CN111732648A

CN111732648A - Xingan larch LgCCHC-20045 and coding gene and application thereof

Info

Publication number: CN111732648A
Application number: CN202010707147.XA
Authority: CN
Inventors: 亢燕; 林晓飞; 赵泉胜; 铁英; 祁智
Original assignee: Inner Mongolia Hesheng Ecological Technology Research Institute Co ltd; Mengshu Ecological Construction Group Co ltd
Current assignee: Inner Mongolia Hesheng Ecological Technology Research Institute Co ltd; Mengshu Ecological Construction Group Co ltd
Priority date: 2020-07-21
Filing date: 2020-07-21
Publication date: 2020-10-02
Anticipated expiration: 2040-07-21
Also published as: CN111732648B

Abstract

The invention provides an LgCCHC-20045 protein, and a coding gene and application thereof. The invention introduces the DNA molecule of the coding protein LgCCHC-20045 into the plant to obtain the transgenic plant, and the root length, the fresh weight and/or the content of calcium, iron and zinc of the transgenic plant are obviously increased under the condition of iron deficiency; under the condition of excessive zinc, the root length, fresh weight and/or calcium, iron and zinc content of the transgenic plant are/is obviously increased. The invention provides a new direction for cultivating nutrition-enhanced plants by utilizing a genetic engineering technology, and has important significance for molecular breeding work related to nutrition. The LgCCHC-20045 gene can be applied to plant genetic engineering to improve the calcium, iron and zinc content of plants.

Description

Xingan larch LgCCHC-20045 and coding gene and application thereof

Technical Field

The invention relates to the field of plant genetic engineering, in particular to dahurian larch LgCCHC-20045 and a coding gene and application thereof.

Background

Genes related to mineral nutrition absorption and accumulation of plants with proprietary intellectual property rights are the basis for realizing bioaugmentation and are the focus of competition in the field of plant molecular breeding. For the development and utilization of genes related to mineral nutrition absorption and accumulation of plants, the current research is mainly focused on model plants such as arabidopsis thaliana or crops such as rice and wheat. Wild plants grown in different natural environmental conditions, even harsh environments, are natural libraries of functional genes, and these plants likely have in vivo genes associated with mineral nutrient uptake and accumulation that are not possessed by the model plants. At present, the excavation of mineral nutrition related genes which are abundant in wild plants is lacked.

The colony-forming tree species in Larix dahurica and Daxing AnLing region of inner Mongolia have the excellent characteristics of cold resistance, moisture resistance, strong adaptability and the like, and are widely applied to a plurality of fields of forestation, paper making and the like. Most of the research on the dahurian larch mainly centers on two aspects of ecological significance and economic value. At present, the mineral nutrition related gene of the dahurian larch is not excavated and researched.

Disclosure of Invention

The technical problem to be solved by the invention is how to regulate the biomass and/or the content of nutrient components of plants.

In order to solve the technical problems, the invention firstly provides a protein related to plant biomass; the protein related to plant biomass provided by the invention is named as LgCCHC-20045 and is the protein of a) or b) or c) as follows:

a) the amino acid sequence is shown as SEQ ID NO. 2;

b) 2, the N end and/or the C end of the protein shown in SEQ ID NO. 2 is connected with a label to obtain fusion protein;

c) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO. 2.

Wherein, SEQ ID NO 2 consists of 373 amino acid residues.

In order to facilitate the purification of the protein of a), a tag as shown in Table 1 may be attached to the amino terminus or the carboxy terminus of the protein shown in SEQ ID NO. 2 of the sequence Listing.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein LgCCHC-20045 in c) above, wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein LgCCHC-20045 in the step c) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

The gene encoding the protein LgCCHC-20045 in c) above can be obtained by deleting one or several amino acid residues of the codon in the DNA sequence shown in SEQ ID NO. 1, and/or by performing missense mutation of one or several base pairs, and/or by attaching the coding sequence of the tag shown in Table 1 above to the 5 'end and/or 3' end thereof.

In order to solve the technical problems, the invention also provides a biological material related to the protein.

The biological material related to the protein provided by the invention is any one of the following A1) to A12):

A1) nucleic acid molecules encoding the above proteins;

A2) an expression cassette comprising the nucleic acid molecule of a 1);

A3) a recombinant vector comprising the nucleic acid molecule of a 1);

A4) a recombinant vector comprising the expression cassette of a 2);

A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);

A6) a recombinant microorganism comprising the expression cassette of a 2);

A7) a recombinant microorganism comprising a3) said recombinant vector;

A8) a recombinant microorganism comprising a4) said recombinant vector;

A9) a transgenic plant cell line comprising the nucleic acid molecule of a 1);

A10) a transgenic plant cell line comprising the expression cassette of a 2);

A11) a transgenic plant cell line comprising the recombinant vector of a 3);

A12) a transgenic plant cell line comprising the recombinant vector of a 4).

In the above biological material, the nucleic acid molecule of A1) is a gene represented by the following 1) or 2) or 3):

1) the coding sequence is a DNA molecule shown in SEQ ID NO. 1;

2) a cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) and encoding the protein;

3) a cDNA molecule or a genome DNA molecule which is hybridized with the nucleotide sequence limited by 1) or 2) under strict conditions and codes the protein.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA.

Wherein, SEQ ID NO 1 consists of 1122 nucleotides and encodes the amino acid sequence shown in SEQ ID NO 2.

The nucleotide sequence encoding lgCCHC-20045 of the present invention can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified and have 75% or more identity to the nucleotide sequence of LgCCHC-20045 isolated in the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode LgCCHC-20045 and have the same function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence that has 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the present invention encoding the protein consisting of the amino acid sequence shown in SEQ ID NO. 2. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

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

In the above biological material, the stringent conditions are hybridization and membrane washing at 68 ℃ for 2 times, 5min each, in a solution of 2 XSSC, 0.1% SDS, and hybridization and membrane washing at 68 ℃ for 2 times, 15min each, in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

In the above-mentioned biological material, the expression cassette containing a nucleic acid molecule encoding lgCCHC-20045 (LgCCHC-20045 gene expression cassette) described in A2) means a DNA capable of expressing LgCCHC-20045 in a host cell, and the DNA may include not only a promoter which promotes transcription of LgCCHC-20045 but also a terminator which terminates transcription of LgCCHC-20045. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: a constitutive promoter; tissue, organ and development specific promoters and inducible promoters. Examples of promoters include, but are not limited to: constitutive promoter of cauliflower mosaic virus 35S: the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1(PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoters (U.S. patent 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5, 057,422); seed-specific promoters, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beachy et al (1985) EMBO J.4: 3047-3053)). They can be used aloneOr in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminators (see, e.g., Odell et al (I)⁹⁸⁵) Nature 313: 810; rosenberg et al (1987) Gene,56: 125; guerineau et al (1991) mol.gen.genet,262: 141; proudfoot (1991) Cell,64: 671; sanfacon et al Genes Dev.,5: 141; mogen et al (1990) Plant Cell,2: 1261; munroe et al (1990) Gene,91: 151; ballad et al (1989) Nucleic Acids Res.17: 7891; joshi et al (1987) Nucleic Acid Res, 15: 9627).

The recombinant vector containing the LgCCHC-20045 gene expression cassette can be constructed by using the existing expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Co., Ltd.), etc. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound capable of producing a color change (GUS gene, luciferase gene, etc.), a marker gene for antibiotics (e.g., nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to phosphinothricin as an herbicide, hph gene conferring resistance to hygromycin as an antibiotic, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or a marker gene for chemical resistance (e.g., herbicide resistance), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose, which can be expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

In the above biological material, the vector may be a plasmid, a cosmid, a phage, or a viral vector.

In the above biological material, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium.

In the above biological material, the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ do not comprise propagation material.

In order to solve the above technical problems, a method for regulating the biomass and/or nutrient content of a plant increases the yield of a protein having an amino acid sequence represented by SEQ ID NO. 2 in the plant.

Further, the above regulation is either increase or decrease.

Furthermore, the protein shown in SEQ ID NO. 2 is over-expressed in the plant to obtain the transgenic plant.

Furthermore, the overexpression of the protein shown as SEQ ID NO. 2 in the plant is specifically to introduce a DNA molecule encoding the protein LgCCHC-20045 into the plant through a recombinant vector containing the DNA molecule encoding the protein LgCCHC-20045, so as to obtain a transgenic plant. The recombinant vector containing the DNA molecule of the coding protein LgCCHC-20045 is a recombinant vector pGWB 617-LgCCHC-20045; the recombinant vector pGWB617-LgCCHC-20045 is obtained by inserting LgCCHC-20045 gene (SEQ ID NO:1) between KPn I enzyme cutting sites and EcoRV enzyme cutting sites of pGWB617 plant expression vector in the forward direction and keeping other sequences of pGWB617 plant expression vector unchanged.

Further, the plant biomass is root length and/or fresh weight;

the nutrient components are mineral nutrients; preferably, the mineral nutrients are calcium, iron and/or zinc elements; more preferably, the mineral nutrient content is the calcium, iron and/or zinc element content of the whole plant seedlings;

further, according to the method for regulating and controlling the plant biomass and/or the content of the nutrient components, under the condition of iron deficiency, the root length, the fresh weight and the content of calcium, iron and/or zinc elements of the transgenic plant are obviously increased;

further, the Fe is Fe in the culture solution or the culture medium under the condition of the lack of Fe²⁺The salt concentration is 0-50 μ M; preferably 0 μ M to 5 μ M; more preferably 0. mu.M to 0.5. mu.M; or 0.5. mu.M-5. mu.M; more preferably 0.5. mu.M or 5. mu.M;

under the condition of excessive zinc, the root length, fresh weight and calcium, iron and/or zinc element content of the transgenic plant are obviously increased;

further, the zinc content is 7.5-150 μ M under the condition of excess zinc; preferably 75 μ M to 150 μ M; more preferably 75 μ M or 150 μ M;

more preferably, the content of calcium, iron and/or zinc elements is the content of calcium, iron and/or zinc elements in the whole plant seedlings;

the application of the protein or the related biological materials in regulating and controlling the biomass and/or the content of the nutrient components of the plants also belongs to the protection scope of the invention.

Further, the plant biomass is specifically root length and/or fresh weight; the nutrient components are mineral nutrients; preferably, the mineral nutrients are calcium, iron and/or zinc elements.

Further, the plant is an angiosperm or a gymnosperm, and the angiosperm is a monocotyledon or a dicotyledon; the dicotyledonous plant is Arabidopsis thaliana; the gymnosperm is Pinaceae plant, specifically Larix dahurica.

The technical scheme of the invention has the following advantages:

the invention provides an LgCCHC-20045 protein, and a coding gene and application thereof. The invention introduces the DNA molecule of the coding protein LgCCHC-20045 into the plant to obtain the transgenic plant, and the root length, the fresh weight, the content of calcium element, iron element and/or zinc element of the transgenic plant are obviously increased under the condition of iron deficiency; under the condition of excessive zinc, the root length, fresh weight, calcium element, iron element and/or zinc element content of the transgenic plant are obviously increased. The invention provides a new direction for cultivating nutrition-enhanced plants by utilizing a genetic engineering technology, and has important significance for molecular breeding work related to nutrition. The LgCCHC-20045 gene can be applied to plant genetic engineering to improve the content of calcium, iron and zinc elements in plants.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a technical flow chart of the overall experiment of the present invention;

FIG. 2 shows the restriction enzyme-digestion-verified electrophoresis of pGWB617-LgCCHC-20045 expression vector (M:200bp DNA Ladder, 1: before restriction enzyme and 2: after restriction enzyme);

FIG. 3 comparison of root length and fresh weight of Arabidopsis thaliana wild type Col-0 and Arabidopsis thaliana containing empty vector pGWB617 under conditions of iron deficiency and zinc excess; FIG. 3A is a photograph showing the root length of wild type Col-0, Arabidopsis T3 generation homozygous seeds transformed with empty vector (GV3101-pGWB617) cultured in 1 XFe (CK), 0 XFe and 10 XZn medium for 14 days; (B) the root length of the arabidopsis T3 generation homozygous seeds which are wild type Col-0 and transferred into an empty vector (GV3101-pGWB617) after being cultured in a CK culture medium and 0 xFe for 14 days; (C) the fresh weight of the arabidopsis T3 generation homozygous seeds which are wild type Col-0 and transferred into an empty vector (GV3101-pGWB617) after being cultured for 14 days in a culture medium of 1 xFe (CK) and 0 xFe; (D) the seed is the wild Col-0, the T3 generation homozygous seed of Arabidopsis thaliana transferred to the empty vector (GV3101-pGWB617) and the root length after being cultured for 14 days in the culture medium of 1xZn (CK) and 10 xZn. (E) The fresh weight of arabidopsis T3 generation homozygous seeds which are wild type Col-0 and transferred into an empty vector (GV3101-pGWB617) after being cultured in a 1xZn culture medium (CK) and 10 xZn for 14 days;

FIG. 4 shows comparison of root length and fresh weight of Arabidopsis thaliana wild type Col-0 and Arabidopsis thaliana transformed with LgCCHC-20045 gene under the condition of iron deficiency; FIG. 4(A) is a photograph showing the root length of wild type Col-0, LgCCHC-20045 transgenic Arabidopsis thaliana (20045-7 line) T3 generation homozygous seeds after being cultured in 1 xFe (CK), 0 xFe, 0.01 xFe, 0.1 xFe medium for 14 days; (B) the root length of T3 generation homozygous seeds of wild type Col-0 and LgCCHC-20045 Arabidopsis thaliana (20045-6, 20045-7, 20045-8 and 20045-9 strains) after being cultured for 14 days in CK, 0 xFe, 0.01 xFe and 0.1 xFe culture media; (C) fresh weights of T3 generation homozygous seeds of wild type Col-0 and LgCCHC-20045 transgenic Arabidopsis thaliana (20045-6, 20045-7, 20045-8 and 20045-9 strains) after being cultured for 14 days in CK, 0 xFe, 0.01 xFe and 0.1 xFe culture media;

FIG. 5 shows the comparison between the root length and fresh weight of Arabidopsis thaliana wild type Col-0 and Arabidopsis thaliana transformed with LgCCHC-20045 gene under the condition of excessive zinc; FIG. 5(A) is a photograph showing the root length of wild type Col-0, LgCCHC-20045 transgenic Arabidopsis thaliana (20045-6 line) T3 generation homozygous seeds after being cultured in 1XZn (CK), 10 XZn, 5 XZn medium for 14 days; (B) the root length of T3 generation homozygous seeds of wild type Col-0 and LgCCHC-20045 Arabidopsis thaliana (20045-6, 20045-8 and 20045-9 strains) after being cultured for 14 days in CK, 10 xZn and 5 xZn culture media; (C) fresh weights of T3 generation homozygous seeds of wild type Col-0 and LgCCHC-20045 transgenic Arabidopsis thaliana (20045-6, 20045-8 and 20045-9 strains) after being cultured for 14 days in CK, 10 xZn and 5 xZn culture media;

FIG. 6 shows the comparison of the contents of Ca, Fe and Zn elements in the whole seedlings of wild Col-0 and LgCCHC-20045 transgenic Arabidopsis under the condition of iron deficiency; (A) the content of Ca element in T3 generation homozygous seeds of wild Col-0 and LgCCHC-20045 Arabidopsis thaliana (20045-6, 20045-7, 20045-8 and 20045-9 strains) after being cultured for 14 days in a1 xFe (CK), 0 xFe, 0.01 xFe and 0.1 xFe culture medium; (B) the content of Fe element of T3 generation homozygous seeds of wild type Col-0 and LgCCHC-20045 Arabidopsis thaliana (four strains of 20045-6, 20045-7, 20045-8 and 20045-9) after being cultured for 14 days in a culture medium of 1 xFe (CK), 0 xFe, 0.01 xFe and 0.1 xFe; (C) the zinc Zn element content of T3 generation homozygous seeds of wild type Col-0 and LgCCHC-20045 Arabidopsis thaliana (four strains of 20045-6, 20045-7, 20045-8 and 20045-9) after being cultured for 14 days in a1 xFe (CK), 0 xFe, 0.01 xFe and 0.1 xFe culture medium;

FIG. 7 shows the comparison of the contents of Ca, Fe and Zn elements in the whole seedlings of wild type Col-0 and LgCCHC-20045 transgenic Arabidopsis; (A) the calcium Ca element content of T3 generation homozygous seeds of wild type Col-0 and LgCCHC-20045 Arabidopsis thaliana (20045-6, 20045-7, 20045-8 and 20045-9 strains) in a culture medium of 1xZn (CK), 10 xZn and 5 xZn; (B) the zinc and Zn element contents of T3 generation homozygous seeds of wild type Col-0 and transgenic LgCCHC-20045 Arabidopsis thaliana (four strains of 20045-6, 20045-7, 20045-8 and 20045-9) are cultured in CK, 10 xZn and 5 xZn culture media for 14 days; (C) the Fe element content of T3 generation homozygous seeds of wild type Col-0 and LgCCHC-20045 Arabidopsis thaliana (20045-6, 20045-7, 20045-8 and 20045-9 strains) after being cultured for 14 days in CK, 10 xZn and 5 xZn culture media.

Detailed Description

The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.

The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

The MQA medium (as CK medium) used in this example had the following composition:

macroelements: 1M KNO₃、1M MgSO₄、1M H₃PO₄、1M CaCl₂

Trace elements: 1/2MS trace (including Zn)²⁺Salt)

Fe²⁺Salt: 1/2MS Fe²⁺Salt (salt)

Mn²⁺Salt: 1/2MS Mn²⁺Salt and 0.5M MES buffer

Carbon source: 1% sucrose

The pH was 5.7

Agar 1% in solid MQA medium

1xF e (CK) in the following examples represents MQA medium as the CK medium, Fe²⁺The salt concentration was 50. mu.M.

1xZn (CK) in the following examples indicates MQA medium as the CK medium, in which Zn is present²⁺The salt concentration was 15. mu.M,

example 1 acquisition of LgCCHC-20045 Gene of Larix dahurica

First, the acquisition of the LgCCHC-20045 gene of dahurian larch

Soaking dahurian larch seeds (provided by seed gardens of great Khingan mountain and river forestry bureau) in water, putting the soaked seeds into a refrigerator at 4 ℃, soaking the seeds for 3 to 4 days, sowing the seeds into nutrient soil, germinating for about one week, and extracting total RNA after 3 to 4 weeks for cloning LgCCHC-20045 genes. The nucleotide sequence of the LgCCHC-20045 gene is shown as SEQ ID NO. 1, and the amino acid sequence of the LgCCHC-20045 protein coded by the LgCCHC-20045 gene is shown as SEQ ID NO. 2.

Expression characteristic of Larix dahurica LgCCHC-20045 gene in Arabidopsis thaliana

1. Construction of pGWB617-LgCCHC-20045 expression vector

CCHC-20045 gene is synthesized by the company, the gene is connected with pENTR3C entry vector to obtain pENTR3C-CCHC-20045, pENTR3C-CCHC-20045 is transformed into Escherichia coli DH5 alpha to obtain Escherichia coli DH5 alpha expressing pENTR3C-CCHC-20045, the resistance of the entry vector is kanamycin, and the processes are all completed by Shanghai Shangxi biological Limited company.

1.1 plasmid extraction

a) Activation of bacteria: taking the Escherichia coli DH5 alpha bacterial liquid for expressing pENTR3C-CCHC-20045, dipping a little with a gun head, and lightly streaking on LB solid culture medium containing 50ug/mL Kan resistance; meanwhile, Escherichia coli DH5 alpha containing pGWB617 vector and stored in a laboratory is streaked onto a culture medium containing 50ug/mL Spe; after sealing, the mixture was placed upside down in a 37 ℃ incubator and cultured overnight.

b) Expanding and culturing bacteria: in a clean bench, a single colony was picked from a) overnight-cultured plate, and placed in a shake tube to which the corresponding antibiotic (Kan/Spe) and 5mL of liquid LB had been added, and cultured overnight at 37 ℃ with shaking at 200 rpm.

c) Plasmid extraction: 2mL of the bacterial liquid obtained in the step b) are respectively put in a centrifugal tube, and plasmids are extracted according to the requirements of the instructions of the small plasmid extraction kit of the Botryote bioengineering (Dalian) Co.

1.2 obtaining of recombinant expression vector pGWB617-LgCCHC-20045

By using Gateway cloning technology, an entry vector pENTR3C-CCHC-20045 connected with a target gene is subjected to LR reaction with an expression vector pGWB617, the target gene CCHC-20045 on the entry vector is connected to the target vector in a homologous recombination mode, the construction of the expression vector is completed, and a recombinant expression vector is obtained, wherein the recombinant expression vector is described as follows: the SEQ ID NO. 1 is inserted between KPn I and EcoRV enzyme cutting sites of pGWB617 plant expression vector in the forward direction, and other sequences of pGWB617 plant expression vector are kept unchanged, so that the recombinant vector pGWB617-LgCCHC-20045 is obtained.

The LR reaction system is as follows:

and adding the components in the 10 mu L reaction system into a microcentrifuge tube according to the sequence and the content, gently mixing the components uniformly, and reacting the mixture for 1h at 25 ℃ in a PCR instrument.

1.3 transformation of competent cells of E.coli DH5 alpha by Heat shock

Adding 10 mu L of LR reaction product (recombinant vector pGWB617-LgCCHC-20045) into 100 mu L of Escherichia coli DH5 alpha competent cells which are just unfrozen, carrying out ice bath for 30min, carrying out water bath heat shock at 42 ℃ for 30s, immediately placing on ice for 2min, adding 500 mu L of liquid LB culture medium, and carrying out shake culture at 37 ℃ and 180rpm for 1h to obtain the Escherichia coli containing the recombinant vector pGWB 617-LgCCHC-20045. The Escherichia coli liquid containing the recombinant vector pGWB617-LgCCHC-20045 is evenly coated on a solid LB culture medium containing 50ug/mLSpe in a clean bench, and is inversely cultured overnight in a constant-temperature incubator at 37 ℃.

1.4 restriction enzyme digestion verification of recombinant plasmid pGWB617-LgCCHC-20045

A single colony on LB solid medium of 1.3 was picked and transferred to LB liquid medium containing 50ug/mL Spe for expansion. And (3) carrying out plasmid extraction on the amplified monoclonal (by adopting a small plasmid extraction kit of Boehringer Bioengineering (Dalian) Co., Ltd.). Positive clones were identified by double digestion with KPn I and EcoRV. The double enzyme digestion system is as follows:

the components are added into a micro centrifugal tube according to the sequence and the content, immediately and gently mixed evenly, and then are subjected to metal bath at the constant temperature of 37 ℃ for 30 min. The enzyme digestion product is detected by 1% agarose gel electrophoresis, the plasmid containing a target band (about 1000 bp) and a carrier band in an electrophoresis band is a correct plasmid, and corresponding bacterial liquid (1mL bacterial liquid +1mL 40% glycerol) is stored in a refrigerator at the temperature of-80 ℃.

2. Acquisition of Agrobacterium GV3101-pGWB617-LgCCHC-20045

a) The constructed expression vector (plasmid) pGWB 617-LgCCHC-200455 μ L is taken and added into the ice-precooled agrobacterium-infected state (GV3101) and kept still for 30min on ice.

b) The agrobacterium liquid mixed with expression vector (plasmid) (pGWB617-LgCCHC-20045) is added into a precooled electric rotating cup and immediately put into a multifunctional cell electroporator for electric shock transformation, and the conditions are set as follows: 2500V, 5 ms.

c) Returning to a clean bench, adding 1mL of liquid LB culture medium into the electric rotating cup, fully mixing, transferring into a 2mL sterile centrifuge tube, and standing and culturing at a constant temperature of 28 ℃ for 1.5 h.

d) And uniformly coating 500 mu L of bacterial liquid on LB solid culture medium containing 50ug/mL Rif and 50ug/mL Spe, and carrying out inverted culture in a constant-temperature incubator at 28 ℃ for 1-2d to grow a single colony.

e) Picking d) single colony, transferring to LB liquid medium containing 50ug/mL Rif and 50ug/mL Spe for expansion culture. And (3) carrying out plasmid extraction on the amplified monoclonal (by adopting a small plasmid extraction kit of Boehringer Bioengineering (Dalian) Co., Ltd.). Identifying positive clone (see figure 2) by KpnI and EcoRV double enzyme digestion method (see figure 1.4), identifying correct single clone and reserving for later use, obtaining agrobacterium GV3101-pGWB 617-LgCCHC-20045. Meanwhile, pGWB617 was transferred to Agrobacterium GV3101 to obtain GV3101-pGWB617 as an empty vector control.

3. Construction of LgCCHC-20045 gene transferred Arabidopsis thaliana

3.1 Agrobacterium-mediated inflorescence dip-dyeing method for transforming Arabidopsis thaliana Col-0

a) The activated Agrobacterium GV3101-pGWB617-LgCCHC-20045 single colony (GV3101-pGWB617 as empty vector control) was picked up and inoculated into 5mL liquid LB medium containing Spe and Rif, and shake-cultured at 28 ℃ and 250rpm to OD₆₀₀＝0.8；

b) Centrifuging at room temperature for 5min at 5000g, and removing supernatant;

c) adding 20mL of dip-dyed suspension to resuspend the thalli; the dip-dyeing suspension comprises the following components: sterile water + 5% (0.5g/L) sucrose + 0.02% (volume percent) Silwet-77;

d) the resuspended strain is dropped on the just exposed arabidopsis inflorescence, and all the opened flowers and siliques of the plant are cut off before the first dip dyeing. And dip-dyeing once every 3-5 days, and continuously dip-dyeing until the plants do not bloom any more. T1 generation seeds were harvested.

3.2 screening of LgCCHC-20045 Gene transgenic Arabidopsis homozygous lines

a) Harvested seeds of T1 generations were evenly sown on MQA solid medium containing 120ug/ml phosphinothricin, vernalized and then grown in a growth chamber (light intensity: 75-100. mu. mol. m^-2·s^-1(ii) a The illumination period is as follows: 12h light/12 h dark; temperature: 22 +/-2 ℃) horizontally culturing for 7-10 days. And (3) taking the survived plant with true leaves as a positive seedling, transplanting the T1 generation positive seedling into nutrient soil to continue to grow, and harvesting the T2 generation seeds from a single plant.

b) Screening T2 generation seeds by the same method, and counting the proportion of transgenic successful plants to transgenic unsuccessful plants, namely the proportion of green plants which can survive and grow true leaves and albino plants which only grow two cotyledons, wherein the proportion is 3: line 1 is a single copy insertion line, these lines are cultivated in soil and the individual plants are harvested for seeds of T3 generations.

c) The sowing of T3 generation seeds is also carried out by the method, all the surviving plants which grow healthily are inserted into a homozygous line in a single copy mode, and finally four lines of 20045-6, 20045-7, 20045-8 and 20045-9 are obtained, and T3 generation seeds corresponding to the four lines are reserved as experimental materials for phenotype analysis.

4. Phenotypic analysis of transgenic Arabidopsis

The lack of culture conditions were: removal of Fe in MQA medium²⁺The nutrient components except salt are unchanged, and Fe is reduced²⁺the salt concentration was set at 0 × Fe (i.e., Fe)²⁺at a concentration of 0. mu.M), 0.01 × Fe (i.e., at a concentration of 0.5. mu.M Fe), or 0.1 × Fe (i.e., at a concentration of 5. mu.M Fe).

The zinc over-culture conditions were: removal of Zn from MQA medium²⁺The other nutrients except salt are unchanged, and Zn is added²⁺the salt concentration was set at 5 × Zn (i.e., Zn)²⁺at a concentration of 75. mu.M), 10 × Zn (i.e., Zn)²⁺At a concentration of 150. mu.M). Each concentration was repeated three times, and transgenic Arabidopsis thaliana (i.e., the T3 seed obtained in the above step) and Arabidopsis thaliana wild type (Col-0) were sown together, cultured, and then data collection was performed.

5. Determination of total calcium, iron and zinc contents of transgenic arabidopsis

sowing transgenic Arabidopsis thaliana and Arabidopsis thaliana wild type Col-0 seeds in a solid medium of CK (MQA medium), 0 × Fe, 0.01 × Fe, 0.1 × Fe, 10 xZn and 5 xZn, vernalizing at 4 ℃ for 2-3d, vertically illuminating and culturing in a growth chamber for 14d, and firstly using 0.5mM GuSO to sample arabidopsis thaliana whole seedling₄Rinsed 3 times and then washed 3 times with sterile water. Drying in a constant temperature oven at 80 ℃, weighing and recording the dry weight. Grinding and crushing by using a high-throughput tissue grinder, and then adding 1.8mL of 5% (volume percentage content) HNO₃Mixing well. Shaking at 200rpm at room temperature for 2-3 d. Following high speed centrifugation for 30min, the supernatant was taken and filtered through a 0.4 μm sterile filter. The contents of calcium, iron and zinc in the sample can be measured by means of an inductively coupled plasma emission spectrometer (ICP-OES).

Arabidopsis T3 generation homozygous seeds, wild type Col-0, transferred into an empty vector (GV3101-pGWB617), were sown on MQA medium (CK as control) and 0 xFe and 10 xZn medium for 14d growth. Statistical analysis of the root length and fresh weight of the plants showed no difference between wild type Col-0 and empty vector phenotypes under iron-deficient and high zinc conditions (see FIG. 3).

T3 generation homozygous seeds of Arabidopsis wild type Col-0 and LgCCHC-20045 gene-transferred Arabidopsis (four 20045-6, 20045-7, 20045-8 and 20045-9 lines) were sown in MQA medium (CK) and 0 xFe, 0.01 xFe, 0.1 xFe medium for 14d growth. Statistical analysis of the root length and fresh weight of the plants, the transgenic plants had significantly higher root length and fresh weight than wild-type Col-0 in the absence of iron, see in particular figure 4 (1 in the upper column of the figure indicates P <0.05 compared to wild-type Col-0, 2 indicates P <0.01 compared to wild-type Col-0).

Arabidopsis wild type Col-0 and Arabidopsis transformed with LgCCHC-20045 gene (three lines 20045-6, 20045-8 and 20045-9) were homozygous seeds at T3 generation, sowed on MQA medium (CK) and 10 xZn, 5 xZn medium for 14 d. The root length and the fresh weight of the plant are statistically analyzed, and under the condition of excessive zinc, the root length and the fresh weight of the transgenic plant are obviously higher than those of the wild Col-0. See figure 5 in particular (upper 1 of the bar graph in the figure indicates P <0.05 compared to wild type Col-0, 2 indicates P <0.01 compared to wild type Col-0).

Arabidopsis wild type Col-0 and Arabidopsis transformed with LgCCHC-20045 gene (four strains 20045-6, 20045-7, 20045-8 and 20045-9) were cultured using the culture conditions of FIG. 4. The contents of calcium Ca (A), iron Fe (B) and zinc Zn (C) in the whole seedlings are measured by adopting ICP-OES, and particularly, the contents are shown in figure 6 (1 in the upper part of a column diagram in the figure indicates that P is less than 0.05 compared with the wild type Col-0, 2 indicates that P is less than 0.01 compared with the wild type Col-0), and DW is dry weight. Under the condition of iron deficiency, the content of Ca, Fe and Zn elements of the transgenic plant is obviously higher than that of the wild Col-0.

Arabidopsis wild type Col-0 and Arabidopsis transformed with LgCCHC-20045 gene (four strains 20045-6, 20045-7, 20045-8 and 20045-9) were cultured using the culture conditions of FIG. 5. The contents of Ca (A), Fe (B) and Zn (C) elements in the whole seedlings are measured by ICP-OES, and are shown in figure 7 (1 in the upper part of a bar chart in the figure indicates that P is less than 0.05 compared with the wild type Col-0, 2 indicates that P is less than 0.01 compared with the wild type Col-0), and DW is dry weight. Under the condition of excessive zinc, the content of calcium, iron and zinc in the transgenic plant is obviously higher than that of the wild Col-0.

The foregoing examples further illustrate the present invention and are not to be construed as limiting thereof. It is within the scope of the present invention to modify or replace methods, steps or conditions of the present invention without departing from the spirit and substance of the present invention.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Sequence listing

<110> Mongolian tree ecological construction group Limited

<120> Xingan larch LgCCHC-20045 and coding gene and application thereof

<130>HA202001533

<160>3

<170>PatentIn version 3.5

<210>1

<211>1122

<212>DNA

<213> Artificial sequence

<400>1

atgccggcca ctgcgggacg ggtgcggatg cccgcaaaca acagggttca cagcagtgca 60

gctcttcaaa cccacggcat ctggcagagc gcaatcggct acgaccccta cgcccccaac 120

cacgacagca acgacaacaa tagagacaac ggccacgggg gagttccggg gcccgaaggc 180

gacggagaga ataatgccta cgacagcttc cagggcctcc tggccttggc ccggctcacc 240

tcctccaccg ctgacgaggc tcgtggcgcc tgcaaaaaat gcaacagggt tggacattta 300

acctttcagt gccggaattt tctcagcggc aaagaagtcg atgctttgca gacggttatg 360

gaaaaggaga ataataataa tagcaataaa tccagtggta aagatcgggt gaaattccct 420

aaggatgccg ttgctccggt gaccggggac agttccgatg aagatgagga ggaatccgag 480

tctgaagtta cggatagcga cgaagactct gaaattgaga agcttatcgc ggcgagggtt 540

ggtaaaagct ctggcaagag caagtcccgt gtaaatgaca agggttctag ggtttactcc 600

cagaaaacca aggtttctag gccttcatct gagaaatcta gcgttcccag ggtctcttcc 660

gagaaatcta gggatcgtag ggtttcatct gagaaacata gggtttctaa gtcttcatct 720

cgcaaaatat cgactcataa gttgaaaaaa cggtccagga gtgattcgga gcccgaagac 780

ggtgaatttt ttgatgattc cgacgtggaa gagaaaaaga tgaggaagag gccgagaagg 840

tcaaggaagg attattctga ttcatcggat gatagcagtg acgatgattc tgatgatgat 900

gatcggaaga gcaggaagga gaggaggaag aaaaagagga ctagaggttc gtccgatagc 960

agttctgaga aaatgaggag gagtaagcat tcagaaagga gacacaggaa acacgagagc 1020

aggattcgtc gcgtggaaac agatgaggat aattcagcca acaaagatga acggaagcat 1080

tataagcatg gccacaagca cggctccagg agaagcgatt aa 1122

<210>2

<211>373

<212>PRT

<213> Artificial sequence

<400>2

Met Pro Ala Thr Ala Gly Arg Val Arg Met Pro Ala Asn Asn Arg Val

1 5 10 15

His Ser Ser Ala Ala Leu Gln Thr His Gly Ile Trp Gln Ser Ala Ile

20 25 30

Gly Tyr Asp Pro Tyr Ala Pro Asn His Asp Ser Asn Asp Asn Asn Arg

35 40 45

Asp Asn Gly His Gly Gly Val Pro Gly Pro Glu Gly Asp Gly Glu Asn

50 55 60

Asn Ala Tyr Asp Ser Phe Gln Gly Leu Leu Ala Leu Ala Arg Leu Thr

65 70 75 80

Ser Ser Thr Ala Asp Glu Ala Arg Gly Ala Cys Lys Lys Cys Asn Arg

85 90 95

Val Gly His Leu Thr Phe Gln Cys Arg Asn Phe Leu Ser Gly Lys Glu

100 105110

Val Asp Ala Leu Gln Thr Val Met Glu Lys Glu Asn Asn Asn Asn Ser

115 120 125

Asn Lys Ser Ser Gly Lys Asp Arg Val Lys Phe Pro Lys Asp Ala Val

130 135 140

Ala Pro Val Thr Gly Asp Ser Ser Asp Glu Asp Glu Glu Glu Ser Glu

145 150 155 160

Ser Glu Val Thr Asp Ser Asp Glu Asp Ser Glu Ile Glu Lys Leu Ile

165 170 175

Ala Ala Arg Val Gly Lys Ser Ser Gly Lys Ser Lys Ser Arg Val Asn

180 185 190

Asp Lys Gly Ser Arg Val Tyr Ser Gln Lys Thr Lys Val Ser Arg Pro

195 200 205

Ser Ser Glu Lys Ser Ser Val Pro Arg Val Ser Ser Glu Lys Ser Arg

210 215 220

Asp Arg Arg Val Ser Ser Glu Lys His Arg Val Ser Lys Ser Ser Ser

225 230 235 240

Arg Lys Ile Ser Thr His Lys Leu Lys Lys Arg Ser Arg Ser Asp Ser

245 250 255

Glu Pro Glu Asp Gly Glu Phe Phe Asp Asp Ser Asp Val Glu Glu Lys

260 265270

Lys Met Arg Lys Arg Pro Arg Arg Ser Arg Lys Asp Tyr Ser Asp Ser

275 280 285

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

290 295 300

Arg Lys Glu Arg Arg Lys Lys Lys Arg Thr Arg Gly Ser Ser Asp Ser

305 310 315 320

Ser Ser Glu Lys Met Arg Arg Ser Lys His Ser Glu Arg Arg His Arg

325 330 335

Lys His Glu Ser Arg Ile Arg Arg Val Glu Thr Asp Glu Asp Asn Ser

340 345 350

Ala Asn Lys Asp Glu Arg Lys His Tyr Lys His Gly His Lys His Gly

355 360 365

Ser Arg Arg Ser Asp

370

Claims

1. The protein is the protein of a) or b) or c) as follows:

a) the amino acid sequence is shown as SEQ ID NO. 2;

2. The protein-related biomaterial according to claim 1, which is any one of the following a1) to a 12):

A1) a nucleic acid molecule encoding the protein of claim 1;

A2) an expression cassette comprising the nucleic acid molecule of a 1);

A3) a recombinant vector comprising the nucleic acid molecule of a 1);

A4) a recombinant vector comprising the expression cassette of a 2);

A5) a recombinant microorganism comprising the nucleic acid molecule of a 1);

A6) a recombinant microorganism comprising the expression cassette of a 2);

A7) a recombinant microorganism comprising a3) said recombinant vector;

A8) a recombinant microorganism comprising a4) said recombinant vector;

A9) a transgenic plant cell line comprising the nucleic acid molecule of a 1);

A10) a transgenic plant cell line comprising the expression cassette of a 2);

A11) a transgenic plant cell line comprising the recombinant vector of a 3);

A12) a transgenic plant cell line comprising the recombinant vector of a 4).

3. The related biological material according to claim 2, wherein: A1) the nucleic acid molecule is a gene shown in the following 1) or 2) or 3):

1) the coding sequence is a DNA molecule shown in SEQ ID NO. 1;

2) a DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) and encoding the protein of claim 1;

3) a DNA molecule which hybridizes under stringent conditions with a nucleotide sequence defined in 1) or 2) and which encodes a protein according to claim 1.

4. A method of regulating plant biomass and/or nutrient content, comprising: the yield of the protein with the amino acid sequence shown as SEQ ID NO. 2 is improved in plants.

5. The method of modulating plant biomass and/or nutrient content of claim 4, wherein the plant biomass and/or nutrient content is selected from the group consisting of: the protein shown in SEQ ID NO. 2 is over-expressed in the plant to obtain the transgenic plant.

6. The method of modulating plant biomass and/or nutrient content of claim 5, wherein the plant biomass and/or nutrient content is selected from the group consisting of: the plant biomass is specifically root length and/or fresh weight;

the nutrient components are mineral nutrients; preferably, the mineral nutrients are calcium, iron and/or zinc elements.

7. Method for regulating the biomass and/or the nutrient content of plants according to any one of claims 4 to 6, characterized in that: under the condition of iron deficiency, the root length, fresh weight and calcium, iron and/or zinc element content of the transgenic plant are obviously increased;

under the condition of excessive zinc, the root length, fresh weight and calcium, iron and/or zinc element content of the transgenic plant are obviously increased.

8. Use of a protein according to claim 1 or a related biomaterial according to claim 2 or 3 for modulating plant biomass and/or nutrient content.

9. Use according to claim 8 for modulating plant biomass and/or nutrient content, wherein: the plant biomass is root length and/or fresh weight; the nutrient components are mineral nutrients; preferably, the mineral nutrients are calcium, iron and/or zinc elements.

10. Use according to claim 8 or 9, characterized in that: the plant is an angiosperm or gymnosperm.