CN115851783A - HvSRLP gene and application thereof in regulating and controlling cadmium tolerance and cadmium accumulation of plants - Google Patents

Abstract

The invention discloses a HvSRLP gene and application thereof in regulation and control of cadmium tolerance and cadmium accumulation of plants, belonging to the technical field of genetic engineering. The CDS region nucleotide sequence of the HvSRLP gene is shown in SEQ ID NO. 1. The barley HvSRLP gene is cloned and analyzed, and the homologous genetic transformation overexpression and RNA interference technology is combined for functional verification, so that the HvSRLP silencing obviously reduces the transfer of Cd from a plant root system to the overground part, and reduces the cadmium accumulation of the plant.

Description

HvSRLP gene and application thereof in regulating and controlling cadmium tolerance and cadmium accumulation of plants

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a HvSRLP gene and application thereof in regulating and controlling cadmium tolerance and cadmium accumulation of plants.

Background

Cadmium (cadmii, cd) is a highly toxic, non-bio-essential heavy metal element that has high soil-plant mobility and accumulates readily in plant tissues (Song et al, 2015), resulting in crop reduction and threatening human health through the food chain (Clemens et al, 2013 sun et al, 2013. Cadmium entering the body accumulates mainly in the kidneys, with a biological half-life of up to about 20 years (Clemens et al, 2013 aziz et al, 2015). Excessive intake of cadmium can lead to various serious health problems including anemia, cancer, cardiovascular disease and renal tubular injury (Satarug et al, 2003, 2009). In view of this, cadmium contamination in soil has been a hot concern (Arthur et al, 2000 wu et al, 2004 cao et al, 2014).

The phytoremediation technology is a low-cost and effective soil cadmium pollution treatment technology, researches on mining cadmium absorption, transportation and accumulation of related genes of plant crops, clarifying the molecular genetic mechanism of regulation and control of the genes and the like are carried out, and the phytoremediation technology has important significance for developing phytoremediation plant varieties and restoring ecological environment. Luo et al (2018) discovered that defensin-like protein CAL1 is involved in cadmium accumulation in rice leaves, promoting cadmium secretion to the extracellular space by chelation and loading into the xylem, which, after long-distance transport through vascular bundles, eventually results in the deposition of most of the cadmium in leaves rather than kernels. Wang et al (2022) found that HvNAT2, a member of the nucleoside-vitamin C transporter (NAT) family, also enhances cadmium resistance by activating antioxidant capacity in barley and also promotes cadmium accumulation in barley. The cadmium-enriched crop cultivation medium can be applied to cultivation of 'restoration type' crop varieties with high cadmium accumulation in straws and up-to-standard cadmium content in grains, and provides a new ideal mechanism for plant restoration work of cadmium-contaminated soil. But the major challenge facing today is still to identify more novel, effective cadmium-resistant genes.

Barley (Hordeum vulgare l.) is the fourth largest cereal crop that is commonly cultivated worldwide, and is also one of the major sources of cadmium uptake by humans either directly or indirectly (Hayes et al, 2020 lei et al, 2020. Meanwhile, barley is a diploid self-pollinated crop, has a small number of chromosomes, can be used as a model plant of other wheat crops, and is very suitable for physiological and genetic mechanism research (Forster et al, 2000). Therefore, the deep research on the function and the molecular mechanism of the barley cadmium accumulation related gene in cadmium accumulation and tolerance has important significance.

Plant-like receptor protein kinases (RLKs) are molecular recognition receptors located at the plasma membrane of cells, transmitting extracellular molecular signals into the cell, which in turn trigger a downstream series of immune responses (He and Wu, 2016). Typical RLKs generally comprise three domains: extracellular receptor, transmembrane and intracellular kinase domains (Guo et al, 2019), and receptor-like proteins (RLPs) if the intracellular kinase domain is deleted, which typically interact with RLKs to participate in signal transduction (Lv et al, 2020). RLKs can be divided into 6 types based on differences in extracellular domains, where lectin receptor-like protein kinases (LecRLKs) are a subfamily of RLKs whose extracellular domains contain one or more lectin motifs that can reversibly bind to monosaccharides or oligosaccharides. LecRLKs are widely present in plants and are reported to be involved in various biotic/abiotic stresses and also play an important role in plant growth and development (Vaid et al, 2013). LecRLKs can be further classified into L-, G-, and C-types according to the diversity of the domains of extracellular lectins (Zhang et al, 2020), wherein the extracellular domains of G-type LecRLKs are usually complex and, in addition to comprising globular lectins, also comprise S-locus glycoprotein domains (S-domains), plasminogen/Apple/Nematode (PAN) domains and Epidermal Growth Factor (EGF) domains. Wherein the S-locus domain plays an important role in the self-incompatibility of flowering plants, so LecRLKs containing S-domains can also be called S-Site RLKs (SRKs). At present, no relevant report of the barley receptor protein in the aspects of regulating cadmium tolerance and cadmium accumulation is seen.

Disclosure of Invention

The invention aims to provide a gene with cadmium tolerance, which is cloned from barley and is applied to cadmium-polluted plant repair and cadmium-tolerant plant breeding by utilizing the functions of the gene in the aspects of regulating cadmium tolerance and cadmium accumulation.

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

the method takes Suyuanmai No.2 (cadmium sensitive genotype) and withered unknown (cadmium tolerant genotype) as parent hybridization F1, DH groups and parents containing 108 strains constructed by microspore culture are taken as materials, soil culture cadmium stress tests are carried out, QTLs (quantitative trait loci) of the barley grain hundredfold weight and the cadmium and mineral element contents are detected, and a QTLs related to the grain Cd content is detected and named as qKCd5H. qKCd5H is located on chromosome 5H, and 31 annotated genes are found in the QTL interval based on BarleyMap. Among them, the annotation of the HORVU5Hr1G118510 gene is spring/threonine-protein kinase, which is presumed to be related to cadmium accumulation. The nucleic acid and protein sequences were subsequently obtained from NCBI and the barley database IPK, and the results showed that the protein contained the S _ loop _ glyco domain, but did not contain the intracellular kinase domain, and was therefore named HvSRLP.

HvSRLP gene cloning and analysis: the HvSRLP gene is cloned from unknown barley atrophy, and the nucleotide sequence of the CDS region of the gene is shown in SEQ ID NO. 1.

The CDS region of the HvSRLP gene has the full length of 1314bp, and the coded protein sequence of the HvSRLP gene comprises 438 amino acid residues, and the amino acid sequence is shown in SEQ ID NO. 2. The molecular weight of the protein is 45.63kDa; isoelectric point pI =8.19. Analysis and prediction results of functional domains and protein structures show that the protein has 4 conserved functional domains: the 1 st-31 st amino acid sequence is a signal peptide; the 40 th to 153 th amino acid sequences are B _ lectin domains and are related to the specific binding of monosaccharide or oligosaccharide; the 197-312 amino acid sequence is Pfam.S _ locus _ glycop and participates in the self-incompatibility of the flowering plant; PAN _2 domain of amino acid sequence 332-398, which is rich in cysteines involved in disulfide bond formation.

Candidate proteins homologous with HvSRLP are screened from the OneKP transcriptome database and subjected to evolutionary tree analysis, and the result shows that the homology relationship between the protein and TaSRLP is nearest, and the sequence of the two proteins has 91.08 percent of similarity.

Analysis of expression pattern of HvSRLP Gene: the expression level of the HvSRLP gene in the stem and the leaf of the overground part is higher than that of the root system, and the cadmium stress obviously induces the expression of the HvSRLP gene in the overground part. Further analysis of response changes in cadmium stress time showed that: the HvSRLP gene can rapidly respond to cadmium stress in a short time, the relative expression quantity of the HvSRLP gene is obviously increased and reaches a peak value after Cd is treated for 3 hours, and then the HvSRLP gene is gradually reduced until the HvSRLP gene falls back to an initial level after Cd is treated for 1 d. HvSRLP is mainly expressed in vascular bundles, analyzed by in situ PCR using Digoxin (DIG) as a marker. Subcellular localization analysis showed that the HvSRLP protein was localized to the barley cytoplasmic membrane.

The invention utilizes agrobacterium-mediated barley embryo genetic transformation technology to cultivate and screen to obtain over-expression and RNAi silent plants of HvSRLP genes. The phenotype identification is carried out on the genetically transformed plant through a hydroponic cadmium stress test. The results show that:

under the cadmium-free normal growth condition, the growth vigor of the HvSRLP overexpression plant is not obviously different from that of a wild type (GP), but the width and the plant height of an inverted two-leaf of the HvSRLP-RNAi strain are obviously lower than those of the GP, the dry weight of the overground part and the underground part of the RNAi-4 strain is also obviously lower than that of the wild type, and the root length of the HvSRLP-RNAi strain is obviously higher than that of the wild type.

Under the condition of cadmium stress, the growth vigor of the HvSRLP overexpression plant is still similar to that of the wild type, but the growth vigor of the HvSRLP-RNAi plant is obviously weaker than that of the wild type, and is mainly expressed in plant height, aboveground and root biomass accumulation. Silencing the HvSRLP gene of barley enhances the cadmium sensitivity of plants.

The result shows that the HvSRLP gene participates in the normal growth of barley plants and regulates and controls the tolerance of the barley plants under the condition of cadmium stress.

Therefore, the invention provides an application of the HvSRLP gene in regulating and controlling the cadmium stress tolerance and cadmium accumulation of plants. Specifically, silencing of the HvSRLP gene reduces the tolerance of the plant to cadmium.

Research shows that under the condition of cadmium stress, significant Cd is generated in the mature region of the root tip of the HvSRLP gene overexpression plant ²⁺ An inflow, the cadmium content of the aerial parts of which is significantly higher than the wild type. After the HvSRLP gene is silenced, the instantaneous Cd absorption capacity of the barley plant rhizosphere is obviously reduced, and the cadmium content in the barley plant is obviously reduced. The HvSRLP gene is shown to regulate the absorption of the barley to cadmium, and the over-expression of the HvSRLP gene can improve the transport coefficient of the cadmium in the root system to the overground part.

Therefore, the invention provides the application of the HvSRLP gene overexpression plant in phytoremediation of cadmium-contaminated soil. The HvSRLP gene overexpression promotes the absorption and accumulation of cadmium by plants, and cadmium ions are absorbed and transported from soil, so that the aim of phytoremediation is fulfilled.

Further, the application includes: inserting the HvSRLP gene into an over-expression vector to construct recombinant plasmid, then introducing a target gene segment into a receptor plant by utilizing an agrobacterium-mediated technology, and screening to obtain a functionally obtained transgenic plant.

The recombinant vector can be constructed by a conventional method. If a Gateway system is adopted, the full length or the gene fragment of the HvSRLP gene is firstly connected into an entry vector pDONR (Zeo) through a BP reaction, and then connected into an overexpression vector through an LR reaction.

The recipient plant may be, but is not limited to, barley, ramie, cotton. The invention can also adopt a heterologous expression technical means to express HvSRLP in inedible plants.

Preferably, the overexpression vector is pBract214.

Preferably, the host bacterium adopted by the agrobacterium-mediated technology is agrobacterium AGL1.

The invention has the following beneficial effects:

according to the invention, through cloning and analyzing the barley HvSRLP gene, and combining with homologous genetic transformation overexpression and RNA interference technology to carry out functional verification on the HvSRLP gene, the HvSRLP silencing obviously reduces the cadmium tolerance of plants, and the HvSRLP gene participates in regulating and controlling the cadmium stress tolerance of the plants, so that theoretical basis and related genes are provided for barley cadmium tolerance breeding. Furthermore, the invention obviously reduces net Cd in the mature region of the plant root tip based on HvSRLP gene silencing ²⁺ The flow rate, the overground part and the whole cadmium accumulation in the seedling stage are also obviously reduced, and the HvSRLP gene overexpression obviously improves the overground part and the whole cadmium accumulation amount of the plant, so that the HvSRLP gene can be applied to the plant restoration of the cadmium-polluted soil, and a feasible technical scheme is provided for the plant restoration of the cadmium-polluted soil.

Drawings

FIG. 1 is a CDS sequence alignment of the HvSRLP gene in three barley genotypes (atrophic, cadmium-tolerant, threo-Mai No.2, cadmium-sensitive, huangjin-hope, GP).

FIG. 2 is an alignment of the amino acid sequences of the HvSRLP proteins in three barley genotypes (an unknown atrophic, cadmium-tolerant barley genotype; suyunmi No.2, cadmium-sensitive barley; huangjin hope, GP).

FIG. 3 is a phylogenetic and protein structure analysis of HvSRLP of barley, wherein (A) is a phylogenetic analysis of SRLP proteins in representative species of major terrestrial plants, constructed using maximum likelihood, and clades are represented by different colors. (B) Predicted 3D structure of SRLP protein in six representative plants, depicted in cartoon fashion and colored iridescently, blue at the N-terminus and red at the C-terminus. (C) is HvSRLP protein functional domain predicted by SMART. (D) Alignment of the domains of SRLP proteins in six representative plants.

FIG. 4 shows the expression pattern analysis and subcellular localization of HvSRLP gene, wherein (A) is the expression level of HvSRLP gene in different tissues of barley. (B) Is the expression quantity of the HvSRLP gene under different Cd stress treatment time. (C) For subcellular localization of HvSRLP in barley protoplasts, plasma membrane nuclear membrane marker was used in protoplasts, in order from left to right: GFP channel, RFP channel, bright field and fusion channel, scale represents 50 μm. (D) HvSRLP in situ PCR verification of barley leaf (first, second line) and root (third, fourth line), positive control of Actin gene on the left; negative control without reverse transcription is arranged in the middle; hvSRLP specific expression is shown on the right.

FIG. 5 shows the expression level and phenotype analysis of HvSRLP transgenic lines under Cd stress, where (A, B) are the growth phenotypes of wild-type GP, hvSRLP-OE and HvSRLP-RNAi lines under control (A) and Cd treatment, respectively, and the scale represents 5cm. (C) The relative expression level of the HvSRLP gene in the overground part of each strain under control and Cd treatment is shown. (D) The width of the leaves (inverted two leaves) of each strain after Cd treatment for 14d is shown. And (E) the plant height and root length of each strain under control and Cd treatment. (F) The difference in lower case letters represents significant differences (P < 0.05) for the dry weight of the above-ground, underground parts of each line under control and Cd treatment.

FIG. 6 shows the transient Cd at root tip of HvSRLP transgenic line ²⁺ Ion flow chemical analysis, wherein (A) is transient Cd ²⁺ The ion current varies. (B) Is maximum Cd ²⁺ The flow of ions. (C) Is average Cd ²⁺ The flow of ions. (D) Is in steady state Cd ²⁺ The flow of ions. (E) Is total Cd ²⁺ Ion flux, data expressed as mean ± sem of 6-8 biological replicates, with significant differences represented by different lower case letters (P)<0.05)。

FIG. 7 shows the effect of Cd stress treatment on Cd content in different tissues of HvSRLP transgenic lines, where (A, B, C) are 10 μ M CdCl hydroponically grown in 3 weeks old wheat seedlings ₂ After 2 weeks of treatment, the cadmium concentration in the aerial parts (A) and root system (B) of wild-type GP, hvSRLP-OE and HvSRLP-RNAi strains and the cadmium accumulation of the whole strain (C) were determined. (D) The Cd transfer rate from the root of each strain to the overground part has obvious difference represented by different lower case letters (P)<0.05)。

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 present invention provides a theoretical explanation of the mechanism of repairing cadmium stress-resistant genotype atrophy in barley previously selected from the group consisting of cadmium stress-resistant genotypes and related cadmium stress-resistant genotypes (published in Chen F, dong J, wang F, et al. Identification of cadmium stress genes with cadmium graft Cd amplification and bits interaction with cadmium stress microorganisms, 2007, 67.

Example 1 cloning and analysis of CDS region of HvSRLP Gene

1. Conditions for barley growth

Seed of Hordeum vulgare, suyumai No.2 and Huangjinhope (GP) with 2% of H ₂ O ₂ Sterilizing for 30min, and thenWashing with distilled water for 5-6 times, placing sterilized seeds on a germination box paved with double-layer wet filter paper, modifying the germination box from a storage box, punching a hole on a cover, adding distilled water into the germination box, ventilating, performing dark culture (22 deg.C/18 deg.C) in a growth room, and performing light supplement (22 deg.C/18 deg.C, day/night) after germination. On day 6, seedlings with consistent growth were selected and transferred to a 1L black plastic bucket containing basic barley culture solution, covered with a 5-hole plastic cover, two seedlings per hole were fixed with sponge, and cultured in a barley culture room using 1/5Hogland formula. Plants pre-cultured for 3d were subjected to cadmium stress (10. Mu.M CdCl) ₂ Dissolved in basic nutrient solution, pH 5.8) and the basic nutrient solution is used as a control.

2. Analysis of genes associated with cadmium accumulation

A DH group and parents containing 108 strains, which are constructed by hybridizing F1 with a Brassian wheat No.2 (cadmium sensitive genotype) and an agnostic wheat No.2 (cadmium resistant genotype) which have different cadmium resistance as parents (the Brassian wheat No.2 multiplied by the agnostic wheat) through microspore culture, are used as materials to carry out a soil culture cadmium stress test, and a QTL related to the Cd content of grains is detected and named as qKCd5H. qKCd5H is located on chromosome 5H, LOD value is 2.59, which can explain 10.5% of phenotypic variation, and 31 genes with explanation are found in the QTL interval based on BarleyMap. Among them, the HORVU5Hr1G118510 gene was annotated as Serine/threonine-protein kinase, whose nucleic acid and protein sequences were subsequently obtained through NCBI and barley database IPK, and as a result, the protein was shown to contain S _ loop _ glycp domain but not intracellular kinase domain, and thus named HvSRLP.

3. Cloning of CDS region sequence of HvSRLP Gene

Total RNA from the leaf of Okinawa, suyunrei No.2 and GP was extracted 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. Designing a specific primer according to a blast sequence, wherein the specific primer sequence is as follows:

HvSRLP-CDS-F：5′-ATGATGCGGCATCTACGGGGA-3′(SEQ ID NO.3)；

HvSRLP-CDS-R：5′-TCATCCATGGAGGCTGGAGGTC-3′(SEQ ID NO.4)；

and (3) carrying out gel electrophoresis verification on the amplification product, then carrying out gel recovery and connecting with a pMD18-T vector, transforming escherichia coli DH5 alpha, sending positive clones to a company for sequencing, carrying out plasmid extraction and glycerol preservation respectively when the sequencing is correct, and naming the obtained plasmid as a pMD18-T-HvSRLP plasmid.

Nucleotide sequence alignment of CDS regions of three barley genotypes of the unknown atrophic, threo-mai No.2 and GP revealed no difference between the CDS sequences of threo-mai No.2 and GP, but they had 4 SNPs with the CDS sequence of the unknown atrophic (FIG. 1), in which 2 is missense mutation to convert the 108 th amino acid residue from leucine to valine and the 372 nd amino acid residue from threonine to serine (FIG. 2).

The PCR primers were prepared by Biotechnology engineering (Shanghai) Inc., and the gene sequencing work was performed by platinum-Heng Biotechnology Inc., shanghai. The CDS region nucleotide sequence of the HvSRLP gene unknown in barley atrophy is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.

4. HvSRLP protein sequence analysis

The HvSRLP encoded protein sequence contains 438 amino acid residues, and the basic physicochemical properties of the protein were analyzed on line by Expasy (http:// www.expasy.org/tools/protparam.html), and the molecular weight of the protein was 45.63kDa; isoelectric point pI =8.19.

The HvSRLP protein sequence was analyzed by functional domain prediction using SMART (http:// SMART. Embl-heidelberg. De /) website, and the results are shown in FIG. 3C, which shows that the protein has 4 conserved functional domains: the 1 st-31 st amino acid sequence is a signal peptide; the 40 th to 153 th amino acid sequences are B _ lectin domains and are related to the specific binding of monosaccharide or oligosaccharide; the 197-312 amino acid sequence is Pfam.S _ loop _ glycop and participates in the self-incompatibility of the flowering plant; PAN _2 domain of amino acid sequence 332-398, which is cysteine-rich and is involved in disulfide bond formation. The amino acid sequence of the HvSRLP protein is shown in SEQ ID NO. 2.

Candidate proteins homologous to HvSRLP were screened from the OneKP transcriptome database and subjected to evolutionary tree analysis, and the results showed that the protein has the closest homology relationship with TaSRLP (FIG. 3A), and the two protein sequences have 91.08% similarity. Furthermore, the HvSRLP homologous candidate protein was only present in terrestrial plants, but not in algal plants, and the HvSRLP homologous protein was only identified in the four bryophytes, suggesting that the SRLP protein may originate from the bryophytes. The prediction of the 3D structure of representative SRLP proteins in six phyla of angiosperms during their evolution by SWISS showed that the 3D structure difference between angiosperms and gymnosperms SRLP proteins was small, but significant (fig. 3B) with ferns, lycopodium and bryozoans, suggesting that HvSRLP may have evolved from gymnosperms. Multiple sequence alignments of the B _ lectin, S _ loop _ glycop and PAN _2 domains of representative SRLP homologues from 6 phyla showed that the extracellular 3 conserved domains are not conserved across different representative species (FIG. 3D), with very low sequence homology, which may be related to the diversity of their binding substrates.

5. Analysis of expression Pattern of HvSRLP Gene

Analysis of expression pattern of HvSRLP Gene: as shown in fig. 4A, under control conditions, the HvSRLP gene was predominantly expressed in the atrophied, unknown aerial part and in the highest amount in leaves; after 10 mu M Cd is treated, the expression level in leaves is the highest, meanwhile, cadmium stress has no obvious influence on the expression level of roots and stems, but the expression level in the induced leaves is obviously up-regulated. The HvSRLP gene is induced by cadmium stress and mainly occurs in barley leaves.

And (3) selecting withered and unknown leaf tissues at 8 time points of cadmium treatment, namely 0h, 1h, 3h, 6h, 12h, 1d, 3d and 7d, and further analyzing the response change of the HvSRLP gene to cadmium stress time. As shown in FIG. 4B, the HvSRLP gene can rapidly respond to cadmium stress in a short time, and the relative expression level of the HvSRLP gene is remarkably increased and reaches a peak value after Cd treatment for 3h, and then is gradually reduced until the HvSRLP gene falls back to the initial level after Cd treatment for 1 d. The validation primers used for qRT-PCR were:

Actin-F：5′-CCAAAAGCCAACAGAGAGAA-3′(SEQ ID NO.5)；

Actin-R：5′-GCTGACACCATCACCAGAG-3′(SEQ ID NO.6)；

GAPDH-F：5′-AAGCATGAAGATACAGGGAGTGTG-3′(SEQ ID NO.7)；

GAPDH-R：5′-AAATTTATTCTCGGAAGAGGTTGTACA-3′(SEQ ID NO.8)；

HvSRLP-qRT-F：5′-ATGGCAGTCATTCGACCACC-3′(SEQ ID NO.9)；

HvSRLP-qRT-R：5′-AGTTAAGCGTGAGGGTGCC-3′(SEQ ID NO.10)；

specific primers are designed to amplify the HvSRLP gene full length (stop codon is removed) containing BamHI and XbaI enzyme cutting sites, pCAMBIA 1300-35S-sGFP is used as a vector, and a 35S-HvSRLP-sGFP fusion expression vector is constructed by adopting a double enzyme cutting enzyme linking method to perform subcellular localization on HvSRLP protein. The subcellular localization of HvSRLP showed (FIG. 4C) that the HvSRLP protein was localized to the cell membrane. Specific primers used to construct the subcellular localization vectors were as follows (BamH I + Xba I):

35S-HvSRLP-GFP-F:5′-ATTGGATCCATGATGCGGCATCTACGGG-3′(SEQ ID NO.11)；

35S-HvSRLP-GFP-R:5′-ATTTCTAGATCCATGGAGGCTGGAGGTC-3′(SEQ ID NO.12)；

the tissue localization of the HvSRLP gene was analyzed by in situ PCR using Digoxin (DIG) as a marker, and specific primers used for in situ PCR assay were as follows:

HvActin-IS-F:5′-AATGGTCAAGGCTGGTTTCG-3′(SEQ ID NO.13)；HvActin-IS-R:5′-AGAACGATACCAGTAGTACG-3′(SEQ ID NO.14)；HvSRLP-IS-F:5′-GACGGCTCCCTCGTCAACCC-3′(SEQ ID NO.15)；HvSRLP-IS-R:5′-GGCAGTCCTGGAGCTCGGTC-3′(SEQ ID NO.16)；

as shown in FIG. 4D, the HvSRLP gene transcript (blue signal) was expressed in both leaf and root system, and the abundance of expression was higher in leaf, and the signal was concentrated in mesophyll and phloem parenchyma cells, compared to the negative control without reverse transcription; in the root system, although the signal is also detected in the epidermal cells, the intensity is significantly lower than that of the pericycle. The DIG signal is concentrated in xylem parenchyma cells and pericycle cells around the vessel.

Example 2 Gene overexpression and RNAi silencing in barley GP verification of HvSRLP Gene function

1. Construction of overexpression vectors and RNAi silencing vectors

The method for constructing the over-expression vector in the experiment is Gateway. Total RNAs of barley atrophied unknown and Golden hope (Golden Promise) are extracted respectively, and are reversely transcribed into cDNAs, overexpression primers for HvSRLP genes are designed, and CDS region amplification of target genes is carried out by using the atrophied cDNAs as templates. The primer sequence of the 1314bp target gene CDS region amplified by the overexpression primer of the HvSRLP gene is as follows:

HvSRLP-OE-F：

5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTATGATGCGGCATCTAC GGGGA-3′(SEQ ID NO.17)；

HvSRLP-OE-R：5′-GGGGACCACTTTGTACAAGAAAGCTGGGTTCATCCATGGAGGCTGG AGGTC-3′(SEQ ID NO.18)；

the cDNA of GP is taken as a template to amplify the RNAi fragment of HvSRLP gene, a primer is designed to amplify a 319bp fragment (679 to 997bp sequence) of HvSRLP gene with a linker, and the RNAi primer sequence is as follows:

HvSRLP-RNAi-F：

5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTGACGGCTCCCTCGTC AACCC-3′(SEQ ID NO.19)；

HvSRLP-RNAi-R：5′-GGGGACCACTTTGTACAAGAAAGCTGGGT GGCAGTCCTGGAGCTCGGTC-3′(SEQ ID NO.20)；

detecting the PCR amplification product by using 1% agarose gel electrophoresis, carrying out gel recovery and purification on the target product, and measuring the concentration of the recovered product. The recovered gene product at a known concentration was then subjected to BP recombination reaction as follows.

BP recombination reaction system:

reacting at 25 ℃ for 4h, adding 1 mu L of protease K, continuing the reaction at 37 ℃ for 10min, immediately converting the reaction product into escherichia coli DH5 alpha competent cells, coating a bleomycin resistant LB solid culture medium, culturing at 37 ℃ for about 16h, selecting monoclonals, shaking bacteria for carrying out bacteria liquid PCR, sending the bacteria liquid of positive clones to a company for sequencing, carrying out amplification on the monoclonals with correct sequencing, storing the bacteria liquid glycerol and preserving quality-improved particles, wherein the plasmids are named as Pdon (Zeo) -HvSRLP-OE or pDONR (Zeo) -HvSRLP-RNAi respectively. The above-mentioned plasmids of known concentration were subjected to LR reaction as follows (wherein pBract214 vector was an overexpression vector and pANDA vector was an RNAi vector).

LR reaction system:

reacting for 4 hours at 25 ℃, adding 1 mu L of protease K, continuing to react for 10 minutes at 37 ℃, immediately converting a reaction product into escherichia coli DH5 alpha competent cells, coating a kanamycin-resistant LB solid culture medium, culturing for about 16 hours at 37 ℃, selecting a monoclonal antibody, shaking the bacterial antibody until the bacterial antibody is turbid, carrying out bacterial liquid PCR, sending the bacterial liquid of a target strip to a company for sequencing, carrying out amplification and propagation on the monoclonal antibody with correct sequencing, preserving the glycerol of the bacterial liquid and preserving quality-improved particles, and respectively naming the plasmids as pBract214-HvSRLP-OE or pANDA-HvSRLP-RNAi.

Agrobacterium-infected cells AGL1 (pSoup) were transformed with the correctly sequenced plasmid, coated with rifampicin + kanamycin resistant YEB plates, incubated at 28 ℃ for about 40-48h, and positive clones were verified by PCR. 100. Mu.L of the positive clone was added to 10mL of MG broth (pH =7.2, containing 25. Mu.g/mL rifampicin, 50. Mu.g/mL kanamycin) and cultured at 28 ℃ and 180rpm, and shaken to OD ₆₀₀ =0.6-0.7, adding 30% of aseptic glycerol with the same volume, mixing uniformly, quickly freezing by liquid nitrogen, and storing at-80 ℃ for later use.

2. Genetic transformation of young barley embryos

2.1 preparation of genetic transformation Material

Genetic transformation takes young embryos of Golden wheat variety (GP; H.vulgare L.) as explants, seeds are generally harvested 2-3 weeks after barley blossoms, and ears are harvested when the diameter of the young embryos is 1.5-2 mm.

2.2 separation and Sterilization of immature embryos

Selecting barley immature embryo seeds meeting the standard, stripping the barley immature embryo seeds from the ears, removing awns, surface-sterilizing the seeds with 70% alcohol for 30s, and washing with sterilized water for several times. Soaking in 10% sodium hypochlorite for 4min, and washing with sterilized water several times. Separating immature embryo on sterile filter paper, removing embryo axis, placing on callus induction culture medium with shield plate facing upwards, and culturing at 23-24 deg.C in dark for 1-2 days.

2.3 Agrobacterium infection and Co-cultivation

mu.L of the preserved Agrobacterium culture fluid was added to 10mL of MG broth containing no antibiotic, and cultured overnight at 28 ℃ at 180rpm ₆₀₀ And the pesticide is used for infection of immature embryos, wherein the = 1.8-2.0. And dripping the prepared agrobacterium tumefaciens staining solution on each young embryo, and airing. Sealing the plate with sealing film, culturing at 23-24 deg.C in dark for 3d.

2.4 selection culture

After 3d of co-culture with agrobacterium, the young embryos are transferred to a fresh callus induction medium plate for selective culture. At this point the medium contained 50mg/L hygromycin to select positive calli and 160mg/L Terminactina to inhibit Agrobacterium growth. After 56 days of dark culture at 23-24 ℃ (medium plates were changed every 14 days), the calli isolated from the young embryos were transferred to transfer medium and cultured for 21 days at 24 ℃ in low light, at which time green spots would be produced.

2.5 transgenic plant regeneration

The green spot was transferred to subculture medium for further culture, and when the leaves at the upper part of the ground reached 2-3cm, the roots began to build up. The plantlets were transferred to rooting medium without any growth regulator added and without antibiotic change. Then taking out the plantlets with the established root systems, washing the culture medium, transferring the plantlets into a small pot containing vermiculite and nutrient medium, and growing the plantlets in an artificial climate room (22 ℃/18 ℃ and day/night) until the seeds are harvested.

3. Verification of transgenic plants and phenotypic identification of HvSRLP transgenic lines

Dormancy of the transgenic barley seeds is broken through a pre-freezing and heating drying mode, and the seeds germinate on a wet sand bed. After the emergence of seedlings for 7d, the seedlings are transferred to a hydroponic container containing a basic nutrient solution to continue growing. Selecting transgenic plant leaves at two leaf stages, extracting DNA, taking wild type GP as negative control and pBract214-HvSRLP-OE or pANDA-HvSRLP-RNAi plasmid as positive control, verifying whether a vector carrying a target fragment is transferred into a barley genome, and screening to obtain an over-expressed plant of the HvSRLP gene: OE-9, OE-13, and RNAi plants: RNAi-4, RNAi-14.

And (3) carrying out RNA extraction and reverse transcription on the overground part and the root system of the plant with positive DNA verification, and verifying the expression quantity of the HvSRLP gene by RT-PCR semi-quantitative and fluorescent quantitative PCR by taking the expression quantity of the HvSRLP gene in wild GP as a reference.

The DNA verification primers for the over-expressed plants were:

pBract-F:5′-GCATATGCAGCAGCTATATGTG-3′(SEQ ID NO.21)；

HvSRLP-CDS-R：5′-TCATCCATGGAGGCTGGAGGTC-3′(SEQ ID NO.4)；

the DNA verification primers of RNAi plants are:

HvSRLP-RNAi-YZ-F:5′-GACGGCTCCCTCGTCAACCC-3′(SEQ ID NO.22)；

Gluslnk-R:5′-GTCGTCGGTGAACAGGTATGG-3′(SEQ ID NO.23)；

as shown in FIG. 5, the growth phenotypes of wild-type and HvSRLP-overexpressed plants under normal growth conditions (Control) and cadmium treatment conditions (Cd) were not significantly different from those of the wild-type and the overexpressed plants under Control conditions (FIG. 5A), but the inverted leaf width and plant height of the HvSRLP-RNAi strain were significantly lower than those of GP (FIG. 5D), and the above-ground and below-ground dry weights of the RNAi-4 strain were also significantly lower than those of the wild-type strain (FIGS. 5E and 5F), while the root length of the HvSRLP-RNAi strain was significantly higher than that of the wild-type strain.

Under the cadmium stress condition, the growth vigor of HvSRLP overexpression plants is still similar to that of wild type plants, but the growth vigor of HvSRLP-RNAi plants is obviously weaker than that of wild type plants, mainly shown in the plant height, the ground and the dry weight of roots are obviously lower than GP (figures 5E and 5F). Under the conditions of control and Cd stress, the expression level of HvSRLP genes in each over-expression strain is obviously improved, for example, the average expression level of the overground part of OE-9 is 5.6 times of wild type GP, the average expression level of the overground part of OE-13 is 5.6 times of GP, and the average expression level of the root system is 28.1 times of GP; in contrast, the expression level of the HvSRLP gene in the aerial part of the HvSRLP silencing strain was significantly reduced, e.g., the expression level of the HvSRLP gene in the two HvSRLP-RNAi strains under the control condition was only 0.33 times that of GP, and the expression level of the HvSRLP gene in the two HvSRLP-RNAi strains under cadmium treatment was only 0.29 times that of GP (FIG. 5C).

4. HvSRLP on barley rhizosphere Cd ²⁺ Influence of ion flow

Barley wild type GP, hvSRLP-OE and HvSRLP-RNAi strains T subjected to germination accelerating treatment ₁ Seeds are first placed in BSM solution (1mM KCl +0.05mM CaCl) ₂ ) After 5 days of medium-aeration culture, selecting seedlings with the same size, and carrying out Cd in a root tip mature region (about 30mm away from the root tip) by adopting a microelectrode ion flux assay (MIFE) technology ²⁺ And (4) measuring the ion current.

Horizontally fixing the seed roots of the seedlings on a glass slide by using a Parafilm sealing film, and placing the glass slide in a culture dish containing a BSM solution for dark culture for 60min to stabilize ions on the surface of the root system; then placed in a detection tank and filled with Cd ²⁺ The microelectrodes of the ion exchanger were fixed at a distance of 40 μm from the root surface and the electrodes were oscillated back and forth during the measurement process by a micromotor (Patchman NP2, eppendorf, hamburg, germany) with an amplitude of 40 μm and a period of 6 s. The first 5min of the assay was performed under control conditions (BSM solution) to confirm that the root system was in an initial steady state; the BSM solution was then exchanged for a solution containing 10. Mu.M CdCl ₂ The measurement was continued for 25min. After completion of the assay the rhizosphere net ion flux was calculated using the MIFEFLUX program (Newman, 2001) and repeated 8 times per line.

The results are shown in FIG. 6, and the MIFE test results show that the net Cd at the root tip of each line of barley under the control condition ²⁺ The flow is about zero; while at 10. Mu.M CdCl ₂ Under the treatment, significant Cd occurs in the root tip maturation regions of both wild type GP and HvSRLP transgenic lines ²⁺ Net Cd of internal flow, hvSRLP overexpression lines and wild type GP ²⁺ The ion flow rate is similar, but HvSRLP-RNAi can obviously reduce the maximum Cd in the mature region of barley root tip ²⁺ Ion flow Rate, average Cd ²⁺ Cd at ion flow rate and steady state ²⁺ Ion flow rate and rhizosphere absorbed Cd ²⁺ Total amount of ions, two RNAi strains and GP is reduced by 35.6%, 31.6%, 30.9% and 31.6% on average, respectively.

5. HvSRLP-RNAi significantly reduces endogenous Cd accumulation in barley

After the water culture cadmium is stressed for 14 days, each plant line is taken out, and the root system passes through 20mM EDTANa ₂ Chelating for 30min, and then washing with deionized water to remove ions adsorbed on the rhizosphere surface; the plants were subsequently cut into root systems and aerial parts, placed in an oven respectively, and dried to constant weight at 70 ℃. Weighing the sample, shearing, transferring to a digestion tube, and adding 2mL concentrated HNO ₃ The digestion was carried out electro-thermally using a constant temperature metal bath (DTU-2CN, TAITEC, japan). The digestion solution was made up to 20mL with deionized water and diluted 10 times, and then the endogenous Cd content in different tissues of each strain was determined by ICP-MS (Inductively coupled Plasma Mass Spectrometry, agilent 7900).

As shown in FIG. 7A, the Cd content in the overground part of both HvSRLP-RNAi strains was significantly lower than that of the wild type, while the Cd content in the overground part of the OE-9 strain was significantly higher than that of GP (FIG. 7B). Therefore, the cadmium content of the entire HvSRLP-RNAi strain is significantly lower than that of the wild type, the average of the two RNAi strains is reduced by 24.1% compared with GP, and the cadmium content of the entire OE-9 strain is increased by 20.2% compared with GP (FIG. 7C). This is probably due to the fact that the HvSRLP gene silencing significantly reduced the transport coefficient of Cd to the overground part of the barley root system, and the transport coefficients of the two RNAi strains were reduced by 26.9% relative to GP (FIG. 7D).

In conclusion, the functional verification of the gene on barley GP by cloning and analyzing barley HvSRLP and combining overexpression and RNA interference technology shows that the cadmium content in HvSRLP silent plants is obviously reduced, and the cadmium accumulation amount of the overground parts and the whole plants of the plants is obviously improved by overexpression of the HvSRLP gene, especially in HvSRLP-OE9 strains. The invention provides a theoretical basis and related candidate genes for cadmium-polluted plant restoration.

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.

Claims (8)

1. The application of the HvSRLP gene in regulating and controlling the cadmium stress tolerance and cadmium accumulation of plants is characterized in that the CDS region nucleotide sequence of the gene is shown as SEQ ID NO. 1.
2. 2. The use according to claim 1, wherein the HvSRLP gene encodes a protein having the amino acid sequence set forth in SEQ ID No. 2.
3. 3. The use of claim 1, wherein the silencing of the HvSRLP gene reduces cadmium tolerance in the plant.
4. 4. The use of claim 1, wherein HvSRLP gene overexpression promotes cadmium uptake and accumulation in plants.
5. 5. The use of claim 4 for phytoremediation of cadmium contaminated soil using HvSRLP gene overexpressing plants.
6. 6. The use according to claim 5, wherein the plant is barley, ramie or cotton.
7. 7. The application of claim 5, wherein the application comprises: inserting the HvSRLP gene into an over-expression vector to construct recombinant plasmids, then introducing a target gene segment into a receptor plant by utilizing an agrobacterium-mediated technology, and screening to obtain a functionally obtained transgenic plant.
8. 8. The use according to claim 7, wherein the overexpression vector is pBract214.

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