CN102604971B - Rice moderate leaf-rolling mutator RL14 and application thereof - Google Patents

Abstract

The invention discloses a nucleotide sequence and a coding protein sequence of a rice moderate leaf-rolling mutator RL14, thus providing a powerful tool for rice transgenic research and promoting the breeding research on high-yield and high-quality rice.

Description

Paddy rice moderate leaf roll mutant gene RL14 and application thereof

Technical field

The invention belongs to gene engineering technology field, relate to a kind of paddy rice mutator gene, also relate to the application of this mutator gene.

Background technology

Paddy rice (Orvza sativa L.) is one of most important food crop in the world, and there is the population of nearly half in the whole world take rice as staple food.Along with the continuous increase of world population and the continuous decrease of cultivated area, grain security becomes worldwide problem.Paddy rice is C3 plant, and photorespiration is stronger, and effectively photosynthetic rate is lower, and especially in the filling stage of high temperature dry season district paddy rice, because temperature is high, its photorespiration will be wasted a large amount of photosynthates, not only can affect output, also can have a strong impact on a meter matter.Therefore, improving effective photosynthetic rate is an effective way that improves rice yield and improve rice matter.Reach this purpose, can realize by the net photosynthesis that increases leaf area index (LAI) or unit leaf area, but because the mutual shading of high leaf area index and blade is a pair of sharp-pointed contradiction, and paddy rice appropriateness leaf roll is conducive to keep upright blade, improves group structure and light transmission, improve optical energy utilization efficiency, therefore, can improve by the polymerization of moderate leaf roll and other high light efficiency gene the effective photosynthetic rate of paddy rice.But there is larger difference in the rice leaf roll material of having found at present in the amount of crimp of blade, and most utility values on producing are not high.Therefore, to have the rolled leaf gene of breeding utility value be a important topic in the rice breeding to screening and identification.

Summary of the invention

In view of this, the object of the present invention is to provide a kind of paddy rice moderate rolled leaf gene, for Transgenic Rice research provides strong instrument, and then promote the breeding research of high yield and high quality paddy rice.

In previous research work, the contriver utilizes the extensive paddy rice moderate leaf roll mutant body (called after that has obtained an inheritance stability for No. 10 of EMS mutagenesis Elite restorer line red silk Rl14), show as blade one side height and be curled into tubular, the little volume of opposite side or do not roll up, with in the rice leaf of having found at present, little volume phenotype is not all identical.Research finds that this proterties is by recessive single-gene (called after RL14) control, this gene major effect sclerenchymatous cell, the formation of mesophyll cell secondary wall and the differentiation of vein conduit, the Water Transportation of remote effect vein conduit → mesophyll cell → leaf cell gap transport pathway causes the bulliform cell variation, thereby causes leaf roll.The molecule marker positioning result shows, in the scope of this assignment of genes gene mapping 24.5kb between the 10th karyomit(e) long-armed Indel mark SID2 and SSR mark SW24.

On the basis of said gene location, the present invention at first by predictive genes, homology search and gene order diversity ratio, and is preliminary definite RL14Gene be 2OG-Fe (II) oxidase gene ( Os10g40960.1); Subsequently, the present invention is with paddy rice moderate leaf roll mutant body Rl14Be material, cloned the cDNA of this gene and check order that the result shows, RL14Full length gene cDNA is 837bp, is comprised of 3 exons, and 278 amino acid of encoding are altogether compared with extensive No. 10 wild type genes of red silk, RL14Gene has the conversion of T-C at the 458th bit base, cause its proteins encoded at the 153rd amino acids generation Isoleucine (Ile, I) to the variation of Threonine (Thr, T), and this mutational site is in 2OG-Fe (II) the oxydase structural domain of high conservative; Again, the present invention has made up RL14Wild type gene recombinant plant overexpression vector and with its rice transformation moderate leaf roll mutant body Rl14, character observation shows that the transgenic positive plant leaf is open and flat, reverts to the wild-type blade profile fully; It is normal that the tissue slice observation shows that transgenic positive plant leaf bulliform cell form is compared obvious recovery with mutant, thereby determined RL14Gene be 2OG-Fe (II) oxidase gene ( Os10g40960.1), its nucleotide sequence is shown in SEQ ID No.1, and coding protein sequence is shown in SEQ ID No.2.

Based on above-mentioned result of study, mutator gene RL14Be paddy rice moderate rolled leaf gene, can be used for for example molecular breeding of extensive No. 10 leaf rolling characteristics of red silk of paddy rice.Based on mutator gene RL14Make up plant expression vector and transform the rice cell of high-quality background, again the rice cell that transforms is cultivated into the leaf roll plant, can realize by transgenosis the rapid polymerization of the excellent genes such as leaf roll, thereby rapidly by the desirable rice varieties of cultivating high yield and high quality.

Beneficial effect of the present invention is: the invention provides a paddy rice moderate leaf roll mutant gene RL14Nucleotide sequence and coding protein sequence thereof, for Transgenic Rice research provides strong instrument, can promote the breeding research of high yield and high quality paddy rice.

Description of drawings

Fig. 1 is RL14The heredity of gene and physical map, wherein A is RL14First location, between the 10th karyomit(e) long-armed SSR mark RM1162 and RM3123; B is RL14Fine Mapping, on BAC clone AC079874, between mark SID2 and the SW24 in the scope of 24.5kb; C is RL14Candidate gene Os10g40960.1Structure and the sudden change position; D is mutant Rl14With wild-type WT's Os10g40960.1Gene expression amount.

Fig. 2 is RL14The agarose gel electrophoresis of gene RT-PCR product is identified figure, and wherein 1 swimming lane is PLAS 2000 DNA Marker, and 2 swimming lanes are the extensive No. 10 RT-PCR product of wild-type red silk, and 3 swimming lanes are mutant Rl14The RT-PCR product, 4 swimming lanes are blank.

Fig. 3 is RL14The sequence alignment figure of gene and wild type gene, RL14Gene cDNA is the 458th transformation that T-C has occured.

Fig. 4 is RL14The sequence alignment of gene coded protein and wild type gene proteins encoded and conserved domain analysis chart, RL14Gene coded protein is the 153rd variation that I-T has occured, and this mutational site is in 2OG-Fe (II) the oxydase structural domain interior (underscore part) of high conservative.

Fig. 5 is RL14The design of graphics of wild type gene recombinant plant overexpression vector.

Fig. 6 is mutant Rl14Complementary phenotype analytical, wherein A is respectively wild-type WT, mutant Rl14, the transgenic positive plant C-rl14B is respectively wild-type WT, mutant Rl14, the transgenic positive plant C-rl14Blade; C is respectively wild-type WT, mutant Rl14, the transgenic positive plant C-rl14The leaf tissue square section; D is respectively wild-type WT, mutant Rl14, the transgenic positive plant C-rl14Blade RL14Gene expression analysis.

Fig. 7 is RL14The phylogenetic analysis of gene coded protein is with paddy rice Q94LP4Affinity nearer, with Chinese sorghum C5WZM7Affinity take second place.

Embodiment

In order to make the purpose, technical solutions and advantages of the present invention clearer, below in conjunction with accompanying drawing the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in the preferred embodiment, usually according to normal condition, the molecular cloning experiment guide (third edition for example, J. the work such as Pehanorm Brooker, Huang Peitang etc. translate, Science Press, 2002) described in condition, or the condition of advising according to manufacturer.

The material that uses in the preferred embodiment: wild-type red silk extensive No. 10 and paddy rice moderate leaf roll mutant body Rl14Cultivated by Southwestern University's rice research; M-MLV reversed transcriptive enzyme, high-fidelity DNA polysaccharase PFU, T4 DNA ligase enzyme, Trizol test kit, dna gel reclaim test kit, plasmid extraction kit available from TaKaRa company; Penbritin (Ampicillin, Amp) is Sigma company product; Primer is synthetic to be finished by the handsome Bioisystech Co., Ltd in Shanghai with dna sequencing; Other chemical reagent is available from Beijing ancient cooking vessel state biotechnology limited liability company.

One, paddy rice moderate leaf roll mutant gene RL14Prediction

Right in early stage RL14On the basis of gene Fine Mapping, the present invention compares (http://blast.ncbi.nlm.nih.gov/) and functional complementation analysis online by online predictive genes (http://mendel.cs.rhul. ac.uk), BLAST, and is preliminary definite RL14Gene be 2OG-Fe (II) oxidase gene ( Os10g40960.1) (Fig. 1).

All there is ortholog albumen in 2OG-Fe (II) oxidase gene family in Chinese sorghum, corn, paddy rice, Arabidopis thaliana, coptis japonica Makino, grape, castor-oil plant, that has wherein reported has Arabidopis thaliana SRG1 and a coptis japonica Makino NCS1.In coptis japonica Makino, NCS1 and PR10 albumen is synthesizing of catalysis berberine together, but its actual functional capability it be unclear that.

Two, Paddy rice moderate leaf roll mutant gene RL14Clone, order-checking and functional verification

1, paddy rice moderate leaf roll mutant gene RL14Clone and sequence

According to paddy rice Japan fine 2OG-Fe (II) the oxidase gene sequence (AK068525) of GenBank login, utilize 1 pair of special primer of Vector NTI software design: upstream primer RL14F:5 '-ggacacaacctgaaagcaggttc-3 ' (SEQ ID No.3); Downstream primer RL14R:5 '-tatgagtcagtttctaagttcgttcg-3 ' (SEQ ID No.4).

The rice moderate of fetching water respectively leaf roll mutant body Rl14With the wild-type red silk spire 2g in extensive No. 10 two weeks of illumination cultivation, put into rapidly the liquid nitrogen grind into powder, extract total RNA according to Trizol test kit specification sheets, electrophoresis result shows, the master tape complete display of gained mutant and the total RNA of wild-type, the band luminance factor of 28S and 18S is about 2:1, illustrates that the concentration of RNA and purity meet requirement of experiment, can be for the synthesis of double-stranded cDNA.

Take gained mutant and the total RNA of wild-type as template, according to M-MLV reversed transcriptive enzyme specification sheets, use Oligo (dT) primer to carry out reverse transcription respectively, obtain cDNA; Take the synthetic cDNA of reverse transcription as template, adopt special primer again RL14F/ RL14R and high-fidelity DNA polymerase PFU carry out pcr amplification, and the PCR reaction conditions is: 94 ℃ of denaturations 5 minutes; Then 94 ℃ of sex change are 30 seconds, 55 ℃ of renaturation 30 seconds, and 72 ℃ were extended totally 35 circulations 1 minute; Last 72 ℃ were extended 10 minutes.Gained RT-PCR product carries out 1.0%(g/mL) agarose gel electrophoresis identifies, the result as shown in Figure 2, mutant and wild-type RT-PCR product all are the single specificity band at about 1000bp place.Reclaim test kit with dna gel and cut glue recovery purifying target DNA fragment, again this dna fragmentation is connected with the pMD-19T carrier and spends the night in 16 ℃ of connections under the effect of T4 dna ligase, connect product and transform the bacillus coli DH 5 alpha competent cell, with the LB plate screening positive colony that contains penbritin, order-checking after PCR identifies.Result's demonstration, RL14Full length gene cDNA is 837bp(SEQ ID No.1), formed 278 amino acid of encoding altogether (SEQ ID No.2) by 3 exons; Compare with wild type gene, RL14Gene is located the conversion (Fig. 3) of T-C at the 458th bit base (i.e. the 190th bit base of the 2nd exon), and causes the variation (Fig. 4) of the 153rd amino acids generation Ile-Thr of coding protein sequence.

2, paddy rice moderate leaf roll mutant gene RL14Functional verification

According to structure shown in Figure 5 RL14Wild type gene recombinant plant overexpression vector, and rice transformation moderate leaf roll mutant body Rl14The result shows, transgenic positive plant (called after C-rl14) blade is open and flat, reverts to wild-type blade profile (Fig. 6 AB) fully.Tissue slice is observed and is shown, the bulliform cell form of transgenic positive plant leaf is compared obvious recovery normal (Fig. 6 C) with mutant.To 6 leaf phase wild-types, mutant Rl14And transgenic positive plant leaf RL14Gene carries out QPCR analyzes, and the result shows the transgenic positive plant RL14Gene expression amount and wild-type be (Fig. 6 D) quite.Thereby determined RL14Gene be exactly 2OG-Fe (II) oxidase gene ( Os10g40960.1).

Three, paddy rice moderate leaf roll mutant gene RL14Bioinformatic analysis

Will RL14Gene order is utilized the ORF Finder(http among the NCBI: //www.ncbi.nlm. nih.gov/gorf/gorf.html) carry out open reading frame identification.Result's demonstration, RL14Gene is comprised of a complete and continuous open reading frame.

Will RL14The gene coded protein sequence is utilized CDD(conserved domain database) (http://www.ncbi. nlm.nih.gov/Structure/cdd/wrpsb.cgi) carry out the domain analysis of protein conserved structure.Result's demonstration, RL14Gene coded protein has 1 high conservative zone (128-228 amino acids), i.e. 2OG-Fe (II) oxydase structural domain, and by RL14The amino acid mutation site that transgenation causes is just in this high conservative zone.

Will RL14The gene coded protein sequence utilizes MEG4 software to carry out the generation of sequence alignment and systematic evolution tree.Sequence alignment result demonstration, RL14Gene and paddy rice (Q 94LP4), Chinese sorghum ( C5WZM7), corn ( C4J2P7) homology of proteins encoded is up to more than 50%.Systematic evolution tree as shown in Figure 7, as seen RL14Gene coded protein and paddy rice Q 94LP4Affinity nearer, with Chinese sorghum C5WZM7Affinity take second place.

Four, paddy rice moderate leaf roll mutant gene RL14Application

Paddy rice moderate leaf roll is the important ideotype of rice high yield.Of the present invention RL14Gene provides important genetic resources for the molecular breeding of paddy rice.Based on RL14Gene constructed plant expression vector also transforms the rice cell of high-quality background, again the rice cell that transforms is cultivated into the leaf roll plant, can realize by transgenosis the rapid polymerization of the excellent genes such as leaf roll, thereby rapidly by the desirable rice varieties of cultivating high yield and high quality.

Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although by invention has been described with reference to the preferred embodiments of the present invention, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and not depart from the spirit and scope of the present invention that appended claims limits.

＜110〉Southwestern University

＜120〉paddy rice moderate leaf roll mutant gene RL14And use

<160> 4

<210> 1

<211> 837

<212> DNA

＜213〉paddy rice ( Oryza SativaL)

<220>

＜223〉paddy rice moderate leaf roll mutant gene RL14

<400> 1

atgcaacaaa aactacagct catcaaccat ggagtgccag atgaagtgat tgcaaatctc 60

aagaacgact tggttgagtt tttcagtcag ccactggatg ccaagaagga atactcacag 120

ctgcccaata accttgaagg ttatggacag gccttcgttg tgtccgacaa ccagaaacta 180

gactgggcag acatgcttta ccttcaagtc tgtcctaccg attcaaggga cttgagattc 240

tggcccaatt accctgcctc tttcaggcat tccattgatg catactcatc agagacagaa 300

aatattggac tctgcctgct gcaattcatg gccaaggccg ttggtgttga gccgaagtcg 360

ttgttaagtg tatttgaagg gcaagctagg ggtttgagga tgaactacta tccaccgtgc 420

ttgaaagctg acaaggtgtt gggtttgtca ccgcacactg atccaggtgg cttgacactg 480

cttctccagg tgaatgatgt acagggcctg cagatcaaca aggatggcaa atggttctcc 540

gtgaatgctc tgaatggtgc actcatcgtt aacattggcg acacacttga gatcctgagt 600

aatggaaagt tcagaagtgt tgagcacagg gccgtggtac atccaagcag agagaggatt 660

tcagcggcac tgttctacta tccgtgccag gatatggtga tcagccctct gccggacttt 720

gtgaaagatg gtaaagtgaa atacaaaaca ataagctacc aggatttgct gacagagtac 780

ttcacagcag agcttgatgg aaggaaccgg ctaaagaaaa tgaagctgga accgtag 837

<210> 2

<211> 278

<212> PRT

＜213〉paddy rice ( Oryza SativaL)

<220>

＜223〉paddy rice moderate leaf roll mutant gene RL14Proteins encoded

<400> 2

Met Gln Gln Lys Leu Gln Leu Ile Asn His Gly Val Pro Asp Glu

1 5 10 15

Val Ile Ala Asn Leu Lys Asn Asp Leu Val Glu Phe Phe Ser Gln

20 25 30

Pro Leu Asp Ala Lys Lys Glu Tyr Ser Gln Leu Pro Asn Asn Leu

35 40 45

Glu Gly Tyr Gly Gln Ala Phe Val Val Ser Asp Asn Gln Lys Leu

50 55 60

Asp Trp Ala Asp Met Leu Tyr Leu Gln Val Cys Pro Thr Asp Ser

65 70 75

Arg Asp Leu Arg Phe Trp Pro Asn Tyr Pro Ala Ser Phe Arg His

80 85 90

Ser Ile Asp Ala Tyr Ser Ser Glu Thr Glu Asn Ile Gly Leu Cys

95 100 105

Leu Leu Gln Phe Met Ala Lys Ala Val Gly Val Glu Pro Lys Ser

110 115 120

Leu Leu Ser Val Phe Glu Gly Gln Ala Arg Gly Leu Arg Met Asn

125 130 135

Tyr Tyr Pro Pro Cys Leu Lys Ala Asp Lys Val Leu Gly Leu Ser

140 145 150

Pro His Thr Asp Pro Gly Gly Leu Thr Leu Leu Leu Gln Val Asn

155 160 165

Asp Val Gln Gly Leu Gln Ile Asn Lys Asp Gly Lys Trp Phe Ser

170 175 180

Val Asn Ala Leu Asn Gly Ala Leu Ile Val Asn Ile Gly Asp Thr

185 190 195

Leu Glu Ile Leu Ser Asn Gly Lys Phe Arg Ser Val Glu His Arg

200 205 210

Ala Val Val His Pro Ser Arg Glu Arg Ile Ser Ala Ala Leu Phe

215 220 225

Tyr Tyr Pro Cys Gln Asp Met Val Ile Ser Pro Leu Pro Asp Phe

230 235 240

Val Lys Asp Gly Lys Val Lys Tyr Lys Thr Ile Ser Tyr Gln Asp

245 250 255

Leu Leu Thr Glu Tyr Phe Thr Ala Glu Leu Asp Gly Arg Asn Arg

260 265 270

Leu Lys Lys Met Lys Leu Glu Pro

275

<210> 3

<211> 23

<212> DNA

＜213〉artificial sequence

<220>

＜223〉amplifying rice moderate leaf roll mutant gene RL14Upstream primer RL14F

<400> 3

ggacacaacc tgaaagcagg ttc 23

<210> 4

<211> 26

<212> DNA

＜213〉artificial sequence

<220>

＜223〉amplifying rice moderate leaf roll mutant gene RL14Downstream primer RL14R

<400> 4

tatgagtcag tttctaagtt cgttcg 26

Claims (4)

1. paddy rice moderate leaf roll mutant gene RL14, its nucleotide sequence is shown in SEQ ID No.1.

2. paddy rice moderate leaf roll mutant gene RL14Proteins encoded, its aminoacid sequence is shown in SEQ ID No.2.

3. the described paddy rice moderate of claim 1 leaf roll mutant gene RL14Application in the molecular breeding of Rolled Leaf Gene in Rice.

4. application according to claim 3 is characterized in that, rice varieties is extensive No. 10 of red silk.

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