CN114989284A - Application of SiPSY1 protein and related biomaterials in regulating and controlling synthesis of millet carotenoids - Google Patents

Abstract

The invention discloses an application of SiPSY1 protein and related biological materials thereof in regulation and control of synthesis of millet carotenoids. The invention utilizes CRISPR/Cas9 gene editing technology and transgenic technology to verify and discover in millet that: the loss of the function of the SiPSY1 gene can cause the content of yellow pigment or carotenoid in the millet seeds to be reduced, and the color of the seeds is whitened; over-expression of the SiPSY1 gene can result in the increase of the content of yellow pigment or carotenoid in millet seeds, the yellowing of the seeds is deepened, and the transparency is increased. The invention discovers that the change of the function of the SiPSY1 gene can cause the change of the content of yellow pigment or carotenoid in millet seeds for the first time, and further can utilize the SiPSY1 gene to regulate the millet color and strengthen the specific expression technology of the SiPSY1 to improve the millet color. The invention has very important application in millet breeding, variety improvement and new germplasm creation.

Description

Application of SiPSY1 protein and related biological materials thereof in regulation and control of synthesis of millet carotenoids

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an application of SiPSY1 protein and related biomaterials thereof in regulation and control of synthesis of millet carotenoids.

Background

The carotenoid is an indispensable nutrient element for human bodies, and has the effects of clearing free radicals in the bodies, delaying the aging of the bodies, enhancing the immunity, resisting cancers and the like; the carotenoid transmits the absorbed light energy to chlorophyll a in the higher plants to assist photosynthesis, and meanwhile, the carotenoid accumulation can enable the higher plants to show rich colors; carotenoids are also important precursors of plant hormones such as abscisic acid and strigolactone, and are closely related to plant growth, so that the enhancement of carotenoid content in crops by a biological enhancement means has important significance.

More than 700 natural carotenoids have been found, and can be classified into two types, namely carotene (the carotene composed of two elements of carbon and hydrogen specifically includes alpha-carotene, beta-carotene, gamma-carotene, lycopene and the like) and lutein (composed of three major elements of carbon, hydrogen and oxygen, and can form various oxygen-containing functional groups such as epoxy group, hydroxyl group, ketone group, carboxyl group, methoxy group and the like, such as lutein, zeaxanthin, violaxanthin, astaxanthin and the like) according to the element composition and physical and chemical properties of the chemical structure.

Millet originates from China, is millet after hulling, and has the characteristics of drought resistance, stress tolerance, wide adaptability, rich nutrition, unique flavor and the like, so that millet becomes the largest coarse cereal crop in China. The quality of millet is an important factor influencing the development of millet industry in China. The millet color, namely the content of the yellow pigment, is an important appearance index and an important nutritional index, and the yellow pigment of the millet is mainly carotenoid such as lutein, zeaxanthin, cryptoxanthin and the like.

The anabolism of the carotenoid is influenced by various factors such as external factors, internal factors and the like, wherein the influence of environmental factors such as illumination, temperature and the like on the carotenoid is obvious; for its own reasons, the expression of related genes involved in carotenoid anabolism, the abundance and activity of proteins regulate the carotenoid content in plants. The biosynthesis and metabolism pathways of carotenoids are relatively conserved, and are widely researched in multiple species such as arabidopsis thaliana, rice, corn, wheat, tomatoes, carrots, oranges and the like at present, but the research on carotenoids in the millet is less, and the research on the synthesis pathways of carotenoids of the millet and the improvement of the content of yellow pigments of the millet have important significance for improving the quality of the millet.

Disclosure of Invention

The invention aims to provide application of SiPSY1 protein in regulating and controlling the anabolism of cereal carotenoid.

In order to achieve the above object, the present invention firstly provides a novel use of the SiPSY1 protein.

The invention provides an application of SiPSY1 protein in any one of the following A1) -A6):

A1) regulating and controlling the content of yellow pigment and/or carotenoid in the millet seeds;

A2) regulating and controlling synthesis and metabolism of yellow pigment and/or carotenoid in the millet seeds;

A3) regulating and controlling the color and/or transparency of the millet seeds;

A4) improving or increasing the quality of the millet;

A5) cultivating transgenic millet;

A6) breeding millet;

the SiPSY1 protein is a protein described in any one of the following (a1) - (a 4):

(a1) a protein shown as SEQ ID No. 3;

(a2) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of the protein of (a 1);

(a3) protein obtained by substituting and/or deleting and/or adding one or more amino acid residues in (a1) and related to the synthesis of millet yellow pigment and/or carotenoid;

(a4) a protein derived from millet and having 98% or more identity to (a1) and involved in millet yellow and/or carotenoid synthesis.

In the protein of (a2), the tag (protein-tag) is a polypeptide or protein expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking or purification of the target protein. Specifically, the tag may be a GFP tag, a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag.

In the protein of (a3), the substitution and/or deletion and/or addition of one or more amino acid residues may be specifically a substitution and/or deletion and/or addition of not more than 10 amino acid residues, a substitution and/or deletion and/or addition of not more than 9 amino acid residues, a substitution and/or deletion and/or addition of not more than 8 amino acid residues, a substitution and/or deletion and/or addition of not more than 7 amino acid residues, a substitution and/or deletion and/or addition of not more than 6 amino acid residues, a substitution and/or deletion and/or addition of not more than 5 amino acid residues, a substitution and/or deletion and/or addition of not more than 4 amino acid residues, or a substitution and/or deletion and/or addition of not more than 3 amino acid residues, or substitution and/or deletion and/or addition of not more than 2 amino acid residues, or substitution and/or deletion and/or addition of not more than 1 amino acid residue.

In the protein according to (a4), the identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

The protein of any one of (a1) to (a4) above may be artificially synthesized, or may be obtained by synthesizing a gene encoding the protein and then performing biological expression.

In order to achieve the above object, the present invention also provides a novel use of the biomaterial related to the SiPSY1 protein described above.

The invention provides application of biological materials related to the SiPSY1 protein in any one of the following A1) -A6):

A1) regulating and controlling the content of yellow pigment and/or carotenoid in the millet seeds;

A2) regulating and controlling synthesis and metabolism of yellow pigment and/or carotenoid in the millet seeds;

A3) regulating and controlling the color and/or transparency of the millet seeds;

A4) improving or increasing the quality of the millet;

A5) cultivating transgenic millet;

A6) breeding millet;

the biological material related to the SiPSY1 protein is a nucleic acid molecule encoding the SiPSY1 protein or an expression cassette, a recombinant vector and a recombinant microorganism containing the nucleic acid molecule.

The nucleic acid molecule is a DNA molecule described in any one of (b1) to (b2) below:

(b1) a DNA molecule shown as SEQ ID No.1 or SEQ ID No. 2;

(b2) a DNA molecule derived from millet and having an identity of 75% or more to (b1) and encoding the SiPSY1 protein.

The nucleotide sequence encoding the SiPSY1 protein of the present invention can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the isolated SiPSY1 nucleotide sequence of the present invention are derived from and identical to the nucleotide sequence of the present invention as long as they encode the SiPSY1 protein and have the same function.

In the nucleic acid molecule according to (b2), the identity refers to sequence similarity with a natural nucleic acid sequence. Identity includes nucleotide sequences that are 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of the present invention encoding the protein consisting of the amino acid sequence shown in SEQ ID No. 3. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

The expression cassette may be DNA capable of expressing SiPSY1 protein in a host cell, which DNA may include not only a promoter that initiates transcription of the SiPSY1 gene, but also a terminator that terminates transcription of the SiPSY1 gene. Further, the expression cassette may also include an enhancer sequence.

The recombinant vector can be a vector containing the DNA molecule shown in SEQ ID No.1 or SEQ ID No.2 and used for coding the SiPSY1 protein. In order to facilitate the identification and screening of the transgenic millet cells or the millet, an expression vector to be used may be processed, for example, a gene encoding an enzyme or a luminescent compound which can produce a color change (GUS gene, luciferase gene, etc.), an antibiotic marker having resistance (gentamicin marker, kanamycin marker, etc.), an anti-chemical agent marker gene (e.g., an anti-herbicide gene), or the like, which can be expressed in the millet, may be added. The recombinant vector can be specifically an SiPSY1 overexpression vector. The SiPSY1 overexpression vector is obtained by removing a stop codon TAG from a CDS sequence of a SiPSY1 gene shown in SEQ ID No.2, inserting the CDS sequence between Kpn I and Spe I enzyme cutting sites of a pCAMBIA-1305 vector, and keeping other sequences of the pCAMBIA-1305 vector unchanged.

The recombinant microorganism can be yeast, bacteria, algae and fungi containing the nucleic acid molecule or the expression cassette or the recombinant vector. The bacterium may in particular be an agrobacterium, such as agrobacterium EHA 105.

In the application a1) -a6), the regulation is improved, and is specifically as follows: when the activity and/or content of SiPSY1 protein in the millet is increased, the yellow pigment content and/or carotenoid content in the millet kernels is increased, the color of the kernels is changed to yellow, and the transparency of the kernels is increased.

The purpose of the millet breeding is to obtain a millet variety with high content of yellow pigment and/or high content of carotenoid or yellow kernel color.

In order to achieve the above object, the present invention also provides a novel use of a substance that inhibits SiPSY 1.

The invention provides an application of a substance inhibiting SiPSY1 in any one of the following B1) -B5):

B1) reducing the yellow pigment content and/or carotenoid content in the millet seeds;

B2) inhibiting synthesis and metabolism of yellow pigment and/or carotenoid in the millet seeds;

B3) whitening the color of the millet seeds;

B4) cultivating transgenic millet;

B5) breeding millet;

the substance for inhibiting SiPSY1 is a substance for inhibiting or reducing the activity and/or content of the SiPSY1 protein in the millet, or a substance for inhibiting the expression of the nucleic acid molecules in the millet, or a substance for knocking out the nucleic acid molecules in the millet.

B1) -B5) in the above application, the substance for inhibiting or reducing the activity and/or content of the SiPSY1 protein in the millet can be any substance capable of reducing or deleting the activity and/or content of the SiPSY1 protein in the millet, such as a protein, polypeptide or small molecule compound (such as a protein activity inhibitor) for inhibiting the synthesis of the SiPSY1 protein, promoting the degradation of the SiPSY1 protein or inhibiting the function of the SiPSY1 protein.

The substance for inhibiting the expression of the nucleic acid molecule encoding the SiPSY1 protein in the millet can be any substance capable of preventing the expression of the SiPSY1 protein encoding gene in the millet, such as a substance (including miRNA, siRNA, dsRNA, shRNA and the like) for silencing the SiPSY1 protein encoding gene in the millet.

The substance encoding the SiPSY1 protein nucleic acid molecule in the knockout millet may be a substance that effects that the host cell does not produce a functional protein product of the SiPSY1 gene in any way, such as removing all or part of the encoding gene sequence, introducing a frameshift mutation such that no functional protein is produced, removing or altering regulatory components (e.g., promoter editing) such that the gene sequence is not transcribed, etc. Typically, the knockout is performed at the genomic DNA level, such that progeny of the cell also permanently carry the knockout.

Further, the substance encoding the SiPSY1 protein nucleic acid molecule in the knockout millet can be a substance editing the SiPSY1 protein encoding gene based on the CRISPR/Cas9 system, such as sgRNA and Cas9 protein, as well as DNA molecules encoding the sgRNA and DNA molecules encoding the Cas9 protein, as well as expression vectors having DNA molecules encoding the sgRNA and DNA molecules encoding the Cas9 protein, or expression vectors having DNA molecules encoding the sgRNA and DNA molecules encoding the Cas9 protein.

Furthermore, the target sequence of the sgRNA is specifically shown as SEQ ID No.4 or SEQ ID No. 5. The expression vector with the DNA molecule encoding the sgRNA and the DNA molecule encoding the Cas9 protein can be specifically a recombinant vector pYLRISPR/Cas 9Pubi-H-SiPSY 1. The recombinant vector pYLCRISPR/Cas9Pubi-H-SiPSY1 is obtained by inserting a DNA molecule shown in SEQ ID No.6 into a Bsa I enzyme digestion site of a pYLCRISPR/Cas9Pubi-H vector and keeping other sequences of the pYLCRISPR/Cas9Pubi-H vector unchanged.

The purpose of the millet breeding is to obtain a millet variety with low yellow pigment content or carotenoid content in grains or white grains.

In order to achieve the purpose, the invention also provides a method for culturing the transgenic millet with the improved content of the yellow pigment and/or the carotenoid.

The method for cultivating transgenic millet with improved yellow pigment content and/or carotenoid content of grains provided by the invention comprises the steps of improving the content and/or activity of SiPSY1 protein in receptor millet to obtain transgenic millet; the transgenic millet kernel has a higher content of yellow pigment and/or carotenoid than the acceptor millet.

Further, the method for increasing the content and/or activity of the SiPSY1 protein in the receptor millet can be to over-express the SiPSY1 protein in the receptor millet.

The overexpression method can be realized by introducing a gene encoding SiPSY1 protein into a receptor millet.

Furthermore, the nucleotide sequence of the SiPSY1 protein coding gene is shown as SEQ ID No.1 or SEQ ID No. 2.

In a specific embodiment of the present invention, the SiPSY1 protein-encoding gene is introduced into a receptor millet via the SiPSY1 overexpression vector described above.

In order to achieve the purpose, the invention also provides a method for culturing the transgenic millet with reduced yellow pigment content and/or carotenoid content.

The method for cultivating the transgenic millet with the reduced content of the yellow pigment and/or the carotenoid comprises the steps of reducing the content and/or activity of SiPSY1 protein in receptor millet to obtain the transgenic millet; the transgenic millet kernel has a lower yellow pigment content and/or carotenoid content than the acceptor millet.

Furthermore, the method for reducing the content and/or the activity of the SiPSY1 protein in the receptor millet is realized by inhibiting or knocking out a gene encoding the SiPSY1 protein in the receptor millet.

Still further, the method for knocking out the gene encoding the SiPSY1 protein in the receptor millet can be used for editing the gene encoding the SiPSY1 protein in the receptor millet based on a CRISPR/Cas9 system.

Still further, the method for editing the SiPSY1 protein encoding gene in the receptor millet based on the CRISPR/Cas9 system can be to introduce a substance for editing the SiPSY1 protein encoding gene based on the CRISPR/Cas9 system into the receptor millet, the substance for editing the SiPSY1 protein encoding gene based on the CRISPR/Cas9 system can be sgRNA and Cas9 protein, a DNA molecule encoding the sgRNA and a DNA molecule encoding the Cas9 protein, and can also be an expression vector having a DNA molecule encoding the sgRNA and an expression vector having a DNA molecule encoding the Cas9 protein, or an expression vector having a DNA molecule encoding the sgRNA and a DNA molecule encoding the Cas9 protein.

In a specific embodiment of the invention, the target sequence of the sgRNA is specifically shown in SEQ ID No.4 or SEQ ID No. 5. The substance for carrying out gene editing on the SiPSY1 protein encoding gene based on the CRISPR/Cas9 system is specifically the recombinant vector pYLCRISPR/Cas9Pubi-H-SiPSY 1.

In order to achieve the above object, the present invention finally provides a method for preparing transgenic millet.

The preparation method of the transgenic millet provided by the invention comprises the following steps: editing the nucleic acid molecules in the receptor millet to obtain a gene-edited millet; the yellow pigment content and/or carotenoid content in the genetically edited millet kernel is lower than that of the acceptor millet.

Further, the method for editing the nucleic acid molecule in the receptor millet can be the method for editing the gene encoding the SiPSY1 protein in the receptor millet based on the CRISPR/Cas9 system.

Further, the gene-edited millet can be SiPSY1 knockout millet homozygous editing lines SiPSY1#1, SiPSY1#8 and SiPSY1# 11.

The SiPSY1 knockout millet homozygous editing line, SiPSY1#1 and the wild-type millet Ci846 only differ in that a deletion of a base T is generated on a gene coding for SiPSY1 protein, and the deletion base T is positioned at the 571 st position of a coding region of the SiPSY1 gene shown in SEQ ID No. 2.

The SiPSY1 knockout millet homozygous editing line, SiPSY1#8 and the wild-type millet Ci846 only have the difference that a 39bp fragment deletion is generated on a gene coding the SiPSY1 protein, and the 39bp deletion fragment is positioned at 85-123 th positions of a coding region of the SiPSY1 gene shown in SEQ ID No. 2.

The SiPSY1 knockout millet homozygous editing line, SiPSY1#11 and the wild-type millet Ci846 only differ in that the insertion of a base T occurs on a gene encoding the SiPSY1 protein, and the insertion position of the base T is between the 116 th position and the 117 th position of a coding region of the SiPSY1 gene shown in SEQ ID No. 2.

In any of the above methods, the introduction may be specifically to transform the millet cells or tissues by using a conventional biological method such as Ti plasmid, Ri plasmid, plant viral vector, direct DNA transformation, microinjection, conductance, agrobacterium-mediated transformation, etc., and to culture the transformed millet tissues into plants.

In any of the uses or methods described above, the yellow pigment or the carotenoid comprises a carotene and a lutein. Further, the carotene may be beta-carotene. The xanthophylls may be lutein and zeaxanthin.

In any of the above applications or methods, the foxtail millet variety may specifically be Ci 846.

The invention verifies that SiPSY1 gene has the function of regulating and controlling the anabolism of millet yellow pigment or carotenoid by using CRISPR/Cas9 gene editing technology and transgenic technology, and the specific expression is as follows: the loss of the gene function can cause the content of yellow pigment or carotenoid in the millet seeds to be reduced, and the color of the seeds to be whitened; the overexpression of the gene can lead to the increase of the content of yellow pigment or carotenoid in millet seeds, the yellowing of the seeds is deepened, and the transparency is increased. The invention discovers that the change of the function of the SiPSY1 gene can cause the change of the content of yellow pigment or carotenoid in millet seeds for the first time, and further can utilize the SiPSY1 gene to regulate the millet color and strengthen the specific expression technology of the SiPSY1 to improve the millet color.

Drawings

FIG. 1 is a schematic structural diagram of 2 sgRNA expression cassettes containing different SiPSY1 targets and a schematic structural diagram of a SiPSY1 knockout vector pYLRISPR/Cas 9Pubi-H-SiPSY 1.

FIG. 2 shows the structural and target sequence alignment of SiPSY1 gene in wild millet Ci846 and SiPSY1 knockout millet homozygous editing lines.

FIG. 3 shows the SiPSY1 expression level detection of SiPSY1 overexpression millet OE13 strain.

FIG. 4 shows the kernel color of wild-type millet Ci846 and SiPSY1 knockout millet homozygous editing lines SiPSY1#1, SiPSY1#8 and SiPSY1# 11.

FIG. 5 shows carotenoid contents of wild-type millet Ci846 and SiPSY1 knockout millet homozygous editing lines SiPSY1#1, SiPSY1#8 and SiPSY1# 11.

FIG. 6 shows the grain color and carotenoid content of wild-type millet Ci846 and SiPSY1 lines overexpressing millet OE 13.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Wild-type millet Ci846 in the examples described below is described in the literature "A transsson insulation drop the loss of natural seed cut shock and fat cereal mile viscosity. mol Biol Evol.2022Apr 7: msac078.doi:10.1093/molbev/msac078. epipb ahead of PMID:35388422.

Example 1 use of SiPSY1 protein in regulating the synthesis of millet carotenoids

The amino acid sequence of the SiPSY1 protein is shown in SEQ ID No. 3. In the cDNA of wild-type millet Ci846, the CDS sequence encoding the SiPSY1 protein is shown in SEQ ID No.2, and in the genomic sequence of wild-type millet Ci846, the genomic sequence encoding the SiPSY1 protein is shown in SEQ ID No. 1.

First, SiPSY1 knockout millet acquisition

1. Construction of SiPSY1 knockout vectors

1) Design of SiPSY1 knockout target

According to the SiPSY1 gene sequence, designing a target by utilizing an online target design website CRISPR-P2.0 (http:// CRISPR. hzau. edu. cn/CRISPR2/), selecting a proper target according to the position of the target and the targeting specificity, and finally screening to obtain the following two SiPSY1 gene knockout targets: target 1 and target 2. The specific sequences of target 1 and target 2 are as follows:

target 1: 5'-CTGGATGCTCTGCTCGCTC-3' (SEQ ID No.4)

Target 2: 5'-AGACTTGAGGATCTATTCGA-3' (SEQ ID No. 5).

2) Construction of SiPSY1 knockout vectors

The DNA molecule shown in SEQ ID No.6 was inserted into the Bsa I cleavage site of pYRCISPR/Cas 9Pubi-H vector (Ma et al, A Robust CRISPR/Cas9 System for convention, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants,2015, Molecular Plant), and the other sequences of pYRCISPR/Cas 9Pubi-H vector were kept unchanged, to obtain SiPSY1 knock-out vector pYRCISPR/Cas 9Pubi-H-SiPSY 1. The SiPSY1 knockout vector pYLCRISPR/Cas9Pubi-H-SiPSY1 contains 2 sgRNA expression cassettes which respectively target a target point 1 and a target point 2, and the structural schematic diagram is shown in FIG. 1A; a schematic structure of the SiPSY1 knock-out vector pYRCISPR/Cas 9Pubi-H-SiPSY1 is shown in FIG. 1B.

2. Obtaining and identifying SiPSY1 knockout millet

Introducing the SiPSY1 knockout vector with correct sequencing into the agrobacterium EHA105 to obtain a recombinant strain; wild millet Ci846 young embryo callus is transformed by using an agrobacterium-mediated genetic transformation method to obtain T0 generation transgenic plants.

DNA detection is carried out on T0 transgenic plants by primers 5'-AGTCATCCACCCATCCC-3' and 5'-TCCTACAATACCACCCTTT-3', the editing mode of the transgenic plants is determined, T1 seeds are obtained, sowing is carried out in a greenhouse, the T1 transgenic plants are amplified and sequenced again, whether the editing mode is stable inheritance or not and pure heterozygosity of editing sites of the plants is detected, and finally 3 SiPSY1 knockout millet homozygous editing strains (namely mutations of two chromosomes are consistent) are obtained and are named as SiPSY1#1, SiPSY1#8 and SiPSY1#11 respectively.

Compared with wild-type millet Ci846, SiPSY1#1 only differs in that a deletion of one base T occurs in the gene coding for the SiPSY1 protein (in exon 3), the deletion base T is located at position 571 of the coding region of the SiPSY1 gene shown in SEQ ID No.2, thereby causing frame shift and leading to premature termination of protein translation, and function of the SiPSY1 protein is deleted. The sequence alignment scheme of SiPSY1 of the wild-type millet Ci846 and SiPSY1 knockout millet homozygous editing line SiPSY1#1 is shown in FIGS. 2B and 2C.

Compared with wild-type millet Ci846, SiPSY1#8 only differs in that a 39bp (TGCTCCTCCTCTGCTCAAGGCGGCGCCGCTGGATGCTC) fragment deletion occurs on the gene coding the SiPSY1 protein (on exon 1), the 39bp deletion fragment is positioned at positions 85-123 of the coding region of the SiPSY1 gene shown in SEQ ID No.2, so that the amino acid sequence of the SiPSY1 protein is deleted from amino acids 29-41 (CSSLLKRRRRWML), and the function of the SiPSY1 protein is deleted. The sequence alignment scheme of SiPSY1 of the wild-type millet Ci846 and SiPSY1 knockout millet homozygous editing line SiPSY1#8 is shown in FIGS. 2B and 2C.

Compared with wild-type millet Ci846, SiPSY1#11 only differs in that an insertion of one base T occurs on the gene encoding the SiPSY1 protein (on exon 1) between position 116 and position 117 of the coding region of the SiPSY1 gene shown in SEQ ID No.2, thereby causing a frame shift and leading to premature termination of protein translation, and the function of the SiPSY1 protein is deleted. Sequence alignment schemes of SiPSY1 of wild-type millet Ci846 and SiPSY1 knockout millet homozygous editing line SiPSY1#11 are shown in fig. 2B and 2C.

II, obtaining the over-expressed millet of SiPSY1

1. Construction of SiPSY1 overexpression vector

The CDS sequence of the SiPSY1 gene shown in SEQ ID No.2 is inserted between Kpn I and Spe I enzyme cutting sites of a pCAMBIA-1305 vector (the nucleotide sequence of the vector is shown in SEQ ID No.7, and the vector carries a GFP TAG protein sequence), and other sequences of the pCAMBIA-1305 vector are kept unchanged, so that the SiPSY1 overexpression vector is obtained. The SiPSY1 overexpression vector expresses a fusion protein of SiPSY1 and GFP, wherein the GFP is used for detecting whether the vector is successfully transferred into a plant.

2. Obtaining and identifying SiPSY1 over-expression millet

Introducing the SiPSY1 overexpression vector into agrobacterium EHA105 to obtain a recombinant strain; wild millet Ci846 young embryo callus is transformed by using an agrobacterium-mediated genetic transformation method to obtain a transgenic plant.

PCR identification is carried out on the transgenic plant by using the primers 5'-CCCTGCCTTCATACGCT-3' and 5'-AAGATGGTGCGCTCCTG-3', and the transgenic plant with 1698bp bands obtained by amplification is a transgenic positive plant.

3. SiPSY1 overexpression plant expression level detection

The expression level of SiPSY1 in transgenic positive plants and wild foxtail Ci846 is detected, and the specific steps are as follows:

1) RNA extraction

Taking transgenic positive plant leaves, sampling, putting the leaves into prepared tin foil paper, and putting the leaves into liquid nitrogen, wherein the speed of the whole sampling process is high, and RNA degradation in a sample is avoided. RNA extraction adopts Trizol method, instruments (a mortar, a grinding rod, a pair of tweezers, scissors and the like) used in the experiment are sprayed and poured with 95% ethanol, combustion is carried out, RNase is removed, and all instruments are immersed into liquid nitrogen for precooling after combustion and cooling. The test bench was wiped clean with 75% alcohol and then the bench was wiped with RNase Zap to remove RNase from the bench. In the whole operation process, gloves and masks are needed to be worn, and RNA degradation by RNase is avoided as much as possible.

2) Reverse transcription of RNA

RNA was inverted into cDNA using the Prime Script II 1st strand cDNA Synthesis Kit (Catno.6210A, Takara, OtsuShiga, Japan) from TaKaRa, and the test bed was wiped with alcohol and sprayed with RNase Zap, with a mask and gloves, before operation. The operation steps are that sample adding is carried out according to the system specification in the kit, thus obtaining cDNA, the cDNA is diluted by 10 times to be working solution, and the working solution is preserved at the temperature of minus 20 ℃.

3) Fluorescent quantitative PCR

qRT-PCR was performed using cDNA as template using primers qPSY1-F (5'-AGGCAACAACTGAAAGCGTGTA-3') and qPSY1-R (5'-AGATGTCCTCATCAGAGAGCCC-3') and Taq Pro Universal SYBR qPCR Master Mix reagent (Vazyme, Catno. Q712-02), and the experiment was performed by configuring a qRT-PCR reaction system according to the instructions, and performing 4 independent replicates for each cDNA sample. The qRT-PCR reaction system was configured and added to a qRT-PCR96 well plate and the qRT-PCR reaction program was run using an Applied Bio systems 7300analyzer fluorescent quantitative PCR instrument. The qRT-PCR reaction program was: pre-denaturation at 95 deg.C for 2min, then 40 cycles at 95 deg.C for 1min, 55 deg.C for 1min, 72 deg.C for 1min, and finally extension at 72 deg.C for 7 min. Finally, EXCEl is used for calculating the relative expression of SiPSY1 in each test plant.

The results are shown in FIG. 3, which shows: the relative expression amount of SiPSY1 in the strain of the SiPSY1 overexpression millet OE13 is 4.5 times of that of wild millet Ci 846.

Application of SiPSY1 in regulation and control of cereal carotenoid anabolism

Test plants: wild type foxtail millet Ci 846; SiPSY1 knockout millet homozygous editing strains SiPSY1#1, SiPSY1#8 and SiPSY1# 11; SiPSY1 overexpresses the millet OE13 strain.

Observing the color of the test plant grains, and detecting the carotenoid component content in the test plant grains by adopting a liquid chromatography tandem mass spectrometry (LC-MS/MS) technology. Each material is selected for 3 biological repetitions, samples are guaranteed to be normally grouted and mature, and the carotenoid content change is detected after the sampled material seeds are peeled. Data were processed using the SPSS diagnostics 20.0 software and the results were expressed as mean ± standard deviation, with P < 0.05 (. star) indicating significant differences and P < 0.001 (. star) indicating very significant differences. The data acquisition instrument system for Liquid Chromatography tandem mass spectrometry (LC-MS/MS) detection mainly comprises Ultra Performance Liquid Chromatography (UPLC) (ExionLC) ^TM AD, https:// science.com.cn /) and Tandem Mass Spectrometry (MS/MS) ((MS/MS)

6500+, https:// sciex. com. cn /). The liquid phase conditions detected by liquid chromatography tandem mass spectrometry (LC-MS/MS) are as follows:

1) column YMC C30(3 μm, 100 mm. times.2.0 mm i.d.);

2) mobile phase A, methanol/acetonitrile (1:3, v/v) with 0.01% BHT and 0.1% formic acid; adding 0.01 percent of BHT into the methyl tert-butyl ether in the phase B;

3) gradient elution procedure, 0min A/B is 100:0(V/V), 3min is 100:0(V/V), 5min is 30:70(V/V), 9min is 5:95(V/V), 10min is 100:0(V/V), and 11min is 100:0 (V/V);

4) the flow rate is 0.8 mL/min; the column temperature is 28 ℃; the sample size was 2. mu.L.

The results of grain color and carotenoid content detection of wild-type millet Ci846 and SiPSY1 knockout millet homozygous editing lines are shown in FIGS. 4-5. Compared with wild millet Ci846, the kernels of SiPSY1 knockout millet homozygous editing lines, i.e., the SiPSY1#1, the SiPSY1#8 and the SiPSY1#11, are white in color, and the yellow pigment is remarkably reduced. The contents of lutein, zeaxanthin and beta-carotene in grains of wild-type millet Ci846 and 3 SiPSY1 knockout millet homozygous editing lines SiPSY1#1, SiPSY1#8 and SiPSY1#11 are measured at the same time, and the contents of lutein in grains of wild-type millet Ci846 and 3 SiPSY1 knockout millet homozygous editing lines SiPSY1#1, SiPSY1#8 and SiPSY1#11 are respectively 9.375 mu g/g, 2.493 mu g/g, 2.331 mu g/g and 1.821 mu g/g; the zeaxanthin content of grains of wild type millet Ci846 and 3 SiPSY1 knockout millet homozygous editing lines SiPSY1#1, SiPSY1#8 and SiPSY1#11 is 1.474 mu g/g, 0.542 mu g/g, 0.414 mu g/g and 0.331 mu g/g respectively; the beta-carotene contents in seeds of wild-type millet Ci846 and 3 SiPSY1 knockout millet homozygous editing lines, namely, SiPSY1#1, SiPSY1#8 and SiPSY1#11 are respectively 0.102 mu g/g, 0.053 mu g/g, 0.070 mu g/g and 0.056 mu g/g. It can be seen that: the lutein and zeaxanthin contents in 3 SiPSY1 knockout millet homozygous editing lines, namely, SiPSY1#1, SiPSY1#8 and SiPSY1#11 grains are extremely lower than those of wild-type millet Ci846, wherein the lutein contents are respectively reduced by 73.4%, 67.5% and 74.8%, the zeaxanthin contents are respectively reduced by 63.2%, 61.6% and 69.4%, and the beta-carotene content is also obviously reduced. The above results indicate that SiPSY1 knock-out results in a reduction in the amount of millet carotenoids, eventually leading to a whitening of the rice color.

The results of the color and carotenoid content detection of the kernels of the over-expressed foxsy 1 millet OE13 strain are shown in fig. 6. Compared with the wild millet Ci846, the kernel of the SiPSY1 overexpression millet OE13 strain has the advantages of deeper yellowing and higher transparency. Meanwhile, the contents of lutein, zeaxanthin and beta-carotene in seeds of wild-type millet Ci846 and SiPSY1 overexpression millet OE13 strains are measured, the contents of lutein in the seeds of the wild-type millet Ci846 and SiPSY1 overexpression millet OE13 strains are respectively 9.375 mu g/g and 14.107 mu g/g, the contents of zeaxanthin in the seeds of the wild-type millet Ci846 and SiPSY1 overexpression millet OE13 strains are respectively 1.474 mu g/g and 1.688 mu g/g, and the contents of beta-carotene in the seeds of the wild-type millet Ci846 and SiPSY1 overexpression millet OE13 strains are respectively 0.102 mu g/g and 0.121 mu g/g. It can be seen that: compared with wild foxtail millet Ci846, the contents of lutein and zeaxanthin in kernels of SiPSY1 over-expressed foxtail millet OE13 strains are remarkably increased and increased by 1.5 times and 1.17 times respectively. The above results indicate that SiPSY1 overexpression results in increased millet carotenoid content, a deepened beige yellowing and an increased transparency.

In conclusion, the SiPSY1 protein and the encoding gene thereof can regulate the anabolism of the millet carotenoid, so as to regulate the color of millet grains and influence the quality of the millet.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is made possible within the scope of the claims attached below.

Sequence listing

<110> institute of crop science of Chinese academy of agricultural sciences

Application of <120> SiPSY1 protein and related biological materials thereof in regulation and control of synthesis of millet carotenoids

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 2916

<212> DNA

<213> Artificial Sequence

<400> 1

ataacaacag gcgacagaca catggccatc atccttgtac gagcagcgtc gcccgggctc 60

tccgacgccg cccaccatgg gagcgggagc gggagcctcg gccagtgctc ctccctgctc 120

aagaggcggc gccgctggat gctctgctcg ctccggtacg cctgcctcgg cctgggcccg 180

ggggagaggg agattggccg ggcccccgcc gtgtactcca gcctggccgt aaacccggcg 240

ggagaggccg tcgtctcctc cgagcagaag gtctacgacg tcgtgatgaa gcaggccgca 300

ttgctcaagc gccagctgcg cacgcccgtc cctcttgacg tcagcaggcc ccgggacctg 360

gagatgccgc gcaacggcct cagggaggcc tacgcccgct gcggagagat ctgcgaggag 420

tatgccaaga cgttttacct cggtatgctg ccgtagctct tccatcactt tgttcctctt 480

gatcacatga caaattaaat ggcacgctcg tcatttgatt tcattcatac atgtctgcca 540

ggaactttgc tgatgacaga ggagcggcgc cgcgccatat gggccatcta tggtatggta 600

tctctcaaag tgacaactgt tccatataat gcttcaattc cctagtagta cagtatgcta 660

cttctcttca ccgtaatcaa tattatcatc atctaataag tacgcctaaa acttcatgca 720

ccgtgcgcgg gcctcccggg cctcccgtgc tgcaataaca aagaaaaaat catgctttta 780

aagtaatggt gccaactaac tagctctcgg tgggaattaa cattggaaag gaagcattgt 840

tgagttccaa tggcagtatc tgtgggagca ctaaaatatg ctttcggtag cctaaaacgc 900

tactaaatgg cttcatgaat actttaagac catgtggaaa gttatgagaa catatatata 960

ttcttcacgc ttcaaactac cttagggata tgaatctcaa gtgttggcaa ttagttttaa 1020

atgacctact tcaggtatga ccaaggaaat ctgatgggta gtgctttctg cagtgtggtg 1080

taggaggacg gatgagctcg tggatgggcc aaacgccaac tacattacgc cgacagcctt 1140

ggaccggtgg gagaagagac ttgaggatct attcgaggga cgcccttacg atatgcttga 1200

tgccgctctt tctgatacca tctcaaggtt ccccatagac attcaggtag tagctgctag 1260

actacacttg ttgctattcc tgatataatt attagcatta gactcccttg gcctcacaat 1320

tttagtgcaa gatgactaga cctactaagt tgacaagaac aaaatcatgt aattgcctgg 1380

gtaaaataaa aaaggaccag tagagtcatt gggtcaaagc tgtttaaata aaagggtggt 1440

attgtaggat attttagaat gaactatttc tagaggaaat cttggatcta gaggcaatcg 1500

ttcggaattt aattatatgg catttggtaa tttgacatta tcaattggtc tcacctttta 1560

ataaacaaaa ggtaactata tgaccaacac aagaacagat ataagaatct gcataatgat 1620

ctaaaaggaa aaggttcagc caagtaaagt gacttgaaaa ttacatgtgt tacttagtgg 1680

tttgacaaat tatgttgtac ataccaatat tctaacatca ctaactctta tgcaatgcac 1740

gtatgtggca gcccttcagg gacatgattg aagggatgcg gagcgacctt agaaaggcaa 1800

ggtataagaa ctttgacgag ctctacatgt actgctacta tgttgctgga actgtgggat 1860

taatgagtgt acctgtgatg ggcatcgcac ctgagtctaa ggcaacaact gaaagcgtgt 1920

acagtgctgc cttggctctt ggaattgcga accaactcac aaatatacta cgggatgtag 1980

gagaggagta agtaaccaat tcctttcaag ttcttgctag aacgaggcat ggacgcgtaa 2040

aaatagtata tggctctaaa catttacact gtggcttttt catttttctt ttagtgctag 2100

aagaggaagg gtgtatttac cacaagatga actggcacag gcagggctct ctgatgagga 2160

catcctcaat ggtgtcgtca caaaccgatg gagaaaattc atgaagcagc agatcaagag 2220

ggccaggatg ttttttgagg aggcggaaag aggggtgact gagctctcac aagctagcag 2280

atggccagta agttgaaggg cttggcactg aacagcagaa caccctcttc ttcttcttta 2340

gcatatatag tgaaagggtt tcctttttcc attgcaggta tgggcctcac tactgttgta 2400

ccggcaaatc ctggatgaga ttgaagcaaa cgactacaac aacttcacaa agagggcgta 2460

tgtaggcaaa gggaagaaat tgctagcact gcctgttgca tacgggaaat cgctactgct 2520

gccatgctca ctgagaaata gccagaccta gccgccagaa gaaacaggac aagatgatca 2580

ggttaggcta ggtagaaaat agggcaatgt ctggtggagt tgttgttcag gtgtatcata 2640

tgaaatgttg ggggcaggca ccgataaagc actgcaacca acctagatat gatggatatg 2700

gaatgcttgc ctgtatatcc tcttaggagc atatgcatgt ctctgaagga aagaagcaac 2760

atgtatactg ttttctttgt tatatcatcc caaacacaga ttggggggga aacacaactg 2820

atgttgttct gtaaatggca atgctaaatg ttgtcgtata ggaagcttgt ggaattgaaa 2880

tatagaattt ctgatggtgt tgatcgagat tgaaac 2916

<210> 2

<211> 1248

<212> DNA

<213> Artificial Sequence

<400> 2

atggccatca tccttgtacg agcagcgtcg cccgggctct ccgacgccgc ccaccatggg 60

agcgggagcg ggagcctcgg ccagtgctcc tccctgctca agaggcggcg ccgctggatg 120

ctctgctcgc tccggtacgc ctgcctcggc ctgggcccgg gggagaggga gattggccgg 180

gcccccgccg tgtactccag cctggccgta aacccggcgg gagaggccgt cgtctcctcc 240

gagcagaagg tctacgacgt cgtgatgaag caggccgcat tgctcaagcg ccagctgcgc 300

acgcccgtcc ctcttgacgt cagcaggccc cgggacctgg agatgccgcg caacggcctc 360

agggaggcct acgcccgctg cggagagatc tgcgaggagt atgccaagac gttttacctc 420

ggaactttgc tgatgacaga ggagcggcgc cgcgccatat gggccatcta tgtgtggtgt 480

aggaggacgg atgagctcgt ggatgggcca aacgccaact acattacgcc gacagccttg 540

gaccggtggg agaagagact tgaggatcta ttcgagggac gcccttacga tatgcttgat 600

gccgctcttt ctgataccat ctcaaggttc cccatagaca ttcagccctt cagggacatg 660

attgaaggga tgcggagcga ccttagaaag gcaaggtata agaactttga cgagctctac 720

atgtactgct actatgttgc tggaactgtg ggattaatga gtgtacctgt gatgggcatc 780

gcacctgagt ctaaggcaac aactgaaagc gtgtacagtg ctgccttggc tcttggaatt 840

gcgaaccaac tcacaaatat actacgggat gtaggagagg atgctagaag aggaagggtg 900

tatttaccac aagatgaact ggcacaggca gggctctctg atgaggacat cctcaatggt 960

gtcgtcacaa accgatggag aaaattcatg aagcagcaga tcaagagggc caggatgttt 1020

tttgaggagg cggaaagagg ggtgactgag ctctcacaag ctagcagatg gccagtatgg 1080

gcctcactac tgttgtaccg gcaaatcctg gatgagattg aagcaaacga ctacaacaac 1140

ttcacaaaga gggcgtatgt aggcaaaggg aagaaattgc tagcactgcc tgttgcatac 1200

gggaaatcgc tactgctgcc atgctcactg agaaatagcc agacctag 1248

<210> 3

<211> 415

<212> PRT

<213> Artificial Sequence

<400> 3

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

1 5 10 15

Ala His His Gly Ser Gly Ser Gly Ser Leu Gly Gln Cys Ser Ser Leu

20 25 30

Leu Lys Arg Arg Arg Arg Trp Met Leu Cys Ser Leu Arg Tyr Ala Cys

35 40 45

Leu Gly Leu Gly Pro Gly Glu Arg Glu Ile Gly Arg Ala Pro Ala Val

50 55 60

Tyr Ser Ser Leu Ala Val Asn Pro Ala Gly Glu Ala Val Val Ser Ser

65 70 75 80

Glu Gln Lys Val Tyr Asp Val Val Met Lys Gln Ala Ala Leu Leu Lys

85 90 95

Arg Gln Leu Arg Thr Pro Val Pro Leu Asp Val Ser Arg Pro Arg Asp

100 105 110

Leu Glu Met Pro Arg Asn Gly Leu Arg Glu Ala Tyr Ala Arg Cys Gly

115 120 125

Glu Ile Cys Glu Glu Tyr Ala Lys Thr Phe Tyr Leu Gly Thr Leu Leu

130 135 140

Met Thr Glu Glu Arg Arg Arg Ala Ile Trp Ala Ile Tyr Val Trp Cys

145 150 155 160

Arg Arg Thr Asp Glu Leu Val Asp Gly Pro Asn Ala Asn Tyr Ile Thr

165 170 175

Pro Thr Ala Leu Asp Arg Trp Glu Lys Arg Leu Glu Asp Leu Phe Glu

180 185 190

Gly Arg Pro Tyr Asp Met Leu Asp Ala Ala Leu Ser Asp Thr Ile Ser

195 200 205

Arg Phe Pro Ile Asp Ile Gln Pro Phe Arg Asp Met Ile Glu Gly Met

210 215 220

Arg Ser Asp Leu Arg Lys Ala Arg Tyr Lys Asn Phe Asp Glu Leu Tyr

225 230 235 240

Met Tyr Cys Tyr Tyr Val Ala Gly Thr Val Gly Leu Met Ser Val Pro

245 250 255

Val Met Gly Ile Ala Pro Glu Ser Lys Ala Thr Thr Glu Ser Val Tyr

260 265 270

Ser Ala Ala Leu Ala Leu Gly Ile Ala Asn Gln Leu Thr Asn Ile Leu

275 280 285

Arg Asp Val Gly Glu Asp Ala Arg Arg Gly Arg Val Tyr Leu Pro Gln

290 295 300

Asp Glu Leu Ala Gln Ala Gly Leu Ser Asp Glu Asp Ile Leu Asn Gly

305 310 315 320

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

325 330 335

Ala Arg Met Phe Phe Glu Glu Ala Glu Arg Gly Val Thr Glu Leu Ser

340 345 350

Gln Ala Ser Arg Trp Pro Val Trp Ala Ser Leu Leu Leu Tyr Arg Gln

355 360 365

Ile Leu Asp Glu Ile Glu Ala Asn Asp Tyr Asn Asn Phe Thr Lys Arg

370 375 380

Ala Tyr Val Gly Lys Gly Lys Lys Leu Leu Ala Leu Pro Val Ala Tyr

385 390 395 400

Gly Lys Ser Leu Leu Leu Pro Cys Ser Leu Arg Asn Ser Gln Thr

405 410 415

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 4

ctggatgctc tgctcgctc 19

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 5

agacttgagg atctattcga 20

<210> 6

<211> 1086

<212> DNA

<213> Artificial Sequence

<400> 6

gaatcggcag caaaggattt tttcctgtag ttttcccaca accatttttt accatccgaa 60

tgataggata ggaaaaatat ccaagtgaac agtattccta taaaattccc gtaaaaagcc 120

tgcaatccga atgagccctg aagtctgaac tagccggtca cctgtacagg ctatcgagat 180

gccatacaag agacggtagt aggaactagg aagacgatgg ttgattcgtc aggcgaaatc 240

gtcgtcctgc agtcgcatct atgggcctgg acggaatagg ggaaaaagtt ggccggatag 300

gagggaaagg cccaggtgct tacgtgcgag gtaggcctgg gctctcagca cttcgattcg 360

ttggcaccgg ggtaggatgc aatagagagc aacgtttagt accacctcgc ttagctagag 420

caaactggac tgccttatat gcgcgggtgc tggcttggct gccgctggat gctctgctcg 480

ctcgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa 540

agtggcaccg agtcggtgct ttttttcaag agcttggagt ggatcaggga gcggataaca 600

atttcacaca ggcacgaatc ggcagcaaag gatgcaagaa cgaactaagc cggacaaaaa 660

aaaaaggagc acatatacaa accggtttta ttcatgaatg gtcacgatgg atgatggggc 720

tcagacttga gctacgaggc cgcaggcgag agaagcctag tgtgctctct gcttgtttgg 780

gccgtaacgg aggatacggc cgacgagcgt gtactaccgc gcgggatgcc gctgggcgct 840

gcgggggccg ttggatgggg atcggtgggt cgcgggagcg ttgaggggag acaggtttag 900

taccacctcg cctaccgaac aatgaagaac ccaccttata accccgcgcg ctgccgcttg 960

tgttgagact tgaggatcta ttcgagtttt agagctagaa atagcaagtt aaaataaggc 1020

tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttca agagcttgga 1080

gtggat 1086

<210> 7

<211> 13935

<212> DNA

<213> Artificial Sequence

<400> 7

aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc 60

gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca 120

ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa 180

caatttcaca caggaaacag ctatgaccat gattacgaat tcgagctcgt gcagtgcagc 240

gtgacccggt cgtgcccctc tctagagata atgagcattg catgtctaag ttataaaaaa 300

ttaccacata ttttttttgt cacacttgtt tgaagtgcag tttatctatc tttatacata 360

tatttaaact ttactctacg aataatataa tctatagtac tacaataata tcagtgtttt 420

agagaatcat ataaatgaac agttagacat ggtctaaagg acaattgagt attttgacaa 480

caggactcta cagttttatc tttttagtgt gcatgtgttc tccttttttt ttgcaaatag 540

cttcacctat ataatacttc atccatttta ttagtacatc catttagggt ttagggttaa 600

tggtttttat agactaattt ttttagtaca tctattttat tctattttag cctctaaatt 660

aagaaaacta aaactctatt ttagtttttt tatttaataa tttagatata aaatagaata 720

aaataaagtg actaaaaatt aaacaaatac cctttaagaa attaaaaaaa ctaaggaaac 780

atttttcttg tttcgagtag ataatgccag cctgttaaac gccgtcgacg agtctaacgg 840

acaccaacca gcgaaccagc agcgtcgcgt cgggccaagc gaagcagacg gcacggcatc 900

tctgtcgctg cctctggacc cctctcgaga gttccgctcc accgttggac ttgctccgct 960

gtcggcatcc agaaattgcg tggcggagcg gcagacgtga gccggcacgg caggcggcct 1020

cctcctcctc tcacggcacc ggcagctacg ggggattcct ttcccaccgc tccttcgctt 1080

tcccttcctc gcccgccgta ataaatagac accccctcca caccctcttt ccccaacctc 1140

gtgttgttcg gagcgcacac acacacaacc agatctcccc caaatccacc cgtcggcacc 1200

tccgcttcaa ggtacgccgc tcgtcctccc cccccccccc tctctacctt ctctagatcg 1260

gcgttccggt ccatggttag ggcccggtag ttctacttct gttcatgttt gtgttagatc 1320

cgtgtttgtg ttagatccgt gctgctagcg ttcgtacacg gatgcgacct gtacgtcaga 1380

cacgttctga ttgctaactt gccagtgttt ctctttgggg aatcctggga tggctctagc 1440

cgttccgcag acgggatcga tttcatgatt ttttttgttt cgttgcatag ggtttggttt 1500

gcccttttcc tttatttcaa tatatgccgt gcacttgttt gtcgggtcat cttttcatgc 1560

ttttttttgt cttggttgtg atgatgtggt ctggttgggc ggtcgttcta gatcggagta 1620

gaattctgtt tcaaactacc tggtggattt attaattttg gatctgtatg tgtgtgccat 1680

acatattcat agttacgaat tgaagatgat ggatggaaat atcgatctag gataggtata 1740

catgttgatg cgggttttac tgatgcatat acagagatgc tttttgttcg cttggttgtg 1800

atgatgtggt gtggttgggc ggtcgttcat tcgttctaga tcggagtaga atactgtttc 1860

aaactacctg gtgtatttat taattttgga actgtatgtg tgtgtcatac atcttcatag 1920

ttacgagttt aagatggatg gaaatatcga tctaggatag gtatacatgt tgatgtgggt 1980

tttactgatg catatacatg atggcatatg cagcatctat tcatatgctc taaccttgag 2040

tacctatcta ttataataaa caagtatgtt ttataattat tttgatcttg atatacttgg 2100

atgatggcat atgcagcagc tatatgtgga tttttttagc cctgccttca tacgctattt 2160

atttgcttgg tactgtttct tttgtcgatg ctcaccctgt tgtttggtgt tacttctgca 2220

ctaggtacct gcaactagtc ttaagtccgg agctagctct agagacgtct cgaggaccgg 2280

tcccggggga tccatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct 2340

ggtcgagctg gacggcgacg tgaacggcca caagttcagc gtgtccggcg agggcgaggg 2400

cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt 2460

gccctggccc accctcgtga ccaccttcac ctacggcgtg cagtgcttca gccgctaccc 2520

cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga 2580

gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga 2640

gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa 2700

catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga 2760

caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag 2820

cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct 2880

gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg 2940

cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactcacg gcatggacga 3000

gctgtacaga tcttaaagcg gccgcccggc tgcagttcaa acatttggca ataaagtttc 3060

ttaagattga atcctgttgc cggtcttgcg atgattatca tataatttct gttgaattac 3120

gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg ggtttttatg 3180

attagagtcc cgcaattata catttaatac gcgatagaaa acaaaatata gcgcgcaaac 3240

taggataaat tatcgcgcgc ggtgtcatct atgttactag atcgggaatt cgatatcaag 3300

cttatcgata ccgtcgacct gcaggcatgc aagctctagt ctgtacccga tcaacaccga 3360

gacccgtggc gtcttcgacc tcaatggcgt ctggaacttc aagctggact acgggaaagg 3420

actggaagag aagtggtacg aaagcaagct gaccgacact attagtatgg ccgtcccaag 3480

cagttacaat gacattggcg tgaccaagga aatccgcaac catatcggat atgtctggta 3540

cgaacgtgag ttcacggtgc cggcctatct gaaggatcag cgtatcgtgc tccgcttcgg 3600

ctctgcaact cacaaagcaa ttgtctatgt caatggtgag ctggtcgtgg agcacaaggg 3660

cggattcctg ccattcgaag cggaaatcaa caactcgctg cgtgatggca tgaatcgcgt 3720

caccgtcgcc gtggacaaca tcctcgacga tagcaccctc ccggtggggc tgtacagcga 3780

gcgccacgaa gagggcctcg gaaaagtcat tcgtaacaag ccgaacttcg acttcttcaa 3840

ctatgcaggc ctgcaccgtc cggtgaaaat ctacacgacc ccgtttacgt acgtcgagga 3900

catctcggtt gtgaccgact tcaatggccc aaccgggact gtgacctata cggtggactt 3960

tcaaggcaaa gccgagaccg tgaaagtgtc ggtcgtggat gaggaaggca aagtggtcgc 4020

aagcaccgag ggcctgagcg gtaacgtgga gattccgaat gtcatcctct gggaaccact 4080

gaacacgtat ctctaccaga tcaaagtgga actggtgaac gacggactga ccatcgatgt 4140

ctatgaagag ccgttcggcg tgcggaccgt ggaagtcaac gacggcaagt tcctcatcaa 4200

caacaaaccg ttctacttca agggctttgg caaacatgag gacactccta tcaacggccg 4260

tggctttaac gaagcgagca atgtgatgga tttcaatatc ctcaaatgga tcggcgccaa 4320

cagcttccgg accgcacact atccgtactc tgaagagttg atgcgtcttg cggatcgcga 4380

gggtctggtc gtgatcgacg agactccggc agttggcgtg cacctcaact tcatggccac 4440

cacgggactc ggcgaaggca gcgagcgcgt cagtacctgg gagaagattc ggacgtttga 4500

gcaccatcaa gacgttctcc gtgaactggt gtctcgtgac aagaaccatc caagcgtcgt 4560

gatgtggagc atcgccaacg aggcggcgac tgaggaagag ggcgcgtacg agtacttcaa 4620

gccgttggtg gagctgacca aggaactcga cccacagaag cgtccggtca cgatcgtgct 4680

gtttgtgatg gctaccccgg agacggacaa agtcgccgaa ctgattgacg tcatcgcgct 4740

caatcgctat aacggatggt acttcgatgg cggtgatctc gaagcggcca aagtccatct 4800

ccgccaggaa tttcacgcgt ggaacaagcg ttgcccagga aagccgatca tgatcactga 4860

gtacggcgca gacaccgttg cgggctttca cgacattgat ccagtgatgt tcaccgagga 4920

atatcaagtc gagtactacc aggcgaacca cgtcgtgttc gatgagtttg agaacttcgt 4980

gggtgagcaa gcgtggaact tcgcggactt cgcgacctct cagggcgtga tgcgcgtcca 5040

aggaaacaag aagggcgtgt tcactcgtga ccgcaagccg aagctcgccg cgcacgtctt 5100

tcgcgagcgc tggaccaaca ttccagattt cggctacaag aacgctagcc atcaccatca 5160

ccatcacgtg tgaattggtg accagctcga atttccccga tcgttcaaac atttggcaat 5220

aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata taatttctgt 5280

tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt atgagatggg 5340

tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac aaaatatagc 5400

gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat cgggaattaa 5460

actatcagtg tttgacagga tatattggcg ggtaaaccta agagaaaaga gcgtttatta 5520

gaataacgga tatttaaaag ggcgtgaaaa ggtttatccg ttcgtccatt tgtatgtgca 5580

tgccaaccac agggttcccc tcgggatcaa agtactttga tccaacccct ccgctgctat 5640

agtgcagtcg gcttctgacg ttcagtgcag ccgtcttctg aaaacgacat gtcgcacaag 5700

tcctaagtta cgcgacaggc tgccgccctg cccttttcct ggcgttttct tgtcgcgtgt 5760

tttagtcgca taaagtagaa tacttgcgac tagaaccgga gacattacgc catgaacaag 5820

agcgccgccg ctggcctgct gggctatgcc cgcgtcagca ccgacgacca ggacttgacc 5880

aaccaacggg ccgaactgca cgcggccggc tgcaccaagc tgttttccga gaagatcacc 5940

ggcaccaggc gcgaccgccc ggagctggcc aggatgcttg accacctacg ccctggcgac 6000

gttgtgacag tgaccaggct agaccgcctg gcccgcagca cccgcgacct actggacatt 6060

gccgagcgca tccaggaggc cggcgcgggc ctgcgtagcc tggcagagcc gtgggccgac 6120

accaccacgc cggccggccg catggtgttg accgtgttcg ccggcattgc cgagttcgag 6180

cgttccctaa tcatcgaccg cacccggagc gggcgcgagg ccgccaaggc ccgaggcgtg 6240

aagtttggcc cccgccctac cctcaccccg gcacagatcg cgcacgcccg cgagctgatc 6300

gaccaggaag gccgcaccgt gaaagaggcg gctgcactgc ttggcgtgca tcgctcgacc 6360

ctgtaccgcg cacttgagcg cagcgaggaa gtgacgccca ccgaggccag gcggcgcggt 6420

gccttccgtg aggacgcatt gaccgaggcc gacgccctgg cggccgccga gaatgaacgc 6480

caagaggaac aagcatgaaa ccgcaccagg acggccagga cgaaccgttt ttcattaccg 6540

aagagatcga ggcggagatg atcgcggccg ggtacgtgtt cgagccgccc gcgcacgtct 6600

caaccgtgcg gctgcatgaa atcctggccg gtttgtctga tgccaagctg gcggcctggc 6660

cggccagctt ggccgctgaa gaaaccgagc gccgccgtct aaaaaggtga tgtgtatttg 6720

agtaaaacag cttgcgtcat gcggtcgctg cgtatatgat gcgatgagta aataaacaaa 6780

tacgcaaggg gaacgcatga aggttatcgc tgtacttaac cagaaaggcg ggtcaggcaa 6840

gacgaccatc gcaacccatc tagcccgcgc cctgcaactc gccggggccg atgttctgtt 6900

agtcgattcc gatccccagg gcagtgcccg cgattgggcg gccgtgcggg aagatcaacc 6960

gctaaccgtt gtcggcatcg accgcccgac gattgaccgc gacgtgaagg ccatcggccg 7020

gcgcgacttc gtagtgatcg acggagcgcc ccaggcggcg gacttggctg tgtccgcgat 7080

caaggcagcc gacttcgtgc tgattccggt gcagccaagc ccttacgaca tatgggccac 7140

cgccgacctg gtggagctgg ttaagcagcg cattgaggtc acggatggaa ggctacaagc 7200

ggcctttgtc gtgtcgcggg cgatcaaagg cacgcgcatc ggcggtgagg ttgccgaggc 7260

gctggccggg tacgagctgc ccattcttga gtcccgtatc acgcagcgcg tgagctaccc 7320

aggcactgcc gccgccggca caaccgttct tgaatcagaa cccgagggcg acgctgcccg 7380

cgaggtccag gcgctggccg ctgaaattaa atcaaaactc atttgagtta atgaggtaaa 7440

gagaaaatga gcaaaagcac aaacacgcta agtgccggcc gtccgagcgc acgcagcagc 7500

aaggctgcaa cgttggccag cctggcagac acgccagcca tgaagcgggt caactttcag 7560

ttgccggcgg aggatcacac caagctgaag atgtacgcgg tacgccaagg caagaccatt 7620

accgagctgc tatctgaata catcgcgcag ctaccagagt aaatgagcaa atgaataaat 7680

gagtagatga attttagcgg ctaaaggagg cggcatggaa aatcaagaac aaccaggcac 7740

cgacgccgtg gaatgcccca tgtgtggagg aacgggcggt tggccaggcg taagcggctg 7800

ggttgtctgc cggccctgca atggcactgg aacccccaag cccgaggaat cggcgtgacg 7860

gtcgcaaacc atccggcccg gtacaaatcg gcgcggcgct gggtgatgac ctggtggaga 7920

agttgaaggc cgcgcaggcc gcccagcggc aacgcatcga ggcagaagca cgccccggtg 7980

aatcgtggca agcggccgct gatcgaatcc gcaaagaatc ccggcaaccg ccggcagccg 8040

gtgcgccgtc gattaggaag ccgcccaagg gcgacgagca accagatttt ttcgttccga 8100

tgctctatga cgtgggcacc cgcgatagtc gcagcatcat ggacgtggcc gttttccgtc 8160

tgtcgaagcg tgaccgacga gctggcgagg tgatccgcta cgagcttcca gacgggcacg 8220

tagaggtttc cgcagggccg gccggcatgg ccagtgtgtg ggattacgac ctggtactga 8280

tggcggtttc ccatctaacc gaatccatga accgataccg ggaagggaag ggagacaagc 8340

ccggccgcgt gttccgtcca cacgttgcgg acgtactcaa gttctgccgg cgagccgatg 8400

gcggaaagca gaaagacgac ctggtagaaa cctgcattcg gttaaacacc acgcacgttg 8460

ccatgcagcg tacgaagaag gccaagaacg gccgcctggt gacggtatcc gagggtgaag 8520

ccttgattag ccgctacaag atcgtaaaga gcgaaaccgg gcggccggag tacatcgaga 8580

tcgagctagc tgattggatg taccgcgaga tcacagaagg caagaacccg gacgtgctga 8640

cggttcaccc cgattacttt ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg 8700

cacgccgcgc cgcaggcaag gcagaagcca gatggttgtt caagacgatc tacgaacgca 8760

gtggcagcgc cggagagttc aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa 8820

atgacctgcc ggagtacgat ttgaaggagg aggcggggca ggctggcccg atcctagtca 8880

tgcgctaccg caacctgatc gagggcgaag catccgccgg ttcctaatgt acggagcaga 8940

tgctagggca aattgcccta gcaggggaaa aaggtcgaaa aggtctcttt cctgtggata 9000

gcacgtacat tgggaaccca aagccgtaca ttgggaaccg gaacccgtac attgggaacc 9060

caaagccgta cattgggaac cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag 9120

gcgatttttc cgcctaaaac tctttaaaac ttattaaaac tcttaaaacc cgcctggcct 9180

gtgcataact gtctggccag cgcacagccg aagagctgca aaaagcgcct acccttcggt 9240

cgctgcgctc cctacgcccc gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa 9300

aaatggctgg cctacggcca ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac 9360

tcgaccgccg gcgcccacat caaggcaccc tgcctcgcgc gtttcggtga tgacggtgaa 9420

aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg 9480

agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg 9540

acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga 9600

ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat 9660

accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 9720

tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 9780

ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 9840

ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 9900

gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 9960

gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 10020

ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 10080

tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 10140

gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 10200

tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 10260

tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 10320

tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 10380

ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 10440

ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 10500

gttaagggat tttggtcatg cattctaggt actaaaacaa ttcatccagt aaaatataat 10560

attttatttt ctcccaatca ggcttgatcc ccagtaagtc aaaaaatagc tcgacatact 10620

gttcttcccc gatatcctcc ctgatcgacc ggacgcagaa ggcaatgtca taccacttgt 10680

ccgccctgcc gcttctccca agatcaataa agccacttac tttgccatct ttcacaaaga 10740

tgttgctgtc tcccaggtcg ccgtgggaaa agacaagttc ctcttcgggc ttttccgtct 10800

ttaaaaaatc atacagctcg cgcggatctt taaatggagt gtcttcttcc cagttttcgc 10860

aatccacatc ggccagatcg ttattcagta agtaatccaa ttcggctaag cggctgtcta 10920

agctattcgt atagggacaa tccgatatgt cgatggagtg aaagagcctg atgcactccg 10980

catacagctc gataatcttt tcagggcttt gttcatcttc atactcttcc gagcaaagga 11040

cgccatcggc ctcactcatg agcagattgc tccagccatc atgccgttca aagtgcagga 11100

cctttggaac aggcagcttt ccttccagcc atagcatcat gtccttttcc cgttccacat 11160

cataggtggt ccctttatac cggctgtccg tcatttttaa atataggttt tcattttctc 11220

ccaccagctt atatacctta gcaggagaca ttccttccgt atcttttacg cagcggtatt 11280

tttcgatcag ttttttcaat tccggtgata ttctcatttt agccatttat tatttccttc 11340

ctcttttcta cagtatttaa agatacccca agaagctaat tataacaaga cgaactccaa 11400

ttcactgttc cttgcattct aaaaccttaa ataccagaaa acagcttttt caaagttgtt 11460

ttcaaagttg gcgtataaca tagtatcgac ggagccgatt ttgaaaccgc ggtgatcaca 11520

ggcagcaacg ctctgtcatc gttacaatca acatgctacc ctccgcgaga tcatccgtgt 11580

ttcaaacccg gcagcttagt tgccgttctt ccgaatagca tcggtaacat gagcaaagtc 11640

tgccgcctta caacggctct cccgctgacg ccgtcccgga ctgatgggct gcctgtatcg 11700

agtggtgatt ttgtgccgag ctgccggtcg gggagctgtt ggctggctgg tggcaggata 11760

tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg gacgttttta 11820

atgtactgaa ttaacgccga attaattcgg gggatctgga ttttagtact ggattttggt 11880

tttaggaatt agaaatttta ttgatagaag tattttacaa atacaaatac atactaaggg 11940

tttcttatat gctcaacaca tgagcgaaac cctataggaa ccctaattcc cttatctggg 12000

aactactcac acattattat ggagaaactc gagcttgtcg atcgacagat ccggtcggca 12060

tctactctat ttctttgccc tcggacgagt gctggggcgt cggtttccac tatcggcgag 12120

tacttctaca cagccatcgg tccagacggc cgcgcttctg cgggcgattt gtgtacgccc 12180

gacagtcccg gctccggatc ggacgattgc gtcgcatcga ccctgcgccc aagctgcatc 12240

atcgaaattg ccgtcaacca agctctgata gagttggtca agaccaatgc ggagcatata 12300

cgcccggagt cgtggcgatc ctgcaagctc cggatgcctc cgctcgaagt agcgcgtctg 12360

ctgctccata caagccaacc acggcctcca gaagaagatg ttggcgacct cgtattggga 12420

atccccgaac atcgcctcgc tccagtcaat gaccgctgtt atgcggccat tgtccgtcag 12480

gacattgttg gagccgaaat ccgcgtgcac gaggtgccgg acttcggggc agtcctcggc 12540

ccaaagcatc agctcatcga gagcctgcgc gacggacgca ctgacggtgt cgtccatcac 12600

agtttgccag tgatacacat ggggatcagc aatcgcgcat atgaaatcac gccatgtagt 12660

gtattgaccg attccttgcg gtccgaatgg gccgaacccg ctcgtctggc taagatcggc 12720

cgcagcgatc gcatccatag cctccgcgac cggttgtaga acagcgggca gttcggtttc 12780

aggcaggtct tgcaacgtga caccctgtgc acggcgggag atgcaatagg tcaggctctc 12840

gctaaactcc ccaatgtcaa gcacttccgg aatcgggagc gcggccgatg caaagtgccg 12900

ataaacataa cgatctttgt agaaaccatc ggcgcagcta tttacccgca ggacatatcc 12960

acgccctcct acatcgaagc tgaaagcacg agattcttcg ccctccgaga gctgcatcag 13020

gtcggagacg ctgtcgaact tttcgatcag aaacttctcg acagacgtcg cggtgagttc 13080

aggctttttc atatctcatt gccccccggg atctgcgaaa gctcgagaga gatagatttg 13140

tagagagaga ctggtgattt cagcgtgtcc tctccaaatg aaatgaactt ccttatatag 13200

aggaaggtct tgcgaaggat agtgggattg tgcgtcatcc cttacgtcag tggagatatc 13260

acatcaatcc acttgctttg aagacgtggt tggaacgtct tctttttcca cgatgctcct 13320

cgtgggtggg ggtccatctt tgggaccact gtcggcagag gcatcttgaa cgatagcctt 13380

tcctttatcg caatgatggc atttgtaggt gccaccttcc ttttctactg tccttttgat 13440

gaagtgacag atagctgggc aatggaatcc gaggaggttt cccgatatta ccctttgttg 13500

aaaagtctca atagcccttt ggtcttctga gactgtatct ttgatattct tggagtagac 13560

gagagtgtcg tgctccacca tgttatcaca tcaatccact tgctttgaag acgtggttgg 13620

aacgtcttct ttttccacga tgctcctcgt gggtgggggt ccatctttgg gaccactgtc 13680

ggcagaggca tcttgaacga tagcctttcc tttatcgcaa tgatggcatt tgtaggtgcc 13740

accttccttt tctactgtcc ttttgatgaa gtgacagata gctgggcaat ggaatccgag 13800

gaggtttccc gatattaccc tttgttgaaa agtctcaata gccctttggt cttctgagac 13860

tgtatctttg atattcttgg agtagacgag agtgtcgtgc tccaccatgt tggcaagctg 13920

ctctagccaa tacgc 13935

Claims (10)

1. Use of the SiPSY1 protein in any one of the following A1) -A6):

   A1) regulating and controlling the content of yellow pigment and/or carotenoid in the millet seeds;

   A2) regulating and controlling synthesis and metabolism of yellow pigment and/or carotenoid in the millet seeds;

   A3) regulating and controlling the color and/or transparency of the millet seeds;

   A4) improving or increasing the quality of the millet;

   A5) cultivating transgenic millet;

   A6) breeding millet;

   the SiPSY1 protein is a protein described in any one of the following (a1) - (a 4):

   (a1) a protein shown as SEQ ID No. 3;

   (a2) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of the protein of (a 1);

   (a3) protein obtained by substituting and/or deleting and/or adding one or more amino acid residues in (a1) and related to the synthesis of millet yellow pigment and/or carotenoid;

   (a4) a protein derived from millet and having 98% or more identity to (a1) and involved in the synthesis of millet yellow and/or carotenoids.
2. 2. Use of a biomaterial related to the SiPSY1 protein as claimed in claim 1 in any of the following A1) -A6):

   A1) regulating and controlling the content of yellow pigment and/or carotenoid in the millet seeds;

   A2) regulating and controlling synthesis and metabolism of yellow pigment and/or carotenoid in the millet seeds;

   A3) regulating and controlling the color and/or transparency of the millet seeds;

   A4) improving or increasing the quality of the millet;

   A5) cultivating transgenic millet;

   A6) breeding millet;

   the biological material related to the SiPSY1 protein in claim 1 is a nucleic acid molecule encoding the SiPSY1 protein in claim 1 or an expression cassette, a recombinant vector, or a recombinant microorganism containing the nucleic acid molecule.
3. 3. Use according to claim 2, characterized in that: the nucleic acid molecule is a DNA molecule described in any one of (b1) to (b2) below:

   (b1) DNA molecule shown in SEQ ID No.1 or SEQ ID No. 2;

   (b2) a DNA molecule derived from millet and having 75% or more identity to (b1) and encoding the SiPSY1 protein of claim 1.
4. 4. Use of a substance inhibiting SiPSY1 in any one of the following B1) -B5):

   B1) reducing the yellow pigment content and/or carotenoid content in the millet seeds;

   B2) inhibiting synthesis and metabolism of yellow pigment and/or carotenoid in the millet seeds;

   B3) whitening the millet seeds;

   B4) cultivating transgenic millet;

   B5) breeding millet;

   the substance for inhibiting SiPSY1 is a substance for inhibiting or reducing the activity and/or content of SiPSY1 protein in claim 1 in the millet, or a substance for inhibiting the expression of the nucleic acid molecule in claim 2 or 3 in the millet, or a substance for knocking out the nucleic acid molecule in claim 2 or 3 in the millet.
5. 5. A method for breeding transgenic millet with increased yellow pigment content and/or carotenoid content, comprising the steps of increasing the content and/or activity of SiPSY1 protein in claim 1 in receptor millet to obtain transgenic millet; the transgenic millet kernel has a higher content of yellow pigment and/or carotenoid than the acceptor millet.
6. 6. A method for breeding transgenic millet with reduced yellow grain content and/or carotenoid content, comprising the steps of reducing the content and/or activity of SiPSY1 protein in claim 1 in receptor millet to obtain transgenic millet; the transgenic millet kernel has a lower yellow pigment content and/or carotenoid content than the acceptor millet.
7. 7. A method for preparing transgenic millet comprises the following steps: a step of editing the nucleic acid molecule of claim 2 or 3 in a receptor millet to obtain a gene-edited millet; the yellow pigment content and/or carotenoid content in the genetically edited millet kernel is lower than that of the acceptor millet.
8. 8. Use according to any one of claims 1 to 4 or method according to any one of claims 5 to 7, wherein: the yellow pigment or the carotenoid includes carotene and lutein.
9. 9. Use according to claim 8, characterized in that: the carotene is beta-carotene.
10. 10. Use according to claim 8, characterized in that: the xanthophyll is lutein and zeaxanthin.

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