CN103695436B - The application of ZmHINT gene in flower development - Google Patents
The application of ZmHINT gene in flower development Download PDFInfo
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Abstract
The invention discloses the application of a kind of ZmHINT gene in flower development.The invention discloses following arbitrary material and promoting the application in plant early flowering: (1) SEQ? ID? albumen shown in No.2; (2) SEQ? ID? the encoding gene of albumen shown in No.2; (3) recombinant vectors containing (2) described encoding gene, expression cassette, transgenic cell line or recombinant bacterium.ZmHINT gene provided by the invention process LAN in Arabidopis thaliana can induce Arabidopis thaliana Blooming, illustrates that ZmHINT gene has vital role in flower development.
Description
Technical field
The present invention relates to the application of a kind of ZmHINT gene in flower development.
Background technology
Histidine trimer protein superfamily is a family with nucleotidyl transferase and hydrolytic enzyme activities, wherein, Histidine tripolymer nucleotide binding protein (Histidine triad nucleotide binding protein, HINT) be the most a kind of albumen that distributes in Histidine trimer protein superfamily member, this albumen all has very high structure and function similarity in lower plant and higher animal.Crystalline structure result of study shows, HINT is the ancestors of other four kinds of Histidine trimer protein superfamily members, and the conservative property of structure and function plays important biological function predictive of HINT.At present, studies have reported that and in apoptosis, tumorigenesis, played vital role about Mammals HINT.The research of relative animal HINT, the functional study of plant HINT is less, and especially to the research of corn HINT, there is not been reported.
Summary of the invention
The object of this invention is to provide the application of a kind of ZmHINT gene in flower development.
The invention provides following arbitrary material and promote the application in plant early flowering:
(1) albumen shown in SEQ ID No.2;
(2) encoding gene of albumen shown in SEQ ID No.2;
(3) recombinant vectors containing (2) described encoding gene, expression cassette, transgenic cell line or recombinant bacterium.
In above-mentioned application, described encoding gene is as shown in SEQ ID No.1.
In above-mentioned arbitrary described application, described plant is Arabidopis thaliana.
The method preparing transgenic plant also belongs to a protection scope of the present invention, comprises the steps: the encoding gene of albumen shown in SEQ IDNo.2 to import to set out in plant, obtains transgenic plant; Compared with the plant that sets out, the flowering time of transgenic plant in advance.
In aforesaid 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 pCAMBIA-1304 that sets out obtains.
In above-mentioned arbitrary described method, described encoding gene is as shown in SEQ ID No.1;
Described plant is specially Arabidopis thaliana.
A kind of albumen also belongs to protection scope of the present invention, 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.
The encoding gene of above-mentioned albumen also belongs to protection scope of the present invention.
In above-mentioned encoding gene, described encoding gene is following middle at least one:
1) DNA molecular shown in SEQ ID No.1;
2) under strict conditions with 1) DNA molecule hybridize that limits and the DNA molecular of code for said proteins;
3) with 1) or 2) DNA molecular that limits has the identity of more than 90% and the DNA molecular of code for said proteins.
Recombinant vectors containing above-mentioned arbitrary described encoding gene, expression cassette, transgenic cell line or recombinant bacterium also belong to protection scope of the present invention.
ZmHINT gene provided by the invention process LAN in Arabidopis thaliana can induce Arabidopis thaliana Blooming, illustrates that ZmHINT gene has vital role in flower development.
Accompanying drawing explanation
Fig. 1 is the PCR qualification of recombinational agrobacterium and contrast Agrobacterium.
Fig. 2 is that the Totomycin of different concns is to turning the screening of ZmHINT gene T1 for Arabidopis thaliana.
To be the T2 that turns ZmHINT gene screen for the hygromycin resistance of Arabidopis thaliana for Arabidopis thaliana and the T2 that turns empty carrier pCAMBIA-1304 Fig. 3.
Fig. 4 be the T2 that turns ZmHINT gene for Arabidopis thaliana, turn empty carrier pCAMBIA-1304 T2 for the PCR qualification of Arabidopis thaliana and wildtype Arabidopsis thaliana.
Fig. 5 is Arabidopis thaliana phenotype analytical.
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.
GC Buffer is purchased from precious biotechnology (Dalian) company limited, and catalog number is 9154.
B73 corn inbred line (Zea mays ssp.mays L.) is disclosed in document " Patrick S.Schnable; etal.The B73Maize Genome:Complexity; Diversity; and Dynamics.Science326; 1112-1115 (2009) ", and the public can obtain from Agricultural University Of He'nan.
Arabidopis thaliana (Arabidopsis thaliana) Colombia's type (Columbia-0) is at document " Xiao Luo, Xi Bai, Xiaoli Sun, Dan Zhu, Baohui Liu, Wei Ji, Hua Cai, Lei Cao, Jing Wu, Mengran Hu, Xin Liu, Lili Tang and Yanming Zhu.Expression of wild soybeanWRKY20in Arabidopsis enhances drought tolerance and regulates ABA signaling.Journal of Experimental Botany.2013. " in be disclosed, the public can obtain from Agricultural University Of He'nan.
MS salt purchased from American Sigma company, article No. is M5524-50L.
MS salts solution is prepared as follows: this solution is made up of solvent and solute; Solute is MS salt, sucrose and Silwet L-77, and solvent is distilled water.The concentration of MS salt in MS salts solution is the concentration of 2.15g/L, Silwet L-77 in MS salts solution is 200 μ l/L, and the concentration of sucrose in MS salts solution is 5g/100ml, adjusts pH=5.8.
YEP liquid nutrient medium is prepared as follows:
Extractum carnis 5.0g/L, peptone 5.0g/L, yeast extract 1.0g/L, sucrose 5.0g/L, MgSO
47H
2o0.5g/L, surplus is water, and after adjusting pH=7.0, sterilizing is preserved.
Agrobacterium tumefaciens (Agrobacterium tumefaciens) GV3101 is disclosed in document " Wei Tang; RonSederoff; Ross Whetten.Regeneration of transgenic loblolly pine (Pinus taedaL.) from zygotic embryos transformed with Agrobacterium tumefaciens.Planta (2001) 213:981-989. ", and the public can obtain from Agricultural University Of He'nan.
PMD18-T Simple carrier is purchased from precious biotechnology (Dalian) company limited, and catalog number is 6011.
Carrier pCAMBIA-1304 is disclosed in document " Ruchi Pandey; Avinash Mishra; G.K.Garg.Plantpromoter driven heterologous expression of HMW glutenin gene (s) subunit in E.coli.Mol Biol Rep (2008) 35:153 – 162. ", and the public can obtain from Agricultural University Of He'nan.
The structure of embodiment 1, ZmHINT gene overexpression carrier and recombinational agrobacterium
One, design of primers
(1) according to ZmHINT gene coding region full length cDNA sequence, following primer is designed and synthesized:
F6：5'-ATGTCGTCGGAGAAGGAGGC-3'；
R6：5'-TTAGCCTGGGGGCCAGTTCA-3'；
F7：5'-AC
CCATGGATGTCGTCGGAGAAGGAGGC-3'；
R7：5'-CGGC
ACTAGTTTAGCCTGGGGGCCAGTTCA-3'。
Wherein 5'-CCATGG-3' is NcoI restriction enzyme site, and 5'-ACTAGT-3' is SpeI restriction enzyme site.
(2) design and synthesize as follows for the detection primer of empty carrier pCAMBIA-1304:
F8:5'-AACAGAACTCGCCGTAA-3'；
R8:5'-GTCAAGAGTCCCCCGTGTT-3'。
Two, extract the RNA of B73 corn inbred line, and reverse transcription is cDNA.With the cDNA of B73 corn inbred line for template, carry out pcr amplification with F7 and R7 for primer, obtain pcr amplification product, be ZmHINT gene.
PCR reaction system:
PCR reaction conditions:
Three, pcr amplification product is connected to pMD18-T Simple carrier and obtains recombinant plasmid, by its called after pMD-ZmHINT-2, send order-checking by pMD-ZmHINT-2 plasmid, result is correct.
Four, NcoI and SpeI double digestion pMD-ZmHINT-2, obtains gene fragment; NcoI and SpeI double digestion pCAMBIA-1304, obtains carrier large fragment; Gene fragment be connected with carrier large fragment, obtain recombinant plasmid pCAMBIA1304-ZmHINT-2, send order-checking by pCAMBIA1304-ZmHINT-2 plasmid, result is correct.
The nucleotide sequence of ZmHINT gene is as shown in SEQ ID No.1.
The aminoacid sequence of ZmHINT albumen is as shown in SEQ ID No.2.
Five, the structure of recombinational agrobacterium
By pCAMBIA1304-ZmHINT-2 Plastid transformation agrobacterium tumefaciens (Agrobacterium tumefaciens) GV3101, obtain recombinational agrobacterium.Simultaneously by empty carrier pCAMBIA-1304 transform Agrobacterium tumefaciens (Agrobacterium tumefaciens) GV3101, obtain contrasting Agrobacterium.
Concrete steps are as follows:
(1) recombinant plasmid pCAMBIA1304-ZmHINNT-2 and empty carrier pCAMBIA-1304 transform Agrobacterium tumefaciens
Respectively get the plasmid of pCAMBIA1304-ZmHINNT-2 recombinant plasmid that 1 μ g builds and pCAMBIA-1304 empty carrier, adopt first liquid nitrogen freezing, then the method for thermal shock is transferred in agrobacterium tumefaciens GV3101.Then the agrobacterium tumefaciens GV3101 after conversion is coated on the YEP plate culture medium containing microbiotic 100 μ g/ml Kan and grow 48 hours.Then respectively select 5 mono-clonals, be inoculated in containing in corresponding antibiotic YEP (100 μ g/ml Kan, 125 μ g/ml Rifampins) liquid nutrient medium, 28 DEG C, 200rpm/min shaking culture is spent the night, and gets overnight culture and does pcr amplification qualification.
(2) PCR qualification
1, mono-clonal bacterium liquid PCR identification system after pCAMBIA1304-ZmHINNT-2 recombinant plasmid transformed agrobacterium tumefaciens GV3101:
PCR reaction conditions:
2, mono-clonal bacterium liquid PCR identification system after pCAMBIA-1304 Plastid transformation agrobacterium tumefaciens GV3101:
PCR reaction conditions:
The agrobacterium tumefaciens GV3101 not turning any plasmid is set simultaneously and carries out this experiment as negative control.
3, the pcr amplification product of step 1 and step 2 is carried out the agarose gel electrophoresis of 1.0%, result as shown in Figure 1.
Figure 1A is the PCR qualification of recombinational agrobacterium (turning the agrobacterium tumefaciens GV3101 of pCAMBIA1304-ZmHINT-2 plasmid), and Figure 1B is the PCR qualification of contrast Agrobacterium (turning the agrobacterium tumefaciens GV3101 of pCAMBIA-1304 plasmid).
In Figure 1A, M is DNA marker, 1-3 is three recombinational agrobacterium clones.
In Figure 1B, M is DNA marker, 1-4 is four contrast Agrobacterium colonies, and 5 is the agrobacterium tumefaciens GV3101 not turning any plasmid.
Figure 1A shows, containing goal gene (ZmHINT gene) in recombinational agrobacterium, its length is about 400bp, and recombinational agrobacterium successfully constructs.
Figure 1B shows, be the object band of 514bp containing length in empty carrier pCAMBIA-1304 in contrast Agrobacterium, this sequence is as shown in SEQ ID No.3, and Agrobacterium successfully constructs in contrast.
The preparation of embodiment 2, transgenic arabidopsis
One, the preparation of Arabidopis thaliana to be transformed
(1) seed of Arabidopis thaliana (Arabidopsis thaliana) Colombia's type (Columbia-O) (hereinafter referred to as wildtype Arabidopsis thaliana) is placed in 1.5ml centrifuge tube, 6.25%NaClO (6.25ml NaClO stoste, the Triton X-100 of 0.01ml, uses ddH
2o constant volume is to 100ml) sterilize 15 minutes.
(2) ddH
2o cleans seed 6 times, after seed avales, abandons upper strata ddH
2o.
(3) by planting seed on 1/2MS culture medium flat plate, lucifuge 4 DEG C of vernalization 72h.
(4) flat board is placed in illumination box (21 DEG C, illumination every day 16 hours, dark 8 hours), growth 8-10 days, obtains the Arabidopsis thaliana Seedlings sprouted.
(5) Arabidopsis thaliana Seedlings is moved into (turfy soil: vermiculite=1:1, volume ratio) in Nutrition Soil, grow under greenhouse (21 DEG C, illumination every day 16 hours, dark 8 hours) condition, during full-bloom stage, carry out genetic transformation.
Two, Agrobacterium-mediated Transformation Arabidopis thaliana
(1) Arabidopis thaliana step one obtained waters sufficient water in conversion the day before yesterday.
(2) recombinational agrobacterium embodiment 1 prepared respectively and contrast Agrobacterium inoculation are in YEP liquid medium (Kan50 μ g/ml, Rifampin 50 μ g/ml), and 28 DEG C of shaking culture spend the night (the several different clone of Simultaneous vaccination).
(3) the recombinational agrobacterium bacterium liquid of the difference of overnight incubation being cloned mixes, and 500ml, is denoted as recombinant bacterium mixed solution altogether.The contrast Agrobacterium bacterium liquid of simultaneously difference of overnight incubation being cloned mixes, altogether 500ml, is denoted as contrast bacterium mixed solution.Getting recombinant bacterium mixed solution respectively or contrasting bacterium mixed solution 5ml adds in the 1L triangular flask containing 500ml YEP liquid nutrient medium (Kan50 μ g/ml), 28 DEG C of shaking culture 6-12 hour (OD
600=0.8-1.0).
(4) 4000rpm/min, centrifugal 10 minutes of room temperature, collects thalline.
(5) with 200mlMS salts solution suspension thalline, the suspension of recombinational agrobacterium and contrast Agrobacterium is obtained respectively.
(6) Arabidopis thaliana inflorescence is immersed in respectively recombinational agrobacterium and contrast Agrobacterium suspension in about 1 minute, put moisturizing one day with freshness protection package.
Arabidopis thaliana was taken out from freshness protection package in (seven) second days, dark placement was put back on illumination cultivation frame after one day, cultivate plant (21 DEG C, illumination every day 16 hours, dark 8 hours) to solid, until ripe the turning ZmHINT gene and turn the Arabidopis thaliana T0 of empty carrier pCAMBIA-1304 for seed of results respectively, individual plant during results, to be divided to gather in the crops.
Three, the screening of transgenic arabidopsis positive plant
(1) the Arabidopis thaliana T0 turning ZmHINT gene and turn empty carrier pCAMBIA-1304 after dry 2 weeks is seeded on the MS substratum containing 40mg/L, 35mg/L, 30mg/L, 25mg/L, 20mg/L, 15mg/L Totomycin (Hygromycin) respectively for seed.
(2) seed 4 DEG C of vernalization 72h hour are placed on 21 DEG C, illumination every day 16 hours, dark 8 hours, light intensity 150 μMs of m
-2s
-1, within 8-10 days, observe the cotyledon of Arabidopis thaliana T1 for seedling and the development condition of root that turn ZmHINT gene He turn empty carrier pCAMBIA-1304 afterwards.
(3) identify through preliminary screening: the T1 that the positive turns ZmHINT gene or transforms empty carrier pCAMBIA-1304 for Arabidopsis thaliana Seedlings can on the MS Selective agar medium containing 30mg/L Totomycin normal growth, and the T1 that feminine gender does not turn ZmHINT gene or unconverted empty carrier pCAMBIA-1304 can not grow on the MS Selective agar medium containing 30mg/L Totomycin for Arabidopsis thaliana Seedlings, be turned into Albino Seedling.
The T1 that the positive turns ZmHINT gene for Arabidopsis thaliana Seedlings hygromycin resistance the selection result as shown in Figure 2.
In Fig. 2, a:40mg/L Hygromycin MS substratum; B:35mg/L Hygromycin MS substratum;
C:30mg/L Hygromycin MS substratum; D:25mg/L Hygromycin MS substratum;
E:20mg/L Hygromycin MS substratum; F:15mg/L Hygromycin MS substratum.
Fig. 2 shows, the T1 turning ZmHINT gene for Arabidopsis thaliana Seedlings can on the MS Selective agar medium containing 30mg/L Totomycin normal growth.
(4) after 8-10d, respectively the positive is turned ZmHINT gene and the T1 that turns empty carrier pCAMBIA-1304 to be transplanted in substrate soil for Arabidopsis thaliana Seedlings and to grow, when plant grows to 10 leaf, get blade and extract geneome RNA reverse transcription becomes cDNA, the T1 obtaining turning ZmHINT gene for Arabidopis thaliana cDNA and turn the cDNA of T1 for Arabidopis thaliana of empty carrier pCAMBIA-1304.
With the T1 turning ZmHINT gene for the cDNA of Arabidopis thaliana for template, with R6 and F6 for primer carries out PCR qualification, with the T1 turning empty carrier pCAMBIA-1304 for the cDNA of Arabidopis thaliana for template, with R8 and F8 for primer carries out PCR qualification, positive judging criterion is as the step 5 of embodiment 1.
T1 is collected for seed with the Arabidopis thaliana T1 turning empty carrier pCAMBIA-1304 for dividing individual plant after seedling maturation by being accredited as the positive ZmHINT gene that turns.After planting seed by T1 generation, obtain T2 for seedling, carried out the hygromycin resistance screening of 30mg/L, result as shown in Figure 3.
In Fig. 3 A, black arrow mark for turning the T2 of ZmHINT gene for Arabidopsis thaliana Seedlings.
In Fig. 3 B, black arrow mark for turning the T2 of empty carrier pCAMBIA-1304 for Arabidopsis thaliana Seedlings.
Fig. 3 shows, the T2 turning ZmHINT gene for Arabidopsis thaliana Seedlings and the T2 that turns empty carrier pCAMBIA-1304 for Arabidopsis thaliana Seedlings can on the MS Selective agar medium containing 30mg/L Totomycin normal growth.
The T2 turning ZmHINT gene of hygromycin resistance screening for Arabidopsis thaliana Seedlings, turn empty carrier pCAMBIA-1304 T2 for Arabidopsis thaliana Seedlings and wildtype Arabidopsis thaliana sprigging in substrate soil, when growing to 10 leaves, individual plant extracts the geneome RNA of blade and reverse transcription becomes cDNA, the T2 obtaining turning ZmHINT gene for Arabidopis thaliana cDNA and turn the cDNA of T2 for Arabidopis thaliana and the cDNA of wildtype Arabidopsis thaliana of empty carrier pCAMBIA-1304.
Respectively with the T2 turning ZmHINT gene for the cDNA of Arabidopis thaliana and the cDNA of wildtype Arabidopsis thaliana for template, with R6 and F6 for primer carries out PCR qualification, respectively with the T2 turning empty carrier pCAMBIA-1304 for the cDNA of Arabidopis thaliana and the cDNA of wildtype Arabidopsis thaliana for template, with R8 and F8 for primer carries out PCR qualification, positive judging criterion is as the step 5 of embodiment 1, and result as shown in Figure 4.
Fig. 4 A is the T2 that turns ZmHINT gene for the PCR qualification of Arabidopis thaliana and wildtype Arabidopsis thaliana.
Fig. 4 B is the T2 that turns empty carrier pCAMBIA-1304 for the PCR qualification of Arabidopis thaliana and wildtype Arabidopsis thaliana.
In Fig. 4 A, the M T2 that to be DNA marker, 1-4 be turns ZmHINT gene is for Arabidopis thaliana, and 5 is wildtype Arabidopsis thaliana.
In Fig. 4 B, the M T2 that to be DNA marker, 1-4 be turns empty carrier pCAMBIA-1304 is for Arabidopis thaliana, and 5 is wildtype Arabidopsis thaliana.
Fig. 4 A shows, the T2 turning ZmHINT gene detects the target stripe of about 400bp for Arabidopis thaliana, and wildtype Arabidopsis thaliana does not amplify object band.
Fig. 4 B shows, the T2 turning empty carrier pCAMBIA-1304 detects the object band of 514bp for Arabidopis thaliana, and wildtype Arabidopsis thaliana does not amplify object band.
Fig. 4 shows, the T2 turning ZmHINT gene successfully constructs for Arabidopis thaliana for Arabidopis thaliana and the T2 turning empty carrier pCAMBIA-1304.
According to the method described above until obtain the T3 that turns ZmHINT gene for Arabidopsis plant and turn empty carrier pCAMBIA-1304 T3 for Arabidopsis plant.In addition, individual plant is divided to gather in the crops seed after being often accredited as positive plant maturation for PCR.
Embodiment 3, turn ZmHINT gene Arabidopis thaliana, turn the flowering time of empty carrier pCAMBIA-1304 Arabidopis thaliana and wildtype Arabidopsis thaliana and compare
By the T3 turning ZmHINT gene for Arabidopis thaliana, turn empty carrier pCAMBIA-1304 T3 for after the planting seed of Arabidopis thaliana and wildtype Arabidopsis thaliana at 21 DEG C, illumination every day 16 hours, cultivate under the condition of dark 8 hours, the phenotype of observing Arabidopis thaliana records flowering time (flowering time refers to start to bloom in which sky by after planting seed) simultaneously, as shown in Figure 5, flowering time is as shown in table 1 for phenotypic results.
In Fig. 5, A be turn ZmHINT gene T3 for Arabidopis thaliana; B be turn empty carrier pCAMBIA-1304 T3 for Arabidopis thaliana; C is wildtype Arabidopsis thaliana.
Table 1 Arabidopis thaliana flowering time
Fig. 5 shows, the T3 turning ZmHINT gene for Arabidopis thaliana flowering time far early than the flowering time of the T3 turning empty carrier pCAMBIA-1304 for Arabidopis thaliana and wildtype Arabidopsis thaliana.
Table 1 shows, the T3 turning ZmHINT gene is 35 days for the average flowering time of Arabidopis thaliana, and the T3 turning empty carrier pCAMBIA-1304 is 41 days for the average flowering time of Arabidopis thaliana and wildtype Arabidopsis thaliana, illustrates that the process LAN of ZmHINT gene in Arabidopis thaliana can induce Arabidopis thaliana Blooming.
Claims (5)
1. following arbitrary material is promoting the application in Arabidopis thaliana early flowering:
(1) albumen shown in SEQ ID No.2;
(2) encoding gene of albumen shown in SEQ ID No.2;
(3) recombinant vectors containing (2) described encoding gene, expression cassette or recombinant bacterium.
2. application according to claim 1, is characterized in that: described encoding gene is as shown in SEQ ID No.1.
3. prepare a method for transgenic arabidopsis, comprise the steps: the encoding gene of albumen shown in SEQ ID No.2 to import to set out in plant, obtain transgenic plant; Compared with the plant that sets out, the flowering time of transgenic plant in advance.
4. method according to claim 3, 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 pCAMBIA-1304 that sets out obtains.
5. the method according to claim 3 or 4, is characterized in that: described encoding gene is as shown in SEQ ID No.1.
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| WO2009113814A2 (en) * | 2008-03-14 | 2009-09-17 | 씨비에스바이오사이언스 주식회사 | Protein marker for early diagnosis of liver cancer |
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| WO2009113814A2 (en) * | 2008-03-14 | 2009-09-17 | 씨비에스바이오사이언스 주식회사 | Protein marker for early diagnosis of liver cancer |
| CN101519655A (en) * | 2009-04-02 | 2009-09-02 | 浙江大学 | Abalone HINT gene and protein and application of same for preparing anticarcinogenic drug |
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| Molecular cloning and expression analysis of an HINT1 homologue from maize (Zea mays L.);Liuji Wu et al.;《Plant Mol Biol Rep》;20110308;第29卷;第1006-1012页 * |
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