CN1537941A - Method for breading paddy rice having high content of amylose - Google Patents
Method for breading paddy rice having high content of amylose Download PDFInfo
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Abstract
A method for breeding the paddy rice with high content of amylose includes such steps as preparing the chimeric gene including coding branching enzyme gene or its fragment, whose upstream side is connected to the nucleic acid fragment of the promoter expressed in endosperm tissue of rice seed and whose downstream side is linked to the nucleic acid fragment of the regulation sequence for terminating transcription, and using the chimeric gent to transform paddy rice.
Description
Technical field
The present invention relates to the plant subject, relate in particular to crop breeding and plant genetic engineering field, belong to the utilisation technology in biological and modern agricultural technology field.More particularly, the present invention relates to change the biosynthetic genetic expression of paddy endosperm starch, thereby produce the starch of tool high amylose starch molecular components at paddy endosperm.
Background technology
In recent decades, because breeding wheat for semidwarfness and heterotic utilization, the per mu yield of China's rice varieties and gross output have had significantly to be increased, and edible rice production is superfluous relatively.China's rice quality breeding started to walk at the beginning of the eighties, and two have obtained the progress of attracting attention during the last ten years, have bred a collection of fine quality that has their own characteristics each.But at present the high yield and high quality kind is few, and the production of good quality rice all also fails to satisfy the needs that living standards of the people improve day by day in quality or quantitatively.In addition, said the rice quality breeding in the past, just noted the eating quality aspect, industrial rice quality was lacked research or even blank.The quality of the non-glutinous rice varieties (as the grain purposes) of large-area applications from the present production, amylose content relatively concentrates on two classes at present: a class contains low or low straight chain powder content, between 12～18%, as the japonica rice (16～18%) of routine, important indica hybrid rice parent (to recover is in the majority, 12～16%).Next is the higher type of amylose content, and as Xian kind morning of routine, important indica hybrid rice parent (sterile line that is become by Xian kind transformation morning), their amylose content is generally more than 22%, have up to more than 25%.In addition, along with food and other industrial expansions, the demand of various particular type starch is constantly increased.Therefore, should pay attention to carrying out the seed selection of special purpose, special purpose rice variety, as the seed selection of industrial use kinds such as utmost point low amylose content, wine rice, high amylose content.
In the various purposes of rice, suitable amylose content is the objective trait of overriding concern.Requirement to amylose content often requires different according to characteristics, the industrial use difference of food Mi Renqun.As edible grain, people generally tend to medium amylose content on the low side.But different areas or crowd's requirement is different again, this existing in front explanation.The north is based on polished rice, and amylose content is generally on the low side, and at 16-18%, the meal that boils is relatively sticking softer; Rice district, south is based on long-grained nonglutinous rice, and amylose content requires higher slightly, is more satisfactory between 20-22%.On food industry applications, the rice of different amylose contents also has different purposes.The rice of low amylose content generally can be used for making infant food, and amylose content high be applicable to powder process, throwing, monosodium glutamate, wine beer, steamed millet etc.Tradition pudding requires based on glutinous rice, and this is because its viscosity is better; And also there is certain requirement in when wine brewing to amylose content.The rice wine of traditional shaohsing wine and so on all is raw material with glutinous rice.In candy is produced, heavy demand be higher starch of amylose content and acid-hydrolyzed starch.On pure industrial utilization, the paddy rice for high amylose content requires comparatively urgent at present.Particularly in starch-containing Biodegradable plastic is produced, need the starch of high amylose content, resulting product has mechanical property preferably.But often lack this germ plasm resource in the paddy rice, and in seeding corn and other crops, have the specific breed of tool extra-high-speed amylose content.Therefore, the paddy rice by certain technological line cultivation high amylose content meets market demand.
Technical scheme
The purpose of this invention is to provide and a kind ofly suppress the expression of the endogenous branching enzyme gene of paddy rice, cultivate the method for cultivation that can be used for industrial a kind of high amylose content paddy rice by genetic engineering technique.
Technical solution of the present invention is achieved in that it comprises:
(1) preparation comprises coding paddy rice branching enzyme gene or its segmental mosaic gene, branching enzyme gene wherein or its segmental upstream one side are operably connected to the direction of antisense can be in the rice paddy seed endosperm tissue on the promoter nucleic acid fragment of efficient specifically expressing, and its downstream one side is connected on the nucleic acid fragment of the appropriate regulation sequence that can be used for Transcription Termination;
(2) use the mosaic gene rice transformation again.
The gene of q enzyme of wherein encoding can be cDNA, also can be the genome structure gene, and this gene or its segmental nucleic acid fragment are derived from paddy rice itself.
Described paddy rice q enzyme comprises two classes, i.e. paddy rice q enzyme SBE1 and paddy rice q enzyme SBE3, more than two kinds of enzymes respectively by branching enzyme gene sbe1 and branching enzyme gene sbe3 coding.
All or part of of coding branching enzyme gene wherein or its segmental nucleic acid fragment coding water branch enzyme SBE1 and SBE3.
Coding branching enzyme gene wherein or its segmental nucleic acid fragment be structure construction with antisense in mosaic gene, perhaps be structured in the mosaic gene with the form that can form based on the hair clip shape structure of double-stranded RNA.
The mosaic gene of member prepares sbe1 or sbe3 gene or its segmental mosaic gene respectively, or preparation contains above-mentioned two genes or its segmental mosaic gene simultaneously.
The present invention has the method for high amylose content rice varieties by the genetic engineering technique seed selection, can overcome the restriction of high amylose content paddy rice inadequate resource in the conventional breeding, what amylose content was the highest reaches 55.9%, surpasses one times of existing rice varieties or more.And can produce the rice special kind that is applicable to the special industry purposes.
Description of drawings
Accompanying drawing 1 is the full length cDNA sequence that the present invention cloned and be used to make up the paddy rice sbe1 gene of antisense mosaic gene.
Accompanying drawing 2 is full length cDNA sequences that the present invention cloned and be used to make up the paddy rice sbe3 gene of antisense mosaic gene.
Accompanying drawing 3 is the plasmid structural representations that contain the antisense branching enzyme gene that make up among the present invention.
Accompanying drawing 4 is to import the influence of antisense branching enzyme gene to endogenous branching enzyme activity in the transgenic paddy rice seed.
Embodiment
Schedule of operation and technical essential that the present invention is concrete are as follows:
(1) clone of branching enzyme gene or its gene fragment.Nucleotide sequence according to paddy rice branching enzyme gene sbe1 that has published and sbe3 gene, the design primer, adopt from oryza sativa genomic dna, the increase part fragment of these two gene coding regions of round pcr, perhaps adopt the RT-PCR technology from the total RNA of paddy rice immature seed endosperm, to clone the total length or the part fragment of these two gene coding regions.In clone said gene or gene fragment, connect specific restriction endonuclease sites being designed respectively and add, so that the structure of antisense mosaic gene subsequently by 5 ' and 3 ' end of clone gene or gene fragment.The applicant as stated above, full-length cDNA or its part fragment of from No. 7, the military fortune of rice varieties round-grained rice, having cloned coding sbe1 and sbe3 successfully.
(2) structure of antisense vector.Utilize sbe1 and sbe3 gene or its part fragment of having cloned, oppositely be connected to can be in the rice paddy seed endosperm promotor downstream of specifically expressing, but and connect the terminator (as the terminator NOS of Agrobacterium rouge alkali synthetase gene) that terminator is transcribed in the downstream of antisense sbe1 or sbe3 gene fragment, form the antisense mosaic gene.And then this antisense mosaic gene is cloned on the carrier applicable to rice transformation.Its main points are:
1. being used to the branching enzyme gene or its fragment that make up, can be the full-length cDNA of this enzyme of coding, part nucleotide fragments that also can the coding region, or contain part segmental nucleotide sequence in coding region in the structure gene.
2. be used to instruct the promotor of antisense branching enzyme gene, be to use can be in paddy endosperm the promotor of specificity overexpression gene, for example promotor of paddy rice waxy gene, paddy rice glutenin gene or paddy rice branching enzyme gene.
3. when making up antisense vector, inverted defined gene or its fragment of sbe1 and two branching enzyme genes of sbe3 can be structured in respectively on two carriers, when transforming, can adopt cotransformation to import in the same rice varieties or import respectively in the different rice plants; Also two inverted defined genes can be structured on the identical carrier, once transform and two inverted defined genes can be imported in the same rice varieties simultaneously so that adopt.
4. use the RNAi technology.Except that adopting Antisense RNA Technique, also can utilize the RNAi technology, structure can form the mosaic gene member of dsRNA.
(3) the antisense branching enzyme gene is to the conversion of paddy rice.Adopt the method for transformation of mediations such as Agrobacterium, particle gun or pollen tube channel, the antisense q enzyme mosaic gene that makes up in (2) is imported in the rice varieties respectively.Its main points are:
1. when the antisense mosaic gene of two branching enzyme genes of sbe1 and sbe3 is structured on the different carriers respectively, can respectively these two inverted defined genes be imported in the different rice plants earlier, and then go in the same transgenic rice plant by two inverted defined genes of conventional hybridization polymerization;
2. or adopt co-transformation method of particle, two antisense branching enzyme genes are imported in the same transgenic rice plant simultaneously.
(4) expression analysis of endogenous branching enzyme gene in the transgenic paddy rice.After obtaining transgenic rice plant, get the blade sample vegetative period, extract total DNA, adopt PCR and Southern hybridization technique to differentiate the integration of antisense branching enzyme gene at transgenic rice plant.Then, after paddy rice blooms, from the paddy rice immature seed, extract total RNA and carry out the Northern hybridization analysis, whether be suppressed to detect transcribing of endogenous branching enzyme gene; Further can from immature seed or mature seed, extract protein, differentiate by measuring the Q enzymic activity whether the activity of q enzyme has decline, perhaps look into method reliably, promptly the antibody by q enzyme immunity preparation adopts immuning hybridization technical Analysis specific branch enzyme is whether expression amount changes.
(5) analysis of starch ingredients and structure in the transgenic paddy rice mature seed.Adopt classical starch content analytical procedure,, do not change with unconverted having compared to determine the starch composition in the transgenic paddy rice as the amylose content in the iodine colorimetry mensuration paddy rice mature seed;
Illustrate below in conjunction with concrete most preferred embodiment:
Embodiment 1:
Utilize paddy rice branching enzyme gene coding region part fragment to make up the antisense mosaic gene, behind the importing paddy rice starch synthetic in the transgenic paddy rice seed is influenced.
(1) the segmental clone of paddy rice branching enzyme gene coding region part.With No. 7 genome DNA of japonica rice variety military fortune round-grained rice is template, uses a pair of primer B with q enzyme sbe1 gene specific
1P
1(5 '-GATGGTGTACACTGGGATCCT-3 ') and B
1P
2(5 '-GATCCCGGGTATAGCATTGATGTAAC-3 ') carry out pcr amplification, and the genomic dna of having isolated length and be 683bp is called B
1F
1Fragment includes paddy rice sbe1 gene the 5th exon 3 ' hold 21bp, the 5th intron complete sequence (108 bp) and the 6th exon 5 ' to hold 554 bp sequences.Simultaneously, use a pair of primer B special again with sbe1 cDNA
1P
3(5 '-GAGGATCCGTGGCAATGTTCGCCTGAG-3 ') and B
1P
4(5 '-TAACCCGGGAATACGATCAACCCATG-3 ') or a pair of primer B special with sbe3 cDNA
3P
3(5 '-GCGGATCCTGAGTAGCACGGAGCCAAAG-3 ') and B
3P
4(5 '-AGTCCCGGGTAGGCATTCCACTGACATC-3 '), from the cDNA library in rice varieties IR36 endosperm source, amplified specific PCR product respectively, the molecular weight size is about 519bp and 640bP respectively, is called B
1F
2Fragment and B
3F
1Fragment.B
1F
2Fragment is positioned at the 5 ' end of sbe1 cDNA, behind the initiator codon ATG between 92bp and 610bp (sequence that has comprised the 2nd to the 5th exon); B
3F
1Fragment is positioned at the centre of sbe3 cDNA coding region, behind the initiator codon ATG between 965bp and 1604bp.
(2) structure of antisense q enzyme mosaic gene expression vector.For structure contains the segmental mosaic gene of antisense branching enzyme gene and is suitable for the agrobacterium mediation converted paddy rice, with above-mentioned clone's B
1F
1, B
1F
2And B
3F
1Insert respectively among the Agrobacterium binary vector p1301B HS (Cai Yi etc., 2002) after fragment is reverse.Be built into respectively drive by paddy rice q enzyme sbe1 gene promoter, contain segmental 3 the engineering plasmid p13B of antisense branching enzyme gene part
1, p13B
2And p13B
3, the structure in their T-DNA districts as shown in Figure 2.Simultaneously, made up again by paddy rice waxy gene promotor control, contain reverse B
1F
2And B
3F
1Segmental engineering plasmid p13WB
1, p13WB
3(accompanying drawing 2).
(3) acquisition of antisense branching enzyme gene rice plant.(Liu Qiaoquan etc. 1998 for the program of the agrobacterium mediation converted paddy rice of having set up by the applicant, Chen Xiuhua etc. 2001), nascent callus with No. 9, the military fragrant round-grained rice of japonica rice variety and rice variety Long Tefu B and special blue or green immature embryo source is a transformation receptor, by agriculture bacillus mediated the acquisition 5 engineering plasmids that contain the antisense branching enzyme gene of above-mentioned structure are imported respectively, and obtained more transgenic rice plant.After most of transgenic rice plants are transplanted into the land for growing field crops, most can normal growths, bloom and solid.
(4) analysis of branching enzyme activity in the transgenic rice plant endosperm.Bloomed back 10 days, 20 days and 30 days at transgenic rice plant, from the part plant, get 20 seeds respectively, the branching enzyme activity in its endosperm is measured, the results are shown in Table .15 with Fig. 4.By in the table as seen, in the more unconverted adjoining tree of the activity of q enzyme obvious decline is arranged in the transgenic rice plant seed endosperm, but decline scope difference between different transfer-gen plant and between the different seeds of same transfer-gen plant.By the analysis of branching enzyme activity, illustrate change the antisense branching enzyme gene over to after, the expression activity of branching enzyme gene has been subjected to inhibition.
(5) variation of amylose content in the transgenic paddy rice mature seed.Press individual plant results seed after the transgenic rice plant maturation, from each transformant, select an individual plant, the amylose content of its mature seed is analyzed.With the amylose content in the iodine colorimetry mensuration paddy rice mature seed.At first an individual plant to each transformant mixes survey, and each sample is done two repetitions, therefrom selects the bigger plant of amylose content increase rate then, again its seed is carried out simple grain and measures.The results are shown in Table 1 for the amylose content determination of transgenic rice plant mature seed for part T0.From table 1 data as seen, in japonica rice variety, change antisense q enzyme sbe3 gene (engineering plasmid is p13WB3, and code name is B3) over to after, the amylose content of seed has had raising to a certain degree, exists compared with the control as B3-47, B3-60 to significantly improve (t
0.05), improved 19.62% and 14.50% respectively.And the raising of amylose content is not obvious in the plant of antisense q enzyme Sbe1 gene (engineering plasmid is p13WB3, and code name the is B1) seed.After changing antisense sbe1 gene over to rice variety, the amylose content of transfer-gen plant mature seed is compared with unconverted adjoining tree, does not only improve to have descended to some extent on the contrary.Wherein code name be B1-2 (20.32%) descend at most, reduced by 27.09% than unconverted contrast (27.87%).B1-5 (24.54%), B1-3 (22.47%), B1-4 (23.27%) compare also with unconverted contrast and have descended in addition, and fall is respectively: 11.95%, 18.94% and 16.05%.After changing antisense sbe3 gene over to long-grained nonglutinous rice, the amylose content of transfer-gen plant mature seed is compared no considerable change with unconverted adjoining tree.
Transgenic paddy rice T after the table 1 importing antisense branching enzyme gene part fragment
0For the amylose starch in the plant mature seed
| The receptor parent title | The plant code name | Amylose content (%) |
| Repeat two mean values | ||
| Military No. 9, fragrant round-grained rice (round-grained rice) | The unconverted contrast of B1-27 B1-36 B3-47 B3-24 B3-60 B3-14 | ????19.7756????21.0634????20.419 ????19.3736????20.6316????20.003 ????22.1055????22.4331????22.269 *????17.4978????19.7905????18.644 ????21.1602????22.6936????21.927 *????18.8749????16.6641????17.769 ????18.4804????18.8228????18.652 |
| Long Tefu (Xian) | The unconverted contrast of B1-2 B1-5 B3-3 B3-5 | ????20.1233????20.5243????20.324 ????24.9231????24.1584????24.541 ????29.0033????28.9206????28.962 ????27.7180????29.2202????28.469 ????28.4324????27.3092????27.871 |
| Special blue or green (Xian) | The unconverted contrast of B1-3 B1-4 B3-1 | ????23.0259????21.9155????22.471 ????24.1984????22.3457????23.272 ????26.7571????25.4502????26.104 ????27.7679????27.6682????27.718 |
*Expression is compared with unconverted contrast, amylose content be significantly increased (5% conspicuous level).
Embodiment 2:
Utilize paddy rice branching enzyme gene full length cDNA sequence to make up the antisense mosaic gene, behind the importing paddy rice starch synthetic in the transgenic paddy rice seed is influenced.
(1) clone of paddy rice branching enzyme gene Sbe1 and Sbe3 cDNA.RT-PCR method by improvement synthesized paddy rice immature seed endosperm cDNA storehouse, and as template, from No. 7, the military fortune of japonica rice variety round-grained rice, cloned two gene Sbe1 and the Sbe3 of encode respectively SBE1 and SBE1II with round pcr.Sequential analysis shows that the size of clone Sbe1 and Sbe3 gene is respectively 2490bp and 2481bp, has comprised the encoding sequence of gene complete.Accompanying drawing 1 is the Sbe1 and the Sbe3 cDNA nucleotide sequence of total length.
(2) structure of total length antisense q enzyme mosaic gene expression vector and the acquisition of transgenic rice plant.The long promoter region sequence of paddy rice gluten GT1 gene translation upstream from start codon 1.3kb that utilizes us to clone.This Gt1 promotor and reverse sbe1 and sbe3 full length gene cDNA and Nos terminator are built into fusion gene, again this fusion gene are structured in the multiple clone site of binary vector pCAMBIA1300, obtained binary vector pYH698 and pYH612.Identify that correct binary vector imports among the agrobacterium tumefaciens bacterial strain EHA105 through freeze-thaw method, is used for the rice conversion of agricultural bacterium mediation.Press method for transformation described in the embodiment 1, respectively the antisense branching enzyme gene sbe1 of total length and sbe3 are imported in No. 9, the military fragrant round-grained rice of rice varieties, obtain transgenic rice plant.
(3) variation of amylose content in the transgenic paddy rice mature seed.Analyzed amylose content in the transgenic rice plant mature seed by measuring method described in the embodiment 1.Part T
0Amylose content determination for the transgenic rice plant mature seed the results are shown in table 2.After changing the antisense branching enzyme gene over to, the amylose content of seed is improved to some extent, as the transgenic rice plant 612-7, the 612-11 that import total length antisense sbe3 gene (transfer-gen plant is numbered 612) compare with unconverted contrast with 612-31, the amylose content (t that is significantly increased
0.05), improved 18.32%, 16.77% and 13.19% respectively.And the amylose content that changes in transfer-gen plant (code name the is 698) seed of total length antisense Sbe1 gene does not have obvious raising.
The military fragrant round-grained rice of table 2 changes total length antisense branching enzyme gene paddy rice T for No. 9
0Amylose starch for part plant mature seed
Amylose content (%)
The plant code name
Repeat two mean values
698-12????????19.9021????????18.5771????????19.240
698-17????????20.2342????????19.4217????????19.828
698-15????????19.1429????????19.6491????????19.396
698-6?????????19.7160????????17.5201????????18.618
698-8?????????20.4083????????18.3985????????19.403
612-7?????????21.8651????????22.2869????????22.076
*
612-25????????18.6441????????19.2322????????18.938
612-9?????????20.3116????????19.3588????????19.835
612-11????????21.5797????????22.1130????????21.846
*
612-31????????20.4009????????21.8227????????21.112
*
Unconverted contrast 18.4804 18.8228 18.652
*Expression is compared with unconverted contrast, amylose content be significantly increased (5% conspicuous level).
Embodiment 3:
After the sbe1 of antisense and two mosaic genes of sbe3 are imported same rice plant simultaneously, to the transgenic paddy rice kind middle starch synthetic influence.
(1) antisense sbe1 and sbe3 mosaic gene are to the polymerization of same rice plant.Adopt the method for conventional hybridization, with importing the transgenic rice plant of antisense sbe1 and sbe3 mosaic gene being arranged respectively is that father and mother originally carry out group gas-mixing hybridization, and the F1 plant is planted in the F1 that obtains of institute hybridization.This hybrid strain is identified through PCR and Southern hybridization, is contained two antisense mosaic genes really simultaneously.
(2) the Q-enzyme activation analysis in the transgenic paddy rice immature seed of polymerization antisense sbe1 and sbe3 mosaic gene.Table 3 shows that polymerization has the Q-enzyme activity in the immature seed on the transgenosis hybrid plant of two antisense branching enzyme genes will be starkly lower than Q-enzyme activity in the unconverted adjoining tree immature seed.After explanation suppressed sbe1 and sbe3 gene simultaneously, the Q-enzyme activity can obviously reduce in the immature seed.
(3) amylose content in the transgenosis hybrid paddy rice mature seed.After the hybrid rice paddy seed maturation, press the individual plant results.The seed of each individual plant is carried out the mensuration of amylose content, and result such as table 4 are listed.As can be seen from the table, the more unconverted contrast of amylose content of transgenosis hybrid paddy rice mature seed is significantly improved, and what wherein simple grain was the highest reaches 55.90%.Illustrate suppress SBE1 and SBE1II expression of gene simultaneously after, can improve the relative content of amylose starch in the rice paddy seed really greatly.
Q-enzyme activity in table 3 part antisense sbe1 and the sbe3 gene hybrid paddy rice immature seed
Mean value
Hybrid numbering combination simple grain immature seed branching enzyme activity (%)
(％)
19.54??13.70??13.51??13.04??12.16
Blue or green morning-sbe1/ Long Tefu-the sbe3 10.96 of C2905-1 association
10.39??9.59???8.86???7.23???1.54
51.72??43.40??36.76??28.95??26.98
Blue or green morning-sbe1/ Long Tefu-the sbe3 28.79 of C2913-1 association
26.98??22.73??21.54??15.38??13.46
5.71???60.29??60.00??59.38??57.14
Blue or green morning-sbe1/ Long Tefu-the sbe3 51.87 of C2918-4 association
50.00??44.44??42.63??37.04??32.08
50.00??27.78??22.39??20.99??18.75
Blue or green morning-sbe1/ Long Tefu-the sbe3 20.30 of C2946 association
15.94??10.14??9.43???7.25
75.41??67.31??66.67??65.71??62.30
Blue or green morning/the Long Tefu 60.78 of C2933 association
60.98??55.00??55.00??50.91??48.48
Amylose content in the table 4 transgenosis hybrid paddy rice mature seed
Mix and survey
Hybrid numbering combination single seed amylose content (%)
(％)
36.95??35.87??35.84??35.81??34.85
Blue or green morning-sbe1/ Long Tefu-the sbe3 30.00 of C2905-1 association
34.58??34.15??33.97??33.20??29.72
37.00??36.26??36.21??36.10??35.53
Blue or green morning-sbe1/ Long Tefu-the sbe3 24.39 of C2913-1 association
34.53??34.04??33.16??32.98??31.39
32.29??31.81??31.29??30.88??30.70
Blue or green morning-sbe1/ Long Tefu-the sbe3 26.47 of C2918-4 association
30.60??30.14??29.68??28.84??28.09
55.90??50.90??45.25??45.00??42.19
Special green grass or young crops-the sbe3 32.17 of C2910-4 Long Tefu-sbe1/
35.88??35.50??35.00??32.50??30.94
30.13??29.98??29.50??29.26??28.28
The special green grass or young crops of C2942-3-sbe1/ Long Tefu-sbe3 25.02
26.69??24.50??23.13??22.03??21.54
31.31??29.38??29.10??29.10??28.06
The unconverted contrast of C2878: Long Tefu/association blue or green early 26.07
27.77??27.54??27.32??27.08??26.07
31.97??31.78??31.64??31.10??30.56
The unconverted contrast of D2918: special green grass or young crops/Long Tefu 27.46
30.18??30.16??29.61??29.00??28.61
Claims (6)
1. method of cultivation with high amylose content paddy rice is characterized in that it comprises:
(1) preparation comprises coding paddy rice branching enzyme gene or its segmental mosaic gene, branching enzyme gene wherein or its segmental upstream one side are operably connected to the direction of antisense can be in the rice paddy seed endosperm tissue on the promoter nucleic acid fragment of efficient specifically expressing, and its downstream one side is connected on the nucleic acid fragment of the regulating and controlling sequence that can be used for Transcription Termination;
(2) use the mosaic gene rice transformation again.
2. the method for cultivation with high amylose content paddy rice according to claim 1 is characterized in that, the gene of the q enzyme of wherein encoding can be cDNA, also can be the genome structure gene, and this gene or its segmental nucleic acid fragment are derived from paddy rice itself.
3. the method for cultivation with high amylose content paddy rice according to claim 2, it is characterized in that, described paddy rice q enzyme comprises two classes, i.e. paddy rice q enzyme SBE1 and paddy rice q enzyme SBE3, more than two kinds of enzymes respectively by branching enzyme gene sbe1 and branching enzyme gene sbe3 coding.
4. the method for cultivation with high amylose content paddy rice according to claim 3 is characterized in that, all or part of of coding branching enzyme gene wherein or its segmental nucleic acid fragment coding water branch enzyme SBE1 and SBE3.
5. the method for cultivation with high amylose content paddy rice according to claim 1, it is characterized in that, coding branching enzyme gene wherein or its segmental nucleic acid fragment be structure construction with antisense in mosaic gene, perhaps be structured in the mosaic gene with the form that can form based on the hair clip shape structure of double-stranded RNA.
6. the method for cultivation with high amylose content paddy rice according to claim 5, it is characterized in that, the mosaic gene that makes up is the mosaic gene for preparing the water section of sbe1 or sbe3 gene or its sheet seed respectively, or preparation contains above-mentioned two genes or its segmental mosaic gene simultaneously.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146746A (en) * | 2013-03-18 | 2013-06-12 | 扬州大学 | Transgenic rice with excellent eating quality and cultivation method |
| CN108192911A (en) * | 2017-12-31 | 2018-06-22 | 青岛袁策生物科技有限公司 | A kind of method of starch quality in improvement rice |
| CN108239655A (en) * | 2017-12-31 | 2018-07-03 | 青岛袁策生物科技有限公司 | The method for promoting rice grain starch quality |
-
2003
- 2003-09-25 CN CNA031583512A patent/CN1537941A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146746A (en) * | 2013-03-18 | 2013-06-12 | 扬州大学 | Transgenic rice with excellent eating quality and cultivation method |
| CN108192911A (en) * | 2017-12-31 | 2018-06-22 | 青岛袁策生物科技有限公司 | A kind of method of starch quality in improvement rice |
| CN108239655A (en) * | 2017-12-31 | 2018-07-03 | 青岛袁策生物科技有限公司 | The method for promoting rice grain starch quality |
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