CN107325161B - Protein related to low-nitrogen stress and high-salt stress resistance as well as encoding gene and application thereof - Google Patents

Abstract

The invention discloses a protein related to tolerance to low nitrogen stress and high salt stress, its encoding gene and application. The protein provided by the present invention is the protein of the following a) or b) or c): a) the amino acid sequence is the protein shown in sequence 1; b) a tag is attached to the N-terminus and/or C-terminus of the protein shown in sequence 1 The obtained fusion protein; c) a protein with the same function obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID NO: 1. Experiments have shown that the ZmCCT gene provided by the present invention can increase the proportion of plants that are resistant to low nitrogen stress and high salt stress in the progeny of the transgenic ZmCCT gene plants, and at the same time, the transgenic ZmCCT gene-positive plants in the progeny are compared with negative plants. The resistance to Fusarium stem rot has been greatly improved.

Description

Protein related to low-nitrogen stress and high-salt stress resistance as well as encoding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a protein related to low-nitrogen stress resistance and high-salt stress resistance, and a coding gene and application thereof.

Background

Soil impoverishment is a major factor affecting worldwide corn yield instability. In 14 billion hectares of arable soil in the world, 22.5% of the soil is severely stressed by nutrients, and only about 10.0% of the soil is under no or mild stress. In corn production, nitrogen fertilizer application is one of the important measures for increasing the yield of corn, but two problems exist in production practice. Firstly, excessive application of nitrogen fertilizer in high-yield developed areas not only reduces the utilization rate of nitrogen fertilizer and improves production cost, but also possibly causes the overproof of underground water nitrate; and secondly, the yield level is lower in underdeveloped areas with low yield due to insufficient application of nitrogen fertilizer. In view of the further reduction of the space for increasing the yield of the application of the nitrogen fertilizer and the quality and environmental problems caused by the application of the nitrogen fertilizer, the method for breeding the nitrogen-efficient corn variety by means of genetics and the like is a fundamental solution for solving the problems of grain production safety and environmental protection.

Moll et al (Moll R H, Kamprath E J, Jackson W A. analytes and interpretional factors in the which control to influence of nitrogen utilization [ J ]. Agronom journal,1982,74(3): 562-) -564.) have classically defined nitrogen efficiencies, i.e., crop yields per unit of nitrogen supply. The nitrogen efficiency can be divided into two parts, namely the efficiency of the plant root system for absorbing nitrogen from soil, and the nitrogen assimilation utilization efficiency in the plant body. Moll et al believe that nitrogen utilization efficiency plays a major role under low nitrogen conditions, while nitrogen absorption efficiency plays a major role under high nitrogen conditions. Ortiz-Monasterio et al (Ortiz-Monasterio R, Sayre K D, Rajaram S, et al. genetic progress in the world itself and nitrogen use efficiency under urea nitrogen rates [ J ]. Crop Science 1997,37(3):898-904.) consider that the difference in nitrogen efficiency under low nitrogen conditions is mainly due to absorption efficiency, while the utilization rate under high nitrogen conditions plays a major role. Lafit and Edmeasles (Lafit HR, Edmes G O. improvement for achieving range to low oil nitrogen in tropicalize I. selection criterion [ J ]. Field Crops Research,1994,39(1):1-14.) consider that the efficiency of nitrogen utilization correlates better with yield under low nitrogen conditions. Results from mihua et al (mihua, Liujian an. physiological and biochemical basis of nitrogen efficiency and genetic improvement progress in maize [ J ] corn science,1997, 5(2):9-13.) show that both absorption and utilization efficiency are balanced under high nitrogen conditions. And Mizhou Hua et al believe that different research results that contribute to the relative importance of nitrogen uptake efficiency and nitrogen utilization efficiency may be due to different environments without genotypes.

Nitrogen efficiency in corn is a complex process. The physiological mechanisms of the high-efficiency absorption of nitrogen in corn at present comprise: (1) good root system configuration (morphology and spatial distribution) and root system characteristics; (2) good physiological metabolic activity (respiration and the like) of the root system; (3) the aerial parts have excellent properties; (4) the single circulation in the plant body in the seedling stage promotes the absorption of nitrogen by the root system. The physiological mechanism of high nitrogen utilization of corn includes: (1) nitrogen metabolism key enzyme with high activity; (2) interactions between nutrient elements and the influence of some substances; (3) the feedback function of the storage capacity; (4) full utilization of vacuole stored NO 3-and reduction of volatilization of nitrogen in the aerial part; (5) the nitrogen has strong retransfer capability to the seeds. Most important agronomic traits of crops such as yield, quality and stress resistance are quantitative traits controlled by multiple Quantitative Trait Loci (QTL). Many current research results indicate that crop nitrogen efficiency is also controlled by multiple quantitative genetic loci.

According to the statistics of the second national soil general survey data, the salinized soil area in China is 3487 ten thousand hectares and about 5 hundred million acres on the premise of not including coastal mudflats, and the exploitable area can reach 2 hundred million acres. Improving the saline-alkali tolerance of crops and having important significance for developing marginal land and improving grain yield. The existing corn cultivation method has the defects of large field water demand and relatively low saline-alkali adaptability. The seedling stage is more sensitive to salt stress, the limit salt concentration (the salt concentration of the plant growth which is inhibited by yield reduction) is only about 1.7dsm-1 and about 0.1% NaCl, and the salt tolerance of the plant is quantitative character controlled by multiple genes in genetics, has complex genetic mechanism and is easily influenced by environmental conditions. The positioning and cloning of salt tolerance have important significance for the elucidation of plant salt tolerance mechanism and breeding application. There have been significant advances in the investigation make internal disorder or usurp of salt tolerance QTL for crops, such as wheat, barley, soybean, rice, etc., of which rice research is the most systematic. However, QTL studies on salt tolerance in corn have progressed slowly.

Disclosure of Invention

The invention aims to solve the technical problem of how to regulate and control the stress resistance of plants.

In order to solve the technical problems, the invention firstly provides a protein related to plant stress resistance.

The protein related to plant stress resistance provided by the invention is named as ZmCCT and is the protein of a) or b) or c) as follows:

a) the amino acid sequence is a protein shown in a sequence 1;

b) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein shown in the sequence 1;

c) and (b) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 1.

Wherein, the sequence 1 consists of 238 amino acid residues.

In order to facilitate the purification of the protein in a), the amino terminal or the carboxyl terminal of the protein shown in the sequence 1 in the sequence table can be connected with a label shown in the table 1.

TABLE 1 sequence of tags

The protein of 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 in the 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 of c) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence No. 2, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 to the 5 'end and/or 3' end thereof.

In order to solve the above technical problems, it is another object of the present invention to provide a biomaterial related to the above 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-mentioned related biological material, the nucleic acid molecule according to A1) is a gene represented by the following 1) or 2) or 3):

1) the coding sequence is a cDNA molecule shown in a sequence 2 or a genome DNA molecule shown in a sequence 3;

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

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

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, the sequence 2 consists of 717 nucleotides, and the coding sequence 1 shows the amino acid sequence.

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

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 1 of the present invention. 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 zmcc (zmcc gene expression cassette) according to a2) is D capable of expressing zmcc in a host cellNA, which DNA may include not only a promoter that initiates transcription of zmcc but also a terminator that terminates transcription of zmcc. 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 alone or 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 existing expression vector can be used for constructing a recombinant vector containing the ZmCCT gene expression cassette. 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 technical problems, the invention also provides a new application of the protein or the related biological materials.

The present invention provides the use of the above-mentioned protein or the above-mentioned related biomaterial in at least one of the following (1) to (6):

(1) regulating and controlling the resistance of the plant to fusarium graminearum stem rot;

(2) regulating and controlling the stress resistance of the plants;

(3) regulating and controlling the total root length and/or trunk root length and/or main embryo root length and/or side root length and/or underground part dry weight and/or overground part dry weight and/or plant height of the plant;

(4) regulating the expression level of a gene associated with plant resistance;

(5) cultivating a transgenic plant resistant to fusarium graminearum stem rot;

(6) and (3) cultivating the transgenic plant with improved stress resistance.

In the above application, the regulation is improvement; the stress resistance is low nitrogen resistance and/or salt resistance; the low nitrogen is specifically 0.04 mmoleL^-1NO₃ ^-(ii) a The high salt is 50mmol L^-1NaCl; the stress resistance of the regulated plant is specifically 0.04 mmoleL^-1NO₃ ^-And/or 50 mmoleL^-1Under NaCl conditions, the total root length of the ZmCCT-transferred plant is longer than the main root length of the receptor plant and/or the ZmCCT-transferred plant and/or the main radicle length of the ZmCCT-transferred plant is longer and/or the lateral root length of the ZmCCT-transferred plant is longer and/or the underground dry weight of the ZmCCT-transferred plant is increased and/or the overground dry weight of the ZmCCT-transferred plant is increased and/or the plant height of the ZmCCT-transferred plant is increased and/or the expression level of the resistance related gene of the ZmCCT-transferred plant is increased.

In the method, the resistance related genes are ZmABA2 and ZmMPK 5.

In order to solve the technical problems, the invention also provides a method for cultivating the transgenic plant with improved stress resistance.

The method for cultivating the transgenic plant with improved stress resistance comprises the steps of introducing the coding gene of the protein into a receptor plant to obtain the transgenic plant; the transgenic plant has higher stress resistance than the recipient plant.

In the above-mentioned method, the first step of the method,

the stress resistance is low nitrogen resistance and/or salt resistance;

the transgenic plant has higher stress resistance than the recipient plant does in any of the following (D1) - (D7):

(D1) the transgenic plant has a higher total root length than the recipient plant;

(D2) the transgenic plant has a higher stem root growth than the recipient plant;

(D3) the transgenic plant has a higher main radicle than the recipient plant;

(D4) the transgenic plant has higher lateral root growth than the recipient plant;

(D5) the underground dry weight of the transgenic plant is higher than that of the recipient plant;

(D6) the transgenic plant has a higher above-ground dry weight than the recipient plant;

(D7) the transgenic plant is higher than the recipient plant;

(D8) the transgenic plant has a higher expression level of the resistance-associated gene than the recipient plant; the resistance related genes are ZmABA2 and ZmMPK 5.

The invention also provides a method for cultivating the transgenic plant resisting fusarium graminearum stem rot.

The method for breeding the transgenic plant resistant to the fusarium graminearum stem rot comprises the step of introducing the coding gene of the protein into a receptor plant to obtain the transgenic plant; the transgenic plant has higher resistance to fusarium graminearum stem rot than the recipient plant.

In the above-mentioned method, the first step of the method,

the nucleotide sequence of the coding gene of the protein is the 1 st to 717 th nucleotide molecules of the sequence 2.

In the embodiment of the invention, the coding gene of the protein (namely, the DNA molecule shown in the sequence 3 in the sequence table) is introduced into the receptor plant through a ZmCCT gene recombinant expression vector containing a ZmCCT gene expression cassette. The ZmCCT gene recombinant expression vector containing the ZmCCT gene expression box is a recombinant expression vector pCAMBIA 3301-ZmCCT; the pCAMBIA3301-ZmCCT is a DNA molecule which is inserted between Sac I enzyme cutting sites of a pCAMBIA3301 vector in a forward direction and is shown as a sequence 3 in a sequence table, and other sequences of the pCAMBIA3301 vector are kept unchanged to obtain the vector.

In the above method, the transgenic plant is understood to include not only the first generation transgenic plant obtained by transforming a plant of interest with the zmcc gene, but also its progeny. For transgenic plants, the gene can be propagated in the species, and can also be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

In the above method, the recipient plant is a monocotyledon or dicotyledon.

In the method, the recipient plant is a monocotyledon, is a gramineae plant, is corn, and specifically is a corn (Zea mays L.) variety Hi II.

The primer pair for amplifying the full length of the ZmCCT gene or any fragment thereof also belongs to the protection scope of the invention.

Experiments prove that the ZmCCT gene provided by the invention can improve the proportion of low nitrogen stress and high salt stress resistant plants in the progeny of ZmCCT gene-transferred plants, and meanwhile, compared with negative plants, the ZmCCT gene-transferred positive plants in the progeny have greatly improved resistance to fusarium graminearum stem rot.

Drawings

FIG. 1 is T₀Transferring ZmCCT gene corn plant to carry out PCR identified electrophoresis pattern. Lane 1 is D2000Marker, and the band sizes from bottom to top are 100bp, 250bp, 500bp, 750bp, 1kb and 2kb in this order; lanes 2, 3, 4, 5, 6, 8, 9, 10, 12, 13, 14, 15, 17, 18, 19, 21, 23, 24, 25 and 26 are all transgenic unsuccessful individuals; lanes 7, 11, 16, 20, 22 are all positive transgenic individuals (band size 518 bp).

FIG. 2 shows the disease resistance of ZmCCT transgenic positive plant (P) and negative plant (N). Wherein, fig. 2A is the plant surface of positive plant (P) and negative plant (N) of the ZmCCT gene; fig. 2B shows positive (P) and negative (N) plants of zmcc gene after stem splitting.

FIG. 3 is T₀Transferring ZmCCT gene corn plant progeny to carry out qRT-PCR identified electrophoresis pattern. Lane 1 is D2000Marker, with the upper and lower bands being 250bp and 100bp, respectively; lanes 2, 3, 4, 5, 6, 7 are all positive transgenic individuals (P); 8. 9, 10, 11, 12 and 13 are all negative transgenic individuals (N); 28 and 32 are both PCR reaction cycles; GAPDH is the reference gene.

FIG. 4 is T₁And (5) counting the disease resistance of the substitute material. Wherein, P represents positive plants transformed with ZmCCT genes, and N represents negative plants.

FIG. 5 is T₂/T₃And (5) counting the disease resistance of the substitute material. Wherein, P represents positive plants transformed with ZmCCT genes, and N represents negative plants.

FIG. 6 shows transgene T₄And (3) generation of a low-nitrogen high-salt stress growth index statistical chart. FIG. 6A is a total root length statistic; FIG. 6B shows the statistics of the length of the main radicle; FIG. 6C is a trunk root length statistic; FIG. 6D is a lateral root length statistic; FIG. 6E is a subsurface dry weight statistic; figure 6F is a geosynthetic dry weight statistic. Wherein TL-23 (+): ZmCCT-transgenic positive plants, TL-23 (-): negative plant, lns (lownitogen stress): 0.04mmol/L nitrogen element stress, HSS (high salinity stress): 50mmol/L NaCl stress, Significant difference: a, P<0.05；**，P<0.01。

FIG. 7 shows the growth status of ZmCCT transgenic positive (TL +) and negative (TL-) plants after normal (Mock) and Low Nitrogen Stress (LNS) treatments. Wherein TL-23 (+): ZmCCT-transgenic positive plants, TL-23 (-): negative plants.

FIG. 8 shows the growth status of ZmCCT-transgenic positive (TL +) and negative (TL-) plants after normal (Mock) and High Salt Stress (HSS) treatments. Wherein TL-23 (+): ZmCCT-transgenic positive plants, TL-23 (-): negative plants.

Figure 9 is the expression change of zmcc after low nitrogen and high salt stress. FIG. 9A is a control root; FIG. 9B is a control aerial portion; FIG. 9C is a salt stressed root; FIG. 9D is salt stressed aerial parts; FIG. 9E is nitrogen stressed roots; FIG. 9F is nitrogen stressed aerial parts. Wherein TL-23 (+): ZmCCT-transgenic positive plants, TL-23 (-): negative plants.

Figure 10 is the expression change of ZmABA2 after low nitrogen and high salt stress. FIG. 10A is a control root; FIG. 10B is a control aerial part; FIG. 10C is a salt stressed root; FIG. 10D is salt stressed aerial parts; FIG. 10E is nitrogen stressed roots; FIG. 10F is nitrogen stressed aerial parts. Wherein TL-23 (+): ZmCCT-transgenic positive plants, TL-23 (-): negative plants.

FIG. 11 is the expression change of ZmMPK5 after low nitrogen and high salt stress. FIG. 11A is a control root; FIG. 11B is a control aerial part; FIG. 11C is a salt stressed root; FIG. 11D is salt stressed aerial parts; FIG. 11E is nitrogen stressed roots; FIG. 11F is nitrogen stressed aerial parts. Wherein TL-23 (+): ZmCCT-transgenic positive plants, TL-23 (-): negative plants.

Detailed Description

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

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

Maize (Zea mays L.) inbred line 1145 in the following example: the national center for crop germplasm preservation, which is 0L010346, is a high low nitrogen stress and salt stress tolerant variety (see "Qin Yang, guang Yin, Yanling Guo, et al. A major QTL for resistance to Gibberella talk about rest in the same Gene, (2010)121: 673-.

Maize (Zea mays L.) variety Hi ii in the following examples: "mail Hi-II" is described in "Lorena Moeller, Qingleigan, Kan Wang. overview and characterization of a mail Hi-II endolumenal. in Viro Cellular & development Biology-Plant,2012(48): 283-.

Plasmid pCAMBIA3301 in the following examples: described in "Huixia Shou, Reid G. Palmer, KanWang. Irreproductivity of the Soybean Pollen-Tube Path transformation procedure. plant Molecular Biology Reporter,2002(20): 325-.

Agrobacterium LBA4404 in the following examples is Agrobacterium tumefaciens LBA4404 Electro-Cells from Clontech, cat #: 9115.

example 1 acquisition of Gene ZmCCT associated with Low Nitrogen stress and high salt stress tolerance

Acquisition of full-length cDNA sequence of gene ZmCCT related to low nitrogen stress resistance and high salt stress resistance

Fusarium graminearum (Fusarium graminearum Schw.) conidia are artificially inoculated into a plant of a high low nitrogen stress resistant maize (Zea mays L.) inbred line 1145 in the male drawing stage by a root-injured soil burying method (see' Songzouheng et al, health cultivation measures for the control effect of maize stalk rot, Liaoning agricultural science, 1993, 05). After inoculation for 16h, the leaves were removed. Total RNA was extracted using TriZol reagent supplied by Invitrogen corporation. Using BD SMART^TMRACE cDNAamplification Kit, using gene specific primers, 5'RACE primer (5' GSPB) and 3'RACE primer (3' GSPA) (see Table 1), and universal primers provided in the Kit, and referring to Kit instructions, to amplify and sequence the 5'RACE product and the 3' RACE product. And (3) carrying out sequence splicing on the 5 'end sequence and the 3' end sequence of the obtained target gene ZmCCT to obtain the full-length cDNA sequence of the ZmCCT gene, wherein the nucleotide sequence of the full-length cDNA sequence is shown as a sequence 2 in a sequence table.

TABLE 1 sequence information of all primers used for Gene function identification

In order to confirm the accuracy of the full-length zmcc gene cDNA sequence (seq id No. 2) obtained by the above splicing, specific primers for amplifying the full-length zmcc gene cDNA sequence were designed as follows.

Primer 1: 5'-ATGTCGTCGGGGCCAGCAGC-3' (1 st to 20 th position of sequence 2 in the sequence table);

primer 2: 5'-TTGCCAAGGTAACCGAATGA-3' (reverse complement of position 698-717 of sequence 2 in the sequence listing).

The cDNA reverse transcription of the total RNA is used as a template, the primer 1 and the primer 2 are used for amplification, and the amplification product is subjected to 1% agarose gel electrophoresis detection. The results showed that a fragment of about 720bp in length was obtained by PCR amplification. The product was recovered and purified, and ligated to pEASY-T1 vector (Beijing Quanjin Biotechnology Co., Ltd.) for sequencing and identification. Sequencing results show that the PCR amplification product is completely the same as the sequence 2 obtained by splicing, namely the full-length cDNA sequence of the ZmCCT gene is shown as the sequence 2 in a sequence table, ORF is the 1 st to 717 th site of the sequence 2, the protein shown as the sequence 1 in the sequence table is coded, and the protein is named as ZmCCT.

II, obtaining of gene ZmCCT genome DNA sequence related to low nitrogen stress and high salt stress tolerance

Fusarium graminearum (Fusarium graminearum Schw.) conidia are artificially inoculated into a maize (Zea mays L.) inbred line 1145 plant with high low nitrogen stress resistance in the male extraction stage by a root-injured soil burying method (see' Songzouheng et al, health cultivation measures for controlling effect on maize stalk rot, Liaoning agricultural science, 1993, 05). And (3) taking leaves 16h after inoculation, and extracting the genome DNA by using an SDS alkaline lysis method.

The genomic DNA obtained above was used as a template, PCR amplification was carried out using the above primer 1 and primer 2, and the amplified product was subjected to 1% agarose gel electrophoresis. The results show that PCR amplification yielded a fragment of approximately 2600bp in length. The product was recovered and purified, and ligated to pEASY-T1 vector (Beijing Quanjin Biotechnology Co., Ltd.) for sequencing and identification. The sequencing result shows that the nucleotide sequence of the product is the 5136-position 7682 of the sequence 3 in the sequence table, wherein the 5136-position 5609 is the first exon sequence, the 5610-position 7439 is the intron sequence, and the 7440-position 7682 is the second exon sequence.

Thirdly, obtaining genome segment containing ZmCCT genome DNA sequence

A "genome segment sequence containing a ZmCCT genome DNA sequence" with restriction enzyme Sac I recognition sites at both ends, namely "GAGCTC + sequence 3+ GAGCTC", is prepared and is marked as a DNA fragment A. The DNA fragment A was ligated to pEASY-T1 vector (Beijing Quanjin Biotechnology Co., Ltd.) and the resulting recombinant plasmid was named pEASY-ZmCCT. Sequencing and identifying pEASY-ZmCCT.

The sequencing result shows that the sequence of the exogenous gene inserted into the pEASY-T1 vector is just 'GAGCTC + sequence 3+ GAGCTC'. Wherein, the 1 st-5135 th site of the sequence 3 is a promoter sequence; the genome sequence of the ZmCCT gene at the position 5136-7682 (the first exon sequence at the position 5136-5609, the second exon sequence at the position 7440-7682 and the intron sequence at the position 5610-7439); no. 7683 and 8147 are untranslated region sequences.

Example 2 obtaining of ZmCCT Gene-transferred maize and functional identification thereof

Construction of recombinant expression vector pCAMBIA3301-ZmCCT

The recombinant plasmid pEASY-ZmCCT obtained in step three of example 1 was digested with a restriction enzyme Sac I, and the desired fragment (a genome segment containing a ZmCCT genomic DNA sequence, about 8.1K) was recovered and ligated to the backbone fragment of pCAMBIA3301 vector, which was also digested with Sac I, to obtain a recombinant plasmid pCAMBIA 3301-ZmCCT.

Sac I enzyme digestion identification is carried out on the obtained recombinant plasmid, the recombinant plasmid which is shown by the enzyme digestion identification to contain a target band with the size of about 8.1kb is sequenced, and a sequencing result shows that: pCAMBIA3301-ZmCCT is a DNA molecule which is inserted between Sac I enzyme cutting sites of pCAMBIA3301 vector and is shown in sequence 3 in the sequence table, and the other sequences of pCAMBIA3301 vector are kept unchanged to obtain the vector. In the recombinant expression vector pCAMBIA3301-ZmCCT, the promoter for starting the transcription of ZmCCT genome DNA sequence is 1-5135 th site of sequence 3.

Second, obtaining of ZmCCT transgenic corn

1. Transformation and identification of Agrobacterium

And (3) introducing the recombinant expression vector pCAMBIA3301-ZmCCT constructed in the step one into agrobacterium LBA 4404. The specific operation is as follows:

(1) mu.l of pCAMBIA3301-ZmCCT plasmid DNA at a concentration of 100 ng/. mu.l was added to 50. mu.l of LBA4404 Agrobacterium-sensitive cells, gently mixed, and placed on ice for 30 minutes.

(2) The mixture of (1) was frozen in liquid nitrogen for 1 minute.

(3) Incubating the frozen mixture of (2) in a water bath at 37 ℃ for 5 minutes.

(4) To the mixture obtained in (3), 1ml of YEP liquid medium was added. The cells were incubated at 28 ℃ for 4 hours at 120 rpm.

(5) The culture solution obtained in (4) was centrifuged at 1000rpm for 30 seconds, and the supernatant was discarded to obtain a bottom layer cell suspension.

(6) Adding 100ul YEP liquid medium to the cell suspension obtained in (5), resuspending the cells, and plating the resuspension mixture on solid medium containing kanamycin and rifampicin.

(7) Culturing the solid culture medium coated in the step (6) at 28 ℃ for 36-48 hours under a dark condition.

(8) And (4) carrying out thallus PCR identification and sequencing identification on the bacterial colony obtained by culturing in the step (7) to obtain a positive transformation strain.

And carrying out PCR identification on the transformed recombinant agrobacterium by using a primer pair consisting of a primer 3 and a primer 4. Agrobacterium LBA4404 identified as containing the ZmCCT genome segment shown in sequence 3 (the size of the PCR band is about 8.1kb) was designated LBA4404/pCAMBIA 3301-ZmCCT. Meanwhile, an agrobacterium contrast transferred with an empty vector of pCAMBIA3301 is set, and agrobacterium LBA4404 transferred with the empty vector of pCAMBIA3301 is named LBA4404/pCAMBIA 3301.

Primer 3: 5'-GAGCTCTTGTTGCGACTTGT-3' (1 st to 20 th position of sequence 3 in the sequence table);

primer 4: 5'-GAGCTCGACAAACAGTACAT-3' (reverse complement of position 8128-8147 of sequence 3 in the sequence Listing).

2. Transformation and identification of recombinant agrobacterium to maize plants

The recombinant Agrobacterium LBA4404/pCAMBIA3301-ZmCCT (or the empty vector control LBA4404/pCAMBIA3301) obtained above was transformed into maize (Zea mays L.) variety Hi II.

Soaking corn callus with the recombinant agrobacterium activated in advance to obtain transformed callus, and after transformation, performing resistance screening with herbicide to obtain transgenic seedlings, namely corn plants transformed with pCAMBIA3301-ZmCCT and corn plants transformed with pCAMBIA3301 empty vectors.

Further on the transgenic maize plants (T) obtained above₀Generation) and screening PCR positive strain lines. Extracting genome DNA of the transgenic corn plant, using the genome DNA as a template, carrying out PCR identification on the corn plant into which pCAMBIA3301-ZmCCT is transferred, using a ZmCCT genome segment shown in a sequence 3 and a pCAMBIA3301 carrier self sequence as target genes, and carrying out PCR amplification by using a primer pair (LBCCT F/R). The recipient parent maize (Zea mays L.) variety Hi II, which was not transgenic, was also set as a control.

LBCCT FP: 5'-TAGCTAGCTCCACCACAGCA-3' (positions 7693-7712 of SEQ ID NO: 3);

LBCCT RP: 5'-TGTGGAATTGTGAGCGGATA-3' (the sequence corresponds to the self-contained sequence on the pCAMBIA3301 vector).

The results of PCR identification of maize plants transformed with pCAMBIA3301-ZmCCT (FIG. 1) showed that transgenic maize amplified with a band of the expected size (518bp) was positive, of which 5T were obtained₀Transgenic positive plants were designated Y3-1, Y3-14, Y3-18, Y3-23 and Y3-25.

Disease resistance identification of ZmCCT transgenic corn

(I) Experimental method

5T obtained in the second step₀Transgenic ZmCCT gene plants Y3-1, Y3-14, Y3-18, Y3-23 and Y3-25 are subjected to selfing to obtain 5 transgenic T₁Generation group, will T₁The generation group is planted in the test field of Beijing Shanzhuang, and each transgenic T₁137 plants are planted in the generation group for identifying the resistance character of each single plant to fusarium graminearum stem rot, and the function of the ZmCCT gene is identified by combining the genotype analysis. The experiment was repeated 3 times and the results averaged. The specific operation is as follows:

1. genotype analysis and ZmCCT gene expression quantity determination:

(1) genotyping analysis

As the non-homozygous transgenic positive plants are separated in the offspring, namely transgenic positive and negative plants appear in the offspring population, the 5T transgenic positive and negative plants can be separated₀The progeny population of the ZmCCT transgenic plant is divided into two genotypes, namely positive (P) and negative (N))。

PCR identification of the above 5T genes by using a specific pair of primers (LBCCT F/R, sequence as above) of pCAMBIA3301-ZmCCT vector₀The genotype of the progeny population of the ZmCCT transgenic plant is transferred, an individual capable of amplifying a band (518bp) is a transgenic positive individual (P), and an individual incapable of amplifying a corresponding band is a transgenic negative individual (N).

(2) ZmCCT gene expression amount assay

And (2) respectively taking the plant population (P) with positive ZmCCT gene transfer and the plant population (N) with negative ZmCCT gene transfer in the step (1) as experimental materials. Total RNA was extracted from each experimental material. The extraction method of the TIANGEN RNAprep pure plant total RNA extraction kit for RNA extraction is the same as the specification.

The RNA was reverse transcribed into cDNA using reverse transcription kit (Fermentas) and stored at-80 ℃ until use.

qRT-PCR is carried out by adopting a Kit SYBR Premix EX Taq Kit (precious bioengineering) according to a Kit instruction, and the expression quantity of the ZmCCT gene is detected.

A forward primer: 5'-ATGAGAACGACGACCAGCCT-3' (5455-5474 of SEQ ID NO: 3);

reverse primer: 5'-GACGACTGATCTACCGGCAT-3' (reverse complement of position 7418-7537 of SEQ ID NO: 3). (Note: primers spanning the intron, cDNA as template)

Reaction system: 20 ul.

Reaction procedure: step 1: 94 ℃ for 3 min;

Step 2：94℃for 30s；

Step 3：60℃for 30s；

Step 4：72℃for 30s；

step 5: go to step 2for 28or 32 cycles; (here, the number of repeated cycles)

Step 6：72℃for 10min；

Step 7：5℃forever。

GAPDH is used as the reference gene. The amplification primers of the internal reference gene are 5'-ATCAACGGCTTCGGAAGGAT-3' and 5'-CCGTGGACGGTGTCGTACTT-3'

Meanwhile, a corn positive plant which is transferred into the pCAMBIA3301 empty vector and is identified in the step two is set as an empty vector Control (CK), and a corn (Zea mays L.) variety Hi II of a non-transgenic receptor parent is set as a parent control (WT).

2. Analysis of resistance traits to fusarium graminearum stem rot:

a method for burying and damaging roots by soil (see' Songzoujing, etc.. health care cultivation measures for controlling effect research on corn stalk rot, Liaoning agricultural science, 1993, 05 th year) comprises the following steps of inoculating fusarium graminearum to an unhygrosy transgenic progeny group plant: after a corn plant silking device, cutting off a part of hairy roots vertically downwards at a position 5-10 cm away from the corn plant, throwing away soil, burying 60-80 g of Fusarium graminearum propagated by corn seeds, watering and moisturizing.

Further, disease-resistant plant proportion statistics is respectively carried out on the plant population (P) which is identified as positive by the ZmCCT gene transfer in the step 1 and the plant population (N) which is identified as negative by the ZmCCT gene transfer. The method comprises the following specific steps: after about 45 days of artificial inoculation with fusarium graminearum, maize plants were split into stems, and the diseased plants were identified as those with hollow stem base and root and rotting, and the disease-resistant plants were identified as those with intact stem base and root (fig. 2). Respectively calculating the proportion of disease-resistant plants in a ZmCCT gene positive plant population (P) and a ZmCCT gene negative plant population (N).

Meanwhile, a corn positive plant which is transferred into the pCAMBIA3301 empty vector and is identified in the step two is set as an empty vector Control (CK), and a corn (Zea mays L.) variety Hi II of a non-transgenic receptor parent is set as a parent control (WT).

In addition, T identified as positive for the transgene by the above-described genotyping₁Selfing the plant to obtain T₂Generation group; t identified as positive for the transgene by the above genotype analysis₂Selfing the plant to obtain T₃And (4) generation groups. For T₂Generation group and T₃The generation population was also subjected to genotype analysis and disease resistance trait analysis using the above methods.

(II) results of the experiment

1. 5T₀Transgenic ZmCCT gene plantProgeny ZmCCT gene expression quantity determination

The results are shown in FIG. 3, from which it can be seen that there are 5 Ts₀In the progeny population of the transgenic ZmCCT plant, the ZmCCT gene expression level of the positive transgenic plant (P) is far higher than that of the negative transgenic material (N). Compared with the negative transgenic material (N), the ZmCCT gene expression level in the two corn plants serving as the parent control (WT) and the empty vector Control (CK) is basically consistent and has no statistical difference.

2. 5T₀Determination of disease resistance of progeny of ZmCCT gene transgenic plants

The results show that: at T₁On the generation group level (figure 4), the disease-resistant plant proportion of the positive plants (P) in Y3-1, Y3-23 and Y3-25 is obviously improved compared with that of the negative plants (N), wherein the disease-resistant plant proportion of the positive plants (P) in Y3-1 is improved by 33 percent compared with that of the negative plants (N), the disease-resistant plant proportion of the positive plants (P) in Y3-23 is improved by 13 percent compared with that of the negative plants (N), and the disease-resistant plant proportion of the positive plants (P) in Y3-25 is improved by 10 percent compared with that of the negative plants (N).

At T₂On the generation group level (figure 5), the disease-resistant plant proportion of Y3-1, Y3-18, Y3-23 and Y3-25 middle positive plants (P) is obviously improved relative to negative plants (N), wherein the disease-resistant plant proportion of Y3-1 middle positive plants (P) is improved by 35% compared with negative plants (N), the disease-resistant plant proportion of Y3-18 middle positive plants (P) is improved by 17% compared with negative plants (N), the disease-resistant plant proportion of Y3-23 middle positive plants (P) is improved by 25% compared with negative plants (N), and the disease-resistant plant proportion of Y3-25 middle positive plants (P) is improved by 9% compared with negative plants (N).

Selecting T₂Selfing transgenic positive Y3-23 to obtain T₃And (4) generation groups. The results show that at T₃On the generation group level (figure 5), the disease-resistant plant proportion of the positive plants (P) in the Y3-23 is still obviously improved relative to the negative plants (N), and the disease-resistant plant proportion of the positive plants (P) in the Y3-23 is improved by 7 percent compared with the negative plants (N).

3. Parental control and empty vector control disease resistance assays

Compared with the above 5T₀The identification results of the progeny of the ZmCCT transgenic plants (FIGS. 4 and 5) were not obtainedThe disease-resistant plant proportion in the transgenic corn (Zea mays L.) variety Hi II (WT) is only 5 percent and is far lower than the 5T₀The disease-resistant plant proportion in the progeny of the ZmCCT gene plant is transferred. The experimental results of the empty vector control were essentially identical to the parental control with no statistical difference.

From the above results 1-3, it can be seen that the above 5T's are compared to the parental control and the empty vector control that are not transgenic₀The disease-resistant plant proportion in the progeny of the plant transformed with the ZmCCT gene is greatly improved, the disease-resistant plant proportion in the positive plant (P) in the progeny is far higher than that in the negative plant (N), and the expression quantity of the ZmCCT gene in the positive plant (P) is also far higher than that in the negative plant (N).

Low nitrogen and high salt stress tolerance analysis of ZmCCT transgenic corn

ZmCCT transgenic corn seedling-stage root property and dry weight measurement

(1) Experimental methods

T identified as positive for the transgene by the above genotype analysis₄The ZmCCT transgenic corn seeds are subjected to low-nitrogen and high-salt stress in a seedling stage under the condition of water culture in an artificial climate chamber, the root characters, the plant height, the SPAD value and the like are measured after the stress treatment for one week, and the dry weight is measured after the seeds are dried to constant weight. Statistical analysis was performed using the t-test method.

(2) Experimental procedure

Will T ₄10% (v/v) H for ZmCCT transgenic corn seeds₂O₂Sterilizing for 30 min, washing with deionized water, and saturating with CaSO₄Soaking for 6 hr, growing in a dark place in a phytotron for 2 days, selecting the seeds with uniform growth when the seeds germinate about 1-2cm, rolling the seedlings with filter paper, rolling into a cylindrical roll from one side, transferring into a small water bucket to grow to 1 leaf 1 heart stage, selecting the seedlings with uniform growth, and transferring into 1L of nutrient solution (Hoagland's nutrient solution: 0.75mmol L)^-1K₂SO₄、0.1mmolL^-1KCl、0.25mmolL^{- 1}KH₂PO₄、0.65mmolL^-1MgSO₄、0.13mmolL^-1EDTA-Fe、1.0μmolL^-1MnSO₄、1.0μmolL^-1ZnSO₄、0.1μmolL^-1CuSO₄、0.005μmolL^-1(NH₄)₆Mo₇O₂₄)4 strains per pot in the culture pot of (1). The growth conditions were controlled at 28 deg.C/22 deg.C for 16/8h light/dark, daily cycle. Luminous flux density of 250-^-2s^-1. After the seedlings are transferred into a culture tank, the seedlings are pre-cultured for two days by using 1/2(c/c) culture solution, then are cultured by using complete nutrient solution until 3 leaves reach 1 heart stage, and low nitrogen (0.04mmol L) is respectively carried out^-1NO₃ ^-) High salt (50mmol L)^-1NaCl) stress treatment and further culture for 7 days. The N element is Ca (NO)₃)₂Providing, in the treatment of low nitrogen stress, Ca²⁺With CaCl₂Form supplement to 4mmolL^-1NO₃ ^-The level of (c). The pH was adjusted to 6.0 with 1mmol/L NaOH. The electric air pump provides oxygen, the nutrient solution is replaced every two days, and each treatment is carried out for three times.

(3) Measurement index

The corn seedlings after being stressed for 7 days are washed by deionized water and divided into an overground part and an underground part, root scanning pictures are measured by Image J software, and the measured characters comprise: total Root Length (TRL), primary germ root length (PRL), primary root length (MRL), and Lateral Root Length (LRL); drying the overground part and the underground part to constant weight at 70 ℃/48h, and weighing the dry weight (SDW) of the overground part and the dry weight (RDW) of the underground part; the Plant Height (PH) was measured with a ruler. 10 seedlings were tested for each trait and the mean value was taken.

(4) Results of the experiment

The statistical measurement result of the T test shows that T₄Compared with a negative plant, the total root length, the lateral root length and the plant height of the ZmCCT transgenic maize plant have obvious difference in a normal control environment (Mock), and the main root length and the underground dry weight have extremely obvious difference; after low nitrogen stress treatment (LNS), there were very significant differences in total root length, lateral root length, dry underground, dry overground and plant height, significant differences in trunk root length, T₄Total root length, lateral root length, underground dry weight and ground of ZmCCT transgenic corn plantThe dry weight of the upper part, the plant height and the trunk root length are obviously higher than those of a negative plant; after high salt treatment (HSS), all the measured traits, transgenic positive plants were significantly different from negative plants, T₄The total root length, the main stem root length, the main embryo root length, the lateral root length, the underground dry weight, the overground dry weight and the plant height of the ZmCCT transgenic maize plant are all obviously higher than those of a negative plant; t is₄The total root length, the main stem root length, the lateral root length, the underground dry weight, the overground dry weight and the plant height of the ZmCCT transgenic corn plant are obviously higher than those of a negative plant. T is₄The ZmCCT transgenic maize plants showed strong growth adaptability under low nitrogen and salt stress conditions (FIG. 6 and Table 2).

TABLE 2, T₄Low-nitrogen high-salt stress growth index statistical table for ZmCCT gene-substituted corn

Due to T₄The ZmCCT-transgenic maize plant and the negative plant have larger growth potential difference, and multi-factor variance analysis is carried out, and the analysis result is shown in table 3. The total root length, the trunk root length and the underground dry weight have significant differences under the low nitrogen stress condition, and the total root length, the main embryo root length, the lateral root length and the plant height have significant differences under the salt stress condition.

TABLE 3, T₄Multi-factor variance analysis table for ZmCCT (ZmCCT) transgenic corn low-nitrogen high-salt stress growth indexes

To sum up, the ZmCCT gene-transferred positive plant has developed root growth and strong adaptability under the conditions of low nitrogen and salt stress in the seedling stage, and the ZmCCT has the effect of improving the low nitrogen and high salt stress resistance of the corn in the seedling stage (fig. 7 and 8).

(II) complementation test transgenic material seedling-stage low-nitrogen and high-salt stress condition expression research

(1) Content of the experiment

Taking T of three leaves in one heart period₄The ZmCCT gene maize plants and negative plants are transformed, expression analysis is carried out on roots and leaves at different time points (0, 3, 6 and 9 hours) after 50mmol of NaCl high salt stress treatment and 0.04mmol of low nitrogen stress, 5 plants are randomly taken from each time point of each sample and mixed together for expression research, 3 times of repetition are set for each sample, and the experiment is repeated for 3 times.

(2) Procedure of experiment

The extraction method of the TIANGEN RNAprep pure plant total RNA extraction kit for RNA extraction is the same as the specification. TransScript First-Strand cDNA SuperMix (AT301-03) from Takara was used for RNA reverse transcription in the same manner as described in the specification. DEPC-ddH for synthesized cDNA₂O is diluted to a proper concentration, and the product is stored at-20 ℃ for later use.

SYBR Premix Ex Taq from TaKaRa for qRT-PCR^TMII (RR820A), detection was performed using RoterGene 6000. Using comparative CT value method (2)^-ΔΔCt) Relative quantification of gene expression was performed (Livak and Schmittgen 2001). The internal reference gene GAPDH and the expression amount measurement primers are shown in Table 4.

qRT-PCR procedure: 95 ℃ for 5 min; 95 ℃, 10S, 60 ℃, 20S, 40 cycles; a stimulating assay.

qRT-PCR reaction system (20. mu.l): SYBR Primix Ex Taq^TMII(2×)10μl、Forward Primer(10μM)0.4μl、Reverse Primer(10μM)0.4μl、cDNA 2μl、ddH₂O 7.2μl。

TABLE 4 internal reference gene GAPDH and expression amount determination primer

(III) results of the experiment

T₄The ZmCCT transgenic maize plant has the phenomenon of induced expression under the conditions of low nitrogen and salt stress (figure 9), T₄ZmCCT gene-substituted maize plant (TL-23(+)) the expression level of roots and overground parts is increased under the conditions of low nitrogen and high salt stress, the peak value is reached after 3 hours, and the expression level is about 0 hour T after treatment₄The transformation of ZmCCT gene maize plants 5.86 times and 9.71 times, then, a decline phenomenon occurs, which does not occur in negative plants.

ZmABA2 and ZmMPK5 were reported to be involved in antioxidant defense responses induced by corn ABA. The increase of the expression levels of ZmABA2 and ZmMPK5 can enhance the capability of abiotic stress such as salt stress resistance, drought stress and oxidative stress resistance of corn and enhance the adaptability of corn to the environment (Ma, Ni et al 2015).

ZmABA2 at T under conditions of low nitrogen and high salt stress₄The expression level of roots and overground parts of the ZmCCT transgenic corn plants (TL-23(+)) is increased (figure 10), the expression level reaches the peak value within 3 hours except for the roots stressed by low nitrogen, the expression level is about 2-4 times of that of the roots stressed by stress, then the phenomenon of decline occurs, the phenomenon of induced expression occurs on the roots stressed by salt, the roots stressed by low nitrogen and the overground parts of negative plants (TL-23(-), but the increase range of the expression level is far smaller than that of ZmCCT transgenic positive plants.

ZmMPK5 at T under conditions of low nitrogen and high salt stress₄The expression level of roots and overground parts of the transgenic ZmCCT gene positive plants (TL-23(+)) is increased (figure 11), and the expression level is 4-6 times higher than that of the negative plants (TL-23(-)) after stress treatment for three hours.

In conclusion, the ZmCCT expression level of the ZmCCT transgenic maize plant is increased after low-nitrogen and high-salt stress treatment, and reaches a peak value in 3 hours. After low nitrogen and high salt stress treatment, the expression quantity of genes ZmABA2 and ZmMPK5 related to improving the resistance of the corn is greatly increased in ZmCCT gene transferred positive plants. The ZmCCT gene has the function of improving the low nitrogen and high salt stress resistance of the corn in the seedling stage.

Claims (4)

1. The use of any one of the following in improving stress resistance of a plant or breeding a transgenic plant with improved stress resistance; 1) the protein has an amino acid sequence shown as SEQ ID NO: 1 is shown in the specification;

2) in SEQ ID NO: 1, the N end and/or the C end of the protein shown in the formula 1 are connected with a label to obtain a fusion protein;

3) a nucleic acid molecule encoding the protein of 1);

4) an expression cassette comprising 3) the nucleic acid molecule;

5) a recombinant vector comprising 3) said nucleic acid molecule;

6) a recombinant vector comprising 4) said expression cassette;

7) a recombinant microorganism containing 3) said nucleic acid molecule;

8) a recombinant microorganism comprising 4) said expression cassette;

9) a recombinant microorganism containing 5) the recombinant vector;

10) a recombinant microorganism containing 6) the recombinant vector;

the plant is a monocot;

the stress resistance is low nitrogen resistance and/or salt resistance.

2. Use according to claim 1, characterized in that: the coding sequence of the nucleic acid molecule is SEQ ID NO: 2.

3. A method for producing a transgenic plant having improved stress resistance, which comprises the step of introducing a gene encoding the protein of claim 1 into a recipient plant to obtain a transgenic plant; the transgenic plant has higher stress resistance than the recipient plant; the stress resistance is low nitrogen resistance and/or salt resistance; the nucleotide sequence of the coding gene of the protein is shown as SEQ ID NO: 2, bits 1-717;

the recipient plant is a monocot.

4. The method of claim 3, wherein:

the transgenic plant has higher low nitrogen tolerance and/or salt resistance than the recipient plant does in any one of the following (D1) - (D8):

(D1) the transgenic plant has a higher total root length than the recipient plant;

(D2) the transgenic plant has a higher stem root growth than the recipient plant;

(D3) the transgenic plant has a higher main radicle than the recipient plant;

(D4) the transgenic plant has higher lateral root growth than the recipient plant;

(D5) the underground dry weight of the transgenic plant is higher than that of the recipient plant;

(D6) the transgenic plant has a higher above-ground dry weight than the recipient plant;

(D7) the transgenic plant is higher than the recipient plant;

(D8) of transgenic plantsZmABA2AndZmMPK5the expression level of the gene is higher than that of the recipient plant.

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