CN102181477A

CN102181477A - Application of anti-sense expression of rice OsPDCD5 (Oryza sativa Programmed Cell Death 5) gene to improving yield character of rice

Info

Publication number: CN102181477A
Application number: CN2011100751632A
Authority: CN
Inventors: 孙凡; 杨金水
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2011-03-28
Filing date: 2011-03-28
Publication date: 2011-09-14

Abstract

The invention belongs to the field of a transgenic technology, particularly relates to application of anti-sense expression of a rice OsPDCD5 (Oryza sativa Programmed Cell Death 5) gene to improving yield character of rice. Indica type rice subspecies of rice are transformed by using the anti-sense expression of the rice OsPDCD5 and a homologous gene thereof, so that the yield character of the rice is improved; in addition, the anti-sense expression of the rice OsPDCD5 gene and the homologous gene thereof are performed in maize, wheat, sorghum and other cereal crops for improving the yield character of the cereal crops. Further, the anti-sense expression of the rice OsPDCD5 gene and the homologous gene thereof are performed in dicotyledonous crops for improving the yield character of the dicotyledonous crops, such as increasing number of branches, grains and fruit number and increasing volume and weight of the grains and the fruits.

Description

The application of antisense expression rice Os PDCD5 gene aspect the improvement rice yield traits

Technical field

The invention belongs to field of transgenic technology, be specifically related to a kind of improvement rice yield traits method.

Background technology

Paddy rice is one of important crops in the world, and have an appointment 2/3 population of the whole world is staple food with the paddy rice.In order to satisfy the demand of growing population to grain, improving the significant yield traits of Rice Production power is the Main Agronomic Characters of describing Rice Production power, mainly is made up of the every fringe Number of kernels of number of productive ear and several parts of thousand seed weight.These yield traitses all belong to quantitative character (QTL), are controlled by the gene that many effects are different.

The technology of utilizing at present transgene improvement farm crop economic characters both at home and abroad mainly is the relation according to known functional gene and phenotypic character, adopt constitutive expression carrier or tissue specificity expression vector that goal gene is imported target plant, in the hope of obtaining the improvement of specific economic characters of crop or resistance proterties.

Apoptosis (PCD) is the dead form of a kind of active of autogenous control, is the part of multicellular organism normal activities, is to reach the important procedure that self starts under the environment-stress situation in the development of plants process. OsPDCD5Gene is an apoptosis gene in the paddy rice. OsPDCD5Expression level in mature tissue is apparently higher than tender tissue, and OsPDCD5Cross the death that expression can cause transgenic regenerated plant.

Blade is the most important organ of paddy rice, there are some researches show the photosynthesis of the 60%-80% of paddy rice heading post-grouting desired nutritional material from blade.Rice leaf old and feeble speed between productive phase directly influences the output of paddy rice, has research then to show the every delay aging of blade one day, and output can improve about 1%.The most tangible performance of leaf senile is exactly that chlorophyll fades away, and the yellowing and shedding phenomenon occurs, and the aging degree of blade is serious more, and chlorophyllous content is just low more.Yet, exist a kind of " holding green " phenomenon in the plant, hold the green blade that is meant and in the long period in plant-growth later stage, still keep green a kind of phenotypic characteristic.Thereby the paddy rice transgenic research shows that anti-ageing gene importing paddy rice can be delayed leaf senile is improved output.There are some genes to be suppressed and low expression level in the leaf senile process, even do not express fully; Other genes then are activated, and express thereby strengthen.Last genoid is called as old and feeble down-regulated gene, and (senescence down-regulation genes SDGs), sharply descends with the aging of blade as the transcript abundance of coding with photosynthetic process proteins associated matter; The back one class then be called as senescence-associated gene (senescence-associated genes, SAGs).SAGs can be divided into two classes again: a class is only just expressed in the old and feeble stage, be called as old and feeble specific gene (senescence-specific genes, SSGs), as SAG12, SAG13; Another kind ofly just can detect low expression level, begin the back expression amount in aging and rise rapidly at the leaf growth initial stage.

Summary of the invention

The objective of the invention is to propose antisense expression paddy rice in the paddy rice OsPDCD5The application of gene aspect the improvement rice yield traits.

The present invention utilizes antisense expression paddy rice programmed death gene OsPDCD5And the long-grained nonglutinous rice subspecies of homogenic carrier rice transformation, in order to the yield traits of improvement paddy rice;

Among the embodiment, the present invention is with the antisense expression paddy rice OsPDCD5The carrier of gene transforms rice variety gold 23B, makes the yield traits of long-grained nonglutinous rice gold 23B take place obviously to improve.Promptly adopting long-grained nonglutinous rice gold 23B is transgene receptor, makes up OsPDCD5The antisense expression transfer-gen plant of gene.

It is a kind of that the present invention also provides OsPDCD5The gene antisense expression vector, it is expression vector that this carrier adopts pCAMBIA1304, with the precious Shan 97 of rice variety OsPDCD5Gene oppositely is inserted into pCAMBIA1304 carrier 35S constitutive promoter downstream and obtains.

The present invention also utilizes antisense expression paddy rice programmed death gene OsPDCD5And homogenic carrier transforms and comprises corn, wheat, and Chinese sorghum and other cereal crop are used for the improvement of yield traits.

The present invention also utilizes antisense expression paddy rice programmed death gene OsPDCD5And homogenic carrier conversion dicotyledonous crops, be used for the improvement of yield traits.

The improvement of described yield traits comprises increasing branch amount, seed and fruit number, improves seed and volume of fruits and weight.

The present invention also comprises: paddy rice programmed death gene OsPDCD5Separation, clone and transgenosis functional study etc.

1) OsPDCD5Gene and protein structure are formed

Adopt the SSH technology, from the rice callus tissue of differentiation separation be cloned into one section with the relevant EST of human cell's programmed death gene TFAR19.On this basis, according to EST in the international data center of delivering login and rice genome data, design PCR primer has been cloned into the homogenic full-length cDNA of TFAR19, called after from precious Shan 97 seedling of paddy rice rice variety OsPDCD5Also be separated in this external rice seedling tissue OsPDCD5The cDNA of two alternative splicings of gene.These three cDNA have signed in to international data center NCBI, and accession number is respectively AY327105, AY749431 and AY749431. OsPDCD5The full-length cDNA of gene is made up of 7 exons.Long 416 bp of the cDNA of normal encoding, sequence shown in SEQ.ID NO1., 128 amino acid of encoding, sequence is shown in SEQ.ID NO2..

The structure of this gene and its encoded protein matter are composed as follows:

Paddy rice OsPDCD5Gene structure is seen shown in Figure 1, and protein structure is seen shown in Figure 2.

2) antisense expression OsPDCD5The structure of expression carrier

Most of genetically engineereds all are that the nucleotide coding sequence forward of gene is inserted carrier, and the present invention be with OsPDCD5Oppositely insert carrier.Use in the embodiment of the invention OsPDCD5Reverse sequence is shown in SEQ.ID NO3..(ACT Australia) is expression vector, utilizes restriction enzyme site for Center for the Application of Molecular Biology of International Agriculture, Canberra to adopt pCAMBIA1304 BglII and SpeI will OsPDCD5Reverse sequence is inserted into pCAMBIA1304 carrier 35S constitutive promoter downstream, obtains antisense expression OsPDCD5Expression vector.Its structure iron as shown in Figure 3.

3) OsPDCD5Reverse sequence gene transformation experiment

Adopting golden 23B is transgene receptor, makes up antisense expression OsPDCD5The transfer-gen plant of gene.Golden 23B mature seed shelled be seeded in the dedifferentiation plant tissue after the sterilization and cultivate base and go up evoked callus.After callus induction 2-3 week, adopt particle gun micropellet bombardment method, will contain OsPDCD5The purified pCAMBIA1304 plasmid DNA of reverse sequence imports golden 23B callus cell.Cultured continuously 3-4 week screening resistant cell line on the MS substratum that adds the 30ppm Totomycin is transferred to the clone of screening division culture medium then and is induced and sprout and take root.

4) conversion processing test-tube seedling transplanting and transformed plant offspring's Molecular Detection

Will OsPDCD5The golden 23B test-tube seedling transplanting field of reverse sequence conversion processing, and adopt pcr amplification technology for detection candidate's resistant plant, obtain positive T0 for plant in the summer in 2008.Winter in 2008 at the Hainan Island adding generation, obtained transgenosis T1 generation, amounted to 5 strain systems.

5) OsPDCD5The real-time quantitative PCR (RealTime PCR) that reverse sequence changes golden 23B plant detects

Gene OsPDCD5Expression amount in golden 23B transfer-gen plant reduces than wild-type plant.See shown in Figure 4.

6) OsPDCD5Reverse sequence transgenosis T2 is for the investigation and the statistics of population yield proterties

Will OsPDCD5Reverse sequence transgenosis T2 plants for sowing in spring in 2009, and four leaf phases were planted in Fudan University experimental plot with 3 transgenosis T2 for strain system, and 20 strains are respectively planted by each strain system, and distance between rows and hills is 6 cun of 6 cun x, and triplicate is set up the golden 23B parent contrast of long-grained nonglutinous rice simultaneously.Add up the number of productive ear that each strain is 20 individual plants in the ripening stage, main fringe spike length, main fringe primary tiller stalk and secondary branch stalk number, main total grain panicle number and thousand seed weight, calculating mean value, statistics is as follows:

Strain system	Spike length (centimetre)	Number of productive ear	Primary tiller stalk number	Secondary branch stalk number	Main total grain panicle number	Thousand seed weight (gram)
							Contrast golden 23B	19.63	9.80	8.08	23.06	131.83	22.50
OsPDCD5 Reverse sequence transgenic line 1	22.08	10.80	11.53	36.95	184.26	25.50
							OsPDCD5Reverse sequence transgenic line 2	23.31	11.13	10.92	34.33	173.42	25.50

Paddy rice OsPDCD5The spike length of reverse sequence transfer-gen plant, number of productive ear, branch stalk number and grain number per spike change to be seen shown in Fig. 5-8.According to above-mentioned field investigation and species test data, compared with the control, OsPDCD5The yield traits of reverse sequence transgenosis long-grained nonglutinous rice gold 23B is better than contrast:; Thousand seed weight increases by 13.3%.Paddy rice OsPDCD5The major cause that the reverse sequence transgenosis increases output is to increase number of productive ear, branch stalk number and grain number per spike.

7) OsPDCD5The leaf senile of reverse sequence transfer-gen plant delays

With paddy rice OsPDCD5Seedling is sent out in the seed vernalization simultaneously of reverse sequence transfer-gen plant and the golden 23B of contrast, and when growing to 30 days, second leaf that contrasts golden 23B plant yellow is fully withered, OsPDCD5Second leaf of reverse sequence transfer-gen plant then still is green, as shown in Figure 9.During heading stage, the leaf senile of transfer-gen plant obviously is later than contrast, as shown in figure 10.Because antisense expression OsPDCD5In transfer-gen plant, cause and hold green and the postponement at heading stage, prolonged the photosynthetic time, therefore increased the accumulation of dry-matter greatly, finally caused significantly improving of output.

Description of drawings

Fig. 1 is a paddy rice OsPDCD5Gene structure.

Fig. 2 is a paddy rice OsPDCD5The gene protein structure.

Fig. 3 is the antisense expression paddy rice OsPDCD5The expression carrier structure iron.

Fig. 4 is OsPDCD5Gene in golden 23B transfer-gen plant with the wild-type plant in the contrasting of expression amount.

Fig. 5 is an antisense expression OsPDCD5The spike length of transfer-gen plant change.

Fig. 6 is an antisense expression OsPDCD5The number of productive ear of transfer-gen plant change.

Fig. 7 is an antisense expression OsPDCD5The branch stalk number of transfer-gen plant change.

Fig. 8 is an antisense expression OsPDCD5The main fringe Number of kernels of transfer-gen plant change.

Fig. 9 is an antisense expression OsPDCD5Transfer-gen plant and the old and feeble contrast of seedling leaf of golden 23B contrast.

Figure 10 is an antisense expression OsPDCD5Transfer-gen plant and the leaf senile contrast at heading stage of golden 23B contrast.

Figure 11 is an antisense expression OsPDCD5Transfer-gen plant and the spike of rice contrast of golden 23B contrast.

Figure 12 is an antisense expression OsPDCD5Transfer-gen plant and the seed contrast of golden 23B contrast.

Embodiment

1, clones paddy rice programmed death gene with the primer of band restriction enzyme site OsPDCD5Full-length cDNA, cDNA and plant expression vector pCAMBIA1304 are used BglII and SpeThe I enzyme is cut the back and is connected, like this OsPDCD5Just oppositely be inserted into the downstream of 35s promoter. OsPDCD5Antisense sequences expression vector pCAMBIA1304/antisense- OsPDCD5Structure is finished.See Fig. 3.

2, inducing paddy rice callus culture base

(1) induces and subculture medium: MS+2 mg/L 2 4-D.

(2) height oozes substratum: MS+2 mg/L 2,4-D+46.67 g/ sorbyl alcohols+46.67 g/ N.F,USP MANNITOL.

(3) first round screening culture medium: MS+2 mg/L 2,4-D+30 mg/L Totomycin.

(4) second take turns screening culture medium: MS+2 mg/L 2,4-D+50 mg/L Totomycin.

(5) division culture medium: MS+3 mg/L 6-BA+0.5 mg/L NAA+50mg/L Totomycin.

(6) strong plantlets and rootage substratum: 1/2 MS+0.1 mg/L NAA.

(annotate: 1. above substratum all contains 30g/L sucrose+2.5 g/L agar, and pH 5.8.2. callus of induce, subculture, screening and culturing condition are 26-28 ℃ of dark the cultivation, and differentiation, strong plantlets and rootage are 26-28 ℃ and 16 hour photoperiod).

3, callus induction and processing

(1) gets 12-15 days the immature seed in pollination back, under aseptic condition, embathe 10 min with 70% ethanol earlier, change 0.1% mercuric chloride over to and soak 20 min, sterile water wash 3 times.

(2) peel off rataria under aseptic condition, be inoculated on the callus of induce substratum, 26-28 ℃ of dark the cultivation after about 20 days cut bud, and subculture once.

(3) in subculture medium, select growth vigorous, flaxen callus 30-50 piece (about every 3 mm), place height to ooze substratum central authorities, line up in the circle of about 2.5 cm of diameter, cultivate and be used for conversion behind about 4-5 h.

4, particle gun transforms

(1) particle gun: from the high pressure gas particle gun that Xin Zhi Science and Technology Ltd. in Ningbo purchases, model: GJ-1000.

(2) particulate bullet preparation.

(3) take by weighing 60mg tungsten powder (about 1 um of diameter), join in the 1.5 ml sterilization centrifuge tube, add the 1ml dehydrated alcohol again, 1 min that vibrates in centrifugal 10 s of 10000 rpm, abandons supernatant, after repeating to wash once, bronze is suspended in existing using or-20 ℃ of preservations in the 1 ml sterilized water.

(4) draw 50 ul tungsten powder suspension in 1.5 ml centrifuge tubes, add 5 ug DNA, 50 ul, 2.5 M CaCl successively ₂, 20 ul, 0.1 M spermidine, vibrated 5 minutes, centrifugal 20 s of 10000 rpm abandon supernatant, with 140 ul dehydrated alcohol rinsings twice, add 60 ul dehydrated alcohols, it is stand-by to suspend.

5, bombardment receptor material

(1) particle gun is put on the Bechtop, clean vacuum chamber with 70% alcohol, and can split film, carry granulosa, metal baffle screen (by the supply of material of Ningbo Xin Zhi Science and Technology Ltd.) sterilization 30 minutes in 70% alcohol, blot or blow off residual alcohol then with aseptic filter paper.

(2) turn on the power switch vacuum pump and helium cylinder valve.

(3) can split film pack into fixing, screw.

(4) get 10 ul bag by the tungsten powder dehydrated alcohol suspension of DNA, evenly coat and carry a granulosa center, be placed on the super clean bench and dry up.

What (5) will be loaded with little bullet carries granulosa and backstop little bullet launching device of packing into, and particulate one is faced down.

(6) culture dish is placed on the pallet, makes callus concentrate on culture dish central authorities.

(7) open gas cylinder, regulate pressure 1100 Psi.

(8) vacuumize, when vacuum tightness reaches desired value, forward the VAC key to the Hold position.

(9) bombardment, every ware bombardment 2 times (after the bombardment culture dish revolved for the first time turn 90 degrees then carry out bombardment second time) makes the vacuum meter reading return zero by the venting key, takes out sample, and the bombardment back is oozed in height and is continued cultivation 12-16 h on the substratum.

6, transformed calli screening

(1) the back height of will shooting oozes callus on the substratum and changes over to and recover growth 5-7 days on the inducing culture that does not contain selective agent.

(2) callus is forwarded on the screening culture medium that contains 30 mg/L Totomycin, evenly put, secretly cultivate and carried out the resistance screening first time in 14-17 days.

(3) kanamycin-resistant callus tissue is changed on the screening culture medium that contains 50 mg/L Totomycin, secretly cultivate and carried out the resistance screening second time in 8-12 days.

7, transformed plant screening and detection

(1) the callus illumination on division culture medium that will screen the back survival was cultivated 30 days.

(2) wait to differentiate plantlet after, change plantlet over to the strong plantlets and rootage substratum, the back of growing up moves into the greenhouse.

(3) adopt pcr amplification to detect candidate's transformed plant, obtain 5 and contain OsPDCD5The plant of reverse sequence.

SEQUENCE?LISTING

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＜120〉application of antisense expression rice Os PDCD5 gene aspect the improvement rice yield traits

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Claims

1. paddy rice OsPDCD5The application aspect the improvement rice yield traits of gene and homologous gene thereof is characterized in that utilizing antisense expression paddy rice programmed death gene OsPDCD5And the long-grained nonglutinous rice subspecies of homogenic carrier rice transformation, with the yield traits of improvement paddy rice.

2. application according to claim 1, it is characterized in that adopting long-grained nonglutinous rice gold 23B is transgene receptor, makes up OsPDCD5The antisense expression transfer-gen plant of gene.

3. one kind OsPDCD5The gene antisense expression vector, it is characterized in that adopting pCAMBIA1304 is expression vector, with the precious Shan 97 of rice variety OsPDCD5Gene oppositely is inserted into pCAMBIA1304 carrier 35S constitutive promoter downstream and obtains.

4. paddy gene OsPDCD5And homogenic carrier comprises corn in conversion, wheat, and Chinese sorghum and other cereal crop are improved the application of its yield traits aspect.

5. paddy gene OsPDCD5And homogenic carrier is improved the application of its yield traits aspect transforming dicotyledonous crops.