CN114805520B - Stress resistance related protein IbGT1, encoding gene and application thereof - Google Patents

Abstract

The invention discloses a plant stress resistance related protein IbGT1, and a coding gene and application thereof. The invention specifically discloses proteins or substances regulating the activity and/or content of the proteins to regulate plant stress resistance. The protein IbGT1 is any one of the following proteins: a1 Amino acid sequence is a protein shown as SEQ ID No. 1; a2 Protein which is obtained by substituting and/or deleting and/or adding amino acid residues of the protein of A1), has more than 80 percent of identity with the protein shown in A1) and has the function of regulating and controlling plant stress resistance; a3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of A1) or A2). Experiments prove that the IbGT1 gene has the capability of positively regulating and controlling plant stress resistance, and the stress resistance of sweet potatoes can be obviously improved by over-expressing the IbGT1 gene in the sweet potatoes.

Description

Stress resistance related protein IbGT1, encoding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to stress resistance related protein IbGT1, and a coding gene and application thereof.

Background

Sweet potato (Ipomoea batatas (l.) lam.) is a dicotyledonous plant of class, order of tubular flowers, family of Convolvulaceae, genus sweet potato, and is an important food, feed, industrial raw material and novel energy crop. The sweet potato vine cutting disease and soft rot become main diseases of main planting areas of sweet potatoes in China, so that the sweet potatoes are reduced in large area and reduced in quality, and serious economic loss is caused. The sweet potato vine cutting disease is also called fusarium wilt (Fusarium oxysporum f.sp batatas), which is a fungal disease, germs are invaded into seedlings from soil through wounds of seedling bases or roots or from seed-bearing potatoes through pipes and propagate in the tissues of the pipes, so that the diseased plants are subjected to full-plant wilt and death, the field is represented by yellowing and falling off of aerial part leaves from bottom to top, stem vascular bundles become brown, and finally, the stems are cracked and the whole plant dies. After the plants are infected, longitudinal cracking symptoms are visible in different positions such as roots, stems and tendrils, and the longitudinal cracking symptoms frequently occur in stems close to soil.

Sweet potato soft rot (Rhizopus soft rot), caused by rhizopus nigricans (Rhizopus nigricans ehrend.), is one of the main diseases in the storage period of sweet potatoes, and is widely distributed and occurs in all sweet potato production areas throughout the country. After the disease, the pathogenic bacteria secrete pectase, and the pectic substances in the cell walls are dissolved, so that the tissues are soft-decayed and spread rapidly, and the whole kiln is often decayed, so that serious squeezing loss is caused.

Disease resistance can be effectively reduced by planting disease-resistant varieties, typical disease plants are difficult to see in fields, and durable resistance can be shown even under continuous cropping conditions. Therefore, breeding new varieties of sweet potatoes with high yield, high quality and high resistance becomes a main goal of breeding in China.

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 resistance of the sweet potato can be directionally improved on a molecular level. Therefore, cloning and regulating the gene related to sweet potato disease resistance creates a new high-yield high-quality high-resistance sweet potato material, and has very important theoretical reference significance and application value for high-quality, high-yield and high-resistance breeding work of sweet potatoes.

Disclosure of Invention

The technical problem to be solved by the invention is how to regulate the stress resistance of plants and/or how to improve the stress resistance of plants.

To solve the above technical problems, the present invention provides a protein or a biological material related to the protein, wherein the protein is named IbGT1, and the protein IbGT1 can be any of the following:

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

a2 Protein which is obtained by substituting and/or deleting and/or adding amino acid residues 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 function of regulating and controlling plant stress resistance;

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 protein-related biomaterial may be any of the following:

b1 A nucleic acid molecule encoding said protein;

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 nucleotide sequence encoding the protein IbGT1 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 IbGT1 isolated by the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the protein IbGT1 and have the function of the protein IbGT1.

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 IbGT1 may be derived from sweet potato (Ipomoea batatas (l.) lam.).

The invention also provides the use of the protein IbGT1 or a substance regulating the activity and/or content of the protein IbGT1 and/or any of the following of the biological material:

u1) use of said protein or of a substance regulating the expression of a gene encoding said protein or of a substance regulating the activity or content of said protein for regulating the resistance of a plant to stress;

u2) the use of said protein or of a substance regulating the expression of a gene encoding said protein or of a substance regulating the activity or the content of said protein for the preparation of a product regulating the stress resistance of a plant;

u3) the use of said protein or of an expression substance or a substance regulating the activity or the content of said protein for breeding plants with altered resistance to stress, said gene encoding said protein;

u4) the use of said protein or of a substance regulating the expression of a gene encoding said protein or of a substance regulating the activity or the content of said protein for the preparation of a product for breeding plants with altered resistance to stress;

u5) the use of said protein or of a substance regulating the expression of a gene encoding said protein or of a substance regulating the activity or the content of said protein in plant breeding.

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 IbGT1.

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).

In the above protein or application, the nucleic acid molecule 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 (IbGT 1 gene) encodes a protein IbGT1 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 encoding the protein IbGT1 (CDS).

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 molecule may also include a nucleic acid molecule having a nucleotide sequence identity of 95% or more with the nucleotide sequence shown in SEQ ID No.2 and being 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 and/or the vector pEASY-Blunt simple may be used.

Recombinant expression vectors containing the IbGT1 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 IbGT1 gene is used for constructing a recombinant plant expression vector, any one of an enhanced promoter or a constitutive promoter can be added before the transcription initiation nucleotide, and the enhanced promoter comprises, but is not limited to, a cauliflower mosaic virus (CAMV) 35S promoter and a ubiquitin promoter (ubiquitin) of corn, 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 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.

By using any vector capable of guiding the expression of exogenous genes in plants, the IbGT1 gene or the gene fragment provided by the invention is introduced into plant cells or receptor plants, and transgenic cell lines and transgenic plants with improved stress resistance (such as salt tolerance) can be obtained. Expression vectors carrying the IbGT1 gene can be transformed into plant cells or tissues by using conventional biological methods such as Ti plasmid, ri plasmid, plant viral vector, direct DNA transformation, microinjection, electric conduction, agrobacterium mediation, etc., and the transformed plant tissues are cultivated 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-IbGT1-GFP, wherein the recombinant vector pCAMBIA1300-IbGT1-GFP 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-IbGT1-GFP expresses IbGT1 protein shown in SEQ ID No.1 in the sequence table.

The recombinant microorganism can be specifically recombinant Agrobacterium EHA105/pCAMBIA1300-IbGT1-GFP.

The recombinant agrobacterium EHA105/pCAMBIA1300-IbGT1-GFP is obtained by introducing the recombinant vector pCAMBIA1300-IbGT1-GFP into the agrobacterium tumefaciens EHA105.

The invention also provides a method for improving the stress resistance of a plant, which comprises enhancing or improving or up-regulating the activity and/or content of the IbGT1 protein in the target plant, or/and the expression level of the coding gene of the IbGT1 protein, so as to improve the stress resistance of the plant.

In the above method, the activity and/or content of the IbGT1 protein in the target plant is enhanced or increased or up-regulated, or/and the expression level of the gene encoding the IbGT1 protein can be higher than that of the target plant obtained by introducing the IbGT1 gene into the recipient plant. The IbGT1 gene encodes the IbGT1 protein.

In one embodiment of the invention, the method of growing a stress resistant plant 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 recipient plant (such as crops or sweet potatoes);

(3) The stress resistant plants with higher stress resistance than the receptor plants are obtained 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.

In the above applications or methods, the stress may be biotic or abiotic.

The biotic stress may be a disease stress. The disease may in particular be gummy stem rot and/or soft rot.

The abiotic stress may be water stress. The water stress may specifically be salt stress and/or drought stress.

In the present invention, the modulation may be up-regulation or enhancement or improvement.

In the present invention, the object of plant breeding includes growing stress-resistant plants. The plant described herein may be any one of the following: c1 Dicotyledonous or monocotyledonous plants; c2 Tubular flower plants; c3 A plant of the family Convolvulaceae; c4 Sweet potato plant; c5 Sweet potato.

The invention provides an IbGT1 protein and a coding gene thereof, and the gene is introduced into sweet potato to obtain a sweet potato plant over-expressing the IbGT1 gene. The transgenic sweet potato plants are subjected to salt stress treatment, and the over-expression strain is found to be enhanced in salt tolerance and drought resistance compared with the wild sweet potato. The transgenic sweet potato plants were inoculated with vine cutting bacteria and soft rot bacteria, and the over-expressed strain was found to have enhanced vine cutting disease resistance and soft rot resistance compared to wild sweet potato. The results show that the IbGT1 gene and the protein encoded by the gene play an important role in the process of resisting abiotic and biotic stress of plants. The IbGT1 protein and the coding gene thereof provided by the invention have important application value in the research of improving the plant stress resistance. The invention has wide application space and market prospect in the agricultural field.

Drawings

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

FIG. 2 shows the results of the transcript level detection of the IbGT1 gene in different transgenic lines.

FIG. 3 shows the growth state of sweet potato plants after inoculating tendril-cutting bacteria.

FIG. 4 shows the growth state of sweet potato plants after inoculation with soft rot fungi.

FIG. 5 is a graph showing the statistical results of the growth status and phenotype index of sweet potato plants under 86mM NaCl.

FIG. 6 is a graph showing the statistical results of growth status and phenotype index of sweet potato plants after drought stress.

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 experimental methods in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Sweet potato salt tolerance mutant ND98 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.

Chestnut flavor (Wang Yuping, etc., chinese agricultural science, 2003, 36 (9): 1000-1005) is a sweet potato variety, which is publicly available from the national agricultural university sweet potato genetic breeding laboratory to repeat the experiment.

The cloning vector pMD19-T is a product of Bao bioengineering (Dalian) company with a catalog number of 6013. The vector pCAMBIA1300-GFP is a product of Vaccinium, and the product number is Beijing Vaccinium organism VECT0460. The total RNA extraction kit is a product of Tiangen Biochemical technology (Beijing) limited company, and the catalog number is DP432. The pEASY-Blunt simple carrier is a product of Beijing full gold biotechnology Co. PrimeScript ^TM 1st Strand cDNA Synthesis Kit is a product of Takara Bio-engineering (Dalian) Co., ltd, product catalog number 6110A.

The SY strains of the sweet potato vine cutting bacteria in the following examples are described in the following documents: lei Jian, yang Xinsun, su Wen, wang Lianjun, identification of resistance of Tex sweet potato varieties to vine cutting disease [ J ]. Hubei agricultural science, 2014,53 (22): 5422-5423, offered by the institute of food crops at the national academy of agricultural sciences, hubei province, saved by the institute of genetic breeding of sweet potato at the university of agriculture, china.

Sweet potato soft rot fungi in the following examples are described in the following documents: yang Dongjing, xu Zhen, zhao Yongjiang, zhang Chengling, sun Houjun, xie Yiping. Sweet potato soft rot resistance identification method research and evaluation of sweet potato germplasm resource resistance [ J ]. North China agricultural journal, 2014,29 (S1): 54-56, supplied by Jiangsu Xuzhou sweet potato research center, saved by Chinese agricultural university sweet potato genetic breeding laboratory.

Example 1 use of the IbGT1 Gene for modulating sweet Potato stress resistance

The IbGT1 gene is derived from sweet potato salt-tolerant mutant ND98, the nucleotide sequence (coding sequence (CDS)) of the gene is shown as SEQ ID No.2, the coded protein is named IbGT1 protein or protein IbGT1, and the amino acid sequence of the coded protein is shown as SEQ ID No. 1.

1. Construction of recombinant plasmids

A. Construction of recombinant plasmid pCAMBIA1300-IbGT1-GFP

1. The double-stranded DNA molecule (IbGT 1 gene) shown in positions 1 to 1335 of the sequence SEQ ID No.2 was synthesized from the 5' -terminus. Taking the double-stranded DNA molecule as a template, and taking OE-F-KpnI:5' -GGGGTACCATGGAATCTAATGGTATGTATTCG-3' and OE-R-SalI:5' -GCGTCGACTCATCCCGTCACAGAAGACGG-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-IbGT1.

3. After the step 2 is completed, the recombinant plasmid pEASY-IbGT1 is digested with restriction enzymes KpnI and SalI, and a fragment containing the IbGT1 gene, which is abbreviated as fragment 1, is recovered.

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

5. The fragment 1 was ligated with pCAMBIA1300-GFP vector backbone 2 to obtain recombinant plasmid pCAMBIA1300-IbGT1-GFP.

Based on the sequencing results, the recombinant plasmid pCAMBIA1300-IbGT1-GFP was structurally described as follows: the small fragment between the recognition sequences of restriction enzymes KpnI and SalI of the recombinant plasmid pCAMBIA1300-GFP is replaced by DNA molecules shown in the 1st to 1335 th positions from the 5' end of the sequence 2 in the sequence table, and other sequences of the pCAMBIA1300-GFP vector are kept unchanged, so that the recombinant expression vector is obtained. Recombinant plasmid pCAMBIA1300-IbGT1-GFP expresses the IbGT1 protein shown in SEQ ID No. 1.

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-IbGT1-GFP is transformed into agrobacterium tumefaciens EHA105 to obtain recombinant agrobacterium, and the recombinant agrobacterium is named EHA105/pCAMBIA1300-IbGT1-GFP.

2. The stem tip meristem with chestnut fragrance of about 0.5mm in length is stripped and placed on embryogenic callus induction solid culture medium (MS solid culture medium containing 2.0 mg/L2, 4-D and 3.0% sucrose, also called MS+2.0 mg/L2, 4-D+3.0% sucrose solid culture medium) to be cultured for 8 weeks at 27+/-1 ℃ to obtain embryogenic callus, then the embryogenic callus is placed on embryogenic callus induction liquid culture medium (MS liquid culture medium containing 2.0 mg/L2, 4-D and 3.0% sucrose, also called MS+2.0 mg/L2, 4-D+3.0% sucrose liquid culture medium) to be cultured for 3D (specific conditions: 100r/min;27 ℃ C., light-dark time: 11h per day; light-dark time: 500 lx) alternately culturing period) on a horizontal shaking table to obtain embryogenic cell mass with a diameter of 0.7-1.3 mm.

3. After step 2 was completed, EHA105/pCAMBIA1300-IbGT1-GFP was transformed into embryogenic cell masses by Agrobacterium-mediated method, and then placed on co-culture medium (MS solid medium containing 30mg/L AS, 2.0 mg/L2, 4-D, also known AS MS+2.0 mg/L2, 4-D+30mg/L AS solid medium) for dark culture at 28℃for 3D.

4. After completion of step 3, embryogenic cell masses were washed 2 times with MS liquid medium (also known as MS+900mg/L CS+2.0 mg/L2, 4-D) containing 900mg/L cefotaxime sodium (cefotaxime sodium, CS) and 2.0 mg/L2, 4-D, and then placed on selection medium (solid MS medium containing 2.0 mg/L2, 4-D, 300mg/L CS and 5mg/L 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 219 sweet potato quasi-transgenic plants are obtained, and the transgenic plants are named IbGT1-OX1 to IbGT1-OX219 in sequence.

7. Respectively extracting genome DNA of young leaves of the sweet potato quasi-transgenic plant obtained in the step 6, and taking the genome DNA as a template and 35S-F:5'-TCAGAAAGAATGCTAACCCACA-3' and IbGT1-R:5'-TCATCCCGTCACAGAAGACG-3' as a primer for PCR amplification to obtain a PCR amplification product; if the PCR amplification product contains 1400bp bands, 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 plants was replaced with equal volume of water for PCR amplification as a blank (W). The genomic DNA of the young leaves of the wild plant of the sweet potato variety chestnut flavor is used for replacing the genomic DNA of the young leaves of the quasi-transgenic plant of the sweet potato, and PCR amplification is carried out to serve as a negative control (WT). The recombinant plasmid pCAMBIA1300-IbGT1-GFP was used to replace the IbGT1 genomic DNA of young leaves of the sweet potato quasi-transgenic plant, and PCR amplification was performed as a positive control.

The results of the experiment are shown in FIG. 1A (M is DNA molecular Marker, W is blank control, P is positive control, and WT is negative control. The results show that IbGT1-OX1, ibGT1-OX2, ibGT1-OX9, ibGT1-OX15, ibGT1-OX27, ibGT1-OX38, ibGT1-OX44, ibGT1-OX56, ibGT1-OX71, ibGT1-OX128, ibGT1-OX156 are sweet potato pseudo-transgenic plants.

8、RT-qPCR

Extracting total RNA of the transgenic sweet potato positive plant, carrying out reverse transcription to obtain cDNA, and carrying out qRT-PCR by taking the wild sweet potato plant as a control. The cDNA concentration of the sample was homogenized using the constitutively expressed sweetpotato Actin (Actin) gene as an internal reference. Then, real-time fluorescent quantitative PCR (RT-qPCR) analysis was performed using specific primers for the IbGT1 gene, using 2 ^-△△CT Method (Livak KJ, schmittgen TD.2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2) ^-△△CT method.Methods.25:402-408 Analysis of IbGT1 gene expression, 3 replicates per group of samples.

The specific primer sequences of the sweet potato Actin (action) gene are as follows:

IbActin-F：5′-AGCAGCATGAAGATTAAGGTTGTAGCAC-3′

IbActin-R：5′-TGGAAAATTAGAAGCACTTCCTGTGAAC-3′

the IbGT1 specific primer sequence is as follows:

IbGTD1-F：5′-CAAGCCCAAAATCACCCC-3′

IbGTD1-R：5′-CCACCATAATCAACAGCCTCAC-3′

the results are shown in FIG. 2, and the results show that the IbGT1 gene is expressed in the sweet potato transgenic positive plants to different degrees. The transgenic sweet potato plants L15 (i.e. IbGT1-OX 15), L27 (i.e. IbGT1-OX 27) and L38 (i.e. IbGT1-OX 38) are selected for tissue culture (asexual propagation), and plants obtained by propagation of a transgenic seedling are taken as a plant line to obtain IbGT1 over-expression transgenic plant lines L15, L27 and L38 (short transgenic plant lines L15, L27 and L38), and the subsequent vine cutting disease resistance test, salt resistance test and drought resistance test are carried out. Transgenic sweet potato plants L2 (i.e., ibGT1-OX 2), L128 (i.e., ibGT1-OX 128) and L156 (i.e., ibGT1-OX 156) are selected for tissue culture (asexual propagation), and plants obtained by propagation of a transgenic seedling are taken as a strain to obtain IbGT1 over-expression transgenic strains L2, L128 and L156 (simply called transgenic strains L2, L128 and L156) for subsequent soft rot resistance test.

5. Identification of stress resistance

1. Identification of resistance to vine cutting disease

The sweet potato strain to be detected is Wild Type (WT) of sweet potato variety chestnut fragrance, ibGT1 over-expression transgene strain L15, L27 and L38.

Experiments were repeated three times, 20 plants per line were planted, and the steps for each repetition were as follows:

a. inoculating the sweet potato vine cutting bacteria into PDA culture medium, culturing at 28deg.C in light-dark alternation (light-dark alternation cycle is that light time is 13h each day and dark time is 11h, light intensity is 500 lx) for 3d, and culturing at 28deg.C in dark for 7d to obtain mycelium.

b. After the step a is completed, transferring hypha into a triangular flask, adding 100mL of sterile distilled water, oscillating for 30min at 100r/min, filtering with double-layer sterile gauze, and counting under a microscope with a blood cell counting plate to obtain the sweet potato vine cutting germ spore with the content of 1 multiplied by 10 ⁷ cfu/mL spore suspension.

c. Cutting the seedlings of the sweet potato plants to be detected with basically consistent growth vigor, aligning, placing in spore suspension for 30min, then planting in a basin filled with sterile sandy soil (3 plants are planted in each basin), and then culturing normally. And observing the growth state of the sweet potato plants at the 0 th d, the 3 rd d, the 5 th d, the 7 th d, the 9 th d and the 11 th d of the planting respectively. Phenotypic indicators (dry fresh weight (g)) of planted sweet potato plants of 11d were measured and counted.

The incidence rate and the disease index of the tendril-cutting disease are identified according to the level 0-6, and the tendril-cutting disease is characterized in that the tendril-cutting disease is taken as a plant unit, wherein:

level 0: the plants grow normally, and no disease symptoms are seen; stage 1: plants grew normally and the basal stem vascular bundles turned brown within 5 cm. 2 stages: the plant growth is basically normal, and the stem basal vascular bundle turns brown within 1/3. 3 stages: the basal leaves of the plants turn yellow, and the vascular bundles turn brown within 2/3. 4 stages: most of plant leaves become yellow and dead, and vascular bundles become brown and spread to the whole plant; 5 stages: the whole plant dies. And evaluating disease resistance according to the disease index.

The disease index is calculated according to the following formula: disease index = [ (0.1×n1+0.2×n2+0.5×n3+0.8×n4+1.0×n5)/N ] ×100 (where N is the number of plants in each stage, and N is the total number of plants tested)

The resistance of the sweet potato vine cutting disease is detected according to the disease index, and the specific classification is as follows:

high Resistance (HR): the disease index is 0.0-20.5; anti (R): the disease index is 20.6-40.5; neutralizing antibody (MR): the disease index is 40.6-60.5; feel (MS): the disease index is 60.6-80.5; mesoscopy (S): the disease index is 80.6-90.5; high Sense (HS): the disease index is 90.6-100. Disease Index (DI) = [ (Σ number of disease plants at each stage×representative value at each stage)/total number of disease plants×representative value at the highest stage) ]×100.

The data were processed using SPSS statistical software and experimental results were expressed as mean ± standard deviation, with P < 0.05 (x) representing significant differences and P < 0.01 (x) representing very significant differences using the t-test.

The growing state of the sweet potato plants in the basin is shown in fig. 3 (0 d is the planted 0d,3d is the planted 3d,5d is the planted 5d,7d is the planted 7d,9d is the planted 9d,11d is the planted 11d, and B is the planted 11d sweet potato plants which are cleaned out of the basin). The experimental results are as follows: when inoculated for 3d, WT plants had more obvious symptoms of disease (yellowing of leaves), and the transgenic lines L15, L27 and L38 all had good growth status; when 9d is inoculated, the leaves of the WT plant are almost completely yellow, part of old leaves fall off, the stem part is brown and soft, and part of the leaves of the over-expression plant are yellow, so that a lighter anaphylactic reaction is shown; when the strain is inoculated for 11 days, WT leaves wither and fall off, stem segments brown, the whole strain dies, the over-expression strain leaves turn yellow less, part of the strain stems brown to a small extent, and the plant can still grow normally. The results of the disease index calculation are shown in Table 1, the WT plants are hypersensitive plants, the transgenic lines L15 are middle resistance, the transgenic lines L27 are disease resistance, and the transgenic lines L38 are disease resistance, which indicates that the vine cutting resistance of the transgenic lines is improved to different degrees. Thus, overexpression of the IbGT1 gene in sweetpotato can increase vine-cutting resistance of sweetpotato.

Table 1 investigation of the condition of tendril-cutting disease

2. Identification of Soft rot resistance

The strain to be tested is wild plant (WT) of sweet potato variety chestnut fragrance, ibGT1 over-expression transgene strain L2, L128, L156.

Experiments were repeated three times, 20 per inoculation per strain, each repetition being as follows:

a. inoculating sweet potato soft rot fungus into PDA culture medium, culturing at 28deg.C for 3d (light-dark alternate culture period is 13 hr for each day, 11 hr for dark time, and 500lx for light intensity), and culturing at 28deg.C for 7d to obtain mycelium.

b. Transferring the mycelium obtained in step a to 500mL triangular flask, adding 100mL sterile distilled water, oscillating for 30min at 100r/min, filtering with double-layer sterile gauze, and counting under microscope with blood cell counting plate to obtain sweet potato soft rot fungus spore with content of 1×10 ⁷ cfu/mL spore suspension.

c. 20 plants per line were taken. And (3) punching small holes with the diameter of 1cm on the potato blocks by using a puncher, and placing the potato block thin rod brought out during punching aside for standby.

d. 1mL of bacterial liquid is taken by a liquid-transferring gun and injected into the small hole, potato blocks with proper length are taken and inserted into the small hole, and then Vaseline and paraffin are sequentially smeared from inside to outside for sealing.

e. After culturing for 10 days at the constant temperature of 28 ℃, the disease condition of the potato blocks is observed.

The disease condition of the sweet potato blocks after soft rot inoculation is shown in figure 4, the potato blocks of the WT are seriously infected, and hyphae almost spread to the whole section; the potato blocks of the over-expression strain only have hyphae near the inoculation area, so that the spread of soft rot is effectively controlled. Thus, overexpression of the IbGT1 gene in sweetpotato can increase the soft rot resistance of sweetpotato.

3. Salt resistance identification

The strain to be tested is a wild plant (WT) of sweet potato variety chestnut flavor, and IbGT1 over-expression transgene strains L15 and L38.

Experiments were repeated three times, 20 strains per treatment per line, each repetition being as follows:

(1) The stem sections (about 25cm long and at least 3 stem sections) of each strain to be tested were fixed with a hard foam plate, and cultured in a 1/2 Hoagland+86 mM NaCl liquid medium (a liquid medium obtained by adding NaCl to a content of 86mM NaCl to a 1/2 Hoagland culture solution (Liu Degao. Obtaining sweet potato plants overexpressing IbP CR, ibERD3, ibELT, ibNFU1 genes and identifying salt tolerance)) at least over 1 stem section.

(2) After completion of step (1), 1/2 Hoagland+86 mM NaCl liquid medium was changed once a week. After 4 weeks, the growth state of the sweet potato plants was observed, and the average Fresh Weight (FW) and Dry Weight (DW) of the sweet potato plants per plant were measured and counted.

The 1/2 Hoagland+86 mM NaCl liquid medium from step (1) was replaced with 1/2 Hoagland nutrient solution, and the other steps were unchanged, as a blank (Normal) according to the above method. Data were processed using SPSS statistical software, experimental results were expressed as mean ± standard deviation, P < 0.05 (x) indicated significant differences and P < 0.01 (x) indicated very significant differences using the t-test.

Statistical results of growth states and phenotype indexes of sweet potato plants are shown in A1, A2 and B1 and B2 in FIG. 5 (A1 and A2 in FIG. 5 are blank controls, B1 and B2 in FIG. 5 are salt stress, FW represents fresh weight, and DW represents dry weight). The result shows that after a period of salt stress, the growth state of wild plants of sweet potato variety chestnut flavor is obviously deteriorated, and the growth states and phenotype indexes of IbGT1 over-expression transgenic lines L15 and L38 are good. Therefore, overexpression of the IbGT1 gene in sweetpotato can improve the salt resistance of sweetpotato.

4. Drought resistance identification

The strain to be detected is a wild plant (WT) of sweet potato variety chestnut fragrance, and IbGT1 over-expression transgene strains L15, L27 and L38, and water planting experiments and potting experiments are respectively carried out.

Experiments were repeated three times, 20 strains per treatment per line, each repetition being as follows:

(1) Stem segments (about 25cm long and at least 3 stem segments) of sweet potato plants were planted in pots filled with artificial soil (mixed from 1 part by volume of vermiculite and 1 part by volume of nutrient soil), and 3 plants were planted in each pot.

(2) After the step (1) is completed, each basin is irrigated with 1/2 Hoagland nutrient solution for 2 weeks.

(3) After step (2) is completed, each basin is subjected to natural Drought stress (draw) for 8 weeks (i.e., without any treatment, including without irrigation of any water and nutrient solution). After 8 weeks, the growth state of the sweet potato plants was observed, and the phenotypic indicators (such as the average fresh weight of individual plants (FW), the average dry weight of individual plants (DW)) of the sweet potato plants were measured and counted. As a control (Normal), 1/2 Hoagland nutrient solution was normally irrigated without drought stress.

The data were processed using SPSS statistical software and experimental results were expressed as mean ± standard deviation, with P < 0.05 (x) representing significant differences and P < 0.01 (x) representing very significant differences using the t-test.

The growth state of the sweet potato plants is shown as A1, A2 and B1, B2 in FIG. 6 (A1 in FIG. 6 and B1 in FIG. 6 are the growth state of the sweet potato plants in the basin, and A2 in FIG. 6 and B2 in FIG. 6 are the cleaned sweet potato plants taken out of the basin). The statistical results of the phenotype index of the sweet potato plants are shown as A3 in FIG. 6 and B3 in FIG. 6. The result shows that the growth state of wild plants of sweet potato variety chestnut flavor is obviously deteriorated after drought stress for a period of time, and the growth states and phenotype indexes of IbGT1 over-expression transgenic lines L15, L27 and L38 are all good. Therefore, overexpression of the IbGT1 gene in sweetpotato can improve drought resistance of the sweetpotato.

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> stress resistance related protein IbGT1, coding gene and application thereof

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 444

<212> PRT

<213> sweet potato (Ipomoea batatas)

<400> 1

Met Glu Ser Asn Gly Met Tyr Ser Asn Met Gly Ser Gly Met Leu Gly

1 5 10 15

Leu Glu Met Ser Leu His His Val Pro Pro Gln Gln Asn Pro Met Gln

20 25 30

His Gln Ser His Pro Pro Met Val Ser Tyr Val Asp His Arg Gln Gln

35 40 45

Ser Gln Pro Pro Leu Arg Pro Gly Ser Gly Gly Gly Ala Tyr Pro Ser

50 55 60

Gly Asn Lys Pro Lys Ile Thr Pro Gly Leu Thr Leu Ser Asp Glu Asp

65 70 75 80

Asp Pro Gly Gly Pro Thr Ala Asp Gln Asn Ser Ala Asp Asp Gly Lys

85 90 95

Arg Lys Thr Cys Pro Trp Gln Arg Met Lys Trp Thr Asp Asn Met Val

100 105 110

Arg Leu Leu Ile Met Val Val Tyr Tyr Ile Gly Asp Glu Val Gly Ser

115 120 125

Glu Gly Asn Ser Asn Asp Pro Ala Ala Gly Asn Lys Lys Lys Ala Gly

130 135 140

Ala Gly Ala Gly Leu Leu Gln Lys Lys Gly Lys Trp Lys Ser Val Ser

145 150 155 160

Arg Ala Met Met Glu Arg Gly Phe Tyr Val Ser Pro Gln Gln Cys Glu

165 170 175

Asp Lys Phe Asn Asp Leu Asn Lys Arg Tyr Lys Arg Val Asn Asp Ile

180 185 190

Ile Gly Lys Gly Thr Ala Cys Lys Val Val Glu Asn Gln Thr Leu Leu

195 200 205

Glu Thr Leu Asp Leu Ser Pro Lys Met Lys Glu Glu Ala Lys Lys Leu

210 215 220

Leu Asn Ser Lys His Leu Phe Phe Arg Glu Met Cys Ala Tyr His Asn

225 230 235 240

Ser Cys Ala His Gly Gly Ala Ser Gly Ser Ala Ala Ala Asp Gly Gly

245 250 255

Ser Asp Pro Thr Ser Gln Thr Asn Asn His His Gln Lys Cys Met His

260 265 270

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

275 280 285

Glu Glu Pro Lys Asp Asn Asn Tyr Glu Asp Asp Glu Asp Ser Asp Asp

290 295 300

Asp Glu Asp Glu Glu Ser Glu Glu Asp Glu Glu Asp Glu Lys Ser Arg

305 310 315 320

Lys Lys Ala Lys Lys Thr Glu Pro Trp Ser Pro Leu Leu Glu Gln Met

325 330 335

Ser Gly Glu Leu Thr Asn Val Cys Glu Asp Ser Thr Arg Ser Pro Gly

340 345 350

Glu Lys Arg Gln Trp Ile Lys Ala Arg Thr Met Gln Leu Glu Glu Gln

355 360 365

Arg Val Glu Phe Gln Ser Gln Ala Leu Glu Leu Glu Lys Gln Arg Leu

370 375 380

Lys Trp Glu Lys Phe Ser Ser Lys Lys Glu Arg Glu Met Glu Arg Glu

385 390 395 400

Lys Met Met Asn Gln Arg Lys Lys Leu Glu Asn Glu Arg Met Val Leu

405 410 415

Leu Leu His Gln Lys Glu Leu Glu Leu Asn Asp Val His His Gln Gly

420 425 430

Tyr Asn Arg Thr Ser Asp Pro Ser Ser Val Thr Gly

435 440

<210> 2

<211> 1335

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

atggaatcta atggtatgta ttcgaacatg ggttctggaa tgttagggct agaaatgtca 60

cttcaccatg tcccacctca acaaaacccc atgcagcacc aatcccaccc tcccatggtg 120

tcctacgttg accaccgtca acaaagtcaa ccgccgttga ggccaggcag cggcggcggc 180

gcttaccctt ccgggaataa gcccaaaatc accccaggct tgaccctcag cgacgaagat 240

gatcccggag ggcccaccgc cgatcagaac agcgctgatg atgggaagag gaaaacatgc 300

ccgtggcagc gaatgaaatg gacggataat atggtgaggc tgttgattat ggtggtgtat 360

tatatcggcg atgaggttgg atccgaaggg aatagcaacg acccggccgc cgggaacaag 420

aaaaaggccg gcgccggcgc cggccttttg cagaagaaag ggaagtggaa atcggtgtcg 480

cgggcgatga tggagagggg attctacgtg tccccccaac aatgcgagga taaattcaat 540

gatctgaaca aaaggtacaa aagggttaac gatatcatcg gaaaaggcac cgcgtgtaag 600

gttgtcgaga atcaaacctt gctggaaaca ttggatttat cgccaaagat gaaagaggaa 660

gccaagaaac tgctaaactc taaacacttg tttttccggg aaatgtgcgc ttaccataac 720

agctgcgccc acggcggcgc tagcggaagc gccgccgccg acggaggctc cgatcccacc 780

tctcagacta ataatcatca ccagaagtgt atgcattcat ctgagaatgt cagaatcgga 840

cccaatttgg ggcccgcaga ggtagaggaa ccaaaagaca acaactacga agacgacgag 900

gatagcgatg atgacgagga cgaggaatcc gaggaggacg aagaagacga aaaatcaaga 960

aagaaggcta aaaagacgga accttggtcg cccctgctag aacagatgag cggggaatta 1020

acaaacgtgt gtgaagacag tacgaggagt ccgggggaaa agcggcagtg gataaaagca 1080

agaacgatgc aattggagga gcagcgcgtg gagttccaat cccaagcatt ggagctggaa 1140

aagcagcgat tgaaatggga aaagttcagc agcaagaagg agagggagat ggagagggag 1200

aagatgatga atcaacggaa gaaattggag aacgagagaa tggttcttct gcttcaccag 1260

aaagagctgg aattgaacga tgttcatcac caaggttaca acagaactag cgatccgtct 1320

tctgtgacgg gatga 1335

Claims (9)

1.A protein, characterized in that the protein is IbGT1 protein, which is the following protein:

a1 Amino acid sequence is a protein shown as SEQ ID No. 1;

a2 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of A1).

2. A biomaterial characterized in that the biomaterial is any one of the following:

b1 A nucleic acid molecule encoding the protein of claim 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), a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3).

3. The biomaterial according to claim 2, wherein the nucleic acid molecule is a cDNA molecule or a DNA molecule whose coding sequence of the coding strand is SEQ ID No. 2.

4. An application, characterized in that the application is any one of the following:

u1) use of the protein of claim 1 or of an expression-enhancing substance of a gene encoding the protein of claim 1 or of an agent that enhances the activity or content of said protein for enhancing the resistance of a plant to stress;

u2) use of the protein of claim 1 or of an expression-enhancing substance of a gene encoding the protein of claim 1 or of an agent that enhances the activity or content of said protein for growing plants with altered resistance to stress;

u3) use of the protein of claim 1 or an expression enhancing substance of a gene encoding the protein of claim 1 or an agent that enhances the activity or content of said protein in plant breeding;

the substance is the biological material of claim 2;

the stress is soft rot, vine disease, drought stress or salt stress.

5. The use according to claim 4, wherein the nucleic acid molecule is a cDNA molecule or a DNA molecule whose coding sequence of the coding strand is SEQ ID No. 2.

6. The use according to claim 4 or 5, wherein the plant is any one of the following:

c1 Dicotyledonous or monocotyledonous plants;

c2 Tubular flower plants;

c3 A plant of the family Convolvulaceae;

c4 Sweet potato plant;

c5 Sweet potato.

7. A method for increasing the resistance of a plant to stress, which is a soft rot, a vine disease, a drought stress or a salt stress, by increasing, increasing or up-regulating the activity and/or content of the protein of claim 1 in the plant of interest, or/and increasing, increasing or up-regulating the expression level of the gene encoding the protein of claim 1.

8. The method according to claim 7, wherein said step S provides a plant of interest having a higher resistance to stress than said recipient plant by introducing a gene encoding said protein into said recipient plant.

9. The method of claim 7 or 8, wherein the plant is any one of the following:

c1 Dicotyledonous or monocotyledonous plants;

c2 Tubular flower plants;

c3 A plant of the family Convolvulaceae;

c4 Sweet potato plant;

c5 Sweet potato.

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