CN1625939A - Method for poducing transgene seeds of hybrid paddy rice in multiple resistances - Google Patents
Method for poducing transgene seeds of hybrid paddy rice in multiple resistances Download PDFInfo
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- CN1625939A CN1625939A CN 200310107230 CN200310107230A CN1625939A CN 1625939 A CN1625939 A CN 1625939A CN 200310107230 CN200310107230 CN 200310107230 CN 200310107230 A CN200310107230 A CN 200310107230A CN 1625939 A CN1625939 A CN 1625939A
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Pretreatment Of Seeds And Plants (AREA)
Abstract
A process for preparing the seeds of multi-resistance transgenic rice includes transferring the anti-herbicide gene and the anti-borer gene to the restoring line of three-line or two-line crossed rice, transferring the plant hopper-resistant gene to the maintenance line of three-line crossed rice, hybridizing between sterile line and maintenance line, and hybridizing between transgenic restoring line and transgenic sterile line.
Description
Technical field
The present invention relates to a kind of production method of hybrid rice seed, particularly a kind of production method of how anti-transgenosis hybrid rice seed.
Background technology
Paddy rice is nearly half people's in the whole world a staple food.The whole world is annual to be seized about 1/3rd by disease pest and weed.According to estimates, the economic loss that causes reaches more than 1,200 ten thousand dollars every year.In order to tackle disease pest and weed, also to produce about more than 200 ten thousand tons agricultural chemicals every year.For example: striped rice borer and rice fulgorid are the two big main insect pests in northern rice field, can make the paddy rice underproduction 10%-20% every year, and severe patient also can cause total crop failure.The major measure of current pest control is to use insecticides such as dimehypo and furans pellet in a large number, annual consumption can reach more than 1,000 ten thousand kilograms, value is about 2,000 ten thousand yuan, not only cause the waste of manpower and financial resources, but also cause environmental pollution, As time goes on, the residual hazard of agricultural chemicals, the pesticide resistance of insect also can become increasingly conspicuous, in promoting the hybrid rice plantation, run into above-mentioned insect pest problem equally now, also run into the impure problem of hybrid rice seed, the two has seriously restricted the popularization and the application of hybrid rice.Be that resistance to insects is poor, can not guarantee high and stable yields; Seed rice is impure, unsuitable establishing in large scale.These two problems are difficult to effectively solve with the conventional breeding method, mix as very easily cause biology and mechanicalness in the hybrid rice production of hybrid seeds, and especially two-line hybrid rice self mixes.For this reason, people are exploring its solution and are obtaining achievement, as having related in Chinese patent application number 99115125.9 application documents anti-herbicide gene is imported the ternary hybrid rice maintenance line so that it is the acquisition anti-herbicide gene, make it to possess the antiweed characteristic.And for example: Chinese patent application number: relate in the 97109814.X file and change anti-herbicide gene over to three and be or two be hybrid rice restoring line, make it to obtain anti-herbicide gene, possesses the antiweed characteristic, only remove grass when protecting in cultivation with the weed killer herbicide weeding, and do not injure the hybridization rice seedling, to produce the high seed rice of purity, in this two application case, all do not relate to the problem that the hybrid rice production of hybrid seeds imports anti-insecticide gene.
Summary of the invention
The objective of the invention is at the above-mentioned problems in the prior art, a kind of production method of how anti-transgenosis hybrid rice seed is provided, can overcome the problem that hybrid rice seed inadequately greatly can not large tracts of land be produced because of purity, can strengthen the resistance to insects of hybrid rice again, improve output.
Technical scheme of the present invention is the recovery system that anti-herbicide gene and anti-snout moth's larva gene is changed over to ternary hybrid rice or two-line hybrid rice, makes to recover system and obtain anti-herbicide gene and anti-striped rice borer gene, possesses antiweed and striped rice borer characteristic; Change against-plant hopper gene the maintenance line of ternary hybrid rice over to, make maintenance line obtain against-plant hopper gene, possess the against-plant hopper characteristic; By male sterile line and maintenance line hybridization, make male sterile line obtain against-plant hopper gene, possess the against-plant hopper characteristic; Utilize the hybridization of resulting recovery system and male sterile line again, produce hybrid rice seed.That is to say: with Snowdrop lectin (the Galanthus nivalis agglutinin of against-plant hopper, GNA) gene imports among maintenance line early blossoming two B of hybrid rice, with the moisture resistance mycin (Hygromycinphosphotransferase, Hyg) gene is as the selection markers gene; Cowpea Trypsin Inhibitor (modified cowpea trypsin inhibitor with the anti-snout moth's larva after modifying, modifiedCpTI=SCK) gene imports in super excellent No. one of the hybrid rice restoring line, with the bar gene of antiweed Basta as the selection markers gene, simultaneously also as genes of interest.In numerous kind of process of male sterile line, male sterile line early blossoming two A have just obtained the against-plant hopper characteristic like this; By hybrid rice production of hybrid seeds process, hybrid rice has just obtained two kinds of resistance to insectss and the antiweed characteristic among the parent.
The assembly parent of many anti-transgenosis Japonica Hybrid
The gene that 1 transgenic restorer line transforms:
1.1 anti-striped rice borer gene
Genes of interest.Used SCK gene is Cowpea Trypsin Inhibitor (the Cowpea trypsin inhibitor after modifying, CpTI.Modified CpTI=SCK) gene, derive from cultivation cowpea variety California cowpea (Vigna unguiculata L.Walp, cv.CaliforniaBlack-eye), be a kind of of serpin, the double end protease inhibitors that belongs to the Bowman-Birk type, promptly inhibitor molecules can suppress the catalytic activity (Hilder of two trypsase molecules, people such as V.A, 1989).CpTI mainly is present in the edible part seed of cowpea, belongs to the seed storage protein of albumin type.The CpTI gene begins to express in seed grouting in early days, and synthetic rapidly CpTI, and the content of CpTI can reach about 10% (Hilder, people such as V.A, 1990) of total protein content in the final mature seed.
Target gene sequences total length 415bps, encode 136 amino acid and a terminator, front end is 25 amino acid whose endoplasmic reticulum framing signal Signal, end is 4 amino acid whose KDEL KDEL, the centre is the CpTI gene of 324bps, 107 amino acid residues of encoding.Comprising 27 amino acid leader peptide sequences and 80 amino acid whose maturation protein sequences.
SCK gene primer sequence:
5′：5′-AAA?ATC?AAG?AGC?ACC?ATC?TTC?3′
3′：5′-TCT?AGA?GTT?CAT?CTT?TCT?CAT?C?3′
1.2 anti-herbicide gene
Marker gene and genes of interest.It is the bar gene of the anti-PPT (Phosphinotricin) that separates from streptomyces hygroscopicus (StrepTomyceshygroscopicus), PPT is a kind of inhibitor of glutamine synthelase, it is generally acknowledged that the PPT that generates in the plant corpus can suppress glutamine synthelase and cause the ammonia accumulation, plant is poisoned to death because of the ammonia accumulation.Bar gene code transacetylase PAT (Phosphinotricin acetyltransferase), PAT make acetyl combine with the free amine group of PPT, and PPT is lost activity, and can not suppress glutamine synthelase.Therefore, can provide resistance to PPT.Bar gene code transacetylase PAT mainly is present in the leaf part of plant, does not express in the seed part of plant.
Bar gene primer sequence:
5′：5′-TCA?AAT?CTC?GGT?GAC?GGG?C?3′
3′：5′-CAC?CAT?CGT?CAA?CCA?CTA?C?3′
2, the gene of transgenic maintainer line conversion:
2.1 against-plant hopper gene
Genes of interest.Used gene is that (galanthus nivalisagglutinin, GNA) gene derives from snowdrop to Snowdrop lectin.Snowdrop (galanthus nivalis) belongs to Amaryllidaceae (wmarvillidaceace) ornamental plants, contains GNA in the great majority tissue in snowdrop certain growth stage, the only single-minded identification of GNA and in conjunction with α-1,3 and α-1,6 mannose.To the glycoconjugate of insect gut, this combination has influenced normal absorption (powell, 1993 of nutriment to the toxicity of lectin based on their specific bond; Van damme, 1988). in addition, also have the research report, lectin can promote bacterial reproduction in the insect digestive tract, and brings out aura (liener, 1986), finally insect is caused serious harm, suppresses the insect growth and breeding, thereby reaches the purpose of desinsection.
Target gene sequences total length 484bps, encode 157 amino acid and two terminators, the N-end is 23 amino acid whose signal peptides, end is 29 amino acid whose C-terminal sequences, 105 amino acid whose maturation proteins of intermediate code.
The GNA primer sequence is:
5′：5′-CAA?AAT?GGC?TAA?GGC?AAG?TC-3′
3′：5′-CGG?TCA?TTA?CTT?TGC?CGT?CA-3′
2.2 the mould plain gene of moisture resistance
Marker gene.(hygromycin Bphosphotransferase, hyg) gene claim aph (4)-Ta or hpt again, and the hph gene is from streptomyces hygroscopicus (Gritz, L.﹠amp for hygromycin B phosphotransferase; Davies, J., 1983).Aph (4)-Ta coded amino acid glucosides 4-phosphotransferase [APH (4)-Ta] claims hygromix phosphotransferase again.But hygromycin B Profilin matter is synthetic, and APH (4)-Ta can be transferred to ATPXa-position phosphorus on the 4th hydroxyl of hygromycin B, and it is active to make it lose inhibition.Therefore, it can provide the resistance to hygromycin.
The method for transformation of above gene is the particle gun blast technique.
3, the gene of transgenosis male sterile line conversion:
The mould plain gene of against-plant hopper gene and moisture resistance is obtained by the transgenic maintainer line breeding.
The compound formulation of many anti-transgenosis Japonica Hybrid
1, numerous kind of the transgenosis male sterile line
Process is as shown in the figure:
Common male sterile line * transgenic maintainer line (containing the GNA gene)
↓ (against-plant hopper)
↓
Transgenosis male sterile line (containing the GNA gene)
(against-plant hopper)
2, the production of hybrid seeds of how anti-transgenosis Japonica Hybrid
Process is as shown in the figure:
Transgenosis male sterile line * transgenic restorer line
(containing the GNA gene) ↓ (containing SCK, bar gene)
(against-plant hopper) ↓ (anti-striped rice borer, antiweed)
↓
Many anti-transgenosis Japonica Hybrid
(containing SCK, GNA, three foreign genes of bar)
(anti-striped rice borer, against-plant hopper, antiweed)
The assembly advantage of many anti-transgenosis Japonica Hybrid
1, use method of the present invention, as long as the random sampling sowing, seedling spraying weed killer herbicide Basta just can easy, accurately count the purity of each batch production of hybrid seeds hybrid rice within one week, and qualification time is short, identifies the efficient height.
2, use hybrid rice seed that method of the present invention produces and can in time dispose pseudostationary by herbicide spraying Basta, also can eliminate weeds simultaneously, thereby improve the purity in production field, embody hybrid vigour better in the sowing nursery period.
3, the hybrid rice seed of application method production of the present invention has the characteristic of anti-striped rice borer and against-plant hopper; can resist the harm of snout moth's larva and rice fulgorid; improve rice yield; and saved expense because of the use that has reduced agricultural chemicals in a large number; alleviated agricultural chemicals murdering and, also protected environment simultaneously to beneficial insect to the murder by poisoning (residual) that product produces.Therefore planting pest-resistant hybrid rice can obtain better social benefit and bigger economic benefit than conventional hybridization rice.
4, the hybrid rice of application the present invention production can be society provides more high quality, of low pollution rice.Along with improving constantly of China's living standards of the people, people are more and more higher to the quality requirements of rice, particularly high-quality, very vast market will not arranged by the rice of pesticide pollution.The production of hybrid seeds of hybrid rice itself and numerous kind of process have been given hybrid rice with wide industrialization prospect.
Embodiment
Three line method is produced the hybrid rice seed sub-instance
1, parent
1.1 recover system
1.1.1 genetic transformation acceptor
Recovering for the high quality japonica of Tianjin Inst of Paddy Rice's seed selection is super excellent No. one (crying Tianjin rice 1229 again).In the 145 days time of infertility of Beijing-Tianjin regional culture, per mu yield 500-600Kg.About plant height 110cm, fringe is big, spike length 22cm, 116 of average every fringes; Grain is big, thousand kernel weight 26-27g, ripening rate 91%.Through Ministry of Agriculture's grain quality supervision, the check of verification test center, every index all reaches country-level superior rice standard.
1.1.2 transgenic restorer line
The time of infertility same transformation receptor, about 145 days.About plant height 115cm, about spike length 26cm, 163 of average every fringes; About thousand kernel weight 25g, ripening rate about 81%.Resistance to insects and antiweed characteristic have been increased.
1.2 maintenance line
1.2.1 genetic transformation acceptor
High quality japonica maintenance line early blossoming two B for Tianjin Inst of Paddy Rice's seed selection.About the 160 days time of infertility, be suitable for doing morning, middle rice culture.General per mu yield 550Kg.The stem stalk is short strong, hard, and lodging resistant force is strong.About plant height 85cm, bar-shaped fringe, it is closeer grain.About 135 of average every fringes, about spike length 17cm, ripening rate 94%, grain type ellipse, thousand kernel weight 23g.No awns or short tip awn is arranged.
1.2.2 transgenic maintainer line
The time of infertility isoacceptor material.About plant height 90cm, higher slightly than acceptor; The fringe type looses slightly than acceptor, other proterties isoacceptors.Against-plant hopper and hygromycin characteristic have been increased.
1.3 male sterile line
1.3.1 transgenosis transformation receptor
High quality japonica male sterile line early blossoming two A for Tianjin Inst of Paddy Rice's seed selection.About the 160 days time of infertility.The stem stalk is short strong, hard, and lodging resistant force is strong.About plant height 85cm, bar-shaped fringe, it is closeer grain.About 130 of average every fringes, about spike length 16cm, self-fruitful rate is 0, outcrossing rate about 30%, grain type ellipse, no awns or short tip awn is arranged.
1.3.2 transgenosis male sterile line
Breed by transgenic maintainer line.Its breeding time and other economical characters are with early blossoming two A.Self-fruitful rate is 0.About outcrossing rate 30%.Against-plant hopper and hygromycin characteristic have been increased.
2, compound formulation
2.1 numerous kind of transgenosis male sterile line
Process is as shown in the figure:
Common male sterile line early blossoming two A * transgenic maintainer line T early blossoming two B (containing the GNA gene)
↓ (against-plant hopper)
↓
Transgenosis male sterile line T early blossoming two A (containing the GNA gene)
(against-plant hopper)
2.2 the production of hybrid seeds of how anti-transgenosis hybrid rice
Process is as shown in the figure:
Super excellent No. one of transgenosis male sterile line T early blossoming two A * transgenic restorer line T
(containing the GNA gene) ↓ (containing SCK, bar gene)
(against-plant hopper) ↓ (anti-striped rice borer, antiweed)
↓
Many anti-transgenosis Japonica Hybrid combinations
(containing SCK, GNA, three foreign genes of bar)
(anti-striped rice borer, against-plant hopper, antiweed)
3, herbicide treatment result
| Combination | The rice seedling bed number of seedling | Anti-Basta seedling | Quick to Basta | The hybrid rice production of hybrid seeds |
| (true hybrid rice strain) | Sense seedling (false hybrid rice strain) | Purity (%) | ||
| Combination 1 | ?52 | ?43 | ?9 | ?83 |
| Combination 2 | ?180 | ?167 | ?13 | ?93 |
| Combination 3 | ?41 | ?23 | ?18 | ?56 |
Claims (7)
1, a kind of production method of how anti-transgenosis hybrid rice seed, it is characterized in that anti-herbicide gene and anti-snout moth's larva gene are changed over to the recovery system of ternary hybrid rice or two-line hybrid rice, make and recover system's acquisition anti-herbicide gene and anti-striped rice borer gene, possess antiweed and striped rice borer characteristic; Change against-plant hopper gene the maintenance line of ternary hybrid rice over to, make maintenance line obtain against-plant hopper gene, possess the against-plant hopper characteristic; By male sterile line and maintenance line hybridization, make male sterile line obtain against-plant hopper gene, possess the against-plant hopper characteristic; Utilize the hybridization of resulting recovery system and male sterile line again, produce hybrid rice seed.
2, the production method of a kind of how anti-transgenosis hybrid rice seed according to claim 1 is characterized in that its anti-careless agent gene is for being the bar gene.
3, the production method of a kind of how anti-transgenosis hybrid rice seed according to claim 1 is characterized in that its anti-snout moth's larva gene is SCK gene (CpTI after the modification, a cowpea trypsin inhibitor gene).
4, the production method of a kind of how anti-transgenosis hybrid rice seed according to claim 1 is characterized in that its against-plant hopper gene is GNA gene (a Snowdrop lectin gene).
5, the production method of a kind of how anti-transgenosis hybrid rice seed according to claim 1 is characterized in that: the transgenosis male sterile line of its ternary hybrid rice is T early blossoming two A systems, breeds acquisition by transgenic maintainer line T early blossoming two B that show against-plant hopper.
6, the production method of a kind of how anti-transgenosis hybrid rice seed according to claim 1 is characterized in that: its transgenic restorer line is super excellent No. one of the T of the anti-Basta of performance.
7, the production method of a kind of how anti-transgenosis hybrid rice seed according to claim 1 is characterized in that: its method for transformation can be the particle gun blast technique.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310107230 CN1625939A (en) | 2003-12-08 | 2003-12-08 | Method for poducing transgene seeds of hybrid paddy rice in multiple resistances |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310107230 CN1625939A (en) | 2003-12-08 | 2003-12-08 | Method for poducing transgene seeds of hybrid paddy rice in multiple resistances |
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| Publication Number | Publication Date |
|---|---|
| CN1625939A true CN1625939A (en) | 2005-06-15 |
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| CN 200310107230 Pending CN1625939A (en) | 2003-12-08 | 2003-12-08 | Method for poducing transgene seeds of hybrid paddy rice in multiple resistances |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100445383C (en) * | 2005-12-13 | 2008-12-24 | 华中农业大学 | Rice starter expressed by induction of chilo simplex eating speciality and its use |
| CN103229712A (en) * | 2013-05-06 | 2013-08-07 | 福建农林大学 | Breeding method of polyclonal antibody cold-resistant two-line sterile line |
-
2003
- 2003-12-08 CN CN 200310107230 patent/CN1625939A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100445383C (en) * | 2005-12-13 | 2008-12-24 | 华中农业大学 | Rice starter expressed by induction of chilo simplex eating speciality and its use |
| CN103229712A (en) * | 2013-05-06 | 2013-08-07 | 福建农林大学 | Breeding method of polyclonal antibody cold-resistant two-line sterile line |
| CN103229712B (en) * | 2013-05-06 | 2014-02-19 | 福建农林大学 | Breeding method of multi-resistance cold-resistant two-line sterile line |
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