CN114591927A - IbPRX17 related to development of sweet potato tuber streak tendon and coding gene and application thereof - Google Patents

Abstract

The invention discloses a sweet potato tuber streak development related protein IbPRX17 and a coding gene and application thereof. The invention specifically discloses application of protein with an amino acid sequence of SEQ ID No.1 or substances for regulating the activity and/or content of the protein in regulation of development of plant (such as sweet potato) striated tendons. According to the invention, the coding gene (IbPRX17 gene) of the protein is introduced into a receptor plant (sweet potato chestnut flavor) to obtain a transgenic plant over-expressing the IbPRX17 gene, compared with a wild sweet potato, the transgenic plant has obviously stronger development of the striated tendon than the wild sweet potato, and shows a phenotype of promoting the development of the striated tendon of a potato tuber. The IbPRX17 protein and the coding gene thereof provided by the invention have important theoretical significance and application value in the research of influence of sweet potatoes on the development of the striated tendon.

Description

IbPRX17 related to development of sweet potato tuber streak tendon and coding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a sweet potato tuber streak development related protein IbPRX17, and a coding gene and application thereof.

Background

Sweet potatoes (Ipomoea batatas (L.) Lam.) are an important crop of grains, feed, industrial raw materials and new energy. China is the biggest sweet potato producing country in the world. Under the influence of cultivation conditions such as soil, water and fertilizer, planting density, growth period and the like, the sweet potato variety is easy to dedifferentiate parenchyma cells of secondary phloem of the potato block to form a new meristem and further develop to form a layer, thereby showing the strip-rib phenotype. The growth of the ribs on the surface of the potato blocks not only affects the appearance quality of the potato blocks, but also seriously reduces the commodity value of the potato blocks and reduces the economic benefit of the sweet potatoes. Therefore, the cultivation of new species of sweet potato with excellent variety is one of the important measures for promoting the development of sweet potato industry.

In addition, the sweet potato has the problems of self-incompatibility, unstable filial generation, deficient germplasm resources, long breeding period and the like, the traditional hybrid breeding method is difficult to select and breed a new sweet potato variety with excellent quality, and the characteristics of the sweet potato can be directionally improved by using genetic engineering means such as gene editing and the like, so that the method is a feasible way for breeding high-quality sweet potato varieties at present. The sweet potato gene causing the striation is excavated, knocked out and edited by using a gene editing method, and the good main cultivated species of the sweet potato which is easy to generate striation is improved, so that the sweet potato gene has important value for the development of the sweet potato industry. The invention identifies the protein related to the growth of the sweet potato tuber streak tendon and the coding gene thereof for the first time, provides a basis for further clarifying the related mechanism causing the sweet potato streak tendon, provides scientific basis for cultivating new sweet potato varieties, and has important theoretical guidance significance and practical application value for high yield and high yield of the sweet potato.

Disclosure of Invention

The technical problem to be solved by the invention is how to regulate or improve the development of the striated tendon of a plant (such as sweet potato). The technical problem to be solved is not limited to the technical subject described, and other technical subject not mentioned herein may be clearly understood by those skilled in the art through the following description.

In order to solve the above technical problems, the present invention provides, in a first aspect, an application of a protein or a substance that regulates an activity and/or a content of the protein, wherein the application may be any one of the following:

D1) the use of a protein or a substance which modulates the activity and/or content of said protein in the modulation of the development of plant striations;

D2) the application of protein or substance for regulating and controlling the activity and/or content of the protein in preparing products for regulating and controlling the development of plant striae fascicularis;

D3) use of a protein or a substance modulating the activity and/or content of said protein for growing plants with altered tendon development;

D4) the use of a protein or a substance which modulates the activity and/or content of said protein for the preparation of a product for the cultivation of plants with altered tendon development;

D5) the application of protein or substance for regulating and controlling the activity and/or content of the protein in the improvement of the species of the sweet potato or the preparation of products for improving the species of the sweet potato;

D6) use of a protein or a substance modulating the activity and/or content of said protein in plant breeding;

the protein is IbPRX17, and can be any one of the following:

A1) a protein having an amino acid sequence of SEQ ID No. 1;

A2) a protein which is obtained by substituting and/or deleting and/or adding amino acid residues to the amino acid sequence shown in SEQ ID No.1, has more than 80% of identity with the protein shown in A1), and has the same function;

A3) a fusion protein with the same function obtained by connecting labels at the N end and/or the C end of A1) or A2).

In order to facilitate the purification or detection of the protein in A1), a tag protein may be attached to the amino terminus or the carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No.1 of the sequence Listing.

Such tag proteins include, but are not limited to: GST (glutathione mercaptotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

The nucleotide sequence of the protein IbPRX17 of the invention can be easily mutated by a person skilled in the art by known methods, for example directed evolution or point mutation. Those nucleotides which are artificially modified and have 75% or more identity to the nucleotide sequence of the protein IbPRX17 isolated in the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode the protein IbPRX17 and have the function of the protein IbPRX 17.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

Herein, identity refers to the identity of amino acid sequences or nucleotide 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, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as a Matrix, the Gap existence cost, the Per residual Gap cost and the Lambda ratio are set to 11, 1 and 0.85 (default values), respectively, and a search is performed to calculate the identity (%) of the amino acid sequences, and then the value (%) of identity can be obtained.

Herein, the 80% or greater identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

Herein, the substance regulating the activity and/or content of the protein may be a substance regulating the expression of a gene encoding the protein IbPRX 17.

As above, the substance that regulates gene expression may be a substance that performs at least one of the following 6 controls: 1) regulation at the level of transcription of said gene; 2) regulation after transcription of the gene (i.e., regulation of splicing or processing of a primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of nuclear to cytoplasmic transport of mRNA of the gene); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; 6) post-translational regulation of the gene (i.e., regulation of the activity of a protein translated from the gene).

The substance for regulating gene expression may specifically be any one of the following biomaterials B1) -B4).

In the above application, the protein IbPRX17 can be derived from Ipomoea batatas (L.) Lam.).

The invention also provides an application of biological materials related to the protein IbPRX17, wherein the application can be any one of the following:

E1) the application of biological materials related to the protein IbPRX17 in regulating and controlling the development of plant striated tendons;

E2) the application of the biological material related to the protein IbPRX17 in preparing products for regulating and controlling the development of plant striated tendons;

E3) the use of a biological material related to said protein IbPRX17 for growing plants with altered fascicular development;

E4) the use of a biological material related to said protein IbPRX17 for the preparation of a product for the cultivation of plants with altered fibrilar development;

E5) the application of the biological material related to the protein IbPRX17 in improving the variety of the sweet potato or preparing the product of the improved variety of the sweet potato;

E6) the use of a biological material related to the protein IbPRX17 in plant breeding;

the biomaterial may be any one of the following B1) to B8):

B1) a nucleic acid molecule encoding the protein IbPRX 17;

B2) a nucleic acid molecule that inhibits or reduces or silences the expression of a gene encoding said protein IbPRX 17;

B3) an expression cassette comprising the nucleic acid molecule of B1) and/or B2);

B4) a recombinant vector containing the nucleic acid molecule according to B1) and/or B2), or a recombinant vector containing the expression cassette according to B3);

B5) a recombinant microorganism containing the nucleic acid molecule according to B1) and/or B2), or a recombinant microorganism containing the expression cassette according to B3), or a recombinant microorganism containing the recombinant vector according to B4);

B6) a transgenic plant cell line comprising B1) and/or B2) said nucleic acid molecule or a transgenic plant cell line comprising B3) said expression cassette;

B7) transgenic plant tissue comprising the nucleic acid molecule according to B1) and/or B2), or transgenic plant tissue comprising the expression cassette according to B3);

B8) a transgenic plant organ containing B1) and/or B2) said nucleic acid molecule, or a transgenic plant organ containing B3) said expression cassette.

In the above application, the nucleic acid molecule of B1) may be any one of the following:

C1) the coding sequence is a DNA molecule of SEQ ID No. 2;

C2) the nucleotide sequence is the DNA molecule of SEQ ID No. 2.

The DNA molecule shown in SEQ ID No.2 (gene IbPRX17 for regulating plant striate development) encodes protein IbPRX17 (sweet potato tuber striate development related protein IbPRX17) with the amino acid sequence of SEQ ID No. 1.

The nucleotide sequence shown in SEQ ID NO.2 is the nucleotide sequence of a coding gene (CDS) of the protein IbPRX 17. The gene of the protein IbPRX17 (IbPRX17 gene) of the present invention may be any nucleotide sequence that can encode the protein IbPRX 17. In view of the degeneracy of the codons and the preference of codons for different species, one skilled in the art can use codons suitable for the expression of a particular species as needed.

B1) The nucleic acid molecule also can comprise a nucleic acid molecule obtained by codon preference modification on the basis of the nucleotide sequence shown in SEQ ID No. 2.

The nucleic acid molecule also comprises a nucleic acid molecule which has more than 95 percent of identity with the nucleotide sequence shown in SEQ ID No.2 and is of the same species as the source.

The nucleic acid molecule described herein may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be an RNA, such as a gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

Vectors described herein are well known to those skilled in the art and include, but are not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), Ti plasmids, or viral vectors. Specifically, the vector may be pMD19-T vector and/or pCAMBIA1300 vector.

The existing plant expression vector can be used for constructing a recombinant expression vector containing the IbPRX17 gene. The plant expression vector includes but is not limited to binary agrobacterium vector, plant microprojectile bombardment vector and other vectors. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylic acid to the 3 'end of the mRNA precursor, and the untranslated regions transcribed from the 3' end of genes including but not limited to Agrobacterium crown gall inducible (Ti) plasmid genes (e.g., nopalin synthase Nos), plant genes (e.g., soybean storage protein genes) all have similar functions.

When the IbPRX17 gene is used to construct a recombinant plant expression vector, any one of an enhanced promoter or a constitutive promoter can be added before the transcription initiation nucleotide, including but not limited to, for example, cauliflower mosaic virus (CAMV)35S promoter, maize ubiquitin promoter (ubiquitin), which can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct plant expression vectors, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of the transgenic plant cells or plants, the plant expression vectors used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic markers having resistance (gentamicin marker, kanamycin marker, etc.), or chemical-resistant agent marker genes (e.g., herbicide-resistant gene), etc., which are expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

Any vector capable of guiding the expression of the exogenous gene in the plant is utilized to introduce the IbPRX17 gene or the gene segment provided by the invention into a plant cell or a receptor plant, so that a transgenic cell line and a transgenic plant with enhanced striate development can be obtained. The expression vector carrying the IbPRX17 gene can be obtained by transforming a plant cell or tissue using a Ti plasmid, a Ri plasmid, a plant viral vector, direct DNA transformation, microinjection, conductance, Agrobacterium mediation, or the like, and culturing the transformed plant tissue into a plant.

The microorganism described herein may be a yeast, bacterium, algae or fungus. Among them, the bacteria may be derived from the genera Escherichia (Escherichia), Erwinia (Erwinia), Agrobacterium (Agrobacterium), Flavobacterium (Flavobacterium), Alcaligenes (Alcaligenes), Pseudomonas (Pseudomonas), Bacillus (Bacillus), etc. Specifically, it may be Escherichia coli DH5 alpha and/or Agrobacterium tumefaciens EHA 105.

The recombinant vector can be specifically a recombinant vector pCAMBIA1300-IbPRX 17. The recombinant vector pCAMBIA1300-IbPRX17 is a recombinant expression vector obtained by replacing a fragment (small fragment) between KpnI recognition sites and SalI recognition sites of a pCAMBIA1300 vector with a DNA fragment of which the nucleotide sequence is SEQ ID No.2 in a sequence table and keeping other sequences of the pCAMBIA1300 vector unchanged. The recombinant vector pCAMBIA1300-IbPRX17 expresses a protein IbPRX17 shown in SEQ ID No. 1.

The invention also provides a method for cultivating the plant with the changed striated tendon development, which comprises the step of improving the content and/or the activity of the protein IbPRX17 in a target plant to obtain the plant with the changed striated tendon development which is stronger than that of the target plant.

In the above method, the increase in the content and/or activity of the protein IbPRX17 in the plant of interest is achieved by increasing the expression level of a gene encoding the protein IbPRX17 in the plant of interest.

In the above method, the improvement of the expression level of the gene encoding the protein IbPRX17 in the target plant is achieved by introducing the gene encoding the protein IbPRX17 into the target plant.

In the above method, the gene encoding the protein IbPRX17 may be any of the following genes:

H1) the coding sequence is a DNA molecule of SEQ ID No. 2;

H2) the nucleotide sequence is the DNA molecule of SEQ ID No. 2.

Specifically, in one embodiment of the invention, the improvement of the expression level of the gene encoding the protein IbPRX17 in the target plant is realized by introducing a DNA molecule shown as SEQ ID No.2 into the target plant.

In one embodiment of the present invention, the method for growing a plant with altered development of the striated tendon comprises the steps of:

(1) constructing a recombinant expression vector containing a DNA molecule shown in SEQ ID NO. 2;

(2) transferring the recombinant expression vector constructed in the step (1) into a target plant (such as crops or sweet potatoes);

(3) and obtaining the plant with the development of the striated tendon stronger than that of the target plant through screening and identification.

The introduction refers to introduction by recombinant means including, but not limited to, Agrobacterium-mediated transformation, biolistic methods, electroporation, in planta techniques, and the like.

The invention also provides a method for cultivating the plant with the changed striated tendon development, which comprises the step of reducing the content and/or the activity of the protein IbPRX17 in a target plant to obtain the plant with the changed striated tendon development which is weaker than that of the target plant.

In the above method, the reduction of the content and/or activity of the protein IbPRX17 in the plant of interest can be achieved by reducing the expression level and/or activity of a gene encoding the protein IbPRX17 in the plant of interest.

In the above method, the reduction of the expression level and/or activity of the gene encoding the protein IbPRX17 in the target plant may be the reduction or inactivation of the activity of the gene encoding the protein IbPRX17 in the genome of the target plant by gene mutation, gene knock-out, gene editing, or gene knock-down techniques.

Herein, the plant may be any one of the following

G1) A monocot or dicot;

G2) a plant of the family Convolvulaceae;

G3) a plant of the genus Ipomoea;

G4) sweet potato group plants;

G5) sweet potato.

The protein IbPRX17 and/or the nucleic acid molecule are also within the scope of the present invention.

Herein, the modulating plant striated development may be up-regulating (enhancing or promoting) plant striated development or down-regulating (attenuating or inhibiting) plant striated development.

Herein, the plant with altered fascicular development may be a plant with enhanced fascicular development or a plant with reduced fascicular development.

The plant with the growth enhancement of the tendon can be a multi-tendon sweet potato variety or a tendon sweet potato variety.

The plant with weakened development of the tendon can be a sweet potato variety with less tendon or a sweet potato variety without tendon.

In the present invention, the plant with altered striated tendon development is understood to include not only the plant of interest transformed with the IbPRX17 gene or the first generation transgenic plant obtained by knocking out the IbPRX17 gene, but also the progeny thereof. The gene may be propagated in the species, or transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The stress-resistant plant comprises seeds, callus, complete plants and cells.

The IbPRX17 gene provided by the invention codes a peroxidase protein, and the gene is introduced into sweet potatoes to obtain a transgenic sweet potato plant over-expressing the IbPRX17 gene, wherein the transgenic sweet potato plant presents a phenotype of promoting the development of tuberous striatus. The IbPRX17 protein and the coding gene thereof provided by the invention have important theoretical significance and application value in the research of influence of sweet potatoes on the development of the striated tendon.

Drawings

FIG. 1 shows PCR detection of transgenic plants.

FIG. 2 is a phenotypic observation of transgenic potato tubers planted in Guangzhou city.

FIG. 3 is a phenotypic observation of transgenic potato tubers planted in Haikou.

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 specified, were carried out in a conventional manner according to the techniques or conditions described in the literature in this field or according to the product instructions. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The pMD19-T vector in the following examples is a product of Takara Bio Inc. (Dalian) under catalog number 6013. The pCAMBIA1300 vector is a product of Cambia corporation.

The plant total RNA extraction kit in the following examples is a product of Tiangen Biochemical technology (Beijing) Ltd, and the catalog number of the product is DP 432. The HiFiScript gDNA Removal RT MasterMix kit is Kangji scientific Biotechnology (Beijing) Inc., product catalog number CW 2020M. The KpnI enzyme is a product of Saimer Feishale science and technology (China) and has a product catalog number of FD 0524. The SalI enzyme is a product of Saimer Feishel science and technology (China) and has a product catalog number of FD 0644. Escherichia coli DH5 alpha is a product of Shenzhen Kangsheng Life technologies, Inc., the product catalog number is KTSM101L, Agrobacterium tumefaciens EHA105 is a product of Beijing Ongshenke Biotechnologies, Inc., the product catalog number is TSC-A03.

The chestnut aroma of sweet potato variety in the following examples is described in the following documents: in vitro screening and identification of drought-resistant mutants of sweet potatoes [ J ] Chinese agricultural science 2003,36(9): 1000-.

The sweet potato variety ND98 in the following examples is described in the following documents: he Shaozhen, in vitro screening of salt-tolerant mutants of sweet potatoes and cloning of salt-tolerant candidate genes [ D ] A doctor academic thesis of Chinese university of agriculture, 2008, publicly available from the research laboratory of sweet potato genetic breeding of Chinese university of agriculture, to repeat the experiment.

LB solid/liquid medium, MS solid/liquid medium are described in the following documents: zhuan, sweet potato salt-tolerant transcriptome analysis and clone and function verification of stress-resistant related genes IbBBX24 and IbCPK28 [ D ]. Doctor's academic paper of chinese university of agriculture, 2017.

Example 1 obtaining of IbPRX17 Gene

1. Extracting total RNA of the sweet potato: taking 1g of young leaves of the sweet potato strain ND98, grinding into powder in liquid nitrogen, adding into a 2mL centrifuge tube, extracting the total RNA of the sweet potato by using a plant total RNA extraction kit, and reversely transcribing to obtain a first strand cDNA by using a HiFiScript gDNA Removal RT MasterMix kit.

2. The interaction gene IbPRX17 is obtained by screening a sweet potato yeast two-hybrid library by means of a sweet potato stress-resistant gene IbBBX24, and is compared in a sweet potato to Garden library (http:// sweet potato-Garden. kazusa. or. jp) to obtain an EST sequence shown as SEQ ID No.3 in a sequence table. Primers IbPRX17-F and IbPRX17-R were designed and artificially synthesized based on the nucleotide sequence of the EST sequence, the sequence being:

IbPRX17-F：5’-ATGATGTATATAACGCTCGTCATCT-3’

IbPRX17-R：5’-TCAATATGAAGCAAACAACTTTGCAG-3’

3. and (3) carrying out PCR amplification by using the cDNA obtained in the step (1) as a template and the IbPRX17-F and IbPRX17-R synthesized in the step (2) as primers to obtain a PCR amplification fragment product of about 996bp and sequencing the PCR amplification fragment product.

The result shows that the nucleotide sequence of the PCR amplification product obtained in the step 3 is shown as SEQ ID No.2 in the sequence table, the gene shown by the sequence is named IbPRX17 gene, the protein coded by the gene is named IbPRX17 protein or protein IbPRX17, and the amino acid sequence is shown as SEQ ID No.1 in the sequence table.

Example 2 application of IbPRX17 protein in regulation and control of development of sweet potato tuber streak

1. Construction of plant expression vectors

According to the coding sequence (SEQ ID No.2) of IbPRX17 protein nucleotide of the sweet potato, a primer sequence for amplifying a complete coding sequence (CDS) is designed, forward and reverse primers are respectively introduced into KpnI and SalI enzyme cutting sites, and the primer sequences are as follows:

IbPRX17-FF-KpnI：5’-TACGAATTCGAGCTCGGTACCATGATGTATATAACGCTCGTCATCT-3' (the underlined part indicates KpnI cleavage site)

IbPRX17-RR-SalI：5’-CTTGCATGCCTGCAGGTCGACTCAATATGAAGCAAACAACTTTGCAG-3' (SalI cleavage site is underlined)

Taking a double-stranded DNA molecule shown in an artificially synthesized SEQ ID No.2 as a template, connecting a product to a pMD19-T vector after PCR amplification to obtain a recombinant vector, namely pMD-IbPRX17, sequencing M13-F/R, and ensuring the correctness of a reading frame and an enzyme digestion site of IbPRX17 protein nucleotides of sweet potatoes.

M13-F:5’-GTAAAACGACGGCCAGT-3’，

M13-R:5’-CAGGAAACAGCTATGAC-3’。

The recombinant vector pMD-IbPRX17 was double-digested with restriction enzymes KpnI and SalI, and about 996bp of DNA fragment 1 was recovered.

And carrying out double enzyme digestion on the pCAMBIA1300 vector by KpnI and SalI, recovering a large vector fragment, and connecting the recovered large vector fragment with the DNA fragment 1 to obtain a recombinant vector pCAMBIA1300-IbPRX17, namely the target plasmid. Transforming the target plasmid into escherichia coli DH5 alpha, culturing for 20h at 37 ℃, performing PCR analysis and enzyme digestion identification on the recombinant vector, and performing sequencing verification. The sequencing result shows that the sequence shown by SEQ ID No.2 in the sequence table is inserted between the KpnI enzyme cutting site and the SalI enzyme cutting site of the vector pCAMBIA1300, which indicates that the recombinant vector is constructed correctly.

The recombinant vector pCAMBIA1300-IbPRX17 is a recombinant expression vector obtained by replacing a fragment (small fragment) between KpnI and SalI recognition sites of the pCAMBIA1300 vector with a DNA fragment having a nucleotide sequence of SEQ ID No.2 in the sequence table, and keeping the other sequences of the pCAMBIA1300 vector unchanged. The recombinant vector pCAMBIA1300-IbPRX17 expresses protein IbPRX17 shown in SEQ ID No. 1.

The recombinant vector pCAMBIA1300-IbPRX17 has an expression cassette, and the nucleotide sequence of the expression cassette contains CaMV35S promoter, IbPRX17 protein coding gene and NOS terminator.

2. Plant expression vector transformation agrobacterium tumefaciens

(1) Thawing Agrobacterium tumefaciens EHA105 competent cells on ice, adding 2 μ g of the extracted pCAMBIA1300-IbPRX17 plasmid, flicking the tube wall, mixing well, ice-cooling for 10 min;

(2) quickly freezing with liquid nitrogen for 5min, water-bathing at 37 deg.C for 10min, and ice-bathing for 5 min;

(3) adding 600 μ L liquid LB culture medium, culturing at 28 deg.C and 200rpm for 5 hr;

(4) 200 mu L of bacterial liquid is coated on LB solid culture medium containing 100ug/ml kanamycin and 100ug/ml rifampicin;

(5) performing inverted dark culture at 28 deg.C for 2d, culturing appropriate amount of Agrobacterium with liquid LB culture medium to obtain Agrobacterium liquid introduced with pCAMBIA1300-IbPRX17 vector, and naming recombinant Agrobacterium as

EHA105/pCAMBIA1300-IbPRX17。

3. Genetic transformation and regeneration of sweet potatoes

EHA105/pCAMBIA1300-IbPRX17 was introduced into the chestnut aroma of sweet potato variety by Agrobacterium-mediated method. The specific method comprises the following steps:

(1) stripping the stem tip meristem of chestnut flavor of sweet potato variety, placing on MS solid culture medium containing 2.0 mg/L2, 4-D, culturing at 27 + -1 deg.C for 8 weeks to obtain embryonic callus;

(2) placing the embryogenic callus into MS liquid culture medium containing 2.0 mg/L2, 4-D, and horizontally shaking and culturing on shaking table for 8 weeks to obtain embryogenic cell mass with diameter of 0.7-1.3 mm;

(3) screening the embryonic cell mass through a 20-mesh screen, transferring the larger cell mass to a 30-mesh screen, slightly grinding to enable the embryonic cell mass to be wound, and carrying out shaking culture on the ground larger embryonic cell mass for 3 days;

(4) transforming the EHA105/pCAMBIA1300-IbPRX17 into an embryonic cell mass by adopting an agrobacterium-mediated method, then placing the embryonic cell mass on a co-culture medium (MS solid culture medium containing 30mg/L AS and 2.0 mg/L2, 4-D), and carrying out dark culture at 28 ℃ for 3D;

(5) washing the embryogenic cell mass once in MS liquid culture medium containing 400mg/L Cefotaxime Sodium (CS) and 2.0 mg/L2, 4-D, and then performing shake culture in MS liquid culture medium containing 2.0 mg/L2, 4-D for 1 week;

(6) placing the embryogenic cell mass on a screening medium (MS solid medium containing 100mg/L CS, 5mg/L hygromycin (Hyg) and 2, 4-D), and dark-culturing at 28 deg.C for 10-12 weeks, wherein the medium is replaced every two weeks;

(7) placing the embryogenic cell mass on a somatic embryo induction culture medium (MS solid culture medium containing 100mg/L CS and 1.0mg/L ABA), and alternately culturing in dark and light at 28 ℃ for 2-4 weeks to obtain resistant callus;

(8) and placing the resistant callus on an MS solid culture medium, and culturing for 4-8 weeks in a light-dark alternating mode at 28 ℃ to obtain 14 transgenic plants to be identified (pseudotransgenic plants), which are sequentially named as OE-P1, OE-P2, OE-P3, OE-P5, OE-P6, OE-P7, OE-P9, OE-P10, OE-P11, OE-P12, OE-P13, OE-P14, OE-P15 and OE-P16.

(9) Extracting genome DNA of a pseudotransgenic plant leaf by using a CTAB method, taking the extracted genome DNA as a template, water and a wild plant (chestnut fragrance) as negative controls, taking a plasmid pCAMBIA1300-IbPRX17 as a positive control, and taking CaMV35S (5'-TGACGCACAATCCCACTATCCT-3') and IbPRX17-RR-SalI as primers to perform PCR amplification to obtain a PCR amplification product; if the PCR amplification product contains a band of about 1234bp, the corresponding transgenic plant of the sweet potato to be identified is the transgenic positive plant of the sweet potato.

The results of electrophoresis detection amplification are shown in FIG. 1 (in FIG. 1, lane M is shown as a marker band, lane W is shown as a band of a negative control (water), lane P is shown as a band of a positive control (recombinant plasmid pCAMBIA1300-IbPRX17), lane WT is shown as a band of a sweet potato chestnut plant, lanes OE-P1, OE-P2, OE-P3, OE-P5, OE-P6, OE-P7, OE-P9, OE-P10, OE-P11, OE-P12, OE-P13, OE-P14, OE-P15 and OE-P16 are shown as bands of a sweet potato pseudotransgenic plant transformed with pCAMBIA1300-IbPRX17, and lanes OE-P9, OE-P10, OE-P11, OE-P87452, OE-P9358, and PRIbPRX 72 are shown as bands integrated into the target gene genome of sweet potato, and the lanes are shown as a positive control, and proves that the regenerated plants are transgenic positive plants. The sweet potato plants (OE-P9, OE-P10, OE-P11, OE-P14, OE-P15 and OE-P16) which are identified as transgenic positives are propagated by adopting a vegetative propagation method, and the plants obtained by propagation of one transgenic seedling are used as a strain for further carrying out the phenotypic observation of the development of the tuberous striations.

4. Potato streak development phenotype observation of transgenic plants

Sweet potato blocks planted in the isolated field for 130 days are harvested from Guangzhou city, Guangdong province in 2020, the phenotypic growth conditions of the sweet potato blocks are observed, and the sweet potato blocks are photographed and recorded. The results of the gain phenotype amplification of 1 potato piece per line are shown in FIG. 2. The results show that the development of the striations of each transgenic potato block (line number is OE-P14, OE-P15 and OE-P16) which overexpresses IbPRX17 gene is obviously stronger than that of the wild type chestnut potato block (line number is LZX).

Sweet potato blocks planted in isolated fields for 150 days were harvested from Haikou City in Hainan province in 2021, observed for phenotypic growth of the sweet potato blocks, and photographed for recording. The results of the enlargement of the striae phenotype of 1 potato piece per line of 1 plant are shown in FIG. 3. The result shows that the development of the streak of each potato piece of the transgenic potato piece (line number is OE-P14, OE-P15 and OE-P16) which overexpresses the IbPRX17 gene is obviously stronger than that of the wild type chestnut sweet potato piece (line number is LZX).

The results show that the sweet potato tuber over-expressing IbPRX17 gene shows obvious development phenotype of the striated tendon.

The IbPRX17 protein and the coding gene IbPRX17 of the invention can regulate and control the striate development of plants (such as sweet potatoes), and can obviously enhance the striate development of target plants by improving the content and/or activity of the IbPRX17 protein (such as over-expression of IbPRX17 gene) in the target plants. Conversely, the striated muscle development of the target plant can be significantly reduced by reducing the content and/or activity of the IbPRX17 protein (such as inhibiting the expression of the IbPRX17 gene) in the target plant.

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 within 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 examples, it will be appreciated that the invention may 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 possible within the scope of the claims attached below.

SEQUENCE LISTING

<110> university of agriculture in China

<120> sweet potato tuber streak development related protein IbPRX17, and coding gene and application thereof

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 331

<212> PRT

<213> sweet potato (Ipomoea batatas (L.) Lam.)

<400> 1

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

1 5 10 15

Ile Gln Ala Glu Ile Val Glu Leu Arg Pro Gly Phe Tyr Ser Asp Thr

20 25 30

Cys Pro Glu Ala Glu Asp Ile Val Arg Gly Val Ile Lys Arg Asn Met

35 40 45

Glu Arg Glu Pro Arg Ser Ala Ala Ser Val Met Arg Leu Gln Phe His

50 55 60

Asp Cys Phe Val Asn Gly Cys Asp Ala Ser Leu Leu Leu Asp Asp Thr

65 70 75 80

Pro Glu Met Leu Gly Glu Lys Leu Cys Leu Ser Asn Ile Asn Ser Leu

85 90 95

Arg Ser Tyr Glu Val Val Asp Glu Ala Lys Glu Ala Val Glu Met Ala

100 105 110

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

115 120 125

Asp Ala Val Val Leu Ser Gly Gly Pro Asn Trp Glu Val Lys Leu Gly

130 135 140

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

145 150 155 160

Pro Ser Pro Arg Ala Asn Ala Asp Thr Leu Ile Asp Leu Phe Asn Arg

165 170 175

Phe Asn Leu Ser Val Lys Asp Leu Val Ala Leu Ser Gly Ser His Ser

180 185 190

Ile Gly Gln Gly Arg Cys Phe Ser Ile Val Phe Arg Leu Tyr Asn Gln

195 200 205

Ser Gly Thr Gly Arg Pro Asp Pro Thr Ile Glu Pro Asn Phe Arg Glu

210 215 220

Lys Leu Asp Asn Leu Cys Pro Leu Gly Gly Asp Gly Asn Val Thr Gly

225 230 235 240

Asp Leu Asp Ala Thr Pro Gln Val Phe Asp Asn Gln Tyr Phe Lys Asp

245 250 255

Leu Val Asn Gly Arg Gly Phe Leu Asn Ser Asp Glu Thr Leu Phe Thr

260 265 270

Asn Pro Glu Thr Arg Gly Tyr Val Val Gln Tyr Arg Arg Asn Glu Ser

275 280 285

Ala Phe Phe Glu Ala Phe Val Glu Gly Met Ile Lys Met Gly Asp Leu

290 295 300

Gln Ser Gly Arg Pro Gly Glu Ile Arg Arg Asn Cys Arg Val Val Asn

305 310 315 320

Ser Trp Glu Pro Ala Lys Leu Phe Ala Ser Tyr

325 330

<210> 2

<211> 996

<212> DNA

<213> sweet potato (Ipomoea batatas (L.) Lam.)

<400> 2

atgatgtata taacgctcgt catcttcttc ctcctcaact tgggcgcgat ccaggcggag 60

atcgtggagc tccggcccgg attttactcc gatacgtgtc cagaagcgga ggatatcgtg 120

aggggcgtga tcaagaggaa catggaaaga gaacccagga gcgccgcctc agtgatgcgc 180

ttgcagtttc acgattgctt tgttaacgga tgcgatgcgt cgttgttgtt ggatgatacg 240

ccggagatgt tgggagagaa gctttgtttg tcgaatataa attcgctgag gtcgtatgaa 300

gttgttgatg aagctaagga agctgtggag atggcctgtc ctggtgttgt ttcctgtgct 360

gatatcataa tcatggctgc cagagatgct gttgttctga gtggaggacc taactgggaa 420

gtaaagctgg gaaggataga cagcttaaca gcaagccaag aagatgcaga caatatcatg 480

ccaagcccaa gagcaaatgc agacaccctc attgatctgt ttaacagatt caatctgtca 540

gtgaaagatc tggtggcact ttcagggtct cactccattg gccagggaag gtgtttttcc 600

atcgtgtttc ggctctacaa ccagtcggga acaggccggc ctgacccgac catcgagcca 660

aacttcagag aaaaactgga caacctttgc ccgctgggcg gggatggaaa tgtaacgggg 720

gacttggacg caacccctca agtattcgac aaccagtact tcaaggactt ggtgaatggg 780

agaggatttc tgaactcaga tgaaacactt ttcactaacc ctgagaccag agggtatgtg 840

gtgcagtata gaagaaatga gagtgcattc tttgaggcat ttgttgaggg gatgataaaa 900

atgggtgatc ttcaatctgg gaggcctgga gagattagga gaaactgcag agtggtcaat 960

agctgggaac ctgcaaagtt gtttgcttca tattga 996

<210> 3

<211> 996

<212> DNA

<213> sweet Potato (Ipomoea batatas (L.) Lam.)

<400> 3

atgatgtata taacgctcgt catcttcttc ctcctcaact tgggcgcgat ccaggcggag 60

atcgtggagc tccggcccgg attttactcc gagacgtgtc cagaagcgga ggatatcgtg 120

aggggcgtga tcaagaggaa catggaaaga gaacccagga gcgccgcctc agtgatgcgc 180

ttgcagtttc acgattgctt tgttaacgga tgcgatgcgt cgttgttgtt ggatgatacg 240

ccggagatgt tgggagagaa gctttgtttg tcgaatataa attcgctgag gtcatatgaa 300

gttgttgatg aagctaagga agctgtggag atggcctgtc ctggtgttgt ttcctgtgct 360

gatatcataa tcatggctgc cagagatgct gttgttctga gtggaggacc taactgggaa 420

gtaaagctgg gaaggataga cagcttaaca gcaagccaag aagatgcaga caatatcatg 480

ccaagcccaa gagcagatgc aaccaccctc attgatctgt ttagcaaatt caatctgtca 540

gtgaaagatc tggtggcact ttcagggtct cactccattg gccagggaag gtgtttttcc 600

atcgtgtttc gcctctacaa ccagtcgggg acaggccggc ctgacccgac catcgagcca 660

aacttcagag aaaaactgga caacctttgc ccgctgggcg gggatgggaa tgtaacgggg 720

gacttggacg caacccctca agtattcgac aaccagtact tcaaggactt ggtgaatggg 780

agaggatttc tgaactcaga tgaaacactt ttcactaacc ctgagaccag agggtatgtg 840

gtgcagtata gaagaaatga gagtgcattc tttgaggcat ttgttgaggg gatgataaaa 900

atgggtgatc ttcaatctgg gaggcctgga gagattagga gaaactgcag agtggtcaat 960

agctgggaac ctgcaaagtt gtttgcttca tattga 996

Claims (10)

1. Use of a protein or a substance modulating the activity and/or content of said protein, wherein said use is any one of:

D1) the use of a protein or a substance which modulates the activity and/or content of said protein in the modulation of the development of plant striations;

D2) the application of protein or substance for regulating and controlling the activity and/or content of the protein in preparing products for regulating and controlling the development of plant striae fascicularis;

D3) use of a protein or a substance modulating the activity and/or content of said protein for the cultivation of a plant with altered tendon development;

D4) use of a protein or a substance modulating the activity and/or content of said protein for the preparation of a product for the cultivation of a plant with altered tendon development;

D5) the application of the protein or the substance for regulating and controlling the activity and/or the content of the protein in the improvement of the species of the sweet potato with the gluten or the preparation of the product for improving the species of the sweet potato with the gluten;

D6) use of a protein or a substance modulating the activity and/or content of said protein in plant breeding;

the protein is any one of the following:

A1) a protein having an amino acid sequence of SEQ ID No. 1;

A2) a protein which is obtained by substituting and/or deleting and/or adding amino acid residues to the amino acid sequence shown in SEQ ID No.1, has more than 80% of identity with the protein shown in A1), and has the same function;

A3) a fusion protein with the same function obtained by connecting labels at the N end and/or the C end of A1) or A2).

2. The use of claim 1, wherein the protein is derived from sweet potato.

3. Use of a biological material related to a protein as claimed in claim 1 or 2, wherein said use is any of the following:

E1) use of a biological material related to a protein as claimed in claim 1 or 2 for modulating the development of plant striated tendons;

E2) use of a biomaterial related to a protein as claimed in claim 1 or 2 in the manufacture of a product for modulating the development of plant streak tendon;

E3) use of a biological material related to a protein as defined in claim 1 or 2 for growing plants with altered fibrilar development;

E4) use of a biological material related to a protein as defined in claim 1 or 2 for the preparation of a product for growing plants with altered tendon development;

E5) use of a biological material related to the protein of claim 1 or 2 for the improvement of a species of a sweet potato or for the preparation of a product for the improvement of a species of a sweet potato;

E6) use of a biological material related to a protein as defined in claim 1 or 2 in plant breeding;

the biomaterial is any one of the following B1) to B8):

B1) a nucleic acid molecule encoding the protein of claim 1 or 2;

B2) a nucleic acid molecule that inhibits or reduces or silences the expression of a gene encoding a protein of claim 1 or 2;

B3) an expression cassette comprising the nucleic acid molecule of B1) and/or B2);

B4) a recombinant vector containing the nucleic acid molecule according to B1) and/or B2), or a recombinant vector containing the expression cassette according to B3);

B5) a recombinant microorganism containing the nucleic acid molecule according to B1) and/or B2), or a recombinant microorganism containing the expression cassette according to B3), or a recombinant microorganism containing the recombinant vector according to B4);

B6) a transgenic plant cell line which contains the nucleic acid molecule described in B1) and/or B2), or a transgenic plant cell line which contains the expression cassette described in B3);

B7) transgenic plant tissue comprising the nucleic acid molecule according to B1) and/or B2), or transgenic plant tissue comprising the expression cassette according to B3);

B8) a transgenic plant organ containing B1) and/or B2) said nucleic acid molecule, or a transgenic plant organ containing B3) said expression cassette.

4. The use according to claim 3, wherein B1) the nucleic acid molecule is any one of:

C1) the coding sequence is a DNA molecule of SEQ ID No. 2;

C2) the nucleotide sequence is the DNA molecule of SEQ ID No. 2.

5. A method for producing a plant with altered striated development, comprising increasing the protein content and/or activity of claim 1 or 2 in a plant of interest to produce a plant with altered striated development that is greater than the striated development of said plant of interest.

6. The method according to claim 5, wherein the increase in the content and/or activity of the protein of claim 1 or 2 in the plant of interest is achieved by increasing the expression level of a gene encoding the protein in the plant of interest.

7. The method according to claim 6, wherein the increase in the expression level of the gene encoding the protein in the target plant is achieved by introducing the gene encoding the protein according to claim 1 or 2 into the target plant.

8. A method for producing a plant with altered striated development, comprising reducing the protein content and/or activity of claim 1 or 2 in a plant of interest to produce a plant with altered striated development that is less extensive than the plant of interest.

9. Use according to any one of claims 1 to 4, and/or method according to any one of claims 5 to 8, wherein the plant is any one of

G1) A monocot or dicot;

G2) a plant of the family Convolvulaceae;

G3) a plant of the genus Ipomoea;

G4) a sweet potato group plant;

G5) sweet potato.

10. The protein of claim 1 or 2, and/or the nucleic acid molecule of claim 3 or 4.

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