CN107325161A - A kind of albumen related with high-salt stress to resistance to Low nitrogen stress and its encoding gene and application - Google Patents
A kind of albumen related with high-salt stress to resistance to Low nitrogen stress and its encoding gene and application Download PDFInfo
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Abstract
The invention discloses a kind of albumen related with high-salt stress to resistance to Low nitrogen stress and its encoding gene and application.The protein that the present invention is provided, is following protein a) or b) or c)：A) amino acid sequence is the protein shown in sequence 1；B) fused protein obtained in N-terminal and/or C-terminal the connection label of the protein shown in sequence 1；C) protein with identical function that substitution and/or missing and/or addition by the amino acid sequence shown in sequence 1 by one or several amino acid residues is obtained.Experiment is proved, ZmCCT genes provided by the present invention can improve the ratio for turning resistance to Low nitrogen stress and high-salt stress resistant plant in ZmCCT gene plant offsprings, turn ZmCCT gene masculines plant compared with negative plant in offspring simultaneously, the resistance to Fusarium graminearum stem rot improves a lot.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of albumen related with high-salt stress to resistance to Low nitrogen stress and its encoding gene and application.
Background technology
Soil depletion is to influence the unstable principal element of corn yield in world wide.In the arable soil that 1,400,000,000 hectares of the world, 22.5% soil is by nutrient condition of serious stress of soil, and only about 10.0% soil is without nutrient stress or mild stress.In Maize Production, applied nitrogen is one of important measures of corn yield increasing, but there are problems that in production practices both sides.One is that high yield developed regions nitrogenous fertilizer is excessively applied, and not only causes utilization rate of nitrogen fertilizer to decline, production cost is improved, but also it is exceeded to be likely to cause groundwater azotate；Two be low yield under-developed area often due to nitrogen application is not enough, yield level is relatively low.In view of the space of nitrogen application volume increase is further reduced, and many qualities and environmental problem that applied nitrogen is brought, the basic solution route for solving grain-production safety and environmental protection is only by the efficient corn variety of the means seed selection nitrogen such as science of heredity.
(the Moll R H such as Moll, Kamprath E J, Jackson W A.Analysis and interpretation of factors which contribute to efficiency of nitrogen utilization [J] .Agronomy Journal, 1982,74 (3):The definition of classics, i.e. crop yield under the conditions of unit applying Namount 562-564.) have been done to nitrogen efficiency.Nitrogen can efficiently be divided into two parts, and one is the efficiency that root system of plant absorbs nitrogen from soil, and two be nitrogen assimilation utilization ratio in plant.It is main that Moll etc. thinks that nitrogen use efficiency serves under the conditions of low nitrogen, and nitrogen absorption efficiency plays a major role under the conditions of high nitrogen.(the Ortiz-Monasterio R such as Ortiz-Monasterio, Sayre K D, Rajaram S, et al.Genetic progress in wheat yield and nitrogen use efficiency under four nitrogen rates [J] .Crop Science, 1997,37 (3):898-904.) then think that the difference of nitrogen efficiency under the conditions of low nitrogen is derived mainly from absorption efficiency, and utilization rate plays a major role under the conditions of high nitrogen.Lafitte and Edmeades (Lafitte H R, Edmeades G O.Improvement for tolerance to low soil nitrogen in tropical maize I.Selection criteria [J] .Field Crops Research, 1994,39 (1):1-14.) think that nitrogen use efficiency and the correlation of yield are more preferable under the conditions of low nitrogen.(meter Guo Hua, Liu builds peace Nitrogen Efficiency in Maize physiological and biochemical basis and genetic improvement progress [J] Maize Sciences, 1997,5 (2) to meter Guo Hua etc.:9-13.) result is shown, is absorbed under the conditions of high nitrogen and both utilization ratios are laid equal stress on.And meter Guo Hua etc. think to cause the different achievements in research of nitrogen absorption efficiency and nitrogen use efficiency relative importance be probably due to without genotype in different environments caused by.
The nitrogen of corn is efficiently a complicated process.The physiological Mechanism that current nitrogen efficient maize absorbs includes：(1) good root system configuration (form and spatial distribution) and Root Characteristics；(2) physiological metabolism of good root system is active (respiration etc.)；(3) aerial part possesses merit；(4) single cycle promotes absorption of the root system to nitrogen in seedling stage plant body.And the physiological mechanism of Efficient Nitrogen Utilization in Maize includes：(1) there are the N metabolic key enzymes of high activity；(2) interaction and the influence of some materials between nutrient；(3) feedback effect of storage capacity；(4) vacuole storage NO3- make full use of and aerial part nitrogen volatilization reduction；(5) nitrogen to seed to retransfer ability strong.Most of important economical characters such as yield, quality and the resistance of crop are all the quantitative characters controlled by multiple quantitative character gene locus therefors (quantitative trait loci, QTL).Current many results of study show that the nitrogen efficiency of crop is also to be controlled by multiple quantitative trait locis.
Counted according to Second National soil survey information, on the premise of coastal tidal is not included, China's salinized soil area is 34,870,000 hectares, and about 500,000,000 mu, the area that can be developed is up to 200,000,000 mu.The salt tolerant alkali ability of crop is improved, for exploitation marginal land, grain yield is improved, all has great importance.Current cultivated maize is big to field water demand, relatively low to saline and alkaline adaptability.Seedling stage is more sensitive to salt stress, its limit salinity (salinity that the suppressed yield of plant strain growth referred to declines) only has about 1.7dsm-1, about 0.1%NaCl, for from science of heredity, the salt tolerance of plant is the quantitative character by controlled by multiple genes, Genetic Mechanisms are complicated, and easily influenced by environmental conditions.Positioning and clone's salt tolerant are respectively provided with important meaning for illustrating plant salt tolerance mechanism and Breeding Application.At present for salt tolerance of crop QTL grind make internal disorder or usurp in existing remarkable break-throughs, such as wheat, barley, wherein soybean, paddy rice etc., rice research system the most.But the QTL progress for salt tolerance of corn is slow.
The content of the invention
The technical problems to be solved by the invention are how to regulate and control stress resistance of plant.
In order to solve the above technical problems, present invention firstly provides a kind of and plant adversity resistance related protein.
Entitled ZmCCT provided by the present invention with plant adversity resistance related protein, is following protein a) or b) or c)：
A) amino acid sequence is the protein shown in sequence 1；
B) fused protein obtained in N-terminal and/or C-terminal the connection label of the protein shown in sequence 1；
C) protein with identical function that substitution and/or missing and/or addition by the amino acid sequence shown in sequence 1 by one or several amino acid residues is obtained.
Wherein, sequence 1 is made up of 238 amino acid residues.
In order that the protein in a) is easy to label as shown in table 1 in purifying, amino terminal or the carboxyl terminal connection of protein that can be in sequence table shown in sequence 1.
The sequence of table 1, label
It is above-mentioned c) in protein, the substitution of one or several amino acid residues and/or missing and/or be added to substitution and/or missing and/or addition no more than 10 amino acid residues.
It is above-mentioned c) in protein can be artificial synthesized, also can first synthesize its encoding gene, then carry out biological expression and obtain.
It is above-mentioned c) in the encoding gene of protein can pass through the codon by one or several amino acid residues are lacked in the DNA sequence dna shown in sequence 2, and/or the missense mutation of one or several base-pairs is carried out, and/or obtained in the coded sequence that its 5 ' end and/or 3 ' ends connect the label shown in table 1.
In order to solve the above-mentioned technical problem, it is a further object to provide the biomaterial with above-mentioned albumen qualitative correlation.
Any of what the present invention was provided is following A 1 with the biomaterial of above-mentioned albumen qualitative correlation) to A12)：
A1 the nucleic acid molecules of above-mentioned protein) are encoded；
A2 A1) is contained) expression cassettes of the nucleic acid molecules；
A3 A1) is contained) recombinant vectors of the nucleic acid molecules；
A4 A2) is contained) recombinant vector of the expression cassette；
A5 A1) is contained) recombinant microorganisms of the nucleic acid molecules；
A6 A2) is contained) recombinant microorganism of the expression cassette；
A7 A3) is contained) recombinant microorganism of the recombinant vector；
A8 A4) is contained) recombinant microorganism of the recombinant vector；
A9 A1) is contained) the transgenic plant cells systems of the nucleic acid molecules；
A10 A2) is contained) the transgenic plant cells system of the expression cassette；
A11 A3) is contained) the transgenic plant cells system of the recombinant vector；
A12 A4) is contained) the transgenic plant cells system of the recombinant vector.
In above-mentioned relevant biological material, A1) nucleic acid molecules for it is following 1) or 2) or 3) shown in gene：
1) its coded sequence is the genomic DNA molecule shown in the cDNA molecules or sequence 3 shown in sequence 2；
2) there is 75% or more than 75% homogeneity, and the cDNA molecules or genomic DNA molecule of the protein described in coding claim 1 with the nucleotide sequence of 1) restriction；
3) under strict conditions with 1) or 2) nucleotide sequence hybridization limited, and the cDNA molecules or genomic DNA molecule of the protein described in coding claim 1.
Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also be RNA, such as mRNA or hnRNA.
Wherein, sequence 2 is made up of 717 nucleotides, the amino acid sequence shown in coded sequence 1.
Those of ordinary skill in the art can be easily using known method, and the method for such as orthogenesis and point mutation is mutated to the coding ZmCCT of present invention nucleotide sequence.Those are by manually modified, nucleotide sequence 75% or the nucleotides of higher homogeneity with the ZmCCT isolated with the present invention, as long as encoding ZmCCT and with identical function, it is the nucleotide sequence derived from the present invention and is equal to sequence of the invention.
Term " homogeneity " used herein refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes nucleotide sequence of the nucleotide sequence with 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher homogeneity with the protein of the amino acid sequence composition shown in the coded sequence 1 of the present invention.Homogeneity can with the naked eye or computer software is evaluated.Using computer software, the homogeneity between two or more sequences can be represented with percentage (%), and it can be for the homogeneity between evaluation correlated series.
Above-mentioned 75% or more than 75% homogeneity, can be 80%, 85%, 90% or more than 95% homogeneity.
In above-mentioned biomaterial, the stringent condition is, in 2 × SSC, in 0.1%SDS solution, to hybridize at 68 DEG C and wash film 2 times, each 5min, and in 0.5 × SSC, 0.1%SDS solution, hybridizes at 68 DEG C and wash film 2 times, each 15min；Or, in 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS solution, hybridize under the conditions of 65 DEG C and wash film.
In above-mentioned biomaterial, A2 the expression cassette (ZmCCT expression casettes) of the nucleic acid molecules containing coding ZmCCT described in), it is the DNA for referring to express ZmCCT in host cell, the DNA not only may include the promoter for starting ZmCCT transcriptions, may also include the terminator for terminating ZmCCT transcriptions.Further, the expression cassette may also include enhancer sequence.Promoter available for the present invention includes but is not limited to：Constitutive promoter；Tissue, organ and the special promoter of development and inducible promoter.The example of promoter includes but is not limited to：The constitutive promoter 35S of cauliflower mosaic virus：Wound-inducible promoter from tomato, leucine aminopeptidase (" LAP ", Chao et al. (1999) Plant Physiol 120：979-992)；Chemical inducible promoter from tobacco, pathogenesis correlation 1 (PR1) (is induced) by salicylic acid and BTH (diazosulfide -7- carbothioic acid S-methyl esters)；Tomato protease inhibitors II promoters (PIN2) or LAP promoters (can use methyl jasmonate induction)；Heat-shock promoters (United States Patent (USP) 5,187,267)；Tetracycline inducible promoter (United States Patent (USP) 5,057,422)；Seed specific promoters, such as Millet Seed specificity promoter pF128 (CN101063139B (Chinese patent 200710099169.7)), the special promoter of seed storage protein matter is (for example, (Beachy et al. (1985) EMBO is J.4 for phaseolin, napin, oleosin and soybean beta conglycin promoter：3047-3053)).They can be used alone or are used in combination with other plant promoters.All references cited herein is quoted in full.Suitable transcription terminator includes but is not limited to：Agrobacterium nopaline syntase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminators, tml terminators, pea rbcS E9 terminators and nopaline and octopine synthase terminator (see, e.g.：Odell et al. (I985)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 of the ZmCCT expression casettes can be contained with existing expression vector establishment.The plant expression vector includes double base agrobacterium vector and the carrier available for plant micropellet bombardment etc..Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA companies).The plant expression vector can also include 3 ' end untranslated regions of foreign gene, i.e., the DNA fragmentation comprising polyadenylation signals and any other participation mRNA processing or gene expression.The bootable polyadenylic acid of polyadenylation signals is added to 3 ' ends of mRNA precursor, and such as Agrobacterium crown gall nodule induction (Ti) plasmid gene (such as rouge alkali synthetase gene Nos), the non-translational region of the end of plant gene (such as soybean storage protein genes) 3 ' transcription is respectively provided with similar functions.During using gene constructed plant expression vector of the invention, it it is also possible to use enhancer, including translational enhancer or transcriptional enhancer, these enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., but must be identical with the reading frame of coded sequence, to ensure the correct translation of whole sequence.The source of the translation control signal and initiation codon is extensive, can be natural or synthesis.Translation initiation region can come from transcription initiation region or structural gene.For the ease of transgenic plant cells or plant are identified and screened, plant expression vector used can be processed, the enzyme of color change or the gene (gus gene of luminophor can be produced as added the coding that can be expressed in plant, luciferase genes etc.), the marker gene of antibiotic is (as assigned to kanamycins and the nptII genes of associated antibiotic resistance, assign the bar genes to herbicide phosphinothricin resistance, assign the hph genes to antibiotic hygromycin resistance, with dhfr gene of the imparting to methotrexate resistance, assign to the EPSPS genes of glyphosate) or anti-chemical reagent marker gene etc. (such as anti-herbicide gene), the mannose-6-phosphate isomerase gene of metabolism mannose ability is provided.From the security consideration of genetically modified plants, any selected marker can be not added with, transformed plant is directly screened with adverse circumstance.
In above-mentioned biomaterial, the carrier can be plasmid, sticking grain, bacteriophage or viral vector.
In above-mentioned biomaterial, the microorganism can be yeast, bacterium, algae or fungi, such as Agrobacterium.
In above-mentioned biomaterial, the transgenic plant cells system, Transgenic plant tissue and genetically modified plants organ do not include propagating materials.
In order to solve the above-mentioned technical problem, present invention also offers above-mentioned protein or the new application of above-mentioned relevant biological material.
The invention provides the application of above-mentioned protein or above-mentioned relevant biological material at least one of following (1)-(6)：
(1) resistance of the regulation and control plant to Fusarium graminearum stem rot；
(2) stress resistance of plant is regulated and controled；
(3) the regulation and control total root length of plant and/or backbone root length and/or main radicle length and/or lateral root length and/or underground part dry weight and/or overground part dry weight and/or plant height；
(4) regulation and control and the expression of plant resistance to environment stress related gene；
(5) genetically modified plants of anti-Fusarium graminearum stem rot are cultivated；
(6) genetically modified plants that resistance is improved are cultivated.
It is described to be regulated to improve in above-mentioned application；The resistance is resistance to low nitrogen and/or salt-resistance；The low nitrogen is specially 0.04mmolL-1NO3 -；The high salt is specially 50mmolL-1NaCl；The regulation and control stress resistance of plant is specially in 0.04mmolL-1NO3 -And/or 50mmolL-1Under the conditions of NaCl, turn total root length of ZmCCT plants higher than the recipient plant and/or turn ZmCCT plants backbone root length it is elongated and/or turn that the main radicle length of ZmCCT plants is elongated and/or to turn the lateral root length of ZmCCT plants elongated and/or turn the underground part dry weight of ZmCCT plants and improve and/or turn the overground part dry weights of ZmCCT plants to improve and/or turn the expression of the resistance related gene that the plant heights of ZmCCT plants uprised and/or turned ZmCCT plants and improve.
It is described resistance related because ZmABA2 and ZmMPK5 in the above method.
In order to solve the above-mentioned technical problem, present invention also offers a kind of method for cultivating the genetically modified plants that resistance is improved.
The method for the genetically modified plants that the cultivation resistance that the present invention is provided is improved includes importing the encoding gene of above-mentioned protein in recipient plant, the step of obtaining genetically modified plants；The resistance of the genetically modified plants is higher than the recipient plant.
In the above method,
The resistance is resistance to low nitrogen and/or salt-resistance；
The resistance of the genetically modified plants is embodied in any of (D1)-(D7) as follows higher than the recipient plant：
(D1) total root length of genetically modified plants is higher than the recipient plant；
(D2) the backbone root length of genetically modified plants is higher than the recipient plant；
(D3) the main radicle length of genetically modified plants is higher than the recipient plant；
(D4) the lateral root length of genetically modified plants is higher than the recipient plant；
(D5) the underground part dry weight of genetically modified plants is higher than the recipient plant；
(D6) the overground part dry weight of genetically modified plants is higher than the recipient plant；
(D7) plant height of genetically modified plants is higher than the recipient plant；
(D8) expression of the resistance related gene of genetically modified plants is higher than the recipient plant；The resistance related gene is specially ZmABA2 and ZmMPK5.
Present invention also offers a kind of method for the genetically modified plants for cultivating anti-Fusarium graminearum stem rot.
The method of what the present invention was provided educate genetically modified plants of anti-Fusarium graminearum stem rot includes importing the encoding gene of above-mentioned protein in recipient plant, the step of obtaining genetically modified plants；The genetically modified plants are higher than the recipient plant to the resistance of Fusarium graminearum stem rot.
In the above method,
The nucleotide sequence of the encoding gene of the protein is 1-717 nucleic acid molecules of sequence 2.
In an embodiment of the present invention, the encoding gene (DNA molecular i.e. in sequence table shown in sequence 3) of the protein is imported in the recipient plant by the ZmCCT gene recombinant vectors containing ZmCCT expression casettes.The ZmCCT gene recombinant vectors containing ZmCCT expression casettes are recombinant expression carrier pCAMBIA3301-ZmCCT；The pCAMBIA3301-ZmCCT is the DNA molecular shown in sequence 3 in the positive insetion sequence table between the Sac I restriction enzyme sites of pCAMBIA3301 carriers, and keeps the other sequences of pCAMBIA3301 carriers are constant to obtain carrier.
In the above method, the genetically modified plants are interpreted as not only comprising the first generation genetically modified plants for obtaining the ZmCCT genetic transformation purpose plant, also including its filial generation.For genetically modified plants, the gene can be bred in the species, it is also possible to which traditional breeding method enters the gene transfer other kinds of same species, particularly including in commercial variety.The genetically modified plants include seed, callus, intact plant and cell.
In the above method, the recipient plant is monocotyledon or dicotyledon.
In the above method, the recipient plant is monocotyledon, is grass, is corn, specially corn (Zea mays L.) kind Hi II.
The primer pair for expanding the ZmCCT full length genes or its any fragment falls within protection scope of the present invention.
Experiment is proved, ZmCCT genes provided by the present invention can improve the ratio for turning resistance to Low nitrogen stress and high-salt stress resistant plant in ZmCCT gene plant offsprings, turn ZmCCT gene masculines plant compared with negative plant in offspring simultaneously, the resistance to Fusarium graminearum stem rot improves a lot.
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Fig. 1 is T0In generation, turns the electrophoresis pattern that ZmCCT gene corn plant enter performing PCR identification.Swimming lane 1 is D2000Marker, and stripe size from top to bottom is followed successively by 100bp, 250bp, 500bp, 750bp, 1kb and 2kb；Swimming lane 2,3,4,5,6,8,9,10,12,13,14,15,17,18,19,21,23,24,25 and 26 is that transgenosis is individual not successfully；Swimming lane 7,11,16,20,22 is that positive transgenic is individual (stripe size is 518bp).
Fig. 2 is the disease-resistant performance for turning ZmCCT gene masculines plant (P) and negative plant (N).Wherein, Fig. 2A is the plant surface for turning ZmCCT gene masculines plant (P) and negative plant (N)；Fig. 2 B turn ZmCCT gene masculines plant (P) and negative plant (N) to split after stem.
Fig. 3 is T0In generation, turns the electrophoresis pattern that ZmCCT gene corns progeny of plants carries out qRT-PCR identifications.Swimming lane 1 is D2000Marker, and two bands are respectively 250bp and 100bp up and down；Swimming lane 2,3,4,5,6,7 is that positive transgenic is individual (P)；8th, 9,10,11,12,13 be negative trans genie individual (N)；28 and 32 be PCR reaction cycle numbers；GAPDH is reference gene.
Fig. 4 is T1For the disease-resistant rate statistical result of material.Wherein, P, which is represented, turns ZmCCT gene masculine plant, and N represents negative plant.
Fig. 5 is T2/T3For the disease-resistant rate statistical result of material.Wherein, P, which is represented, turns ZmCCT gene masculine plant, and N represents negative plant.
Fig. 6 is transgenosis T4For low nitrogen high-salt stress growth indexes statistical chart.Fig. 6 A count for total root length；Fig. 6 B count for main radicle length；Fig. 6 C count for backbone root length；Fig. 6 D count for lateral root length；Fig. 6 E count for underground part dry weight；Fig. 6 F count for overground part dry weight.Wherein, TL-23 (+)：Turn ZmCCT gene masculine plant, TL-23 (-)：Negative plant, LNS (low nitrogen stress)：0.04mmol/L nitrogens are coerced, HSS (High salty stress)：50mmol/L NaCl are coerced, Significant difference：*, P<0.05；*, P<0.01.
Fig. 7 is to turn ZmCCT gene masculines (TL+) and negative (TL-) the plant growth conditions after normal (Mock) and Low nitrogen stress (LNS) processing.Wherein, TL-23 (+)：Turn ZmCCT gene masculine plant, TL-23 (-)：Negative plant.
Fig. 8 is the growth conditions for turning ZmCCT gene masculines (TL+) and negative (TL-) plant after normal (Mock) and high-salt stress (HSS) processing.Wherein, TL-23 (+)：Turn ZmCCT gene masculine plant, TL-23 (-)：Negative plant.
Fig. 9 is expression changes of the ZmCCT after low nitrogen and high-salt stress.Fig. 9 A are the root of control；Fig. 9 B are the overground part of control；Fig. 9 C are the root of salt stress；Fig. 9 D are the overground part of salt stress；Fig. 9 E are the root of N stress；Fig. 9 F are the overground part of N stress.Wherein, TL-23 (+)：Turn ZmCCT gene masculine plant, TL-23 (-)：Negative plant.
Figure 10 is expression changes of the ZmABA2 after low nitrogen and high-salt stress.Figure 10 A are the root of control；Figure 10 B are the overground part of control；Figure 10 C are the root of salt stress；Figure 10 D are the overground part of salt stress；Figure 10 E are the root of N stress；Figure 10 F are the overground part of N stress.Wherein, TL-23 (+)：Turn ZmCCT gene masculine plant, TL-23 (-)：Negative plant.
Figure 11 is expression changes of the ZmMPK5 after low nitrogen and high-salt stress.Figure 11 A are the root of control；Figure 11 B are the overground part of control；Figure 11 C are the root of salt stress；Figure 11 D are the overground part of salt stress；Figure 11 E are the root of N stress；Figure 11 F are the overground part of N stress.Wherein, TL-23 (+)：Turn ZmCCT gene masculine plant, TL-23 (-)：Negative plant.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
Corn (Zea mays L.) self-mating system 1145 in following embodiments：National crops preserving seed center, its numbering is 0L010346, the corn for high resistance to Low nitrogen stress and salt stress kind (referring to " Qin Yang; Guangming Yin; Yanling Guo; et al.A major QTL for resistance to Gibberella stalk rot in maize.Theor Appl Genet, (2010) 121:673-687. " text).
Corn (Zea mays L.) kind Hi II in following embodiments：It is recorded in " Lorena Moeller; Qinglei Gan; Kan Wang.Establishment and characterization of a maize Hi-II endosperm culture.In Viro Cellular＆Developmental Biology-Plant, 2012 (48):" maize Hi-II " in the texts of 283-294. " one.
Plasmid pCAMBIA3301 in following embodiments：It is recorded in " Huixia Shou; Reid G.Palmer; Kan Wang.Irreproducibility of the Soybean Pollen-Tube Pathway Transformation Procedure.Plant Molecular Biology Reporter, 2002 (20):325-334. " text.
Agrobacterium LBA4404 is the Agrobacterium tumefaciens LBA4404 Electro-Cells of Clontech companies, article No. in following embodiments：9115.
The acquisition of embodiment 1, the gene ZmCCT related with high-salt stress to resistance to Low nitrogen stress
First, the acquisition of the gene ZmCCT full length cDNA sequence related with high-salt stress to resistance to Low nitrogen stress
Using hindering root soil buries method (referring to " the health cares cultivation step such as Song Zuoheng is to corn stalk rot disease control effect research, the phases of Liaoning agricultural sciences .1993 the 05th ") by Fusarium graminearum (Fusarium graminearum Schw.) conidium artificial infection in the plant of corn (Zea mays L.) self-mating system 1145 of the high resistance to Low nitrogen stress in tasseling stage.16h after inoculation, takes its blade.The TriZol reagents provided using Invitrogen companies extract total serum IgE.Use BD SMARTTMRACE cDNA Amplification Kit, utilize the primer -5'RACE primers (5'GSPB) and 3'RACE primers (3'GSPA) (being shown in Table 1) of gene specific, and the universal primer provided in kit, and expand 5 ' RACE products and 3 ' RACE products with reference to kit specification, and it is sequenced.Obtained target gene ZmCCT 5 ' terminal sequences and 3 ' terminal sequences are subjected to sequence assembly, the full length cDNA sequence of ZmCCT genes are obtained, its nucleotide sequence is as shown in sequence 2 in sequence table.
All primer sequence information used in table 1, Functional identification of genes
In order to further confirm the correctness of the ZmCCT full length genes cDNA sequence (sequence 2) obtained by above-mentioned splicing, the following specific primer for being used to expand ZmCCT full length gene cDNA sequences of design.
Primer 1：1-20 of sequence 2 in 5'-ATGTCGTCGGGGCCAGCAGC-3'(sequence tables)；
Primer 2：The reverse complementary sequence of 698-717 of sequence 2 in 5'-TTGCCAAGGTAACCGAATGA-3'(sequence tables).
Using the cDNA obtained by above-mentioned total serum IgE reverse transcription as template, expanded using above-mentioned primer 1 and primer 2, amplified production is subjected to 1% agarose gel electrophoresis detection.As a result show, the fragment that length is about 720bp is obtained through PCR amplifications.Reclaim and purify the product, be connected on pEASY-T1 carriers (Beijing Quanshijin Biotechnology Co., Ltd), carry out sequencing identification.Sequencing result shows, the pcr amplification product is identical with the sequence 2 that above-mentioned splicing is obtained, i.e. the full length cDNA sequence of ZmCCT genes is as shown in sequence 2 in sequence table, and ORF is 1-717 of sequence 2, albumen in polynucleotide shown in sequence 1, ZmCCT is named as by the albumen.
2nd, the acquisition of the gene ZmCCT genomic dna sequence related with high-salt stress to resistance to Low nitrogen stress
Using hinder root soil buries method (referring to " the health cares cultivation step such as Song Zuoheng is to corn stalk rot disease control effect research, the phases of Liaoning agricultural sciences .1993 the 05th ") by Fusarium graminearum (Fusarium graminearum Schw.) conidium artificial infection be in tasseling stage high resistance to Low nitrogen stress corn (Zea mays L.) self-mating system 1145 plant.16h after inoculation, takes its blade, and genomic DNA is extracted using SDS alkali formulas cracking process.
Using the genomic DNA of above-mentioned acquisition as template, enter performing PCR amplification using above-mentioned primer 1 and primer 2, amplified production is subjected to 1% agarose gel electrophoresis detection.As a result show, the fragment that length is about 2600bp is obtained through PCR amplifications.Reclaim and purify the product, be connected on pEASY-T1 carriers (Beijing Quanshijin Biotechnology Co., Ltd), carry out sequencing identification.Sequencing result shows that the nucleotides sequence of the product is classified as 5136-7682 of sequence 3 in sequence table, wherein 5136-5609 are First Exon sequence, 5610-7439 are intron sequences, and 7440-7682 are Second Exon sequence.
3rd, the acquisition of the genomic segment containing ZmCCT genomic dna sequences
" the genomic segment sequence containing ZmCCT genomic dna sequences " that two ends carry restriction enzyme Sac I recognition sites is prepared, i.e., " GAGCTC+ sequence 3+GAGCTC ", are designated as DNA fragmentation first.DNA fragmentation first is connected on pEASY-T1 carriers (Beijing Quanshijin Biotechnology Co., Ltd), gained recombinant plasmid is named as pEASY-ZmCCT.Sequencing identification is carried out to pEASY-ZmCCT.
Sequencing result shows that the exogenous gene sequence for inserting pEASY-T1 carriers is just being " GAGCTC+ sequences 3+GAGCTC ".Wherein, 1-5135 of sequence 3 are promoter sequence；5136-7682 be ZmCCT genes genome sequence (5136-5609 are First Exon sequence, 7440-7682 be Second Exon sequence, 5610-7439 be intron sequences)；7683-8147 are non-translational region sequence.
Embodiment 2, the acquisition for turning ZmCCT gene corns and its Function Identification
First, recombinant expression carrier pCAMBIA3301-ZmCCT structure
With the recombinant plasmid pEASY-ZmCCT obtained by the step 3 of restriction enzyme Sac I digestions embodiment 1, reclaim the purpose fragment (genomic segment containing ZmCCT genomic dna sequences, about 8.1K), it is connected with the skeleton fragment of the pCAMBIA3301 carriers equally through Sac I digestions, recombinant plasmid pCAMBIA3301-ZmCCT is obtained.
Sac I digestions identification is carried out to the recombinant plasmid of gained, and to showing to be sequenced containing the recombinant plasmid that size is about 8.1kb purpose bands through digestion identification, sequencing result shows：PCAMBIA3301-ZmCCT is the DNA molecular shown in sequence 3 in the positive insetion sequence table between the Sac I restriction enzyme sites of pCAMBIA3301 carriers, and keeps the other sequences of pCAMBIA3301 carriers are constant to obtain carrier.In recombinant expression carrier pCAMBIA3301-ZmCCT, the promoter for starting the transcription of ZmCCT genomic dna sequences is 1-5135 of sequence 3.
2nd, the acquisition of ZmCCT gene corns is turned
1st, the conversion and identification of Agrobacterium
The recombinant expression carrier pCAMBIA3301-ZmCCT that step one is built imports Agrobacterium LBA4404.Concrete operations are as follows：
(1) 5 μ l concentration are added in 50 μ l LBA4404 Agrobacterium competent cells for 100ng/ μ l pCAMBIA3301-ZmCCT DNAs, flick mixing, be placed in 30 minutes on ice.
(2) mixture in (1) is freezed 1 minute in liquid nitrogen.
(3) mixture by freezing in (2) is incubated 5 minutes under 37 DEG C of water bath conditions.
(4) 1ml YEP fluid nutrient mediums are added in the mixed liquor obtained in (3).Under the conditions of 28 DEG C, 120rpm is cultivated 4 hours.
(5) the nutrient solution 1000rpm obtained in (4) is centrifuged 30 seconds, abandoning supernatant obtains bottom cell suspending liquid.
(6) 100ul YEP fluid nutrient mediums are added in the cell suspending liquid obtained in (5), cell is resuspended, and by be resuspended mixed liquor be coated in received containing card mycin and rifampin solid medium on.
(7) by the solid medium coated in (6) under dark condition, cultivated 36-48 hours in 28 DEG C.
(8) bacterium colony that culture is obtained in (7) is subjected to thalline PCR identifications and sequencing identification obtains positive transformants bacterial strain.
Performing PCR identification is entered to the primer pair that the recombinational agrobacterium primer 3 after conversion and primer 4 are constituted.Show that the Agrobacterium LBA4404 containing ZmCCT genomic segments shown in sequence 3 (PCR purpose band sizes are about 8.1kb) is named as LBA4404/pCAMBIA3301-ZmCCT by identified.The Agrobacterium control for being transferred to pCAMBIA3301 empty carriers is set simultaneously, the Agrobacterium LBA4404 for being transferred to pCAMBIA3301 empty carriers is named as LBA4404/pCAMBIA3301.
Primer 3：1-20 of sequence 3 in 5'-GAGCTCTTGTTGCGACTTGT-3'(sequence tables)；
Primer 4：The reverse complementary sequence of 8128-8147 of sequence 3 in 5'-GAGCTCGACAAACAGTACAT-3'(sequence tables).
2nd, conversion and identification of the recombinational agrobacterium to plant
By recombinational agrobacterium LBA4404/pCAMBIA3301-ZmCCT (or empty vector control LBA4404/pCAMBIA3301) maize transformation (Zea mays L.) kind Hi II of above-mentioned gained.
With the recombinational agrobacterium soaking corn callus activated in advance, transformed calli is obtained, after conversion, resistance screening is carried out with herbicide, transgenic seedling is obtained, that is, is transferred to pCAMBIA3301-ZmCCT plant and is transferred to the plant of pCAMBIA3301 empty carriers.
Further to the transgenic corn plant (T of above-mentioned acquisition0Generation) enter performing PCR identification, the positive strains of screening PCR.Extract the genomic DNA of transgenic corn plant, it is used as template, enter performing PCR identification to the plant for being transferred to pCAMBIA3301-ZmCCT, using ZmCCT genomic segments shown in sequence 3 and pCAMBIA3301 carriers its own sequence as target gene, performing PCR amplification is entered with primer pair (LBCCT F/R).Receptor parent corn (Zea mays L.) kind Hi II of non-transgenosis is set simultaneously as control.
LBCCT FP：5 '-TAGCTAGCTCCACCACAGCA-3 ' (7693-7712 of sequence 3)；
LBCCT RP：5 '-TGTGGAATTGTGAGCGGATA-3 ' (sequence pair is answered and carries sequence on pCAMBIA3301 carriers).
The result (Fig. 1) for entering performing PCR identification to the plant for being transferred to pCAMBIA3301-ZmCCT shows that it is the positive to amplify the transgenic corns of expected size purpose band (518bp), wherein 5 T obtained0Y3-1, Y3-14, Y3-18, Y3-23 and Y3-25 are designated as transgenic positive plant.
3rd, the Disease Resistance Identification of ZmCCT gene corns is turned
(1) experimental method
5 T that step 2 is obtained0In generation, turns ZmCCT gene plant Y3-1, Y3-14, Y3-18, Y3-23 and Y3-25, and 5 transgenosis T are obtained after carrying out selfing1For colony, by T1Planted for colony in Bei Jingshang village experimental plot, each transgenosis T1137 plants are planted for colony, for identifying resistance trait of each individual plant to Fusarium graminearum stem rot, and combines the analysis of genotype to identify the function of ZmCCT genes.Experiment is repeated 3 times, results averaged.Concrete operations are as follows：
1st, genotyping and ZmCCT gene expression amounts are determined：
(1) genotyping
Because the transgenic positive plant of non-homozygosis can separate in offspring, i.e., transgenic positive and negative plant occur in progeny population, so can be 5 T above0In generation, turns ZmCCT gene plant progeny populations and is divided into two kinds of genotype, i.e., positive (P) and negative (N).
The method for taking pcr gene parting, 5 T above are identified with the special pair of primers of pCAMBIA3301-ZmCCT carriers (LBCCT F/R, sequence is ibid) PCR0In generation, turns the genotype of ZmCCT gene plant progeny populations, can expand the individual of band (518bp) individual (P) for transgenic positive, it is impossible to expand the individual of respective strap individual (N) for transgene negative.
(2) ZmCCT gene expression amounts are determined
The plant population (P) of ZmCCT gene masculines is turned with step (1) respectively and turns the plant population (N) of ZmCCT gene negatives for experiment material.Extract the total serum IgE of each experiment material.RNA, which is extracted, uses TIANGEN RNAprep pure plant total RNA extraction reagent boxes, the same specification of extracting method.
Using reverse transcription reagent box (Fermentas), by RNA reverse transcriptions into cDNA, be stored in -80 DEG C it is standby.
Using kit SYBR Premix EX Taq Kit (precious bioengineering), qRT-PCR is carried out according to kit specification, the expression quantity of ZmCCT genes is detected.
Forward primer：5 '-ATGAGAACGACGACCAGCCT-3 ' (5455-5474 of sequence 3)；
Reverse primer：5 '-GACGACTGATCTACCGGCAT-3 ' (reverse complementary sequence of 7418-7537 of sequence 3).(note：Across the primer of introne, using cDNA as masterplate)
Reaction system：20ul.
Response procedures：Step 1：94℃for 3min；
Step 2：94℃for 30s；
Step 3：60℃for 30s；
Step 4：72℃for 30s；
Step 5：Go to step 2for 28or 32cycles；(period to repeat here)
Step 6：72℃for 10min；
Step 7：5℃forever.
Reference gene uses GAPDH.The amplimer of reference gene is 5 '-ATCAACGGCTTCGGAAGGAT-3 ' and 5 '-CCGTGGACGGTGTCGTACTT-3 '
Identify that the obtained corn positive plant for being transferred to pCAMBIA3301 empty carriers, as empty vector control (CK), sets corn (Zea mays L.) kind Hi II of the receptor parent of non-transgenosis to be used as parent control (WT) in setting steps two simultaneously.
2nd, to the analysis of the resistance trait of Fusarium graminearum stem rot：
Take soil to bury the method for hindering root (referring to " the health cares cultivation step such as Song Zuoheng is to corn stalk rot disease control effect research; the phases of Liaoning agricultural sciences .1993 the 05th "), the transgenic progeny colony plant of non-homozygosis is carried out with Fusarium graminearum to connect bacterium processing, specific steps：After plant fusulus, soil apart from plant 5-10 centimeters cut-off parts capillary root straight down, is being cast aside, numerous Fusarium graminearum Fusarium graminearum, and moisturizing of watering are expanded in 60-80 grams of embedment with corn kernel.
Further, the plant population (P) for turning ZmCCT gene masculines to being accredited as through step 1 and the plant population (N) for turning ZmCCT gene negatives carry out disease resistant plant ration statisticses respectively.It is specific as follows：After Fusarium graminearum artificial infection about 45 days, plant is split into stem processing, basal part of stem and root are hollow and have the susceptible strain of regarding as of putrefactive phenomenon, basal part of stem and root are complete and normally regard as disease-resistant strain (Fig. 2).The ratio of disease-resistant plant in the plant population (P) for turning ZmCCT gene masculines and the plant population (N) for turning ZmCCT gene negatives is calculated respectively.
Identify that the obtained corn positive plant for being transferred to pCAMBIA3301 empty carriers, as empty vector control (CK), sets corn (Zea mays L.) kind Hi II of the receptor parent of non-transgenosis to be used as parent control (WT) in setting steps two simultaneously.
In addition, the T for transgenic positive being accredited as through said gene type analysis1For plant selfing, T is obtained2For colony；The T of transgenic positive will be accredited as through said gene type analysis2For plant selfing, T is obtained3For colony.To T2For colony and T3Genotyping and disease resistance character analysis are also carried out using method as above for colony.
(2) experimental result
1st, 5 T0In generation, turns ZmCCT gene plant offspring ZmCCT gene expression amounts measure
As a result as shown in figure 3, it can be seen that 5 T0In generation, turns in ZmCCT gene plant progeny populations, and the ZmCCT gene expression amounts of positive transgenic plant (P) are significantly larger than negative transgenic line (N).And as ZmCCT gene expression amounts in two plants of parent control (WT) and empty vector control (CK) compared with negative transgenic line (N), basically identical, no difference of science of statistics.
2nd, 5 T0In generation, turns ZmCCT gene plant offspring's resistance screenings
As a result show：In T1For (Fig. 4) on population level, the disease-resistant plant ratio of the positives plant of Y3-1, Y3-23 and Y3-25 (P) has the raising of conspicuousness relative to negative plant (N), the more negative plant of disease-resistant plant ratio (N) of the positives plant of wherein Y3-1 (P) is improved up to 33%, the more negative plant of disease-resistant plant ratio (N) that the more negative plant of disease-resistant plant ratio (N) of the positives plant of Y3-23 (P) is improved up to the positives plant of 13%, Y3-25 (P) is improved up to 10%.
In T2For (Fig. 5) on population level, Y3-1, Y3-18, the disease-resistant plant ratio of the positives plant of Y3-23 and Y3-25 (P) has the raising of conspicuousness relative to negative plant (N), the more negative plant of disease-resistant plant ratio (N) of the positives plant of wherein Y3-1 (P) is improved up to 35%, the more negative plant of disease-resistant plant ratio (N) of the positives plant of Y3-18 (P) is improved up to 17%, the more negative plant of disease-resistant plant ratio (N) of the positives plant of Y3-23 (P) is improved up to 25%, the more negative plant of disease-resistant plant ratio (N) of the positives plant of Y3-25 (P) is improved up to 9%.
Choose T2For the Y3-23 selfings of transgenic positive, T is obtained3For colony.As a result show, in T3For (Fig. 5) on population level, the disease-resistant plant ratio of the positives plant of Y3-23 (P) still has the raising of conspicuousness relative to negative plant (N), and the more negative plant of disease-resistant plant ratio (N) of the positives plant of Y3-23 (P) is improved up to 7%.
3rd, parent control and empty vector control resistance screening
5 T compared to more than0The disease-resistant plant ratio that generation turns in the qualification result (Fig. 4 and Fig. 5) of ZmCCT gene plant offsprings, corn (Zea mays L.) kind Hi II (WT) of non-transgenosis is only 5%, 5 T far below more than0In generation, turns the disease-resistant plant ratio in ZmCCT gene plant offsprings.The experimental result of empty vector control and parent control are basically identical, no difference of science of statistics.
In summary 1-3 result, it is seen that relative to the parent control and empty vector control of non-transgenosis, 5 T of the above0The disease-resistant plant ratio that generation turns in ZmCCT gene plant offsprings is greatly improved, and disease-resistant plant ratio is far above negative plant (N) in the positive plant (P) in offspring, while the expression quantity of ZmCCT genes is also far above negative plant (N) in positive plant (P).
4th, resistance to low nitrogen and the high-salt stress analysis of ZmCCT gene corns is turned
(1) ZmCCT transgenic corns seedling stage Analysing Root Characters and dry weight measurement
(1) experimental method
The T of transgenic positive will be accredited as through said gene type analysis4In generation, turns ZmCCT gene corns seed in the controlled environment chamber under water planting planting conditions, and seedling stage carries out low nitrogen and high-salt stress, and Stress treatment is measured after one week to Analysing Root Characters, plant height, SPAD values etc., dries and measures dry weight to constant weight.Statistical analysis is carried out with t testing methods.
(2) experimental procedure
By T4In generation, turns ZmCCT gene corns seed with 10% (v/v) H2O2Sterilization 30 minutes, after deionized water rinsing, saturation CaSO4Immersion 6 hours, lucifuge grows 2 days in the controlled environment chamber, when germination general 1-2cm, select the consistent seed of growing way, seedling is rolled up with filter paper, cylinder paper roll is rolled into since side, moves into breaker and grows to the heart stage of 1 leaf 1, the consistent seedling of growing way is selected, 1L nutrient solution (Hoagland ' s nutrient solutions are moved into：0.75mmolL-1K2SO4、0.1mmolL-1KCl、0.25mmolL-1KH2PO4、0.65mmolL-1MgSO4、0.13mmolL-1EDTA-Fe、1.0μmolL-1MnSO4、1.0μmolL-1ZnSO4、0.1μmolL-1CuSO4、0.005μmolL-1(NH4)6Mo7O24) culture tank in, per 4 plants of tank.Growth conditions control is 28 DEG C/22 DEG C, 16/8h illumination/dark, day circulation.16h illumination periods pharosage is 250-300 μm of olm-2s-1.Seedling is moved into after culture tank, first with 1/2 (c/c) nutrient solution preculture two days, then is finished pancebrin culture to the heart stage of 3 leaf 1, low nitrogen (0.04mmolL is carried out respectively-1NO3 -), high salt (50mmolL-1NaCl) Stress treatment, is further cultured for 7 days.N element is by Ca (NO3)2There is provided, in Low nitrogen stress processing, Ca2+With CaCl2Form adds to 4mmolL-1NO3 -Level.PH value is adjusted to 6.0 with 1mmol/L NaOH.Electric air pump provides oxygen, and nutrient solution is changed once for every two days, and each processing carries out three repetitions.
(3) measurement index
Maize seedling of the Stress treatment after 7 days, with deionized water rinsing, is divided into two parts of overground part and underground part, and root scanned picture is measured with Image J softwares, and the character of measure has：Total root length (TRL, Total root length), main radicle length (PRL, total primary radicle length), backbone root length (MRL, main root length) and lateral root length (LRL, lateral root length)；70 DEG C/48h dries overground part and underground part to constant weight, weighs overground part dry weight (SDW, shoot dry weight) and underground part dry weight (RDW, root dry weight)；With ruler measurement plant height (PH, Plant height).Each character surveys 10 young plants, averages.
(4) experimental result
T quiz statistics measurement results show, T4In generation, turns ZmCCT gene corns plant compared with negative plant, and total root length, lateral root length and plant height in normal control environment (Mock) have significant difference, and backbone root length and underground part dry weight have pole significant difference；After Low nitrogen stress processing (LNS), total root length, lateral root length, underground part dry weight, overground part dry weight and plant height have pole significant difference, and backbone root length has significant difference, T4In generation, turns total root length, lateral root length, underground part dry weight, overground part dry weight, plant height and the backbone root length of ZmCCT gene corn plant apparently higher than negative plant；After high salt treatment (HSS), all characters of measurement, transgenic positive plant has significant difference, T with negative plant4In generation, turns total root length, backbone root length, main radicle length, lateral root length, underground part dry weight, overground part dry weight and the plant height of ZmCCT gene corn plant obviously higher than negative plant；T4In generation, turns total root length, backbone root length, lateral root length, underground part dry weight, overground part dry weight and the plant height of ZmCCT gene corn plant obviously higher than negative plant.T4In generation, turns ZmCCT gene corns plant and very strong growth adaptability (Fig. 6 and table 2) is shown under low nitrogen and condition of salt stress.
Table 2, T4In generation, turns the low nitrogen high-salt stress growth indexes statistical form of ZmCCT gene corns
Due to T4In generation, turns ZmCCT gene corns plant and is differed greatly with negative plant growth potential itself, carries out the multifactor analysis of variance, analysis result is as shown in table 3.Total root length, backbone root length and underground part dry weight have significant difference under the conditions of Low nitrogen stress, and total root is long, and main radicle is long, and lateral root length and plant height have significant difference under condition of salt stress.
Table 3, T4In generation, turns the low nitrogen high-salt stress growth indexes multifactor analysis of variance table of ZmCCT gene corns
To sum up result, turns ZmCCT gene masculine plant seedling stages under low nitrogen and condition of salt stress, root growth is more flourishing, and with very strong adaptability, ZmCCT has the effect (Fig. 7, Fig. 8) for improving Maize at Seedling Stage low nitrogen resisting and high-salt stress.
(2) complementation test transgenic line seedling stage low nitrogen and high-salt stress condition expression study
(1) experiment content
Take the T of the heart stage of three leaf one4In generation, turns ZmCCT gene corns plant and negative plant, the root and leaf of different time points (0,3,6 and 9 hours) carry out expression analysis after 50mmol NaCl high-salt stress process and 0.04mmol Low nitrogen stress, each sample each time point takes 5 plants to mix carry out expression study at random, each sample sets 3 repetitions, and experiment is repeated 3 times.
(2) experimental implementation process
RNA, which is extracted, uses TIANGEN RNAprep pure plant total RNA extraction reagent boxes, the same specification of extracting method.The TransScript First-Strand cDNA SuperMix (AT301-03) of RNA reverse transcriptions Quan Shi King Companies, the same specification of operating method.The cDNA DEPC-ddH of synthesis2O is diluted to debita spissitudo, and product is standby in -20 DEG C of preservations.
The SYBR Premix Ex Taq of qRT-PCR TaKaRa companiesTMII (RR820A), is detected with RoterGene6000.Using comparing CT values method (2- ΔΔ Ct) carry out gene expression relative quantification (Livak and Schmittgen2001).It is as shown in table 4 that reference gene selects GAPDH and expression quantity to determine primer.
QRT-PCR programs：95 DEG C, 5min；95 DEG C, 10S, 60 DEG C, 20s, 40cycles；mealting curve analysis.
QRT-PCR reaction systems (20 μ l)：SYBR Primix Ex TaqTM II(2×)10μl、Forward Primer(10μM)0.4μl、Reverse Primer(10μM)0.4μl、cDNA 2μl、ddH2O 7.2μl。
Table 4, reference gene select GAPDH and expression quantity to determine primer
(3) experimental result
T4In generation, turns ZmCCT gene corns plant under conditions of low nitrogen and salt stress, the phenomenon (Fig. 9) of induced expression, T occurs4In generation, turns ZmCCT gene corns plant (TL-23 (+)) root and overground part expression quantity under conditions of low nitrogen and high-salt stress and raises, and reaches within 3 hours peak value, and expression quantity is approximately 0 hour T of processing4In generation, turns 5.86 times and 9.71 times of ZmCCT gene corn plant, the phenomenon declined then occurs, negative plant is then without such phenomenon.
ZmABA2 and ZmMPK5, which are reported, participates in the anti-oxidative defense reaction of corn ABA inductions.The rise of ZmABA2 and ZmMPK5 expression quantity can strengthen the ability of the abiotic stress such as the salt stress-resistant, drought stress and oxidative stress of corn, adaptability (Ma, Ni et al.2015) of the enhancing corn to environment.
Under conditions of low nitrogen and high-salt stress, ZmABA2 is in T4In generation, turns ZmCCT gene corns plant (TL-23 (+)) root and overground part expression quantity is raised (Figure 10), except Low nitrogen stress root, reach peak value within 3 hours, expression quantity is about 2-4 times when Stress treatment starts, then there is the phenomenon declined, there is the phenomenon of induced expression in negative plant (TL-23 (-)) root of salt stress, the root of Low nitrogen stress and overground part, but expression quantity ascensional range is much smaller than and turns ZmCCT gene masculine plant.
Under conditions of low nitrogen and high-salt stress, ZmMPK5 is in T4In generation, turns ZmCCT gene masculines plant (TL-23 (+)) root and overground part expression quantity is raised (Figure 11), and three hours expression quantity of Stress treatment are higher than negative 4-6 times of plant (TL-23 (-)).
To sum up experimental result, turns ZmCCT gene corns plant after low nitrogen and high-salt stress process, the rise of ZmCCT expression quantity, reaches peak value within 3 hours.After low nitrogen and high-salt stress process, to improving expression quantity amplification of the related gene ZmABA2 and ZmMPK5 of corn resistance in ZmCCT gene masculine plant are turned also far above feminine gender plant.Illustrate that ZmCCT genes have the function of improving Maize at Seedling Stage low nitrogen resisting and high-salt stress.
Claims (10)
1. protein, is following protein a) or b) or c)：
A) amino acid sequence is the protein shown in sequence 1；
B) fused protein obtained in N-terminal and/or C-terminal the connection label of the protein shown in sequence 1；
C) by the amino acid sequence shown in sequence 1 by one or several amino acid residues substitution and/or missing and/or
Add the obtained protein with identical function.
2. the biomaterial with the albumen qualitative correlation described in claim 1, is following A 1) to A12) in any
Kind：
A1) the nucleic acid molecules of the protein described in coding claim 1；
A2 A1) is contained) expression cassettes of the nucleic acid molecules；
A3 A1) is contained) recombinant vectors of the nucleic acid molecules；
A4 A2) is contained) recombinant vector of the expression cassette；
A5 A1) is contained) recombinant microorganisms of the nucleic acid molecules；
A6 A2) is contained) recombinant microorganism of the expression cassette；
A7 A3) is contained) recombinant microorganism of the recombinant vector；
A8 A4) is contained) recombinant microorganism of the recombinant vector；
A9 A1) is contained) the transgenic plant cells systems of the nucleic acid molecules；
A10 A2) is contained) the transgenic plant cells system of the expression cassette；
A11 A3) is contained) the transgenic plant cells system of the recombinant vector；
A12 A4) is contained) the transgenic plant cells system of the recombinant vector.
3. relevant biological material according to claim 2, it is characterised in that：A1) nucleic acid molecules is such as
It is lower 1) or 2) or 3) shown in gene：
1) its coded sequence is the genomic DNA molecule shown in the cDNA molecules or sequence 3 shown in sequence 2；
2) there is 75% or more than 75% homogeneity, and coding claim 1 institute with the nucleotide sequence of 1) restriction
The cDNA molecules or genomic DNA molecule for the protein stated；
3) under strict conditions with 1) or 2) nucleotide sequence hybridization limited, and described in coding claim 1
The cDNA molecules or genomic DNA molecule of protein.
4. the relevant biological material described in protein or Claims 2 or 3 described in claim 1 following (1)-
At least one of (6) application in：
(1) resistance of the regulation and control plant to Fusarium graminearum stem rot；
(2) stress resistance of plant is regulated and controled；
(3) the regulation and control total root length of plant and/or backbone root length and/or main radicle length and/or lateral root length and/or underground part dry weight
And/or overground part dry weight and/or plant height；
(4) regulation and control and the expression of plant resistance to environment stress related gene；
(5) genetically modified plants of anti-Fusarium graminearum stem rot are cultivated；
(6) genetically modified plants that resistance is improved are cultivated.
5. application according to claim 4, it is characterised in that：
It is described to be regulated to improve；
The resistance is resistance to low nitrogen and/or salt-resistance.
6. a kind of method for cultivating the genetically modified plants that resistance is improved, including by the protein described in claim 1
Encoding gene is imported in recipient plant, the step of obtaining genetically modified plants；The resistance of the genetically modified plants is higher than institute
State recipient plant.
7. method according to claim 6, it is characterised in that：
The resistance is resistance to low nitrogen and/or salt-resistance；
And/or, the resistance of the genetically modified plants is embodied in (D1)-(D7) as follows higher than the recipient plant
It is any：
(D1) total root length of genetically modified plants is higher than the recipient plant；
(D2) the backbone root length of genetically modified plants is higher than the recipient plant；
(D3) the main radicle length of genetically modified plants is higher than the recipient plant；
(D4) the lateral root length of genetically modified plants is higher than the recipient plant；
(D5) the underground part dry weight of genetically modified plants is higher than the recipient plant；
(D6) the overground part dry weight of genetically modified plants is higher than the recipient plant；
(D7) plant height of genetically modified plants is higher than the recipient plant；
(D8) expression of the ZmABA2 and ZmMPK5 genes of genetically modified plants is higher than the recipient plant.
8. a kind of method for the genetically modified plants for cultivating anti-Fusarium graminearum stem rot, including by described in claim 1
The encoding gene of protein is imported in recipient plant, the step of obtaining genetically modified plants；The genetically modified plants are to cereal
The resistance of corn stalk rot caused by Fusarium is higher than the recipient plant.
9. the method according to claim 6 or 8, it is characterised in that：
The nucleotide sequence of the encoding gene of the protein is 1-717 nucleic acid molecules of sequence 2.
10. according to any described method in claim 6-9, it is characterised in that：The recipient plant is unifacial leaf
Plant or dicotyledon.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111434678A (en) * | 2019-01-10 | 2020-07-21 | 中国农业科学院作物科学研究所 | Plant dehydration response element encoding protein and application of encoding gene thereof in low nitrogen stress resistance |
| CN112795552A (en) * | 2021-03-10 | 2021-05-14 | 河南大学 | Application of Zm0001d024568 gene and encoding protein thereof in drought stress resistance of corn |
| CN116606882A (en) * | 2023-05-25 | 2023-08-18 | 浙江大学海南研究院 | Application of OsbZIP79 gene in enhancing nitrogen deficiency stress resistance of rice |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014055477A2 (en) * | 2012-10-03 | 2014-04-10 | Pioneer Hi-Bred International, Inc. | Genes controlling photoperiod sensitivity in maize and sorghum and uses thereof |
| CN104558128B (en) * | 2013-10-14 | 2017-09-22 | 中国农业大学 | The albumen related to anti-Fusarium graminearum stem rot and its encoding gene and application |
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2016
- 2016-04-29 CN CN201610282306.XA patent/CN107325161B/en active Active
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111434678A (en) * | 2019-01-10 | 2020-07-21 | 中国农业科学院作物科学研究所 | Plant dehydration response element encoding protein and application of encoding gene thereof in low nitrogen stress resistance |
| CN111434678B (en) * | 2019-01-10 | 2022-03-15 | 中国农业科学院作物科学研究所 | Plant dehydration response element encoding protein and application of encoding gene thereof in low nitrogen stress resistance |
| CN112795552A (en) * | 2021-03-10 | 2021-05-14 | 河南大学 | Application of Zm0001d024568 gene and encoding protein thereof in drought stress resistance of corn |
| CN116606882A (en) * | 2023-05-25 | 2023-08-18 | 浙江大学海南研究院 | Application of OsbZIP79 gene in enhancing nitrogen deficiency stress resistance of rice |
| CN116606882B (en) * | 2023-05-25 | 2024-01-23 | 浙江大学海南研究院 | Application of OsbZIP79 gene in enhancing nitrogen deficiency stress resistance of rice |
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