1. TobaccoNtAAP6The application of the gene in the tobacco is characterized in that the tobaccoNtAAP6In connection with amino acid transport, after the amino acid transport is silenced, the tyrosine content, the proline content and the asparagine content in tobacco are obviously reduced, and the total protein content is obviously reduced;

said tobaccoNtAAP6The gene and the specific CDS sequence are shown in SEQ ID No. 3.

2. TobaccoNtAAP6The application of the gene in tobacco is characterized in that the gene NtAAP6 of the tobacco is related to the content regulation of pigment substances in the tobacco,after the tobacco leaves are silenced, the contents of chlorophyll a/b, total chlorophyll and carotenoid in the tobacco leaves are obviously increased;

said tobaccoNtAAP6The gene and the specific CDS sequence are shown in SEQ ID No. 3.

PCR amplification to obtain tobaccoNtAAP6The primer pair for gene is characterized by aiming at the tobacco shown as SEQ ID No.3NtAAP6The gene and primer sequence are designed as follows:

the upstream primer NtAAP 6-F: 5'-ATGGCACCCGAATTTCAGAAGAA-3' the flow of the air in the air conditioner,

downstream primer NtAAP 6-R: 5'-TTATTGTTGAGTTGAGAAAGGC-3' are provided.

4. Tobacco obtained by using the primer pair of claim 3NtAAP6A method for PCR amplification of a gene,

the cDNA of the safflower gold is used as a template, and the primer pair NtAAP6-F, NtAAP6-R is used for PCR amplification.

5. Tobacco for silencingNtAAP6A gene editing vector of a gene is characterized by being specifically constructed by the following steps:

(1) first, toNtAAP6Genes, design editing primer sequences as follows:

NtAAP6-K-F：5’-GATTGTATCAACAGAACTCGAAAG-3’，

NtAAP6-K-R：5’-AAACCTTTCGAGTTCTGTTGATAC-3’；

(2) then, obtaining double-stranded DNA of a target site in an annealing operation mode;

the specific sequence of the target site is as follows: GTATCAACAGAACTCGAAAG, respectively;

(3) next, the CRISPR/Cas9 vector pORE-Cas9/gRNA was usedBsaI, enzyme digestion is carried out, and a digestion product is connected with the annealing product;

(4) and finally, transforming the ligation product into an escherichia coli competent cell, and screening and identifying to ensure correct recombination.

6. Use rightObtaining the gene editing vector of claim 5NtAAP6A method for breeding a new variety of gene-silenced tobacco, comprising subjecting the silenced tobacco to a breeding processNtAAP6Transforming agrobacterium into infection liquid with gene editing vector, transforming tobacco, screening and identifying to obtainNtAAP6A new gene-silenced tobacco variety.

Background

Amino acids, which are basic constituent units of enzymes and proteins, not only are carriers of organic nitrogen and precursors of important secondary metabolites in plants, but also play an important role in the growth and development of plants. Plants can assimilate inorganic nitrogen, which is present in the form of ammonium salts and nitrates, from the soil through the roots to produce amino acids, or can assimilate amino acids directly from the soil, or can autonomously synthesize amino acids in the leaves. The absorption and transport of amino acids in plants depends on amino acid transporters, which are located on biological membranes and are classified according to sequence homology and function as: APC super family (Amino acid, polyamine and Choline transporters super family) and ATF super family (Amino acid transporter family); the ATF superfamily is divided into 6 subfamilies, namely AAPs (amino acid catalysts), LHTs (lysine histone transporters), ProTs (proline transporters), gamma-GATs (gamma-aminobutyric transporters), ANTs (aromatic and neutral amino acid transporters) and AUX (antibiotic-resistant family), wherein the subfamilies of AAPs (amino acid catalysts) are relatively deeply studied.

Amino Acid Permeases (AAPs) were first discovered and isolated in Arabidopsis thalianaAAPsA total of 8 members of the gene family: (AtAAP1～8) Mainly responsible for the transport of acidic and neutral amino acids, among themAtAAP1The gene is highly expressed in endosperm and cotyledon, and has high expression in root valleyThe transport of the salts of the acids and of the neutral amino acids is closely linked, while it is also possible to influence the weight and number of the plant seeds;AtAAP2andAtAAP3the expression level of the gene in the phloem of the stem and the root is higher, and the gene is responsible for the transportation of phloem amino acid;AtAAP5the gene is highly expressed mainly in the root cortex, and mediates the transportation of arginine and lysine;AtAAP6the gene has high expression level at the root and is responsible for adjusting the components and the content of amino acid in the sieve tube molecules;AtAAP8the gene regulates the transport of amino acids into endosperm at the early stage of seed development.

For crops, the research on AAPs has been mainly focused on rice, but there are reports on crops such as corn and potato. Rice (Oryza sativa L.) with improved resistance to stressAAPsUp to 19 members of a gene family, whereinOsAAP3The gene is related to the transport of lysine and arginine, and the inhibition of the expression of the gene can increase the spike number and tiller number of rice, thereby improving the utilization rate of nitrogen of the rice and increasing the yield;OsAAP6the gene participates in regulating and controlling the nutritional quality and the protein content of the rice. Pan et al discovered corn by yeast function complementation experimentZmAAP4The gene can regulate and control the transport of various amino acids, and particularly has strong affinity to glutamic acid and proline. Koch et al discovered potatoes by RNAi techniqueStAAP1The gene plays an important role in the transport of amino acids from leaves to rhizomes.

In the prior art, against tobaccoNtAAPThe family gene research shows that the tobacco amino acid permeaseNtAAP2Gene (tobacco amino acid permease)NtAAP2Gene and application thereof, CN 201711351625.2) has important influence on the regulation of the content of part types of amino acids in tobacco leaves. However, whether other genes in the family have similar functions in tobacco needs to be further researched to be further determined.

Disclosure of Invention

In the tobaccoNtAAP2On the basis of preliminary gene research, to further define the content of tobaccoNtAAPFamily of other genes, the present application aims to further define the tobaccoNtAAP3、NtAAP6The gene function, thereby laying a certain technical foundation for analyzing the genome function of the tobacco and cultivating new varieties of the tobacco.

The technical solution adopted in the present application is detailed as follows.

TobaccoNtAAP3Application of gene in tobacco, tobaccoNtAAP3In connection with amino acid transport, after the amino acid transport is silenced, the tyrosine content, the proline content and the asparagine content in tobacco are obviously reduced, and the total protein content is obviously reduced;

in another aspect, tobaccoNtAAP3The gene is related to the regulation of the content of pigment substances in the tobacco, and after the gene is silenced, the content of pigment substances such as chlorophyll a/b, total chlorophyll, carotenoid and the like in the tobacco leaves is obviously increased;

said tobaccoNtAAP3The specific CDS sequence of the gene is shown as SEQ ID No.1, and the corresponding edited amino acid sequence of the tobacco NtAAP3 protein is shown as SEQ ID No.2, or specifically as follows:

NtAAP3gene CDS sequence (1440 bp):

ATGGGAGAAAACAACAACGTTGCTTCAAAACACCAAGTGTTCGATGTTTCCATTAATGTGACTGAATCCAAGTGCTTTGACGATGATGGCCGTCTCAAAAGAACCGGGAGCGTTTGGACGGCAAGTGCTCATATCATAACAGCTGTGATTGGTTCGGGAGTTTTGTCTTTAGCATGGGCAGTAGCTCAACTTGGTTGGATTGCTGGTCCTATTGTTATGATTTTGTTCTCTTTTGTTACTTATTACACTTCCGCTCTTCTTGCCGATTGTTACCGCTCCGGCGACTCTGTTTCCGGCAAGAGAAACTATACTTACATGGATGCTGTCCAAGCCAATCTTGGTGGGCTCCAAGTCAAGATTTGTGGATGGATTCAGTATGCGAATCTTTTTGGAGTTGCTATCGGATACACCATTGCATCTTCAATTAGCATGATAGCTATTAAAAGGTCTAATTGTTTCCACAAACATGGTGATCAAGCTCCTTGTCAAGTATCCAGCACTCCATACATGATCATGTTTGGAATAATAGAAATCATCTTCTCCCAAATTCCAGATTTTGATCAGATTTGGTGGCTTTCAATTGTGGCTGCCGTTATGTCTTTCACTTACTCTACTATTGGACTAGGATTAGGAGTTGCTAAAGTGGCAGAAACTGGAAAAATCGGAGGAAGTCTCACTGGAATTAGCATCGGAACTGTGACTGAAATGCAAAAGATTTGGAAAAGCTTCCAAGCCCTTGGAGCTATCGCTTTTGCCTATTCTTACTCTCTCATCCTTATTGAGATTCAGGATACACTCAAATCTCCACCGTCAGAATCCAAGACAATGAAAAATGCAACTCTAATTAGTGTAGCAGTAACAACAGTTTTCTACATGCTCTGTGGCTGCTTTGGCTATGCAGCATTTGGAGATCTTGCTCCTGGAAACTTACTAACTGGTTTTGGATTCTACAATCCTTATTGGCTACTCGATATAGCGAACATAGCCATTGTCGTCCACCTTGTTGGTGCATACCAGGTTTACTGCCAGCCCCTTTTCGCCTTCATTGAAAAAACAGCAGCAGAATGGTACCCCGAGAGTAAATTCATTGCCAAAGAGATTAGTGTCCCGATTATAGGCTATAAATCCTTTAAACTCAACCTTTTCCGCATAATTTGGAGGACTATTTTCGTCATCATCACCACGGTCATATCTATGCTATTGCCATTCTTCAATGACATAGTTGGAATTCTTGGAGCCTTTGGGTTTTGGCCGCTAACAGTCTATTTCCCGGTGGAAATGTACATTGTGCAAAAGAAGATAACAAAATGGAGCACAAAATGGATTTGCCTTCAAATGCTTAGTGTTGCTTGCCTTATTATCTCAATTGCTGCAGCTGCTGGTTCTTTTGCTGGCGTTGTATCTGATCTACAAGTTTACAAGCCTTTTAAGACGACTTGA；

NtAAP3 protein sequence (479 AA):

MGENNNVASKHQVFDVSINVTESKCFDDDGRLKRTGSVWTASAHIITAVIGSGVLSLAWAVAQLGWIAGPIVMILFSFVTYYTSALLADCYRSGDSVSGKRNYTYMDAVQANLGGLQVKICGWIQYANLFGVAIGYTIASSISMIAIKRSNCFHKHGDQAPCQVSSTPYMIMFGIIEIIFSQIPDFDQIWWLSIVAAVMSFTYSTIGLGLGVAKVAETGKIGGSLTGISIGTVTEMQKIWKSFQALGAIAFAYSYSLILIEIQDTLKSPPSESKTMKNATLISVAVTTVFYMLCGCFGYAAFGDLAPGNLLTGFGFYNPYWLLDIANIAIVVHLVGAYQVYCQPLFAFIEKTAAEWYPESKFIAKEISVPIIGYKSFKLNLFRIIWRTIFVIITTVISMLLPFFNDIVGILGAFGFWPLTVYFPVEMYIVQKKITKWSTKWICLQMLSVACLIISIAAAAGSFAGVVSDLQVYKPFKTT*。

tobaccoNtAAP6Application of gene in tobacco, tobaccoNtAAP6In connection with amino acid transport, after the amino acid transport is silenced, the tyrosine content, the proline content and the asparagine content in tobacco are obviously reduced, and the total protein content is obviously reduced;

in another aspect, tobaccoNtAAP6The gene is related to the regulation of the content of pigment substances in the tobacco, and after the gene is silenced, the content of pigment substances such as chlorophyll a/b, total chlorophyll, carotenoid and the like in the tobacco leaves is obviously increased;

said tobaccoNtAAP6The specific CDS sequence of the gene is shown as SEQ ID No.3, and the corresponding edited amino acid sequence of the tobacco NtAAP6 protein is shown as SEQ ID No.4, or specifically as follows:

NtAAP6gene CDS sequence (1506 bp):

ATGGCACCCGAATTTCAGAAGAACACTATGTACGTATCAACAGAACTCGAAAGAGGAGATGTTCAAAAAAACTTTGATGATGATGGGCGTGAGAAAAGAACTGGGACGTTACTAACGGCAAGTGCACATATTATCACTGCTGTAATTGGTTCAGGAGTGCTTTCTTTAGCATGGGCTATAGCTCAGTTAGGATGGGTGGCTGGTCCTGCTGTTCTCTTTGCTTTTTCTTTCATTACATACTTCACTTCTACACTTCTTGCCGACTGTTACCGTTCTCCCGGCCCCATCTCCGGCAAGAGAAACTACACTTACATGGACGTTGTTCGTTCTCACTTAGGAGGTGTGAAGGTAACACTGTGTGGACTTGCACAATATGCTAACCTCGTCGGAGTTACCATTGGATACACTATTACAGCATCTATCAGTATGGTCGCAGTAAAGAGATCAAATTGTTTTCACAAACATGGCCACGAAGCCAGCTGCTCAATATCGAGCTACCCATATATGATCATATTTGCAGTCATTCAAGTAGTTCTAAGCCAAATACCAAATTTCCACAAGCTCTCATGGCTATCAATTCTTGCTGCTGTTATGTCTTTTACTTACGCTTCTATTGGTCTTGGACTCTCTATTGCCAAAGCTGCTGGGGTAGGGCACCATGTAAAGACAAGCCTAACAGGGACGACAGTAGGAGTTGATGTGTCTGGATCAGAGAAAATATGGAAAAGCTTCCAAGCCATAGGAGATATTGCATTTGCTTATGCTTATTCCACCGTTCTCATCGAAATACAGGCAAGCACACTGTCATTGATTCTGATTCTGATCTTTTCTAAGATTTTATTACGTCGTAGGGATACATTGAGGTCACAACCTCCAGAAAGCAAGGTTATGAAGAGAGCCTCATTAGCTGGAGTTTCCACCACAACTTTATTCTATATACTATGTGGTACCATTGGTTATGCAGCCTTTGGAAATGATGCTCCTGGAAATTTCCTTACTGGTTTTGGTTTCTATGAACCATTTTGGCTAATTGACTTTGCCAACGTTTGCATTGCCGTCCACCTTGTTGGAGCTTACCAGGTTTTCTGCCAACCTTTATATGGGTTCGTGGAGGCTCGTTGCAACGAGCGATGGTCAGACAGCAAATTCATCACCTCCGAGTACGCTGTGCAAGTTCCATGCTGTGGCGTTTACAACGTCAACTTGTTCAGGTTGGTGTGGAGAACAGCATATGTTGTAGTGACAGCCGTGATTGCCATGATATTCCCCTTCTTCAATGACTTCTTGGGTTTGATCGGGGCAGCATCGTTCTATCCATTAACTGTCTACTTCCCAATAGAGATGCACATTGCCCAGAGAAAGATACCAAAGTATTCTTTCACATGGGTATGGCTGAAAATTTTGAGCTGGACTTGCCTGGTTGTATCACTTGTTGCAGCTGCTGGATCTATCCAGGGTCTTGTCACTTCTCTCAAGCATTACAAGCCTTTCTCAACTCAACAATAA；

NtAAP6 protein sequence (501 AA):

MAPEFQKNTMYVSTELERGDVQKNFDDDGREKRTGTLLTASAHIITAVIGSGVLSLAWAIAQLGWVAGPAVLFAFSFITYFTSTLLADCYRSPGPISGKRNYTYMDVVRSHLGGVKVTLCGLAQYANLVGVTIGYTITASISMVAVKRSNCFHKHGHEASCSISSYPYMIIFAVIQVVLSQIPNFHKLSWLSILAAVMSFTYASIGLGLSIAKAAGVGHHVKTSLTGTTVGVDVSGSEKIWKSFQAIGDIAFAYAYSTVLIEIQASTLSLILILIFSKILLRRRDTLRSQPPESKVMKRASLAGVSTTTLFYILCGTIGYAAFGNDAPGNFLTGFGFYEPFWLIDFANVCIAVHLVGAYQVFCQPLYGFVEARCNERWSDSKFITSEYAVQVPCCGVYNVNLFRLVWRTAYVVVTAVIAMIFPFFNDFLGLIGAASFYPLTVYFPIEMHIAQRKIPKYSFTWVWLKILSWTCLVVSLVAAAGSIQGLVTSLKHYKPFSTQQ*。

obtaining tobacco by PCR amplificationNtAAP3The primer pair for the gene comprises the following specific steps:

the upstream primer NtAAP 3-F: 5'-ATGGGAGAAAACAACAACGTTGC-3' the flow of the air in the air conditioner,

downstream primer NtAAP 3-R: 5'-TCAAGTCGTCTTAAAAGGCTTG-3' are provided.

A method for preparing tobaccoNtAAP3Of genesThe PCR amplification method is to perform PCR amplification on the NtAAP3-F, NtAAP3-R by taking the safflower large gold element cDNA as a template and utilizing a primer pair.

Obtaining tobacco by PCR amplificationNtAAP6The primer pair for the gene comprises the following specific steps:

the upstream primer NtAAP 6-F: 5'-ATGGCACCCGAATTTCAGAAGAA-3' the flow of the air in the air conditioner,

downstream primer NtAAP 6-R: 5'-TTATTGTTGAGTTGAGAAAGGC-3' are provided.

A method for preparing tobaccoNtAAP6The PCR amplification method of the gene takes safflower large gold element cDNA as a template and utilizes a primer pair NtAAP6-F, NtAAP6-R to carry out PCR amplification.

Tobacco for silencingNtAAP3The gene editing vector of the gene is specifically constructed by the following steps:

(1) first, toNtAAP3The gene (target site specific sequence: GTGACTGAATCCAAGTGCTTTG), the design and editing primer sequence is as follows:

NtAAP3-K-F：5’-GATTGTGACTGAATCCAAGTGCTTTG-3’，

NtAAP3-K-R：5’-AAACCAAAGCACTTGGATTCAGTCAC-3’；

(2) then, obtaining double-stranded DNA of a target site in an annealing operation mode;

(3) next, the CRISPR/Cas9 vector pORE-Cas9/gRNA was usedBsaI, enzyme digestion is carried out, and a digestion product is connected with the annealing product;

(4) and finally, transforming the ligation product into an escherichia coli competent cell, and screening and identifying to ensure correct recombination.

Tobacco for silencingNtAAP3The gene editing vector of the gene is specifically constructed by the following steps:

(1) first, toNtAAP6The gene (target site specific sequence: GTATCAACAGAACTCGAAAG), the design and editing primer sequence is as follows:

NtAAP6-K-F：5’-GATTGTATCAACAGAACTCGAAAG-3’，

NtAAP6-K-R：5’-AAACCTTTCGAGTTCTGTTGATAC-3’。

(2) then, obtaining double-stranded DNA of a target site in an annealing operation mode;

（3）next, the CRISPR/Cas9 vector pORE-Cas9/gRNA was usedBsaI, enzyme digestion is carried out, and a digestion product is connected with the annealing product;

(4) and finally, transforming the ligation product into an escherichia coli competent cell, and screening and identifying to ensure correct recombination.

An obtainingNtAAP3A method for cultivating new variety of gene-silenced tobaccoNtAAP3Transforming agrobacterium into infection liquid with gene editing vector, transforming tobacco, screening and identifying to obtainNtAAP3A new gene-silenced tobacco variety.

An obtainingNtAAP6A method for cultivating new variety of gene-silenced tobaccoNtAAP6Transforming agrobacterium into infection liquid with gene editing vector, transforming tobacco, screening and identifying to obtainNtAAP6A new gene-silenced tobacco variety.

In the prior art, aiming at tobaccoNtAAP2Although there have been preliminary studies on gene functions, further studies have been made on gene functionsNtAAP3、NtAAP6It was found that all three genes belong toAAPsMembers of the gene family, but their actual functions are still clearly distinct. Specifically, the method comprises the following steps:NtAAP2the gene is highly expressed in the roots and stems of the tobacco in the full-bloom stage, particularly the expression level in the stems is higher, andNtAAP3the expression level of the gene in the leaves in the vigorous growth stage and the topping stage is obviously higher than that of other organs,NtAAP6the result that the expression level of the gene in roots and leaves, especially in leaves, is significantly higher than that in other organs indicatesNtAAP3、NtAAP6The functions of the two genes in different growth and development stages and different tissues of tobacco are different. On the other hand, from the construction aspect of new gene mutant varieties, as the RNAi silencing technology has the defects of high off-target rate, poor silencing effect, low conversion rate and the like, the application designs a new gene mutant conversion technology by adopting the CRISPR/Cas9 gene editing technology, thereby laying a certain technical foundation for new variety cultivation.

Generally, the method detects and analyzes the physiological characters such as protein content change, photosynthetic index change, pigment content change and the likeNtAAP3Gene, gene,NtAAP6The relationship between gene and amino acid regulation, protein content regulation and pigment content regulation is preliminarily determined. Based on these studies, for elucidationNtAAP3Gene, gene,NtAAP6The gene lays a theoretical foundation for the functions of amino acid absorption, transport, nitrogen utilization, plant stress resistance regulation and control and the like, and also lays a certain technical foundation for improving the high-efficiency utilization of nitrogen by tobacco and further for breeding new tobacco varieties.

Drawings

FIG. 1 shows the results of total RNA electrophoresis, in which: m: trans2K DNA Marker; 1-2 are directed toNtAAP3The electrophoresis results of total RNA extracted during gene cloning (28S, 18S and 5S bands in the figure, wherein 1-2 are total RNA results of different samples), and 3-4 are specificNtAAP6The electrophoresis result of total RNA extracted during gene cloning (28S, 18S and 5S bands in the figure, wherein 3-4 are the total RNA result of different samples);

FIG. 2 shows the result of electrophoresis of PCR products, in which: m: trans2K DNA Marker, left handNtAAP3Electrophoresis results after gene PCR amplification, right panelNtAAP6Electrophoresis results after gene PCR amplification;

FIG. 3NtAAP3Relative expression of the gene in different periods and tissues of the tobacco Honghua Dajinyuan;

FIG. 4NtAAP6Relative expression of the gene in different periods and tissues of the tobacco Honghua Dajinyuan;

FIG. 5 shows the first round of PCR electrophoresis results in the process of identifying the gene-editing mutant, wherein: m: DL5000 DNA Marker; the upper drawing is a partNtAAP3Identifying results of the gene editing mutants (1-8 are numbers of tobacco plant samples to be identified); the lower drawing is a partNtAAP6Identifying results of the gene editing mutants (1-5 are numbers of tobacco plant samples to be identified);

FIG. 6 shows the results of a second round of PCR electrophoresis of a sample of partial gene editing mutants, wherein: m: DL5000 DNA Marker; the upper diagram isNtAAP3Performing second round PCR electrophoresis on the gene editing mutant (1-8 in the figure are tobacco seedling samples to be identified, and correspond to the samples in the figure 5); the lower diagram isNtAAP6Second round PCR electrophoresis results of gene editing mutants (in the figure, 1-5 are tobacco seedling samples to be identified, andFIG. 5 sample by sample)

FIG. 7 shows the results of sequencing analysis of a sample of a partial gene editing mutant, in which: the upper diagram isNtAAP3The results of sequencing analysis of the gene editing mutants (numbers 2, 6 and 7 in the figure are consistent with the numbers of the samples in figure 5); the lower diagram isNtAAP6The sequencing analysis result of the gene editing mutant (the numbers 3 and 4 in the figure are consistent with the sample numbers in the figure 5);

FIG. 8 is a drawing showingNtAAP3Statistical results of agronomic traits for Gene editing mutants (legend from top to bottom in each set of histograms from left to right)

FIG. 9 is a schematic view ofNtAAP6Gene editing mutant agronomic trait statistics;

FIG. 10 shows the change of total protein content of tobacco leaves after gene mutant baking, wherein: the upper diagram isNtAAP3The total protein content of the tobacco leaves is changed after the gene mutant is roasted; the lower diagram isNtAAP6The total protein content of the tobacco leaves is changed after the gene mutant is roasted;

FIG. 11 shows the amino acid content change of the tobacco leaves after gene mutants are baked, wherein: the upper diagram isNtAAP3The amino acid content of the tobacco leaves is changed after the gene mutants are baked; the lower diagram isNtAAP6The amino acid content of the tobacco leaves after the gene mutants are baked is changed (each group of column charts correspond to tyrosine, proline and asparagine from left to right respectively);

FIG. 12 is a drawing showingNtAAP3Fv/Fm values (upper panel), NPQ values (lower panel) of the gene mutants under normal conditions;

FIG. 13 is a drawing showingNtAAP6Fv/Fm values (upper panel), NPQ values (lower panel) of the gene mutants under normal conditions;

FIG. 14 is a drawing showingNtAAP3Fv/Fm value (upper graph) and NPQ value (lower graph) of the gene mutant under strong light stress;

FIG. 15 is a drawing showingNtAAP6Fv/Fm value (upper graph) and NPQ value (lower graph) of the gene mutant under strong light stress;

FIG. 16 is a drawing showingNtAAP3The chlorophyll a content (upper graph) and the chlorophyll b content (lower graph) of the gene mutants;

FIG. 17 is a drawing showingNtAAP6The chlorophyll a content (upper graph) and the chlorophyll b content (lower graph) of the gene mutants;

FIG. 18 is a drawing showingNtAAP3Total chlorophyll content (upper panel) and carotene content (lower panel) of the gene mutant;

FIG. 19 is a drawing showingNtAAP6Total chlorophyll content (upper panel) and carotenoid content (lower panel) of the gene mutants.

Detailed Description

The present application is further illustrated by the following examples. Before describing the specific embodiments, a brief description will be given of some experimental background cases in the following embodiments.

Biological material:

indoor culture of common tobacco Honghua Dajinyuan is carried out in the greenhouse of the national tobacco gene research center (Zhengzhou), and the culture conditions are as follows: the temperature is 23 +/-1) DEG C, the relative humidity is 60 +/-2 percent, and the illumination/darkness is 16 h/8 h; planting and culturing in field in Yuxi Yunan;

the synthesis and sequencing of related primers are completed by the Biotechnology Limited company of the New industry of Beijing Optingkok;

experimental reagent:

EasyPure Plant RNA Kit、EasyPure Plant Genomic DNA Kit、TransStartGreen qPCR SuperMix、TransScript Reverse Transcriptase、2×TransTaq High Fidelity (HiFi) PCR SuperMix、pEASY-T1 Cloning Kit、Trans5α Chemically Competent Cell、Trans2K DNA Marker was purchased from Beijing Quanjin Biotechnology Ltd;

DL5000 DNA Marker was purchased from Biotechnology Ltd of New industry of Beijing Okagaku.

Example 1

The embodiment first of allNtAAP3Gene, gene,NtAAP6The process for obtaining the gene is briefly described below.

(I) designing primers for PCR amplification

Based on the existing tobacco genome sequence and aiming at the target geneNtAAP3、NtAAP6The primer sequences for PCR amplification were designed as follows:

the upstream primer NtAAP 3-F: 5'-ATGGGAGAAAACAACAACGTTGC-3' the flow of the air in the air conditioner,

downstream primer NtAAP 3-R: 5'-TCAAGTCGTCTTAAAAGGCTTG-3', respectively;

the upstream primer NtAAP 6-F: 5'-ATGGCACCCGAATTTCAGAAGAA-3' the flow of the air in the air conditioner,

downstream primer NtAAP 6-R: 5'-TTATTGTTGAGTTGAGAAAGGC-3' are provided.

(II) preparation of template for PCR amplification

Taking tobacco leaves of Honghuadajinyuan as sample source, freezing with liquid nitrogen, grinding, and referringEasyPurePlant RNA Kit instruction, extracting total RNA, detecting the concentration and purity of the RNA by using a NanoDrop 2000 ultramicro spectrophotometer, and referring to the obtained product after ensuring that the use requirement is metTransScriptReverse Transcriptase, further Reverse transcribing the extracted total RNA into cDNA for later use as a template for subsequent PCR amplification.

The electrophoresis results of total RNA extracted from different samples are shown in FIG. 1, and it can be seen that: the 18S and 28S bands of the total RNA of the extracted tobacco leaves are clear and have no obvious degradation. Further, the RNA concentration measured by an ultramicro spectrophotometer NanoDrop 2000 is between 900 and 1000 ng/mu L, OD₂₆₀/OD₂₈₀The temperature is between 1.8 and 2.0, and the requirements of subsequent experiments are met.

(III) PCR amplification

Reference 2TransTaqIn the specification of High Fidelity (High fi) PCR Supermix, the cDNA prepared in the step (two) is used as a template, the primers designed in the step (one) are used for PCR amplification, and a 50-microliter amplification system is designed as follows:

cDNA template, 2. mu.L;

upstream primer, (10. mu. mol/L) 1. mu.L;

downstream primer, (10. mu. mol/L) 1. mu.L;

2×TransTaq HiFi PCR SuperMix ，25 μL；

ultrapure water, added to 50 μ L;

the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30 s, annealing at 56 ℃ for 30 s, extension at 72 ℃ for 90 s, and 35 cycles; extension at 72 ℃ for 5 min.

And detecting the PCR amplification product by 1% agarose gel electrophoresis. The electrophoresis results are shown in fig. 2, and it can be seen that: there is a clear band around 1500 bp.

Subsequently, glueRecovering target fragment, performing ligation transformation according to pEASY-T1 Cloning Kit, further screening, performing colony PCR identification, and sending to Beijing Okagaku Biotech Co., Ltd for sequencing identification (the specific sequence information is shown as SEQ ID No. 1-4, and the expected sequence information is shown as SEQ ID No. 1-4)NtAAP31440 bp gene sequence length,NtAAP6The 1506 bp length of the gene results in agreement).

On the basis of the sequencing result, the inventors further developed the sequence by using the Real-time PCR technologyNtAAP3Gene, gene,NtAAP6The expression pattern of the gene was specifically analyzed, and the specific procedures are briefly described below.

First, based on sequencingNtAAP3、NtAAP6CDS sequence of gene, designing primer sequence for Real-time PCR primer as follows:

upstream primer NtAAP 3-qF: 5'-AGTGTGGTCCATTGAACTCAGAA-3' the flow of the air in the air conditioner,

the downstream primer NtAAP 3-qR: 5'-TATGATATGAGCACTTGCCGTC-3', respectively;

upstream primer NtAAP 6-qF: 5'-ACACACACGCGCGCTAAA-3' the flow of the air in the air conditioner,

the downstream primer NtAAP 6-qR: 5'-GACCAGCCACCCATCCTAAC-3', respectively;

at the same time, the gene is directed to the reference geneL25The gene, the primer sequence for PCR amplification is designed as follows:

L25-F：5’-CCCCTCACCACAGAGTCTGC-3’，

L25-R：5’-AAGGGTGTTGTTGTCCTCAATCTT-3’

then, inL25The gene is used as an internal reference gene, cDNA is used as a template, and reference is carried outTransStartGreen qPCR SuperMix instructions, a 20 μ L amplification system was designed as follows:

cDNA template, 1. mu.L;

upstream primer, (10. mu. mol/L) 0.4. mu.L;

downstream primer, (10. mu. mol/L) 0.4. mu.L;

2×TransStart Green qPCR SuperMix，10 μL；

supplementing the nucleic-free Water to 20 mu L;

the reaction conditions are as follows: 94 ℃ for 30 s; 94 ℃, 5 s, 60 ℃, 30 s, 45 cycles; storing at 4 ℃. Each sample was tested in triplicate, averaged, and used for 2 ^C-△△ _T And calculating the result by the method.

Results of expression patterns showed that:NtAAP3the expression level of the gene in the leaf is obviously higher than that of other organs (P< 0.05), especially in the vigorous and apical stages (see FIG. 3 for specific results); in a similar manner to this, the first and second,NtAAP6the expression level of the gene in roots and leaves, especially in leaves, is also significantly higher than that in other organs (see FIG. 4 for specific results).

Example 2

On the basis of example 1, to determineNtAAP3、NtAAP6The specific functions of the genes, namely the CRISPR/Cas9 technology, are utilized, and the inventor further constructs a gene editing vector and carries out gene silencing so as to specifically determine the functions of the two genes. This example is specifically summarized below with respect to the relevant experimental conditions.

(I) construction of Gene editing vector

(1) First, toNtAAP3The gene (target site specific sequence: GTGACTGAATCCAAGTGCTTTG), the design and editing primer sequence is as follows:

NtAAP3-K-F：5’-GATTGTGACTGAATCCAAGTGCTTTG-3’，

NtAAP3-K-R：5’-AAACCAAAGCACTTGGATTCAGTCAC-3’；

to is directed atNtAAP6The gene (target site specific sequence: GTATCAACAGAACTCGAAAG), the design and editing primer sequence is as follows:

NtAAP6-K-F：5’-GATTGTATCAACAGAACTCGAAAG-3’，

NtAAP6-K-R：5’-AAACCTTTCGAGTTCTGTTGATAC-3’。

(2) subsequently, with reference to the Annealing Buffer for DNA oligonucleotides (5 ×) kit instructions, target site double-stranded DNA was obtained by Annealing operation; in a specific reaction, a 20. mu.L reaction system was designed as follows:

upstream primer, 4. mu.L (50. mu. mol/L);

downstream primer, 4. mu.L (50. mu. mol/L);

Annealing Buffer for DNA Oligos (5×)， 4 μL；

nuclear-free Water, to 20 μ L;

the specific annealing reaction conditions are as follows: reducing the temperature of 95 ℃ for 5 min to 25 ℃ every 8 s, wherein the temperature is reduced by 0.1 ℃; the reaction product is directly used or stored at 4 ℃ for later use.

(3) Next, the CRISPR/Cas9 vector pORE-Cas9/gRNA was usedBsaI, enzyme digestion is carried out, and a digestion product is connected with the annealing product;

during specific enzyme digestion, a 20-mu-L enzyme digestion system is designed as follows:

pORE-Cas9/gRNA vector, 3. mu.L;

10× Buffer，2 μL；

Bsaenzyme I, 1 μ L;

supplementing sterile water to 20 μ L;

the enzyme was cleaved at 37 ℃ for 1 h.

For specific ligation, a 20 μ L ligation system was designed as follows:

3 mu L of carrier enzyme digestion product;

annealed product, 6 μ L;

10× T4 DNA Ligase Buffer，2 μL；

T4 DNA Ligase，1 μL；

supplementing sterile water to 20 μ L;

ligation was carried out at 16 ℃ for 30 min.

(4) Finally, the ligation product is convertedTrans5 α chemical Complex Cell (E.coli Competent Cell) and screened and colony PCR identified (forNtAAP3The gene editing vector of (1), when identifying, a primer pair U26-jiance-F, NtAAP3-K-R is adopted; to is directed atNtAAP6The gene editing vector of (1), when identifying, a primer pair U26-jiance-F, NtAAP6-K-R is adopted; U26-jiance-F: 5'-TTAGGTTTACCCGCCAATA-3'); and (3) further sequencing and identifying the positive clone plasmid with correct identification to ensure that the recombination is correct.

(II) preparation of transfection solution

And (3) further amplifying the positive clone (the gene editing vector with correct recombination) which is sequenced and identified in the step (I), transferring the gene editing vector into agrobacterium GV3101 by using an electric transfer method, and further screening and carrying out colony PCR identification to ensure that the transformation is correct. For identification of the correct transformants, Kan was used⁺And Rif⁺Both resistantFurther performing amplification culture on LB liquid culture medium, when OD value is 0.6-0.8, centrifugally collecting thalli, and then using MS to collect thalli₀Liquid Medium (MS)₀Liquid culture medium: 4.4 g/L MS inorganic salt, pH value of 5.8-5.9) to OD value of 0.2-0.3, and using the suspension as transfection solution for standby.

(III) transformation and screening

And (5) further transforming the tobacco by using the transfection solution prepared in the step (II) by adopting a leaf disc transformation method, and screening and identifying positive transformation materials.

Specific transformation operations can be referred to as follows:

cutting the leaves of the tobacco aseptic seedlings into 0.5-1 cm²Immersing a square leaf disc into the transfection solution for 7-9 min, taking out the leaves, absorbing residual bacteria liquid on the leaves by using sterile absorbent paper, and transferring the leaves to an MS solid culture medium;

dark culture at 22-25 deg.C for 2 d, and transfer to MS (Kan)⁺) Differentiating the culture medium, culturing under the conditions of illumination at 28 ℃ for 16 h, darkness at 25 ℃ for 8 h and relative humidity of 60%, and replacing the fresh culture medium every 8-10 days until adventitious buds grow out;

when the adventitious bud grows to about 1 cm, cutting the adventitious bud, transferring the adventitious bud into an elongation culture medium for culture, and transferring the adventitious bud into a rooting culture medium for continuous culture after two weeks; after the root system grows well, transferring the root system from the culture medium to a seedling culture medium for culture.

The specific culture medium formula can be referred as follows:

MS culture medium: 4.4 g/L of MS inorganic salt, 30 g/L of cane sugar and 2.5 g/L of plant gel, wherein the pH value is 5.8-5.9;

MS（Kan⁺) Differentiation medium: 4.4 g/L of MS inorganic salt, 30 g/L of cane sugar, 1 mg/L of 6-BA, 0.1 mg/L of NAA, 150 mg/L of kanamycin, 250 mg/L of cefotaxime sodium, 2.5 g/L of plant gel and the pH value is 5.8-5.9;

elongation culture medium: 4.4 g/L of MS inorganic salt, 30 g/L of cane sugar, 0.1 mg/L of 6-BA, 150 mg/L of kanamycin, 250 mg/L of cefotaxime sodium, 2.5 g/L of plant gel and the pH value of 5.8-5.9;

rooting culture medium: 4.4 g/L of MS inorganic salt, 30 g/L of cane sugar, 0.002 mg/L of NAA, 150 mg/L of kanamycin, 250 mg/L of cefotaxime sodium, 2.5 g/L of plant gel and the pH value of the plant gel is 5.8-5.9.

When the specific screening and identification are carried out:

firstly, extracting the DNA of tobacco leaves to be identified, and taking the DNA as a template for PCR amplification;

subsequently, respectively utilizing primer pairs NtAAP3-K-F/NtAAP3-K-R, NtAAP6-K-F/NtAAP6-K-R to carry out first round PCR amplification, carrying out electrophoresis detection on PCR amplification products, and carrying out gel recovery on the amplification products;

finally, the first round PCR amplification product recovered by the glue is used as a template, a second round PCR amplification is respectively carried out on NtAAP3-JD-F/NtAAP3-K-R (NtAAP 3-JD-F: 5'-ATAACAGCTGTGATTGGTT-3') and NtAAP6-JD-F/NtAAP6-K-R (NtAAP 6-JD-F: 5'-ACTAACGGCAAGTGCACAT-3') by using a target specific primer, and the electrophoresis detection is carried out on the PCR amplification product.

Based on the second round amplification product results, if the product is less or no, the mutant is a potential homozygous mutant, and then the corresponding first round PCR product is clonally transformed and subjected to sequencing analysis to determine whether the mutant is a homozygous mutant.

The electrophoresis results of the first round PCR amplification products are shown in FIG. 5. It can be seen that the samples of the related gene editing mutants are amplified to obtain specific target bands.

The electrophoresis results of the second round PCR amplification products are shown in FIG. 6. It can be seen that the partially edited mutant samples had fewer PCR products and could be potential homozygous mutants for further characterization.

Further sequencing identification results (shown in fig. 7) show that: to is directed atNtAAP3The result that a gene editing mutant in which a base substitution G → A occurred at the target site and the base substitution caused a change in the original amino acid sequence caused a loss of function of the NtAAP3 protein indicates that a positive result was successfully obtainedNtAAP3Editing mutant tobacco seedlings by using genes; to aim atNtAAP6The result of gene sequencing showed that base substitution G → T occurred at the target site and that the base substitution caused a change in the original amino acid sequence, resulting in loss of function of the NtAAP6 protein, indicating that positive was successfully obtainedNtAAP6And (3) editing the mutant tobacco seedlings by using genes.

(IV) tobacco trait Change after Gene editing

Based on the homozygous mutant obtained by screening, field planting is carried out on Yuxi Yu nan, and the agronomic characters such as plant height, leaf number, stem circumference, pitch, maximum waist leaf length/width and the like are subjected to statistical analysis before and after the topping period.

In the planting process, the contents of protein, amino acid and pigment in the tobacco leaves of the editing mutant are synchronously detected (the content of the protein in the tobacco leaves of the tobacco leaves and the baked tobacco leaves is respectively measured by a continuous flow method according to the industry standard method of YC/T249-2008 tobacco and tobacco product protein measurement, the content of various amino acids is detected according to the industry standard method of YCT 282-2009 tobacco free amino acid measurement, and the contents of chlorophyll and carotenoid in the tobacco are detected according to the high performance liquid chromatography method of YC/T382-2010 tobacco and tobacco product plastid pigment measurement).

Meanwhile, in the planting process, an Imaging-PAM (polyacrylamide) -full-leaf fluorescence Imaging system is used for monitoring various photosynthetic physiological indexes of the gene editing mutant subjected to topping.

The following is a brief introduction of specific experimental results.

(1) Statistical results of agricultural characters of field

The statistical results of the agronomic traits of part of the fields are shown in fig. 8 and fig. 9. As can be seen,NtAAP3a gene-editing mutant,NtAAP6The phenotype of the gene editing mutant related traits is similar, namely: the natural plant height and the knockout plant height of the gene-editing mutant were reduced compared with those of the control group (HD), presumably because ofNtAAP3Gene, gene,NtAAP6The mutation of the gene affects the amino acid transport and nitrogen metabolism, thereby reducing the total biological yield, but the length/width of the maximum waist leaf has no obvious change, and the total tobacco yield is not affected. In general, the agronomic characters of the gene editing mutant are more in line with the requirements of high-quality flue-cured tobacco.

(2) Tobacco leaf protein and amino acid content change

The total protein content and amino acid content of the tobacco leaf after baking the gene editing mutant are measured asFig. 10 and 11 show the drawings. Analysis can see that: compared with the control group of safflower Honghuadajinyuan (HD),NtAAP3gene, gene,NtAAP6The total protein content of the tobacco leaves after the gene editing mutant is baked is reduced,NtAAP3the total protein content of the mutant was reduced by 19.8%,NtAAP6the total protein content of the mutant was reduced by 8.4%; the contents of tyrosine, proline and asparagine are lower than those of the control, wherein the reduction of proline and asparagine is particularly remarkable,NtAAP3the proline of the mutant is reduced by 19.0 percent, the asparagine is reduced by 68.1 percent,NtAAP6proline of the mutant is reduced by 36.3%, and asparagine is reduced by 62.9%; show thatNtAAP3Gene, gene,NtAAP6Mutation of the gene reduces the transport efficiency of amino acids and accumulation of proteins to some extent.

It should be explained that, in view of manufacturing of cigarette products, obtaining cigarette raw materials with low phenol release amount during smoking is a main technical objective of related variety improvement, and therefore, when the content of the amino acid is measured, only the flue-cured tobacco which is a direct raw material for preparing the cigarette products is taken as a standard, and the content of part of typical amino acids in the flue-cured tobacco is measured, so that the technical effects of the application can be more directly reflected.

(3) Physiological changes of photosynthesis

The results of the photo-biological analysis (the results are shown in FIG. 12, FIG. 13, FIG. 14 and FIG. 15),NtAAP3a gene-editing mutant,NtAAP6The related change trends of the gene editing mutants are consistent, and specifically: compared with the HD of the control group,NtAAP3a gene-editing mutant,NtAAP6The Fv/Fm values of the gene editing mutants under normal conditions are not obviously different, while the NPQ values are increased, which indicates thatNtAAP3Gene, gene,NtAAP6After the gene is edited, the photosynthetic efficiency of the plant is not greatly influenced, and the photoprotective capacity of the plant is improved.

Further, the measurement result under the strong light condition (60000-70000 Lux) shows that compared with the HD of the control group,NtAAP3a gene-editing mutant,NtAAP6The Fv/Fm and NPQ values of the gene editing mutant are obviously reduced under the condition of strong light hypochondrium. Photosynthetic efficiency theory of plants when illumination is enhancedShould be elevated, while the plants also initiate a heat dissipation, i.e. photoprotective mechanism, andNtAAP3mutant,NtAAP6The Fv/Fm and NPQ values of the gene editing mutant do not increase or decrease reversely, which indicates that the gene is edited, the transport rate of amino acid is influenced, the photosynthetic efficiency of the plant is possibly reduced, and the photoprotective ability of the plant is weakened.

(4) Change of pigment content

The measurement results of different types of pigment contents (fresh tobacco leaves at harvest time) are shown in fig. 16, 17, 18 and 19.NtAAP3A gene-editing mutant,NtAAP6The related change trends of the gene editing mutants are consistent, and specifically:NtAAP3a gene-editing mutant,NtAAP6The chlorophyll a/b, total chlorophyll and carotenoid content of the gene editing mutant are all increased, whereinNtAAP3The chlorophyll a of the mutant is increased by 10.5 percent, the chlorophyll b is increased by 17.5 percent, the total chlorophyll is increased by 12.6 percent, the carotenoid is increased by 6.4 percent,NtAAP6the chlorophyll a of the mutant is increased by 15.8%, the chlorophyll b is increased by 15.4%, the total chlorophyll is increased by 15.7%, and the carotenoid is increased by 21.4%. This indicates that the mutation of the gene, although affecting amino acid transport and protein accumulation, did not reduce the pigment content of tobacco. The appearance and the internal quality of the tobacco leaves can be influenced by the content of the carotenoid, degradation and thermal cracking products of the carotenoid contain a plurality of aroma substances and are closely related to the aroma of the tobacco leaves, and the mutant also ensures the internal and external excellent quality of the tobacco leaves on the basis of reducing the content of amino acid and protein.

Amino acids are very important nitrogen-containing compounds in plants, play a very important role in the processes of growth, development, metabolic regulation and the like of plants, and amino acid transporters play an indispensable role in the process of amino acid transport in plants. Amino Acid Permeases (AAPs), a subfamily of amino acid transporters, are widely involved in the transport and absorption of amino acids in various tissues and organs throughout plant growth and development.

In the present application, through the above-mentioned pairsNtAAP3Gene, gene,NtAAP6The preliminary study of gene function can preliminarily confirm that:

NtAAP3the gene is highly expressed in leaves, and has direct influence on the transfer rate of amino acid after mutation, particularly obviously reduces the reduction of the content of amino acid such as proline, asparagine and the like, and further reduces the content of total protein; on the other hand, in the case of a liquid,NtAAP3the content of pigment substances such as chlorophyll a/b, total chlorophyll, carotenoid and the like in the mutant is obviously increased;

andNtAAP3the genes are similar to each other and can be used,NtAAP6the gene is highly expressed in roots and leaves, has direct influence on the transfer rate of amino acid after being mutated, and particularly obviously reduces the reduction of the content of amino acid such as proline, asparagine and the like, and further leads to the reduction of the content of total protein; on the other hand, in the case of a liquid,NtAAP6the content of pigment substances such as chlorophyll a/b, total chlorophyll, carotenoid and the like in the mutant is obviously increased.

In combination with the results of the relevant assays, will furtherNtAAP3AndNtAAP6the statistics of the changes in some components in the gene editing mutants are shown in table 1 below.

In the context of Table 1, the following examples are,NtAAP3andNtAAP6statistics of changes in Components of Gene editing mutants

Note: the change trends indicated by ↓and ↓ in the table with respect to the control.

By combining the data in the table, the following can be seen visually:NtAAP3andNtAAP6in comparison between the gene-editing mutants,NtAAP3the reduction of total protein and asparagine of the mutant is larger than that of the mutantNtAAP6The mutant is a mutant of a microorganism,NtAAP3respectively isNtAAP62.36 times and 1.08 times of the total weight of the composition; chlorophyll b has higher amplification than that of chlorophyll bNtAAP6The mutant is a mutant of a microorganism,NtAAP3is increased byNtAAP61.14 times of. WhileNtAAP6The proline of the mutant is reduced more thanNtAAP3The mutant is a mutant of a microorganism,NtAAP6is reduced in amplitudeNtAAP31.91 times of; the chlorophyll a, total chlorophyll and carotenoid are increased more thanNtAAP3The mutant is a mutant of a microorganism,NtAAP6respectively isNtAAP31.50 times, 1.25 times and 3.34 times of the total weight of the composition.

These data further show that it is possible to identify,NtAAP3andNtAAP6although genes perform similar functions in some respects, they are somewhat different.

SEQUENCE LISTING

<110> Zhengzhou tobacco institute of China tobacco general Co

<120> application of NtAAP6 gene of tobacco in tobacco

<130> none

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1440

<212> DNA

<213> Nicotiana tabacum

<400> 1

atgggagaaa acaacaacgt tgcttcaaaa caccaagtgt tcgatgtttc cattaatgtg 60

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tcccaaattc cagattttga tcagatttgg tggctttcaa ttgtggctgc cgttatgtct 600

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atcggaggaa gtctcactgg aattagcatc ggaactgtga ctgaaatgca aaagatttgg 720

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gcagcctttg gaaatgatgc tcctggaaat ttccttactg gttttggttt ctatgaacca 1020

ttttggctaa ttgactttgc caacgtttgc attgccgtcc accttgttgg agcttaccag 1080

gttttctgcc aacctttata tgggttcgtg gaggctcgtt gcaacgagcg atggtcagac 1140

agcaaattca tcacctccga gtacgctgtg caagttccat gctgtggcgt ttacaacgtc 1200

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<213> Nicotiana tabacum

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Met Ala Pro Glu Phe Gln Lys Asn Thr Met Tyr Val Ser Thr Glu Leu

1 5 10 15

Glu Arg Gly Asp Val Gln Lys Asn Phe Asp Asp Asp Gly Arg Glu Lys

20 25 30

Arg Thr Gly Thr Leu Leu Thr Ala Ser Ala His Ile Ile Thr Ala Val

35 40 45

Ile Gly Ser Gly Val Leu Ser Leu Ala Trp Ala Ile Ala Gln Leu Gly

50 55 60

Trp Val Ala Gly Pro Ala Val Leu Phe Ala Phe Ser Phe Ile Thr Tyr

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Phe Thr Ser Thr Leu Leu Ala Asp Cys Tyr Arg Ser Pro Gly Pro Ile

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Ser Gly Lys Arg Asn Tyr Thr Tyr Met Asp Val Val Arg Ser His Leu

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Ala Val Lys Arg Ser Asn Cys Phe His Lys His Gly His Glu Ala Ser

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Ile Leu Ala Ala Val Met Ser Phe Thr Tyr Ala Ser Ile Gly Leu Gly

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Leu Ser Ile Ala Lys Ala Ala Gly Val Gly His His Val Lys Thr Ser

210 215 220

Leu Thr Gly Thr Thr Val Gly Val Asp Val Ser Gly Ser Glu Lys Ile

225 230 235 240

Trp Lys Ser Phe Gln Ala Ile Gly Asp Ile Ala Phe Ala Tyr Ala Tyr

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Ser Thr Val Leu Ile Glu Ile Gln Ala Ser Thr Leu Ser Leu Ile Leu

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

275 280 285

Ser Gln Pro Pro Glu Ser Lys Val Met Lys Arg Ala Ser Leu Ala Gly

290 295 300

Val Ser Thr Thr Thr Leu Phe Tyr Ile Leu Cys Gly Thr Ile Gly Tyr

305 310 315 320

Ala Ala Phe Gly Asn Asp Ala Pro Gly Asn Phe Leu Thr Gly Phe Gly

325 330 335

Phe Tyr Glu Pro Phe Trp Leu Ile Asp Phe Ala Asn Val Cys Ile Ala

340 345 350

Val His Leu Val Gly Ala Tyr Gln Val Phe Cys Gln Pro Leu Tyr Gly

355 360 365

Phe Val Glu Ala Arg Cys Asn Glu Arg Trp Ser Asp Ser Lys Phe Ile

370 375 380

Thr Ser Glu Tyr Ala Val Gln Val Pro Cys Cys Gly Val Tyr Asn Val

385 390 395 400

Asn Leu Phe Arg Leu Val Trp Arg Thr Ala Tyr Val Val Val Thr Ala

405 410 415

Val Ile Ala Met Ile Phe Pro Phe Phe Asn Asp Phe Leu Gly Leu Ile

420 425 430

Gly Ala Ala Ser Phe Tyr Pro Leu Thr Val Tyr Phe Pro Ile Glu Met

435 440 445

His Ile Ala Gln Arg Lys Ile Pro Lys Tyr Ser Phe Thr Trp Val Trp

450 455 460

Leu Lys Ile Leu Ser Trp Thr Cys Leu Val Val Ser Leu Val Ala Ala

465 470 475 480

Ala Gly Ser Ile Gln Gly Leu Val Thr Ser Leu Lys His Tyr Lys Pro

485 490 495

Phe Ser Thr Gln Gln

500