CN114805520A - Stress resistance related protein IbGT1, and coding 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 a protein or a substance for regulating the activity and/or content of the protein to regulate the stress resistance of plants. The protein IbGT1 is a protein shown in any one of the following formulas: A1) the amino acid sequence is protein shown as SEQ ID No. 1; A2) a protein which is obtained by substituting and/or deleting and/or adding amino acid residues to the protein of A1), has more than 80% of identity with the protein of A1), and has plant stress resistance regulation and control functions; A3) a fusion protein obtained by connecting a protein tag to the N-terminus or/and the C-terminus of A1) or A2). Experiments prove that the IbGT1 gene has the capability of positively regulating and controlling the stress resistance of plants, and the IbGT1 gene is overexpressed in sweet potatoes to obviously improve the stress resistance of the sweet potatoes.

Description

Stress resistance related protein IbGT1, and coding 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 potatoes (Ipomoea batatas (L.) Lam.) are plants of dicotyledonae, tubular florales, Convolvulaceae and Ipomoea, and are important crops of grains, feed, industrial raw materials and novel energy resources. Sweet potato vine canker and soft rot become main diseases of main sweet potato growing areas in China, so that the sweet potatoes are reduced in a large area, the quality of the sweet potatoes is reduced, and serious economic loss is caused. Sweet potato vine cutting disease is also called Fusarium oxysporum f.sp batatas, is a fungal disease, pathogenic bacteria invade seedlings from soil through wounds at the base parts or roots of the seedlings or seed-carrying potatoes through a conduit and propagate in conduit tissues, so that the diseased plants are withered and die completely, and the field shows that parts of leaves on the ground turn yellow and fall off from bottom to top, the stem vascular bundles turn brown, finally the stems are cracked, and the whole plants die. After the plant is infected, longitudinal splitting symptoms can be seen at different parts such as roots, stems and tendrils, and the longitudinal splitting symptoms often occur at the stem parts close to the soil.

Sweet potato soft rot (Rhizopus soft rot), caused by Rhizopus nigricans (Rhizopus nigricans Ehrend), is one of the main diseases of sweet potatoes in the storage period, is widely distributed and occurs in each sweet potato production area all over the country. After the disease occurs, the pathogenic bacteria secrete pectinase, and the pectic substance in the cell wall is dissolved, so that the tissue is soft and rotten, the tissue is rapidly spread, and the whole kiln is often rotten, and serious warp extrusion loss is caused.

The disease can be effectively reduced by planting the disease-resistant variety, the typical diseased plant is difficult to see in the field of the high-resistance variety, and the high-resistance variety can show lasting resistance even under the condition of continuous cropping. Therefore, breeding of a new sweet potato variety with high yield, high quality and high resistance becomes a main target of breeding in China.

The sweet potato variety improved by using the genetic engineering technology can overcome the obstacles of reproductive isolation, gene linkage and the like in conventional breeding, and directionally improve the yield, quality and resistance of the sweet potato on the molecular level. Therefore, genes related to disease resistance of the sweet potatoes are cloned and regulated, a new sweet potato material with high yield, high quality and high resistance is created, and the method has very important theoretical reference significance and application value for breeding of the sweet potatoes with high quality, high yield and high resistance.

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.

In order 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 as IbGT1, and the protein IbGT1 can be any one 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 to the amino acid sequence shown in SEQ ID No.1, has more than 80% of identity with the protein shown in A1), and has the function of regulating and controlling plant stress resistance;

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

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

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

The protein-related biological material may be any one of the following:

B1) a nucleic acid molecule encoding the protein;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) said nucleic acid molecule, or a recombinant microorganism containing B2) said expression cassette, or a recombinant microorganism containing B3) said recombinant vector;

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing B1) the nucleic acid molecule or a transgenic plant organ containing B2) the expression cassette.

The nucleotide sequence of the protein IbGT1 of the present invention can be easily mutated by known methods, such as directed evolution or point mutation. Those nucleotides which are artificially modified and have 75% or more identity to the nucleotide sequence of the isolated protein IbGT1 of the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode the protein IbGT1 and have the function of the protein IbGT 1.

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

Herein, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as a Matrix, the Gap existence cost, the Per residual Gap cost and the Lambda ratio are set to 11, 1 and 0.85 (default values), respectively, and a search is performed to calculate the identity (%) of the amino acid sequences, and then the value (%) of identity can be obtained.

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

Further, the protein IbGT1 may be derived from sweet potato (Ipomoea batatas (L.) Lam.).

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

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

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

u3) the use of a substance that regulates the expression of the protein or of a gene that encodes the protein or of a substance that regulates the activity or content of the protein for growing plants with altered resistance to stress;

u4) the use of a substance that regulates the expression of the protein or of a gene that encodes the protein or of a substance that regulates the activity or content of the protein for the preparation of a product for cultivating plants with altered resistance to stress;

u5) the expression substance of the protein or the regulatory gene encoding the protein or the substance regulating the activity or content of the protein.

Herein, the substance which regulates the activity and/or content of the protein may be a substance which regulates the expression of a gene encoding the protein IbGT 1.

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

In the above protein or application, the nucleic acid molecule may be any one of the following:

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

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

The DNA molecule shown in SEQ ID No.2 (IbGT1 gene) encodes 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 coding gene (CDS) of the protein IbGT 1.

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

B1) The nucleic acid molecule also can comprise a nucleic acid molecule which has more than 95 percent of identity with the nucleotide sequence shown in SEQ ID No.2 and is of the same species as the source.

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

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

The recombinant expression vector containing the IbGT1 gene can be constructed by using the existing plant expression vector. The plant expression vector includes but is not limited to binary agrobacterium vector, plant microprojectile bombardment vector, etc. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal can direct polyadenylic acid to the 3 'end of the mRNA precursor, and similar functions can be found in untranslated regions transcribed from the 3' end of genes including but not limited to Agrobacterium crown gall inducible (Ti) plasmid genes (e.g., nopalin synthase Nos), plant genes (e.g., soybean storage protein genes).

When the IbGT1 gene is used to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter may be added before the transcription initiation nucleotide, including but not limited to, for example, cauliflower mosaic virus (CAMV)35S promoter, and maize ubiquitin promoter (ubiquitin), which can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct plant expression vectors, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

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

By using any vector capable of guiding the expression of the exogenous gene in the plant, the IbGT1 gene or the gene segment provided by the invention is introduced into plant cells or receptor plants, and a transgenic cell line and a transgenic plant with improved stress resistance (such as salt tolerance) can be obtained. The expression vector carrying the IbGT1 gene can be used to transform plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant viral vector, direct DNA transformation, microinjection, conductance, agrobacterium-mediated transformation, etc., and to culture the transformed plant tissues into plants.

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

The recombinant vector can be specifically a recombinant vector pCAMBIA1300-IbGT1-GFP, the recombinant vector pCAMBIA1300-IbGT1-GFP is a recombinant expression vector obtained by replacing a fragment (small fragment) between KpnI and SalI recognition sites of the pCAMBIA1300-GFP vector with a DNA fragment of which the nucleotide sequence is SEQ ID No.2 in a sequence table and keeping other sequences of the pCAMBIA1300-GFP vector unchanged. The recombinant vector pCAMBIA1300-IbGT1-GFP expresses the IbGT1 protein shown in SEQ ID No.1 in the sequence table.

The recombinant microorganism can be specifically recombinant agrobacterium EHA105/pCAMBIA1300-IbGT 1-GFP.

The recombinant agrobacterium EHA105/pCAMBIA1300-IbGT1-GFP is a recombinant strain obtained by introducing the recombinant vector pCAMBIA1300-IbGT1-GFP into the Agrobacterium tumefaciens EHA 105.

The present invention also provides a method for increasing stress resistance in a plant, which comprises enhancing or increasing or up-regulating the activity and/or content of the IbGT1 protein, or/and the expression level of a gene encoding the IbGT1 protein in the plant of interest, to increase stress resistance in the plant.

In the method, the activity and/or content of the IbGT1 protein in a target plant are enhanced or increased or up-regulated, or/and the expression level of the IbGT1 protein coding gene can be obtained by introducing the IbGT1 gene into a receptor plant, so that the stress resistance of the plant is higher than that of the receptor plant. The IbGT1 gene encodes the IbGT1 protein.

In one embodiment of the present 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 receptor plant (such as crops or sweet potatoes);

(3) obtaining a stress-resistant plant having higher stress resistance than the recipient plant by screening and identifying.

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

In the above use or method, the stress may be a biotic stress or an abiotic stress.

The biotic stress may be a disease stress. The disease can be specifically a vine cutting disease and/or a soft rot disease.

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 increase.

In the present invention, the purpose of the plant breeding includes breeding of stress-resistant plants. The plant described herein may be any of the following: c1) a dicot or monocot; c2) tubular plants of the order florida; c3) a plant of the family Convolvulaceae; c4) ipomoea plants; c5) sweet potato.

The invention provides IbGT1 protein and a coding gene thereof, and the gene is introduced into sweet potatoes to obtain sweet potato plants over-expressing IbGT1 gene. Salt and drought stress treatment is carried out on the transgenic sweet potato plants, and the salt and drought resistance of the over-expression strains is found to be enhanced compared with that of wild sweet potatoes. The transgenic sweet potato plant is inoculated with the Umbillcaria and soft rot, and the Umbilication strain is found to have enhanced resistance to the Umbilica and soft rot compared with the wild sweet potato. The results show that the IbGT1 gene and the protein coded 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 values in the research of improving the stress resistance of plants. The invention has wide application space and market prospect in the agricultural field.

Drawings

FIG. 1 shows the PCR amplification result of the sweet potato transgenosis-like plant.

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

FIG. 3 shows the growth status of sweet potato plants inoculated with the stem rot fungi.

FIG. 4 shows the growth status of sweet potato plants inoculated with Cordycepsmilitaris.

FIG. 5 shows the statistical results of growth status and phenotype index of sweet potato plants under 86mM NaCl.

FIG. 6 shows the growth status and phenotype index statistics of sweet potato plants subjected to drought stress.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

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

Salt-tolerant sweet potato mutant ND98 is described in the following documents: he Shao Zhen, in vitro screening of sweet potato salt-tolerant mutants and cloning of salt-tolerant candidate genes, doctor academic thesis of Chinese university of agriculture, 2008. The public is available from sweet potato genetic breeding research laboratory of Chinese agriculture university to repeat the experiment.

Chestnut incense (Wang Yu Nu et al, China agricultural science 2003, 36 (9): 1000-.

The cloning vector pMD19-T is a product of Takara Bio-engineering (Dalian) Inc. under the catalog number 6013. The vector pCAMBIA1300-GFP is a product of the Huayuyo company, and the product number is Beijing Huayuyo biological VECT 0460. The total RNA extraction kit of the plant comprisesTiangen Biochemical technology (Beijing) Ltd, catalog number DP 432. The pEASY-Blunt simple vector is a product of Beijing Quanji gold biotechnology, Inc. PrimeScript ^TM The 1st Strand cDNA Synthesis Kit is a product of Takara Bio Inc. (Dalian) under the catalog number 6110A.

The sweet potato secamont SY strains in the following examples are described in the following documents: leishang, poplar new bamboo shoot, a gem of Suwen, Wanlianjun, Chaisha, resistance identification of ten sweet potato varieties to the vine cutting disease [ J ]. Hubei agricultural science, 2014,53(22): 5422-doped 5423, provided by the institute of food crops of the academy of agricultural sciences of Hubei province, and stored by the research laboratory of sweet potato genetic breeding of the university of China agricultural university.

Sweet potato soft rot fungi in the following examples are described in the following documents: yangshuangjing, xu zheng, Zhao Yongqiang, Zhang Ling, Sun Thick, Xiyi Pinna, sweet potato soft rot resistance identification method research and evaluation of sweet potato germplasm resource resistance [ J ]. North China agricultural science report, 2014,29(S1):54-56, provided by Jiangsu Xuzhou sweet potato research center and stored by sweet potato genetic breeding research laboratory of Chinese agriculture university.

Example 1 application of IbGT1 gene in regulation of sweet potato stress resistance

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

Construction of recombinant plasmid

A. Construction of recombinant plasmid pCAMBIA1300-IbGT1-GFP

1.A double-stranded DNA molecule (IbGT1 gene) having a sequence shown in SEQ ID No.2 from the 5' end to the 1st to the 1335 th positions was artificially synthesized. 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 recognition sequences for restriction enzymes KpnI and SalI) as primers, to obtain a double-stranded DNA molecule containing a recognition site for restriction enzymes KpnI at one end and a recognition site for restriction enzymes SalI at the other end.

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

3. After completion of step 2, the recombinant plasmid pEASY-IbGT1 was digested with restriction enzymes KpnI and SalI, and the fragment containing the IbGT1 gene, referred to as fragment 1, was recovered.

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

5. Fragment 1 was ligated to pCAMBIA1300-GFP vector backbone 2 to obtain recombinant plasmid pCAMBIA1300-IbGT 1-GFP.

According to the sequencing results, the recombinant plasmid pCAMBIA1300-IbGT1-GFP was structurally described as follows: replacing a small fragment between recognition sequences of restriction enzymes KpnI and SalI of the recombinant plasmid pCAMBIA1300-GFP with a DNA molecule shown in the 1st to 1335 th positions from the 5' end of a sequence 2 in a sequence table, and keeping other sequences of the pCAMBIA1300-GFP vector unchanged to obtain the recombinant expression vector. The recombinant plasmid pCAMBIA1300-IbGT1-GFP expresses the IbGT1 protein shown in SEQ ID No. 1.

Second, obtaining recombinant agrobacterium and regenerating sweet potato transgenic plant

A. Regeneration of transgenic positive sweet potato plant

1. The recombinant plasmid pCAMBIA1300-IbGT1-GFP is transformed into the agrobacterium tumefaciens EHA105 to obtain the recombinant agrobacterium tumefaciens, and the recombinant agrobacterium tumefaciens is named as EHA105/pCAMBIA1300-IbGT 1-GFP.

2. Peeling stem tip meristem of chestnut fragrance about 0.5mm in length, placing on an embryonic callus induction solid culture medium (MS solid culture medium containing 2.0 mg/L2, 4-D and 3.0% of cane sugar, also called MS +2.0 mg/L2, 4-D and 3.0% of cane sugar), culturing at 27 +/-1 ℃ for 8 weeks to obtain embryonic callus, then placing the embryonic callus in an embryonic callus induction liquid culture medium (MS liquid culture medium containing 2.0 mg/L2, 4-D and 3.0% of cane sugar, also called MS +2.0 mg/L2, 4-D and 3.0% of cane sugar), shaking on a horizontal shaking table and culturing for 3D (specific conditions are 100 r/min; 27 ℃, the period of light and dark alternate culture is 13h per day, the time is 11h, and the illumination intensity is 500lx), obtaining the embryogenic cell mass with the diameter of 0.7-1.3 mm.

3. After step 2 is completed, the embryonic cell mass is transformed with EHA105/pCAMBIA1300-IbGT1-GFP by the Agrobacterium-mediated method, and then placed on a co-culture medium (MS solid medium containing 30mg/L AS and 2.0 mg/L2, 4-D, also called MS +2.0 mg/L2, 4-D +30mg/L AS solid medium) and cultured in the dark at 28 ℃ for 3 days.

4. After step 3 is completed, the embryogenic cell mass is washed 2 times with MS liquid medium containing 900mg/L Cefotaxime Sodium (CS) and 2.0 mg/L2, 4-D (also called MS +900mg/L CS +2.0 mg/L2, 4-D), then placed on selection medium (solid MS medium containing 2.0 mg/L2, 4-D, 300mg/L CS and 5mg/L Hygromycin (Hygromycin, Hyg)), and cultured in the dark at 27 + -1 ℃ for 10-12 weeks (the selection medium needs to be changed every 2 weeks).

5. After 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) and is subjected to light-dark alternate culture at the temperature of 27 +/-1 ℃ (the period of the light-dark alternate culture is 13h of light time, 11h of dark time and 3000lx of light intensity), and the resistant callus is obtained after 2-4 weeks.

6. After the step 5 is completed, the resistant callus tissues are placed on an MS solid culture medium and are cultured alternately in light and dark (the light time is 13h, the dark time is 11h, and the light intensity is 3000lx) at the temperature of 27 +/-1 ℃ for 4-8 weeks, and 219 sweet potato transgenosis-like plants are obtained and are named IbGT1-OX1 to IbGT1-OX219 in sequence.

7. And (3) respectively extracting the genome DNA of the young leaf of the sweet potato transgenosis-like plant obtained in the step (6), taking the genome DNA as a template, and performing 35S-F: 5'-TCAGAAAGAATGCTAACCCACA-3' and IbGT 1-R: 5'-TCATCCCGTCACAGAAGACG-3' performing PCR amplification by using the primer to obtain a PCR amplification product; if the PCR amplification product contains 1400bp of band, the corresponding sweet potato quasi-transgenic plant is the sweet potato transgenic positive plant. The genomic DNA of the young leaf of the sweet potato transgenic-like plant was replaced with equal volume of water and PCR amplification was performed as a blank control (W). The genomic DNA of the young leaf of a sweet potato variety chestnut wild-type plant was used in place of the genomic DNA of the young leaf of a sweet potato transgenic-pseudoplant, and PCR amplification was performed as a negative control (WT). The IbGT1 genome DNA of the young leaf of the sweet potato transgenosis-like plant is replaced by the recombinant plasmid pCAMBIA1300-IbGT1-GFP to carry out PCR amplification as a positive control.

The experimental results are shown in the figure 1, wherein A (M is a DNA molecule Marker, W is a blank control, P is a positive control, and WT is a negative control, and the results show that IbGT1-OX1, IbGT1-OX2, IbGT1-OX9, IbGT1-OX15, IbGT1-OX27, IbGT1-OX38, IbGT1-OX44, IbGT1-OX56, IbGT1-OX71, IbGT1-OX128 and IbGT1-OX156 are sweet potato pseudotransgenic plants.

8、RT-qPCR

Extracting total RNA of the positive plants of the transgenic sweet potatoes, carrying out reverse transcription to obtain cDNA, and carrying out qRT-PCR by taking wild sweet potato plants as a control. The cDNA concentration of the sample is homogenized by using a constitutively expressed sweet potato Actin (Actin) gene as an internal reference. Then real-time fluorescent quantitative PCR (RT-qPCR) analysis is carried out by using specific primers of IbGT1 gene, 2 is used ^-△△CT Method (Livak KJ, Schmitgen TD.2001.analysis of relative gene expression data using real-time quantitative PCR and the 2 ^-△△CT method.25: 402-408) analysis of the expression of IbGT1 gene, each set of samples was repeated 3 times.

The specific primer sequence of the sweet potato Actin (Actin) gene is as follows:

IbActin-F：5′-AGCAGCATGAAGATTAAGGTTGTAGCAC-3′

IbActin-R：5′-TGGAAAATTAGAAGCACTTCCTGTGAAC-3′

the sequence of the specific primer of IbGT1 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 different degrees in the positive transgenic plants of the sweet potato. Selecting transgenic sweet potato plants L15 (namely IbGT1-OX15), L27 (namely IbGT1-OX27) and L38 (namely IbGT1-OX38) to carry out tissue culture (asexual propagation) propagation, using the plant obtained by propagation of one transgenic seedling as a strain to obtain IbGT1 overexpression transgenic strains L15, L27 and L38 (transgenic strains L15, L27 and L38 for short), and carrying out subsequent vine cutting resistance tests, salt resistance tests and drought resistance tests. Transgenic sweet potato plants L2 (namely IbGT1-OX2), L128 (namely IbGT1-OX128) and L156 (namely IbGT1-OX156) are selected for tissue culture (asexual propagation) propagation, and a plant obtained by propagation of a transgenic seedling is used as a line to obtain IbGT1 overexpression transgenic lines L2, L128 and L156 (shortly transgenic lines L2, L128 and L156) for subsequent soft rot resistance tests.

Fifth, identification of stress resistance

1. Identification of vine cutting disease resistance

The sweet potato strains to be detected are sweet potato variety chestnut flavor Wild Type (WT), IbGT1 overexpression transgenic strains L15, L27 and L38.

The experiment was repeated three times, each line was planted with 20 plants each time, and the procedure for each repetition was as follows:

a. inoculating sweet potato vine canker in PDA culture medium, culturing at 28 deg.C under light and dark alternately (the period of light and dark alternately is 13 h/day, 11 h/day, and the illumination intensity is 500lx) for 3d, and culturing at 28 deg.C under dark for 7d to obtain mycelium.

b. After the step a is finished, transferring the hyphae to a triangular flask, adding 100mL of sterile distilled water, oscillating at 100r/min for 30min, filtering by using double-layer sterile gauze, and counting by using a blood counting chamber under a microscope to obtain the sweet potato stem rot fungus spore with the content of 1 × 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 the spore suspension for 30min, planting in the basin filled with sterile sandy soil (3 plants are planted in each basin), and then normally culturing. The growth state of the sweet potato plants was observed at the 0 th planted, the 3 rd planted, the 5 th planted, the 7 th planted, the 9 th planted and the 11 th planted, respectively. Phenotypic indicators (dry fresh weight (g)) of the planted sweet potato plants of 11d were measured and counted.

The incidence and disease index of the vine cutting disease are identified according to 0-6 grades, and plants are taken as units, wherein:

level 0: the plant grows normally and does not have disease symptoms; level 1: the plant grows normally, and the vascular bundles at the base of the stem are browned within 5 cm. And 2, stage: the plant grows basically normally, and the vascular bundles at the base of the stem are browned within 1/3. And 3, level: the leaves at the base of the plant turn yellow, and the vascular bundle turns brown within 2/3. 4, level: most of plant leaves turn yellow and die, and the vascular bundles turn brown and expand to the whole plant; stage 5: the whole plant died. Disease resistance was evaluated according to disease index.

The disease index is calculated according to the following formula: disease index [ (0.1 Xn 1+0.2 Xn 2+0.5 Xn 3+0.8 Xn 4+1.0 Xn 5)/N ]. times 100 (wherein, N is the number of disease strains at each stage, and N is the total number of strains in the test)

Detecting the resistance of the sweet potato vine cutting disease according to the disease index, and specifically grading as follows:

high Resistance (HR): the disease index is 0.0-20.5; anti (R): the disease index is 20.6-40.5; anti-Medium (MR): the disease index is 40.6-60.5; feeling (MS): the disease index is 60.6-80.5; sensing (S): the disease index is 80.6-90.5; high feeling (HS): the disease index is 90.6-100. The Disease Index (DI) [ (Σ number of diseased plants per stage × representative value per stage)/total number of plants per highest representative value) ] × 100.

Data were processed using the SPSS statistical software and the results were expressed as mean ± standard deviation, with P < 0.05 (. star) indicating significant differences and P < 0.01 (. star) indicating very significant differences using the t-test.

The growing state of the sweet potato plant in the basin is shown in figure 3(0d is planted 0d, 3d is planted 3d, 5d is planted 5d, 7d is planted 7d, 9d is planted 9d, 11d is planted 11d, and B is planted 11d for taking out the cleaned sweet potato plant from the basin). The experimental results are as follows: when the strain is inoculated for 3d, the WT plant has obvious disease symptoms (the leaf turns yellow), and the growth states of transgenic strains L15, L27 and L38 are all good; when the strain is inoculated for 9d, the leaves of the WT plant are almost totally yellow, part of old leaves fall off, the stem section is browned and softened, and part of leaves of an overexpression strain are yellow and show light anaphylactic reaction; when the strain is inoculated for 11d, the WT leaves wither and fall off, the stem segments brown, the whole strain dies, the yellowing number of the leaves of the over-expression strain is less, the stem segments of part of the strain brown to a smaller degree, and the plant can still grow normally. The disease index calculation results are shown in table 1, the WT plants are highly susceptible plants, the transgenic line L15 shows resistance, the transgenic line L27 shows susceptible disease, and the transgenic line L38 shows disease resistance, which indicates that the resistance to the vine cutting disease of the transgenic line is improved to different degrees. Therefore, overexpression of the IbGT1 gene in sweet potato can improve the vine cutting disease resistance of sweet potato.

TABLE 1 investigation of the behavior of the vine cutting disease

2. Identification of Soft rot resistance

The strains to be detected are wild type plants (WT) of sweet potato varieties with chestnut flavor, IbGT1 overexpression transgenic lines L2, L128 and L156.

The experiment was repeated three times, each line was inoculated with 20 blocks at a time, and the procedure for each repetition was as follows:

a. inoculating sweet potato soft rot germ into PDA culture medium, performing light-dark alternate culture at 28 deg.C for 3d (the period of light-dark alternate culture is 13 h/day, 11 h/day, and the illumination intensity is 500lx), and performing dark culture at 28 deg.C for 7d to obtain mycelium.

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

c. 20 plants were taken from each line. Punching small holes with the diameter of 1cm on the potato blocks by using a puncher, and placing the potato block fine rods taken out during punching aside for later use.

d. And (3) taking 1mL of bacterial liquid by using a liquid transfer gun, injecting the bacterial liquid into the small hole, cutting and taking a potato block thin rod with a proper length, inserting the potato block thin rod into the small hole, and then smearing vaseline and paraffin from inside to outside in sequence for sealing.

e. And (5) observing the disease condition of the potato blocks after culturing at the constant temperature of 28 ℃ for 10 days.

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

3. Identification of salt resistance

The strains to be tested are wild type plants (WT) of sweet potato varieties with chestnut flavor, IbGT1 overexpression transgenic lines L15 and L38.

The experiment was repeated three times, each strain was treated with 20 strains each time, and the procedure for each repetition was as follows:

(1) the stem segments (about 25cm long and at least 3 stem nodes) of each strain to be tested are taken, fixed by a rigid foam plate, cultured in 1/2 Hoagland +86mM NaCl liquid culture medium (liquid culture medium obtained by adding NaCl to 1/2 Hoagland culture solution (Liu De Gao. obtaining sweet potato plants with over-expression IbP5CR, IbERD3, IbELT and IbNFU1 genes and identifying salt tolerance) until the NaCl content is 86 mM), and at least 1 stem node is penetrated.

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

1/2 Hoagland +86mM NaCl broth in step (1) was replaced with 1/2 Hoagland broth, all other steps being unchanged, as a blank control (Normal) as described above. Data were processed using SPSS statistical software and the results were expressed as mean ± standard deviation, with P < 0.05 (x) indicating significant differences and P < 0.01 (x) indicating very significant differences using t-test.

Statistical results of growth state and phenotype index of sweet potato plants are shown in A1, A2, 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 is fresh weight, and DW is dry weight). The result shows that the growth state of the wild type plant with chestnut fragrance of the sweet potato variety is obviously deteriorated after the salt stress for a period of time, and the growth state and the phenotype indexes of IbGT1 overexpression transgenic lines L15 and L38 are good. Therefore, the salt resistance of the sweet potato can be improved by over-expressing the IbGT1 gene in the sweet potato.

4. Identification of drought resistance

The plant to be tested is a wild type plant (WT) with chestnut fragrance of a sweet potato variety, IbGT1 overexpression transgenic lines L15, L27 and L38, and a water culture experiment and a pot culture experiment are respectively carried out.

The experiment was repeated three times, each strain was treated with 20 strains each time, and the procedure for each repetition was as follows:

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

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

(3) After step (2) was completed, each basin was naturally Drought stressed (Drought) for 8 weeks (i.e., no treatment was performed, including no irrigation of any water and nutrient solution). After 8 weeks, the growth state of the sweet potato plants was observed, and phenotypic indicators (e.g., mean Fresh Weight (FW) per plant, mean Dry Weight (DW) per plant) of the sweet potato plants were measured and counted. As a control, Hoagland nutrient solution was used without drought stress, i.e., normally irrigated with 1/2 (Normal).

Data were processed using SPSS statistical software and the results were expressed as mean ± standard deviation, with P < 0.05 (. lambda.) indicating significant differences and P < 0.01 (. lambda.) indicating very significant differences using the t-test.

The growth state of sweet potato plants is shown in A1, A2 and B1, B2 in FIG. 6 (A1 in FIG. 6 and B1 in FIG. 6 are the growth state of sweet potato plants in the pot pool, and A2 in FIG. 6 and B2 in FIG. 6 are the cleaned sweet potato plants taken out from the pot pool). The statistical results of the phenotypic indicators of sweet potato plants are shown in A3 in FIG. 6 and B3 in FIG. 6. The result shows that the growth state of the wild type plant with chestnut fragrance of the sweet potato variety is obviously deteriorated after a period of drought stress, and the growth state and the phenotype indexes of IbGT1 overexpression transgenic lines L15, L27 and L38 are good. Therefore, the IbGT1 gene is over-expressed in the sweet potato to improve the drought resistance of the sweet potato.

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

SEQUENCE LISTING

<110> university of agriculture in China

<120> stress resistance related protein IbGT1, and 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 (10)

1. Protein or biological material related to said protein, characterized in that said protein is the IbGT1 protein, being the following proteins 1) or 2) or 3):

A1) the amino acid sequence is protein shown as SEQ ID No. 1;

A2) protein which is obtained by substituting and/or deleting and/or adding amino acid residues to the protein of A1), has more than 80% of identity with the protein shown in A1), and has the function of regulating and controlling the stress resistance of plants;

A3) a fusion protein obtained by connecting a protein tag to the N-terminal or/and C-terminal of A1) or A2);

the biological material is any one of the following materials:

B1) a nucleic acid molecule encoding the protein;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) said nucleic acid molecule, or a recombinant microorganism containing B2) said expression cassette, or a recombinant microorganism containing B3) said recombinant vector;

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing B1) the nucleic acid molecule or a transgenic plant organ containing B2) the expression cassette.

2. The protein of claim 1, wherein said protein is derived from sweetpotato.

3. The application is characterized in that: the application is any one of the following:

u1) use of a substance that modulates the expression of the protein of claim 1 or of a gene that encodes the protein of claim 1 or of a substance that modulates the activity or content of the protein for modulating the resistance of a plant to stress;

u2) the use of IbGT1 protein or substances regulating the expression of genes encoding the proteins of claim 1 or substances regulating the activity or content of said IbGT1 protein for the preparation of products regulating the stress resistance of plants;

u3) the use of an expression material for the IbGT1 protein or a regulatory gene encoding the protein of claim 1 or a material which regulates the activity or content of the IbGT1 protein to cultivate plants having altered resistance to stress;

u4) the use of a substance that expresses the IbGT1 protein or regulates the gene that encodes the protein of claim 1 or regulates the activity or content of the IbGT1 protein for the preparation of products for growing plants with altered resistance to stress;

u5) the use of IbGT1 protein or substances regulating the expression of genes encoding the proteins of claim 1 or substances regulating the activity or content of said IbGT1 protein in plant breeding.

4. The protein of claim 1 or 2, or the use of claim 3, wherein: the nucleic acid molecule is a gene shown in the following B1) or B2):

B1) the coding sequence of the coding chain is a cDNA molecule or a DNA molecule of SEQ ID No. 2;

B2) the nucleotides of the coding strand are cDNA molecules or DNA molecules of SEQ ID No. 2.

5. A method of increasing stress resistance in a plant, comprising: the method comprises a step S of enhancing, increasing or up-regulating the activity and/or content of the IbGT1 protein of claim 1 or 2 in a plant of interest, or/and enhancing, increasing or up-regulating the expression level of a gene encoding the IbGT1 protein of claim 1 or 2 to increase the stress resistance of the plant.

6. The method of claim 5, wherein: the step S is to obtain a target plant having higher stress resistance than the recipient plant by introducing the IbGT1 gene into the recipient plant.

7. The use of claim 3 or 4, or the method of claim 5 or 6, wherein: the stress is biotic stress or abiotic stress.

8. The method of claim 7, wherein: the abiotic stress is water stress.

9. The method of claim 7, wherein: the biotic stress is disease stress.

10. The protein of claim 1, or the use of claim 3 or 4, or the method or use of any one of claims 5 to 9, wherein: the plant is any one of the following:

C1) a dicot or monocot;

C2) a plant of the order of the tubulose meshes,

C3) a plant of the family Convolvulaceae,

C4) the plant of the genus Ipomoea,

C5) sweet potato.

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