CN101519660B - Method for improving content of amylose in rice by using RNA interference - Google Patents

Abstract

The invention discloses a method for increasing the content of amylose in rice by using RNA interference in the field of biotechnology. The present invention clones the starch branching enzyme RBE3 gene fragment from rice as an RNA interference fragment, uses an endosperm-specific promoter to initiate expression, constructs an siRNA expression vector of the RBE3 gene, and obtains a transgene that interferes with the RBE3 gene by using the method mediated by Agrobacterium rice. RT-PCR and Southern hybridization were used to test the integration and expression of the target gene, and the amylose content of the endosperm of the transgenic rice plants was determined by iodine chromogenic method. 238%, the transgenic rice plants with significantly increased grain amylose content were obtained after screening. Therefore, a method for increasing the amylose content in rice is provided, and a foundation is laid for the large-scale production of amylose by transgenic rice.

Description

Method of increasing rice amylose content by RNA interference

technical field

The invention relates to a method for increasing the amylose content in rice seeds in the field of biotechnology, in particular to a method for increasing the amylose content in rice seeds by using RNA interference.

Background technique

Starch is the main storage form of carbohydrates in higher plants and the most important component of food crop products. The storage starch of plants mainly consists of two components: amylose and amylopectin. The ratio of amylose and amylopectin determines the use of starch. Amylopectin is mainly used in the food industry, while amylose is widely used in industry, involving more than 30 industries such as food, medical, textile, papermaking, and environmental protection. field. The function of amylose, especially the amylose after physical and chemical modification, is further strengthened. If the amylose is dissolved and combined with hydrogen bonds, it can form a rigid opaque colloid. This feature is used in the candy industry to keep the candy fixed The shape and complete shape of amylose; amylose is also used in thickeners, fixatives, and coating agents in French fries to prevent excessive oil absorption in the food industry; using amylose instead of polystyrene to produce degradable plastics, this This kind of plastic has the potential to be widely used in packaging industry, agricultural film processing industry and fundamentally solve the problem of white pollution.

The synthesis of plant starch is produced by starch synthase through a series of complex processes. At present, it is believed that the biosynthesis of starch mainly involves four types of enzymes—ADPG pyrophosphorylase, starch synthase, starch branching enzyme and debranching enzyme. They catalyze the formation of ADP-glucose, the elongation of glucan chains, and the formation of branched chains, respectively. Amylose is directly catalyzed by granule-bound starch synthase I (GBSS I or WAXY), while amylopectin is catalyzed by starch branching enzyme (SBE), soluble starch synthase It is the product of the joint action of soluble starch synthase (SSS) and starch debranching enzyme (DBE), among which RBE3, an isoform of SBE, is considered to play the most critical role in the formation of amylopectin. Therefore, the strategy of using genetic engineering to improve starch quality focuses on regulating the content of specific enzymes or the coordination relationship between several enzymes in the process of starch synthesis.

In the study of cereal crops, the breakthrough in obtaining high levels of amylose without significantly reducing the total starch content was the discovery of the ae (amylose extender) gene located on the fifth chromosome of maize by Vineyard and Bear in 1952. Genetic research shows that high amylose is mainly controlled by ae gene, and the synergistic effect of ae gene and its modifying genes can increase the amylose content in corn starch to 50-80%. The ae gene located on the fifth linkage group has been marked by SSR linkage. The ae gene has been cloned from high starch maize. The gene has a total of 23449bp, and its accession number in Genebank is AF072725. These provide important germplasm resources and gene resources for the transfer and utilization of this trait, but due to the large size of the gene, it is not easy to operate, and the expression of the gene is accompanied by deterioration of other agronomic traits and low grain yield, so its application is limited. In addition, because the inheritance of high amylose is very complicated, the breeding methods such as conventional crossing, backcrossing and recurrent selection need to be large enough, and the cycle is long and the analysis samples are large, so the investment is also high.

Genetic engineering to change the quality of starch focuses on the operation of three enzymes, GBSS, SSS and SBE, mainly using technologies:

1. Sense and antisense RNA transgenic technology, that is, by introducing the sense structure or antisense structure of an enzyme gene, affecting the content or activity of the enzyme in the cell, causing the overexpression of the target gene or inhibiting its expression, so as to achieve Control over starch structure. Visser et al. (1991) used antisense RNA technology to introduce a reverse-linked GBSS gene into the potato, resulting in a decrease in the content and activity of the GBSS gene, which in turn led to a sharp decrease in the amylose content in potato tubers (reduction of 70%-100%) . Similarly, antisense RNA technology was used to obtain low (or no) amylose transformants in cassava (Salehuzzman, 1993), rice and other plants. International patent WO9722703A2 reported the application of antisense RNA technology to transform the full-length cDNA of maize sbe2b gene into maize. As a result, transgenic maize with higher amylose content in grain starch was obtained. However, the application of antisense technology has certain limitations. Its inhibition of endogenous gene expression is weak, and often produces transitional phenotypes, which will hinder the accurate judgment of the function of the target gene. The genetic stability of its inhibitory effect is poor, and it cannot stably and reliably reduce the expression of the target gene.

2. Gene knockout technology and gene conversion technology mediated by DNA/RNA chimeric molecules, but this method can only study one gene at a time, and cannot effectively knock out multi-gene families. For some genes that play a key role in the development of animals and plants, if gene knockout or mutation is used for heritable modification at the DNA level, the gene will be silenced prematurely, resulting in a lethal phenotype, so it cannot be studied in depth.

Contents of the invention

The technical problem solved by the present invention is to provide a method for overcoming the difficulty in increasing the amylose content of rice grains in the current conventional breeding process, and to increase the amylose content of rice by using RNA interference, so that it can inhibit rice starch by using RNA interference. A method for increasing the amylose content in rice seeds by expressing the key enzyme SBEII b gene in the seed endosperm.

The present invention is achieved through the following technical solutions: a method for increasing the amylose content of rice by using RNA interference, cloning the RBE3 gene fragment from rice as the RNA interference fragment, using an endosperm-specific promoter to initiate expression, and constructing a normal amylose containing RBE3 gene. The expression vector of the reverse fragment was mediated by Agrobacterium tumefaciens, and the positive and negative structure of the RBE3 gene was transferred into rice. RT-PCR and Southern hybridization were used to test the integration and expression of the target gene, and the effect on the amylose content of the endosperm of the transgenic rice plants The iodine chromogenic method is used to measure, and the transgenic rice plants whose grain amylose content is significantly increased are obtained after screening.

Specifically include the following main steps:

(1) According to the sequence of the rice starch synthesis key enzyme RBE3 gene, through sequence comparison, determine the specific RNA interference sequence of the rice amylose gene, design primers according to the interference sequence, and carry out the interference fragment of the rice starch synthesis key enzyme RBE3 gene specific amplification of

(2) Promote expression with an endosperm-specific promoter, and construct a plant expression vector containing the positive and negative fragments of the RBE3 gene;

(3) transforming the vector containing the target interfering segment, and integrating the target interfering segment into the rice genome;

(4) identification of transgenic rice by molecular detection methods such as RT-PCR and Southern hybridization;

(5) Using the iodine chromogenic method to detect the amylose content of the grain, and screen to obtain the transgenic rice with increased amylose content.

In the step (1), through homology search and sequence comparison, it is determined that the RNA interference specific fragment is located at position 66197-66391 of the rice genome, is 195 bp long, and includes the 12th exon (135 bp) of the RBE3 gene sequence, and a 60bp intron sequence.

In the step (2), the high molecular weight glutenin (HMW) promoter is used as the endosperm-specific promoter, and the positive and negative fragments of the RBE3 gene are placed behind the specific promoter to specifically inhibit the expression of the RBE3 gene in the endosperm .

In the step (4), the molecular detection is to use the PCR amplified fragment of the specific primer of the screening marker bar gene as a probe, and use Southern hybridization to accurately verify that the target interference fragment is integrated into the rice genome and its copy and design RT-PCR special primers for RBE3 gene and specific primers for rice actin gene, then conduct RT-PCR on transgenic rice and control respectively, and use semi-quantitative PCR to analyze the expression difference of RBE3 gene.

Compared with the prior art, the beneficial effects of the present invention are reflected in:

1. The present invention utilizes RNA interference technology to improve the quality of rice starch. This is the first time that RNA interference technology has been researched and applied in this field, and it is also the originality of the present invention. RNA interference technology has the characteristics of high efficiency, specificity, heritability, and simple operation, which are unmatched by traditional gene knockout technology and antisense RNA technology.

2. In terms of receptor selection, the receptors to be studied by traditional gene knockout technology and antisense RNA technology are generally complete gene sequences, and there are obvious limitations for receptors with unclear gene sequences. However, using RNA interference technology, it is only necessary to know the gene segment homologous to the receptor to study the receptor, so the research scope is relatively wide.

3. In terms of operation methods, antisense RNA technology generally needs to construct large fragments containing thousands of base pairs, which is difficult to operate, while the vector to be constructed in RNA interference technology only contains tens to hundreds of base pairs. Small fragments of base pairs are simple, convenient and easy to use. In addition, one or more interference fragments can be selected for a gene, and the interference effects of these fragments or combinations can be analyzed and optimized to obtain the most effective fragments.

4. In terms of genetic stability, using traditional antisense RNA technology, because the whole gene transformation has a great impact on the receptor, it will cause a large variation in the receptor, and the genetic stability is poor, it is difficult to obtain excellent transgenic crops, but The RNA interference fragments transformed by this technology are small, have little effect on other agronomic traits of the recipient, and have high genetic stability, making it easy to obtain transgenic plants that interfere with the target gene and have excellent other agronomic traits.

Detailed ways

Embodiments of the present invention will be specifically described below.

1. Obtaining RBE3 gene-specific RNAi fragments

(1), utilize CTAB method to carry out the extraction of rice genome DNA.

Take 0.5g of young rice leaves, add liquid nitrogen to pulverize, add 2mL of 2% 2×CTAB extract solution kept at 65°C, mix well, and keep at 65°C for 30-60min. Add an equal volume of chloroform/isoamyl alcohol (24:1), mix gently by inversion, and centrifuge at 10000 r/min for 5 min. Take the supernatant, add 1/10 volume (about 0.2 mL) of CTAB/NaCl solution at 65°C, and mix by inverting. Extract with an equal volume of chloroform/isoamyl alcohol (24:1), centrifuge at 10,000 r/min for 5 min. Take the supernatant, add (exactly) equal volume of CTAB precipitation solution, invert and mix well, if precipitation is visible, proceed to the next step, otherwise, keep warm at 65°C for 30min. 4°C, 2700r/min, centrifuge for 5min. Remove the supernatant and resuspend with high-salt TE buffer (0.25-0.5mL). (It can be kept at 65°C for 30 minutes until most of it dissolves). Add 0.6 volume of isopropanol to precipitate nucleic acid, mix well, centrifuge at 10000r/min at 4°C for 15min. Remove the supernatant, wash the precipitate with 80% ethanol, dry, and resuspend with as little TE buffer as possible (0.025-0.05mL).

(2) PCR amplification of RBE3 gene fragment.

Referring to the published rice starch branching enzyme gene RBE3 (accession number: D16201) and the nucleotide sequence of the rice genome, the RBE3 gene fragment suitable for RNAi was selected for the construction of the vector, and the fragment was located at position 66197-66391 of the rice genome. It is 195bp long, contains the 12th exon (135bp) sequence of the RBE3 gene, and has a 60bp intron sequence. BamH I and Sal I restriction sites were added to the 5' ends of the primers, and the primers were designed: the 5' end primer was 5'-GCGGATCCGGGAAGTAGCGATTAACGTGTT-3', and the 3' end primer RBE3i-R was 5'-GCGTCGACATAGCTTACCTTTGCCCCTT-3' . The reaction system (50 μL) is 1.0 μL of dNTP (10 mM), 1.0 μL of each primer (10 pmol/μL), 1.0 μL of rice genomic DNA template (1 μg/μL), 5.0 μL of 10×pfu Buffer (+Mg ²⁺ ), pfu DNA Polymase (5U/μL) 1.0μL, make up to 50μL with sterilized double distilled water. PCR reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 1 min, annealing at 56°C for 45 sec, extension at 72°C for 30 sec, a total of 35 cycles; and finally extension at 72°C for 10 min.

2. Establishment of RBE3 gene siRNA expression vector

(1), RNAi (2RBE3i) intermediate vector construction.

The RBE3 interference fragment RBE3i obtained by PCR amplification and cloned on the pGM-T vector was digested with BamH I and Sal I enzymes, connected with the pUCCRNAi vector cut with the same enzymes, and the recombinant (RNAi (RBE3i) identification primer 5' end primer pUCC1 is 5'-GGACCGTACTACTCTATTCGTTTC-3', 3' end primer is RBE3i-R), and then verified by Pst I digestion to obtain recombinant plasmid RNAi (RBE3i); then use Xho I and Recombinant plasmid RNAi (RBE3i) was digested with Bgl II, and then ligated with the BamH I/Sal I fragment of RBE3i to form an intron-containing inverted repeat structure of about 600 bp. The recombinant was initially screened by colony PCR. (RNAi (2RBE3i) identification primer 5' end primer is RBE3i-F, 3' end primer pUCC2 is 5'-GAAACGAATAGAGTAGTACGGTCC-3'), the recombinant was verified by Pst I digestion, and the cloning vector RNAi (2RBE3i) was constructed.

(2) Construction of siRNA expression vector p1300 (2RBE3i).

The RNAi (2RBE3i) vector was digested with Pst I and Sal I, and the DNA fragment of about 600bp in the digested product was recovered, then ligated with the HMW GUS vector digested with Pst I and Sal I, and then digested with Pst I and Sal I The recombinants were cut and verified, and constructed into HMW (2RBE3i) vectors. Primers were designed according to the full sequence from the promoter to the terminator of HMW GUS, restriction sites Sac I and Xba I were added to the 5' ends of the primers, and the primer HMW-F at the 5' end was 5'-CCGGAGCTCGCAAATATGCAACATAATTTCC-3',3 The 'end primer HMW-R is 5'-CCCTCTAGATGATCTTGAAAGATCTTT-3'. In the 50 μL reaction system, there are 1.0 μL of dNTP (10 mM), 1.0 μL of each primer (10 pmol/μL), 1.0 μL of DNA template (HMW (2RBE3i) plasmid 1 μg/μL), 5.0 μL of 10×pfu Buffer (+Mg ²⁺ ) , pyrobest DNA polymase (5U/μL) 1.0μL, make up to 50μL with sterile double distilled water. PCR reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 1 min, annealing at 61°C for 45 sec, extension at 72°C for 3 min, a total of 35 cycles; and finally extension at 72°C for 10 min. A target gene DNA fragment of about 2.6 kb was obtained. The target fragment was purified and recovered and cloned into pGM-T. After the sequence was correct, it was digested with Sac I and Xba I enzymes, and ligated with pCAMBIA1300 digested with Sac I and Xba I enzymes to obtain the siRNA expression vector p1300 (2RBE3i). Recombinants were verified by digestion with Sac I and Xba I.

In this example, the commonly used genetic engineering method for vector construction was used to connect the RBE3 gene fragment forward and reverse to both sides of the potato GA20-oxidase intron 1 fragment, and to start it with the promoter of the wheat HMW glutenin Glu-1D-1 gene. The p1300(2RBE3i) expression vector is constructed, and the expression vector can increase the amylose content of rice by means of genetic engineering.

3. Transformation of Agrobacterium tumefaciens to obtain candidate transgenic rice

(1) The expression vector is introduced into Agrobacterium.

Pick a single colony of LBA4404 or EHA105 and inoculate it into 5ml of YEB liquid medium containing rifampicin 100ug/ml, and incubate with shaking at 28°C and 200r/min for about 48 hours. Take 2ml of the bacterial liquid and transfer it into 50ml of YEB liquid medium, and continue to cultivate until the OD600 reaches about 1.0. Transfer to a sterile centrifuge tube and place on ice for 30 minutes. Centrifuge at 5000r/min for 5min, remove the supernatant. Add 2ml of newly prepared 20mmol/L pre-cooled CaCL2 resuspended bacteria. Take the sterilized centrifuge tubes and aliquot 200ul per tube on ice. Take 2ug of the p1300(2RBE3i) plasmid and add it to 200ul of Agrobacterium competent. ) ice bath for 5 minutes, transferred to liquid nitrogen to freeze for 1 minute, and then 37 ° C water bath for 5 minutes. Add 800ul of YEB liquid culture, 28 ℃, 200r/min shaking culture for about 5 hours. Draw 300 ul of the bacterial solution and spread it on the YEB solid medium (containing 100 ug/ml rifampicin and kanamycin). Pick positive clones for colony PCR and extract plasmid identification.

(2) Genetic transformation of rice mediated by Agrobacterium.

For callus induction, young embryos of Zhonghua 11 were used, and the immature embryos pollinated for about 10 days were cultured on the induction medium, and the induction medium was about 20 days; the induced callus was subcultured for about 30 days, and the subculture medium was the same as Induction medium; then prepare Agrobacterium and callus for co-cultivation, co-cultivation temperature 21°C, 3d solid co-culture medium: NB basic medium + 2,4-D 2.0mg/L + inositol 2.0g/L + AS 100μM/L (liquid co-culture without 2,4-D and agar powder); then wash the bacteria with sterile water, transfer to the screening medium for 40 days: NB basic medium +2,4-D 2.0mg/L+cef. 500mg/L+Hyg 50mg/L (or 20mg/L PPT); pick and screen the resistant callus and culture in light for about 1 month, differentiation medium: NB basic medium+NAA0.25mg/L+6-BA 2mg/L+KT 0.5mg/L+cef.500mg/L+Hyg 50mg/L (or 20mg/L PPT); pick differentiated seedlings for rooting culture, rooting medium: NB basic medium (a large amount of N6 and sucrose Halved)+MET 1mg/L+NAA1mg/L. N6 large amount+MS-Fe salt+B5 organic+proline 500mg/L+glutamine 500mg/L+hydrolyzed casein 300mg/L+sucrose 30g/L+agar powder 2.6g/L.

In this example, genetic transformation mediated by Agrobacterium was used to induce callus from rice immature embryos around 10 days old, and resistant plants were obtained through co-cultivation and PPT screening. The plant will be subjected to molecular identification and testing, providing materials for transgenic rice to increase amylose content.

4. Identification of transgenic rice

(1) Molecular detection of candidate transgenic resistant plants

Southern hybridization detection

The young leaves of the transgenic resistant plants were taken, and the DNA was extracted by the CTAB method. Primers were designed according to the sequence of the bar gene, the 5'-end primer bar-F was 5'-ATGAGCCCAGAAGACG-3', and the 3'-end primer bar-R was 5'-TCAGATCTCGGTGACGG-3'. Use a standard reaction system. The amplification conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 70°C for 30 sec, and extension at 72°C for 1 min, a total of 35 cycles; finally, extension at 72°C for 10 min. The plants that were positive in the PCR test were then tested by Southern hybridization. The probe used for Southern hybridization was the bar gene (550 bp) recovered after PCR amplification with the above-mentioned specific primers (bar-F and bar-R). Probe labeling, hybridization and washing were carried out according to the methods provided by Promega's Prime-a-Gene Labeling System kit. Whether it is transgenic rice can be judged by whether the Southern hybridization band appears.

RT-PCR detection of candidate transgenic resistant plants

Total RNA was extracted from immature seeds (2 weeks after flowering) of wild-type and transgenic plants respectively, and primers were designed according to the published rice Actin1 gene sequence (accession number: AY212324), and the 5' end primer Act-F was 5'-CCCTTGTGTGTGACAATGGAACT -3', 3' end primer Act-R is 5'-GACACGGAGCTCGTTGTAGAAGG-3'; RT-PCR primers are designed according to rice RBE3 gene sequence, 5' end primer RBE3-F is 5'-ATGAGTTCGGACATCCTGAATGG-3', 3' end Primer RBE3-R is 5'-CATTCCGCTGGAGCATAGACAAC-3'. The reverse transcription kit uses First-Strand cDNA Synthesis Kit from Shanghai Shenergy Biotechnology Co., Ltd. Using the Actin1 gene as an internal reference, the PCR reaction program was pre-denaturation at 94°C for 2 minutes; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, a total of 25 cycles, and finally 10 min at 72°C. The agarose electrophoresis bands of the amplified products were quantitatively analyzed. Transgenic rice was obtained through Southern hybridization analysis and RT-PCR detection. Molecular testing of transgenic plants.

(2) Genetic analysis of transgenic strains.

A number of seeds were randomly selected from all transgenic rice lines and the seeds of the control non-transformed rice Zhonghua 11 were germinated in clear water. After the seedlings grow to 2-3cm, replace the clean water and use 20mg/L PPT water solution to irrigate and screen. The screened resistant seedlings are planted in the field, and the separation of their characters is observed and tested, and the compound Mendelian genetic law 3: 1 isolated strain.

5. Determination of Amylose Content in Transgenic Rice

The endosperm part of the seeds was used for the determination of the amylose content, and 30 seeds with varying transparency were randomly selected from each plant, and were randomly divided into three groups. In the control group, 30 seeds of untransformed rice Zhonghua 11 were also randomly selected and randomly divided into three groups. Three groups. Amylose in the sample was determined by iodine color method: the sample was crushed through a 60-mesh sieve, degreased with ether, weighed about 0.1 g (accurate to 1 mg) of the degreased sample, and placed in a 50 ml volumetric flask. Add 10ml of 0.5mol/L KOH solution, heat in a boiling water bath for 10min, take it out, and distill the volume to 50ml with distilled water. If there are foams, use ethanol to eliminate them and let stand. Take two parts of 2.5ml of sample solution (i.e. sample measurement solution and blank solution), add 30ml of distilled water to both, adjust to about pH 3.5 with 0.1mol/L HCI solution, add 0.5ml of iodine reagent to the sample, and add no iodine reagent to the blank solution, then Dilute to 50ml. Let it stand for 20 minutes, take the sample blank solution as a control, measure the respective absorbance with a 1cm cuvette, and obtain the corresponding amylose content from the regression equation, and take the average value as the endosperm amylose content of the strain. The result is that the average amylose content of the transgenic plants is 140% higher than that of the non-transgenic plants, and the highest is 238%. After screening, the transgenic rice plants with significantly increased grain amylose content can be obtained.

The present invention clones the 195bp specific fragment of the starch branching enzyme RBE3 gene from rice as the RNA interference fragment, uses the high molecular weight glutenin promoter as the endosperm-specific promoter, and connects it inversely to the plant expression vector pCAMBIA1300 to construct the RBE3 gene The siRNA expression vector is used to transform the callus induced by rice immature embryos through the method mediated by Agrobacterium, and the transgenic rice that interferes with the RBE3 gene is obtained. After identification by PCR and Southern hybridization, semi-quantitative RT-PCR detection showed that the expression level of RBE3 gene was significantly lower than that of the control. The iodine chromogenic method of the amylose content in the endosperm of the transgenic rice plants showed that the amylose content of the transgenic plants increased by an average of 140% compared with the non-transgenic plants, and the highest reached 238%. Therefore, a method for increasing the amylose content in rice is provided, and a foundation is laid for the large-scale production of amylose by transgenic rice.

sequence listing

<110> Anhui Agricultural University

<120> Method for Improving Rice Amylose Content by RNA Interference

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<170>PatentIn version 3.3

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<221> Forward sequence of the cloned RBE3 gene fragment

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<221> GA20 intron in PUCRNAi vector

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<221> The reverse sequence of the cloned RBE3 gene fragment

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aaccgttcaa cccatctagg gtattatttg acgcatggct gcaaaaggaa aaaaaagggg 180

caaaggtaaa gctatgtacg gaccgtacta ctctattcgt ttcaatatat ttatttgttt 240

cagctgactg caagattcaa aaatttcttt attattttaa attttgtgtc actcaaaacc 300

agataaacaa tttgatatag aggcactata tatatacata ttctcgatta tatatgtaaa 360

tgagttaacc tttttttcca cttaaattat atagtatcga aatggaaacg gggaaaaaaa 420

aggaaaacgt cggtacgcag tttattatgg gatctaccca acttgccaaa actaccatgt 480

ctatgcgtaa tgaaagtatc accaagtgcg ccggtagtaa cttacaccct aagagcggaa 540

aagttgatac ccttaaccct tcattccttg tgcaattagc gatgaaggg 589

Claims (3)

1. method that interfere to improve content of amylose in rice with RNA, it is characterized in that: clone RBE3 gene fragment is as the RNA interference fragment from paddy rice, start expression with endosperm specificity promoter, structure contains the positive and negative segmental expression vector of RBE3 gene, with Agrobacterium tumefaciens mediated, the positive antistructure of RBE3 gene is changed in the paddy rice, the integration and the expression of RT-PCR and Southern hybridization check goal gene, transgenic rice plant endosperm amylose content is adopted the iodine determination of color, and the screening back obtains the transgenic rice plant that the seed amylose content significantly improves; Comprise following key step:

(1) according to the sequence of the synthetic key enzyme RBE3 gene of rice fecula,, determines that the RBE3 specific RNA of amylose in rice enzyme gene is interfered sequence,, carry out specific amplification according to interfering the sequences Design primer by sequence alignment;

(2) start expression with endosperm specificity promoter, structure contains the RBE3 specific RNA and interferes the positive and negative segmental plant expression vector of sequence;

(3) transform the carrier that contains RBE3 specific RNA interference sequence, and interfere sequence to be incorporated in the rice genome RBE3 specific RNA;

(4) identify transgenic paddy rice by RT-PCR and Southern hybridization equimolecular detection method;

(5) utilize the iodine development process to detect the seed amylose content, screening obtains the transgenic paddy rice that amylose content improves;

In described step (1), by homology search and sequence alignment, determine that this RBE3 specific RNA interference sequence is positioned at 66197～66391 positions of rice genome, long 195bp, comprise the 12nd exon sequence 135bp of RBE3 gene, and the intron sequences of 60bp is arranged.

2. the method that improves content of amylose in rice of interfering with RNA according to claim 1, it is characterized in that: in described step (2), with high molecular weight glutenin HMW promotor as endosperm specificity promoter, interfere the RBE3 specific RNA the positive and negative fragment of sequence to place after the HMW promotor the specific RBE3 expression of gene that in endosperm, suppresses.

3. the method that improves content of amylose in rice of interfering with RNA according to claim 2, it is characterized in that: in described step (4), described Molecular Detection is respectively with the pcr amplified fragment of the Auele Specific Primer of selection markers bar gene and makes probe, adopts Southern hybridization to verify that accurately RBE3 specific RNA interference sequence is incorporated in the rice genome and copy number; With the special primer of design RBE3 Gene RT-PCR primer special and paddy rice actin gene, RT-PCR is carried out in transgenic paddy rice and contrast respectively then, adopts sxemiquantitative pcr analysis RBE3 expression of gene difference.