CN100532553C - Amylose content control gene DU1 of rice endosperm and application thereof - Google Patents
Amylose content control gene DU1 of rice endosperm and application thereof Download PDFInfo
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Abstract
the invention discloses a gene DU1, which controls straight-chain starch content of rice endosperm in the rice beauty-water 11, wherein the gene ribonucleotide sequence is selected from (1) the ribonucleotide sequence of amino acid sequence of coded SEQ ID NO: 2; (2) the ribonucleotide sequence to control plant organ starch composition and rice endosperm straight-chain starch content simultaneously. the invention also provides protein to control straight-chain starch content of rice endosperm and plant expressive carrier of gene as well as plant cultivating method, which comprises the following steps: converting the gene of expressive carrier into plant cell; cultivating the converted plant cell into plant.
Description
Technical field
The present invention relates to plant genetic engineering field, more specifically, the present invention relates to amylose content control gene DU 1 of rice endosperm, the protein of this genes encoding and functional analogue thereof, encode its nucleotide sequence contains the carrier of this Nucleotide and contains the host cell of this carrier; In addition, the invention still further relates to the method for controlling plant endosperm amylose content and the method for improvement albumen amylose content breeding.
Background technology
Paddy endosperm starch is made up of amylose starch and amylopectin usually, and content is up to about 90%
[1]Amylose starch is meant between the glucose unit with α-1, that 4 glycosidic links are connected to form, as to be about 1000 glucosyl residues linear long chain molecule, amylopectin then is to derive little chain molecule with α-1,6 glycosidic link on this basis, about 20 glucosyl residues of side chain mean length
[2]The ratio of amylose starch and amylopectin and characteristic determine the attribute of starch, also determine the greatest factor of rice food flavor quality and processing quality.
Wx is the important gene that influences rice quality, and paddy rice Wx gene is at first cloned by Okagaki
[3], Wang etc. has cloned the DNA complete sequence of Wx gene subsequently
[4]Studies show that paddy rice Wx gene transcription ability and GBSS protein content and amylose content are in close relations.Particle closely is combined on the starch small grain in conjunction with starch synthase (GBSS), catalysis paddy endosperm amylose starch synthetic, and its size is about 60kD
[3,4]
1981, Satoh etc.
[5]Find a mutant, seed just shows the cloud feature behind complete drying, is called dark endosperm (dull endosperm is called for short du), and its endosperm amylose content generally descends.Studies show that, this proterties is controlled by a recessive gene, mutant material endosperm amylose content is usually about 6%, the rice outward appearance is glossy and be rich in gloss, the food flavor uniqueness, coldly not bringing back to life, especially be fit to the various instant-rices of exploitation etc., also is the excellent material of improvement rice grain amylose content and food flavor quality
[6,7]
Isshiki etc.
[8]By the research of du mutant being thought thereby the albumen of DU genes encoding may be by influencing the biosynthesizing of paddy endosperm starch to regulating Wx.But do not see the report of this gene of clone so far.
Summary of the invention
At above-mentioned research background, an object of the present invention is to provide a kind of gene of controlling the paddy endosperm amylose content.
Another object of the present invention provides a kind of protein of the coded by said gene by control paddy endosperm amylose content of the present invention.
A further object of the present invention provides a kind of plant expression vector that contains the gene of control paddy endosperm amylose content of the present invention.
Another purpose of the present invention provides a kind of method that cultivates plants, and this method can make the starch of the main storage organ of plant amylum constitute and change.
The invention provides a kind of gene of controlling the paddy endosperm amylose content, the nucleotide sequence of this gene is selected from:
(1) nucleotide sequence of the aminoacid sequence shown in the coding SEQ ID NO:2;
(2) with (1) in nucleotide sequence can under rigorous condition, hybridize and the nucleotide sequence of coding with function of control paddy endosperm amylose content simultaneously.
Rigorous hybridization conditions is meant, Hybond membrane is placed in the prehybridization solution (0.25mol/L sodium phosphate buffer, pH7.2,7% SDS) 65 ℃ of prehybridization 30min; Abandon prehybridization solution, add hybridization solution (0.25mol/L sodium phosphate buffer, pH7.2,7% SDS, isotope-labeled nucleotide fragments), hybridized 12 hours for 65 ℃; Abandon hybridization solution, add film washing liquid I (20mmol/L sodium phosphate buffer, pH7.2,5% SDS), wash film 2 times for 65 ℃, each 30min; Add film washing liquid II (20mmol/L sodium phosphate buffer, pH7.2,1% SDS), wash film 30min for 65 ℃.
The gene of control paddy endosperm amylose content of the present invention preferably has the dna sequence dna shown in Fig. 7 and SEQID NO:1.
It is a kind of by above-mentioned nucleotide sequence coded protein that the present invention also provides.This protein preferably has the aminoacid sequence shown in Fig. 8 and SEQ ID NO:2.SEQ ID NO:2 and protein shown in Figure 8 among the present invention belong to the PRP protein family, with the mankind's Prp1/Zer1 albumen and the proteic homology of Prp6 be 51%, participate in the montage of mRNA precursor and regulate and control the biosynthesizing of amylopectin.
The present invention also provides a kind of plant expression vector that contains the gene of control paddy endosperm amylose content of the present invention.This expression vector can be pCAMDU1 as shown in Figure 5, and this carrier can be expressed the polypeptide by above-mentioned nucleic acid sequence encoding.
The present invention also provides a kind of method that cultivates plants, and this method can make the starch of the main storage organ of plant amylum constitute and change, and comprises with expression vector transformed plant cells of the present invention; With the step that the plant transformed cell culture is become plant.
Description of drawings
Be described in further detail understanding the present invention below in conjunction with accompanying drawing, but be not that the present invention is limited.
Accompanying drawing 1. paddy rice F
2For the seed bearing phenotypic evaluation of plant (A, the common rice seed shows as the positive; B low amylose starch paddy endosperm shows as feminine gender)
The Primary Location figure of accompanying drawing 2.DU1 on the 10th karyomit(e) of paddy rice
The Fine Mapping and the physical positioning of accompanying drawing 3.DU1 gene
Accompanying drawing 4.DU1 gene transformation dul mutant recovers normal phenotype (A.dul mutant endosperm performance low amylose starch feature; B. phenotype recovers normal after changeing the DU1 gene)
Accompanying drawing 5. carrier pCAMDU1 plasmid maps
The 8.3kb genomic fragment that comprises DU1 of accompanying drawing 6. carrier pCAMDU1 plasmid expressions
The nucleotide sequence of accompanying drawing 7.DU1
The aminoacid sequence that accompanying drawing 8.DU1 is coded
Embodiment
Be described in further detail understanding the present invention below in conjunction with accompanying drawing, but be not that the present invention is limited.
The clone of embodiment 1 amylose content control gene DU 1 of rice endosperm
1. rice material
Paddy rice (Oryza sativa ssp.) endosperm low amylose mutant du1 (the use EMS of China Paddy Rice Inst (ethylmethane sulfonate) is mutagenic obtained)
[6,7]With bright extensive 63 (available from the China Paddy Rice Inst) of conventional rice kind.
2. analyze and target group
Rice variety that isozygotys bright extensive 63 and japonica rice mutant dul are hybridized F
1For selfing, obtain 7,150 F altogether
2Individuality, and therefrom select 1,618 individuality as target group.Every strain in seedling stage get 2 the gram about tender leaf, be used for extracting DNA.
3, by SSR (Simple Sequence Repeat), STS (Sequence-tagged Sites), and CAPS (Cleaved amplified polymorphic sequence) mark location DU1 gene
Adopt improved CTAB (Cetyltrimethyl Ammonium Bromide) method
[9]From rice leaf, extract the genomic dna that is used for the assignment of genes gene mapping.Get about 100mg rice leaf, through liquid nitrogen freezing, pulverize in the little mortar of diameter 5cm is transferred in the 1.5ml centrifuge tube and is extracted DNA, and the DNA resolution of precipitate of acquisition is in 100 μ l ultrapure waters.Each SSR, STS or CAPS react with 1 μ l DNA sample.
According to the research of paddy rice classical genetics, the DU1 gene be located in the 10th chromosomal long-armed on.We have chosen two pairs of SSR primers (sequence sees Table 1).We find the DU1 gene and this two marks are chain and between the two.On this basis, our the PCR primer of design is that template is carried out pcr amplification (94 ℃ of pre-sex change 5min, 94 ℃ of 1min with two parents' genomic dna respectively, 55 ℃ of 1min, 72 ℃ of 1min, 35 circulations, 72 ℃ are extended 10mins), with DU1 gene Primary Location between SSR mark M2 and M3.
For DU1 being positioned on the pac clone, we with PAC library (http://rgp.dna.affrc.gojp) sequence construct of the rice varieties Nipponbare that announced near the contig the DU1 site, the PCR primer of design is that template is carried out pcr amplification (94 ℃ of pre-sex change 5min with two parents' (rice variety bright extensive 63 and du1 mutant) genomic dna respectively, 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, 35 circulations, 72 ℃ are extended 10mins).Developed STS and SSR mark (sequence sees Table 1) with this, and be used for colony's Fine Mapping, finally be located on a pac clone AC068923.
4.DU1 the Fine Mapping of gene
According to the sequence (http://rgp.dna.affrc.go.jp) of the pac clone AC068923 that announces, 2 new marks (sequence sees Table 1) are positioned at DU1 within the scope of 66kb by linkage analysis at last.
Table 1 clone DU1 gene the primer sequence
| Title | The forward sequence | Reverse sequence |
| DU1 | atgtataccttggtatgcgtgc | tatgctttcccactcctgcg |
| DU2 | attcacccaaaggtcccaaacc | ctagggatctgcagcatttgg |
| DU3 | aaaggaagctgttggaggaagg | tacgatggcatctcctggaactg |
| M1 | tcagatctacaattccatcc | tcggtgagacctagagagcc |
| M2 | tcgataacacagtattcagccagg | acaaggacaaatgctatgggactc |
| M3 | atgtgcaatacagtgccatgtgg | tgctattgccattgtactgctgc |
| M4 | tgcactttcacctagcagtatgcc | ttccttgtgcctcacagtccatc |
| a | attagccggtaaatggatgagttc | aagcaatactaatccctccaaacc |
| b | aggtcttgggtcgtaccaccctgc | tcgttcgctccctggcttctcc |
| c | tgttccttgtgcggttgtgc | aacacccacctccgaacacacc |
| d | tgtggtgccttttattccctcc | tttcctgcacggcatacagtg |
| e | aacgcgaggacacgtacttac | acgagatacgtacgcctttg |
| f | aatccaacgcatcaaggctggc | acaatgccaaacaccaggaactcg |
| g | tgagctttacctcccctcctaacc | tccacctttctctctcatcccac |
5.DU1 the acquisition of Partial cDNA gene and the prediction of function
At first to the full-length gene group sequence of 66kb with GENSCAN software (
Http:// genes.mit.edu/GENSCAN.html) the possible coding region (ORF) of prediction, find that this interval has 12 ORF.One of them ORF has the proteic open reading frame of typical PRP; Enlarge the prediction of sequence scope, and with gene product with the blastX software prediction (
Http:// www.ncbi.nlm.nih.gov/BLAST).Carry out protein sequence comparison and evolutionary tree analysis with the ClustalW method in DNAStar software (Lasergene) the MegAlign program.Designing simultaneously a pair of primer DUF (5 ' tgtgaagctgtggttgcagg 3 ') and DUR (5 ' ttcatccagaccctctcagtgc 3 ') again is that template is carried out RT-PCR reaction (70 ℃ of 10mins with long-grained nonglutinous rice bright extensive 63 total RNA, 42 ℃ of 60mins, 99 ℃ of 5mins, 4 ℃ of 5mins) and with ABI3730 DNAanalyzers type sequenator check order, obtained the cDNA sequence of DU1 gene.On the basis of above-mentioned research, infer this gene a kind of splicing factor of may encoding, extremely similar to the mankind's Prp1/Zer1 albumen with Prp6 albumen.
6. the comparison of DU1 gene order between different varieties
By between Different Rice Varieties, the comparison of dna sequence dna in the site, DU1 gene place of a plurality of kinds (providing by China Paddy Rice Inst) such as wild-type show water 11, parent bright extensive 63 is provided, and it is corresponding with the phenotype result, found to cause the gene alteration position of phenotypic difference, shown that it is the hereditary basis that causes paddy endosperm straight chain content to change that base is replaced.
Embodiment 2 paddy endosperm amylose content controlling gene SU1 have complementary functions and transgenic research
Sequences Design primer according to the bright extensive 63DU1 gene of long-grained nonglutinous rice, use primer DU1F, DU1R, DU2F, DU2R, DU3F and DU3R (sequence sees Table 1) divide 3 sections high-fidelity PCR, and (sequence sees Table 1,94 ℃ of pre-sex change 5min, 94 ℃ of 1min, 60 ℃ of 1min, 72 ℃ of 1min, 35 circulations, 72 ℃ are extended 10mins and use ABI3730 DNA analyzers type sequenator order-checking (ABI company, the U.S.), select the right-on clone of sequence and utilize total Sal I and Xba I site that they are connected into a 8.3kb fragment, comprise 2 behind 3,055 bases of initiator codon ATG upstream and the terminator codon TAG, the full length sequence of 115 bases, be cloned among the binary vector pCAMBIA1300 (available from CAMIA company, Australia), obtained the plasmid pCAMBIDU1 (Fig. 5) that is used to transform.It is rice transformation among the EHA105 (available from CAMIA company, Australia) that plasmid changes Agrobacterium (AgroBacterium tumefaciens) strain over to by the method that shocks by electricity.With the rataria of dul mutant shelling sterilization, be inoculated into evoked callus the MS substratum [
10] in.The dark cultivation for 3 weeks grows callus from scultellum in 28 ℃ culturing room, and it is vigorous to select growth, and color is pale yellow, and more open embryo callus is as the acceptor that transforms.Infect the rice callus tissue with the Agrobacterium EHA105 bacterial strain that contains the double base plasmid vector, cultivate after 3 days for 25 ℃, containing screening resistant calli and transfer-gen plant on the selection substratum of 50mg/L Totomycin at the dark place.With hygromycin resistance plant hardening in the cool, be transplanted to the paddy field after several days, solid back is received and kind is carried out phenotypic evaluation.Gather in the crops the T of 8 strain systems altogether
0For seed, wherein the endosperm amylose content shows as male 5 strains system, prove that the DU1 gene has been integrated in the acceptor gene group also can correctly express (seeing accompanying drawing 4).
Reference
1. chief editor such as Min Shao pattern,＜＜rice breeding learns 〉, the Chinese Academy of Agricultural Sciences published ISBN7-109-04338-X/S.2689, P:324 in 1996
2.French?D(1984).Organization?of?starch?granules.In：Whistler?RL?BeMillerJN.Paschall?E(eds).Starch：Chemistry?and?Technology.Orlando：Academic，183-247
3.Okagaki, R.J., and Wessler, S.R.Genetics.1988,120:1137-1143.
4.Wang, Z.Y. and Wu, Z.L. .Nucleic Acids Res.1990,18,5898.
5.Satoh and Omura., Japan.J.Breed..1981,316-326.
6. before the money, Zhu Xudong, once waited energetically. Zhejiang agricultural sciences, 1996, (4): 155-156
7. Teng Sheng etc. before the money, once energetically. rice in China science, 2000,14 (3): 173-176
8.Isshiki, M. and Nakajima, M.Plant J.2000,23,451-460.
9.Xueyong?Li，Qian?Qian?and?Jiayang?Li.，Nature，2003，422(6932)：618-21
10.Linsmaier，E.M.and?Skoog，F.，Organic?growth?factor?requirements?oftobacco?tissue?cultures.Physiol.Plant.1965，18，100-127
Sequence table
＜110〉Inst. of Genetics and Development Biology, CAS
＜120〉amylose content control gene DU 1 of rice endosperm and application thereof
<130>IB053806
<160>2
<170>PatentIn?version?3.1
<210>1
<211>3120
<212>DNA
<213>Oryza?sativa
<400>1
<210>2
<211>1039
<212>PRT
<213>Oryza?sativa
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Claims (7)
1. gene of controlling the paddy endosperm amylose content, the nucleotide sequence of this gene are selected from the nucleotide sequence of the aminoacid sequence shown in the coding SEQ ID NO:2.
2. according to the described gene of claim 1, its dna sequence dna is shown in SEQ ID NO:1.
3. protein by claim 1 or 2 described genes encodings.
4. according to the described protein of claim 3, its aminoacid sequence is shown in SEQ ID NO:2.
5. plant expression vector that contains the gene of claim 1 or 2 described control paddy endosperm amylose contents.
6. according to the described expression vector of claim 5, this expression vector is pCAMDU1, and its plasmid map as shown in Figure 5.
7. method that cultivates plants, this method can make the starch of the main storage organ of plant amylum constitute and change, and comprise with claim 5 or 6 described expression vector transformed plant cells; With the step that the plant transformed cell culture is become plant.
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|---|---|---|---|---|
| CN106591435A (en) * | 2016-11-22 | 2017-04-26 | 南京农业大学 | Molecular marker method for site of low-amylose-content gene of dark endosperm mutant w54 of japonica rice variety Koshihikari |
| CN113637688B (en) * | 2021-09-23 | 2023-10-13 | 上海师范大学 | Rice amylose content regulating gene OsACF1 and application thereof |
| CN114262710B (en) * | 2021-12-31 | 2023-10-31 | 西南大学 | Rice plasmodesmata gene, mutant gene thereof, coded protein and application |
| CN114438101B (en) * | 2022-03-10 | 2024-07-19 | 江苏省农业科学院 | Transparent rice appearance low amylose allele and application thereof |
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| AAL58273. NCBI. 2002;水稻3个新性状的胚和胚乳品系的育成及评价. 钱前等.浙江农业科学，第4期. 1996 * |
| 水稻3个新性状的胚和胚乳品系的育成及评价. 钱前等.浙江农业科学，第4期. 1996 |
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