CN112725352A - Barley HvZIFL2 gene and application thereof - Google Patents

Abstract

The invention discloses a barley HvZIFL2 gene and application thereof in regulating and controlling tolerance of barley to drought stress, belonging to the technical field of genetic engineering. The CDS region nucleotide sequence of the barley HvZIFL2 gene is shown in SEQ ID No.1, and the invention verifies the function of the gene on barley XZ5 by cloning and analyzing the barley HvZIFL2 gene and combining the BSMV-VIGS technology. Silencing of the HvZIFL2 gene was found to result in a significant reduction in tolerance of barley plants to drought stress. The invention provides theoretical basis and related genes for barley drought stress tolerance breeding and production.

Description

Barley HvZIFL2 gene and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to cloning and analysis of a barley HvZIFL2 gene and application thereof.

Background

Drought is one of the most common global natural disasters, severely restricts agricultural production, is the first of all abiotic stresses, and reduces crop yield by more than 50% every year due to drought. In recent years, continuous drought has had a major impact on major agricultural production areas worldwide (Wang et al, 2018). In addition to water-source water shortage, with the rapid development of economic society in China, the problem of water pollution is becoming more serious, the water quality is continuously deteriorated, water shortage is caused, and the problem of insufficient water resources in agricultural irrigation in China is further aggravated. Mild drought stress results in a decrease in the rate of plant growth and development, and in severe cases, directly leads to plant death. Under drought conditions, the maximum guarantee of the crop yield can be realized by improving cultivation measures and breeding drought-enduring varieties, wherein the effect of breeding the drought-enduring varieties is more obvious.

Barley (Hordeum vulgare L.) is one of the cereal crops that are widely grown in the world. From a global perspective, barley plants have fourth place to plant area and overall yield, second only to rice, wheat and corn. The barley has high nutritive value and wide adaptability, and has wide application in the aspects of eating, feeding and brewing. More importantly, compared with other cereal crops, the barley has higher drought tolerance and is a good germplasm resource for digging drought-tolerant genes and exploring drought-tolerant mechanisms. However, due to the long-term domestication process, especially modern breeding and cultivation modes, the genetic diversity of the barley is lower and lower, a large number of genes are lost, a monotonous genetic background of the cultivated barley is caused, and the adaptability of the cultivated barley to adverse situations is reduced.

As an ancient crop, barley has two centers of origin, one in the near east gulf of fertile moon and the other in tibetan plateau in china (Dai et al, 2012). Compared with the origin center of the east, the Qinghai-Tibet plateau has strong stress resistance and adaptability to wild barley in the region due to unique features of terrain, climate and ecological environment, has more abundant germplasm resources and higher genetic diversity (Dai and the like, 2014). And the genetic distance between the kindred wild barley and the cultivated barley is short, and the hybridization is completely fertile, so that the kindred wild barley becomes an important gene bank for barley breeding, and particularly, the unique annual wild barley is a valuable resource for adaptability, stress resistance and yield breeding. Therefore, by researching the excellent characteristics of the wild barley, exploring the molecular mechanism behind the wild barley, excavating related genes, introducing the genes into cultivated barley or other crops by utilizing the mode of combining traditional breeding and biotechnology breeding, improving the related characteristics, improving the stress adaptability, forming excellent varieties, being applied to agricultural production, creating economic value and having very wide prospects.

Zinc Induced facilitated transporters (Zinc Induced transporter-Like) are a class of transporters in the Major Facilitator Superfamily (Major Facilitator transporter superfamilies). The ZIFL gene was first discovered and identified in arabidopsis thaliana and is thought to be closely related to plant Zn homeostasis (Haydon et al, 2007). In rice, the ZIFLs gene was identified as a mugineic acid transporter and involved in Fe absorption by root systems (Nozoye et al, 2011, 2015). However, the role and function of ZIFLs in barley is not clear.

Disclosure of Invention

The invention aims to provide a gene with drought stress resistance cloned from barley, which provides a theoretical basis and related genes for barley drought stress resistance breeding and production.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention takes the annual wild barley XZ5 (drought stress tolerant genotype) of Qinghai-Tibet plateau and the internationally recognized drought tolerant barley variety Tadmor screened in the early stage of a subject group as experimental materials to carry out whole genome re-sequencing, comprehensively reveals the mutation types (SNP, InDel, SV and CNV) of the XZ5 and Tadmor whole genomes, identifies the genes affected by the mutations, further screens and clones specific drought tolerant candidate genes in XZ5 by combining with the transcriptome research of the XZ5 and Tadmor responding to the drought stress, and carries out gene function verification.

The invention clones gene HvZIFL2 with drought stress resistance from annual wild barley XZ5 in Qinghai-Tibet plateau, and the nucleotide sequence of CDS region of the gene is shown in SEQ ID No. 1.

Cloning and analysis of the HvZIFL2 gene: based on whole genome re-sequencing comparative analysis and drought response transcriptome studies of two drought tolerant barley genotypes, a drought tolerant candidate gene HORVU3Hr1G043300 was identified, annotated as Zinc Induced facility-Like 2. The full-length CDS sequence of the gene was cloned from XZ5, Tadmor and Hordeum vulgare variety Morex and named HvZIFL 2. The CDS region of the gene has the full length of 1473bp and encodes a 490aa protein sequence.

The invention also provides a protein coded by the HvZIFL2 gene, and the amino acid sequence of the protein is shown in SEQ ID No. 2. The HvZIFL2 amino acid sequence was derived from XZ 5.

The molecular weight of the HvZIFL2 protein is 54.04kDa, and the isoelectric point pI is 9.17. The HvZIFL2 protein sequence was subjected to domain prediction analysis via SMART (http:// SMART. embl-heidelberg. de /) website and InterPro (http:// www.ebi.ac.uk/Interpro /) website, and the results showed that the protein contained 1 domain: major facility supermarket functional domain, comprising 12 transmembrane domains. The sequence of HvZIFL2 protein is subjected to evolutionary tree analysis, and the result shows that the protein and wheat TaZIFL2_ D are located on the same clade.

The invention also provides a BSMV gamma-HvZIFL 2 recombinant vector, which is constructed by connecting the 343bp HvZIFL2 gene fragment shown in SEQ ID No.3 between NheI sites of the BSMV gamma vector. The HvZIFL2 gene fragment used for constructing the vector is derived from XZ 5.

Constructing a BSMV-HvZIFL 2 recombinant vector: a343 bp HvZIFL2 gene fragment is reversely linked between NheI sites of a BSMV gamma vector in a single enzyme digestion connection mode. Sequencing and enzyme digestion verification are carried out on the reverse insertion clone which is positive in PCR verification, so that the accuracy of the recombinant vector is ensured.

Further, the function of the HvZIFL2 gene was verified on wild barley XZ5 by using the BSMV-VIGS system, and the result shows that the expression level of the HvZIFL2 gene in the HvZIFL2 inoculated plants was respectively reduced by 78.55% and 74.94% compared with the plants inoculated with empty vectors under both the control condition and the drought stress condition. Under drought stress, the growth vigor of the inoculated BSMV HvZIFL2 plant is obviously weaker than that of the plant inoculated with the empty carrier, the plant height, the root length and the biomass are also obviously lower than that of the plant inoculated with the empty carrier, and under the control condition, the growth vigor of the two plants is not obviously different. These results indicate that silencing of the HvZIFL2 gene significantly reduced barley tolerance to drought stress.

The invention provides application of the barley HvZIFL2 gene in regulating and controlling the tolerance of barley to drought stress, and the gene enhances the tolerance of barley to drought stress. The barley HvZIFL2 gene silencing leads to the remarkable reduction of the tolerance of the barley to drought stress.

The invention has the following beneficial effects:

the invention discovers that the HvZIFL2 gene silencing causes the barley plant to have obviously reduced tolerance to drought stress by cloning and analyzing the barley HvZIFL2 gene and combining BSMV-VIGS technology to verify the gene function on barley XZ 5. The invention provides theoretical basis and related genes for barley drought stress tolerance breeding and production.

Drawings

FIG. 1 is a sequence alignment of three barley genotypes (XZ5, Tadmor and Morex): (A) nucleotide sequence alignment of HvZIFL2 gene; (B) amino acid sequence alignment of HvZIFL2 protein.

FIG. 2 is a diagram of the prediction of the HvZIFL2 protein domain.

FIG. 3 is a comparison of the amino acid sequences of HvZIFL2 with OsZIFL2 and AtZIFL 2.

FIG. 4 is a phylogenetic tree analysis of HvZIFL2 and the ZIFL2 protein in various plant species.

FIG. 5 shows the construction of the BSMV HvZIFL2 virus. (A) Schematic representation of the BSMV gamma-HvZIFL 2 vector; (B) enzyme digestion verification of BSMV gamma-HvZIFL 2; (C) linearization of BSMV alpha, beta, gamma and gamma-HvZIFL 2; (D) in vitro transcription of BSMV alpha, beta, gamma-HvZIFL 2.

FIG. 6 shows the function of HvZIFL2 gene was verified on wild barley XZ5 using the BSMV-VIGS method. (A) Phenotype identification of HvZIFL2 silenced plants, Vector-inoculated empty Vector plants, VIGS-inoculated BSMV gene silenced plants with HvZIFL 2; (B) qRT-PCR detects the relative expression of HvZIFL2 gene in each treated plant; (C) after 20% PEG-6000 treatment for 30d, the plant height, root length, dry weight of overground part and dry weight of root system of each treated plant are different.

Detailed Description

The technical solution of the present invention will be further specifically described below by way of specific examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.

In the present invention, all parts and percentages are by weight, unless otherwise specified, and the equipment and materials used are commercially available or commonly used in the art. The methods in the following examples are conventional in the art unless otherwise specified.

The invention clones the drought stress-tolerant related gene HvZIFL2 of the barley by taking the drought stress-tolerant genotype XZ5, barley varieties Tadmor and Morex of the annual wild barley of the Qinghai-Tibet plateau screened in the early stage of a subject group as main materials, and has important significance for clarifying the molecular mechanism of barley response to the stress and breeding and production.

Example 1 cloning and analysis of CDS region of HvZIFL2 Gene

1. Conditions for barley growth

Drought stress tolerant genotype XZ5 is the annual wild barley genotype in Qinghai-Tibet plateau screened in the early stage of the subject group and is published in ZHao J, Sun H, Dai H, Zhang G, Wu FB (communication authors). Difference in stress to gravity stress amplification Tibet wibald genetics.Euphytoica 2010,172: 395-403; he XY, Zeng JB, Cao FB, Ahmed IM, Zhang G, Vincze E, Wu FB (Communicator). HvEXPB7, a novel β -excansin gene derived by the root of the root hair transfer of Tibet world wide barley, improves root hair growth under stress journal of Experimental Botany 2015,66(22):7405 eye 7419.

Tadmor is an internationally recognized drought tolerant barley variety (Forster BP, Ellis RP, Moir J, Talame V, Sanguineti MC, Tuberosa R, This D, Telat-Merah B, Ahmed I, Mariy S, Bahri H, El Ouahabi M, Zoumaou-Wallis N, El-Fellah M, Ben Salem M. genotype and phenotype associations with hydro-tolerotic barley Biol 2004,144: 157-168); qia CW, ZHao J, Chen Q, Wu FB (Committee). Genome-wide characterization of stress-reactive non-coding RNAs in bettan-wide barrel, environmental and Experimental Botany,2019,164: 124-.

Morex is a barley variety for cultivation beer.

Seeds of barley XZ5, Tadmor and Morex were seeded with 2% H₂O₂Sterilizing for 30min, washing with distilled water for 5-6 times, placing sterilized seeds on wet sand bed, culturing in dark (22 deg.C/18 deg.C, day/night) in growth room, and supplementing light after germination. At 7d, the seedlings with consistent growth vigor are selected and transferred to a 1L black plastic bucket containing the basic culture solution of barley, covered by a plastic cover with 5 holes, two seedlings in each hole are fixed by using a sponge, and cultured in a barley culture room, and the basic culture solution of barley uses 1/5Hogland formula.

2. Cloning of CDS region sequence of HvZIFL2 Gene

The extraction of total RNA from XZ5, XZ54 and Tadmor leaf was performed according to the instructions of the RNA extraction kit (Takara, Japan), and genomic DNA contamination was removed from the total RNA by DNaseI, and the extracted total RNA was reverse-transcribed into cDNA. Specific Primer design was performed using Primer-BLAST from NCBI with specific Primer sequences:

HvZIFL2-CDS-F：5'-CACGTCCAACGGAGTAGGAG-3'(SEQ ID No.4)

HvZIFL2-CDS-R：5'-TGCGTAGGAGTTTTGACCCC-3'(SEQ ID No.5)

the PCR amplification product was ligated to pMD18-T vector (Takara, Japan), E.coli DH 5. alpha. was transformed, positive clones were selected and sequenced, and plasmid extraction and glycerol storage were carried out, respectively, with the sequencing being correct, and the resulting plasmid was named pMD18-T-HvZIFL 2. The PCR primer synthesis and gene sequencing are completed by Zhejiang Shanghai Biotechnology Ltd. The CDS region nucleotide sequence of HvZIFL2 gene in barley XZ5 is shown in SEQ ID NO. 1. The differences in the nucleotide sequence of the CDS region of the HvZIFL2 gene in XZ5, Tadmor and Morex are shown in FIG. 1.

3. HvZIFL2 protein sequence analysis

The amino acid sequence of the barley HvZIFL2 protein was subjected to prediction and analysis of functional protein domains by the UniProt (https:// www.uniprot.org /) and InterPro (http:// www.ebi.ac.uk/Interpro /) websites, and the result showed that the protein contained 1 functional domain (FIG. 2) and contained 12 transmembrane domains (FIG. 3), consistent with its identity as a transporter protein.

Amino acid sequences of ZIFL2 proteins of various plant species including HvZIFL2 were analyzed in comparison by MEGA-X software, and phylogenetic trees were constructed. The results show that HvZIFL2 was evolutionarily conserved and most closely related to TaZIFL2_ D of wheat, on the same clade (fig. 4).

Example 2 BSMV-VIGS method to verify HvZIFL2 Gene function in wild barley XZ5

1. Construction of BSMV-gamma-HvZIFL 2 vector

Specific Primer design was performed using NCBI's Primer-BLAST to amplify a 343bp HvZIFL2 gene fragment using the pMD18-T-HvZIFL2 plasmid described in example 1 as a template. The sequence of the fragment is shown as SEQ ID No. 3.

The reaction system for PCR amplification is as follows:

the amplification procedure was: 94 ℃ for 2min, (98 ℃ for 10s, 60 ℃ for 30s, 68 ℃ for 1min)35 cycles.

Primer sequences are (restriction sites underlined):

HvZIFL2-γ-F：5'-GTACGCTAGCTTTTCGGCCTTAGCACGACT-3'(SEQ ID No.6)

HvZIFL2-γ-R：5'-GTACGCTAGCATGTGCAGAGTTTCCGGGAG-3'(SEQ ID No.7)

connecting the HvZIFL2 gene fragment to a pMD18-T vector, transforming escherichia coli DH5 alpha, selecting positive clones, sending the positive clones to a company for sequencing, and carrying out shake bacteria extraction on the single clones with correct sequencing and storing glycerol. The extracted plasmid is cut by Nhe I restriction enzyme, and is respectively connected with virus vector BSMV gamma vector which is cut by the same restriction enzyme and dephosphorylated by T4 ligase. The ligated product was transformed into E.coli DH 5. alpha. and reverse insertion was verified using primer γ -stain-F on BSMV: γ vector and forward primer HvZIFL2- γ -F of HvZIFL2 gene, the reverse inserted positive clone was sent to the company for sequencing, the single clone with correct sequencing was shaken to extract plasmid and glycerol for storage, the extracted plasmid was digested again (FIG. 5B), and the plasmid was named BSMV: γ -HvZIFL 2.

γ-stain-F：5'-CAACTGCCAATCGTGAGTAGG-3'(SEQ ID No.8)。

2. BSMV vector linearization and in vitro transcription

Separately digesting BSMV alpha, BSMV gamma and BSMV gamma-HvZIFL 2 with Mlu I restriction enzyme and separately digesting BSMV beta with Spe I restriction enzyme, linearizing the linearized products, performing agarose gel electrophoresis on the linearized products, and recovering and purifying the gel (FIG. 5C). Purifying the purified BSMV alpha, BSMV beta, BSMV gamma and BSMV gamma-HvZIFL 2 at a certain concentration according to RiboMAX^TMLargeScale RNA Production System-T7 kit and Ribo m⁷The G Cap Analog kit instructions were used for reverse transcription (Promega, USA) (FIG. 5D), and all manipulations were required to ensure no RNase contamination.

RNA alpha, RNA beta and RNA gamma/RNA gamma-HvZIFL 2 plasmids after in vitro transcription were mixed at a volume ratio of 1:1:1, diluted with three volumes of RNase-free water, followed by addition of an equal volume of 2 XGKP buffer (1% bentonite, 1% celite, 50mM glycine and 30mM dipotassium hydrogen phosphate pH 9.2) to the diluted product, and mixed well for subsequent inoculation. The resulting product was named BSMV HvZIFL 2.

3. Barley seedling culture before BSMV inoculation

Barley XZ5 seed 2% H₂O₂Sterilizing for 30min, washing with distilled water for 5-6 times, placing sterilized seeds on wet sand bed, culturing in dark (22 deg.C/18 deg.C, day/night) in growth room, and supplementing light after germination. At 7d, the seedlings with consistent growth vigor are selected and transferred to a 1L black plastic bucket containing the basic culture solution of barley, covered by a plastic cover with 5 holes, two seedlings in each hole are fixed by using a sponge, and cultured in a barley culture room, and the basic culture solution of barley uses 1/5Hogland formula. Changing the culture solution every 3d, wherein the pH value of the culture solution is determinedNaOH or HCl is adjusted to 5.8 +/-0.1, and aeration is continued for 24h by using an air pump.

4. Verification of gene function by BSMV vaccination

Selecting the second leaf of the barley in the two-leaf period, carrying out BSMV rubbing inoculation in an RNase-free environment, using 10 mu L of the mixture for each plant, spraying a proper amount of DEPC water on the inoculated plant, moisturizing the plant by using a transparent plastic cover, taking out the glass cover after 3 days of moisturizing, continuously culturing in a barley growth culture room (22 ℃/18 ℃, day/night), and regularly observing the phenotype of the plant. There were 4 treatments in this experiment: inoculation of BSMV: HvZIFL2 and growth in minimal medium (BNS) for 36d (treatment 1), inoculation of BSMV: Vector and growth in minimal medium (BNS) for 36d (treatment 2), inoculation of BSMV: HvZIFL2 and growth in minimal medium (BNS) for 6d, further 20% PEG-6000 treatment for 30d (treatment 3), inoculation of BSMV: Vector and growth in minimal medium (BNS) for 6d, and further 20% PEG-6000 treatment for 30d (treatment 4). Each treatment was 3 replicates, each replicate 8 plants. And after drought treatment for 30d, observing the growth condition of each treated plant, and determining related growth character indexes.

The results show that under drought stress, the growth vigor of the plants inoculated with BSMV HvZIFL2 (treatment 3) was significantly weaker than that of the plants inoculated with the empty vector (treatment 4), and the plant height, root length and biomass were also significantly lower than that of the plants inoculated with the empty vector, whereas under the control conditions, there was no significant difference in growth vigor between the two (FIG. 6). These results indicate that silencing of the HvZIFL2 gene significantly reduced barley tolerance to drought stress.

Fluorescent quantitative PCR detection of HvZIFL2 gene expression level

Separately extracting total RNA from different treated samples with total RNA extraction kit (Takara, Japan), removing genomic DNA contamination from total RNA with DNaseI, and using PrimeScript^TMThe RT reagent Kit reverse transcription Kit (Takara, Japan) reverse transcribes the total RNA of each sample into single-stranded cDNA, respectively. The expression of HvZIFL2 gene in the corresponding sample was subjected to quantitative fluorescent PCR analysis (qRT-PCR) using SYBR Green Fluorogenic enzyme complex (Takara, Japan) and Light Cycler 480 PCR instrument (Roche, Switzerland), and the expression value was corrected using an internal reference gene GAPDH.

The qRT-PCR system is:

the specific procedure for PCR was: 95 ℃ 30s, (95 ℃ 5s, 60 ℃ 30s)40 cycles. The dissolution curve program was: cooling at 95 deg.C for 5s, 60 deg.C for 1min, 95 deg.C, and 50 deg.C for 30 s. By use of 2^-ΔΔCqThe gene expression value change was calculated by a relative quantification method. Each set of experiments was repeated three times. The qRT-PCR primer sequence is:

HvZIFL2-qRT-PCR-F：5'-GAATATGTTTGCGGCTACGATT-3'(SEQ ID No.9)；

HvZIFL2-qRT-PCR-R：5'-CACTCGGCATAGAGAAAAATGG-3'(SEQ ID No.10)；

GAPDH-F：5'-AAGCATGAAGATACAGGGAGTGTG-3'(SEQ ID No.11)；

GAPDH-R：5'-AAATTTATTCTCGGAAGAGGTTGTACA-3'(SEQ ID No.12)。

the results obtained were: HvZIFL2 gene expression was inhibited by 78.6% and 75.0% in the BSMV HvZIFL2 plants inoculated, respectively, compared to the empty vector inoculated plants, both under control and drought conditions (1 vs.2, 3vs.4 treatment) (FIG. 6B).

In conclusion, through cloning and analysis of the HvZIFL2 of the barley and functional verification of the gene on the annual wild barley XZ5 of the Qinghai-Tibet plateau by combining the BSMV-VIGS technology, the tolerance of the XZ5 plant subjected to HvZIFL2 gene silencing to drought stress is remarkably reduced.

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

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<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

caactgccaa tcgtgagtag g 21

<210> 9

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gaatatgttt gcggctacga tt 22

<210> 10

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cactcggcat agagaaaaat gg 22

<210> 11

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

aagcatgaag atacagggag tgtg 24

<210> 12

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

aaatttattc tcggaagagg ttgtaca 27

Claims (6)

1. The barley HvZIFL2 gene is characterized in that the CDS region nucleotide sequence of the gene is shown in SEQ ID No. 1.

2. The barley HvZIFL2 gene-encoded protein according to claim 1, which has the amino acid sequence shown in SEQ ID No. 2.

3. The BSMV gamma-HvZIFL 2 recombinant vector is characterized by comprising a BSMV gamma vector and a target gene fragment inserted between NheI sites of the vector, wherein the nucleotide sequence of the target gene fragment is shown as SEQ ID No. 3.

4. Use of the barley HvZIFL2 gene according to claim 1 for modulating tolerance of barley to drought stress.

5. The use according to claim 4, wherein the silencing of the HvZIFL2 gene results in a significant reduction in barley tolerance to drought stress.

6. The use according to claim 4, wherein the barley is the annual wild barley (Hordeum vulgare L.ssp. spontaneem) XZ5 from Qinghai-Tibet plateau.

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