CN116286864A

CN116286864A - Cadmium-resistant gene IlDTX49 of Iris irica, and encoding protein and application thereof

Info

Publication number: CN116286864A
Application number: CN202310151484.9A
Authority: CN
Inventors: 郭强; 李翠; 张嘉; 侯新村; 赵春桥; 王庆海
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2023-02-22
Filing date: 2023-02-22
Publication date: 2023-06-23

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to a cadmium-resistant gene IlDTX49 of Iris lactea and encoding protein and application thereof. The invention clones IlDTX49 gene from Iris lactea, and the gene expression is obviously induced and regulated under cadmium stress. Compared with a wild type, the gene functional verification experiment shows that the IlDTX49 gene can improve plant biomass under cadmium stress, reduce cadmium content in plants, improve the antioxidant enzyme activity of the plants, inhibit active oxygen burst and reduce membrane lipid peroxidation, thereby enhancing cadmium tolerance of the plants. Can provide a new idea for cultivating new varieties of cadmium-resistant or low-cadmium crops and pastures, and has wide application prospect.

Description

Cadmium-resistant gene IlDTX49 of Iris irica, and encoding protein and application thereof

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a cadmium-resistant gene IlDTX49 of Iris lactea and encoding protein and application thereof.

Background

In recent years, with the rapid development of industry and agriculture, the cultivated area polluted by heavy metals such As cadmium (Cd) and arsenic (As) is increasing, and the cultivated area is a serious environmental problem which cannot be ignored. It is well known that Cd, in comparison with other heavy metal elements ²⁺ Is more soluble in water and has extremely strong biotoxicity. The reason for this is Cd ²⁺ Resulting in oxidative stress, reduced enzymatic activity, and Ca in plants ²⁺ A series of stress reactions such as signal unbalance and the like can further trigger damage of a photosynthetic system, membrane peroxidation, endoplasmic reticulum stress and the like, finally influence crop quality and even lead to death of plants, and more seriously enter human bodies through food chains to form serious threat to human health. Thus, how to control and reduce Cd ²⁺ Accumulation in plants has become a current challenge.

Although phytoremediation is an emerging energy-saving and eco-friendly soil heavy metal pollution remediation technology, cultivation of safe low-cadmium crop varieties is one of the effective ways to achieve maximum utilization of cadmium-contaminated land resources for large-area light and medium-cadmium-contaminated farmlands. While understanding plant Cd ²⁺ The detoxification mechanism is a key point for developing cultivation of low-cadmium crop varieties. Cd (cadmium sulfide) ²⁺ Including cell wall fixation, plant chelation, vacuole isolation, active efflux of roots, induction of antioxidant mechanisms, stress proteins, etc. (Clemens S, palmgren MG,

U.A Long way head understanding and engineering Plant metal collection, trends in Plant Science,2002, 7:309-315.). Research shows that the Phytochelatin (PC) complexes Cd in root cells ²⁺ Forming a PC-Cd complex, and then separating the PC-Cd complex into vacuoles by a vacuole membrane ABCC3 transporter, thereby relieving Cd ²⁺ Toxicity to cellsDeleterious effects (Brunett P, zanella L, paolis AD, di Litta D, cecchetti V, falasca G, barbieri M, altamura MM, costantin P, cardarelli M.Caseium-inducible expression of the ABC-type transporter AtABCC3 increases phytochelatin-mediated cadmium tolerance in Arabidopsis. Journal of Experimental Botany,2015, 66:3815-3829.). Overexpression of tonoplast heavy metal P in rice _1B2 The ATPase enzyme HMA3 can convert a large amount of Cd ²⁺ The division is carried out until the root cell vacuoles are fixed on the root system, so that the detoxification function is realized on one hand, and the Cd restriction is realized on the other hand ²⁺ Transferring to overground part to greatly reduce Cd in rice ²⁺ The content of the food reaches the international stipulated edible standard.

Multidrug and toxic compound efflux transporter MATE (Multidrug and Toxic Compound Extrusion), which is a membrane transporter, also known as detoxification efflux transporter DTXs (Detoxification Efflux Carriers), is widely found in eukaryotes and prokaryotes, and plays a vital role in Plant nutrient absorption, secondary metabolite transport, and detoxification of harmful substances, among other things (Baetz U, martinoia e. Root ex udates: the hidden part of Plant inhibitors. Trends in Plant Science,2014, 19:90-98; uppaphyAy N, kar D, deep Mahajan B, nanda S, rahiman R, panchaksky N, bhagovatula L, datta S.the multitasking abilities of MATE transporters in plants. Journal of Experimental Botany,2019,70:4643-4656, xia JQ, nazish T, javaid A, ali M, liu QQ, wang L, zhang ZY, zhang ZS, huang YJ, wu J, yang ZS, sun LF, chen YX, xiang CB.A gain-of function mutation of the MATE family transporter DTX 6-confers paraquat resistance in Arabidopsis. Molecular Plant,2021,14:2126-2133. Notably, the expression of the Arabidopsis thaliana plasma membrane AtDTX1 transporter gene is expressed in various tissues under the stress of Cd, and the heterogenous expression of the AtDTX1 gene in yeast obviously reduces the Cd of the yeast ²⁺ Concentration and strong tolerance to cadmium, indicating that AtDTX1 may be involved in mediating root Cd ²⁺ Is arranged (Li L, he Z, pandey GK, tsukiya T, luan S.functional cloning and characterization of a plant efflux carrier for multidrug and heavy metal detoxication.journal of Biological Chemistry,2002, 277:5360-5)368.). Thus, the DTXs can be used as a potential excellent candidate gene for molecular design breeding of low-cadmium crops or low-cadmium pastures.

Iris lactea pall.var.chinensis (Fisch.) Koidz.) is a perennial herb of Iridaceae, which is mainly distributed in natural grassland areas in North China, and shows extremely strong tolerance to cadmium, so that it is widely used in urban and mining ecological environment construction (Guo Q, meng L, zhang YN, mao PC, tian XX, li SS, zhang L.stationary systems, metal ion homeostasis and cadmium distribution in Iris lactea exposed to cadmium stress. Ecosystem and Environmental Safety,2017,139:50-55;Guo Q,Tian XX,Mao PC,Meng L.Functional characterization of IlHMA2,a P1B2-ATPase in Iris lactea response to Cd.environmental and Experimental Botany,2019, 157:131-139.). However, the research on the cadmium-resistant mechanism of iris lactea is still limited at present. In view of the above, the invention clones the detoxified efflux transporter gene IlDTX49 from Iris lactea for the first time, and the gene function verification shows that the detoxified efflux transporter gene IlDTX49 can obviously reduce the Cd of transgenic plants ²⁺ Concentration, and enhanced tolerance to cadmium, and increased biomass. Provides a new idea for cultivating new varieties of low-cadmium crops or low-cadmium pastures, and has important application prospect.

Disclosure of Invention

The invention provides a cadmium-resistant related gene IlDTX49 of Iris lactea, and a coding protein and application thereof.

The invention screens a significant up-regulated differential expression gene IlDTX49 by transcriptome sequencing of Iris with cadmium treatment, and the gene has low nucleotide sequence homology (38.68 percent only) with Arabidopsis AtDTX1, which suggests that IlDTX49 is different from AtDTX1 in cadmium-resistant function. Further, the detoxification efflux transporter gene IlDTX49 is obtained by first cloning from Iris, and is introduced into Arabidopsis. Experimental results show that the gene obviously reduces Cd of transgenic arabidopsis thaliana ²⁺ The concentration and the antioxidant enzyme defense system are activated, so that the active oxygen is removed, the peroxidation degree of membrane lipid is reduced, and the tolerance of transgenic plants to cadmium is enhanced, and the biomass of the transgenic plants is improved.

Specifically, the invention provides the following technical scheme:

in a first aspect, the present invention provides an Iris irisquish IlDTX49 protein having any one of the amino acid sequences as follows (1) - (4):

(1) An amino acid sequence as shown in SEQ ID NO. 1;

(2) An amino acid sequence with the same functional protein obtained by replacing, deleting or inserting one or more amino acids into the amino acid sequence shown as SEQ ID NO. 1;

(3) An amino acid sequence having at least 90% homology with the amino acid sequence shown in SEQ ID NO.1 and having an equivalent functional protein;

(4) An amino acid sequence obtained by ligating a tag, an enzyme cleavage site and/or a linker peptide sequence to the N-terminus and/or C-terminus of any one of the amino acid sequences (1) to (3).

The label in the above (4) may be any one of the labels described in table 1.

TABLE 1 tag sequences

Label (Label)	Residues	Sequence(s)
			Poly-Arg	5-6 (usually 5)	RRRRR
Poly-His	2-10 (usually 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

In the invention, the Iris lactea IlDTX49 protein can be obtained by artificial synthesis or by first synthesizing the coding gene and then biologically expressing.

In a second aspect, the present invention provides a nucleic acid molecule encoding the iris lactea 49 protein.

According to the iris lactea il dtx49 protein and the codon rules provided above, a person skilled in the art can obtain a nucleotide sequence of a nucleic acid molecule encoding the iris lactea il dtx49 protein, and the sequence of the nucleic acid molecule is not unique due to the degeneracy of codons, so that all nucleic acid molecules capable of encoding the iris lactea 49 protein are within the scope of the present invention.

Specifically, the nucleic acid molecule has a nucleotide sequence of any one of the following (1) to (4):

(1) A nucleotide sequence as shown in SEQ ID NO. 2;

(2) A nucleotide sequence which is obtained by substituting, deleting or inserting one or more nucleotides into the nucleotide sequence shown as SEQ ID NO.2 and codes for the equivalent functional protein;

(3) A nucleotide sequence having at least 90% homology with the nucleotide sequence shown as SEQ ID NO.2 and encoding a functionally equivalent protein;

(4) A nucleotide sequence which hybridizes to the sequence shown in SEQ ID No.2 and encodes a functionally equivalent protein under stringent conditions, i.e., in a 0.1 XSSPE solution containing 0.1% SDS or in a 0.1 XSSC solution containing 0.1% SDS, at 65℃and washing the membrane with said solution.

The nucleic acid molecule comprises genomic DNA or cDNA encoding the Iris irisquish IlDTX49 protein.

In a third aspect, the invention provides a biological material comprising the nucleic acid molecule, the biological material being an expression cassette, a vector or a host cell.

The above expression cassette may be a recombinant DNA obtained by ligating a regulatory element for driving transcription and/or translation thereof upstream and/or downstream of the above-described nucleic acid molecule.

The vector may be an expression vector or a cloning vector, including but not limited to, plasmid vectors, phage vectors, transposons, and the like.

Among them, expression vectors include, but are not limited to, plant expression vectors pBI121, pCAMBIA1301/1302/2301/3301, pGreen0029, pBin19 or other derived plant expression vectors.

From the viewpoint of transgenic plant safety, the above vectors can be substituted with a 6-phosphomannose isomerase gene, a xylose isomerase gene, or a betaine aldehyde dehydrogenase gene for an antibiotic selection marker or an herbicide resistance marker to obtain transformed plants free of the antibiotic marker or the chemical resistance marker gene.

The host cell may be a microbial cell or a transgenic plant cell, wherein the microorganism includes, but is not limited to, E.coli, agrobacterium, yeast, and the like.

In a fourth aspect, the present invention provides primers for amplifying the above-described nucleic acid molecules.

In a fifth aspect, the present invention provides the use of the irisquinone IlDTX49 protein or the nucleic acid molecule or the biological material described above for improving cadmium tolerance in plants.

The invention provides application of the Iris irisquilla IlDTX49 protein or the nucleic acid molecule or the biological material in reducing cadmium content in plants.

The reduction of plant cadmium content is preferably the reduction of plant cadmium accumulation or the reduction of plant cadmium content under high cadmium conditions.

The invention provides application of the Iris irisquilla IlDTX49 protein or the nucleic acid molecule or the biological material in preparing transgenic plants.

Preferably, the transgenic plant is a cadmium tolerant transgenic plant or a low cadmium transgenic plant.

The invention provides application of the Iris irisquilla IlDTX49 protein or the nucleic acid molecule or the biological material in improving plant biomass, root length and/or relieving plant leaf wilting and yellowing under high cadmium conditions.

The invention provides application of the Iris irisquilla IlDTX49 protein or the nucleic acid molecule or the biological material in improving the activity of plant antioxidant enzyme, reducing the level of plant active oxygen, inhibiting the peroxidation of plant membrane lipid and/or improving the photosynthesis of plants under the condition of high cadmium.

Wherein, the improvement of the plant antioxidant enzyme activity, the reduction of the plant active oxygen level, the inhibition of the plant membrane lipid peroxidation are specifically represented by the reduction of MDA content of plants and the increase of APX enzyme activity; the improvement of photosynthesis in plants is embodied by an increase in chlorophyll content and carotenoid content in the plants.

In some embodiments of the invention, the high cadmium conditions are a cadmium ion concentration of 50. Mu.M or greater, preferably 100. Mu.M or greater, more preferably 200. Mu.M or greater.

In a sixth aspect, the invention provides a method of improving cadmium tolerance or growing a low cadmium plant, the method comprising: the expression quantity and/or activity of the Iris Palatifolia IlDTX49 protein in the plant are improved.

In some embodiments of the present invention, the expression amount and/or activity of iris lactea protein IlDTX49 in the plant is increased by introducing the gene encoding iris lactea protein IlDTX49 into the plant, resulting in a transgenic plant with increased cadmium resistance.

The coding gene of the Iris lac DTX49 protein can be introduced into plants through a plant expression recombinant vector carrying the coding gene, and the recombinant vector can be introduced into target plants through conventional transformation methods such as Ti plasmid, ri plasmid, DNA transformation, plant virus vector, electric conduction, microinjection, agrobacterium mediation and the like.

In some embodiments of the present invention, a recombinant expression vector containing the gene encoding Iris irisquish IlDTX49 protein is transformed into a target plant by using an Agrobacterium-mediated method, and transgenic plants are selected. Wherein the starting vector of the recombinant expression vector is pBI121.

In the present invention, the plant may be a monocot plant including iris, wheat, rice, corn, millet, sorghum, barley, bluegrass, ryegrass, oat, pennisetum, timothy, switchgrass, etc., or a dicot plant including arabidopsis, tobacco, soybean, tomato, rape, cotton, alfalfa, venture, red bean grass, white clover, chicory, etc.

In some embodiments of the invention, the plant is arabidopsis thaliana or iris.

The invention has the beneficial effects that: the detoxification efflux transporter gene IlDTX49 is obtained by first cloning from Iris japonica, and the expression of IlDTX49 in Iris japonica is obviously induced and regulated by cadmium stress. Functional verification experiments prove that the IlDTX49 gene can improve cadmium resistance of plants, improve plant biomass under cadmium stress, and reduce cadmium content of the plants; specifically, in arabidopsis, ilDTX49 is overexpressed, compared with a wild type, the biomass of a transgenic arabidopsis plant under cadmium stress is higher, particularly the cadmium content in the body is obviously reduced, the antioxidant enzyme activity is greatly improved, the active oxygen burst is inhibited, and the membrane lipid peroxidation is reduced, so that the cadmium tolerance of the transgenic plant is enhanced. The Iris lactea gene IlDTX49 and its coded protein have very wide application prospect in the cultivation of new variety of cadmium-resistant or low-cadmium crops and pastures.

Drawings

In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the detection of the PCR product of Iris irisquish IlDTX49 open reading frame in example 1 of the present invention. Wherein, M is DNA Marker DL2000,1 is Iris Palo IlDTX49 open reading frame PCR product.

FIG. 2 is a sequence alignment and structural analysis of Iris irisquish IlDTX49 in example 2 of the present invention. Wherein A is multiple comparisons of amino acid sequences of Iris Palmetto IlDTX49, date PdDTX49 and oil palm EgDTX 49; b is Iris IlDTX49 topology.

FIG. 3 is a phylogenetic tree analysis of Iris lacuna DTX49 and other plant DTX in example 2 of the present invention. Wherein, arabidopsis thaliana (Arabidopsis thaliana, at); date (Phoenix dactylifera, pd); oil palm (Elaeis guineensis, eg); coconut (Cocos nucifera, cn); pineapple (Ac); ginger (Zingiber officinale, zo); rice (Oryza sativa, os); barley (Hordeum vulgare, hv); wheat (Triticum aestivum, ta); corn (Zea mays, zm); hickory nut (Carya illinoinensis, ci); walnut (Juglans regia, jr); populus alba (Pa); china rose (Rc); populus trichocarpa (Populus trichocarpa, pt); apples (Malus domestica, md); grape (grape vinifera, vv); rubber tree (Hevea brasiliensis, hb); pistachio (piscia vera, pv); white pear (Pyrus x bretschneideri, pb); peach (Prunus persica, pp); sweet almond (Prunus dulcis, pd).

FIG. 4 shows abscisic acid treatment (100. Mu.M ABA), salicylic acid treatment (20. Mu.M SA), cadmium stress (400. Mu.M CdCl) in example 3 of the invention ₂ 〃2.5H ₂ O), aluminum stress (30 μm Al), and iron deficiency (-Fe) for 24h, ilDTX49 was analyzed on the relative expression levels of iris root, leaf. Each point in the graph represents mean ± standard deviation (n=3), and the different letters on the column represent the significant differences between the different strains (P<0.05)。

FIG. 5 shows the construction of plant expression vector pBI121-CaMV35S-IlDTX49-Nos in example 4 of the present invention. Wherein A is a plant expression vector construction schematic diagram; b is the product of the double digestion of the constructed plant expression vector by BamH I/Xba I, wherein M1 is DNA Marker DL15000,1 is the product of the double digestion of the plant expression vector by BamH I/Xba I; c is the PCR product detection of the constructed plant expression vector, wherein M2 is DNA Marker DL2000, and 2 is the PCR product of the plant expression vector.

FIG. 6 is a molecular screen and identification of transgenic Arabidopsis plants in example 5 of the present invention. Wherein A is PCR amplification detection of each strain of transgenic arabidopsis thaliana, M: DNA marker DL2000;1: a negative control; 2: WT (wild type); 3-17: each transgenic arabidopsis line of T2 generation; b is qRT-PCR for detecting the relative expression level of each strain of transgenic arabidopsis, and WT: wild type; OE1-15: each transgenic arabidopsis line in T2 generation, each value in the figure represents mean ± standard deviation (n=3), and the different letters on the column represent significant differences between the different lines (P < 0.05), respectively.

FIG. 7 shows the cadmium stress (0, 10, 50, 100, 200. Mu. MCdCl) in example 6 of the present invention ₂ 〃2.5H ₂ O) effect on WT and transgenic arabidopsis lines OE2, OE10 phenotype (a), fresh weight (B) and root length (C) after 14 d. Each point in the graph represents mean ± standard deviation (n=6), and the different letters on the column represent the significant differences between the different strains (P<0.05)。

FIG. 8 is a graph showing the cadmium stress (50, 100, 200. Mu.M CdCl) in example 6 of the present invention ₂ ·2.5H ₂ O) effect on cadmium content of WT and tissues of transgenic arabidopsis OE2, OE10 lines after 14 d. Each value in the figure represents the mean ± standard deviation (n=5), and the different letters on the column represent the significant differences between the different strains (P<0.05)。

FIG. 9 is a graph showing the cadmium stress (0, 50, 100, 200. Mu.M CdCl) in example 6 of the present invention ₂ ) Effects on WT and transgenic arabidopsis OE2, OE10 lines chlorophyll content (a), carotenoid content (B), MDA content (C) and ascorbate peroxidase Activity (APX) (D) after 14D. Each value in the figure represents the mean ± standard deviation (n=6), and the different letters on the column represent the significant differences between the different strains (P<0.05)。

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1 cloning of Iris Palatifolia detoxification efflux Transporter encoding Gene IlDTX49

Culturing Iris Pallas seedling of 8 weeks of water culture in 200 μm cadmium (CdCl) ₂ ·2.5H ₂ The Hoagland nutrient solution of O) is subjected to stress treatment for 24 hours, the root is sheared, washed by sterile water, placed on disinfection filter paper, dried, weighed 100mg of fresh root, fully ground in liquid nitrogen, total RNA is extracted according to the instruction of Baotou TaKaRaMiniBEST Plant RNA Extraction Kit kit, the concentration of the RNA is measured by using a Quawell5000 nucleic acid protein meter, the RNA integrity is detected by 1.2% formaldehyde denaturing gel electrophoresis, 1 mug of total RNA is taken, and reverse transcription is carried out according to the instruction of PrimeScript RTasec DNA first strand synthesis kit (TaKaRa) to synthesize cDNA. Based on early-stage iris root system cadmium-resistant transcriptome analysis, 1 differential expression up-regulating detoxification efflux transporter coding gene IlDTX49 (c 196803 _g1) related to cadmium resistance is obtained through screening. The gene sequence was used to design open reading frame primers P1 and P2, and RT-PCR was used to clone the gene Open Reading Frame (ORF) of IlDTX49 from Iris irica (FIG. 1). Sequencing verification shows that the ORF fragment is 1650bp long and codes 549 amino acids. BLAST analysis shows that the homology of the cloned ORF nucleotide sequence with monocot DTX49 is over 70%, while the homology with dicot Arabidopsis thaliana is only 38.68%. The protein was predicted to have a molecular weight of 58.99kDa and an isoelectric point of 9.1 by the computer pI/Mw program (http:// web. Expasy. Org/computer_pi /), and was designated IlDTX49.

The primer sequences used above were as follows:

P1(SEQ ID NO.3):5’-ATGTGCGAAACCAACACCAACAGC-3’；

P2(SEQ ID NO.4):5’-CTAGCTCGATCGATAGCGTAGAAG-3’。

example 2 structural characteristics of Iris pallidum detoxification efflux Transporter IlDTX49 and related analysis

Analysis of the amino acid sequence of Iris pallidum IlDTX49 with that of other plants in GenBank by DNAMAN8.0 software revealed that IlDTX49 had 70.3% and 71.32% homology with monocot date PdDTX49 and EgDTX49 oil palm proteins, respectively (FIG. 2A). The transmembrane region and topology of the IlDTX49 protein was predicted using TMHMM-2.0 (https:// dtu. Biolib. Com/deep TMHMM) and HMMTOP (http:// www.sacs.ucsf.edu/cgi-bin/HMMTOP. Py). The results show that it contains 12 transmembrane regions at its N-terminus, one hydrophilic tail at its C-terminus, and a typical MATE_LIKE conserved domain at 49-484aa (FIG. 2B). Subcellular predictions were further performed using Plant-mPloc website (http:// www.csbio.sjtu.edu.cn/bioif/Plant-multi/#) to find that the IlDTX49 protein is localized to the plasma membrane.

In addition, in order to analyze the relatedness of Iris irica IlDTX49 to other plants DTXs, a phylogenetic tree analysis was performed on them using MEGA6.0 software. The results show that lDTX49 has a relatively close relationship with monocotyledonous plant date PdDTX49, oil palm EgDTX49, pineapple AaDTX49 and the like, and has a relatively far relationship with dicotyledonous plant Arabidopsis AtDTX49 and the like (figure 3), suggesting that Iris Palmetto IlDTX49 belongs to a plasma membrane detoxification efflux transporter.

Example 3 Effect of different treatments on the expression level of Iris pallidum detoxification efflux transporter gene IlDTX49

The relative quantitative analysis of the expression level of IlDTX49 in the leaves and roots of Iris seed after 24 hours of different treatments (abscisic acid, salicylic acid, aluminum stress, iron deficiency and cadmium stress) is carried out by a qRT-PCR method. Total RNA was extracted according to the protocol of the Baozhen RNA extraction kit, and its concentration was measured using a Quawell5000 ultramicro nucleic acid protein determinator according to TaKaRa PrimeScript ^TM The cDNA was synthesized by reverse transcription using the method described in RT reagent Kit with gDNA Eraser kit. Designing Iris Palno IlDTX49 gene qRT-PCR primers P3 and P4 by using Primer 5.0 software; the internal reference Actin gene qRT-PCR primers P5 and P6. According to TaKaRa

The method of the Premix Ex Taq II kit instruction performs a real-time fluorescent quantitative qRT-PCR reaction on a Bio-Rad CFX96 instrument. By 2 ^-△△CT The method calculates the expression data of Iris IlDTX49, and all experiments are 3 biological replicates. The results showed that the IlDTX49 expression in leaves and roots was unchanged under abscisic acid, salicylic acid, iron deficiency treatment compared to the control, whereas the expression in leaves IlDTX49 was down-regulated under aluminum stress, and the difference in roots was not significant. Notably, under cadmium stress, its rootsThe transcript level of part IlDTX49 was greatly up-regulated (fig. 4). Thus, ilDTX49 expression was shown to be significantly induced and regulated by cadmium stress.

The primer sequences used above were as follows:

P3(SEQ ID NO.5):5’-TCAACCTCGTCCTGCTGCTCTC-3’；

P4(SEQ ID NO.6):5’-AGCGACTTCCACCCTCTGAACG-3’；

P5(SEQ ID NO.7):5’-CATGCTATCCTCCGATTAGACCTTGC-3’；

P6(SEQ ID NO.8):5’-TTATATCCCTGACAATTTCCCGCTCTG-3’。

EXAMPLE 4 construction of plant expression vector pBI121-CaMV35S-IlDTX49-Nos

According to Clontech In-Fusion seamless connection principle, primers P7 and P8 containing BamH I digestion site are designed by combining with the ORF sequence of IlDTX49 gene and the sequence characteristics of plant expression vector pBI121 respectively, using the Online In-Fusion Tools website (https:// www.takarabio.com/learning-centers/cloning/primer-design-and-other-Tools) and using IlDTX49-ORF product as template, and performing high-fidelity by RT-PCR

Max DNA Polymerase polymerase amplification, gel cutting, recovery and purification to obtain the fragment IlDTX49-A. And then BamH I is used for carrying out single enzyme digestion on the pBI121 vector plasmid, and large fragments are recovered by gel cutting to obtain a linearization vector which is named pBI121-B. Finally, according to the corresponding technical system of an In-Fusion kit (3 mu L of IlDTX49-A,1 mu L of pBI121-B,2 mu L of 5 xIn-Fusion HD Enzyme Premix, adding water to 10 mu L, standing at 50 ℃ for 15min after instantaneous centrifugation) to obtain a recombinant vector pBI121-CaMV35S-IlDTX49-Nos (A of FIG. 5), taking 5 mu L of an In-Fusion reaction solution, transforming into E.coli DH5 alpha competence, and using a kit containing 50mg L of ^-1 Screening LB solid medium of Kan (kanamycin), transforming bacteria, extracting plasmids, obtaining a specific band of 1650bp target gene by BamH I single enzyme digestion (B of FIG. 5), and then using primers P1 and P2 to verify that the size of IlDTX49 is consistent with the identification result by taking recombinant vector plasmids as templates (C of FIG. 5), and sending to the sequencing verification of Shanghai.

The primer sequences used above were as follows:

P7(SEQ ID NO.9):

5’-GGACTCTAGAGGATCCATGTGCGAAACCAACACCAAC-3’；

P8(SEQ ID NO.10):

5’-GACCACCCGGGGATCCTAGCTCGATCGATAGCGTAG-3’。

example 5 molecular characterization of transgenic Arabidopsis plants

The pBI121-CaMV35S-IlDTX49-Nos are transformed into the competent Agrobacterium tumefaciens GV3101 by adopting a freeze thawing method, and the transformed bacterial liquid is uniformly coated on a medium containing 50mg L ^-1 Kan and 50mg L ^-1 The single colony is picked up by an inoculating needle and inoculated on 100mL of a solid medium YEB containing 50mg L, and the single colony is grown after 2-3 days of inversion culture at 28 ℃ in dark place ^-1 Kan and 50mg L ^-1 Shake culturing in YEB liquid medium at 200rpm and 28deg.C to OD ₆₀₀ The plasmid was then extracted and positive clones were confirmed by PCR identification. Then, harvesting T in a special Arabidopsis artificial climate chamber according to a transformation method of an Agrobacterium-infected Arabidopsis inflorescence ₀ And (5) replacing transgenic plant seeds. T to be harvested ₀ The seed is sterilized with 5% sodium hypochlorite and contains 50mg L ^-1 After vernalizing for 2d in a 1/2MS culture medium of Kan, placing the culture medium in an illumination incubator for about 5-6d, transplanting transgenic materials with consistent germination into a nutrition pot containing nutrition soil, namely vermiculite (2:1), placing the culture medium in a climatic chamber for 2 weeks, and cutting out leaves. WT (wild type) and T were extracted according to the TaKaRaMiniBEST Universal Genomic DNA Extraction Kit kit instructions ₀ DNA of leaf of transgenic plant is measured by using Quawell5000 nucleic acid protein instrument, and the DNA concentration is used as template, and the primers P1 and P2 are used to molecular detect transgenic positive plant (A of FIG. 6), and the transgenic plant is randomly selected to continuously culture and harvest T ₁ Seed generation, and total RNA of leaves of the seeds are extracted and reverse transcribed into cDNA according to the method in the example 1; the transcription abundance of transgenic Arabidopsis plants (target gene primers P3 and P4; arabidopsis internal reference Actin gene qRT-PCR upstream primer P9 and downstream primer P10) was detected by qRT-PCR. As shown in FIG. 6B, the transcript abundance was varied for each transgenic Arabidopsis line, and IlDTX49 expression was selected to be abundantThe most highly transgenic OE2 and OE10 strains. T is further harvested according to the method described above ₂ And carrying out subsequent cadmium-resistant physiological function evaluation analysis on the seeds of the transgenic arabidopsis strains OE2 and OE 10.

Example 6 Effect of cadmium stress on cadmium tolerance of transgenic Arabidopsis lines

The seeds of WT and T2 generation Arabidopsis lines OE2 and OE10 were inoculated in 1/2MS medium containing different cadmium concentrations (0, 50, 100, 200. Mu.M) and vernalized for 2d, and after 14d cultivation in an illumination incubator, the morphological index, physiological biochemistry and change rule of cadmium concentration in the aerial parts and roots of the WT, OE2 and OE10 lines were tested. The results show that: as shown in fig. 7, WT, OE2, and OE10 did not differ significantly in phenotype without the addition of cadmium. With increasing cadmium treatment concentration, the root length and biomass of WT are significantly reduced, the growth of WT is severely inhibited, and especially obvious leaf wilting yellow cadmium poisoning symptoms occur under 200 mu M cadmium treatment to dying, and the OE2 and OE10 strains grow normally.

As further shown in FIG. 8, overall, cd was increased in all plants with increased cadmium stress concentration ²⁺ The concentration increases significantly. However, in comparison with WT, the upper and root Cd of the OE2 and OE10 strains under 50-200. Mu.M cadmium treatment ²⁺ The concentration is significantly reduced. In particular, the upper parts of the OE2 and OE10 strains were reduced by 36.93% and 40.62% respectively and their roots by 36.22% and 37.62% respectively under 200. Mu.M cadmium treatment. Suggesting that IlDTX49 may be mediating root system Cd ²⁺ The discharge of the plant body plays an important role in reducing cadmium accumulation in the plant body.

In addition, as can be seen from fig. 9, under normal conditions, the MDA, chlorophyll content, carotenoid content and APX enzyme activity of the leaves of WT, OE2 and OE10 plants were not significantly different. However, under 100-200 μm cadmium stress, the MDA content of transgenic arabidopsis lines OE2 and OE10 was significantly reduced by 30.69%, 30.03% and 27.67%, 26.09% compared to WT; whereas the chlorophyll content, the carotenoids content and the APX enzyme activity of OE2 and OE10 were significantly increased. The result shows that the over-expression of IlDTX49 under the cadmium treatment reduces the active oxygen level and reduces Cd by improving the activity of antioxidant enzyme ²⁺ Damage to membrane lipid peroxidation, thereby improving photosynthesis. Thus, ilDTX49 passes through root system Cd ²⁺ The efflux and activation of antioxidant enzyme defense system reduces the cadmium concentration of transgenic plants and inhibits intracellular active oxygen burst and membrane lipid peroxidation, thereby improving the tolerance of the transgenic plants to cadmium. The full explanation shows that IlDTX49 can be used as an excellent candidate gene for molecular design breeding of low-cadmium crops and excellent pasture, and has wide application prospect.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Iris irisquid IlDTX49 protein, characterized in that it has any one of the amino acid sequences (1) - (4) as follows:

(1) An amino acid sequence as shown in SEQ ID NO. 1;

2. A nucleic acid molecule encoding the irisquinone IlDTX49 protein of claim 1.

3. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule has a nucleotide sequence of any one of (1) to (4) as follows:

(1) A nucleotide sequence as shown in SEQ ID NO. 2;

(4) The nucleotide sequence which hybridizes with the sequence shown in SEQ ID No.2 and encodes a functionally equivalent protein under stringent conditions, i.e.in a solution of 0.1 XSSPE containing 0.1% SDS or 0.1 XSSC containing 0.1% SDS, at 65℃and washing the membrane with said solution.

4. A biological material comprising the nucleic acid molecule of claim 2 or 3, wherein the biological material is an expression cassette, a vector or a host cell.

5. Use of an irisquinone IlDTX49 protein according to claim 1 or a nucleic acid molecule according to claim 2 or 3 or a biological material according to claim 4 for increasing cadmium tolerance in plants.

6. Use of an irisquinone IlDTX49 protein according to claim 1 or a nucleic acid molecule according to claim 2 or 3 or a biological material according to claim 4 for reducing cadmium content in plants.

7. Use of an irisquinone IlDTX49 protein according to claim 1 or a nucleic acid molecule according to claim 2 or 3 or a biological material according to claim 4 for the preparation of transgenic plants.

8. Use of an irisquinone IlDTX49 protein of claim 1 or a nucleic acid molecule of claim 2 or 3 or a biological material of claim 4 in increasing plant biomass, root length and/or alleviating wilting and yellowing of plant leaves under high cadmium conditions.

9. Use of an irisquinone IlDTX49 protein of claim 1 or a nucleic acid molecule of claim 2 or 3 or a biological material of claim 4 in increasing plant antioxidant enzyme activity, decreasing plant active oxygen levels, inhibiting plant membrane lipid peroxidation and/or improving plant photosynthesis under high cadmium conditions.

10. A method of improving cadmium tolerance or growing a low cadmium plant, the method comprising: increasing the expression level and/or activity of irisquinone IlDTX49 protein in said plant;

the Iris irisquish IlDTX49 protein is as described in claim 1.