Application of rice variable shear variant NAL1-Del 2EX

Abstract

The invention provides application of a rice variable shear variant NAL1-Del 2EX in regulation and control of rice plant height, leaf width and photosynthesis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and relates to application of a rice variable splicing variant NAL1-Del2EX in regulation of rice plant height and leaf width. BACKGROUND

[0002] Rice is one of the most important crops in the world and is the main food crop for at least 65% of the population in China. With the reduction of arable land and the rapid growth of population, improving the yield of rice has important significance for solving the crisis of food security. Therefore, using modern biological techniques to cultivate crop varieties with higher yield is still the main goal of current breeding work.

[0003] Rice yield is a complex agronomic trait affected by multiple factors, and its constituent factors mainly include plant height, grain number per panicle, heading date, seed setting rate, tiller number and 1000-grain weight. The plant height of rice is an important plant type index affected by the longitudinal elongation of internodes, determines the lodging resistance and biological yield of rice, and acts on the grain yield of rice. The second green revolution is to achieve high yield by reducing the height of rice and improving the lodging resistance. The internode morphology of rice is the result of the joint action of the division of intercalary meristem cells and the longitudinal elongation of cells, and any factor affected may cause the occurrence of plant dwarfing phenotype. In addition, the leaf is the main part of photosynthesis of plants, so the morphology of the leaf, especially the area of the leaf, is an important factor determining the grain yield. Excellent leaf morphology can improve photosynthetic efficiency, increase plant biomass and improve grain yield. Therefore, leaf morphology has become an important target for ideal type breeding of "super rice". In rice, the uppermost three leaves, especially the flag leaf, provide most of the photosynthate accumulated in the grain. Therefore, it is of great significance to mine the genes related to the plant height and leaf type of rice and improve the traits of rice by using genetic engineering techniques to improve the yield and appearance quality of rice.

[0004] Early studies found that NAL1 is an important gene related to rice leaf type control, and its loss-of-function mutant shows reduced plant height, narrower leaves, and increased photosynthetic rate per unit leaf area (Wang et al. 2020). According to whether the 233rd amino acid is arginine or histidine, it is divided into indica NAL1 and japonica NAL1, the latter positively regulates the leaf type and plant type of rice, and is applied to the improvement of indica rice, which can improve rice yield and quality, so the gene has important application potential in rice high-yield breeding. NAL1 gene encodes a kind of trypsin-like serine / cysteine protease, which plays a key regulatory role in leaf size by affecting the polar transport of auxin and cell division (Qi et al., 2008; Cho et al., 2014). In addition, NAL1 also regulates the yield, photosynthesis, source-sink relationship and adventitious root formation of rice (Takai et al., 2013; Zhang et al., 2014; Wang et al., 2015).

[0005] Alternative Splicing (AS) is a molecular mechanism that generates multiple transcripts from a single gene. After it was first discovered in the plant kingdom (related to ribulose-bisphosphate carboxylase / oxygenase activase) (Werneke et al. 1989), it is now considered a widely used means of regulating gene expression in plants (Barbazuk et al. 2008; Reddy 2007). Expressed Sequence Tag (EST) sequencing shows that at least 20% of genes in Arabidopsis and rice are affected by AS (Buell et al. 2006). In rice, grain quality (Prathepha 2007), grain aroma (Chen et al. 2008; Niu et al. 2007) and photosynthetic rate (Isshiki et al. 2000; To et al. 1999) have been shown to be related to AS. Alternative splicing leads to polymorphism in transcript and protein structure and function, and is an important post-transcriptional regulation mechanism that plays a role in regulating gene expression and causing functional differentiation. Alternative splicing is not constant in different tissues or different stages of development, and specific splicing isoforms are produced in specific tissues or conditions, which have specific temporal and spatial effects, so that alternative splicing is associated with specific traits, and therefore research on alternative splicing in different varieties, different tissues and different developmental stages has application potential in breeding plants with specific traits. SUMMARY

[0006] An object of the present application is to provide a rice alternative splicing variant NAL1-Del 2EX.

[0007] According to the NAL1 natural variant derived from rice (Oryza sativa L.), the NAL1 transcript is sequenced and screened to determine the NAL1 second exon deletion transcript (NAL1-Del 2EX), the nucleotide sequence of which is shown as SEQ ID NO: 2, and the amino acid sequence of the encoded protein NAL1-Del 2EX is shown as SEQ ID NO: 4.

[0008] The application provides a rice variable splicing variant and its encoding gene in regulating the plant height, leaf type and / or photosynthesis of rice.

[0009] The application provides a method for regulating the plant height, leaf type and / or photosynthesis of rice, which comprises the step of deleting the second exon of NAL1 gene, the nucleotide sequence of which is shown as SEQ ID NO: 1, and the second exon sequence of which is shown as SEQ ID NO: 7.

[0010] In some embodiments, the method for regulating the plant height, leaf type and / or photosynthesis of rice, wherein the step of deleting the second exon of NAL1 gene is achieved by gene knockout.

[0011] In the above method, the NAL1 second exon is the 157-204 amino acid fragment.

[0012] In some embodiments, the method for regulating the plant height, leaf type and / or photosynthesis of rice, which comprises the step of overexpressing NAL1-Del 2EX gene, wherein the nucleotide sequence of NAL1-Del 2EX gene is shown as SEQ ID NO: 2.

[0013] In some embodiments, wherein the step of overexpressing NAL1-Del 2EX gene comprises transfecting NAL1-Del 2EX gene or a vector containing the NAL1-Del 2EX gene into rice cells or tissues, wherein the nucleotide sequence of NAL1-Del 2EX gene is shown as SEQ ID NO: 2.

[0014] In the above application and method, the regulation of the plant height of rice is to reduce the plant height of rice, the regulation of the leaf type is to narrow the leaf width, and the regulation of the photosynthesis is to improve the photosynthetic rate.

[0015] In order to facilitate the identification and screening of the transgenic plant cells or plants, the Flag tag is added to the C-terminal of the NAL1 amino acid sequence in the processing of the plant expression vector. If other uses are needed, the genes (GUS gene, luciferase gene, etc.) that can produce color change or luminescent compounds, the antibiotic markers (gentamicin marker, kanamycin marker, etc.) with resistance, or the chemical reagent resistant marker genes (such as herbicide resistant genes) that are expressed in plants can also be added.

[0016] The application provides application of the NAL1 alternative splicing variant in breeding of transgenic rice with dwarfed plant height and narrowed leaf width.

[0017] The application also provides application of the NAL1 alternative splicing variant in rice breeding.

[0018] In the method, the rice is preferably Zhonghua 11 (ZH11).

[0019] The application utilizes the mutant with dwarfed plant height and narrowed leaf width obtained by alternative splicing in the transcriptome of rice ZH11, and obtains the gene NAL1-Del 2EX for regulating the plant height and leaf type of rice. Further, by constructing the NAL1-Del 2EX transgenic line (ZH11 background), it is confirmed that NAL1-Del 2EX plays an important role in regulating the plant height, leaf type and photosynthesis of rice. The application not only provides a basis for further elucidating the molecular mechanism for regulating the plant height and leaf type of rice, but also provides a new gene resource for rice breeding. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1A Comparison of growth phenotypes of ZH11, ZH11-CR and ZH11-2EX plants screened for resistance (tillering stage); Figure 1B Comparison of leaf types of ZH11, ZH11-CR and ZH11-2EX plants screened for resistance, wherein ZH11 is Zhonghua 11, ZH11-CR is a mutant of NAL1 knocked out by CRISPR / Cas9, and ZH11-2EX is a NAL1-Del 2EX transgenic plant.

[0021] Figure 2 Comparison of plant heights of ZH11, ZH11-CR and ZH11-2EX plants screened for resistance (tillering stage), 20 plants of each rice.

[0022] Figure 3 Western-blot detection of the expression of the corresponding protein in the NAL1-Del 2EX transgenic material of rice.

[0023] Figure 4Comparison of field unit leaf area photosynthetic rate of ZH11, ZH11-CR and ZH11-2EX plants. DETAILED DESCRIPTION

[0024] The application will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the application are not limited thereto. The reagents, methods and equipment used in the application are conventional reagents, methods and equipment in the technical field unless otherwise specified. The reagents and raw materials used in the application are commercially available unless otherwise specified.

[0025] Example 1: Identification of natural variants of rice NAL1

[0026] Using Zhonghua 11 (ZH11) of japonica rice material cultured in a 25°C greenhouse for 20 days, RNA was extracted and reverse transcribed into cDNA using a TAKARA reverse transcription kit (RR047A), and the cDNA was used as a template for PCR reaction. The amplification primers used are as follows:

[0027] Upstream primer: ATGAAGCCTTCGGACGATAAG

[0028] Downstream primer: TCATTTCTCCAGGTCAAGGCT

[0029] The PCR product was sequenced, and after comparison with the NAL1 sequence (SEQ ID NO: 1), it was found that there was a 144 bp deletion, which encoded 48 amino acids, located at positions 157-204, and the deleted part was the complete NAL1 second exon region. This NAL1 alternative splicing type was named NAL1-Del 2EX (SEQ ID NO: 2).

[0030] Example 2: Obtaining and testing of NAL1-Del 2EX transgenic plant material

[0031] In the process of obtaining NAL1-Del 2EX transgenic plants, the vector used was Actin1 promoter-Flag-p2300, the sequence of which is shown in SEQ ID NO: 5. The above vector was linearized using XbaI restriction enzyme (NEB, Lot#10128088), and NAL1-Del 2EX gene (SEQ ID NO: 2) was added to the homologous arms at both ends of the above p2300 vector insertion site by PCR to obtain Actin1 promoter-NAL1 2EX -Flag-p2300, the amplification primers containing the homologous arms used are as follows:

[0032] Upstream primer: tgttgcgcctccgcggtggaATGAAGCCTTCGGACGATAAG

[0033] Downstream primer: cagccggatctaagcttgcgTCATTTCTCCAGGTCAAGGCT

[0034] The mature embryo callus of rice was transformed by Agrobacterium-mediated method, in which the Agrobacterium strain was EHA105-WM (Unichroma Biotech Co., Ltd.), and the Actinl promoter-NALl 2EX The Flag-p2300 was introduced into the mature embryo of rice, and the specific steps were as follows:

[0035] 1. Callus induction and subculture

[0036] The mature rice seeds (preferably the newly harvested seeds) were selected, the hulls were peeled off, and then were poured into 50 ml centrifuge tubes, 75% ethanol was added for sterilization for 1 min, the ethanol was poured out, and then the callus was washed with sterile water for one time, and then the water was poured out, and then 30% sodium hypochlorite was added for sterilization for 20 min, and then the sodium hypochlorite was poured out and washed with sterile water for 5-6 times. The residual water was removed by using a pipette gun (the filter paper can be used for drying), and then the seeds were transferred to the induction medium, 20-25 seeds per dish. The callus was induced, and then the protocorms were directly used for transformation, the small particles grown beside the protocorms were picked to the new induction medium for subculture, and then the protocorms were directly used for transformation when the size was appropriate.

[0037] 2. Agrobacterium culture

[0038] The Agrobacterium EHA105 containing the target gene vector was streaked on the plate containing the corresponding antibiotic, and then was cultured at 28°C in the dark for 2 days until single colonies appeared.

[0039] 3. Agrobacterium infection

[0040] The infection solution was prepared, the Agrobacterium on the plate was washed down by using a pipette to suck the infection solution, and then the Agrobacterium suspension for co-culture and transformation of rice was obtained. A sufficient amount of callus (the callus was in good condition, the color was bright yellow, the texture was round and hard, and the particle diameter was about 3 mm) was selected and was placed in a 100 ml sterile Erlenmeyer flask, and then an appropriate amount of Agrobacterium suspension was added (the bacteria solution was sufficient to contact with the material), and then the Erlenmeyer flask was placed at room temperature for 20 min, and was shaken from time to time. The bacteria solution was poured out, the callus was placed on the sterile filter paper to remove the residual bacteria solution, and then was immediately transferred to the solid co-culture medium covered with a layer of sterile filter paper, and then was cultured at 26°C in the dark for 3 days.

[0041] 4. Screening culture

[0042] After 3 days of co-cultivation, the callus is washed by pipetting 1 ml of co-cultivation medium onto a sterile flask, washing twice with sterile water, and washing a third time with sterile water containing 500 ul / L carbenicillin. The callus is then transferred to sterile filter paper and dried using a clean bench for about 30 minutes. The callus is then transferred to a selection medium and cultured for 3-4 weeks at 28-30°C in the dark.

[0043] 5. Differentiation and regeneration

[0044] After one month of selection, positive calli with bright yellow color are observed. The positive calli are then picked and placed on differentiation medium for differentiation and regeneration. Each differentiation dish contains 16 positive calli and is placed in a 28-30°C greenhouse under light. After about 10 days, green spots appear on the calli, and after another 10 days, seedlings are differentiated.

[0045] 6. Seedling rooting

[0046] When the differentiated seedlings grow to about 2-3 cm with obvious root systems, they are transferred to rooting medium for growth. The rooting medium is poured into a relatively high bottle or tube to provide sufficient space for the seedlings to grow. The rooting medium is cultured at 28-30°C under sterile light.

[0047] The leaves of the transgenic plants at the seedling stage are cut and subjected to Western blot analysis using Maximum Sensitivity Substrate (Thermo Fisher, Lot#34096) as the chemiluminescent substrate. Figure 3 To detect the expression of the target protein in NAL1 rice transgenic materials using Flag-HRP direct label antibody (Sigma, A2220), it is shown that NAL1-Del 2EX is expressed.

[0048] Example 3: Effect of NAL1-Del 2EX on rice phenotype

[0049] The C-terminal of NAL1-Del 2EX is fused with a Flag tag, and the growth phenotype of rice transgenic materials positive for resistance screening (neomycin, 0.5 mg / ml) is compared (rice tillering stage) by introducing the mature embryo callus of rice material Zhonghua 11 (ZH11) into the transgenic rice material.

[0050] The wild type ZH11 knockout NAL1 mutant (ZH11-CR) was obtained from wild type ZH11 by gene editing, using conventional CRISPR / Cas9 method to knockout NAL1 gene. Sequencing showed that in the genomic DNA of the mutant, a deletion of 4 bases occurred after the start codon ATG, and the deleted bases were AAGC, the sequence of which is shown in SEQ ID NO: 6. The NAL1-Del 2EX plant (ZH11-2EX) was obtained by the transgenic method in Example 2.

[0051] The results are shown in Figures 1 and 2. In Figure 1, the left wild type plant ZH11 has a higher plant type and wider leaf type; the middle is the wild type ZH11 CRISPR / Cas9 knockout NAL1 mutant. Field phenotype shows that after deletion of NAL1, the plant height becomes shorter and the leaf width becomes narrower; the right is the field phenotype of overexpression of NAL1-Del 2EX in the background of wild type ZH11. Compared with the wild type plant ZH11, NAL1-Del 2EX negatively regulates the plant height and leaf width of rice. From the above results, it can be seen that the NAL1-Del 2EX transgenic plants have a shorter plant height and narrower leaf width than the wild type ZH11. Figure 2 The plant height data graph shows that the plant height of ZH11-CR and ZH11-2EX is significantly lower than that of ZH11. According to the above results, it is concluded that deletion of the second exon of NAL1 (NAL1-Del 2EX) negatively regulates the plant height and leaf width of rice.

[0052] The LI-6800 photosynthetic fluorescence measurement instrument (LI-COR, USA) was used to measure the photosynthetic rate of the corresponding transgenic plant leaves, and the results are shown in Figure 3. Figure 4 As shown in Figure 3, the photosynthetic rate per unit leaf area of NAL1-Del 2EX transgenic plants is higher than that of wild type ZH11, thus indicating that deletion of the second exon of NAL1 can regulate the photosynthesis of rice leaves.

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[0072] SEQUENCE LISTING

[0073] SEQ ID NO: 1 Nucleotide sequence of NAL1 gene

[0074] ATGAAGCCTTCGGACGATAAGGCGCAGCTCTCCGGTTTGGCGCAATCAGAAGAATCGTCACTTGATGTGGATCACCAGTCATTTCCTT

[0075] GTTCTCCATCAATCCAACCGGTTGCTTCTGGGTGCACACACACAGAGAACAGCGCAGCATACTTCTTATGGCCGACATCCAACCTACA

[0076] GCATTGTGCAGCCGAGGGACGTGCAAACTACTTTGGAAACCTTCAGAAAGGATTGTTGCCAAGGCACCCTGGTCGGTTGCCCAAAGG

[0077] TCAGCAAGCAAATAGCTTGCTTGACTTGATGACTATAAGAGCTTTCCATAGCAAGATATTGCGGCGTTTTAGCCTCGGGACAGCAGTG

[0078] GGATTCCGCATCAGGAAAGGGGATCTAACAGATATCCCTGCAATCCTTGTCTTTGTTGCTCGCAAGGTTCATAAGAAGTGGCTTAATCC

[0079] AGCACAATGTCTTCCTGCTATTCTTGAGGGTCCAGGAGGTGTTTGGTGTGATGTTGATGTTGTTGAATTTTCGTACTACGGTGCA

[0080] CCGGCTCAAACACCTAAAGAGCAAATGTTCAGTGAGCTTGTTGATAAGTTATGTGGCAGTGACGAATGTATTGGTTCAGGCTC

[0081] TCAGGTTGCAAGCCATGAAACTTTTGGTACTTTGGGTGCAATTGTGAAACGGCGCACTGGCAACAAGCAGGTTGGTTTCCTCACTAA

[0082] CCATCATGTCGCGGTTGACTTGGACTACCCTAATCAGAAGATGTTTCATCCATTACCACCCAATCTTGGGCCTGGCGTTTATCTTGGAGC

[0083] TGTTGAAAGAGCAACTTCTTTCATCACAGATGACGTTTGGTATGGAATCTATGCTGGAACAAACCCAGAGACATTTGTACGAGCTGAC

[0084] GGTGCATTTATCCCATTTGCTGATGACTTTGACATTTCCACCGTCACGACTGTAGTTAGGGGAGTCGGTGACATTGGGGATGTCAAGGT

[0085] TATAGATCTGCAGTGTCCGCTCAATAGCCTCATAGGGAGGCAAGTATGCAAAGTTGGCAGAAGCTCTGGTCACACAACTGGGACTGTG

[0086] ATGGCCTATGCCCTTGAGTACAATGACGAGAAAGGAATATGCTTCTTCACAGACATCCTTGTTGTTGGTGAGAACCGCCAAACATTTGA

[0087] TTTGGAAGGTGATAGCGGAAGCCTTATTATCCTGACTAGCCAAGATGGTGAGAAGCCGCGTCCAATTGGAATTATATGGGGTGGCACA

[0088] GCAAATCGTGGGAGGTTGAAGCTTACAAGTGATCATGGCCCTGAAAACTGGACTAGTGGGGTTGATCTTGGCCGTCTACTCGACCGTC

[0089] TGGAACTTGATATTATCATTACCAATGAATCACTCCAAGATGCCGTGCAGCAGCAAAGATTTGCTTTGGTGGCCGCCGTTACCTCAGCT

[0090] GTTGGGGAGTCTTCCGGGGTGCCTGTCGCCATCCCGGAAGAGAAGATCGAAGAGATCTTCGAGCCATTGGGGATCCAAATCCAGCAA

[0091] CTGCCTCGCCATGACGTGGCGGCCTCTGGAACTGAAGGGGAGGAGGCATCCAACACGGTGGTCAATGTGGAAGAGCACCAGTTCATC

[0092] TCAAACTTCGTCGGTATGTCGCCCGTGCGCGACGACCAAGACGCTCCGAGGAGCATCACCAACCTGAACAACCCCTCCGAGGAAGA

[0093] ACTCGCCATGTCGCTCCATCTGGGTGACCGAGAGCCCAAGCGGCTCCGTTCGGACTCCGGATCAAGCCTTGACCTGGAGAAATGA SEQ ID NO: 2 Nucleotide sequence of NAL1-Del 2 EX gene

[0094] ATGAAGCCTTCGGACGATAAGGCGCAGCTCTCCGGTTTGGCGCAATCAGAAGAATCGTCACTTGATGTGGATCACCAGTCATTTCCTT

[0095] GTTCTCCATCAATCCAACCGGTTGCTTCTGGGTGCACACACACAGAGAACAGCGCAGCATACTTCTTATGGCCGACATCCAACCTACA

[0096] GCATTGTGCAGCCGAGGGACGTGCAAACTACTTTGGAAACCTTCAGAAAGGATTGTTGCCAAGGCACCCTGGTCGGTTGCCCAAAGG

[0097] TCAGCAAGCAAATAGCTTGCTTGACTTGATGACTATAAGAGCTTTCCATAGCAAGATATTGCGGCGTTTTAGCCTCGGGACAGCAGTG

[0098] GGATTCCGCATCAGGAAAGGGGATCTAACAGATATCCCTGCAATCCTTGTCTTTGTTGCTCGCAAGGTTCATAAGAAGTGGCTTAATCC

[0099] AGCACAATGTCTTCCTGCTATTCTTGAGGTTGCAAGCCATGAAACTTTTGGTACTTTGGGTGCAATTGTGAAACGGCGCACTGGCAAC

[0100] AAGCAGGTTGGTTTCCTCACTAACCATCATGTCGCGGTTGACTTGGACTACCCTAATCAGAAGATGTTTCATCCATTACCACCCAATCT

[0101] TGGGCCTGGCGTTTATCTTGGAGCTGTTGAAAGAGCAACTTCTTTCATCACAGATGACGTTTGGTATGGAATCTATGCTGGAACAAACC

[0102] CAGAGACATTTGTACGAGCTGACGGTGCATTTATCCCATTTGCTGATGACTTTGACATTTCCACCGTCACGACTGTAGTTAGGGGAGTC

[0103] GGTGACATTGGGGATGTCAAGGTTATAGATCTGCAGTGTCCGCTCAATAGCCTCATAGGGAGGCAAGTATGCAAAGTTGGCAGAAGCT

[0104] CTGGTCACACAACTGGGACTGTGATGGCCTATGCCCTTGAGTACAATGACGAGAAAGGAATATGCTTCTTCACAGACATCCTTGTTGT

[0105] TGGTGAGAACCGCCAAACATTTGATTTGGAAGGTGATAGCGGAAGCCTTATTATCCTGACTAGCCAAGATGGTGAGAAGCCGCGTCCA

[0106] ATTGGAATTATATGGGGTGGCACAGCAAATCGTGGGAGGTTGAAGCTTACAAGTGATCATGGCCCTGAAAACTGGACTAGTGGGGTTG

[0107] ATCTTGGCCGTCTACTCGACCGTCTGGAACTTGATATTATCATTACCAATGAATCACTCCAAGATGCCGTGCAGCAGCAAAGATTTGCT

[0108] TTGGTGGCCGCCGTTACCTCAGCTGTTGGGGAGTCTTCCGGGGTGCCTGTCGCCATCCCGGAAGAGAAGATCGAAGAGATCTTCGAG

[0109] CCATTGGGGATCCAAATCCAGCAACTGCCTCGCCATGACGTGGCGGCCTCTGGAACTGAAGGGGAGGAGGCATCCAACACGGTGGTC

[0110] AATGTGGAAGAGCACCAGTTCATCTCAAACTTCGTCGGTATGTCGCCCGTGCGCGACGACCAAGACGCTCCGAGGAGCATCACCAAC

[0111] CTGAACAACCCCTCCGAGGAAGAACTCGCCATGTCGCTCCATCTGGGTGACCGAGAGCCCAAGCGGCTCCGTTCGGACTCCGGATCA

[0112] AGCCTTGACCTGGAGAAATGA

[0113] SEQ ID NO:3 Amino acid sequence of NAL1 protein

[0114] MKPSDDKAQLSGLAQSEESSLDVDHQSFPCSPSIQPVASGCTHTENSAAYFLWPTSNLQHCAAEGRANYFGNLQKGLLPRHPGRLPKGQQ

[0115] ANSLLDLMTIRAFHSKILRRFSLGTAVGFRIRKGDLTDIPAILVFVARKVHKKWLNPAQCLPAILEGPGGVWCDVDVVEFSYYGAPAQTPK

[0116] EQMFSELVDKLCGSDECIGSGSQVASHETFGTLGAIVKRRTGNKQVGFLTNHHVAVDLDYPNQKMFHPLPPNLGPGVYLGAVERATSFIT

[0117] DDVWYGIYAGTNPETFVRADGAFIPFADDFDISTVTTVVRGVGDIGDVKVIDLQCPLNSLIGRQVCKVGRSSGHTTGTVMAYALEYNDEK

[0118] GICFFTDILVVGENRQTFDLEGDSGSLIILTSQDGEKPRPIGIIWGGTANRGRLKLTSDHGPENWTSGVDLGRLLDRLELDIIITNESLQDAVQ

[0119] QQRFALVAAVTSAVGESSGVPVAIPEEKIEEIFEPLGIQIQQLPRHDVAASGTEGEEASNTVVNVEEHQFISNFVGMSPVRDDQDAPRSITNLNNPSEEELAMSLHLGDREPKRLRSDSGSSLDLEK*

[0120] SEQ ID NO:4 Amino acid sequence of NAL1-Del 2 EX protein

[0121] MKPSDDKAQLSGLAQSEESSLDVDHQSFPCSPSIQPVASGCTHTENSAAYFLWPTSNLQHCAAEGRANYFGNLQKGLLPRHPGRLPKGQQ

[0122] ANSLLDLMTIRAFHSKILRRFSLGTAVGFRIRKGDLTDIPAILVFVARKVHKKWLNPAQCLPAILEVASHETFGTLGAIVKRRTGNKQVGFLT

[0123] NHHVAVDLDYPNQKMFHPLPPNLGPGVYLGAVERATSFITDDVWYGIYAGTNPETFVRADGAFIPFADDFDISTVTTVVRGVGDIGDVKVI

[0124] DLQCPLNSLIGRQVCKVGRSSGHTTGTVMAYALEYNDEKGICFFTDILVVGENRQTFDLEGDSGSLIILTSQDGEKPRPIGIIWGGTANRGRL

[0125] KLTSDHGPENWTSGVDLGRLLDRLELDIIITNESLQDAVQQQRFALVAAVTSAVGESSGVPVAIPEEKIEEIFEPLGIQIQQLPRHDVAASGTEGEEASNTVVNVEEHQFISNFVGMSPVRDDQDAPRSITNLNNPSEEELAMSLHLGDREPKRLRSDSGSSLDLEK*

[0126] SEQ ID NO: 5 Actinl promoter-Flag-p2300 vector

[0127] catgccaaccacagggttcccctcgggatcaaagtactttgatccaacccctccgctgctatagtgcagtcggcttctgacgttcagtgcagccgtcttctgaaaacgacatgtcgcacaagtcctaagttacgcgacagg

[0128] ctgccgccctgcccttttcctggcgttttcttgtcgcgtgttttagtcgcataaagtagaatacttgcgactagaaccggagacattacgccatgaacaagagcgccgccgctggcctgctgggctatgcccgcgtcagcac

[0129] cgacgaccaggacttgaccaaccaacgggccgaactgcacgcggccggctgcaccaagctgttttccgagaagatcaccggcaccaggcgcgaccgcccggagctggccaggatgcttgaccacctacgccctg

[0130] gcgacgttgtgacagtgaccaggctagaccgcctggcccgcagcacccgcgacctactggacattgccgagcgcatccaggaggccggcgcgggcctgcgtagcctggcagagccgtgggccgacaccaccac

[0131] gccggccggccgcatggtgttgaccgtgttcgccggcattgccgagttcgagcgttccctaatcatcgaccgcacccggagcgggcgcgaggccgccaaggcccgaggcgtgaagtttggcccccgccctaccctc

[0132] accccggcacagatcgcgcacgcccgcgagctgatcgaccaggaaggccgcaccgtgaaagaggcggctgcactgcttggcgtgcatcgctcgaccctgtaccgcgcacttgagcgcagcgaggaagtgacgcc

[0133] caccgaggccaggcggcgcggtgccttccgtgaggacgcattgaccgaggccgacgccctggcggccgccgagaatgaacgccaagaggaacaagcatgaaaccgcaccaggacggccaggacgaaccgtttt

[0134] tcattaccgaagagatcgaggcggagatgatcgcggccgggtacgtgttcgagccgcccgcgcacgtctcaaccgtgcggctgcatgaaatcctggccggtttgtctgatgccaagctggcggcctggccggccagc

[0135] ttggccgctgaagaaaccgagcgccgccgtctaaaaaggtgatgtgtatttgagtaaaacagcttgcgtcatgcggtcgctgcgtatatgatgcgatgagtaaataaacaaatacgcaaggggaacgcatgaaggttatc

[0136] gctgtacttaaccagaaaggcgggtcaggcaagacgaccatcgcaacccatctagcccgcgccctgcaactcgccggggccgatgttctgttagtcgattccgatccccagggcagtgcccgcgattgggcggccgt

[0137] gcgggaagatcaaccgctaaccgttgtcggcatcgaccgcccgacgattgaccgcgacgtgaaggccatcggccggcgcgacttcgtagtgatcgacggagcgccccaggcggcggacttggctgtgtccgcgat

[0138] caaggcagccgacttcgtgctgattccggtgcagccaagcccttacgacatatgggccaccgccgacctggtggagctggttaagcagcgcattgaggtcacggatggaaggctacaagcggcctttgtcgtgtcgcg

[0139] ggcgatcaaaggcacgcgcatcggcggtgaggttgccgaggcgctggccgggtacgagctgcccattcttgagtcccgtatcacgcagcgcgtgagctacccaggcactgccgccgccggcacaaccgttcttgaa

[0140] tcagaacccgagggcgacgctgcccgcgaggtccaggcgctggccgctgaaattaaatcaaaactcatttgagttaatgaggtaaagagaaaatgagcaaaagcacaaacacgctaagtgccggccgtccgagcgc

[0141] acgcagcagcaaggctgcaacgttggccagcctggcagacacgccagccatgaagcgggtcaactttcagttgccggcggaggatcacaccaagctgaagatgtacgcggtacgccaaggcaagaccattaccga

[0142] gctgctatctgaatacatcgcgcagctaccagagtaaatgagcaaatgaataaatgagtagatgaattttagcggctaaaggaggcggcatggaaaatcaagaacaaccaggcaccgacgccgtggaatgccccatgt

[0143] gtggaggaacgggcggttggccaggcgtaagcggctgggttgtctgccggccctgcaatggcactggaacccccaagcccgaggaatcggcgtgacggtcgcaaaccatccggcccggtacaaatcggcgcggc

[0144] GCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCAGGCCGCC CAGCGGCAACGCATCGAGGCGAAGCACGCCCGGTGAACTGTGGC AAGCGGCCGCTGATCGAATCCGCAAAGAATCCCAGCAACC

[0145] CCGGCAGCCGGTGCGCCGTCGAT TAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTCTTTCCGATGCTCTATGACGTGGGCACCCGC GATAGTCGCAGCATCATGGACGTGGCCGTTTTCCGTCTGTCGAA GC

[0146] GTGACC GACGAGCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTA GAGGTTTCCGCAGGGCCGGCCGGCATGGCCAGTGTGTGGGATTACGACCTG GTACTGATGGCGGT TTCCCATCTAACCGAATCCA

[0147] TGAACCGACACC GGG AAGGGAAGGGAGACAAGCCC GGC GCGTGTTC GTC CACACGTTGC GGACGTACTCAAGTTCTGCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGCATTCGGTT

[0148] AAACACCACGCACGTTGCCATGCAGC GTACGAAGAAGGCCAAGAACGGCCGCCTG GTGACGGTATCCGAGGGTGAAGCCTTGAT TAGCCGCTACAAGATCGTAAAGA GC GAAACC GGGCGGCCGGAGTACATCGA

[0149] GATCGAGCTAGCTGATTGGATGTACC GCGAGATCACAGAAGGCAAGACC C GGC GTCGCTGACGGTTCACCCCGATTACTTTTTGATCGATCCC GGC ATCGGCCGTTTCTCTACC GCCTGGCACGCCGC GCCGCAGGC

[0150] aaggcagaagccagatggttgttcaagacgatctacgaacgcagtggcagcgccggagagttcaagaagttctgtttcaccgtgcgcaagctgatcgggtcaaatgacctgccggagtacgatttgaaggaggaggc

[0151] ggggcaggctggcccgatcctagtcatgcgctaccgcaacctgatcgagggcgaagcatccgccggttcctaatgtacggagcagatgctagggcaaattgccctagcaggggaaaaaggtcgaaaaggtctctttc

[0152] ctgtggatagcacgtacattgggaacccaaagccgtacattgggaaccggaacccgtacattgggaacccaaagccgtacattgggaaccggtcacacatgtaagtgactgatataaaagagaaaaaaggcgatttttc

[0153] cgcctaaaactctttaaaacttattaaaactcttaaaacccgcctggcctgtgcataactgtctggccagcgcacagccgaagagctgcaaaaagcgcctacccttcggtcgctgcgctccctacgccccgccgcttcgcg

[0154] tcggcctatcgcggccgctggccgctcaaaaatggctggcctacggccaggcaatctaccagggcgcggacaagccgcgccgtcgccactcgaccgccggcgcccacatcaaggcaccctgcctcgcgcgtttcg

[0155] gtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatga

[0156] GCTTCCTCGC TCACTGACTC GCTGC GCTCGGTCG TTCGGCTGC GCGAGCGGT ATCAGCTCAC TCAAAGGC GGT AATACGGTT ATCCACAGAA TCAGGGGATAACGCAGGA AAGAACATGTGAGCAA AAGGCCAGCAA

[0157] GCTTCCTCGC TCACTGACTC GCTGC GCTCGGTCG TTCGGCTGC GCGAGCGGT ATCAGCTCAC TCAAAGGC GGT AATACGGTT ATCCACAGAA TCAGGGGATAACGCAGGA AAGAACATGTGAGCAA AAGGCCAGCAA

[0158] GCTTCCTCGC TCACTGACTC GCTGC GCTCGGTCG TTCGGCTGC GCGAGCGGT ATCAGCTCAC TCAAAGGC GGT AATACGGTT ATCCACAGAA TCAGGGGATAACGCAGGA AAGAACATGTGAGCAA AAGGCCAGCAA

[0159] GCTTCCTCGC TCACTGACTC GCTGC GCTCGGTCG TTCGGCTGC GCGAGCGGT ATCAGCTCAC TCAAAGGC GGT AATACGGTT ATCCACAGAA TCAGGGGATAACGCAGGA AAGAACATGTGAGCAA AAGGCCAGCAA

[0160] GCTTCCTCGC TCACTGACTC GCTGC GCTCGGTCG TTCGGCTGC GCGAGCGGT ATCAGCTCAC TCAAAGGC GGT AATACGGTT ATCCACAGAA TCAGGGGATAACGCAGGA AAGAACATGTGAGCAA AAGGCCAGCAA

[0161] GCTTCCTCGC TCACTGACTC GCTGC GCTCGGTCG TTCGGCTGC GCGAGCGGT ATCAGCTCAC TCAAAGGC GGT AATACGGTT ATCCACAGAA TCAGGGGATAACGCAGGA AAGAACATGTGAGCAA AAGGCCAGCAA

[0162] CGCTGGTAGCGGTG G TTTTTTTGTTTGCAAGCAGCAGATTACGC GCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGG GTCTGACGCTCAGTGGAACGAAAAC TCACGT TAAGGGATTTTG GT CATGCATTCT

[0163] AGGTACTAAAACAATTCATCCAGTAAAATATAATATTTATTTTCTCCCAATCAGGCTTGATCCCCAGTAAGTCAAAAAATAGCTCGACAT ACTGTTCTTCCCCGATATCCTCCCTGATCGACCGGACGCAGAAGGCAATGTCATA

[0164] CCACTTGTC CGCCCTGCCGCTTCTCCCAAGATCAATAAAGCCACTTACTTTGCCATCTTTCACAAAGATGTTGCTGTCTCCCAGGTCGCCGTGGGAAAAGACAAGTTCCTCTTCGGGCTTTTCCGTCTTTAAAAAATCATA CAGCT

[0165] C GC GC GG ATCTTTAAATGGAGTGTCTTCTTCCCAGTTTTCGCAATCCACATCGGCCAGATCGTTATT CAGTAAGTAATCCAATT CGGCTAAGCGGCTGTCTAAGCTATT CGTATAGGGACAATCCGATATGTCGATGGAGTGAAA

[0166] GAGCCTGATGC ACTCCGCATACAGCTCGATAATCTTTTCAGGGCTTTGTTCATCTTCATACTCTTCCGAGCAAAGGACGCCATCGGCCTC ACTCATGAGCAGATTGCTCCAGCCATCATGCCGTTCAAAGTGCAGGACCTTTGG

[0167] aacaggcagctttccttccagccatagcatcatgtccttttcccgttccacatcataggtggtccctttataccggctgtccgtcatttttaaatataggttttcattttctcccaccagcttatataccttagcaggagacattccttc

[0168] cgtatcttttacgcagcggtatttttcgatcagttttttcaattccggtgatattctcattttagccatttattatttccttcctcttttctacagtatttaaagataccccaagaagctaattataacaagacgaactccaattcactgttcc

[0169] ttgcattctaaaaccttaaataccagaaaacagctttttcaaagttgttttcaaagttggcgtataacatagtatcgacggagccgattttgaaaccgcggtgatcacaggcagcaacgctctgtcatcgttacaatcaacatgc

[0170] taccctccgcgagatcatccgtgtttcaaacccggcagcttagttgccgttcttccgaatagcatcggtaacatgagcaaagtctgccgccttacaacggctctcccgctgacgccgtcccggactgatgggctgcctgtat

[0171] cgagtggtgattttgtgccgagctgccggtcggggagctgttggctggctggtggcaggatatattgtggtgtaaacaaattgacgcttagacaacttaataacacattgcggacgtttttaatgtactgaattaacgccgaat

[0172] TATCTGGGAAC TACTCACACA TTATTATGGA GAAACTCGAG CTTGTGATCG ACTCTAGCTA GAGGATCGAT CC GAACCCCAG AGTCCCCTCA GAAGAAGTCG TCAAGAAGGC GATAGAAGGC GATGC GC TGC GAA TC

[0173] TTATCTGGGAAC TACTCACACA TTATTATGGA GAAACTCGAG CTTGTGATCG ACTCTAGCTA GAGGATCGAT CC GAACCCCAG AGTCCCCTCA GAAGAAGTCG TCAAGAAGGC GATAGAAGGC GATGC GC TGC GAA TC

[0174] GGGAGCGGCG ATACC GTA AAGCACGAGGAAGCGGTCAGCCC ATTCGCCGCC AAGCTCTTCA GCAATATCACGGGTAGCC AACGCTATG TCCTGATAGC GGTCCGCCAC ACCCAGCCGG CCACAGTCGA TGAATCCA

[0175] GAAAAGCGGC C ATTTCCACCA TGATATTCGG CAAGCAGGC ATCGCCATGT GTCACGACGA GATCCTCGCC GTCGGGCATGC GC GCCTTGA GCCTGGCGAAC AGTTCGGCTG GC GCAGCCCCT GA TGC TCTTCGTCC A

[0176] GATCATCCTG ATCGACAAGA CCGGCTTCCA TCCGAGTACG TGCTCGCTCG ATGC GATGTTTC GCTTGGTGGT C GAATGGGCAG GTAGCCGGAT CAAGCGTATG CAGCCGC CGC ATTC ATCAGCC ATGATGGAT ACTTTCT

[0177] CGGCAGGAGC AAGGTGAGAT GACAGGAGAT CCTGCCCGGC ACTTCGCCCA ATAGCAGCCAG TCCCTTCCC GCTTCAGTGA CAACGTCGAG CACAGCTGCG AAGGAACGCC CGTCGTGGCC AGCCACGATAG CC

[0178] GCGCTGCCTCGTCCTGGAGTTCATTCAGGGCACCGGACAGGTCG GTCTTGA CAAAAAGAACC GGGCGCCCCTGC GCTGACAGCCGGAACACGGCGGC ATCAGAGCAGCCGATTGTCTGTTGTGCCCAGT CATAGCCG

[0179] AATAGCCTCTCCACCCAAGCGGCCGGAGAACC TGC GTGCAATCC ATCTTGTTC AATCCCCATGGTCGATCGACAGATCTGCGAAAGCTCGAGAGAGATAGATTTTGTA GAGAGAGACTG GTGATTT C AGCGTGTCCTCTCCA

[0180] AATGAAATGAAC TTCCTTATATAGAGGAAGGTCTTGC GAAGGATAGTGGGATTGTGC GTCATCCCTTACGTCAGTGGAGATATCACATCAATCCACTTGCTTTGAAGACGTGGTT GGAACGTCTTCTTTTTCCACGATGCTCCTCG

[0181] TGGGTGGGGGTCCATCTTTGGGACCAC TGT CGGCAGAGGC ATCTTGAACGATAGCCTTTCCTTTATCGCAATGATGGC ATTTGTA GGTGCCACCTTCCTTTTCTACTGTCCTTTTGATGAAGTGACAGATAGCTGGGCAATGGAAT

[0182] CCGAGGAGGTTTCCC GATATTA CCCTTTGT TGAAGAGTCTCAATAGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTC TGGAGTAGACGAGAGTGT CGTGCTCCACCATGTTATCACATCAATCCACTTGCTTTGAA GACGTGG

[0183] TTGGAACGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCAC TGT CGGCAGAGGC ATCTTGAACGATAGCCTTTCCTTTATCGCAATGATGGC ATTTGTA GGTGCCACCTTCCTTTTCTACTGTCCTTTTGATGAAGTGACAGATAGCTGGGCAATGGAAT

[0184] atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat taccccttgt gttgaaaagt ctcaatagcc ctttggtctt ctgagactgt atctttgata ttcttggagt agacgagagt gtcgtgctcc accatgtt

[0185] ggcaagctgc tctagccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcatta

[0186] ggcaccccag gctttacac tttatgcttc cgctcgtatg ttgtgtggaa ttgtgagcgg at aatttcac acaggaaaca gctatgacc atgattacga ttcgagctcg gtacctcgag gtcattcat atgcttgaga ag

[0187] agagtcggga tagtccaaaa taaacaaagg taagattac ctggtcaaaa gtgaaaacat cagttaaaag gtggtataag taaatatcgg taataaaag gtggcccaaa gtgaaattac tcttttctac tattataaa att

[0188] gaggatgttt tgtcggtact ttgatacgtc atttttgtat gaattggttt ttaagttta ttcgcgattt ggaaatgcat atctgtattt gagtcggttt ttaagttcgt tgcttttgta aatacagagg gatttgtata agaaatatct ttaaaa

[0189] aacccatatgctaatttgacataatttttgagaaaaatatatattcaggcgaattccacaatgaacaataataagattaaaatagcttgcccccgttgcagcgatgggtattttttctagtaaaataaaagataaacttagactcaa

[0190] aacatttacaaaaacaacccctaaagtcctaaagcccaaagtgctatgcacgatccatagcaagcccagcccaacccaacccaacccaacccaccccagtgcagccaactggcaaatagtctccacccccggcactat

[0191] caccgtgagttgtccgcaccaccgcacgtctcgcagccaaaaaaaaaaaaagaaagaaaaaaaagaaaaagaaaaacagcaggtgggtccgggtcgtgggggccggaaaagcgaggaggatcgcgagcagcg

[0192] acgaggcccggccctccctccgcttccaaagaaacgccccccatcgccactatatacatacccccccctctcctcccatccccccaaccctaccaccaccaccaccaccacctcctcccccctcgctgccggacgacg

[0193] agctcctcccccctccccctccgccgccgccggtaaccaccccgcccctctcctctttctttctccgttttttttttcgtctcggtctcgatctttggccttggtagtttgggtgggcgagagcggcttcgtcgcccagatcggtg

[0194] cgcgggaggggcgggatctcgcggctggcgtctccgggcgtgagtcggcccggatcctcgcggggaatggggctctcggatgtagatcttctttctttcttctttttgtggtagaatttgaatccctcagcattgttcatcgg

[0195] tagtttttcttttcatgatttgtgacaaatgcagcctcgtgcggagcttttttgtaggtagaagaggtacccggggatcctctaga ATGGGCGGGGACTACTACCACTCGTGCTGCGGCGAC

[0196] CCCGACCCCGACCTCCGCGCGCCCGAGGGGCCCAAGCTGCCGTACGTCGGGGACAAGGAACCTCTCTCCACTTTAGCCGCCGAGTTT

[0197] CAGTCTGGCAGCCCCATTTTACAGGAGAAAATAAAGTTGCTTGGTGAACAGTATGATGCTTTAAGAAGGACACGAGGAGATGGAAAC

[0198] TGCTTTTATCGAAGCTTTATGTTTTCCTACTTGGAACATATCCTAGAGACACAAGACAAAGCTGAGGTTGAGCGCATTCTAAAAAAAAT

[0199] TGAGCAGTGCAAGAAGACTCTTGCAGATCTTGGATACATTGAGTTCACCTTTGAAGATTTCTTCTCTATATTCATTGATCAGCTGGAAA

[0200] GTGTTCTGCAGGGACATGAATCCTCCATAGGGGCCGAAGAGCTTCTAGAAAGAACCAGGGATCAGATGGTTTCTGATTATGTTGTCAT

[0201] GTTCTTTAGGTTTGTCACCTCTGGTGAAATCCAAAGGAGGGCCGAGTTCTTCGAACCATTCATCTCTGGCTTGACAAATTCGACTGTG

[0202] GTTCAGTTCTGCAAGGCTTCCGTGGAGCCGATGGGCGAGGAAAGTGACCATGTCCACATAATTGCCCTATCAGATGCGTTGGGTGTGC

[0203] CAATCCGTGT GATGTACCTA GACAGAAGCT CATGTGATGC TGGAAATATA AGTGTGAACC ACCCATGATT TCAGCCCTGA GGCCAATTCA

[0204] TCGGACGGTG CTGCTGCTGC TGAGAAACCT TACATTACTT TGCTCTACCG TCCTGGTCACT ACGACATTCT CTACCCGAAG TCTAGAGACT

[0205] ACAAAGACCA TGACGGTGAT TATAAAGATC ATGACATCGA CTACAAGGAT GACGATGACA AGTAGGTCGC CTGCAGGCTG CCAGGGCTCT CAATGGAGT

[0206] ttgaatcaaa tcttccagct gctttaatga gatatgcgag acgcctatga tcgcatgata tttgctttca attctgttgt gcacgttgta aaaaacctga gcatgtgtag ctcagatcct taccgccgtt tcggttcatt ctaatga

[0207] atatatcacc cgttactatc gtatttttat gaataatatt ctccgttca atttactga ttgaagcttg gcactggccg tcgttttaca acgtcgtgac tgggaaaac cctggcgtta cccaacttaa tcgccttgcag cacatccccc

[0208] tttcgccagc tggcgtaata gcgaagaggg ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgct agagcagctt gagcttggat cagattgtcg tttcccgcct tcagtttaaa ctatcagtg tttg

[0209] acaggatat attggcgggt aaacctaaga gaaaagagcg tttagaataa cggatattta aaagggcgtg aaaaggtttat ccgttcgtcc atttgtatgt g

[0210] SEQ ID NO: 6 NAL1 gene nucleotide sequence after gene editing

[0211] ATGCTTCGGACGATAAGGCGCAGCTCTCCGGTTTGGCGCAATCAGAAGAATCGTCACTTGATGTGGATCACCAGTCATTTCCTTGTTCT

[0212] CCATCAATCCAACCGGTTGCTTCTGGGTGCACACACACAGAGAACAGCGCAGCATACTTCTTATGGCCGACATCCAACCTACAGCATT

[0213] GTGCAGCCGAGGGACGTGCAAACTACTTTGGAAACCTTCAGAAAGGATTGTTGCCAAGGCACCCTGGTCGGTTGCCCAAAGGTCAG

[0214] CAAGCAAATAGCTTGCTTGACTTGATGACTATAAGAGCTTTCCATAGCAAGATATTGCGGCGTTTTAGCCTCGGGACAGCAGTGGGATT

[0215] CCGCATCAGGAAAGGGGATCTAACAGATATCCCTGCAATCCTTGTCTTTGTTGCTCGCAAGGTTCATAAGAAGTGGCTTAATCCAGCAC

[0216] AATGTCTTCCTGCTATTCTTGAGGGTCCAGGAGGTGTTTGGTGTGATGTTGATGTTGTTGAATTTTCGTACTACGGTGCACCGGC

[0217] TCAAACACCTAAAGAGCAAATGTTCAGTGAGCTTGTTGATAAGTTATGTGGCAGTGACGAATGTATTGGTTCAGGCTCTCAGG

[0218] TTGCAAGCCATGAAACTTTTGGTACTTTGGGTGCAATTGTGAAACGGCGCACTGGCAACAAGCAGGTTGGTTTCCTCACTAACCATCA

[0219] TGTCGCGGTTGACTTGGACTACCCTAATCAGAAGATGTTTCATCCATTACCACCCAATCTTGGGCCTGGCGTTTATCTTGGAGCTGTTG

[0220] AAAGAGCAACTTCTTTCATCACAGATGACGTTTGGTATGGAATCTATGCTGGAACAAACCCAGAGACATTTGTACGAGCTGACGGTGC

[0221] ATTTATCCCATTTGCTGATGACTTTGACATTTCCACCGTCACGACTGTAGTTAGGGGAGTCGGTGACATTGGGGATGTCAAGGTTATAG

[0222] ATCTGCAGTGTCCGCTCAATAGCCTCATAGGGAGGCAAGTATGCAAAGTTGGCAGAAGCTCTGGTCACACAACTGGGACTGTGATGG

[0223] CCTATGCCCTTGAGTACAATGACGAGAAAGGAATATGCTTCTTCACAGACATCCTTGTTGTTGGTGAGAACCGCCAAACATTTGATTTG

[0224] GAAGGTGATAGCGGAAGCCTTATTATCCTGACTAGCCAAGATGGTGAGAAGCCGCGTCCAATTGGAATTATATGGGGTGGCACAGCAA

[0225] ATCGTGGGAGGTTGAAGCTTACAAGTGATCATGGCCCTGAAAACTGGACTAGTGGGGTTGATCTTGGCCGTCTACTCGACCGTCTGGA

[0226] ACTTGATATTATCATTACCAATGAATCACTCCAAGATGCCGTGCAGCAGCAAAGATTTGCTTTGGTGGCCGCCGTTACCTCAGCTGTTG

[0227] GGGAGTCTTCCGGGGTGCCTGTCGCCATCCCGGAAGAGAAGATCGAAGAGATCTTCGAGCCATTGGGGATCCAAATCCAGCAACTGC

[0228] CTCGCCATGACGTGGCGGCCTCTGGAACTGAAGGGGAGGAGGCATCCAACACGGTGGTCAATGTGGAAGAGCACCAGTTCATCTCA

[0229] AACTTCGTCGGTATGTCGCCCGTGCGCGACGACCAAGACGCTCCGAGGAGCATCACCAACCTGAACAACCCCTCCGAGGAAGAACT

[0230] CGCCATGTCGCTCCATCTGGGTGACCGAGAGCCCAAGCGGCTCCGTTCGGACTCCGGATCAAGCCTTGACCTGGAGAAATGA

[0231] SEQ ID NO: 7 Amino acid sequence of the second exon of the NAL1 gene

[0232] GPGGVWCDVDVVEFSYYGAPAQTPKEQMFSELVDKLCGSDECIGSGSQ

[0233] SEQ ID NO: 8 Nucleotide sequence of the second exon of the NAL1 gene

[0234] GGTCCAGGAGGTGTTTGGTGTGATGTTGATGTTGTTGAATTTTCGTACTACGGTGCACCGGCTCAAACACCTAAAGAGCAAAT

[0235] GTTCAGTGAGCTTGTTGATAAGTTATGTGGCAGTGACGAATGTATTGGTTCAGGCTCTCAG

Claims

1. A variable splicing variant of NAL1, the amino acid sequence of which is shown as SEQ ID NO:

4.

2. The variable splicing variant of NAL1 of claim 1, the nucleotide sequence of the gene encoding which is shown as SEQ ID NO:

2.

3. Use of the variable splicing variant of NAL1 of claim 1 in regulating plant height, leaf type and / or photosynthesis of rice.

4. Use of the gene NAL1-Del2EX encoding the variable splicing variant of NAL1 of claim 1 in regulating plant height, leaf type and / or photosynthesis of rice.

5. Use according to claim 4, characterized in that, wherein the nucleotide sequence of the gene NAL1-Del 2EX is shown as SEQ ID NO:

2.

6. The use of claim 3 or 4, wherein the regulating plant height of rice is to reduce the plant height of rice, the regulating leaf type is to narrow the leaf width, and the regulating photosynthesis is to increase the photosynthetic rate.

7. A method of regulating plant height, leaf type and / or photosynthesis of rice, comprising the step of deleting the second exon of NAL1 gene, the nucleotide sequence of which is shown as SEQ ID NO: 1, and the nucleotide sequence of the second exon of which is shown as SEQ ID NO:

8.

8. The method of claim 7, wherein the step of deleting the second exon of NAL1 gene is achieved by a method of gene knockout.

9. A method of regulating plant height, leaf type and / or photosynthesis of rice, comprising the step of overexpressing NAL1-Del2EX gene, the nucleotide sequence of which is shown as SEQ ID NO:

2.

10. The method of claim 9, wherein the step of overexpressing NAL1-Del 2EX gene comprises transfecting NAL1-Del 2EX gene or a vector containing the NAL1-Del 2EX gene into rice cells or tissues, the nucleotide sequence of which is shown as SEQ ID NO:

2.

11. The method of claim 7 or 9, wherein the regulating plant height of rice is to reduce the plant height of rice, the regulating leaf type is to narrow the leaf width, and the regulating photosynthesis is to increase the photosynthetic rate.

Images