CN103820480B - The application of a kind of rice transcription factor in improvement rice high yield proterties - Google Patents
The application of a kind of rice transcription factor in improvement rice high yield proterties Download PDFInfo
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- CN103820480B CN103820480B CN201410083584.3A CN201410083584A CN103820480B CN 103820480 B CN103820480 B CN 103820480B CN 201410083584 A CN201410083584 A CN 201410083584A CN 103820480 B CN103820480 B CN 103820480B
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Abstract
The present invention relates to the application of a kind of rice transcription factor in improvement rice high yield proterties, belong to genetically engineered field.It utilizes transcription factor activation motif VP64 and rice transcription factor Os01g60960.1 gene fusion to build to obtain composing type transcription factor, and be transformed into farm crop as in paddy rice, thus improve the output of paddy rice.There is important theory value for illustrating adjusting and controlling rice High-yielding Mechanism in detail, and can transgenic approach be passed through, improve the output of paddy rice, therefore also significant in production practice.
Description
Technical field
The present invention relates to genetically engineered field, specifically, relate to the application of rice transcription factor Os01g60960.1 gene.
Background technology
Paddy rice (OryzasativaL.) is one of most important Three major grain crops in China and the whole world, is the staple food of world's population over half, is also the model plant of an important functional gene research.The attention of relative genetics and molecular biology research extremely investigator always, the regulation and control of transcriptional level are the important way of gene expression regulation.The research of current increasing production of rice comparatively depends on limited Rice Germplasm Resources, and traditional cross-breeding advantage weakens gradually, and Transgenic Rice technology likely excavates the potentiality that paddy rice is increased production further.
In vegitabilia, the plant that can form seed accounts for more than 2/3rds of plant total, and as important organ of multiplication, seed is simultaneously also for people provide food source, and paddy rice is exactly important representative wherein, and seed source is in the ovule of after fertilization.From molecular biological angle, the Development and germination of seed be one orderly, optionally genetic expression process.And transcription factor serves critical effect in the accuracy controlling of genetic expression.
Os01g60960.1 is a member of LBD family, but due to LBD gene be a gene family specific to newfound plant, about the exact function of LOB structural domain in development of higher plants it be unclear that.But, still for we understand its function, part clue is provided to the initial analysis of several LBD gene functions of having cloned.LOB is first gene belonging to LBD gene family be separated, and it can not observe obvious phenotypic mutation when transposon inserts the afunction caused, and shows that LOB exists functional redundancy to a certain degree in the growth course of Arabidopis thaliana.But the fusion expression vector transformed wild type plant built with the 35S strong promoter of Caulimovirus and LOB encoding sequence, can be observed that plant is short and small, blade upsweeps, petiole and bennet shortens, the change (Shuaietal., 2002) of the obvious phenotype such as floral organ deformity.Another LBD gene family member---AS2 in Arabidopis thaliana, its mutant as2 presents blade heterauxesis or deformity, vein ateliosis isophenous, then disclose morphogenesis (Serrano-Cartagenaetal., 1999 that part LBD gene may take part in higher plant organ; Orietal., 2000; Sunetal., 2000; Semiartietal., 2001:Xuetal., 2003).The transgenic Arabidopsis plants of dystopy process LAN AS2 has occurred cotyledon, the blade of shorter primary root, narrow and small and upper volume and calyx abaxial side forms burr shape proliferation, bennet is bent downwardly isophenous; Meanwhile, the process LAN organizing microscopic examination also to disclose AS2 disturbs the foundation of the normal nearly axle polarity far away of lateral organ, causes the cell type of abaxial side by cell type Some substitute (Linetal., 2003 of adaxial and its surface; Nakazawaetal., 2003).The homologous gene ASL1 (also known as LBD36) that AS2 sibship in Arabidopis thaliana is nearest is LBD gene family member equally, and its process LAN can cause colored and that angle fruit is sagging phenotype (Chalfun-Junioretal., 2005; Nakazawaetal., 2003).The afunction mutant asl1 of ASL1 does not observe obvious abnormal phenotype, but asl1/as2 double-mutant shows, and narrow, the inner floral organ of calyx exposes, calyx and the petal outwards mutant phenotype such as curling, Blooming, this shows that ASL1, As2 take part in the growth of floral organ simultaneously and there is certain functional redundancy (Chalfun-Junioretal., 2005) in this stage.
First LBD gene of cloning in paddy rice---ARL1 (ADVENTITIOUSROOTLESS1), or claim CRL1 (CROWNROOTLESS1), the sudden change of its afunction shows as and can not form normal adventive root/from taking root, the forfeiture of growth hormone susceptibility, the geotropic forfeiture isophenous of root growth, but overground part does not have obvious paramophia (Liuetal., 2005; Inukaietal., 2005).Nearest research has also been separated a LBD gene family member in paddy rice, preliminary designation is DH1 (DEGENERATEDHULL1), and dh1 mutant presents the clever shell disappearance of grain husk flower or premature termination, Pistil And Stamen numerical abnormality, the sterile isophenous of partial male of glume development; The transgenic paddy rice of overexpression DH1 shows as that plant is short and small, internode and grain husk flower branch stalk shortens, blade hangs low isophenous (Li Aihong, 2006, personal communication) downwards.These results show the LBD gene in paddy rice, compared with Arabidopis thaliana, may have more importantly act in the development of lateral organ.
VP64 is that 4 VP16 functional domain motifs merge composition, is a class enhanser.VP16 finds in animal virus gene, has now been widely applied in plant, is mainly used in the research of the transcriptional control of plant gene.Transcription factor effect in vivo can be divided into two kinds substantially: a kind of is transcriptional enhancer, and another kind is Transcription inhibition.After transcription factor and VP16 functional domain motif merge, it will strengthen the function of transcription factor, thus in transfer-gen plant, occur more obvious character mutation.
Summary of the invention
The invention provides the application of a kind of rice transcription factor in improvement rice high yield proterties.
In order to realize object of the present invention, the present invention provide firstly a kind of fusion rotein, and described fusion rotein is (VP16)
4-Linker-Os01g60960.1;
Wherein, Linker is in series by 39 flexible amino acid, and its sequence is as shown in SEQIDNo.9, and its nucleotide sequence is as shown in SEQIDNo.3; VP16 is the VP16 albumen from hsv; Os01g60960.1 is rice transcription factor Os01g60960.1.
The aminoacid sequence of described rice transcription factor Os01g60960.1 is as shown in SEQIDNo.2, or this sequence is through replacing, lacking or add one or several amino acids formed aminoacid sequence with same function.
Wherein, (VP16)
4i.e. VP64, merge with GlySer two acids apart the enhanser formed by 4 VP16 functional domain motifs (AspAlaLeuAspAspPheAspLeuAspMetLeu), its sequence is as shown in SEQIDNo.10, and its nucleotide sequence is as shown in SEQIDNo.4.
The present invention also provides the carrier of the gene containing encoding said fusion protein.
The construction process of described carrier is as follows:
(1) in plant transcription factor database, Os01g60960.1 gene is found, according to its sequences Design pcr amplification primer pair, it is forward primer F:5'-CAAAAAAGCAGGCTTCATGTCCACAGAGAGGGAAAGAC-3' and reverse primer R:5'-CAAGAAAGCTGGGTCACCAAAGTATGCAGCATGCTC-3';
(2) with the total cDNA of the fine paddy rice of wild Japanese for template, carry out PCR and obtain Os01g60960.1 complete sequence;
(3) PCR primer is cloned on connection pDONER cloning vector, obtains the identical sequence with goal gene through order-checking qualification;
(4) sequence comprised with binary expression vector right boundary is for frame sequence, pass through vitro recombination, ubipromoter-VP64-Gateway is expressed unit, 35Spromoter-asRED expresses unit and 35Spromoter-hyg expression unit constructs with it, obtains the complete sequence of carrier nVP64-hyg-asRED as shown in SEQIDNo.5;
(5) reacting by LR N end Os01g60960.1 being building up to its order ground gene has merged on the plant expression vector nVP64-hyg-asRED of VP64 label, and obtain carrier ubi:VP64-Os01g60960.1, carrier complete sequence is as shown in SEQIDNo.6.
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, MethodforPlantMolecularBiologyVIII, AcademyPress, NewYork, 411-463 page; Geiserson and Corey, 1998, PlantMolecularBiology, 2ndEdition).
The present invention also provides the engineering bacteria of the gene containing encoding said fusion protein.
The present invention also provides a kind of construction process of transgenic rice plant, be specially, adopt agriculture bacillus mediated method, aforementioned bearer is proceeded in Rice Callus, transform with the AAM conversion fluid containing inductor and Agrobacterium, material after conversion through the exercise of Dual culture-screen-break up-take root-transgenic seedling and transplanting, screening transgenic rice plant.
The present invention also provides the gene of encoding said fusion protein in improvement rice high yield proterties.
Present invention also offers the primer pair for amplifying rice transcription factor Os01g60960.1 gene, it is forward primer F:5'-CAAAAAAGCAGGCTTCATGTCCACAGAGAGGGAAAGAC-3' and reverse primer R:5'-CAAGAAAGCTGGGTCACCAAAGTATGCAGCATGCTC-3'.
The present invention also provides the application of rice transcription factor Os01g60960.1 gene in adjusting and controlling rice high-yield character further.
Aforesaid application is by the CDS sequence of rice transcription factor Os01g60960.1 gene by the downstream of Gateway system constructing to 4 transcription factor activation motif VP16, rice transformation, thus improvement transgenic paddy rice output.
The present invention utilizes transcription factor activation motif VP64 (i.e. 4 transcription factor activation motif VP16) and rice transcription factor Os01g60960.1 gene fusion to build first and obtains composing type transcription factor, and be transformed in paddy rice, thus improvement rice high yield proterties, improve the output of paddy rice.For illustrating water transfer rice High-yielding Mechanism in detail, there is important theory value, therefore also significant in production practice.
Accompanying drawing explanation
Fig. 1 is nVP64-hyg-asRED Vector map in the embodiment of the present invention 1;
Fig. 2 is ubi:VP64-Os01g60960.1 Vector map in the embodiment of the present invention 1;
Fig. 3 is that PCR of the present invention detects VP64-Os01g60960.1 transgenic positive strain, and wherein WT is wild rice ' kitaake ', and V2590H-24, V2590H-44 are VP64-Os01g60960.1 transgenic paddy rice strain;
Fig. 4 is that the phenotype of transgenic line high-yield character of the present invention compares, and wherein the left side first basin WT is wild rice ' kitaake ', and the left side second basin V2590H-24, the left side the 3rd basin V2590H-44 are transgenic paddy rice strain;
Fig. 5 is transgenic line high-yield character data statistic analysis of the present invention, and wherein WT is wild rice ' kitaake ', and V2590H-24, V2590H-44 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.
Carrier source used in experimentation:
PDONER is purchased from invitrogen;
35Spromoter-asRED the Chinese Academy of Agricultural Sciences provides;
Ubipromoter-VP64 the Chinese Academy of Agricultural Sciences provides;
35Spromoter-hyg the Chinese Academy of Agricultural Sciences provides;
Ubi:VP64-Os01g60960.1 is building up on ubipromoter-VP64 carrier by Gateway cloning process;
Engineering bacteria EHA105 is provided by the Chinese Academy of Agricultural Sciences.
The separation of embodiment 1Os01g60960.1 gene and plant expression vector construction
Os01g60960.1 gene is found in plant transcription factor database, according to its sequences Design pcr amplification primer, according to its sequences Design pcr amplification primer (F1:5'-CAAAAAAGCAGGCTTCATGTCCACAGAGAGGGAAAGAC-3' and reverse primer R1:5'-CAAGAAAGCTGGGTCACCAAAGTATGCAGCATGCTC-3').With the total cDNA of the wild-type fine paddy rice of Japan for template, carry out PCR and obtain Os01g60960.1 complete sequence, its nucleotide sequence is as shown in SEQIDNo.1.
PCR is carried out according to PrimeSTAR polymeric enzymatic amplification system and response procedures.Two-wheeled PCR is comprised in this process, the primer gene primer (F1 and R1) adding part adaptorattB joint of first round PCR, and the second template of taking turns PCR primer of the first round, and the complete adaptorattB primer (attB5'adaptor:5'-GTGGGGACAAGTTTGTACAAAAAAGCAGGCTTC-3', attB3'adaptor:5'-GTGGGGACCACTTTGTACAAGAAAGCTGGGTC-3') of primer.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, Os01g60960.1 is building up to nVP64-hyg-asRED (Fig. 1, carrier complete sequence is as shown in SEQIDNo.5) on, obtain carrier ubi:VP64-Os01g60960.1 (Fig. 2, carrier complete sequence is as shown in SEQIDNo.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-Os01g60960.1, 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/LNAA+2.0mg/LKinetin (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
Whether be integrated in rice genome to detect Os01g60960.1 gene, extract wild-type and transgenic paddy rice leaf DNA respectively, the plant expression vector at gene two ends designs one section of primer and carries out PCR reaction, qualification transfer-gen plant, PCR primer is detected, found that, transgenic paddy rice all amplifies object band (Fig. 3), and without this object band in wild rice, PCR primer is correct goal gene sequence through sequencing analysis, determines that Os01g60960.1 gene is inserted in rice genome by agriculture bacillus mediated method thus.
Embodiment 4 transgenic paddy rice phenotype analytical and species test analysis
Transgenosis and wild rice are compared, can significantly find out that transfer-gen plant is higher than wild-type phenotype, and show suitably flower in evening (Fig. 4) cycle spending.Significantly can find out that the single plant yield of transgenic paddy rice is significantly improved (Fig. 5) compared with wild-type by the data results of species test.The average single plant yield of one of them strain wild-type is 7.25g, and the average single plant yield of transfer-gen plant is 13.61g.
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 (1)
1. the application of rice transcription factor in improvement rice high yield proterties, it is characterized in that the nucleotide sequence of described rice transcription factor Os01g60960.1 is as shown in SEQIDNo.1, the aminoacid sequence of its proteins encoded is as shown in SEQIDNo.2.
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| CN102786599A (en) * | 2012-08-01 | 2012-11-21 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os05g39950 gene |
| CN103255148A (en) * | 2013-04-28 | 2013-08-21 | 中国农业科学院作物科学研究所 | Application of paddy rice transcription factor Os01g18440 gene |
| CN103266114A (en) * | 2013-05-10 | 2013-08-28 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os01g23760 gene |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102786599A (en) * | 2012-08-01 | 2012-11-21 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os05g39950 gene |
| CN103255148A (en) * | 2013-04-28 | 2013-08-21 | 中国农业科学院作物科学研究所 | Application of paddy rice transcription factor Os01g18440 gene |
| CN103266114A (en) * | 2013-05-10 | 2013-08-28 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os01g23760 gene |
Non-Patent Citations (2)
| Title |
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| LBD 基因家族在高等植物中的研究进展;李爱宏 等;《分子植物育种》;20061231;第4卷(第3期);301-308 * |
| NM_001051204;GenBank;《GenBank》;20100608;CDS和ORIGIN部分 * |
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