CN104250303A - Application of transcription factor Os11g39000.2 in change of grain properties of rice - Google Patents

Abstract

The invention relates to an application of transcription factor Os11g39000.2 gene in change of grain properties of rice. The CDS sequence of the transcription factor geneOs11g39000.2 is constructed to the downstream of each of four VP16 sequences through a Gateway system, and the rice kind kitaake is converted in order to lengthen and widen the grains of the transgenic rice. The above transgenic means lengthens and widens the grains of rice in order to improve the grain properties, and is valuable to the detailed description of the rice grain change regulated molecular mechanism, so the transcription factor geneOs11g39000.2 is highly valuable to the improvement of the rice output.

Description

Transcription factor Os11g39000.2 is changing the application in rice grain proterties

Technical field

The present invention relates to field of genetic engineering, specifically, relate to the application of rice transcription factor Os11g39000.2 gene.

Background technology

Paddy rice (Oryza sativa L.) is the important food crop in China and the whole world, the world more than 1/2 population take paddy rice as staple food, therefore the concern of rice yield traits extremely scientists all the time, rice grain size is then one of principal element affecting output.In addition, paddy rice is as the model plant of farm crop gene functional research, and relative genetics and molecular biology research are subject to the attention of investigators always.Rice grain grow be one orderly, optionally genetic expression process, transcription factor serves critical effect in the accuracy controlling of its genetic expression.

The output of cereal crop depends on the size of its seed to a great extent, and therefore the gene regulating of rice grain proterties is important field of research.In recent years, disclose at least 8 genes by the technique means such as gene clone, QTL, in control grain type, there is vital role.The albumen of a SG1 genes encoding Unknown Function, mainly expressing in the fringe of rice root and growth, there is short grain and Dwarfing phenotypes in the overexpression of this albumen.SG1 reduces the response to brassinolide BR, by reducing cell proliferation thus reducing the elongation of the such as organ such as seed, rachis internode.SGL1 is a class SG1 albumen, has the function similar with SG1, and lowered the expression of SG1 and homologous gene SGL1 thereof by RNAi, the internode of paddy rice grain length and cob is elongated compared with wild-type (Nakagawa etc., 2012).GS3 site is the main effect QTL controlling rice grain weight and grain length, is also the minor effect QTL (Fan, Xing etc., 2006 that control the wide and grain-filling degree of rice grain simultaneously; Mao, Sun etc., 2010).PGL1 be one atypical not in conjunction with the bHLH protein (bHLH) of DNA, overexpression PGL1 adds seed length and weight.APG is that the Antagonism of PGL1 makes the factor mutually, is both positioned in core.The seed length increase of PGL1/APG mediation causes due to the increase of inner glume cell length.APG and PGL1 does not affect the expression of known seed length-adjusting gene GS3 and SRS3.PGL1-APG represents a new seed length and weight regulatory pathway, and APG is negative regulation, and its function is subject to the suppression (Heang etc., 2012) of PGL1.Mi3 (t) gene, derives from Indica Rice Y34, and this gene can make grain length shorten 35.7% ~ 47.4%, and thousand seed weight reduces by 45.9% ~ 62.4%, its major control seed length, produces granule (Liu Mingwei etc., 2005).In addition, the gene controlling rice grain proterties also has gGL7 and gGL7-2.In a word, controlling rice grain growth is a complex process, relate to the cooperation control of polygene many approach, the gene that current discovery regulates and controls this proterties is few, the molecule mechanism of regulation and control Grain Development is not yet illustrated, therefore, find and control gene that grain characters grows and new excavation means to crop improvement and to improve crop yield significant.

Summary of the invention

In order to solve the problems of the technologies described above, the invention provides a kind of fusion rotein, this fusion rotein comprises VP16 albumen and the rice transcription factor Os11g39000.2 of hsv (Herpes simplex virus).

In an embodiment of the present invention, this fusion rotein is (VP16) ₄-Linker-Os11g39000.2; Wherein Linker is in series (SEQ ID No.3) by 39 flexible amino acid, and its nucleotide sequence is as shown in SEQ ID No.12; VP16 is the VP16 albumen (its aminoacid sequence is Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu) from hsv (Herpes simplex virus), (VP16) ₄i.e. VP64, to be merged by Gly Ser interval by 4 VP16 functional domain motifs and form, its nucleotide sequence is as shown in SEQ ID No.4, aminoacid sequence is as SEQ ID No.9,, or this sequence is through replacing, lacking or add one or several amino acids formed aminoacid sequence with same function; Os11g39000.2 is rice transcription factor Os11g39000.2, its nucleotide sequence is as shown in SEQ ID No.1, its aminoacid sequence is as shown in SEQ ID No.2, or this sequence is through replacing, lacking or add one or several amino acids formed aminoacid sequence with same function.

Present invention also offers the primer for the Os11g39000.2 gene that increases, forward primer F1:

5'-CAAAAAAGCAGGCTTC?ATGTCGTCGAGCCGGCG-3'

With reverse primer R1:

5'-CAAGAAAGCTGGGTCCATGAGTAGGCTACGGATGAGG-3'。

The present invention also provides the gene of encoding said fusion protein, and under strict conditions, can with the nucleotide sequence of the nucleotide sequence hybridization of this gene; Wherein, described stringent condition is 65 DEG C, hybridizes and wash film in the solution of 0.1 × SSPE or 0.1 × SSC, 0.1%SDS.

The present invention also provides the carrier of the gene containing encoding said fusion protein.Described carrier is the carrier that any one bootable foreign gene is expressed in host.Preferably, described carrier is plant binary expression vector (such as, pCAMBIA1301).By when encoding said fusion protein of the present invention gene constructed is in plant expression vector, any one strong promoter (such as, corn strong promoter Ubiquitin) or inducible promoter can be added before its transcription initiation Nucleotide.In addition, by when encoding said fusion protein of the present invention gene constructed is in plant expression vector, enhanser can also be used, and these enhanser regions must be identical with the reading frame of encoding sequence, to guarantee the translation of whole piece sequence.

The present invention also provides the transgenic cell line of the gene containing encoding said fusion protein.

The present invention also provides the engineering bacteria of the gene containing encoding said fusion protein.

The present invention also provides the gene of described fusion rotein, encoding said fusion protein increasing the application in the wide and thousand seed weight of paddy rice grain length, grain.

The present invention further provides the application of rice transcription factor Os11g39000.2 gene, it is the downstream CDS sequence of rice transcription factor Os11g39000.2 gene (complete translation district) being building up to 4 transcription factor activation motif VP16, conversion of plant, screening also finally obtains transgenic plant.

Aforesaid application, it is by the downstream of Gateway system constructing to 4 transcription factor activation motif VP16 by the CDS sequence of rice transcription factor Os11g39000.2 gene, rice transformation (such as, rice varieties ' kitaake '), thus render transgenic rice grain elongated, broaden.

The expression vector carrying the gene of encoding said fusion protein imports (Weissbach in vegetable cell by using the standard biologic technological methods such as Ti-plasmids, plant viral vector, directly delivered DNA, microinjection, electroporation, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, 411-463 page; Geiserson and Corey, 1998, Plant Molecular Biology, 2 ^ndedition).

The present invention utilizes transcription factor activation motif VP64(i.e. 4 transcription factor activation motif VP16 first) build obtain composing type transcription factor with rice transcription factor Os11g39000.2 gene fusion, and be transformed into farm crop, and render transgenic rice paddy seed obviously elongated, broaden, thousand seed weight increase, there is good yield potential.

Accompanying drawing explanation

Fig. 1 is nVP64-hyg-asRED Vector map in the embodiment of the present invention 1;

Fig. 2 is ubi:VP64-Os11g39000.2 Vector map in the embodiment of the present invention 1;

Fig. 3 is that Western blot of the present invention detects VP64-Os11g39000.2 transgenic positive strain, and wherein WT is wild rice ' kitaake ', and V1282H-06, V1282H-13 are VP64-Os11g39000.2 transgenic paddy rice strain;

Fig. 4 is the phenotype of transgenic line grain characters of the present invention, and wherein WT is wild rice ' kitaake ', and V1282H-06, V1282H-13 are transgenic paddy rice strain;

Fig. 5 is transgenic line grain characters data statistic analysis of the present invention, and wherein WT is wild rice ' kitaake ', and V1282H-06, V1282H-13 are transgenic paddy rice strain.

Embodiment

Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.

If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.

The separation of embodiment 1Os11g39000.2 gene and plant expression vector construction

Os11g39000.2 gene is found, according to its sequences Design pcr amplification primer in plant transcription factor database http://planttfdb.cbi.edu.cn/index.php sp=Osj:

F1：5'-CAAAAAAGCAGGCTTC?ATGTCGTCGAGCCGGCG-3'

With reverse primer R1:

5'-CAAGAAAGCTGGGTCCATGAGTAGGCTACGGATGAGG-3', with the total cDNA of the wild-type fine paddy rice of Japan for template, carry out PCR and obtain Os11g39000.2 complete sequence, its nucleotide sequence is as shown in SEQ ID NO.1.

PCR is carried out according to PrimeSTAR polymeric enzymatic amplification system and response procedures.Two-wheeled PCR is comprised in this process, primer gene primer F1 and R1 adding part adaptor attB joint of first round PCR, and the second template of taking turns PCR primer of the first round, and the complete adaptor attB primer of primer, attB5'adaptor:5'-GTGGGGACAAGTTTGTACAAAAAAGCAGGCTTC-3', attB3'adaptor:5'-GTGGGGACCACTTTGTACAAGAAAGCTGGGTC-3'.PCR primer is cloned into and connects on pDONER cloning vector (purchased from Invitrogen), obtain the identical sequence with goal gene through order-checking qualification.By LR reaction, Os11g39000.2 is building up to nVP64-hyg-asRED(Fig. 1, carrier complete sequence is as shown in SEQ ID No.5, the sequence that this carrier comprises with binary expression vector pCAMBIA1300 right boundary is for frame sequence, pass through vitro recombination, ubi promoter-VP64-Gateway is expressed unit, 35S promoter-asRED expresses unit and 35S promoter-hyg expression unit constructs with it, obtain carrier nVP64-hyg-asRED) on, obtain carrier ubi:VP64-Os11g39000.2(Fig. 2, carrier complete sequence is as shown in SEQ ID No.6).

The acquisition of embodiment 2 transgenic rice plant

Water intaking rice ' kitaake ' mature seed, artificial or mechanical dejacketing, selects the full bright and clean seed without bacterial plaque and is inoculated on inducing culture after sterilizing and carries out inducing culture.Selection outward appearance is good, the Rice Callus that growing ability is good is acceptor material, agrobacterium-mediated transformation is adopted to proceed in Rice Callus by ubi:VP64-Os11g39000.2, transform with the AAM conversion fluid being the Agrobacterium of 0.7 containing the Syringylethanone of 100 μMs and O.D. value, the callus soaked by conversion fluid is placed on Dual culture base and carries out Dual culture, 25 DEG C of light culture 3d are placed in screening culture medium and cultivate about 30d, and every 10d subculture once.Then transferred on division culture medium by the kanamycin-resistant callus tissue sifted out and break up about 20d, every 10d subculture once.The kanamycin-resistant callus tissue differentiating green seedling is transferred on root media and takes root, grow hardening after flourishing root system until about 7d, and calculating conversion institute obtains transgenic seedling number.Grown in field is transferred to after hardening 7d.

Wherein, inducing culture based formulas is: a large amount of+B5 trace+NB of N6 is organic+and molysite+copper cobalt mother liquor+2.5mg/L2,4D+0.6g/L acid hydrolyzed casein+2.878g/L proline(Pro)+0.5g/L glutamine+30g/L sucrose, with water preparation, adds plant gel 4g/L after adjusting pH to 5.8 ~ 5.9.

Dual culture based formulas is: a large amount of+B5 trace+NB of N6 is organic+and molysite+2.5mg/L2,4D+0.5g/L glutamine+0.6g/L acid hydrolyzed casein+10g/L glucose+30g/L sucrose, with water preparation, after adjusting pH to 5.2, add plant gel 4g/L.After sterilizing, about 50 DEG C add AS(Syringylethanone) 100 ~ 200 μ g/mL.

Screening and culturing based formulas is: a large amount of+B5 trace+NB of N6 is organic+and molysite+copper cobalt mother liquor+2.5mg/L2,4D+0.6g/L acid hydrolyzed casein+2.878g/L proline(Pro)+0.5g/L glutamine+30g/L sucrose, with water preparation, after adjusting pH to 5.8 ~ 5.9, add plant gel 4g/L.35mg/L Totomycin (purchased from Shanghai Niu Jin Bioisystech Co., Ltd) is added after sterilizing.

Differentiation culture based formulas is: MS inorganic+MS-B5 trace+MS is organic+and molysite+MS-copper cobalt mother liquor+0.05mg/L NAA+2.0mg/L Kinetin(kinetin)+30g/L sorbyl alcohol+2g/L caseinhydrolysate+30g/L sucrose, with water preparation, after adjusting pH to 5.8 ~ 5.9, add plant gel 4g/L.

The qualification of embodiment 3 transgenic positive strain

For detecting ubi:VP64-Os11g39000.2 gene at T2 for the process LAN situation in transgenic paddy rice, VP64 antibody is utilized to identify on protein level it, through SDS-PAGE protein electrophoresis → immunoblotting analysis → Immunofluorescence Reactions, Western Blot qualification result shows that transfer-gen plant exists target protein, the band (Fig. 3) and wild-type is not mixed out.

Concrete Western Blot experiment flow is as follows:

Appropriate amount of sample is put into the freezing rear grind into powder of liquid nitrogen, add the mixing of appropriate sample-loading buffer, centrifugal 10 minutes of 12000rpm; Get 5 μ l supernatant application of samples, with 90V voltage SDS-PAGE electrophoresis after 30 minutes, 120V electrophoresis 60-90 minute, the bottom arriving gel when tetrabromophenol sulfonphthalein can stop electrophoresis; Adopt half-dried transfer method transferring film after electrophoresis, and with ponceau staining fluid, film is dyeed, observe transferring film effect; After transferring film, film is put into containing 5% skim-milk PBST solution close room temperature close within 60 minutes or 4 DEG C, spend the night; Under room temperature, primary antibodie (VP64 antibody) is hatched 1 hour or 4 DEG C of overnight incubation, then washs 3 times with PBST, each 5 minutes; Two anti-under room temperature (goat-anti rabbit, purchased from Abmart, article No. M21002S) hatch 1 hour, then wash 3 times with PBST, each 5 minutes; Film adds substrate, exposes.

Wherein, VP64 antibody is that the rabbit source of being prepared as specific antigen according to the aminoacid sequence improvement on synthesis of VP64 by Ai Bimate biological medicine (Shanghai) Co., Ltd. resists more.

Embodiment 4 transgenic paddy rice phenotype analytical and species test analysis

By the present invention obtain transgenic paddy rice seed and wild-type (' kitaake ') rice paddy seed compare, find transgenic line seed obviously elongated, broaden.Species test data analysis shows, the length and width of transgenic paddy rice seed is all significantly greater than wild rice seed.

Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.

Claims (9)

1. a fusion rotein, is characterized in that, this fusion rotein comprises VP16 albumen and the rice transcription factor Os11g39000.2 of hsv (Herpes simplex virus).

2. fusion rotein according to claim 1, is characterized in that, described fusion rotein is (VP16) ₄-Linker-Os11g39000.2, wherein Linker is the aminoacid sequence shown in SEQ ID No.3.

3. the gene of fusion rotein described in coding claim 1 or 2.

4. the carrier containing gene described in claim 3.

5. the engineering bacteria containing gene described in claim 3.

6. gene described in fusion rotein described in claim 1 or 2 or claim 3 makes rice grain elongated, broadens and improves the application in thousand seed weight.

7. the application of rice transcription factor Os11g39000.2 gene in improvement rice grain proterties.

8. application according to claim 7, is characterized in that, it is by the downstream of the CDS sequence construct of rice transcription factor Os11g39000.2 gene to 4 transcription factor activation motif VP16, conversion of plant, and screening also finally obtains transgenic plant.

9. application according to claim 8, it is characterized in that, it is by the downstream of Gateway system constructing to 4 transcription factor activation motif VP16 by the CDS sequence of rice transcription factor Os11g39000.2 gene, rice transformation, thus render transgenic rice grain broadens elongated, improve seed thousand seed weight.