CN104673804B - A kind of paddy gene for adjusting Sucrose synthesis and its application - Google Patents

Abstract

The present invention provides a kind of paddy gene Os ER ANT1 for adjusting Sucrose synthesis, the gene delection causes rice Os ER ANT1 genes to inactivate, Sucrose synthesis is caused to be obstructed, cause rapid dead after the leaf phase of mutant 3, it is the house-keeping gene for having critical function to show Os ER ANT1, has important Pasitive Regulation Effect of Genseng to rice sucrose metabolism.The present invention is not only that the mechanism of Study On Rice Sucrose synthesis lays the foundation, and to study the biological function of Os ER ANT1 albumen and being provided the foundation using rice Os ER ANT1 genetically modified crops kinds.

Description

A kind of paddy gene for adjusting Sucrose synthesis and its application

Technical field

The invention belongs to biology field, more particularly to a kind of paddy gene Os-ER-ANT1 for adjusting Sucrose synthesis And its albumen and the application of coding.

Background technology

Rice is the important cereal crops in China, and rice in China sowing face accounts for the 1/4 of national cereal crops, rice it is effective Fringe, setting percentage, mass of 1000 kernel and grain-filling degree are that these factors have certain pass with glycometabolism an important factor for forming yield System.

Sucrose is one of photosynthetic major end products, is the master of long-distance transportation carbohydrate in most plants body Want form, it be only storehouse organ carry out every physiological activity provide energy, while be also certain plants for example sugarcane, beet, The main compound of the storages such as carrot.

The enzyme relevant with Sucrose Metabolism has three classes in plant, is invertase or G- fructofuranosidases (Inv), sucrose respectively Phosphate synthase (SPS), sucrose synthase (SUSy), wherein sucrose synthase are one of key enzymes of catalysing sucrose metabolism.Remove Outside this, regulation Sucrose synthesis related gene is found, new selection will be provided for crop improvement.

Atriphos (ATP) is energy carrier main in organism.ATP/ADP transport proteins are Eukaryotic one Kind adenylate transport vehicle, it is the main attemperator of plastid ATP concentration.Inventor to coding rice ATP/ADP by transporting egg The research of white encoding gene Os-ER-ANT1 genes, it is found that the gene can adjust the Sucrose synthesis of plant, the gene and light Cooperation is with closely related.

The content of the invention

Mechanism and photosynthetic mechanism for research non-plant sucrose synthesis, the present invention seeks to same regulation sucrose The gene Os-ER-ANT1 and its encoding proteins of synthesis and application.

The invention provides application of the Os-ER-ANT1 genes in regulation non-plant sucrose synthesis, the Os-ER-ANT1 bases The nucleotide sequence of cause is as shown in SEQ ID No.1, and amino acid sequence is as shown in SEQ ID No.2.Os-ER-ANT1 genes are Encode the paddy gene of ATP/ADP transport proteins.

Preferably, described plant is rice.

The invention provides application of the Os-ER-ANT1 genes in plant breeding.

Present invention also offers application of the Os-ER-ANT1 genes in genetically modified plants are cultivated.

The present invention also provides the primer pair for expanding Os-ER-ANT1 coding region genes.

Wherein, the length of primer pair each primer is 15 to 25 bases, such as：

Sense primer：5’-GGATCCAAATCCGCCGTCGACGATGCCA-3’（Nucleotides as shown in SEQ ID No.3 Sequence）

Anti-sense primer：

5’-ACTAGTTCATCTAGATTTCAATGCCCCTTTCATCTTG-3’（Nucleotides as shown in SEQ ID No.4 Sequence）

The beneficial effects of the present invention are：The Os-ER-ANT1 gene delections of the present invention, show as rice Os-ER-ANT1 Gene inactivates, and directly causes Sucrose synthesis to be obstructed, and causes rapid death after the leaf phase of mutant 3, passes through the experiment of the present invention, it was demonstrated that Os-ER-ANT1 is the house-keeping gene for having critical function, can adjust Sucrose synthesis in plant, to sucrose in plant Metabolism has important Pasitive Regulation Effect of Genseng, and genetic resources is provided to cultivate new variety of plant.

Brief description of the drawings

Fig. 1 is Os-ER-ANT1 mutation type surfaces in embodiment 1, water when being respectively 12 days, 15 days and 25 days in figure Rice Os-ER-ANT1 mutation type surfaces, wherein WT spend 11 in being.

Fig. 2 is Os-ER-ANT1 mutant Ds insertion mutations site in embodiment 2, and wherein A figures are that er-ant1 mutant exists Insertion point on genome；B figures are the structural representation of ER-ANT1 genes.

Fig. 3 is the phenotype photo of RNAi strains in embodiment 3.A figures are wild type and the phenotype of RNAi transfer-gen plants, B Figure is Os-ER-ANT1 gene expressions in wild type and RNAi transgenic lines.

Fig. 4 is the phenotype photo of revertant in embodiment 4.A figures are deletion mutant and back mutation sequencing knot Fruit, B figures are the phenotype of back mutation, and C figures are the gene expression abundance of OsER-ANT1 genes in revertant.

Fig. 5 is to sow the 10th day, 13 days and 16 days different time points soluble sugars in WT and mutant in embodiment 5 Content analysis.Fig. 5 A are the change curve of fructose, and Fig. 5 B are the change curve of glucose, and Fig. 5 C are the change curve of sucrose.

Fig. 6 be in embodiment 6 mutant in the phenotype without sucrose and under conditions of having sucrose.

Embodiment

Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Without departing substantially from spirit of the invention In the case of essence, the modifications or substitutions made to the inventive method, step or condition, the protection model of the present invention is belonged to Enclose.

PCUbi1390 carriers are conventional carrier in embodiment, and pEasy-T1 is commercially available；Rice varieties are flower in japonica rice 11；There are preservation in the conventional bacterial strain of Agrobacterium GV3101 and EHA105 bacterial strain, most Molecular Biology Labs.

Main agents in embodiment are：Restriction enzyme, Taq enzyme, T4 ligases, Pyrobest Taq enzymes, KOD, RNase A, M-MLV reverse transcriptase etc. are purchased from the biotech firm such as TAKARA (Dalian), Promega, NEB, Toyobo；dNTPs Purchased from Genestar companies；The small extraction reagent kit of plasmid and Ago-Gel QIAquick Gel Extraction Kit are purchased from Shanghai JaRa bioengineering Company；TRLzol RNA extracts kits are purchased from Invitrogen companies；MS culture mediums, agar powder, agarose, ammonia benzyl mould The antibiotic such as plain (Amp), kanamycins (Kan), gentamicin sulphate (Gen), rifampin (Rif) and DEPC, Galactose, Glucose, BSA, nylon membrane, nitrocellulose filter, LBMedium etc. are purchased from Sigma, nitrocellulose filter purchase From companies such as Amersham, Bio-Rad；Other chemical reagent used in embodiment are that import or domestic analysis are pure.

Primer used in embodiment is synthesized by Invitrogen companies, and is sequenced.

Embodiment 1Os-ER-ANT1 mutation type surfaces are analyzed

WT and Os-er-ant1 mutant plants grow in rice field, 12 days, at 15 days and 25 days, draw materials, clean, clap According to.Mutant phenotype is shown in Fig. 1.Leaf color and wild type no significant difference before the leaf phase of er-ant1 mutant 2（12 sky maps in Fig. 1）, three leaves Mutant is changed into light green after phase（15 sky maps in Fig. 1）, and it is rapid shrivelled (25 sky maps in Fig. 1).

Analyze in embodiment 2Os-ER-ANT1 mutational sites

The genomic DNA in mutant blade is extracted, the 5 ' and 3 ' of Ds insertion mutations is expanded using TAIL-PCR method End, Ds insertion points are located on the gene of the coding OsER-ANT1 on No. 11 chromosomes, refer to Fig. 2.

(1995) such as method Liu using TAIL-PCR【Liu Y G,Mitsukawa N,Oosumi T et al.Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR.Plant Journal,1995,8(3):457- 463】To expand the sequence of Ds introns both sides, concrete operations are as follows：

The special primer design of right boundary uses Primer Premier5.Side of the reaction condition with reference to (1995) such as Liu Method is carried out（Table 1）, react the primer and be shown in Table 2, the third round PCR primer that specific amplified obtains is used for being sequenced.

Ds3 ' ends TAIL-PCR the amplifications of mutant.The flanking sequence amplification of Ds insertion points uses TAIL-PCR, 3 wheel spies Different primer is followed successively by Ds3 ' -1a, Ds3 ' -2a, Ds3 ' -3a, random primer AD3.Public affairs are sent after 3rd wheel PCR primer electrophoresis detection Department's sequencing, primer used is sequenced and takes turns primer for TAIL-PCR the 3rd.

Ds5 ' ends TAIL-PCR the amplifications of mutant.The flanking sequence amplification of Ds insertion points uses TAIL-PCR, expands Condition be shown in Table 1,3 wheel special primers be followed successively by Ds5 ' -1a, Ds5 ' -2a, Ds5 ' -3a, random primer AD1.3rd wheel PCR productions Thing electrophoresis detection Hou Song companies are sequenced, and it is Ds5 ' -3a that primer used, which is sequenced,.

Table 1TAIL-PCR cycling conditions

Table 2TAIL-PCR the primers

L-Primer and R-Primer represents the special nested primers in the end of Ds elements 5 ' and 3 ' ends, AD respectively Primer represents the relatively low arbitrary degenerate primer of melting temperature.

After the adjacent sequence in Ds sides that mutant is obtained by TAIL-PCR, utilize NCBI (www.ncbi.nlm.nih.gov/) Database, plain database is searched using BLASTn programs, to obtain Ds insertion points and corresponding gene.Analysis result shows purpose Gene is located on the BAC clones AC146592 of the chromosome of rice the 11st（A schemes in Fig. 2）.Cloning of full length 19234bp, from 11876-14462 is purpose constant gene segment C, corresponding to rice genome annotations database RGAP（http:// rice.plantbiology.msu.edu/）LOC_Os11g43960 gene locis, this gene is by 3 extrons and two Containing sub- composition, Ds be inserted in First Intron away from downstream exon 652bp at（B schemes in Fig. 2）, it is Os-ER- by the unnamed gene ANT1, its ORF (990bp) encode 329 amino acid.

Embodiment 3Os-ER-ANT1RNAi vector constructions and transfer-gen plant phenotypic analysis

In order to fully understand influence that Os-ER-ANT1 synthesizes to non-plant sucrose, the present invention therefrom spends in 11 rice and cloned The gene Os-ER-ANT1 of coded plant ATP/ADP transport proteins.According to sequence analysis, primer is designed by the partial order of the gene Row（306bp）Amplify and, be connected on the over-express vector pCAMBIA2300-35S-OCS with 35S promoter, build RNAi expression vector, Os-ER-ANT1RNAi.Primer used is：

Sense primer：5’-ACTAGTTTGGGCTGGGCAATAACTAC-3’（Nucleotides sequence as shown in SEQ ID No.3 Row）

Anti-sense primer：5’-CTCGAGATCCCCATTTGCACATCATT-3’（Nucleotides sequence as shown in SEQID No.4 Row）

The tool that will be connected to after Os-ER-ANT1RNAi on the carrier pCAMBIA2300-35S-OCS with 35S promoter Body method is：First using rice cDNA as template, using sense primer and anti-sense primer by Os-ER-ANT1 code areas 306bp's Specific section, which amplifies, to be come（SEQ ID NO.3 and 4 primer）, by PCR primer using XhoI and SpeI restriction enzyme sites with PUCCRNAi intermediate carriers connect, and insert positive fragment, after identification, are named as pUCCRNAi-Os-ER-ANT1-sense.Will PUCCRNAi-Os-ER-ANT1-sense and PCR primer the connection of T4DNA enzymes, are named as with after XbaI and SalI digestions after identification pUCCRNAi-Os-ER-ANT1RNAi.PCAMBIA2300-35S-OCS and pUCCRNAi-Os-ER-ANT1RNAi are used respectively After XhoI and SpeI digestions, the purpose fragment that length is 821bp is building up on pCAMBIA2300-35S-OCS carriers, connected Product is named as pCAMBIA2300-35S-OCS-Os-ER-ANT1RNAi.

PCAMBIA2300-35S-OCS-Os-ER-ANT1RNAi is imported in wild rice using agrobacterium-mediated transformation In spending 11, transfer-gen plant is obtained.Its transcriptional level spends 11 obvious downwards in comparing, it is prominent to show that it is lowered for Os-ER-ANT1 Variant, the gene are suppressed.Phenotypic analysis shows that leaf color turns yellow after the transfer-gen plant tri-leaf period, and rapid shrivelled, with DS Insertion mutation body is similar.

The acquisition of embodiment 4Os-ER-ANT1 revertants and phenotypic analysis

Because mutation type surface and the Ds factors are close linkages, if mutant and plant containing Ac hybridized, because Ds Swivel base again can occur in the presence of Ac transposases for the factor, may cause the gene recovery function being destroyed originally, so mutation Phenotype can produce change, may be returned to wild.Ac/Ds transposons often produces when inserting genome at insertion point 8bp or so repetitive sequence.After these factor swivel bases again, often have repetitive sequence and stay（Mckenzie et al, TheorAppl Genet,2002,105:23-33）.In order to confirm obtained back mutation, Ds factor insertion points are surveyed Sequence, it is possible to find the trace left after Ds factor swivel bases（footprint）.In Ds insertion points both sides, according to paddy gene Group sequencing data design special primer, detects the Ds factors and swivel base trace.

Female parent is made with Ac homozygotes, mutation heterozygote makees paternal hybrid, selects and remain 18 F1 plant, in case screening reply is prominent Become.F2 is planted, emergence investigates field phenotype after 2 weeks, then sprays Basta, transplants normal resistant plant, and transplanting samples after 3 weeks Detection.With Dsyb (ATTGGTGTGAGGCCAACATT), Dsyc (AGCGGGAGTACAACCACAAC), Ds3 ' -1a (GGTTCCCGTCCGATTTCGACT) detection Ds whether there is, and swivel base trace is detected with Dsyb, Dsyc.F₂Generation 18 strains of plantation System, each strain detect 20-80 strains, DNA990 strains are extracted altogether, wherein leaving trace after 14 plants of Ds swivel bases.

11, Ds donors, back mutation strain are spent in being expanded with Dsyb, Dsyc, makees sequencing primer with Dsyc, PCR primer is direct Sequencing, sequencing, which compares, to be found, Ds after swivel base, leaves the trace (tgtggt) of 6 bases again, with Ds insertions caused by 8bp Base repeats (gttgtggt) sequence and compares few two bases（A schemes in Fig. 4）.Phenotype, which is observed, to be shown, back mutation and wild type Phenotype is no different（B schemes in Fig. 4）, and RT-PCR analysis shows back mutation destination gene expressions are recovered（C schemes in Fig. 4）.With Upper result shows that Os-ER-ANT1 is object of this investigation gene, and Ds insertions cause Os-ER-ANT1 genes to inactivate, and are mutant Os-er-ant1 produces the reason for yellow phenotype.

Soluble sugar is analyzed in embodiment 5Os-ER-ANT1 mutant

The leaf phase of rice 3 is the critical period that plant changes from heterotrophic growth to autophyting growth.Due to mutant after 3 leaf phases it is fast It is fast dead, thus it is speculated that it may be caused by energy deficiency.Therefore, the present invention analyzes mutant Soluble sugar contents part content Change.Comprise the following steps that：

In order to analyze leaves of plants piece candy part comprehensively, WT and Os-er-ant1 mutant is planted in rice field, after planting 10 days, The morning 7 of 13 days and 16 days:00 point and afternoon 19:00 point separately sampled, can using the fresh blade of efficient liquid phase chromatographic analysis The content of dissolubility sugar.

Measurement result shows that cyclically-varying is presented in the change of WT lines its fructose, dextrose and saccharose, under it Noon soluble sugar part content is above the morning（Fig. 5 A, Fig. 5 B, Fig. 5 C）.After planting afternoon of 13 days in 6 measurement results （PM）Measured value is maximum, and every gram of blade fructose, dextrose and saccharose content are respectively 6.67mg, 8.39mg and 17.23mg.Mutation Although the fructose of body plant, glucose content are low compared with wild type, soluble sugar part content on the same day is above the morning in afternoon Cyclically-varying is still presented, its maximum also appears in the afternoon of after planting 13 days, respectively 2.83mg/g FW and 3.44mg/g FW, this shows that fructose in mutant, glucose metabolism are still regulated and controled by circadian rhythm, closely related with photosynthesis.And it is mutated Body plant after one day photosynthesis cane sugar content almost without increase, even below morning initial value, with wild type most Big value（17.23mg）Comparatively speaking, the corresponding period cane sugar content of mutant plants（2.34mg）Substantially less than wild type, show Mutant cane sugar content is significantly reduced, and its metabolic patterns is also changed.

In summary, show to have impact on after Os-ER-ANT1 gene knockouts the metabolism of rice soluble sugar, and to sucrose The influence of metabolism is maximum.

The addition experiment of embodiment 6Os-ER-ANT1 organic matters

Sucrose is the most important transport form of photosynthesis of plant assimilation products, for growing to pass for higher plant It is important.The result of embodiment 5 shows that Os-ER-ANT1 gene delections cause mutant blade cane sugar content and significantly reduced.According to This speculates that supplement sucrose ought to can alleviate mutation type surface.In order to analyze the influence that sucrose grows to mutant, the present invention according to Related experiment has been carried out according to step as described below, it is specific as follows：

In order to obtain the mutant seeds of homozygosis, the present invention is obtained by following 2 steps：

（1）Seed to be detected is cut into half endosperm（Without embryo）, DNA is extracted, detects genotype；Part containing embryo preserves It is standby.

（2）According to Rice Genome Sequence pair of primers is designed in Ds insertion points both sides（DsL: CCAATGTCATCCGATACTTCC,DsR:GCCATACAGATAACAAGGGTTC）, their binding site is transposons respectively The Rice Genome Sequence of both sides.In addition a primer (Ds5 ' -1a is designed inside Ds transposons close to end: ACGGTCGGGAAACTAGCTCTAC) detection Ds whether there is.Expanded simultaneously using this three primers, it is pure that mutation can be distinguished simultaneously The plant of fit, mutation heterozygote and wild type three types.

WT the and Os-er-ant1 mutant seeds that above-mentioned detection is confirmed, are seeded in and contain or not contain the 1/ of sucrose In 2MS culture mediums, growth observes phenotype after 15 days.

As a result show, wild type seedlings are in the culture medium containing sucrose（2%）With sucrose free culture medium growing state without Notable difference.But mutant is significantly better than without sucrose culture medium in the culture basal growth containing sucrose, show to supplement sucrose energy Enough alleviate mutation type surface.

In summary, the present invention draws a conclusion：Ds insertions cause rice Os-ER-ANT1 genes to inactivate, and cause Sucrose synthesis It is obstructed, causes rapid death after the leaf phase of mutant 3, and mutation type surface can significantly be alleviated by adding sucrose.It is indicated above, Os-ER- ANT1 is the house-keeping gene for having critical function, has important Pasitive Regulation Effect of Genseng to rice sucrose metabolism.

Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, without departing from the technical principles of the invention, some improvements and modifications can also be made, these improvements and modifications Also it should be regarded as protection scope of the present invention.

Claims (2)

1. Application of the 1.Os-ER-ANT1 genes in regulation rice sucrose synthesis, it is characterised in that the Os-ER-ANT1 genes Nucleotide sequence is as shown in SEQ ID No.1, and the amino acid sequence that it is encoded is as shown in SEQ ID No.2.
2. Application of the 2.Os-ER-ANT1 genes in transgenic paddy rice is cultivated, the Os-ER-ANT1 gene nucleotide series are such as Shown in SEQ ID No.1.