CN114591927B - Sweet potato block bar development related protein IbPRX17, and coding gene and application thereof - Google Patents

Abstract

The invention discloses a sweet potato block bar development related protein IbPRX17, and a coding gene and application thereof. The invention specifically discloses application of a protein with an amino acid sequence of SEQ ID No.1 or a substance for regulating the activity and/or content of the protein in regulating the development of plant (such as sweet potato) streaks. According to the invention, the coding gene (IbPRX 17 gene) of the protein is introduced into a receptor plant (sweet potato chestnut aroma) to obtain a transgenic plant which over-expresses the IbPRX17 gene, and compared with a wild sweet potato, the transgenic plant has obviously stronger bar development than the wild type and presents a phenotype of promoting the bar development of the potato blocks. The IbPRX17 protein and the coding gene thereof provided by the invention have important theoretical significance and application value in research on the influence of sweet potato on the development of streak.

Description

Sweet potato block bar development related protein IbPRX17, and coding gene and application thereof

Technical Field

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

Background

Sweet potato (Ipomoea batatas (l.) lam.) is an important food, feed, industrial raw material and new energy crop. China is the largest sweet potato producer in the world. Under the influence of cultivation conditions such as soil, water and fertilizer, planting density, growth period length and the like, the sweet potato variety can easily dedifferentiate parenchyma cells of the secondary phloem of the sweet potato to form new meristematic tissues, and further develop into a cambium, so that the phenotype of streaks is shown. The development of the ribs on the surface of the potato block not only affects the appearance quality of the potato block, but also seriously reduces the commodity value of the potato block and reduces the economic benefit of the sweet potato. Therefore, a new variety of sweet potato with excellent cultivated quality becomes one of important measures for promoting the development of the sweet potato industry.

In addition, the sweet potato has the problems of self incompatibility, unstable filial generation, lack of germplasm resources, long breeding period and the like, the traditional hybridization breeding method is difficult to breed a new sweet potato variety with excellent quality, and the genetic engineering means such as gene editing and the like can be used for directionally improving the sweet potato character, so that the method is a feasible way for breeding the high-quality sweet potato variety at present. The sweet potato gene causing the streak is excavated, knocked out and edited by using a gene editing method, and the sweet potato excellent main variety which is easy to cause streak is improved, so that the method has important value for the development of the sweet potato industry. The invention identifies the protein related to the development of the sweet potato strips and the coding gene thereof for the first time, provides a basis for further elucidating the related mechanism causing the sweet potato strips, provides a scientific basis for cultivating new varieties of sweet potato, and has important theoretical guiding significance and practical application value for high yield and excellent harvest of sweet potato.

Disclosure of Invention

The technical problem to be solved by the invention is how to regulate or improve the development of the streak of plants (such as sweet potatoes). The technical problems to be solved are not limited to the described technical subject matter, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.

To solve the above technical problems, the present invention provides first an application of a protein or a substance regulating the activity and/or content of the protein, wherein the application may be any of the following:

d1 Use of a protein or a substance regulating the activity and/or content of said protein for regulating plant bar development;

d2 Use of a protein or a substance regulating the activity and/or content of said protein for the preparation of a product regulating the development of plant bars;

d3 Use of a protein or a substance regulating the activity and/or content of said protein for growing plants with altered bar development;

d4 Use of a protein or a substance regulating the activity and/or content of said protein for the preparation of a product for growing plants with altered bar development;

d5 Protein or a substance regulating the activity and/or content of the protein in improving a variety of raised sweet potatoes or in preparing a product of improving a variety of raised sweet potatoes;

d6 Use of a protein or a substance regulating the activity and/or content of said protein in plant breeding;

the protein is named 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 an amino acid residue in 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 Fusion proteins having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of A1) or A2).

In order to facilitate purification or detection of the protein of A1), a tag protein may be attached to the amino-or carboxy-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-sulfhydryl transferase) 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), eFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

The nucleotide sequence encoding the protein IbPRX17 of the present invention can be easily mutated by a person skilled in the art using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the protein IbPRX17 isolated according to the invention are derived from the nucleotide sequence of the invention and are identical to the sequence of the invention, provided that they encode the protein IbPRX17 and function as the protein IbPRX17.

The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

Herein, identity refers to identity of an amino acid sequence or a nucleotide sequence. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in 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 Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and search is performed to calculate the identity of amino acid sequences, and then the value (%) of identity can be obtained.

Herein, the 80% identity or more may 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 that modulates the activity and/or content of the protein may be a substance that modulates the expression of a gene encoding the protein IbPRX17.

In the above, the substance that regulates gene expression may be a substance that performs at least one of the following 6 regulation: 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 the 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 the protein translated by the gene).

The substance for regulating gene expression may specifically be any of the biological materials described in the following B1) to B4).

In the above application, the protein IbPRX17 may be derived from sweet potato (Ipomoea batatas (l.) lam.).

The invention also provides the use of biological material related to the protein IbPRX17, which may be any of the following:

e1 Use of biological material related to said protein IbPRX17 for regulating plant bar development;

e2 Use of a biological material related to said protein IbPRX17 for the preparation of a product for regulating plant bar development;

e3 Use of biological material related to said protein IbPRX17 for growing plants with altered bar development;

e4 Use of a biological material related to said protein IbPRX17 for the preparation of a product for growing plants with altered bar development;

e5 Use of a biological material related to said protein IbPRX17 for improving a variety of raised sweet potato or for preparing a product of improved variety of raised sweet potato;

e6 Use of biological material related to said protein IbPRX17 in plant breeding;

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

b1 A nucleic acid molecule encoding said protein IbPRX 17;

b2 A nucleic acid molecule that inhibits or reduces or silences expression of a gene encoding the protein IbPRX 17;

b3 An expression cassette containing the nucleic acid molecule of B1) and/or B2);

b4 A recombinant vector comprising the nucleic acid molecule of B1) and/or B2) or a recombinant vector comprising the expression cassette of B3);

b5 A recombinant microorganism comprising the nucleic acid molecule of B1) and/or B2), or a recombinant microorganism comprising the expression cassette of B3), or a recombinant microorganism comprising the recombinant vector of B4);

b6 A transgenic plant cell line comprising the nucleic acid molecule of B1) and/or B2) or a transgenic plant cell line comprising the expression cassette of B3);

b7 A) transgenic plant tissue containing the nucleic acid molecule of B1) and/or B2) or a transgenic plant tissue containing the expression cassette of B3);

b8 A transgenic plant organ comprising the nucleic acid molecule of B1) and/or B2) or a transgenic plant organ comprising the expression cassette of B3).

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

c1 A DNA molecule whose coding sequence is SEQ ID No. 2;

c2 A DNA molecule with the nucleotide sequence of SEQ ID No. 2.

The DNA molecule shown in SEQ ID No.2 (gene IbPRX17 for regulating plant bar development) codes for the protein IbPRX17 (sweet potato block bar development related protein IbPRX 17) with the amino acid sequence shown in SEQ ID No. 1.

The nucleotide sequence shown in SEQ ID NO.2 is the nucleotide sequence of the coding gene (CDS) of the protein IbPRX17. The gene of the protein IbPRX17 (IbPRX 17 gene) according to the invention may be any nucleotide sequence capable of encoding the protein IbPRX17. In view of the degeneracy of codons and the preferences of codons of different species, one skilled in the art can use codons appropriate for expression of a particular species as desired.

B1 The nucleic acid molecules may also comprise nucleic acid molecules which have been modified by codon preference on the basis of the nucleotide sequence indicated in SEQ ID No. 2.

The nucleic acid molecules also comprise nucleic acid molecules which have more than 95% nucleotide sequence identity with the nucleotide sequence shown in SEQ ID No.2 and are derived from the same species.

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

Vectors described herein are well known to those of skill 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 a pMD19-T vector and/or a pCAMBIA1300 vector.

Recombinant expression vectors containing the IbPRX17 gene can be constructed using existing plant expression vectors. Such plant expression vectors include, but are not limited to, vectors such as binary Agrobacterium vectors and vectors useful for microprojectile bombardment of plants, and the like. The plant expression vector may also comprise the 3' -untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal may direct the addition of polyadenylation to the 3 'end of the mRNA precursor and may function similarly to untranslated regions transcribed from the 3' end of plant genes including, but not limited to, agrobacterium tumefaciens induction (Ti) plasmid genes (e.g., nopaline synthase Nos genes), plant genes (e.g., soybean storage protein genes).

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

In order to facilitate the identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, such as by adding genes encoding enzymes or luminescent compounds that produce a color change (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin markers, kanamycin markers, etc.), or anti-chemical marker genes (e.g., anti-herbicide genes), etc., which may be expressed in plants. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.

The IbPRX17 gene or the gene fragment provided by the invention is introduced into plant cells or receptor plants by using any vector capable of guiding exogenous genes to express in plants, so that a transgenic cell line and a transgenic plant with enhanced bar development can be obtained. Expression vectors carrying the IbPRX17 gene can be used to transform plant cells or tissues by conventional biological methods using Ti plasmids, ri plasmids, plant viral vectors, direct DNA transformation, microinjection, conductance, agrobacterium-mediated, etc., and the transformed plant tissues are grown into plants.

The microorganism described herein may be a yeast, bacterium, algae or fungus. Wherein the bacteria may be derived from Escherichia, erwinia, agrobacterium (Agrobacterium), flavobacterium (Flavobacterium), alcaligenes (Alcaligenes), pseudomonas, bacillus (Bacillus), etc. Specifically, the bacillus coli DH5 alpha and/or the agrobacterium tumefaciens EHA105 can be used.

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

The present invention also provides a method of growing plants with altered streak development, the method comprising increasing the level and/or activity of the protein IbPRX17 in a plant of interest to obtain a plant with altered streak development that is stronger than the streak development of the plant of interest.

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

In the above method, the increase in 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:

h1 A DNA molecule whose coding sequence is SEQ ID No. 2;

h2 A DNA molecule with the nucleotide sequence of SEQ ID No. 2.

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

In one embodiment of the invention, the method of growing plants with altered bar development 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) Obtaining the plants with the changed bar development stronger than the target plants through screening and identification.

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

The present invention also provides a method of growing plants with altered streak development comprising reducing the level and/or activity of the protein IbPRX17 in a plant of interest to yield a plant with altered streak development that is weaker than the streak development of the plant of interest.

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

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 a reduction or inactivation of the activity of the gene encoding the protein IbPRX17 in the genome of the target plant by using a gene mutation, gene knockout, gene editing or gene knockdown technique.

Herein, the plant may be any one of the following

G1 Monocotyledonous or dicotyledonous plants;

g2 A plant of the family Convolvulaceae;

g3 Sweet potato plant;

g4 Sweet potato group plants;

g5 Sweet potato.

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

Herein, the modulation of plant streak development may be up-regulating (enhancing or promoting) plant streak development or down-regulating (attenuating or inhibiting) plant streak development.

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

The plant with enhanced development of the streak can be a multi-streak sweet potato variety or a streak sweet potato variety.

The plant with reduced development of the streak can be a sweet potato variety with little streak or a sweet potato variety without streak.

In the present invention, the plant with altered streak 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 that species, or may be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The stress-resistant plants include seeds, calli, whole plants and cells.

The IbPRX17 gene provided by the invention codes a peroxidase protein, and the gene is introduced into sweet potato to obtain a transgenic sweet potato plant which over-expresses the IbPRX17 gene, and the transgenic sweet potato plant presents a phenotype of promoting the development of the bar of the sweet potato. The IbPRX17 protein and the coding gene thereof provided by the invention have important theoretical significance and application value in research on the influence of sweet potato on the development of streak.

Drawings

FIG. 1 is a PCR assay for transgenic plants.

FIG. 2 is a view of the bar phenotype of transgenic potato blocks planted in Guangzhou market.

FIG. 3 is a view of the bar phenotype of transgenic potato blocks planted in Haikou City.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The pMD19-T vector in the examples described below is a product of Takara Bio-engineering (Dalian) under the product catalog number 6013. The pCAMBIA1300 vector is a product of Camcia company.

The total RNA extraction kit for plants in the following examples is a product of Tiangen Biochemical technology (Beijing) limited company, and the product catalog number is DP432.HiFiScript gDNA Removal RT MasterMix the kit is a product of century biotechnology (Beijing) limited company, and the product catalog number is CW2020M. KpnI enzyme is the product of Simer Feishul technology (China) with the product catalog number of FD0524.SalI enzyme is available from Siemens technology (China) with catalog number FD0644. Coli DH5 alpha is a product of Shenzhen Kangbody life technology Co., ltd, the product catalog number is KTSM101L, and the Agrobacterium tumefaciens EHA105 is a product of Beijing qingke biological technology Co., ltd, and the product catalog number is TSC-A03.

The sweet potato variety chestnut note in the following examples is described in the following literature: wang Yuping, liu Qingchang, li Aixian, etc. in vitro screening and identification of drought-enduring mutants of sweetpotato [ J ]. Chinese agricultural science, 2003,36 (9): 1000-1005. The public is available from the university of Chinese agriculture, sweet potato genetic breeding laboratory, to repeat the experiment.

The sweet potato variety ND98 in the following examples is described in the following documents: he Shaozhen in vitro screening of sweet potato salt-tolerant mutants and cloning of salt-tolerant candidate genes [ D ]. Doctor's academy of Chinese agricultural university, 2008, public were obtained from the national agricultural university sweet potato genetic breeding laboratory to repeat the experiment.

LB solid/liquid medium, MS solid/liquid medium are described in the following documents: zhang Huan cloning and functional verification of the stress-resistance related genes IbBBX24 and IbCPK28 by sweetpotato salt-tolerant transcriptome analysis [ D ]. Doctor's academic paper, 2017, of the university of agriculture.

Example 1 acquisition of IbPRX17 Gene

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

2. The interactive gene IbPRX17 is obtained by screening a sweet potato stress-resistant gene IbBBX24 through a sweet potato yeast two-hybrid library, and is compared in a Sweetpotato Garden library (http:// sweetpotto-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 are designed and artificially synthesized according to the nucleotide sequence of the EST sequence, and the sequences are as follows:

IbPRX17-F：5’-ATGATGTATATAACGCTCGTCATCT-3’

IbPRX17-R：5’-TCAATATGAAGCAAACAACTTTGCAG-3’

3. and (3) carrying out PCR amplification by taking 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 amplified fragment product of about 996bp and sequencing.

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 in the sequence table is named as IbPRX17 gene, the encoded protein is named as 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 regulating and controlling sweet potato block bar development

1. Construction of plant expression vectors

According to the coding sequence (SEQ ID No. 2) of the sweet potato IbPRX17 protein nucleotide, designing a primer sequence for amplifying a complete coding sequence (CDS), respectively introducing KpnI and SalI restriction sites into forward and reverse primers, wherein the primer sequence is as follows:

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

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

The double-stranded DNA molecule shown in the artificially synthesized SEQ ID No.2 is used as a template, after PCR amplification, the product is connected to a pMD19-T vector to obtain a recombinant vector, which is named pMD-IbPRX17, and the sequencing of M13-F/R is carried out to ensure the accuracy of the reading frame and the enzyme cutting site of the sweet potato IbPRX17 protein nucleotide.

M13-F:5’-GTAAAACGACGGCCAGT-3’，

M13-R:5’-CAGGAAACAGCTATGAC-3’。

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

And (3) 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. The target plasmid is transformed into escherichia coli DH5 alpha, cultured for 20 hours at 37 ℃, and then the PCR analysis and the digestion identification of the recombinant vector are carried out, and the sequencing verification is carried out. Sequencing results show that the sequence shown in SEQ ID No.2 in the sequence table is inserted between KpnI and SalI enzyme cutting sites of the vector pCAMBIA1300, which proves 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 with a nucleotide sequence of SEQ ID No.2 in a sequence table, and keeping other sequences of the pCAMBIA1300 vector unchanged. Recombinant vector pCAMBIA1300-IbPRX17 expresses the protein IbPRX17 shown in SEQ ID No. 1.

The recombinant vector pCAMBIA1300-IbPRX17 is provided with an expression cassette, and the nucleotide sequence of the expression cassette is provided with a CaMV35S promoter, an encoding gene of IbPRX17 protein and a NOS terminator.

2. Agrobacterium transformation with plant expression vectors

(1) Melting competent cells of Agrobacterium tumefaciens EHA105 on ice, adding 2 μg of the extracted pCAMBIA1300-IbPRX17 plasmid, mixing with light elastic tube wall, and ice-bathing for 10min;

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

(3) 600. Mu.L of liquid LB medium is added, and the culture is carried out for 5 hours at 28 ℃ and 200 rpm;

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

(5) Culturing 2d in an inverted dark at 28deg.C, culturing 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 potato

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

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

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

(3) Screening the embryogenic cell mass by a 20-mesh screen, transferring the larger cell mass to a 30-mesh screen, lightly grinding to enable the embryogenic cell mass to generate wounds, and carrying out shaking culture on the grinded larger embryogenic cell mass for 3 days;

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

(5) The embryogenic cell mass was washed once with MS liquid medium containing 400mg/L Cefotaxime Sodium (CS) and 2.0 mg/L2, 4-D, and then shake-cultured in MS liquid medium containing 2.0 mg/L2, 4-D for 1 week;

(6) Placing embryogenic cell masses on a screening medium (MS solid medium containing 100mg/L CS, 5mg/L hygromycin (Hyg), 2, 4-D), and culturing at 28deg.C for 10-12 weeks with medium replacement every two weeks;

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

(8) And (3) placing the resistant callus on an MS solid culture medium, and alternately culturing at 28 ℃ in a light-dark manner for 4-8 weeks to obtain 14 transgenic plants to be identified (quasi-transgenic 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 leaves of a plant to be transgenic by using a CTAB method, taking the extracted genome DNA as a template, taking water and a wild plant (chestnut fragrance) as negative controls, taking plasmid pCAMBIA1300-IbPRX17 as positive controls, and taking CaMV35S (5'-TGACGCACAATCCCACTATCCT-3') and IbPRX17-RR-SalI as primers for PCR amplification to obtain PCR amplification products; if the PCR amplification product contains a band of about 1234bp, the corresponding transgenic plant to be identified of the sweet potato is the transgenic positive plant of the sweet potato.

The result of electrophoresis detection amplification is shown in FIG. 1 (in FIG. 1, lane M is shown as a marker band, and lane W is shown as a band of negative control (water); lane P shows the band of positive control (recombinant plasmid pCAMBIA1300-IbPRX 17), lane WT shows the band of sweetpotato chestnut aromatic plants, 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, OE-P16 shows the band of sweet potato quasi-transgenic plants transformed with pCAMBIA1300-IbPRX17, as seen in FIG. 1, lanes OE-P9, OE-P10, OE-P11, OE-P14, OE-P15, OE-P16 and positive control amplified 1234bp target bands, indicating that the IbPRX17 gene had been integrated into the genome of sweetpotato chestnut aromatic and demonstrated that these regenerated plants were positive for the transgenic seedlings (OE-P9, OE-P10, OE-P11, OE-P14, and OE-P16) were amplified by a further propagation method as a seed line.

4. Phenotype observation of potato block bar development of transgenic plant

Sweet potato blocks planted in 130 days of isolated fields were harvested from Guangzhou city in Guangdong in 2020, and the phenotypic growth status of the sweet potato blocks was observed and photographed for recording. The result of enlarging the 1 potato piece bar phenotype of 3 potato pieces per strain is shown in fig. 2. The result shows that the bar development of each transgenic potato block (strain number is OE-P14, OE-P15 and OE-P16) over-expressing the IbPRX17 gene is obviously stronger than that of a wild chestnut fragrant potato block (strain number is LZX).

Sweet potato blocks planted in the isolated field for 150 days were harvested from Haikou City of Hainan province in 2021, the phenotypic growth condition of the sweet potato blocks was observed, and photographed and recorded. The result of enlarging the 1 potato block bar phenotype of 1 strain is shown in figure 3. The results show that each potato piece of the transgenic potato pieces (strain numbers of OE-P14, OE-P15 and OE-P16) over-expressing the IbPRX17 gene has obviously stronger development than the wild chestnut sweet potato pieces (strain number of LZX).

The above results indicate that sweet potato blocks over-expressing the IbPRX17 gene exhibit a phenotype with pronounced bar development.

The IbPRX17 protein and the coding gene IbPRX17 thereof can regulate and control the development of the plant (such as sweet potato) and can obviously enhance the development of the plant by increasing the content and/or activity of the IbPRX17 protein in the target plant (such as over-expression of the IbPRX17 gene). Conversely, the development of the plant's streak can be significantly reduced by reducing the amount and/or activity of the IbPRX17 protein in the plant of interest (e.g., inhibiting the expression of the IbPRX17 gene).

The present invention is described in detail above. It will be apparent to those skilled in the art that the present 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 respect to specific embodiments, 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 application of some of the basic features may be done in accordance with the scope of the claims that follow.

SEQUENCE LISTING

<110> Chinese university of agriculture

<120> sweet potato block bar 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 (11)

1. Use of a protein or a substance regulating the activity and/or content of said protein, characterized in that said use is any of the following:

d1 Use of a protein or a substance regulating the activity and/or content of said protein for regulating plant bar development;

d2 Use of a protein or a substance regulating the activity and/or content of said protein for the preparation of a product regulating the development of plant bars;

d3 Use of a protein or a substance regulating the activity and/or content of said protein for growing plants with altered bar development;

d4 Use of a protein or a substance regulating the activity and/or content of said protein for the preparation of a product for growing plants with altered bar development;

d5 Protein or a substance regulating the activity and/or content of the protein in improving a variety of raised sweet potatoes or in preparing a product of improving a variety of raised sweet potatoes;

d6 Use of a protein or a substance regulating 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 fusion protein with the same function obtained by connecting a label to the N end and/or the C end of A1);

the substance regulating the activity and/or content of the protein is a gene encoding the protein; the plant is sweet potato plant.

2. The use according to claim 1, wherein the sweet potato plant is a sweet potato group plant.

3. The use according to claim 2, wherein the sweet potato group plant is sweet potato.

4. Use according to any one of claims 1-3, characterized in that the protein is derived from sweet potato.

5. Use of a biological material related to a protein according to any one of claims 1-4, characterized in that the use is any one of the following:

e1 Use of a biological material related to the protein of any one of claims 1-4 for regulating plant bar development;

e2 Use of a biological material related to the protein of any one of claims 1-4 for the preparation of a product for regulating plant bar development;

e3 Use of a biological material related to a protein according to any one of claims 1 to 4 for growing plants with altered bar development;

e4 Use of a biological material related to the protein of any one of claims 1-4 for the preparation of a product for growing plants with altered bar development;

e5 Use of a biological material related to the protein of any one of claims 1-4 for improving a variety of raised sweet potato or for preparing a product of improved variety of raised sweet potato;

e6 Use of a biological material related to the protein of any one of claims 1-4 in plant breeding;

the biomaterial is any one of the following B1) to B7):

b1 A nucleic acid molecule encoding a protein according to any one of claims 1 to 4;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

b5 A transgenic plant cell line comprising the nucleic acid molecule of B1) or a transgenic plant cell line comprising the expression cassette of B2);

b6 A transgenic plant tissue comprising the nucleic acid molecule of B1) or a transgenic plant tissue comprising the expression cassette of B2);

b7 A transgenic plant organ comprising the nucleic acid molecule of B1) or a transgenic plant organ comprising the expression cassette of B2);

the plant is sweet potato plant.

6. The use according to claim 5, wherein the sweet potato plant is a sweet potato group plant.

7. The use according to claim 6, wherein the sweet potato group plant is sweet potato.

8. The use according to any one of claims 5 to 7, wherein the nucleic acid molecule of B1) is any one of the following:

c1 A DNA molecule whose coding sequence is SEQ ID No. 2;

c2 A DNA molecule with the nucleotide sequence of SEQ ID No. 2.

9. A method for growing a plant with altered shoot development, which comprises increasing the expression level of a gene encoding the protein of any one of claims 1 to 4 in a target plant, whereby a plant with altered shoot development stronger than the target plant is obtained, said increasing the expression level of a gene encoding the protein of any one of claims 1 to 4 in a target plant is achieved by introducing the gene encoding the protein of any one of claims 1 to 4 into said target plant, said plant being a plant of the genus sweetpotato.

10. The method of claim 9, wherein the sweet potato plant is a sweet potato group plant.

11. The method of claim 10, wherein the sweet potato group plant is sweet potato.

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