected to the bottom of the tube by short-time centrifugation and is placed at room temperature for 0.5-1 h. The reaction product was transformed into competent cell DH 5. alpha. and the bacterial suspension was spread gently on Amp-resistant plates, inverted and cultured overnight at 37 ℃.

Table 3: recombination reaction system

The clone identification can be carried out by carrying out colony PCR positive detection by matching a common primer T3 with ATG9-2-g-, and carrying out sequencing detection by using the common primer T7 or T3 to verify whether the detection is correct.

T7：5’-TAATACGACTCACTATAGG-3’

T3：5’-ATTAACCCTCACTAAAGGGA-3’

3. Construction to the final vector

The vector pC1300-Cas9 was recovered by digestion with KpnI and BamHI. The SK-gRNA-ATG9 recombinant vector is cut by KpnI and BglII and the fragment is recovered. The reaction systems of the pC1300-Cas9 and SK-gRNA-ATG9 vectors are shown in tables 4-1 and 4-2, and the double enzyme digestion is carried out for 1h at 37 ℃.

Table 4-1: pC1300-Cas9 vector enzyme digestion reaction system

Tables 4-2: SK-gRNA-ATG9 recombinant vector enzyme digestion reaction system

And the recovered fragment was ligated to the linearized pC1300-Cas9 vector to obtain recombinant vector 2. The ligation system is shown in Table 5, the components are gently pipetted and mixed evenly, the reaction solution is collected to the bottom of the tube by short-time centrifugation and is placed at room temperature for 0.5-1 h. The reaction product was transformed into competent cell DH 5. alpha. and the bacterial suspension was spread by a bar on a plate containing Kan resistance, and the plate was inverted and cultured overnight at 37 ℃.

Table 5: final carrier ligation system

Sequencing primer pC1300-F is used for sequencing detection to verify whether the recombinant vector is correct.

pC1300-F：5’-ACACTTTATGCTTCCGGCTC-3’

4. Transforming agrobacterium to rice callus to obtain transgenic plant

Transforming agrobacterium to transform rice callus to obtain transgenic plant T₀Instead, T is added₀The generation self-bred until the single mutant of the homozygous locus is obtained (the process is completed by Baige Gene technology (Jiangsu) Co., Ltd.). The target site of the gene was aligned by sequencing (sequencing was done by Guangzhou Rui Bo Biotechnology Ltd.), and it was confirmed that a T base was inserted into the gene at position 390, which eventually resulted in frame-shift and premature termination.

5. Extraction of total DNA of rice genome

Extracting the genomic DNA of the leaf of the rice plant to be detected, designing a primer near the sequence of the knocked target site, carrying out PCR amplification on the primer, and carrying out sequencing analysis on the product to identify whether the gene target sequence is changed.

(1) Placing a small amount of leaves in a 2mL tube, adding a steel ball, and adding 400-;

(2) pulverizing with pulverizer, and water bathing at 75 deg.C for 30 min;

(3) centrifuging at 12000rpm for 10 min;

(4) taking supernatant (500 mu L) to a 1.5mL tube, adding frozen equal-volume absolute ethyl alcohol, and mixing up and down;

(5) standing at-20 deg.C for 0.5 hr or more;

(6) centrifuging at 12000rpm for 5min, and removing supernatant;

(7) standing for more than 0.5h, removing alcohol, and drying;

(8) add 100. mu.L ddH₂And O, uniformly mixing and dissolving for later use.

6. PCR amplification identification

PCR amplification was carried out using the extracted DNA as a template and ATG9-F/R as a primer in Table 6, and the PCR amplification reaction system is shown in Table 7.

Table 6: target fragment primer for PCR amplification

Table 7: PCR reaction system

The amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension at 68 ℃ for 60s/1kb for 25 cycles; total extension was carried out at 68 ℃ for 5min and at 16 ℃ for 1 min. After the reaction, 5. mu.L of the amplified product was added to 1% agarose gel containing nucleic acid dye for electrophoresis; and (3) after the electrophoresis is finished, the gel is irradiated on an ultraviolet imager, whether the band is amplified or not is detected, and if the band is amplified, the fragment is the target gene fragment which possibly comprises the mutation point. The remaining PCR products were sent to the sequencing company for sequencing (sequencing was done by Guangzhou Rui Bo Biotechnology, Inc.).

7. Sequencing alignment

And (3) putting the sequencing result into a DSDecodeM website of the national university of south China, Liu dazzling academy of sciences, to compare, judging pure heterozygous sites according to a peak diagram, wherein a single peak represents a homozygous site, and a double peak represents a heterozygous site. The results are shown in FIG. 1, where the T is inserted at the mutation site and is a single peak, indicating that the plant is homozygous atg9 mutant.

Example 2: construction of ATG9 overexpression plant

The plant overexpression Vector pRHVcGFP was provided by the Wang Beam project group of Chinese academy of agricultural sciences (the plant overexpression Vector pRHVcGFP is disclosed in supplementary data of He F, Zhang F, Sun W, et al. A Versatile Vector Toolkit for Functional Analysis of Rice Genes [ J ]. Rice,2018,11(1): 27.)

1. Construction of pRHVcGFP-ATG9 overexpression vector

(1) Amplification of target Gene

Taking cDNA of WT leaves as a template (according to the instructions of abm reverse transcription kit), designing primers according to target genes (Table 8), and performing PCR amplification to obtain the target genes, wherein an amplification PCR system is shown in Table 9:

table 8: target fragment primer for PCR amplification

Table 9: target gene PCR reaction system

The amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, and extension at 68 ℃ for 60s, for 32 cycles; total extension was carried out at 68 ℃ for 5min and at 16 ℃ for 1 min. After the reaction is finished, 50 mu L of amplification product is added into 1% agarose gel containing nucleic acid dye for electrophoresis, and after the electrophoresis is finished, the gel is illuminated on an ultraviolet imager to detect whether the band is amplified. If the target band is amplified, the amplified fragment may contain the target gene. The band of interest was cut and the gel was continued to recover the purified product (according to the gel recovery kit instructions) and concentration measurements.

(2) Preparation of linearized vector

Respectively taking 3 mug of overexpression vector pRHVcGFP and target gene (operated according to the specification of a plasmid extraction kit), and adding corresponding restriction endonuclease into both reaction systems for double digestion at 37 ℃ for 20 min. The overexpression vector pRHVcGFP and the target gene are shown in Table 10:

table 10: enzyme digestion reaction system

Adding 50 mu L of reaction product into 1% agarose gel containing nucleic acid dye for electrophoresis, irradiating gel on an ultraviolet imager after the electrophoresis is finished, cutting a target band after the target band is separated, continuing to gel, recovering and purifying the product and measuring the concentration.

(3) Recombination reactions

The insert and the vector were recombined in proportion using T4DNA ligase to give pRHVcGFP-ATG9 overexpression vector. The optimal molar ratio of the vector to the insert is 1 (2-3). The DNA mass corresponding to these mole numbers can be roughly calculated by the following formula: the optimum amount of vector used was [0.02 Xvector base pair ] ng (0.03 pmol). The optimum amount of the insert used was either [0.04 × base pair of insert ] ng (0.06pmol) or [0.06 × base pair of insert ] ng (0.09 pmol). The recombination reaction system is shown in Table 11:

table 11: recombination reaction system

After the system is prepared, gently sucking and beating the components uniformly by using a pipettor, and centrifuging for a short time to collect reaction liquid to the bottom of the tube. Reacting at 16 deg.C for 6-8 h. The reaction product can be directly transformed, or can be stored at-20 ℃ and be thawed and transformed when needed.

(4) Transformation and plating of recombinant products

The clone competent cells DH5 α were thawed on ice. Adding 10 μ L of cooled recombinant product (pRHVcGFP-ATG9 overexpression vector) into 100 μ L of competent cells, flicking tube wall, mixing, and standing on ice for 30 min; heat shock at 42 deg.C for 60s, incubating in ice bath for 2min, adding 900 μ L LB medium, shaking at 37 deg.C and 200rpm for 30min, centrifuging at 5000rpm for 3min, and discarding the supernatant. The cells were resuspended in the remaining medium and gently spread on a kan-resistant plate using a sterile spreading rod. After the bacterial solution was absorbed, the plates were inverted and incubated overnight at 37 ℃.

(5) Cloning and identification

The most convenient and rapid method is colony PCR. And picking a single colony to 500 mu L of LB culture medium by using a sterile gun head or toothpick, uniformly mixing, directly taking 1 mu L of the colony as a PCR template, and subsequently performing sequencing identification on the residual bacteria liquid. The primers used were as in Table 6, and the PCR reaction was carried out as in Table 12:

table 12: colony PCR reaction system

The amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 25 cycles; total extension at 72 ℃ for 5min and holding at 16 ℃ for 1 min. After the reaction is finished, 20 microliter of amplification product is added into 1 percent agarose gel containing nucleic acid dye for electrophoresis; and after the electrophoresis is finished, the gel is irradiated on an ultraviolet imager to detect whether the band is amplified. If the band is amplified, the clone is sent to sequencing company for sequencing (sequencing done by Guangzhou Rui Bo Biotechnology, Inc.).

2. Genetic transformation and identification of transgenic plants

Sending the constructed pRHVcGFP-ATG9 overexpression vector to Baige Gene science and technology (Jiangsu) GmbH for genetic transformation to obtain T₀And (5) plant generation. The obtained transgenic plant is identified, and the pRHVcGFP-ATG9 overexpression vector carries a HYG hygromycin label, so that whether the plant is the transgenic plant or not is judged only by identifying the existence of the hygromycin label. Extracting total genome DNA of plant leaves to be detected, carrying out PCR amplification by adopting a primer HYG (shown in table 13), and judging whether the size of a strip of a PCR product meets the requirement through running agarose gel electrophoresis, wherein the strip is a transgenic plant (namely an ATG9 overexpression plant) containing a target gene. The PCR amplification method refers to a PCR reaction system and a PCR amplification program cloned and identified in the step (5) in the construction method of the overexpression vector of the plant pRHVcGFP-ATG9, after the reaction is finished, 5 mu L of amplification product is added into 1% agarose gel containing nucleic acid dye for electrophoresis, after the electrophoresis is finished, the agarose gel is photographed on an ultraviolet imager, and whether a target band exists or not is observed. T is₀Selfing the plant to obtain T₁Plant generation, T₁Selfing the plant to obtain T₂Generation homozygous ATG9 overexpressing plants.

Table 13: HYG identification primer

Example 3: phenotypic analysis of ATG9

The inventor seeds japonica rice variety Wild Type (WT), mutant (ATG9) and ATG9 overexpression plant (OE) in a test field of a city-increasing experimental base in Guangdong province and a farm of southern China agriculture university in Guangzhou in Guangdong province, counts plant height and spike length in the mature period in the growth process of rice, and measures grain width, grain length and hundred-grain weight when receiving seeds.

And (3) the plant to be detected: wild Type (WT), mutant (ATG9) and ATG9 overexpressing plants (OE).

And respectively taking the plants to be detected, analyzing the plant height, the ear length, the grain width, the grain length and the hundred grain weight in the mature period in detail, and carrying out further measurement and statistics on the plants. The results are shown in FIGS. 2 and 3. Phenotypic analysis found that the atg9 mutant exhibited multiple growth and development-deficient phenotypes compared to Wild Type (WT), including strain shortening, ear shortening, kernel shortening and reduced grain weight. Compared with WT, the plant height and spike length of OE are significantly increased, and the grain width, grain length and hundred grain weight are also significantly increased. The results show that the overexpression of ATG9 positively regulates a plurality of important agronomic traits of rice, and is a potential new target for improving the yield and quality of rice.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> southern China university of agriculture

Application of <120> autophagy gene ATG9 in rice breeding and/or rice grain type mechanism research

<160> 17

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2784

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<223> nucleotide sequence of autophagy gene ATG9

<400> 1

atgatgtcct tccttccgaa gggcaaaact acacaaacag cgtttaagtg gccatggaga 60

ggtgaatcgc agctatcggc gcatcttctc attgatattc cacctgaaat cgaattgtcg 120

gactaccgaa gattgccaag ccctggtaat gagagcccat cagggctact tcatggagaa 180

gactttaagg aggaggtgat ccctgacttg gatattttct ttgagaggct ctatgagtat 240

ttctgcgcga aaggtctgcg gtgcatcatc accaagtgga taattgagat cctcaatgtg 300

acgtttatgg tttgctgtat cgggtttttc ttcttgtttg ttgattggcc tgcccttggc 360

gatttgaaat gtggagtgga agcgctcgag tcaggggcaa aaccttgtga cctaatgaaa 420

ctcattaagt atcgtcccct agatcctttc acatttacaa agtttatcac tattggatcc 480

atggttatac tctcaactta cggaatcatt aactttgtga agttttttgt aaaactgaga 540

agcacactca aggtccgtga cttctactgt aacagtctca aggttacaga tctcgaaatt 600

cagactatat catggcctag agtagttgag aaggtcgtac ttctgcagaa gtcacaacga 660

ctttgtgtcg ttaaagatct cacggagcat gatataatca tgcgaataat gcgtaaacaa 720

aactacctga ttgggatggt taataaaggt atcatcgcat tgccaatccc ctcttggctt 780

cctggagttg gcccaactgt cagctcacgt atgcatggaa aaaaaagcta tctgatgctt 840

ccaaaggccc tggagtggac attgaattgg tgcatcttcc aaaccatgtt tgataggaaa 900

ttttgtgtta gaaaagacat tctgacaagt ccgtctttat tgaagaaacg gcttgtattt 960

atgggtattg ccatgtttct tctgtcacct tgtcttgtta tcttcccatt ggtataccta 1020

tttctgagat atgcagaaga attctacaat caccccagta cggcatcatc tcgaaaatgg 1080

tcgaatcttt caaagtggat tttgagggaa tacaatgagg ttgaccattt cttcaaacat 1140

aggttgaaca acagtagtgt caactcacta aattatttga agcagtttcc aactccacta 1200

gtatctatta ttgcaaaatt catatcgttt gtatctggtg gattggctgg aattcttctc 1260

atccttggat ttctaggtga atccatcctt gagggtcatg tatttggccg aaatcttctc 1320

tggtatacta ttgtttttgg aacaatagca actgttagtc ggaatgttgt ggttgatgaa 1380

ctccaagtga ttgatccaga aggagctatg tcctttgttc tgcagcagac acactacatg 1440

cctaaaaggt ggcgcggtaa agagggcagt gaacttgtac gaagggactt tgaatctctg 1500

tttcagtata ccataacgat gctactagaa gagatggcct caattttcat cactccatat 1560

ctgctaatat ttgtggttcc aaagcgtgtt gatgatattc tacgcttcat atcagacttc 1620

acagtttatg ttgatggagt gggagatgtg tgcagcttaa gcatgttcga cttaagaaga 1680

catggaaata gaaactacgg ttcaccacat aatgcagtta aaagcatgag aagctcccaa 1740

ggaaaaatgg agaaatcact attaagtttc caaagcacct acacatcatg ggagccaaac 1800

gcagatggca agaaatttat ttgcaacctc cagaaattca aggagaaaca aatccgtcaa 1860

catacctttc aaacaacgga atcatcacag cttgggttga gttgtagagg tcaaactgca 1920

gttttccatc gactgctccc tagaaacatc tatcctggca atggggtcat ttttaacttt 1980

gatccattag gactgctcga cacagatcaa agagcttgtc catatatcct ggattggtat 2040

tacacacacc aacacacaaa tagagaagcg ggctcatctt cccaccttaa tgaagcatca 2100

cctgagcaac aggaggagat atggccacct ctgagcaaac cattaacaga aattgaagat 2160

gagcaaatct gggattctga tttatataga agagctcgaa gctacctgga ggcatcaaca 2220

tcaagtgcat tctttcgaca ggccaccacc ttcaagcgcc atggcaggga acaaaattct 2280

accagccatc aatggtgggc tcaggcctcg aggcagcagg ctgatccacg aaacagtttt 2340

caagggcctc ctcaagacag ttttcttgag cctccggact tccgaaatca tttggaagct 2400

agccatgatt ccagccatca aagtgattgt aggctgacct ccagaaggtc aactgatcca 2460

caagacagct tcgttgagcc tcctgatttt ggtgattaca tgtcctgtca tagctctagc 2520

tatcatggcg atgaaacgtc ggatggaaac tcagaactag atcaaagcaa caatagttgg 2580

cgaagccccc atgccctgtc aaagacaagg tacatgggtg atgatgatct tgacctagaa 2640

caaggcccga gtttccattt tactgatgcc cctcagaagg acagtggaag tgaaggagat 2700

ggacatggtg tagccaatat ttacagctct acgccagcca gccttcctgt acggataata 2760

cctagaagca gtgacccagt ttag 2784

<210> 2

<223> amino acid sequence of autophagy gene ATG9

<400> 2

Met Met Ser Phe Leu Pro Lys Gly Lys Thr Thr Gln Thr Ala Phe Lys

1 5 10 15

Trp Pro Trp Arg Gly Glu Ser Gln Leu Ser Ala His Leu Leu Ile Asp

20 25 30

Ile Pro Pro Glu Ile Glu Leu Ser Asp Tyr Arg Arg Leu Pro Ser Pro

35 40 45

Gly Asn Glu Ser Pro Ser Gly Leu Leu His Gly Glu Asp Phe Lys Glu

50 55 60

Glu Val Ile Pro Asp Leu Asp Ile Phe Phe Glu Arg Leu Tyr Glu Tyr

65 70 75 80

Phe Cys Ala Lys Gly Leu Arg Cys Ile Ile Thr Lys Trp Ile Ile Glu

85 90 95

Ile Leu Asn Val Thr Phe Met Val Cys Cys Ile Gly Phe Phe Phe Leu

100 105 110

Phe Val Asp Trp Pro Ala Leu Gly Asp Leu Lys Cys Gly Val Glu Ala

115 120 125

Leu Glu Ser Gly Ala Lys Pro Cys Asp Leu Met Lys Leu Ile Lys Tyr

130 135 140

Arg Pro Leu Asp Pro Phe Thr Phe Thr Lys Phe Ile Thr Ile Gly Ser

145 150 155 160

Met Val Ile Leu Ser Thr Tyr Gly Ile Ile Asn Phe Val Lys Phe Phe

165 170 175

Val Lys Leu Arg Ser Thr Leu Lys Val Arg Asp Phe Tyr Cys Asn Ser

180 185 190

Leu Lys Val Thr Asp Leu Glu Ile Gln Thr Ile Ser Trp Pro Arg Val

195 200 205

Val Glu Lys Val Val Leu Leu Gln Lys Ser Gln Arg Leu Cys Val Val

210 215 220

Lys Asp Leu Thr Glu His Asp Ile Ile Met Arg Ile Met Arg Lys Gln

225 230 235 240

Asn Tyr Leu Ile Gly Met Val Asn Lys Gly Ile Ile Ala Leu Pro Ile

245 250 255

Pro Ser Trp Leu Pro Gly Val Gly Pro Thr Val Ser Ser Arg Met His

260 265 270

Gly Lys Lys Ser Tyr Leu Met Leu Pro Lys Ala Leu Glu Trp Thr Leu

275 280 285

Asn Trp Cys Ile Phe Gln Thr Met Phe Asp Arg Lys Phe Cys Val Arg

290 295 300

Lys Asp Ile Leu Thr Ser Pro Ser Leu Leu Lys Lys Arg Leu Val Phe

305 310 315 320

Met Gly Ile Ala Met Phe Leu Leu Ser Pro Cys Leu Val Ile Phe Pro

325 330 335

Leu Val Tyr Leu Phe Leu Arg Tyr Ala Glu Glu Phe Tyr Asn His Pro

340 345 350

Ser Thr Ala Ser Ser Arg Lys Trp Ser Asn Leu Ser Lys Trp Ile Leu

355 360 365

Arg Glu Tyr Asn Glu Val Asp His Phe Phe Lys His Arg Leu Asn Asn

370 375 380

Ser Ser Val Asn Ser Leu Asn Tyr Leu Lys Gln Phe Pro Thr Pro Leu

385 390 395 400

Val Ser Ile Ile Ala Lys Phe Ile Ser Phe Val Ser Gly Gly Leu Ala

405 410 415

Gly Ile Leu Leu Ile Leu Gly Phe Leu Gly Glu Ser Ile Leu Glu Gly

420 425 430

His Val Phe Gly Arg Asn Leu Leu Trp Tyr Thr Ile Val Phe Gly Thr

435 440 445

Ile Ala Thr Val Ser Arg Asn Val Val Val Asp Glu Leu Gln Val Ile

450 455 460

Asp Pro Glu Gly Ala Met Ser Phe Val Leu Gln Gln Thr His Tyr Met

465 470 475 480

Pro Lys Arg Trp Arg Gly Lys Glu Gly Ser Glu Leu Val Arg Arg Asp

485 490 495

Phe Glu Ser Leu Phe Gln Tyr Thr Ile Thr Met Leu Leu Glu Glu Met

500 505 510

Ala Ser Ile Phe Ile Thr Pro Tyr Leu Leu Ile Phe Val Val Pro Lys

515 520 525

Arg Val Asp Asp Ile Leu Arg Phe Ile Ser Asp Phe Thr Val Tyr Val

530 535 540

Asp Gly Val Gly Asp Val Cys Ser Leu Ser Met Phe Asp Leu Arg Arg

545 550 555 560

His Gly Asn Arg Asn Tyr Gly Ser Pro His Asn Ala Val Lys Ser Met

565 570 575

Arg Ser Ser Gln Gly Lys Met Glu Lys Ser Leu Leu Ser Phe Gln Ser

580 585 590

Thr Tyr Thr Ser Trp Glu Pro Asn Ala Asp Gly Lys Lys Phe Ile Cys

595 600 605

Asn Leu Gln Lys Phe Lys Glu Lys Gln Ile Arg Gln His Thr Phe Gln

610 615 620

Thr Thr Glu Ser Ser Gln Leu Gly Leu Ser Cys Arg Gly Gln Thr Ala

625 630 635 640

Val Phe His Arg Leu Leu Pro Arg Asn Ile Tyr Pro Gly Asn Gly Val

645 650 655

Ile Phe Asn Phe Asp Pro Leu Gly Leu Leu Asp Thr Asp Gln Arg Ala

660 665 670

Cys Pro Tyr Ile Leu Asp Trp Tyr Tyr Thr His Gln His Thr Asn Arg

675 680 685

Glu Ala Gly Ser Ser Ser His Leu Asn Glu Ala Ser Pro Glu Gln Gln

690 695 700

Glu Glu Ile Trp Pro Pro Leu Ser Lys Pro Leu Thr Glu Ile Glu Asp

705 710 715 720

Glu Gln Ile Trp Asp Ser Asp Leu Tyr Arg Arg Ala Arg Ser Tyr Leu

725 730 735

Glu Ala Ser Thr Ser Ser Ala Phe Phe Arg Gln Ala Thr Thr Phe Lys

740 745 750

Arg His Gly Arg Glu Gln Asn Ser Thr Ser His Gln Trp Trp Ala Gln

755 760 765

Ala Ser Arg Gln Gln Ala Asp Pro Arg Asn Ser Phe Gln Gly Pro Pro

770 775 780

Gln Asp Ser Phe Leu Glu Pro Pro Asp Phe Arg Asn His Leu Glu Ala

785 790 795 800

Ser His Asp Ser Ser His Gln Ser Asp Cys Arg Leu Thr Ser Arg Arg

805 810 815

Ser Thr Asp Pro Gln Asp Ser Phe Val Glu Pro Pro Asp Phe Gly Asp

820 825 830

Tyr Met Ser Cys His Ser Ser Ser Tyr His Gly Asp Glu Thr Ser Asp

835 840 845

Gly Asn Ser Glu Leu Asp Gln Ser Asn Asn Ser Trp Arg Ser Pro His

850 855 860

Ala Leu Ser Lys Thr Arg Tyr Met Gly Asp Asp Asp Leu Asp Leu Glu

865 870 875 880

Gln Gly Pro Ser Phe His Phe Thr Asp Ala Pro Gln Lys Asp Ser Gly

885 890 895

Ser Glu Gly Asp Gly His Gly Val Ala Asn Ile Tyr Ser Ser Thr Pro

900 905 910

Ala Ser Leu Pro Val Arg Ile Ile Pro Arg Ser Ser Asp Pro Val

915 920 925

<210> 3

<223> target site

<400> 3

gagtggaagc gctcgagtc 19

<210> 4

<223> pRHVcGFP vector sequence

<400> 4

atgcaggtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga 60

acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa 120

cgggaaacgt cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa 180

tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc 240

gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat 300

gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt 360

atccgtactc ctgatgatgc atggttactc accactgcga tccccggaaa aacagcattc 420

caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc 480

ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt 540

cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat 600

gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca 660

ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac 720

gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag 780

gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt 840

tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc 900

gatgagtttt tctaatcaga attggttaat tggttgtaac actggcagag cattacgctg 960

acttgacggg acggcgcaag ctcatgacca aaatccctta acgtgagttt tcgttccact 1020

gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 1080

taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 1140

aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 1200

ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 1260

catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 1320

ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 1380

ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 1440

agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 1500

taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 1560

atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 1620

cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 1680

ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 1740

accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 1800

gcgagtcagt gagcgaggaa gcggaagagc gcccggcgcg ccgaatgaac gccaagagga 1860

acaagcatga aaccgcacca ggacggccag gacgaaccgt ttttcattac cgaagagatc 1920

gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt ctcaaccgtg 1980

cggctgcatg aaatcctggc cggtttgtct gatgccaagc tggcggcctg gccggccagc 2040

ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt tgagtaaaac 2100

agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag taaataaaca aatacgcaag 2160

gggaacgcat gaaggttatc gctgtactta accagaaagg cgggtcaggc aagacgacca 2220

tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg ttagtcgatt 2280

ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa ccgctaaccg 2340

ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc cggcgcgact 2400

tcgtagtgat cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg atcaaggcag 2460

ccgacttcgt gctgattccg gtgcagccaa gcccttacga catatgggcc accgccgacc 2520

tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa gcggcctttg 2580

tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag gcgctggccg 2640

ggtacgagct gcccattctt gagtcccgta tcacgcagcg cgtgagctac ccaggcactg 2700

ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc cgcgaggtcc 2760

aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt taatgaggta aagagaaaat 2820

gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca gcaaggctgc 2880

aacgttggcc agcctggcag acacgccagc catgaagcgg gtcaactttc agttgccggc 2940

ggaggatcac accaagctga agatgtacgc ggtacgccaa ggcaagacca ttaccgagct 3000

gctatctgaa tacatcgcgc agctaccaga gtaaatgagc aaatgaataa atgagtagat 3060

gaattttagc ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc accgacgccg 3120

tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc tgggttgcct 3180

gccggccctg caatggcact ggaaccccca agcccgagga atcggcgtga gcggtcgcaa 3240

accatccggc ccggtacaaa tcggcgcggc gctgggtgat gacctggtgg agaagttgaa 3300

ggccgcgcag gccgcccagc ggcaacgcat cgaggcagaa gcacgccccg gtgaatcgtg 3360

gcaagcgtcc gctgatcgaa tccgcaaaga atcccggcaa ccgccggcag ccggtgcgcc 3420

gtcgattagg aagccgccca agggcgacga gcaaccagat tttttcgttc cgatgctcta 3480

tgacgtgggc acccgcgata gtcgcagcat catggacgtg gccgttttcc gtctgtcgaa 3540

gcgtgaccga cgagctggcg aggtgatccg ctacgagctt ccagacgggc acgtagaggt 3600

ttccgcaggg ccggccggca tggccagtgt gtgggattac gacctggtac tgatggcggt 3660

ttcccatcta accgaatcca tgaaccgata ccgggaaggg aagggagaca agcccggccg 3720

cgtgttccgt ccacacgttg cggacgtact caagttctgc cggcgagccg atggcggaaa 3780

gcagaaagac gacctggtag aaacctgcat tcggttaaac accacgcacg ttgccatgca 3840

gcgtacgaag aaggccaaga acggccgcct ggtgacggta tccgagggtg aagccttgat 3900

tagccgctac aagatcgtaa agagcgaaac cgggcggccg gagtacatcg agatcgagct 3960

agctgattgg atgtaccgcg agatcacaga aggcaagaac ccggacgtgc tgacggttca 4020

ccccgattac tttttgatcg atcccggcat cggccgtttt ctctaccgcc tggcacgccg 4080

cgccgcaggc aaggcagaag ccagatggtt gttcaagacg atctacgaac gcagtggcag 4140

cgccggagag ttcaagaagt tctgtttcac cgtgcgcaag ctgatcgggt caaatgacct 4200

gccggagtac gatttgaagg aggaggcggg gcaggctggc ccgatcctag tcatgcgcta 4260

ccgcaacctg atcgagggcg aagcatccgc cggttcctaa tgtacggagc agatgctagg 4320

gcaaattgcc ctagcagggg aaaaaggtcg aaaaggtctc tttcctgtgg atagcacgta 4380

cattgggaac ccaaagccgt acattgggaa ccggaacccg tacattggga acccaaagcc 4440

gtacattggg aaccggtcac acatgtaagt gactgatata aaagagaaaa aaggcgattt 4500

ttccgcctaa aactctttaa aacttattaa aactcttaaa acccgcctgg cctgtgcata 4560

actgtctggc cagcgcacag ccgaagagct gcaaaaagcg cctagtttac ccgccaatat 4620

atcctgtcag gcgcgccccg atctagtaac atagatgaca ccgcgcgcga taatttatcc 4680

tagtttgcgc gctatatttt gttttctatc gcgtattaaa tgtataattg cgggactcta 4740

atcataaaaa cccatctcat aaataacgtc atgcattaca tgttaattat tacatgctta 4800

acgtaattca acagaaatta tatgataatc atcgcaagac cggcaacagg attcaatctt 4860

aagaaacttt attgccaaat gtttgaacga tctactcact tagcggccgt tacttgtaca 4920

gctcgtccat gccgagagtg atcccggcgg cggtcacgaa ctccagcagg accatgtgat 4980

cgcgcttctc gttggggtct ttgctcaggg cggactgggt gctcaggtag tggttgtcgg 5040

gcagcagcac ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg gcgagctgca 5100

cgctgccgtc ctcgatgttg tggcggatct tgaagttcac cttgatgccg ttcttctgct 5160

tgtcggccat gatatagacg ttgtggctgt tgtagttgta ctccagcttg tgccccagga 5220

tgttgccgtc ctccttgaag tcgatgccct tcagctcgat gcggttcacc agggtgtcgc 5280

cctcgaactt cacctcggcg cgggtcttgt agttgccgtc gtccttgaag aagatggtgc 5340

gctcctggac gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt 5400

cggggtagcg gctgaagcac tgcacgccgt aggtcagggt ggtcacgagg gtgggccagg 5460

gcacgggcag cttgccggtg gtgcagatga acttcagggt cagcttgccg taggtggcat 5520

cgccctcgcc ctcgccggac acgctgaact tgtggccgtt tacgtcgccg tccagctcga 5580

ccaggatggg caccaccccg gtgaacagct cctcgccctt gctcaccata gcggccgcac 5640

tagtaagctt ggtaccgagc tcacccgggg atcccactgg atctggatat cctgcataag 5700

taacaccaaa caacagggtg agcatcgaca aaagaaacag taccaagcaa ataaatagcg 5760

tatgaaggca gggctaaaaa aatccacata tagctgctgc atatgccatc atccaagtat 5820

atcaagatca aaataattat aaaacatact tgtttattat aatagatagg tactcaaggt 5880

tagagcatat gaatagatgc tgcatatgcc atcatgtata tgcatcagta aaacccacat 5940

caacatgtat acctatccta gatcgatatt tccatccatc ttaaactcgt aactatgaag 6000

atgtatgaca cacacataca gttccaaaat taataaatac accaggtagt ttgaaacagt 6060

attctactcc gatctagaac gaatgaacga ccgcccaacc acaccacatc atcacaacca 6120

agcgaacaaa aagcatctct gtatatgcat cagtaaaacc cgcatcaaca tgtataccta 6180

tcctagatcg atatttccat ccatcatctt caattcgtaa ctatgaatat gtatggcaca 6240

cacatacaga tccaaaatta ataaatccac caggtagttt gaaacagaat tctactccga 6300

tctagaacga ccgcccaacc agaccacatc atcacaacca agacaaaaaa aagcatgaaa 6360

agatgacccg acaaacaagt gcacggcata tattgaaata aaggaaaagg gcaaaccaaa 6420

ccctatgcaa cgaaacaaaa aaaatcatga aatcgatccc gtctgcggaa cggctagagc 6480

catcccagga ttccccaaag agaaacactg gcaagttagc aatcagaacg tgtctgacgt 6540

acaggtcgca tccgtgtacg aacgctagca gcacggatct aacacaaaca cggatctaac 6600

acaaacatga acagaagtag aactaccggg ccctaaccat ggaccggaac gccgatctag 6660

agaaggtaga gagggggggg gggggaggac gagcggcgta ccttgaagcg gaggtgccga 6720

cgggtggatt tgggggagat ctggttgtgt gtgtgtgcgc tccgaacaac acgaggttgg 6780

ggaaagaggg tgtggagggg gtgtctattt attacggcgg gcgaggaagg gaaagcgaag 6840

gagcggtggg aaaggaatcc cccgtagctg ccggtgccgt gagaggagga ggaggccgcc 6900

tgccgtgccg gctcacgtct gccgctccgc cacgcaattt ctggatgccg acagcggagc 6960

aagtccaacg gtggagcgga actctcgaga ggggtccaga ggcagcgaca gagatgccgt 7020

gccgtctgct tcgcttggcc cgacgcgacg ctgctggttc gctggttggt gtccgttaga 7080

ctcgtcgacg gcgtttaaca ggctggcatt atctactcga aacaagaaaa atgtttcctt 7140

agttttttta atttcttaaa gggtatttgt ttaattttta gtcactttat tttattctat 7200

tttatatcta aattattaaa taaaaaaact aaaatagagt tttagttttc ttaatttaga 7260

ggctaaaata gaataaaata gatgtactaa aaaaattagt ctataaaaac cattaaccct 7320

aaaccctaaa tggatgtact aataaaatgg atgaagtatt atataggtga agctatttgc 7380

aaaaaaaaag gagaacacat gcacactaaa aagataaaac tgtagagtcc tgttgtcaaa 7440

atactcaatt gtcctttaga ccatgtctaa ctgttcattt atatgattct ctaaaacact 7500

gatattattg tagtactata gattatatta ttcgtagagt aaagtttaaa tatatgtata 7560

aagatagata aactgcactt caaacaagtg tgacaaaaaa aatatgtggt aattttttat 7620

aacttagaca tgcaatgctc attatctcta gagaggggca cgaccgggtc acgctgcact 7680

gcagtaattc gggggatctg gattttagta ctggattttg gttttaggaa ttagaaattt 7740

tattgataga agtattttac aaatacaaat acatactaag ggtttcttat atgctcaaca 7800

catgagcgaa accctatagg aaccctaatt cccttatctg ggaactactc acacattatt 7860

atggagaaac tcgagcttgt cgatcgacag atcccggtcg gcatctactc tatttctttg 7920

ccctcggacg agtgctgggg cgtcggtttc cactatcggc gagtacttct acacagccat 7980

cggtccagac ggccgcgctt ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg 8040

atcggacgat tgcgtcgcat cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa 8100

ccaagctctg atagagttgg tcaagaccaa tgcggagcat atacgcccgg agtcgtggcg 8160

atcctgcaag ctccggatgc ctccgctcga agtagcgcgt ctgctgctcc atacaagcca 8220

accacggcct ccagaagaag atgttggcga cctcgtattg ggaatccccg aacatcgcct 8280

cgctccagtc aatgaccgct gttatgcggc cattgtccgt caggacattg ttggagccga 8340

aatccgcgtg cacgaggtgc cggacttcgg ggcagtcctc ggcccaaagc atcagctcat 8400

cgagagcctg cgcgacggac gcactgacgg tgtcgtccat cacagtttgc cagtgataca 8460

catggggatc agcaatcgcg catatgaaat cacgccatgt agtgtattga ccgattcctt 8520

gcggtccgaa tgggccgaac ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca 8580

tagcctccgc gaccggttgt agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg 8640

tgacaccctg tgtacggcgg gagatgcaat aggtcaggct ctcgctaaac tccccaatgt 8700

caagcacttc cggaatcggg agcgcggccg atgcaaagtg ccgataaaca taacgatctt 8760

tgtagaaacc atcggcgcag ctatttaccc gcaggacata tccacgccct cctacatcga 8820

agctgaaagc acgagattct tcgccctccg agagctgcat caggtcggag acgctgtcga 8880

acttttcgat cagaaacttc tcgacagacg tcgcggtgag ttcaggcttt ttcattgtcc 8940

tctccaaatg aaatgaactt ccttatatag aggaagggtc ttgcgaagga tagtgggatt 9000

gtgcgtcatc ccttacgtca gtggagatgt cacatcaatc cacttgcttt gaagacgtgg 9060

ttggaacgtc ttctttttcc acgatgctcc tcgtgggtgg gggtccatct ttgggaccac 9120

tgtcggcaga gagatcttga atgatagcct ttcctttatc gcaatgatgg catttgtagg 9180

agccaccttc cttttctact gtcctttcga tgaagtgaca gatagctggg caatggaatc 9240

cgaggaggtt tcccgaaatt atcctttgtt gaaaagtctc aatagccctt tggtcttctg 9300

agactgtatc tttgacattt ttggagtaga ccagagtgtc gtgctccacc atgtttgttt 9360

acaccacaat atatcctgcc a 9381

<210> 5

<223> SK-gRNA vector

<400> 5

gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60

caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120

ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180

gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240

tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300

ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360

tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420

atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480

gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540

caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600

ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660

ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720

ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780

ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840

gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900

ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960

taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020

agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080

atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140

aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200

caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260

ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320

cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380

tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440

gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500

ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560

gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620

caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680

ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740

tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800

ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860

agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920

aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980

gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040

tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100

tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160

ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg 2220

gccgcagatc tgctagcgtc gacgattaag gaatctttaa acatacgaac agatcactta 2280

aagttcttct gaagcaactt aaagttatca ggcatgcatg gatcttggag gaatcagatg 2340

tgcagtcagg gaccatagca caagacaggc gtcttctact ggtgctacca gcaaatgctg 2400

gaagccggga acactgggta cgtcggaaac cacgtgatgt gaagaagtaa gataaactgt 2460

aggagaaaag catttcgtag tgggccatga agcctttcag gacatgtatt gcagtatggg 2520

ccggcccatt acgcaattgg acgacaacaa agactagtat tagtaccacc tcggctatcc 2580

acatagatca aagctgattt aaaagagttg tgcagatgat ccgtggcagc tcgcaggtga 2640

acacaacacc tgcacacgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt 2700

tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ccacataatc ctcgagtcta 2760

gaggatcctc ggtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg 2820

tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag 2880

cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc 2940

aacagttgcg cagcctgaat ggcgaatgga aattgtaagc gttaatattt tgttaaaatt 3000

cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat 3060

cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa 3120

gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg 3180

cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa 3240

agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc 3300

gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag 3360

tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg 3420

cgcgtcag 3428

<210> 6

<223> pC1300-Cas9 vector

<400> 6

aattcgagct cggtaccaag gatccgtacc cctactccaa aaatgtcaaa gatacagtct 60

cagaagacca aagggctatt gagacttttc aacaaagggt aatttcggga aacctcctcg 120

gattccattg cccagctatc tgtcacttca tcgaaaggac agtagaaaag gaaggtggct 180

cctacaaatg ccatcattgc gataaaggaa aggctatcat tcaagatgcc tctgccgaca 240

gtggtcccaa agatggaccc ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa 300

ccacgtcttc aaagcaagtg gattgatgtg acatctccac tgacgtaagg gatgacgcac 360

aatcccaccc ctactccaaa aatgtcaaag atacagtctc agaagaccaa agggctattg 420

agacttttca acaaagggta atttcgggaa acctcctcgg attccattgc ccagctatct 480

gtcacttcat cgaaaggaca gtagaaaagg aaggtggctc ctacaaatgc catcattgcg 540

ataaaggaaa ggctatcatt caagatgcct ctgccgacag tggtcccaaa gatggacccc 600

cacccacgag gagcatcgtg gaaaaagaag acgttccaac cacgtcttca aagcaagtgg 660

attgatgtga catctccact gacgtaaggg atgacgcaca atcccactat ccttcgcaag 720

acccttcctc tatataagga agttcatttc atttggagag gacagcccaa gctagatcca 780

tggcccctaa gaagaagaga aaggtcggta ttcacggcgt tcctgcggcg atggacaaga 840

agtatagtat tggtctggac attgggacga attccgttgg ctgggccgtg atcaccgatg 900

agtacaaggt cccttccaag aagtttaagg ttctggggaa caccgatcgg cacagcatca 960

agaagaatct cattggagcc ctcctgttcg actcaggcga gaccgccgaa gcaacaaggc 1020

tcaagagaac cgcaaggaga cggtatacaa gaaggaagaa taggatctgc tacctgcagg 1080

agattttcag caacgaaatg gcgaaggtgg acgattcgtt ctttcataga ttggaggaga 1140

gtttcctcgt cgaggaagat aagaagcacg agaggcatcc tatctttggc aacattgtcg 1200

acgaggttgc ctatcacgaa aagtacccca caatctatca tctgcggaag aagcttgtgg 1260

actcgactga taaggcggac cttagattga tctacctcgc tctggcacac atgattaagt 1320

tcaggggcca ttttctgatc gagggggatc ttaacccgga caatagcgat gtggacaagt 1380

tgttcatcca gctcgtccaa acctacaatc agctctttga ggaaaaccca attaatgctt 1440

caggcgtcga cgccaaggcg atcctgtctg cacgcctttc aaagtctcgc cggcttgaga 1500

acttgatcgc tcaactcccg ggcgaaaaga agaacggctt gttcgggaat ctcattgcac 1560

tttcgttggg gctcacacca aacttcaaga gtaattttga tctcgctgag gacgcaaagc 1620

tgcagctttc caaggacact tatgacgatg acctggataa ccttttggcc caaatcggcg 1680

atcagtacgc ggacttgttc ctcgccgcga agaatttgtc ggacgcgatc ctcctgagtg 1740

atattctccg cgtgaacacc gagattacaa aggccccgct ctcggcgagt atgatcaagc 1800

gctatgacga gcaccatcag gatctgaccc ttttgaaggc tttggtccgg cagcaactcc 1860

cagagaagta caaggaaatc ttctttgatc aatccaagaa cggctacgct ggttatattg 1920

acggcggggc atcgcaggag gaattctaca agtttatcaa gccaattctg gagaagatgg 1980

atggcacaga ggaactcctg gtgaagctca atagggagga ccttttgcgg aagcaaagaa 2040

ctttcgataa cggcagcatc cctcaccaga ttcatctcgg ggagctgcac gccatcctga 2100

gaaggcagga agacttctac ccctttctta aggataaccg ggagaagatc gaaaagattc 2160

tgacgttcag aattccgtac tatgtcggac cactcgcccg gggtaattcc agatttgcgt 2220

ggatgaccag aaagagcgag gaaaccatca caccttggaa cttcgaggaa gtggtcgata 2280

agggcgcttc cgcacagagc ttcattgagc gcatgacaaa ttttgacaag aacctgccta 2340

atgagaaggt ccttcccaag cattccctcc tgtacgagta tttcactgtt tataacgaac 2400

tcacgaaggt gaagtatgtg accgagggaa tgcgcaagcc cgccttcctg agcggcgagc 2460

aaaagaaggc gatcgtggac cttttgttta agaccaatcg gaaggtcaca gttaagcagc 2520

tcaaggagga ctacttcaag aagattgaat gcttcgattc cgttgagatc agcggcgtgg 2580

aagacaggtt taacgcgtca ctggggactt accacgatct cctgaagatc attaaggata 2640

aggacttctt ggacaacgag gaaaatgagg atatcctcga agacattgtc ctgactctta 2700

cgttgtttga ggatagggaa atgatcgagg aacgcttgaa gacgtatgcc catctcttcg 2760

atgacaaggt tatgaagcag ctcaagagaa gaagatacac cggatgggga aggctgtccc 2820

gcaagcttat caatggcatt agagacaagc aatcagggaa gacaatcctt gactttttga 2880

agtctgatgg cttcgcgaac aggaatttta tgcagctgat tcacgatgac tcacttactt 2940

tcaaggagga tatccagaag gctcaagtgt cgggacaagg tgacagtctg cacgagcata 3000

tcgccaacct tgcgggatct cctgcaatca agaagggtat tctgcagaca gtcaaggttg 3060

tggatgagct tgtgaaggtc atgggacggc ataagcccga gaacatcgtt attgagatgg 3120

ccagagaaaa tcagaccaca caaaagggtc agaagaactc gagggagcgc atgaagcgca 3180

tcgaggaagg cattaaggag ctggggagtc agatccttaa ggagcacccg gtggaaaaca 3240

cgcagttgca aaatgagaag ctctatctgt actatctgca aaatggcagg gatatgtatg 3300

tggaccagga gttggatatt aaccgcctct cggattacga cgtcgatcat atcgttcctc 3360

agtccttcct taaggatgac agcattgaca ataaggttct caccaggtcc gacaagaacc 3420

gcgggaagtc cgataatgtg cccagcgagg aagtcgttaa gaagatgaag aactactgga 3480

ggcaactttt gaatgccaag ttgatcacac agaggaagtt tgataacctc actaaggccg 3540

agcgcggagg tctcagcgaa ctggacaagg cgggcttcat taagcggcaa ctggttgaga 3600

ctagacagat cacgaagcac gtggcgcaga ttctcgattc acgcatgaac acgaagtacg 3660

atgagaatga caagctgatc cgggaagtga aggtcatcac cttgaagtca aagctcgttt 3720

ctgacttcag gaaggatttc caattttata aggtgcgcga gatcaacaat tatcaccatg 3780

ctcatgacgc atacctcaac gctgtggtcg gaacagcatt gattaagaag tacccgaagc 3840

tcgagtccga attcgtgtac ggtgactata aggtttacga tgtgcgcaag atgatcgcca 3900

agtcagagca ggaaattggc aaggccactg cgaagtattt cttttactct aacattatga 3960

atttctttaa gactgagatc acgctggcta atggcgaaat ccggaagaga ccacttattg 4020

agaccaacgg cgagacaggg gaaatcgtgt gggacaaggg gagggatttc gccacagtcc 4080

gcaaggttct ctctatgcct caagtgaata ttgtcaagaa gactgaagtc cagacgggcg 4140

ggttctcaaa ggaatctatt ctgcccaagc ggaactcgga taagcttatc gccagaaaga 4200

aggactggga cccgaagaag tatggaggtt tcgactcacc aacggtggct tactctgtcc 4260

tggttgtggc aaaggtggag aagggaaagt caaagaagct caagtctgtc aaggagctcc 4320

tgggtatcac cattatggag aggtccagct tcgaaaagaa tccgatcgat tttctcgagg 4380

cgaagggata taaggaagtg aagaaggacc tgatcattaa gcttccaaag tacagtcttt 4440

tcgagttgga aaacggcagg aagcgcatgt tggcttccgc aggagagctc cagaagggta 4500

acgagcttgc tttgccgtcc aagtatgtga acttcctcta tctggcatcc cactacgaga 4560

agctcaaggg cagcccagag gataacgaac agaagcaact gtttgtggag caacacaagc 4620

attatcttga cgagatcatt gaacagattt cggagttcag taagcgcgtc atcctcgccg 4680

acgcgaattt ggataaggtt ctctcagcct acaacaagca ccgggacaag cctatcagag 4740

agcaggcgga aaatatcatt catctcttca ccctgacaaa ccttggggct cccgctgcat 4800

tcaagtattt tgacactacg attgatcgga agagatacac ttctacgaag gaggtgctgg 4860

atgcaaccct tatccaccaa tcgattactg gcctctacga gacgcggatc gacttgagtc 4920

agctcggggg ggataagaga ccagcggcaa ccaagaaggc aggacaagcg aagaagaaga 4980

agtagcaatt cggtacgctg aaatcaccag tctctctcta caaatctatc tctctctatt 5040

ttctccataa ataatgtgtg agtagtttcc cgataaggga aattagggtt cttatagggt 5100

ttcgctcatg tgttgagcat ataagaaacc cttagtatgt atttgtattt gtaaaatact 5160

tctatcaata aaatttctaa ttcctaaaac caaaatccag tactaaaatc cagatctcct 5220

aaagtcccta tagatctttg tcgtgaatat aaaccagaca cgagacgact aaacctggag 5280

cccagacgcc gttcgaagct agaagtaccg cttaggcagg aggccgttag ggaaaagatg 5340

ctaaggcagg gttggttacg ttgactcccc cgtaggtttg gtttaaatat gatgaagtgg 5400

acggaaggaa ggaggaagac aaggaaggat aaggttgcag gccctgtgca aggtaagaag 5460

atggaaattt gatagaggta cgctactata cttatactat acgctaaggg aatgcttgta 5520

tttataccct atacccccta ataacccctt atcaatttaa gaaataatcc gcataagccc 5580

ccgcttaaaa attggtatca gagccatgaa taggtctatg accaaaactc aagaggataa 5640

aacctcacca aaatacgaaa gagttcttaa ctctaaagat aaaagatctt tcaagatcaa 5700

aactagagtc gacctgcagg catgcaagct tggcactggc cgtcgtttta caacgtcgtg 5760

actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 5820

gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 5880

atggcgaatg ctagagcagc ttgagcttgg atcagattgt cgtttcccgc cttcagttta 5940

aactatcagt gtttgacagg atatattggc gggtaaacct aagagaaaag agcgtttatt 6000

agaataacgg atatttaaaa gggcgtgaaa aggtttatcc gttcgtccat ttgtatgtgc 6060

atgccaacca cagggttccc ctcgggatca aagtactttg atccaacccc tccgctgcta 6120

tagtgcagtc ggcttctgac gttcagtgca gccgtcttct gaaaacgaca tgtcgcacaa 6180

gtcctaagtt acgcgacagg ctgccgccct gcccttttcc tggcgttttc ttgtcgcgtg 6240

ttttagtcgc ataaagtaga atacttgcga ctagaaccgg agacattacg ccatgaacaa 6300

gagcgccgcc gctggcctgc tgggctatgc ccgcgtcagc accgacgacc aggacttgac 6360

caaccaacgg gccgaactgc acgcggccgg ctgcaccaag ctgttttccg agaagatcac 6420

cggcaccagg cgcgaccgcc cggagctggc caggatgctt gaccacctac gccctggcga 6480

cgttgtgaca gtgaccaggc tagaccgcct ggcccgcagc acccgcgacc tactggacat 6540

tgccgagcgc atccaggagg ccggcgcggg cctgcgtagc ctggcagagc cgtgggccga 6600

caccaccacg ccggccggcc gcatggtgtt gaccgtgttc gccggcattg ccgagttcga 6660

gcgttcccta atcatcgacc gcacccggag cgggcgcgag gccgccaagg cccgaggcgt 6720

gaagtttggc ccccgcccta ccctcacccc ggcacagatc gcgcacgccc gcgagctgat 6780

cgaccaggaa ggccgcaccg tgaaagaggc ggctgcactg cttggcgtgc atcgctcgac 6840

cctgtaccgc gcacttgagc gcagcgagga agtgacgccc accgaggcca ggcggcgcgg 6900

tgccttccgt gaggacgcat tgaccgaggc cgacgccctg gcggccgccg agaatgaacg 6960

ccaagaggaa caagcatgaa accgcaccag gacggccagg acgaaccgtt tttcattacc 7020

gaagagatcg aggcggagat gatcgcggcc gggtacgtgt tcgagccgcc cgcgcacgtc 7080

tcaaccgtgc ggctgcatga aatcctggcc ggtttgtctg atgccaagct ggcggcctgg 7140

ccggccagct tggccgctga agaaaccgag cgccgccgtc taaaaaggtg atgtgtattt 7200

gagtaaaaca gcttgcgtca tgcggtcgct gcgtatatga tgcgatgagt aaataaacaa 7260

atacgcaagg ggaacgcatg aaggttatcg ctgtacttaa ccagaaaggc gggtcaggca 7320

agacgaccat cgcaacccat ctagcccgcg ccctgcaact cgccggggcc gatgttctgt 7380

tagtcgattc cgatccccag ggcagtgccc gcgattgggc ggccgtgcgg gaagatcaac 7440

cgctaaccgt tgtcggcatc gaccgcccga cgattgaccg cgacgtgaag gccatcggcc 7500

ggcgcgactt cgtagtgatc gacggagcgc cccaggcggc ggacttggct gtgtccgcga 7560

tcaaggcagc cgacttcgtg ctgattccgg tgcagccaag cccttacgac atatgggcca 7620

ccgccgacct ggtggagctg gttaagcagc gcattgaggt cacggatgga aggctacaag 7680

cggcctttgt cgtgtcgcgg gcgatcaaag gcacgcgcat cggcggtgag gttgccgagg 7740

cgctggccgg gtacgagctg cccattcttg agtcccgtat cacgcagcgc gtgagctacc 7800

caggcactgc cgccgccggc acaaccgttc ttgaatcaga acccgagggc gacgctgccc 7860

gcgaggtcca ggcgctggcc gctgaaatta aatcaaaact catttgagtt aatgaggtaa 7920

agagaaaatg agcaaaagca caaacacgct aagtgccggc cgtccgagcg cacgcagcag 7980

caaggctgca acgttggcca gcctggcaga cacgccagcc atgaagcggg tcaactttca 8040

gttgccggcg gaggatcaca ccaagctgaa gatgtacgcg gtacgccaag gcaagaccat 8100

taccgagctg ctatctgaat acatcgcgca gctaccagag taaatgagca aatgaataaa 8160

tgagtagatg aattttagcg gctaaaggag gcggcatgga aaatcaagaa caaccaggca 8220

ccgacgccgt ggaatgcccc atgtgtggag gaacgggcgg ttggccaggc gtaagcggct 8280

gggttgtctg ccggccctgc aatggcactg gaacccccaa gcccgaggaa tcggcgtgac 8340

ggtcgcaaac catccggccc ggtacaaatc ggcgcggcgc tgggtgatga cctggtggag 8400

aagttgaagg ccgcgcaggc cgcccagcgg caacgcatcg aggcagaagc acgccccggt 8460

gaatcgtggc aagcggccgc tgatcgaatc cgcaaagaat cccggcaacc gccggcagcc 8520

ggtgcgccgt cgattaggaa gccgcccaag ggcgacgagc aaccagattt tttcgttccg 8580

atgctctatg acgtgggcac ccgcgatagt cgcagcatca tggacgtggc cgttttccgt 8640

ctgtcgaagc gtgaccgacg agctggcgag gtgatccgct acgagcttcc agacgggcac 8700

gtagaggttt ccgcagggcc ggccggcatg gccagtgtgt gggattacga cctggtactg 8760

atggcggttt cccatctaac cgaatccatg aaccgatacc gggaagggaa gggagacaag 8820

cccggccgcg tgttccgtcc acacgttgcg gacgtactca agttctgccg gcgagccgat 8880

ggcggaaagc agaaagacga cctggtagaa acctgcattc ggttaaacac cacgcacgtt 8940

gccatgcagc gtacgaagaa ggccaagaac ggccgcctgg tgacggtatc cgagggtgaa 9000

gccttgatta gccgctacaa gatcgtaaag agcgaaaccg ggcggccgga gtacatcgag 9060

atcgagctag ctgattggat gtaccgcgag atcacagaag gcaagaaccc ggacgtgctg 9120

acggttcacc ccgattactt tttgatcgat cccggcatcg gccgttttct ctaccgcctg 9180

gcacgccgcg ccgcaggcaa ggcagaagcc agatggttgt tcaagacgat ctacgaacgc 9240

agtggcagcg ccggagagtt caagaagttc tgtttcaccg tgcgcaagct gatcgggtca 9300

aatgacctgc cggagtacga tttgaaggag gaggcggggc aggctggccc gatcctagtc 9360

atgcgctacc gcaacctgat cgagggcgaa gcatccgccg gttcctaatg tacggagcag 9420

atgctagggc aaattgccct agcaggggaa aaaggtcgaa aaggtctctt tcctgtggat 9480

agcacgtaca ttgggaaccc aaagccgtac attgggaacc ggaacccgta cattgggaac 9540

ccaaagccgt acattgggaa ccggtcacac atgtaagtga ctgatataaa agagaaaaaa 9600

ggcgattttt ccgcctaaaa ctctttaaaa cttattaaaa ctcttaaaac ccgcctggcc 9660

tgtgcataac tgtctggcca gcgcacagcc gaagagctgc aaaaagcgcc tacccttcgg 9720

tcgctgcgct ccctacgccc cgccgcttcg cgtcggccta tcgcggccgc tggccgctca 9780

aaaatggctg gcctacggcc aggcaatcta ccagggcgcg gacaagccgc gccgtcgcca 9840

ctcgaccgcc ggcgcccaca tcaaggcacc ctgcctcgcg cgtttcggtg atgacggtga 9900

aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg 9960

gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat 10020

gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag 10080

attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa 10140

taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 10200

ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 10260

gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 10320

gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 10380

cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 10440

ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 10500

tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 10560

gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 10620

tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 10680

ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 10740

ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 10800

ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 10860

accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 10920

tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 10980

cgttaaggga ttttggtcat gcattctagg tactaaaaca attcatccag taaaatataa 11040

tattttattt tctcccaatc aggcttgatc cccagtaagt caaaaaatag ctcgacatac 11100

tgttcttccc cgatatcctc cctgatcgac cggacgcaga aggcaatgtc ataccacttg 11160

tccgccctgc cgcttctccc aagatcaata aagccactta ctttgccatc tttcacaaag 11220

atgttgctgt ctcccaggtc gccgtgggaa aagacaagtt cctcttcggg cttttccgtc 11280

tttaaaaaat catacagctc gcgcggatct ttaaatggag tgtcttcttc ccagttttcg 11340

caatccacat cggccagatc gttattcagt aagtaatcca attcggctaa gcggctgtct 11400

aagctattcg tatagggaca atccgatatg tcgatggagt gaaagagcct gatgcactcc 11460

gcatacagct cgataatctt ttcagggctt tgttcatctt catactcttc cgagcaaagg 11520

acgccatcgg cctcactcat gagcagattg ctccagccat catgccgttc aaagtgcagg 11580

acctttggaa caggcagctt tccttccagc catagcatca tgtccttttc ccgttccaca 11640

tcataggtgg tccctttata ccggctgtcc gtcattttta aatataggtt ttcattttct 11700

cccaccagct tatatacctt agcaggagac attccttccg tatcttttac gcagcggtat 11760

ttttcgatca gttttttcaa ttccggtgat attctcattt tagccattta ttatttcctt 11820

cctcttttct acagtattta aagatacccc aagaagctaa ttataacaag acgaactcca 11880

attcactgtt ccttgcattc taaaacctta aataccagaa aacagctttt tcaaagttgt 11940

tttcaaagtt ggcgtataac atagtatcga cggagccgat tttgaaaccg cggtgatcac 12000

aggcagcaac gctctgtcat cgttacaatc aacatgctac cctccgcgag atcatccgtg 12060

tttcaaaccc ggcagcttag ttgccgttct tccgaatagc atcggtaaca tgagcaaagt 12120

ctgccgcctt acaacggctc tcccgctgac gccgtcccgg actgatgggc tgcctgtatc 12180

gagtggtgat tttgtgccga gctgccggtc ggggagctgt tggctggctg gtggcaggat 12240

atattgtggt gtaaacaaat tgacgcttag acaacttaat aacacattgc ggacgttttt 12300

aatgtactga attaacgccg aattaattcg ggggatctgg attttagtac tggattttgg 12360

ttttaggaat tagaaatttt attgatagaa gtattttaca aatacaaata catactaagg 12420

gtttcttata tgctcaacac atgagcgaaa ccctatagga accctaattc ccttatctgg 12480

gaactactca cacattatta tggagaaact cgagcttgtc gatcgacaga tccggtcggc 12540

atctactcta tttctttgcc ctcggacgag tgctggggcg tcggtttcca ctatcggcga 12600

gtacttctac acagccatcg gtccagacgg ccgcgcttct gcgggcgatt tgtgtacgcc 12660

cgacagtccc ggctccggat cggacgattg cgtcgcatcg accctgcgcc caagctgcat 12720

catcgaaatt gccgtcaacc aagctctgat agagttggtc aagaccaatg cggagcatat 12780

acgcccggag tcgtggcgat cctgcaagct ccggatgcct ccgctcgaag tagcgcgtct 12840

gctgctccat acaagccaac cacggcctcc agaagaagat gttggcgacc tcgtattggg 12900

aatccccgaa catcgcctcg ctccagtcaa tgaccgctgt tatgcggcca ttgtccgtca 12960

ggacattgtt ggagccgaaa tccgcgtgca cgaggtgccg gacttcgggg cagtcctcgg 13020

cccaaagcat cagctcatcg agagcctgcg cgacggacgc actgacggtg tcgtccatca 13080

cagtttgcca gtgatacaca tggggatcag caatcgcgca tatgaaatca cgccatgtag 13140

tgtattgacc gattccttgc ggtccgaatg ggccgaaccc gctcgtctgg ctaagatcgg 13200

ccgcagcgat cgcatccata gcctccgcga ccggttgtag aacagcgggc agttcggttt 13260

caggcaggtc ttgcaacgtg acaccctgtg cacggcggga gatgcaatag gtcaggctct 13320

cgctaaactc cccaatgtca agcacttccg gaatcgggag cgcggccgat gcaaagtgcc 13380

gataaacata acgatctttg tagaaaccat cggcgcagct atttacccgc aggacatatc 13440

cacgccctcc tacatcgaag ctgaaagcac gagattcttc gccctccgag agctgcatca 13500

ggtcggagac gctgtcgaac ttttcgatca gaaacttctc gacagacgtc gcggtgagtt 13560

caggcttttt catatctcat tgccccccgg gatctgcgaa agctcgagag agatagattt 13620

gtagagagag actggtgatt tcagcgtgtc ctctccaaat gaaatgaact tccttatata 13680

gaggaaggtc ttgcgaagga tagtgggatt gtgcgtcatc ccttacgtca gtggagatat 13740

cacatcaatc cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc 13800

tcgtgggtgg gggtccatct ttgggaccac tgtcggcaga ggcatcttga acgatagcct 13860

ttcctttatc gcaatgatgg catttgtagg tgccaccttc cttttctact gtccttttga 13920

tgaagtgaca gatagctggg caatggaatc cgaggaggtt tcccgatatt accctttgtt 13980

gaaaagtctc aatagccctt tggtcttctg agactgtatc tttgatattc ttggagtaga 14040

cgagagtgtc gtgctccacc atgttatcac atcaatccac ttgctttgaa gacgtggttg 14100

gaacgtcttc tttttccacg atgctcctcg tgggtggggg tccatctttg ggaccactgt 14160

cggcagaggc atcttgaacg atagcctttc ctttatcgca atgatggcat ttgtaggtgc 14220

caccttcctt ttctactgtc cttttgatga agtgacagat agctgggcaa tggaatccga 14280

ggaggtttcc cgatattacc ctttgttgaa aagtctcaat agccctttgg tcttctgaga 14340

ctgtatcttt gatattcttg gagtagacga gagtgtcgtg ctccaccatg ttggcaagct 14400

gctctagcca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg 14460

gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta 14520

gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg 14580

aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acg 14633

<210> 7

<223> ATG9-2-g + + primer

<400> 7

ggcagagtgg aagcgctcga gtc 23

<210> 8

<223> ATG9-2-g- -primer

<400> 8

aaacgactcg agcgcttcca ctc 23

<210> 9

<223> pC1300-F

<400> 9

acactttatg cttccggctc 20

<210> 10

<223> ATG9-F

<400> 10

ggctacttca tggagaagac tt 22

<210> 11

<223> ATG9-R

<400> 11

ttcgagatct gtaaccttga ga 22

<210> 12

<223> ATG9-cGFP-F

<400> 12

ggggtaccat gatgtccttc cttccgaag 29

<210> 13

<223> ATG9-cGFP-R

<400> 13

ttgctcacca tagcggccgc aactgggtca ctgcttctag 40

<210> 14

<223> HYG-F

<400> 14

aagatgttgg cgacctcgta ttgg 24

<210> 15

<223> HYG-R

<400> 15

gatcgttatg tttatcggca ctttgc 26

<210> 16

<223> T7

<400> 16

taatacgactcactatagg 19

<210> 17

<223> T3

<400> 17

attaaccctcactaaaggga 20

Claims (6)

1. Autophagy geneATG9The application in rice breeding regulation and control is characterized in that the autophagy geneATG9The amino acid sequence of the encoded protein is shown in SEQ ID NO. 2.

2. The use of claim 1, wherein said autophagy gene isATG9The nucleotide sequence of (A) is shown as SEQ ID NO. 1 in the sequence table.

3. The use of claim 1, wherein said modulation is positive modulation.

4. The use of claim 3, wherein the positive regulation is overexpression of autophagy genes in riceATG9Promoting the growth of riceATG9The over-expression plant has high plant height, high ear length, wide grain, long grain and increased grain weight.

5. The use of claim 4, wherein the rice is japonica rice.

6. The use according to claim 5, wherein the japonica rice is Zhonghua 11.

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