CN1112098C

CN1112098C - Method for raising rice yield potentiality

Info

Publication number: CN1112098C
Application number: CN99116561A
Authority: CN
Inventors: 张启发; 曹孟良; 林拥军; 徐才国
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 1999-07-23
Filing date: 1999-07-23
Publication date: 2003-06-25
Anticipated expiration: 2019-07-23
Also published as: CN1281636A

Abstract

The present invention relates to a method for enhancing the yield potentiality of rice, which belongs to the technical field of the breeding of new plant varieties. The method is characterized in that A P<SAG12>-IPT gene is transferred in rice by a transgenic method, transgenic plants are identified by report genes, the detection of polymerase chain reaction (PCR) and molecular hybridization, the photosynthetic property and the agronomic character of descendants are researched for breeding transgenic plant lines with obvious retardation of leaf senescence, obvious improvement of photosynthetic property and larger enhancement of single-plant yield so as to culture high-yield varieties and create new breeding resources.

Description

A kind of method that improves rice yield potentiality

Technical field

The invention belongs to the rice breeding technology field.Be specifically related to a kind of applying transgene method with self-control blade suppressor (P _SAG12-IPT) import paddy rice, improve yield potentiality by delaying leaf senile, cultivate the method for new high-yielding rice varieties or creation new resources.Its international Patent classificating number is A01H1/00.

Background technology

The main method that conventional hybridization and seed selection are still current rice high yield breeding.Because it is subjected to the restriction of sexual hybrid strain resource, uses difficult increasing that this breeding method breeds high-yield variety.The application of gene engineering method makes people be able to break through the boundary of species biologically in the utilization of genetic resources.Though plant genetic engineering has been obtained bigger success at aspects such as disease-resistant, pest-resistant, degeneration-resistant, the antiweed of improvement crop, quality, cross-breeding technologies, do not finding effective way so far yet aspect the output improving.

Aging is the process of programmed cell death.Although the blade sorrow can promote the nutrition of each organ of plant to redistribute always, make plant be able in adverse circumstance, survive and raise up seed, the forfeiture of the assimilation that aging course is followed usually is the factor of restriction crop yield.As in paddy rice, the rice varieties that production is at present upward used is the early ageing phenomenon of various degrees mostly.Because blade later stage photosynthesis deficiency, organic matter accumulation is restricted, and causes the heavy decline of ripening rate and dry granular, finally influences output.There is statistics to show that the ripening rate of most of kinds is between 70%-75%; Also there is the not full phenomenon of grouting in various degree in solid grain husk flower because photosynthetic product is under-supply.Therefore, the yield potentiality of kind is brought into play far away.In large-spike cultivar and breeding intermediate materials, this phenomenon is even more serious.Therefore, increase photosynthetic capacity by the adjusting vane aging and improve rice yield, have great potentialities and to dig.

The years of researches result shows and can delay the process of leaf senile by the basic element of cell division (Richmond etc. 1957, Science125,650-651; Van staden etc. 1988, (Eds) such as In L D Nooden, Senescence and aging in plant, Academic Press, San Diego:281-328; Smart etc. 1991, Plant Cell 3,647-656).Therefore people's analog of once attempting dosed cells mitogen or synthetic delays senility.Ludford etc. (1987, In P J Davies (Eds), Plant hormones and their role in plant growth and development, Martinus Nijhoff, Boston:574-592) can delay stripped senescence of leaves time of leaf vegetables in this way effectively, Borochov etc. (1989, In J Janick (Eds), Horticultural Reviews, Timber Press Portland:15-43) has delayed the old and feeble time of cut-flower in the flowers in this way effectively.Yet because cost is high and big for environment pollution, this method fails to be extensive use of.People's using gene engineering method is also arranged, make basic element of cell division excess synthetic to delay leaf senile.Its basic way is with various different promoters and prenyltransferase (isopentenyl transferase, IPT) gene fusion chimeric genetic construct, conversion plant.Because employed promotor all causes genetically modified plants to show as basic element of cell division overexpression, though the some of them plant shows the feature that leaf senile is delayed, but all there be the undesired of form and growth in various degree in these transfer-gen plants, so the potentiality of practical application and little.

(1995, Science 270,1986-1988) at above-mentioned IP T genetically modified plants weak point, designed a kind of self regulating and control and delayed the leaf senile system for Gan and Amasino.They are cloned into the gene promoter P of specifically expressing in the old and feeble blade from arabidopsis _SAG12(its international patent is PCT/US96,02313) is built into mosaic gene P with itself and IPT gene fusion _SAG12-IPT, layout strategy is: when blade senesces, the promotor P of old and feeble leaf specific expressino _SAG12Being activated gives expression to IPT, promotes the synthetic of the basic element of cell division, the level of endogenous cell mitogen is improved, thereby delay leaf senile; And the leaf senile process is obstructed, and the expression of the mosaic gene of old and feeble leaf specific expressino is obstructed, and the short-term training amount of the basic element of cell division reduces.Its result had both delayed leaf senile, had avoided the excessive synthetic of the basic element of cell division again and the form that causes and growth unusual.They are with the mosaic gene transformation of tobacco, genetically modified plants with compare, plant height, the stem number of blade and early flowering season basically identical, average individual plant flower number, seed amount and the dry matter of transfer-gen plant compared respectively according to increasing more than 80%, 50% and 40%, and the main cause that causes above-mentioned proterties to be improved is that the photosynthetic efficiency several times ground of the lower blade of transfer-gen plant is higher than the leaf of the same age that contrasts strain.But up to now, Shang Weijian utilizes this method to improve the report of crop yield potentiality.

Summary of the invention

The objective of the invention is to overcome the deficiency of prior art, with a self-control blade suppressor (P _SAG12-IPT) import the paddy rice acceptor, to delay leaf senile, correspondingly prolong the leaf photosynthesis time, increase accumulation of photosynthate, thereby reach the raising rice yield potentiality, the breeding high-yield kind is created the breeding new resources.The present invention is achieved through the following technical solutions:

A kind of method that improves rice yield potentiality comprises and adopts a self-control blade suppressor (P _SAG12-IPT), and utilize molecular biology method, physiological and biochemical analysis method and conventional breeding method are by transgenic method, with genes of interest P _SAG12-IPT changes in the paddy rice acceptor, obtain a large amount of transfer-gen plants, in conjunction with polymerase chain reaction (PCR) (PCR) and molecular hyridization method, the examination of physiological and biochemical analysis method and economical character, the seed selection leaf senile obviously delays in the self progeny, photosynthetic performance significantly improves, the transgenic line breeding high-yield kind of the bigger raising of single plant yield and creation breeding new resources, and its operating procedure is as follows:

A. utilize transgenic method with self-control blade suppressor (P _SAG12-IPT) import the paddy rice acceptor, obtain a large amount of transformed plants;

B. by the positive transformant of reporter gene and polymerase chain reaction (PCR) (PCR) Analysis and Identification, and further identify the integration copy number of positive transformant with the molecular hyridization method, with a transgenosis strain self-fertility to a few copy number, individual plant is received kind;

C. will go up the seed kind of selecting and remain in season and become strain system.Do the investigation of photosynthesis characteristics and economical character, select a plurality of strains likely to be, each strain system selects 10 individual plant selfing to reserve seed for planting;

Continuation will be gone up the seed of season results by strain system and the plantation of individual plant numbering.Further investigate photosynthesis characteristics and agronomy, the individual plant that isozygotys that select therefrom that phenotype is stable, photosynthetic performance and economical character has 2-3 strain of bigger improvement to be;

Further cultivate into new lines or breeding new resources.

The invention has the advantages that:

Utilize P _SAG12A kind of new method of rice high yield breeding is created in delaying leaf senile, increasing the leaf photosynthesis time and improve the characteristic of output of-IPT gene.

This method can directly apply to the SOYBEAN IN HIGH-YIELD BREEDING practice of other crop.

Adopt transgenic method, the binding molecule biology techniques, the cycle of breed breeding is short, efficient is high.

More detailed technological invention details is provided by following examples, but is not the protection domain that limits this invention:

Embodiment

Embodiment 1:

The foundation of the structure of double base Ti-plasmids carrier and conversion Agrobacterium:

To be with self-control blade suppressor (P _SAG12-IPT gene) plasmid pSG516 does not separate the purpose fragment after cutting with the SpeI enzyme, and directly the vector plasmid pCAMBIA1301 with XbaI enzyme cutting and dephosphorylation processing is connected;

Connect product and transform DH5 α, on the resistance medium of the kanamycin that contains X-Gal, select white single bacterium colony;

With pick out and enrichment single bacterium colony be numbered, insert anti-selection the on the medium contain ampicillin in turn;

1) the white single bacterium colony extracting plasmid that can not grow on the amicillin resistance medium, restriction enzyme digestion and electrophoresis detect, and in conjunction with the sequence analysis of joint, select the new structure product of single copy of two different direction of insertion, respectively called after pC35-1 and pC35-2;

2) two new products that make up are imported respectively in Agrobacterium EHA105 and the AGL-1 bacterial strain; Bacterial strain after the conversion is called after A1 and A2, A7 and A8 respectively.

Embodiment 2:

The genetic transformation of Agrobacterium Ti-plasmids mediation:

1), and the 2N6 successive transfer culture there are 20 days with mature embryo evoked callus on the 2N6 medium of No. 2, rice varieties Millin and Wu Yu round-grained rice;

2) will the grow particle callus of vigorous, foresythia inserts pre-the cultivation 4-5 days on the 1/2N6 medium that contains 1% glucose and 100 μ M acetosyringones;

3) will newly activate, to be in exponential phase, concentration be that the conversion agrobacterium strains of 1--1.5OD soaks through pre-incubated callus 30 minutes, blotting paper with sterilization blots callus then, inserts to put under 19 ℃ of-21 ℃ of temperature on the pre-culture medium and cultivates 2-3 days;

4) fully wash callus after the common cultivation with sterile water, blot the back and insert the resistance that contains 250mg/L carbenicillin and 50mg/L hygromycin and select to cultivate base and go up and cultivate, every fortnight subculture once;

5) kanamycin-resistant callus tissue that will newly grow changes regeneration plant on the differential medium that contains the 50mg/L hygromycin over to;

6) regeneration plant moves on the 1/2MS medium that contains 40mg/L and takes root, and moves in the alms bowl potted plant then.

Embodiment 3:

It is as follows to utilize Agrobacterium Ti-plasmids mediated method to transform the result of No. 2, Millin and Wu Yu round-grained rice:

Rice genetic transformation efficiency rice varieties callus sum/kanamycin-resistant callus tissue number/regeneration plant number/transformation efficiency that table 1 is agriculture bacillus mediated

A1 A2 A7 adds up to TotalMillin 475/22/11/2.3 426/26/16/3.8 961/139/78/8.1 1862/187/105/5.6 forces to educate No. 2 1,79/,14/,0/0 137/32/4/2.9 129,/8/,0/0 445/54/4/0.9 notes of round-grained rice: A1, A2 and A7 representative transform bacterial strain

Embodiment 4:

The screening of transfer-gen plant and the cultivation of desirable transgenic material:

1) getting the blade extracting DNA of the resistance regeneration plant of No. 2, the Millin that moves into the greenhouse and Wu Yu round-grained rice, detect and the molecular hyridization method is identified transfer-gen plant with gus reporter gene analysis, PCR, is 1-3 transgenosis strain self-fertility with copy number, and individual plant is received kind;

2) will go up the seed kind of selecting and remain in season and become strain system.In conjunction with the investigation of photosynthesis characteristics and economical character, obtain the transformation plant that the transformation plant of 3 Millin and 2 forces are educated No. 2, round-grained rice, select 10 individual plant selfing to reserve seed for planting in each strain system;

3) continue to go up the seed of season results by strain system and the plantation of individual plant numbering.Further photosynthesis characteristics and economical character are investigated, from 1-2 the individual plant that isozygotys that each selects when roguing system that phenotype is stable, photosynthetic performance and economical character have bigger improvement;

4) further cultivate into new lines or be used for the donor parents of back cross breeding.Example 5: the 5 strain positive plants and the 2 strain negative controls of the transgenosis force of seed selection being educated No. 2, round-grained rice are under equal conditions potted plant, and booting stage is as follows to the photosynthetic rate and the measuring chlorophyll content result of blade: table 2P _SAG12-IPT transgenic paddy rice force is educated the photosynthesis of No. 2 positives of round-grained rice and negative plant

The comparison of related parameter is arranged

Photosynthetic rate (μ molCO ₂/ M ²/ Sec) chlorophyll content (ChlII%)

The leaf that falls falls two leaves and falls three leaves and fall a leaf and fall two leaves and fall three Yeyang property plant mean values 16.04 10.81 9.52 0.644 0.462 0.413, (5 strain) standard deviation 2.50 2.70 2.36 0.065 0.116 0.092 negative plant mean values 10.01 5.94 4.90 0.456 0.410 0.324, (2 strain) standard deviation 0.37 0.93 0.58 0.028 0.099 0.013

Embodiment 5:

Transgenosis Millin, the transgenosis force of seed selection are educated No. 2, round-grained rice plant under same field condition with negative control, the every material in ripe back is got 5 strains at random and is carried out indoor species test, obtains following result: table 3 P _SAG12The economical character of-IPT transgenic paddy rice and negative control relatively

The plant height individual plant has the biological product of thousand kernel weight single-strain grain weight ripening rate

Grain number per spike

(cm) imitate fringe (g) (g) (%) amount (g) force educate round-grained rice mean value 84.80 14.00 153.7 22.29 41.39 85.97 81.67

Positive No. 2 standard deviations 3.70 2.55 28.5 1.72 11.37 2.33 18.97

Plant

Mean value 79.80 8.50 147.8 22.58 25.11 88.38 49.09

Negative

Standard deviation 3.11 0.71 13.7 1.13 3.83 2.38 5.11

Plant Millin mean value 87.9 13.1 62.67 25.12 20.49 82.19 48.07

Positive

Standard deviation 4.8 4.6 11.69 1.84 6.66 5.68 13.08

Plant

Mean value 86.3 10.9 51.22 23.19 11.94 76.07 34.93

Negative

Standard deviation 5.5 5.6 15.29 2.56 4.34 10.54 11.34

Plant

Claims

1, a kind of method that improves rice yield potentiality is characterized in that:

A, utilize transgenic method with P _SAG12One IPT imports the paddy rice acceptor, obtains transformed plant;

B, identify positive transformant, and further identify the integration copy number of positive transformant that with one to the several copy transformant of minority self-fertility, individual plant is received kind with the molecular hyridization method by reporter gene and polymerase chain reaction (PCR) (PCR) method;

C, will go up the seed kind of selecting and remain in season and become strain system, do the investigation of photosynthesis characteristics and economical character, select a plurality of strains likely system, and select 10 individual plant selfing to reserve seed for planting in each strain system;

D, the seed that continues to go up the season results are further investigated photosynthesis characteristics and economical character by strain system and the plantation of individual plant numbering, the individual plant that isozygotys that select therefrom that phenotype is stable, photosynthetic performance and economical character has 2-3 strain of bigger improvement to be;

E, further cultivate into new lines or breeding new resources.