CN104650201A - ZmAGO1a protein as well as coding gene and application thereof - Google Patents
ZmAGO1a protein as well as coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses ZmAGO1a protein as well as a coding gene and application thereof. The protein disclosed by the invention is represented as follows: (1), protein represented by SEQ ID No.2; and (2), protein obtained by substitution and/or deletion and/or addition of an amino acid sequence represented by SEQ ID No.2 through one or more amino acid residues and having same functions. The ZmAGO1a gene disclosed by the invention is capable of obviously influencing growth and development of plants, such as fecundity, a maturation period (early maturation), florescence, the number of leaves, the sizes of the leaves, the shapes of the leaves and the colors of the leaves; most of the characteristics belong to important agronomic characteristics and are related to the yield, growth period and plant type of plants; and the ZmAGO1a protein disclosed by the invention has important application value in cultivation of high-yield, stress-tolerant and high-efficiency plants (such as corns).
Description
Technical field
The present invention relates to a kind of ZmAGO1a albumen and encoding gene thereof and application, belong to biological technical field.
Background technology
AGO albumen induces the core parts of silencing complex as RNA, in RNA silence, play keying action.Prokaryotic organism AGO albumen is the key ingredient in the systems of defense such as defence foreign genetic element invasion, and archeobacteria has similar function in RNAi system.Eukaryote AGO albumen plays a significant role at Gene expression and regulation, wide participation regulate several biological processes, comprises (Wei KF, et al. such as growing sequential, cytodifferentiation, cell proliferation, necrocytosis, Metabolism control, immunity and transposon silencing, 2012, Pumplin N, et al., 2013), in addition, AGO albumen transcribing, variable sheer, even the aspect such as DNA reparation plays a significant role (Meister G, 2013).AGO is a kind of Multidomain, larger albumen (about 90-100kDa).Eukaryote AGO albumen is made up of (Tolia NH, et al., 2007) the structural domain that N terminal domains, PAZ structural domain, MID structural domain and PIWI structural domain four is basic usually.Bohmert etc. are when a series of ago1 mutant of research Arabidopis thaliana, and because its mutant phenotype is the spitting image of little cuttlefish, therefore by this type of mutant called after Argonaute albumen, the gene corresponding with it is called AGO1 (Bohmert K, et al., 1998).(the Yigit E such as Yigit, et al., 2006) according to AGO protein system evolutionary relationship and they are in conjunction with the ability of sRNA, be divided three classes from the AGO protein family member representing species in plant, animal and fungi: Ago subtribe, PIWI (P-elementinducedwimpytestis) subtribe and WAGO subtribe (Worm-specificargonaute).AGO subtribe member protein is similar to Arabidopis thaliana AtAGO1, and its member in conjunction with miRNA and siRNA, can instruct the target site that RISC target is suitable.PIWI subtribe member protein is then similar to fruit bat DmPIWI, and PIWI subtribe member can combine with piRNA (PIWI-interacting RNA).WAGO member exists only in worm, can in conjunction with secondary siRNA.
AGO1 mainly participates in the cutting of miRNA target mRNAs, also relates to the maintenance etc. of genetically modified PTGS (SchuckJ, et al., 2013) and chromatin Structure.Initial ago1 mutant performance organ polarity defect, proves that Arabidopis thaliana AGO1 participates in miRNA path (A.KC, et al., 2004).In ago1 mutant, miRNA accumulates decline, and contrary miRNA target gene accumulation increases, and shows that the integrity of AGO1 to maintenance miRNA path is required (Vaucheret H, et al., 2004).AGO1 also participates in the PTGS defensive raction of siRNA mediation, hinders from virus and genetically modified exogenous RNA (Martinez de Alba AE, et al., 2011).
Report two kinds of AGO1 gene TaAGO1a-1b (Liu T-l, 2011, Meng F, et al., 2013) in wheat, correlative study has been done to genomic structure and expression feature etc., but still has lacked biological evidences.Same on corn, corn AGO1 gene identification still lacks experiment compared with system and analytical evidence, and research still concentrates in model plant Arabidopis thaliana and paddy rice.Qian Yexiong etc. utilize bioinformatics method to predict 18 ZmAGO genes, wherein ZmAGO1 member totally five, but still lack evidence on a molecular scale.
Corn is China's sown area and the first crop of ultimate production.Current China corn year sown area is up to 5.3 hundred million mu.Extensively adopt cross-fertilize seed owing to producing, planting in industry and have great Development volue, is the crop planting industry business development Maximum Value in China food crop.Cultivate high yield, resistance toly inverse with efficient corn variety, there is important production and plant industry development significance and value.
Summary of the invention
Technical problem to be solved by this invention cultivates high yield, resistance to inverse with efficient plant (as corn).
In order to solve above technical problem, the invention provides a kind of albumen, shown in following (1) or (2):
(1) albumen shown in SEQ ID No.2;
(2) by the aminoacid sequence shown in SEQ ID No.2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and the identical protein of function.
In order to solve above technical problem, present invention also offers the encoding gene of described albumen.
In above-mentioned encoding gene, described encoding gene is following middle at least one:
1) DNA molecular shown in SEQ ID No.1;
2) DNA molecular shown in the 150th to the 3458th or cDNA molecule in SEQ ID No.1;
3) under strict conditions with 1) or 2) DNA molecular that limits or cDNA molecular hybridization and the DNA molecular of code for said proteins or cDNA molecule;
4) with 1) or 2) or 3) DNA molecular that limits or cDNA molecule have the identity of more than 90% and the DNA molecular of code for said proteins or cDNA molecule.
In order to solve above technical problem, present invention also offers the recombinant vectors containing above-mentioned arbitrary described encoding gene, expression cassette, transgenic cell line or recombinant bacterium.
In order to solve above technical problem, present invention also offers a kind of method of Promoting plant growth and/or growth, comprising the steps: above-mentioned arbitrary described encoding gene to import to set out in plant, obtaining transgenic plant; Compared with the plant that sets out, the growth of transgenic plant and/or developmental process are accelerated or are grown and/or developmental level raising.
In aforesaid method, described growth and/or developmental process are accelerated to be presented as flowering period and/or ripening stage in advance;
Described growth and/or developmental level raising are presented as that biological yield improves;
Described biological yield raising is embodied as number of blade increase, leaf long growth, the loose increase of leaf, bear pods number and/or seed number raising.
In above-mentioned arbitrary described method, described encoding gene is imported by recombinant expression vector, and described recombinant expression vector is that the multiple clone site described encoding gene being inserted the carrier pCPB that sets out obtains;
Described recombinant expression vector tool is for inserting the BamHI site of pCPB by the sequence in SEQ ID No.1 shown in the 123rd to the 3610th Nucleotide, all the other sequences of pCPB remain unchanged and obtain;
Described plant is specially corn or Arabidopis thaliana;
Described corn or Arabidopis thaliana are specially the corn or Arabidopis thaliana with growth and/or developmental defect;
Described Arabidopis thaliana is specially Arabidopis thaliana ago1-27 mutant.
In order to solve above technical problem, present invention also offers above-mentioned albumen or above-mentioned arbitrary described encoding gene in Promoting plant growth and/or developmental application;
Described plant is specially corn or Arabidopis thaliana;
Described corn or Arabidopis thaliana are specially the corn or Arabidopis thaliana with growth and/or developmental defect;
Described Arabidopis thaliana is specially Arabidopis thaliana ago1-27 mutant;
Described Promoting plant growth and/or growth are embodied as growth and/or the developmental process quickening of plant or grow and/or developmental level raising;
Described growth and/or developmental process quickening are embodied as flowering period and/or the ripening stage shifts to an earlier date;
Described growth and/or developmental level raising are embodied as biological yield and improve;
Described biological yield raising is embodied as number of blade increase, leaf long growth, the loose increase of leaf, bear pods number and/or seed number raising.
ZmAGO1a gene provided by the invention can the growth of remarkably influenced plant and growth: comprise fecundity, ripening stage (precocity), flowering period, the number of blade, leaf blade size, blade shape, leaf color etc., these proterties mostly belong to important economical character, relevant with plant type to the output of plant, breeding time, cultivation high yield, resistance to inverse with efficient plant (as corn) in there is major application be worth.
Accompanying drawing explanation
Fig. 1 is plant over-express vector pCPB-35S::ZmAGO1a structural representation.
Fig. 2 is the detection of Bar gene and ZmAGO1a gene in transgenic arabidopsis.
Fig. 3 is Arabidopis thaliana ago1-27 mutant strain (ago1-27), wild-type Columbia ecotype Arabidopis thaliana (wt), T
3for seedling stage of positive Arabidopis thaliana ago1-27 mutant transfer-gen plant pure lines (line16, line19 and line21), flowering period and ripening stage form.
Fig. 4 is that the real-time fluorescence quantitative PCR of ZmAGO1a gene and AtAGO1 gene in transgenic arabidopsis detects.
Fig. 5 is the tissue specificity analysis of ZmAGO1a genetic expression in corn Different Organs.
Fig. 6 is the differential expression response characteristic of ZmAGO1a gene under 5 kinds of abiotic stress conditions in Maize leaf.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Corn inbred line tucks in 478 (Zea mays ssp L.) at document " Xie C., Zhang S., Li M., Li X., Hao Z, Bai L, Zhang D.i, Liang Y. (2007) Inferring genome ancestry andestimating molecular relatedness among 187Chinese maize inbred lines.J.Genet. & Genomics 34 (8): 738-748 " in be disclosed, the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science, this selfing is domestic corn breeding conventional parental inbred line upper with production.
PCPB plasmid is at document " Nan Wu; Piwu Wang; Dan Li; Liqiang Dai, ChengzhongZheng, Shi Lu; Yuan Cai; Zhuo Zhang, Jing Qu, Haifeng Xia Yi Chuan.Functionof chalcone reductase gene CHR1in soybean.2014; 36 (7): 707-12. " be disclosed in, the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science.
Arabidopis thaliana ago1-27 mutant is at document " Morel JB:Fertile Hypomorphic ARGONAUTE (ago1) Mutants Impaired in Post-Transcriptional Gene Silencing and VirusResistance.The Plant Cell Online 2002, 14:629-639. " in be disclosed, the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science, this mutant has obvious developmental defect, leaf look is deep green, spination blade, compared with Columbia ecotype Arabidopis thaliana, to bloom delay 7 to 12 days, setting percentage only has the 45-60% of Columbia ecotype Arabidopis thaliana, this mutant strain only has AtAGO1 transgenation.
Columbia ecotype Arabidopis thaliana (A.thanliana ecoype Colombia0, Col-0) at document " MorelJB:Fertile Hypomorphic ARGONAUTE (ago1) Mutants Impaired inPost-Transcriptional Gene Silencing and Virus Resistance.The Plant Cell Online2002; be disclosed in 14:629-639., the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science.
Corn B73 self-mating system (Zea mays ssp L.) is at document " Xie C.; Zhang S.; Li M.; Li X.; Hao Z; Bai L, Zhang D.i, Liang Y. (2007) Inferring genome ancestry andestimating molecular relatedness among 187Chinese maize inbred lines.J.Genet. & Genomics 34 (8): 738-748 " in be disclosed, the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science.
The preparation of embodiment 1, plant over-express vector pCPB-35S::ZmAGO1a
One, corn inbred line tucks in the full length cDNA sequence of the ZmAGO1a gene of 478 as shown in SEQ ID No.1, and in SEQ ID No.1, the 150th to the 3458th is the coding gene sequence of ZmAGO1a albumen.The aminoacid sequence of ZmAGO1a albumen is as shown in SEQ ID No.2.
Two, the structure of pCPB-35S::ZmAGO1a
Sequence in SEQ ID No.1 shown in the 123rd to the 3610th Nucleotide is inserted the BamHI site of pCPB, all the other sequences of pCPB remain unchanged, obtain recombinant plasmid, by its called after pCPB-35S::ZmAGO1a, order-checking is sent by pCPB-35S::ZmAGO1a, result is correct, and sequencing result to show in SEQ ID No.1 that the 123rd to the 3610th sequence shown in Nucleotide correctly inserts the BamHI site of pCPB, can express the ZmAGO1a albumen shown in SEQ ID No.2.
As shown in Figure 1, plant over-express vector pCPB-35S::ZmAGO1a take 35S as promotor process LAN ZmAGO1a to plant over-express vector pCPB-35S::ZmAGO1a structural representation, and screening-gene is Bar gene.
The qualification that has complementary functions of embodiment 2, ZmAGO1a gene pairs Arabidopis thaliana ago1 mutant
One, by after the seed disinfection sterilizing of Columbia ecotype Arabidopis thaliana and Arabidopis thaliana ago1-27 mutant, be placed in 1/2MS (Murashige andSkoog) substratum respectively, in short day (8h illumination/16h is dark), cultivate 10 days under 23 ± 1 DEG C of conditions, then be transferred in soil, short day (8h illumination/16h is dark), 23 ± 1 DEG C of CMC model.
Two, plant over-express vector pCPB-35S::ZmAGO1a embodiment 1 prepared is transformed into Agrobacterium GV3301, obtain recombinational agrobacterium, recombinational agrobacterium is adopted inflorescence soaking method (Clough SJ, et al., 1998) Colombia's Arabidopsis thaliana ecotype and the Arabidopis thaliana ago1-27 mutant of step one cultivation is infected, obtain Columbia ecotype Arabidopis thaliana transfer-gen plant and Arabidopis thaliana ago1-27 mutant transfer-gen plant respectively, and gather in the crops Columbia ecotype Arabidopis thaliana transfer-gen plant T
0for seed and Arabidopis thaliana ago1-27 mutant transfer-gen plant T
0for seed.
Three, the qualification of positive transgenic Arabidopis thaliana
(1) sterilizing and vernalization
By the Colombia Arabidopsis thaliana ecotype transfer-gen plant T of results
0for seed and Arabidopis thaliana ago1-27 mutant transfer-gen plant T
0be placed in 1.5ml centrifuge tube respectively for seed, with the aqueous ethanolic solution surface sterilization 1min of 1ml volumn concentration 70%, period constantly rocks, and subsequently with aqua sterilisa cleaning twice, then the aqueous sodium hypochlorite solution adding 1.5g/100ml soaks 1min, last ddH
2o cleans 5-8 time.Be placed in 4 DEG C of refrigerators, vernalization 2-3d.
(2) preliminary screening
By the T of step () through sterilizing and vernalization treatment
0cultivate 7-10d for planting seed at Selective agar medium (2.2g/L MS, 50 μ g/mL Glufosinate ammoniums, 8g/L agar, 30g/L sucrose, surplus is water, pH5.8-6.0), grow up to T
1for seedling, screening T
1for positive Columbia ecotype Arabidopis thaliana transfer-gen plant and T
1for positive Arabidopis thaliana ago1-27 mutant transfer-gen plant.Wherein will can grow the T of normal root system and green blade
1be T for seedling preliminary judgement
1for positive Columbia ecotype Arabidopis thaliana transfer-gen plant and T
1for positive Arabidopis thaliana ago1-27 mutant transfer-gen plant, wherein screen the T obtained
1for totally 21 strains of positive Arabidopis thaliana ago1-27 mutant transfer-gen plant.
(3) PCR qualification
1, by the T of step (two) preliminary screening
1for positive Columbia ecotype Arabidopis thaliana transfer-gen plant and T
1after cultivating 2 weeks for positive Arabidopis thaliana ago1-27 mutant transfer-gen plant on Selective agar medium, individual plant gets leaf sample, extract each transfer-gen plant genomic dna.
2, with T
1genomic dna for positive Columbia ecotype Arabidopis thaliana transfer-gen plant is template, with Bar-F and Bar-R for primer, carries out pcr amplification, detects Bar gene; With T
1genomic dna for positive Columbia ecotype Arabidopis thaliana transfer-gen plant is template, with ZmAGO-1-F and ZmAGO-1-R for primer, carries out pcr amplification, detects ZmAGO1a gene;
Template is replaced with T
1for the genomic dna of positive Arabidopis thaliana ago1-27 mutant transfer-gen plant, all the other conditions remain unchanged, and carry out above-mentioned experiment, detect its Bar gene and ZmAGO1a gene;
Template is replaced with sterilized water, all the other conditions remain unchanged simultaneously, carry out above-mentioned experiment, detect its Bar gene and ZmAGO1a gene, as negative control.
Template is replaced with the genomic dna of Columbia ecotype Arabidopis thaliana, all the other conditions remain unchanged simultaneously, carry out above-mentioned experiment, detect its Bar gene and ZmAGO1a gene, as blank.
Bar-F：5’-CCATCGTCAACCACTACATCG-3’(SEQ ID No.3)
Bar-R：5’-AGCTGCCAGAAACCCACGT-3’(SEQ ID No.4)
ZmAGO-1-F：5’-ATTCAGCACACCACTTTACCTTG-3’(SEQ ID No.5)
ZmAGO-1-R：5’-CCTTGGAGGAGGCAGAACAC-3’(SEQ ID No.6)
The detected result of Arabidopis thaliana ago1-27 mutant transfer-gen plant as shown in Figure 2.
In Fig. 2, A is Bar gene test result; B is ZmAGO1a gene test result.M is DNA marker (GeneRuler
tM100bp ladder marker); CK
-for negative control; CK is blank; 1-21 is the T of step (two) primary election screening
1for positive Arabidopis thaliana ago1-27 mutant transfer-gen plant.
Fig. 2 shows, detects through PCR, except the sample that label is 18 and 20, and the T of step (two) primary election screening
1for in positive remaining 19 sample of Arabidopis thaliana ago1-27 mutant transfer-gen plant, all there is Bar gene and ZmAGO1a gene object band, be defined as positive transgenic plant further, 5 wherein are recovered completely the T of (recovering completely is mainly refer to that blade reverts to subcircular completely by spination, and leaf look reverts to normal green leaf look by deep green)
1for positive Arabidopis thaliana ago1-27 mutant transfer-gen plant, continue breeding screening pure lines, filter out 3 pure lines and test for follow-up phenotype.
(4) expression of ELISA test strip Bar albumen
The T that selecting step (three) PCR detects
1for positive Columbia ecotype Arabidopis thaliana transfer-gen plant, get fresh blade respectively, according to Bar test strip (QuickStix
tMkit for
(bar) Cotton, EnviroLogix, Portland, ME, USA, catalog number is AS 013LS) specification sheets detects the protein expression situation of Bar gene.
Detected result is all positive for selected plant, and Bar gene successful expression is wherein described.
(5) identify until obtain T according to the method for step (three)
2for positive Columbia ecotype Arabidopis thaliana transfer-gen plant and T
2for positive Arabidopis thaliana ago1-27 mutant transfer-gen plant, results T
2for seed, by T
2for seed culture screening pure lines, often kind of transfer-gen plant generally screens 3-5 strain, each strain screens 10 individual plants, every strain screening about 100 seeds, statistics percentage of germination, by percentage of germination 100% and screening culture medium (2.2g/L MS, 50 μ g/mL Glufosinate ammoniums, 8g/L agar, 30g/L sucrose, surplus is water, pH5.8-6.0) above can grow normal strain and be defined as pure lines, i.e. T
3for Columbia ecotype Arabidopis thaliana transfer-gen plant pure lines and T
3be sheerly for Arabidopis thaliana ago1-27 mutant transfer-gen plant.
Arabidopis thaliana ago1-27 mutant strain (ago1-27), wild-type Columbia ecotype Arabidopis thaliana (wt), T
3for Arabidopis thaliana ago1-27 mutant transfer-gen plant pure lines seedling stages of (line16, line19 and line21), flowering period and ripening stage form respectively as shown in A, B and C in Fig. 3.
Fig. 3 shows, Arabidopis thaliana ago1-27 mutant strain has obvious developmental defect, and leaf look is deep green, spination blade.By transforming the Arabidopis thaliana ago1-27 mutant transfer-gen plant that ZmAGO1a gene obtains to Arabidopis thaliana ago1-27 mutant strain, phenotype is obviously recovered, and Seedling Stage point is narrow, spination blade returns to initial closely round; By the statistical study (as shown in table 1) to the number of blade, leaf length, Ye Kuan, long-width ratio, setting percentage and breeding time etc., show that the physical signs of Arabidopis thaliana ago1-27 mutant transfer-gen plant returns to wild-type (wild-type Columbia ecotype Arabidopis thaliana) level substantially.
The phenotype analytical of table 1 Arabidopis thaliana
In table, significantly (P<0.05), statistical method is Tukey's HSD method to the different strain difference of the different letter representations of " * " mark.
In jaggedness, more the bright jaggedness of multilist is larger for "+" number;
" number of days " in flowering period or ripening stage in " wt+ number of days " represents the number of days of flowering period or ripening stage delay compared with wild-type Columbia ecotype Arabidopis thaliana.
Table 1 shows, the growth that the proceeding to of ZmAGO1a gene makes Arabidopis thaliana ago1-27 mutant strain and/or developmental process are accelerated, be embodied as compared with Arabidopis thaliana ago1-27 mutant strain, flowering period or ripening stage are in advance, flowering period shifts to an earlier date 10-11 days, ripening stage shifts to an earlier date 11 days, with flowering period of wild-type Columbia ecotype Arabidopis thaliana or the ripening stage close; And, the growth that the proceeding to of ZmAGO1a gene makes Arabidopis thaliana ago1-27 mutant strain and/or developmental level improve, be embodied as biological yield to improve, number of blade increase, leaf long growth, the wide increase of leaf, bear pods number and seed number raising, these indexs are all close with wild-type Columbia ecotype Arabidopis thaliana.
(6) real-time fluorescence quantitative PCR analysis
Extract Arabidopis thaliana ago1-27 mutant strain (ago1-27), wild-type Columbia ecotype Arabidopis thaliana (wt), T respectively
3for Arabidopis thaliana ago1-27 mutant transfer-gen plant pure lines (line16, line19 and line21) total serum IgE, and reverse transcription obtains cDNA, respectively to extract the cDNA that obtains for template, with ZmAGO1a forward primer and ZmAGO1a reverse primer for primer carries out real-time fluorescence quantitative PCR, detect the relative expression quantity of ZmAGO1a gene, or with AtAGO1 forward primer and AtAGO1 reverse primer for primer carries out real-time fluorescence quantitative PCR, detect the relative expression quantity of AtAGO1 gene, or with alpha-Tubulin 4 forward primer and alpha-Tubulin 4 reverse primer for primer carries out real-time fluorescence quantitative PCR, detect the relative expression quantity of reference gene Tubulin 4.The method of calculation of the relative expression quantity of each gene are 2
-Δ Δ CTmethod (Livak KJ, et al., 2001).
The dyestuff of above-mentioned real-time fluorescence quantitative PCR is SYBRPremixExTaq
tMiI (TliRnaseHPlus) (TaKaRa product).
Real-time fluorescence quantitative PCR reaction system (20 μ L) is: 10 μ L2 × SYBRPremixExTaq
tMiI, 0.4 μ LRoxReferenceDyeII (50 ×), the reverse primer of the forward primer of 2 μ L template cDNA, 0.4 μ L10 μM, 0.4 μ L10 μM, moisturizing is to cumulative volume 20 μ L.
Real-time fluorescence quantitative PCR response procedures is: 94 DEG C of 30s; 95 DEG C of 15s, 60 DEG C of 30s, 72 DEG C of 34s, 40 circulations.
ZmAGO1a forward primer: 5 '-GCTTGACTTGCTGATGGTAATACT-3 '; (SEQ ID No.7)
ZmAGO1a reverse primer: 5 '-CTGATGCTTGTTCGCCTTGA-3 '; (SEQ ID No.8)
AtAGO1 forward primer: 5 '-ATCAGACAGTGGCTCAAT-3 '; (SEQ ID No.9)
AtAGO1 reverse primer: 5 '-GACACGCTTCACATTCTC-3 '; (SEQ ID No.10)
Alpha-Tubulin 4 forward primer: 5 '-GAGGGAGCCATTGACAACATCTT-3 '; (SEQ ID No.11)
Alpha-Tubulin 4 reverse primer: 5 '-GCGAACAGTTCACAGCTATGTTCA-3 '.(SEQ ID No.12)
Result as shown in Figure 4.
Fig. 4 A shows, T
3expression amount for Arabidopis thaliana ago1-27 mutant transfer-gen plant pure lines (line16, line19 and line21) ZmAGO1a gene is significantly higher than Arabidopis thaliana ago1-27 mutant strain (ago1-27), wild-type Columbia ecotype Arabidopis thaliana (wt); Fig. 4 B shows, the expression amount of the endogenous AtAGO1 gene of Arabidopis thaliana ago1-27 mutant strain (ago1-27) is significantly higher than T
3the expression amount of (line16, line19 and line21) and wild-type Columbia ecotype Arabidopis thaliana (wt) is sheerly for Arabidopis thaliana ago1-27 mutant transfer-gen plant.The expression amount of the mRNA of the AtAGO1 of mutant strain ago1-27 higher than wild-type, result consistent with the result of the immunoblotting of the AGO1 albumen of Mallory (Mallory AC, et al., 2009).
The tissue specificity analysis of ZmAGO1a genetic expression in embodiment 3, corn Different Organs
One, the corn inbred line gathering large Tanaka's normal growth tucks in the tasseling stage tassel of 478, phase filigree of weaving silk, the phase bract that weaves silk, phase of weaving silk female fringe, and the corn inbred line of normal water planting tucks in leaf sheath, young root, the spire in the tri-leaf period of 478.
Two, the total serum IgE of each tissue of extraction step one collection, and reverse transcription obtains cDNA, respectively to extract the cDNA that obtains for template, with ZmAGO1a forward primer (SEQ ID No.7) and ZmAGO1a reverse primer (SEQ ID No.8) for primer carries out real-time fluorescence quantitative PCR, detect the relative expression quantity of ZmAGO1a gene, or, carry out real-time fluorescence quantitative PCR with GAPDH forward primer and GAPDH reverse primer for primer, detect the relative expression quantity of reference gene GAPDH.
GAPDH forward primer: 5 '-ACTGTTCATGCCATCACTGC-3 '; (SEQ ID No.13)
GAPDH reverse primer sequences: 5 '-GAGGACAGGAAGCACTTTGC-3 '.(SEQ ID No.14)
Real-time fluorescence quantitative PCR reaction system (20 μ L) is: 10 μ L2 × SYBRPremixExTaq
tMiI, 0.4 μ LRoxReferenceDyeII (50 ×), the reverse primer of the forward primer of 2 μ L template cDNA, 0.4 μ L10 μM, 0.4 μ L10 μM, moisturizing is to cumulative volume 20 μ L.
Real-time fluorescence quantitative PCR response procedures is: 94 DEG C of 30s; 95 DEG C of 15s, 60 DEG C of 30s, 72 DEG C of 34s, 40 circulations.
Result as shown in Figure 5.
Fig. 5 shows, ZmAGO1a gene has expression in different tissues, but gene expression abundance has tissue specificity.ZmAGO1a gene expression abundance in Maize leaf, phase of weaving silk female fringe is the highest, relative to corn leaf sheath, ZmAGO1a gene 34.19 times and 11.89 times of being respectively at spire and gene expression abundance in phase female fringe of weaving silk, phase filigree gene expression abundance of weaving silk is 4.84 times of leaf sheath, and the gene expression abundance in young root and tasseling stage tassel is minimum.ZmAGO1a gene gene expression abundance in the tissue relevant with growth, organ is higher.
The differential expression response characteristic of ZmAGO1a gene under 5 kinds of abiotic stress conditions in embodiment 4, Maize leaf
One, corn B73 self-mating system was cultured to for 3 leaf phases, obtain 3 the leaf phase seedling, by 3 the leaf phase seedling carry out following packet transaction:
(1) at low phosphorus solution (2m M Ca (NO
3)
24H
2o, 0.75mM K
2sO
4, 0.35mM KCl, 0.25 μM of KH
2pO
4, 0.001mM H
3bO
3, 0.65mM MgSO
47H
2o, 0.2mM Fe-EDTA, 1.0 μMs of MnSO
4h
2o, 1.0 μMs of ZnSO
47H
2o, 0.1 μM of CuSO
45H
2o, 0.005 μM of (NH
4)
6mo
7o
244H
2o, surplus is water) middle cultivation, carry out low-phosphorous (0.25 μM of KH
2pO
4) process, as low-phosphorous treatment group;
(2) at low nitrogen solution (0.02mMCa (NO
3)
24H
2o, 1.98mM CaCl
2, 0.75mM K
2sO
4, 0.1mMKCl, 0.25mM KH
2pO
4, 0.001mM H
3bO
3, 0.65mM MgSO
47H
2o, 0.2mM Fe-EDTA, 1.0 μMs of MnSO
4h
2o, 1.0 μMs of ZnSO
47H
2o, 0.1 μM of CuSO
45H
2o, 0.005 μM of (NH
4)
6mo
7o
244H
2o, surplus is water) middle cultivation, carry out low nitrogen (0.04mMNO
3 -) (
liu J, et al., 2008) process, as low nitrogen treatment group;
(3) at high level salt solution (2mM Ca (NO
3)
24H
2o, 0.75mM K
2sO
4, 0.1mM KCl, 0.25mM KH
2pO
4, 0.001mM H
3bO
3, 0.65mM MgSO
47H
2o, 0.2mM Fe-EDTA, 1.0 μMs of MnSO
4h
2o, 1.0 μMs of ZnSO
47H
2o, 0.1 μM of CuSO
45H
2o, 0.005 μM of (NH
4)
6mo
7o
244H
2o, 200mM NaCl, surplus is water) in cultivate, carry out high salt (200mMNaCl) (
zhu C, et al., 2005) process, as high Ficus caricaL group;
(4) at PEG-6000 solution (2mMCa (NO
3)
24H
2o, 0.75mM K
2sO
4, 0.1mM KCl, 0.25mMKH
2pO
4, 0.001mM H
3bO
3, 0.65mM MgSO
47H
2o, 0.2mM Fe-EDTA, 1.0 μMs of MnSO
4h
2o, 1.0 μMs of ZnSO
47H
2o, 0.1 μM of CuSO
45H
2o, 0.005 μM of (NH
4)
6mo
7o
244H
2o, 15g/100mlPEG-6000, surplus is water) in cultivate, carry out dewatering (PEG-6000 of 15g/100ml) (
ashraf M, et al., 2001) process, as processed group;
(5) at ABA solution (2mM Ca (NO
3)
24H
2o, 0.75mM K
2sO
4, 0.1mM KCl, 0.25mM KH
2pO
4, 0.001mM H
3bO
3, 0.65mM MgSO
47H
2o, 0.2mM Fe-EDTA, 1.0 μMs of MnSO
4h
2o, 1.0 μMs of ZnSO
47H
2o, 0.1 μM of CuSO
45H
2o, 0.005 μM of (NH
4)
6mo
7o
244H
2o, 100 μMs of ABA, surplus is water) in cultivate, carry out ABA (100 μMs) (
jiang M, et al., 2002) process, as ABA treatment group;
(6) at normal nutrition liquid (2mM Ca (NO
3)
24H
2o, 0.75mM K
2sO
4, 0.1mM KCl, 0.25mMKH
2pO
4, 0.001mM H
3bO
3, 0.65mM MgSO
47H
2o, 0.2mM Fe-EDTA, 1.0 μMs of MnSO
4h
2o, 1.0 μMs of ZnSO
47H
2o, 0.1 μM of CuSO
45H
2o, 0.005 μM of (NH
4)
6mo
7o
244H
2o, surplus is water) middle cultivation, as a control group;
The treatment time of each group is 24h above, often organizes 3 repetitions.
Two, the total serum IgE of each group of plant is extracted, and reverse transcription is cDNA, be template respectively with cDNA, with ZmAGO1a forward primer (SEQ ID No.7) and ZmAGO1a reverse primer (SEQ ID No.8) for primer carries out real-time fluorescence quantitative PCR, detect the relative expression quantity of ZmAGO1a gene, or, carry out real-time fluorescence quantitative PCR with GAPDH forward primer (SEQ ID No.13) and GAPDH reverse primer (SEQ ID No.14) for primer, detect the relative expression quantity of reference gene GAPDH.
Real-time fluorescence quantitative PCR reaction system (20 μ L) is: 10 μ L2 × SYBRPremixExTaq
tMiI, 0.4 μ LRoxReferenceDyeII (50 ×), the reverse primer of the forward primer of 2 μ L template cDNA, 0.4 μ L10 μM, 0.4 μ L10 μM, moisturizing is to cumulative volume 20 μ L.
Real-time fluorescence quantitative PCR response procedures is: 94 DEG C of 30s; 95 DEG C of 15s, 60 DEG C of 30s, 72 DEG C of 34s, 40 circulations.
Result as shown in Figure 6.
In Fig. 6, CK represents control group; NaCl represents high Ficus caricaL group; ABA represents ABA treatment group; PEG represents processed group; LN represents low nitrogen treatment group; LP represents low-phosphorous treatment group.
Fig. 6 shows, ZmAGO1a gene expression amount after high salt and ABA process 24h is only 22.47% and 74.99% of control group ZmAGO1a gene expression amount; And under low nitrogen treatment condition, compared with control group, ZmAGO1a expresses rising, expression amount is 1.69 times of control group ZmAGO1a gene expression amount; Under processed and low-phosphorous process, the expression amount of ZmAGO1a gene is then without noticeable change.
Claims (8)
1. an albumen, shown in following (1) or (2):
(1) albumen shown in SEQ ID No.2;
(2) by the aminoacid sequence shown in SEQ ID No.2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and the identical protein of function.
2. the encoding gene of albumen described in claim 1.
3. encoding gene according to claim 2, is characterized in that: described encoding gene is following middle at least one:
1) DNA molecular shown in SEQ ID No.1;
2) DNA molecular shown in the 150th to the 3458th or cDNA molecule in SEQ ID No.1;
3) under strict conditions with 1) or 2) DNA molecular that limits or cDNA molecular hybridization and encode protein DNA molecule described in claim 1 or cDNA molecule;
4) with 1) or 2) or 3) DNA molecular that limits or cDNA molecule have more than 90% identity and encode protein DNA molecule described in claim 1 or cDNA molecule.
4. the recombinant vectors containing encoding gene described in Claims 2 or 3, expression cassette, transgenic cell line or recombinant bacterium.
5. a method for Promoting plant growth and/or growth, comprises the steps: the encoding gene described in Claims 2 or 3 to import to set out in plant, obtains transgenic plant; Compared with the plant that sets out, the growth of transgenic plant and/or developmental process are accelerated or are grown and/or developmental level raising.
6. method according to claim 5, is characterized in that: described growth and/or developmental process are accelerated to be presented as flowering period and/or ripening stage in advance;
Described growth and/or developmental level raising are presented as that biological yield improves.
7. the method according to claim 5 or 6, is characterized in that: described encoding gene is imported by recombinant expression vector, and described recombinant expression vector is that the multiple clone site described encoding gene being inserted the carrier pCPB that sets out obtains.
8. encoding gene described in albumen according to claim 1 or Claims 2 or 3 is in Promoting plant growth and/or developmental application.
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| CN116064644A (en) * | 2022-10-10 | 2023-05-05 | 中国农业科学院生物技术研究所 | SbAGO1b protein and application of coding gene thereof in regulation and control of plant insect resistance |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110527741A (en) * | 2019-09-11 | 2019-12-03 | 东北农业大学 | A kind of molecular labeling, primer and application with american pumpkin mildew-resistance biological strain 2F gene close linkage |
| CN116064644A (en) * | 2022-10-10 | 2023-05-05 | 中国农业科学院生物技术研究所 | SbAGO1b protein and application of coding gene thereof in regulation and control of plant insect resistance |
| CN116064644B (en) * | 2022-10-10 | 2023-10-10 | 中国农业科学院生物技术研究所 | SbAGO1b protein and application of coding gene thereof in regulation and control of plant insect resistance |
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