CN117164686A

CN117164686A - Stress resistance related protein IbRCD1, related biological material and application thereof

Info

Publication number: CN117164686A
Application number: CN202210508582.9A
Authority: CN
Inventors: 何绍贞; 张欢; 刘庆昌; 翟红; 高少培; 赵宁; 张铅
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2022-05-11
Filing date: 2022-05-11
Publication date: 2023-12-05

Abstract

The application discloses stress-resistance related protein IbRCD1, and related biological materials and application thereof. 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 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). The application obtains the over-expressed transgenic plant by introducing the coding gene of the protein into the receptor plant (sweet potato chestnut flavor). Compared with wild sweet potato, the transgenic plant has obviously enhanced salt tolerance. The IbRCD1 protein and the coding gene thereof provided by the application can be used for creating stress-resistant plants and/or plant breeding and/or plant germplasm resource improvement.

Description

Stress resistance related protein IbRCD1, related biological material and application thereof

Technical Field

The application belongs to the technical field of biology, and particularly relates to stress-resistance related protein IbRCD1, and related biological materials and application thereof.

Background

Sweet potatoes (Ipomoea batatas (l.) lam.) are important foodstuffs, feeds, industrial raw materials and new energy crops. China is the largest sweet potato producing country in the world, southeast coastal hillside fields and the vast drought areas in the west are main sweet potato planting areas, and soil salinization and drought become important factors restricting sweet potato production in the areas. Salt stress is the decrease in water potential in the cells of the affected plants, and the damage to plants caused by excessive sodium ions affecting the key biochemical pathways of the plant cells, mainly in osmotic stress and ion stress, etc., which destroy plant cell membrane structures, affect the activities of many enzymes and the functions of photosynthesis organs, and also generate active oxygen in the plants, thereby causing oxidative stress, and preventing plant growth. Along with the gradual perfection of the transgenic technology, the genetic engineering means is utilized to improve the salt tolerance of plants, and important salt tolerance genetic resources are excavated, so that the method plays a key role in cultivating new varieties of salt tolerance plants, and the development and utilization of the salt tolerance plants have immeasurable ecological benefits, economic benefits and social benefits.

The sweet potato has the characteristics of difficult flowering and self-hybridization incompatibility, and simultaneously, the germplasm resources are relatively deficient and the genetic basis is narrow, so that the breeding of new varieties of high-quality high-yield high-resistance sweet potato by utilizing the conventional breeding means is severely restricted. The genetic engineering technology is used for improving the sweet potato variety, so that the barriers of reproductive isolation, gene linkage and the like existing in conventional breeding can be overcome, and the yield, quality and stress resistance of the sweet potato can be directionally improved on a molecular level. The breeding of new sweet potato varieties with high yield, high quality and high stress resistance is a main goal of sweet potato breeding. Therefore, the gene related to the stress resistance of the sweet potato is cloned and regulated, a new high-yield high-quality high-stress-resistance sweet potato material is created, and the gene has very important theoretical reference significance and application value for high-quality, high-yield and stress-resistance breeding work of the sweet potato.

Disclosure of Invention

The technical problem to be solved by the application is how to regulate the stress resistance of plants and/or how to improve the salt tolerance of plants. 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 application provides a protein, i.e., ibRCD1, where IbRCD1 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 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 IbRCD1 of the present application 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 IbRCD1 isolated according to the application are derived from the nucleotide sequence of the application and are identical to the sequence of the application, provided that they encode the protein IbRCD1 and function as the protein IbRCD1.

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.

Further, the protein IbRCD1 may be derived from sweet potato (Ipomoea batatas (l.) lam.).

The protein can be IbRCD1.

The present application also provides a biomaterial, which may be any one of the following B1) to B7):

b1 A nucleic acid molecule encoding said protein IbRCD 1;

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).

In the above biological material, 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 (IbRCD 1 gene) encodes a protein IbRCD1 whose amino acid sequence is SEQ ID No. 1.

The nucleotide sequence shown in SEQ ID NO.2 is the nucleotide sequence of the gene (CDS) encoding the protein IbRCD1.

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.

B1 The nucleic acid molecules also include nucleic acid molecules which have more than 95% identity to the nucleotide sequence shown in SEQ ID No.2 and are of 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 pCAMBIA1300-GFP and/or the vector pEASY-blue simple can be used.

Recombinant expression vectors containing the IbRCD1 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 IbRCD1 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 application 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 IbRCD1 gene or the gene fragment provided by the application is introduced into plant cells or receptor plants by using any vector capable of guiding the expression of exogenous genes in plants, and transgenic cell lines and transgenic plants with improved stress resistance (such as salt tolerance) can be obtained. Expression vectors carrying the IbRCD1 gene can be obtained by transforming plant cells or tissues using conventional biological methods such as Ti plasmid, ri plasmid, plant viral vector, direct DNA transformation, microinjection, electric conductance, agrobacterium mediation, etc., and cultivating the transformed plant tissues 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, agrobacterium tumefaciens EHA105.

The recombinant vector can be specifically a recombinant vector pCAMBIA1300-IbRCD1-GPF, wherein the recombinant vector pCAMBIA1300-IbRCD1-GPF is a recombinant expression vector obtained by replacing a fragment (small fragment) between KpnI and SalI recognition sites of a pCAMBIA1300-GFP 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-GFP vector unchanged. Recombinant vector pCAMBIA1300-IbRCD1-GPF expresses IbRCD1 protein shown in SEQ ID No.1 of the sequence Listing.

The recombinant microorganism can be specifically recombinant agrobacterium tumefaciens EHA105/pCAMBIA1300-IbRCD1-GPF, and the recombinant agrobacterium tumefaciens EHA105/pCAMBIA1300-IbRCD1-GPF is obtained by introducing the recombinant vector pCAMBIA1300-IbRCD1-GPF into the agrobacterium tumefaciens EHA105.

The application also provides a method for cultivating stress-resistant plants, which comprises the step of increasing the content and/or activity of the protein IbRCD1 in target plants to obtain stress-resistant plants with stress resistance higher than that of the target plants.

In the above method, the increase in the content and/or activity of the protein IbRCD1 in the target plant can be achieved by increasing the expression level of the gene encoding the protein IbRCD1 in the target plant.

In the above method, the increase in the expression level of the gene encoding the protein IbRCD1 in the target plant can be achieved by introducing the gene encoding the protein IbRCD1 into the target plant.

In the above method, the gene encoding the protein IbRCD1 may be any of the following:

f1 A DNA molecule whose coding sequence is SEQ ID No. 2;

f2 A DNA molecule with the nucleotide sequence of SEQ ID No. 2.

Specifically, in one embodiment of the present application, the increase in the expression level of the gene encoding the protein IbRCD1 in the plant of interest is achieved by introducing a DNA molecule shown in SEQ ID No.2 into the plant of interest.

In one embodiment of the application, the method of growing stress-tolerant plants 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 screening and identifying to obtain the stress-resistant plant with stress resistance higher than that of the target plant.

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

The application also provides the use of the protein IbRCD1 or a substance that modulates the activity and/or content of the protein IbRCD1, and/or any of the following of the biological materials:

d1 Application in regulating plant stress resistance;

d2 The application of the plant stress resistance regulating agent in the preparation of products for regulating and controlling plant stress resistance;

d3 Use in the cultivation of stress-tolerant plants;

d4 The application of the plant strain in the preparation of products for cultivating stress-resistant plants;

d5 Use in plant breeding.

In the above applications, the stress resistance includes salt resistance, drought resistance, heat resistance, cold resistance and/or light resistance.

The stress-resistant plants include, but are not limited to, salt-tolerant plants, drought-tolerant plants, heat-tolerant plants, cold-tolerant plants, and/or strong-light-tolerant plants.

Specifically, the stress resistance may specifically be salt resistance.

The application also provides application of the method for cultivating the stress-resistant plant in creating the stress-resistant plant and/or plant breeding and/or plant germplasm resource improvement.

Herein, the substance that modulates the activity and/or content of the protein IbRCD1 may be a substance that modulates the expression of a gene encoding the protein IbRCD1.

Herein, 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 regulating gene expression may specifically be a biological material as described in any one of B1) to B3) herein.

Further, the agent that modulates gene expression may be an agent (including a nucleic acid molecule or vector) that increases or upregulates gene expression of the protein IbRCD1 encoding gene.

Herein, the plant may be a crop (e.g., a crop).

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.

Specifically, the sweet potato can be of sweet potato variety chestnut flavor.

The plant breeding described herein may be crop anti-reverse gene breeding, and in particular, crop salt tolerance transgenic breeding.

The regulation of plant stress resistance may be increasing (up-regulating) plant stress resistance or decreasing (down-regulating) plant stress resistance, in particular increasing plant salt tolerance or decreasing plant salt tolerance.

In the present application, the stress-tolerant plant is understood to include not only the first generation transgenic plant obtained by transforming the IbRCD1 gene into the plant of interest, 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 present application provides a transgenic sweetpotato plant over-expressing a gene IbRCD1 by introducing a gene encoding a stress-resistance related protein IbRCD1 (IbRCD 1 gene) derived from sweetpotato (Ipomoea batatas (l.) lam) into a recipient plant (sweetpotato chestnut flavor). Salt stress treatment is carried out on the transgenic sweet potato plants, and the over-expression strain is found to have obviously enhanced salt tolerance compared with the wild sweet potato. The results show that the IbRCD1 gene and the protein encoded by the gene play an important role in the process of resisting salt stress of plants. The IbRCD1 protein and the coding gene thereof provided by the application have important application value in the research of improving plant stress resistance. The application has wide application space and market prospect in the agricultural field.

Drawings

FIG. 1 shows the PCR amplification results of transgenic plants of sweetpotato.

FIG. 2 shows the results of the transcript level detection of the IbRCD1 gene in different transgenic lines.

FIG. 3 shows the growth status and phenotype index statistics (dry fresh weight) of sweet potato plants.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application 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 application 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.

Sweet potato salt-tolerant mutant ND98 in the following examples is described in the following literature: he Shaozhen screening of sweet potato salt-tolerant mutants ex vivo and cloning of salt-tolerant candidate genes. Doctor's treatises, 2008, of Chinese university of agriculture. The public is available from the national agricultural university sweet potato genetic breeding laboratory to repeat the experiment.

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 Chinese agricultural science, 2003, 36 (9): 1000-1005. The public is available from the national agricultural university sweet potato genetic breeding laboratory to repeat the experiment.

The vector pCAMBIA1300-GFP in the examples described below is a product from Va. Beijing Va. VECT0460. The pEASY-Blunt simple carrier is a product of Beijing full gold biotechnology Co.

The total RNA extraction kit for plants in the following examples is available from Tiangen Biochemical technology (Beijing) Co., ltd, catalog number DP432.PrimeScript ^TM 1st Strand cDNA Synthesis Kit is a product of Takara Bio-engineering (Dalian) Co., ltd, product catalog number 6110A.

The 1/2 Hoagland nutrient solutions in the examples below are described in the following documents: liu Degao obtaining sweet potato plants overexpressing IbP CR, ibERD3, ibELT, ibNFU1 genes and salt tolerance identification Beijing 2014 doctor graduation paper, university of agriculture in China.

The following examples used SPSS 17.0 statistical software to treat the data, and the experimental results were expressed as mean.+ -. Standard deviation, with significant differences indicated by P < 0.05 (x) and extremely significant differences indicated by P < 0.01 (x) using Student's t-test. The quantitative tests in the following examples were carried out in three replicates, and the results were averaged unless otherwise indicated.

Example 1 obtaining IbRCD1 Gene

The IbRCD1 gene was obtained as follows:

1. template acquisition

Extracting total RNA of young leaves of sweet potato salt-tolerant mutant ND98 with plant total RNA extraction kit, and subjecting the total RNA to PrimeScript ^TM 1st Strand cDNA Synthesis Kit reverse transcribes the first strand cDNA.

2. And obtaining a homologous sequence shown as SEQ ID No.3 in the sequence table according to a transcriptome sequencing result. Based on the nucleotide sequence of the sequence, BLAST analysis was performed in the sweet potato closely related wild species (I.trigida, 2n=2x=30) database (http:// sweetpo tat-garden. Kazusa. Or. Jp/BLAST. Html), and primers RCD1-F and RCD1-R were designed and artificially synthesized using this as templates.

3. After the step 2 is completed, PCR amplification is carried out by taking the cDNA obtained in the step 1 as a template and the RCD1-F and the RCD1-R synthesized in the step 2 as primers, so that a PCR amplification product of about 1000bp is obtained and sequenced.

The nucleotide sequence information of RCD1-F and RCD1-R are detailed in Table 1.

TABLE 1 primer sequence information

Primer name	Sequence information 5'-3'
		RCD1-F	5′-ATGGCAAAAATGGAGGCAT-3′
RCD1-R	5′-TCACTTCTTACACATAATTCCA-3′

The result shows that the nucleotide sequence of the PCR amplification product obtained in the step 3 is shown as SEQ ID No.2 (1050 bp), the gene shown in the sequence is named as IbRCD1 gene, the encoded protein is named as IbRCD1 protein or protein IbRCD1, and the amino acid sequence is shown as SEQ ID No.1 in the sequence table.

Example 2, application of IbRCD1 protein in regulating stress resistance of sweet potato

1. Construction of recombinant plasmids

1. The double-stranded DNA molecule (IbRCD 1 gene) shown in positions 1 to 1050 from the 5' -end of SEQ ID No.2 was synthesized. Taking the double-stranded DNA molecule as a template, and taking OE-F-KpnI:5' -GGGGTACCATGGC AAAAATGGAGGCAT-3' and OE-R-SalI:5' -GCGTCGACTCACTTCTTACACATAATTCC A-3' (underlined are recognition sequences of restriction enzymes KpnI and SalI) as primers to obtain a double-stranded DNA molecule having a recognition site of restriction enzyme KpnI at one end and a recognition site of restriction enzyme SalI at the other end.

2. The double-stranded DNA molecule of step 1 was ligated to pEASY-Blunt simple vector to obtain recombinant plasmid pEASY-IbRCD1.

3. After the step 2 is completed, the recombinant plasmid pEASY-IbRCD1 is digested with restriction enzymes KpnI and SalI, and a fragment containing the IbRCD1 gene, abbreviated as fragment 1, is recovered.

4. The pCAMBIA1300-GFP vector was digested with restriction enzymes KpnI and SalI to recover about 1.1Kb fragment, abbreviated as pCAMBIA1300-GFP vector backbone 2.

5. The fragment 1 was ligated with pCAMBIA1300-GFP vector backbone 2 to give recombinant plasmid pCAMBIA1300-IbRCD1-GPF.

Based on the sequencing results, the structure of the recombinant plasmid (recombinant vector) pCAMBIA1300-IbRCD1-GPF is described as follows: the recombinant vector pCAMBIA1300-IbRCD1-GPF is a recombinant expression vector obtained by replacing a segment (small segment) between KpnI and SalI recognition sites of the pCAMBIA1300-GFP 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-GFP vector unchanged. Recombinant vector pCAMBIA1300-IbRCD1-GPF expresses IbRCD1 protein shown in SEQ ID No.1 of the sequence Listing.

2. Acquisition of recombinant Agrobacterium and regeneration of sweet potato transgenic plants

A. Regeneration of sweet potato transgenic positive plants

1. The recombinant plasmid pCAMBIA1300-IbRCD1-GPF is transformed into agrobacterium tumefaciens EHA105 to obtain recombinant agrobacterium tumefaciens A, and the recombinant agrobacterium tumefaciens A is named EHA105/pCAMBIA1300-IbRCD1-GPF.

2. Picking stem tip meristem with chestnut fragrance of about 0.5mm length, placing on embryogenic callus induction solid culture medium (MS solid culture medium containing 2.0 mg/L2, 4-D and 3.0% sucrose), culturing at 27+ -1deg.C for 8 weeks to obtain embryogenic callus, then placing embryogenic callus in embryogenic callus induction liquid culture medium (MS liquid culture medium containing 2.0 mg/L2, 4-D and 3.0% sucrose), and culturing alternately with shaking light and darkness on horizontal shaker for 3D (specific conditions: 100r/min;27 deg.C, light and darkness alternate culture period: 13h, darkness time 11h; light intensity 500 lx), to obtain embryogenic cell mass with diameter of 0.7-1.3 mm.

3. After completion of step 2, embryogenic cell masses were transformed with EHA105/pCAMBIA1300-IbRCD1-GPF using Agrobacterium-mediated methods, and then placed on co-culture medium (MS solid medium containing 30mg/L AS, 2.0 mg/L2, 4-D) and dark cultured at 28℃for 3D.

4. After completion of step 3, the embryogenic cell mass was washed 2 times with MS liquid medium containing 900mg/L of cefotaxime sodium (cefotaxime sodium, CS) and 2.0mg/L of 2,4-D, and then placed on selection medium (solid MS medium containing 2.0mg/L of 2,4-D, 300mg/L of CS and 0.5mg/L of Hygromycin (Hyg)) and dark-cultured at 27.+ -. 1 ℃ for 10-12 weeks (selection medium needs to be changed every 2 weeks).

5. After the step 4 is completed, the embryogenic cell mass is placed on a somatic embryo induction culture medium (MS solid culture medium containing 1.0mg/L ABA, 300mg/L CS and 5mg/L Hyg) for 2-4 weeks, and the resistant callus is obtained by alternate light and dark culture at the temperature of 27+/-1 ℃ for 13 hours in the light and dark time of 11 hours and 3000lx in the light and dark time period.

6. After the step 5 is completed, the resistant callus is placed on an MS solid culture medium and is alternately cultivated in light and dark at the temperature of 27+/-1 ℃ for 4-8 weeks (the illumination time is 13 hours, the dark time is 11 hours, the illumination intensity is 3000 lx), and 188 sweet potato quasi-transgenic plants are obtained, and the IbRCD1-OX1 to IbRCD1-OX188 are named in sequence.

7. Extracting genomic DNA of young leaves of the sweet potato quasi-transgenic plants (IbRCD 1-OX1 to IbRCD1-OX 188) obtained in the step 6 respectively, and taking the genomic DNA as a template and 35S-F:5'-TCAGAAAGAATGCTAACCCACA-3' and IbRCD1-T-R:5'-TCACTTCTTACACATAATTCCA-3' as a primer for PCR amplification to obtain a PCR amplification product; if the PCR amplification product contains a band of about 1000bp, the corresponding sweet potato quasi-transgenic plant is the sweet potato transgenic positive plant. The genomic DNA of young leaves of the sweet potato quasi-transgenic plant is replaced by equal volume of water, and PCR amplification is carried out to serve as negative control. The genome DNA of the young leaves of the wild plant with sweet potato variety chestnut fragrance is used to replace the genome DNA of the young leaves of the quasi-transgenic plant with sweet potato, and PCR amplification is carried out to serve as a control. The recombinant plasmid pCAMBIA1300-IbRCD1-GPF is used for replacing the genomic DNA of young leaves of the sweet potato quasi-transgenic plant, and PCR amplification is carried out to serve as a positive control.

The experimental results are shown in FIG. 1 (M is a DNA molecular Marker, w is a negative control, p is a positive control, ck is a wild plant of sweet potato variety chestnut aroma, and the results show that IbRCD1-OX5, ibRCD1-OX16, ibRCD1-OX23, ibRCD1-OX27, ibRCD1-OX47, ibRCD1-OX58, ibRCD1-OX92, ibRCD1-OX97, ibRCD1-OX122, ibRCD1-OX137, ibRCD1-OX141, ibRCD1-OX147, ibRCD1-OX153, ibRCD1-OX167, ibRCD1-OX171, ibRCD1-OX176 and IbRCD1-OX183 are all transgenic positive plants of sweet potato.

The results of the transcriptional level detection of the IbRCD1 gene in the different transgenic lines are shown in FIG. 2 and are detected by RT-qPCR method. 3 independent transgenic lines L58 (i.e., ibRCD1-OX 58), L92 (i.e., ibRCD1-OX 92) and L122 (i.e., ibRCD1-OX 122) were selected (FIG. 2).

The method of asexual propagation is adopted to propagate the transgenic positive plants of sweet potatoes, and the plants obtained by propagating a transgenic seedling are used as a plant line.

3. Identification of salt resistance of transgenic plants

The experiment was repeated three times, each time with the following steps:

1. salt resistance identification

The sweet potato plants are wild type plants (WT) of the sweet potato variety chestnut flavor, plants of L58 (i.e., ibRCD1-OX 58), plants of L92 (i.e., ibRCD1-OX 92), and plants of L122 (i.e., ibRCD1-OX 122).

The experiment was repeated three times, each time with the following steps:

(1) Stem segments (about 25cm long and at least 3 stem nodes) of sweet potato plants were harvested, fixed with rigid foam plates, and cultured in 1/2 Hoagland medium containing 86mM NaCl, at least 1 stem node was spread. 20 plants per line.

(2) After completion of step (1), 1/2 Hoagland medium containing 86mM NaCl was changed once a week. After 4 weeks, the growth state of the sweet potato plants was observed, and the average fresh weight (g) of the single sweet potato plants was measured and counted.

The 1/2 Hoagland nutrient solution containing 86mM NaCl in step (2) was replaced with 1/2 Hoagland nutrient solution, and the other steps were unchanged, as a blank.

Statistical results of growth states and phenotype indexes of sweet potato plants are shown in A and B in FIG. 3, wherein A in FIG. 3 is blank control, and B in FIG. 3 is salt stress (NaCl). FW represents fresh weight and DW represents dry weight. The results show that after a period of salt stress treatment, the growth state of wild plants with chestnut flavor of the sweet potato variety is obviously deteriorated, and the growth state and the phenotype indexes of the plants of L58, the plants of L92 and the plants of L122 are all good. Under 86mM NaCl treatment conditions, the Fresh Weight (FW) and the Dry Weight (DW) of the L58, L92 and L122 plants over-expressing the IbRCD1 gene are obviously higher than those of wild plants, and the growth state is obviously better than that of the wild plants, so that the salt tolerance of the transgenic plants is obviously enhanced, and the over-expression of the IbRCD1 gene in sweet potatoes can obviously improve the salt tolerance of the sweet potatoes. The IbRCD1 protein and the coding gene IbRCD1 thereof can regulate and control the stress resistance (such as salt tolerance) of plants, and the stress resistance of target plants can be obviously improved by improving the content and/or activity of the IbRCD1 protein in the target plants (such as over-expression of the IbRCD1 gene).

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application 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> stress-resistance-associated protein IbRCD1, and related biological materials and applications thereof

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 349

<212> PRT

<213> sweet potato (Ipomoea batatas (l.) lam.)

<400> 1

Met Ala Lys Met Glu Ala Phe Ala Asn Gln Phe Ala Ala Ile Phe Val

1 5 10 15

Val Phe Phe Val Thr Ser Phe Val Met Met Lys Ser Thr Glu Ala Ala

20 25 30

His His Asn Lys Val Asp His Val Thr Cys Ser Asn Arg Gly Ser Lys

35 40 45

Cys Phe Tyr Lys Tyr Leu Thr Cys Pro Ser Gln Cys Pro Glu Ile His

50 55 60

Pro Lys Asp Pro Thr Ala Gln Ala Cys Phe Leu Asp Cys Tyr Ser Pro

65 70 75 80

Lys Cys Glu Ala Val Cys Lys Ser Arg Lys Pro Asn Cys Asp Gly Pro

85 90 95

Gly Ala Ala Cys Tyr Asp Pro Arg Phe Ile Gly Gly Asp Gly Ile Val

100 105 110

Phe Tyr Phe His Gly Lys Lys Asn Glu His Phe Thr Leu Ile Ser Asp

115 120 125

Thr Asn Leu Gln Ile Asn Ala His Phe Ile Gly Leu Arg Pro Leu Asn

130 135 140

Arg Thr Arg Asp Phe Thr Trp Ile Gln Ala Leu Gly Ile Met Phe Gly

145 150 155 160

Pro His Asn Phe Thr Ile Ala Ala Ala Ala Ala Glu Thr Trp Asp Asp

165 170 175

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

180 185 190

Pro Gln Gly Gln Ser Ser Gln Trp Thr Ser Pro Asp Gly Ser Leu Lys

195 200 205

Leu Glu Arg Thr Phe Pro Ser Asn Ser Ala Thr Val Thr Val Ser Asp

210 215 220

Ile Ala Glu Val Ser Ala Asn Val Val Pro Val Ala Glu Gln Glu Ser

225 230 235 240

Ala Ile His Gly Tyr Gly Ile Pro Ala Asn Asn Ser Phe Ala His Leu

245 250 255

Glu Val Gln Phe Arg Phe Phe Ser Leu Ser Pro Arg Val Asp Gly Ile

260 265 270

Leu Gly Arg Thr Tyr Arg Pro Asp Phe Gln Asn Pro Ala Lys Pro Gly

275 280 285

Val Glu Met Ala Val Val Gly Gly Glu Asp Lys Phe Arg Thr Ser Ser

290 295 300

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

305 310 315 320

Thr Ala Gly Val Glu Arg Glu Tyr Tyr Gly Thr Val Asp Cys Ala Thr

325 330 335

Gly Gly Thr Asp Gly Gly Tyr Gly Ile Met Cys Lys Lys

340 345

<210> 2

<211> 1050

<212> DNA

<213> sweet potato (Ipomoea batatas (l.) lam.)

<400> 2

atggcaaaaa tggaggcatt tgccaatcaa tttgctgcaa tctttgttgt cttctttgtg 60

accagttttg tgatgatgaa atctactgaa gctgcccatc ataataaggt tgatcatgtc 120

acgtgcagta atcgtggaag taagtgcttt tataagtact taacttgccc atctcagtgc 180

ccagaaattc accccaaaga tcccactgct caagcctgct ttcttgattg ttactcccct 240

aaatgtgaag ctgtctgcaa aagtagaaaa ccaaactgcg acggtccggg agcagcctgt 300

tacgacccac ggttcatcgg cggcgacggc attgttttct actttcacgg caagaaaaac 360

gagcacttca ccttaatatc cgacaccaat cttcaaatca acgcccattt catcggcctt 420

cgtcccctca acagaacccg agatttcaca tggatccagg ctttaggcat aatgttcggc 480

ccccacaact tcaccatcgc cgccgccgcc gccgagacct gggacgatca aaccgaccac 540

ctagaattcc gctacgacgg cgcgtccgcc gtcatcccgc aaggccaatc ctcccaatgg 600

acttcccccg acgggagcct gaaactggag aggacgtttc cgagtaacag cgcgacggta 660

accgtttccg acatcgccga ggtttcggct aacgttgttc cggtggcgga acaggaaagc 720

gcgattcacg gatacgggat tccggcgaat aacagctttg cgcacttgga agtgcagttc 780

agattcttca gtttgtcgcc tagggttgac gggattctcg gccggactta ccggccggat 840

ttccagaatc cggcgaagcc tggagtggaa atggcggttg tcggcggcga agataagttc 900

aggactagtt cgcttctcgc ggcggactgc aattcttgtg tgttcgctcc cggaaatgtg 960

accgccggcg tggagaggga atactacggc acggttgact gcgccaccgg agggaccgac 1020

gggggatatg gaattatgtg taagaagtga 1050

<210> 3

<211> 1047

<212> DNA

<213> sweet potato (Ipomoea batatas (l.) lam.)

<400> 3

atggcaaaaa tggaggcatt tgccaatcaa tttgctgcaa tctttgttct cttctttctc 60

accagttttg tgatgatgaa atctactgaa gctgcccatc ataataaggt tgatcatgtc 120

acgtgcagta atcgtggaag taaatgcttt ctcaagtact taacttgccc atctcagtgc 180

ccacaaactc accccaaaga tcccactgct caagcctgct ttcttgattg ttactcccct 240

aaatgtgaag ctgtctgcaa aagtagaaaa ccaaactgcg acggtccggg agcagcctgt 300

tacgacccac ggttcatcgg cggcgacggc attgttttct actttcacgg caagaaaaac 360

gagcacttca ccttaatatc cgacaccaat cttcaaatca acgcccattt catcggcctt 420

cgtcccctca acagaacccg agatttcaca tggatccagg ctttaggcat aatgttcggc 480

ccccacaact tcaccatcgc cgccgccgcc gccgagacct gggacgatca aaccgaccac 540

ctagaattcc gctacgacgg cgcgtccgcc gtcatcccgc aaggccaatc ctcccaatgg 600

acttcccccg acgggagcct gaaactggag aggacgtttc cgaggaacag cgcgacggta 660

accgtttccg acatcgccga ggtttcggct aacgttgttc cggtgacgga acaggaaagc 720

gcgattcacg gatacgggat tccggcgaat aacagctttg cgcacttgga agtgcagttc 780

agattcttca gtttgtcgcc tagggttgac gggattctcg gccggactta ccggccggat 840

ttccagaatc cggcgaagcc tggagtggaa atggcggttg tcggcggcga agataagttc 900

aggactagtt cgcttctcgc ggcggactgc aattcttgtg tgttcgctcc cggaaatgtg 960

accgccggcg tggagaggga atactacggc acggttgact gcgccaccgg agggaccgac 1020

gggggatatg gaattatgtg taagaag 1047

Claims

1. A protein, characterized in that the protein is any one of the following:

a1 A protein having an amino acid sequence of SEQ ID No. 1;

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

3. A biomaterial, characterized in that the biomaterial is any one of the following B1) to B7):

b1 A nucleic acid molecule encoding the protein of claim 1 or 2;

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

4. The biomaterial of claim 3, 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.

5. A method of growing a stress-tolerant plant, comprising increasing the amount and/or activity of a protein according to claim 1 or 2 in a plant of interest to obtain a stress-tolerant plant having a stress-tolerance higher than that of the plant of interest.

6. The method according to claim 5, wherein the increase in the content and/or activity of the protein according to 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. The protein of claim 1 or 2 or a substance that modulates the activity and/or content of said protein, and/or the use of any of the following biological materials of claim 3 or 4:

d1 Application in regulating plant stress resistance;

d3 Use in the cultivation of stress-tolerant plants;

d5 Use in plant breeding.

9. The use according to claim 8, wherein the stress resistance comprises salt resistance, drought resistance, heat resistance, cold resistance and/or light resistance.

10. Use of the method according to any one of claims 5-7 for creating stress-tolerant plants and/or plant breeding and/or plant germplasm resource improvement.