CN103343139A - Novel method for enhancing drought resistance of plants - Google Patents

Abstract

The invention provides a novel method for enhancing drought resistance of plants, which comprises the following steps: 1) SHR-1 overexpression vector construction and agrobacterium transformation: extracting RNA (ribonucleic acid) of the whole Arabidopsis thaliana strain, carrying out reverse transcription to obtain cDNA (complementary deoxyribonucleic acid), and carrying out PCR (polymerase chain reaction) to obtain a gene complete segment of the SHR-1 by using 5'ATGGATACTCTCTTTAGACTAG3' and SHR-R:5'TTACGTTGGCCGCCACGCACTAG3' as primers; selecting forward clone, cutting off the SHR-1 gene segment from a KS vector by utilizing two digestion sites BamHI and SalI, connecting into a plant expression vector pCAMBIA1301 to construct pCAMBIA1301-SHR-1, and transforming into agrobacterium GV3101 for later use; 2) Arabidopsis thaliana drought treatment and water loss determination; 3) RT-PCR (reverse transcription-polymerase chain reaction) and Real-Time PCR analysis; 4) genetic transformation of rice; and 5) PEG simulated drought treatment of rice to express the enhanced resistance.

Description

A kind of novel method that improves plant drought resistance

Technical field

The present invention relates to plant genetic engineering field, especially the gene SHR-1 that a control plant Casparian strip is formed changes Arabidopis thaliana over to, under the prerequisite that does not influence its normal growth growth, reduces plant transpiration rate in water-stressed conditions, thereby improves its drought resistance.

Background technology

In recent years, the arid that lack of water causes causes a large amount of underproduction of grain even total crop failure in the global range, it is one of regional unstable inducement that the agricultural-food scarcity causes substantial appreciation of prices, utilize the existing farm crop of modern genetic engineering means transformation improve its tolerance to arid be the future of agriculture must be by selection, searching has the adversity gene of potential use value, explore its possible purposes in agriculture production, it is the active demand of modern agriculture, fast development along with molecular biology of plants, the genome of a large amount of species obtains order-checking, thereby has paved road for carrying out of reverse genetics.

Salt stress is the important factor of restriction plant distribution range and output, and root is the major organs that the control salinity enters plant materials, betides Casparian strip on the endodermis and is considered to play a significant role in the control salinity enters the process of center pillar by the apoplast approach.SHR and the distinctive transcription factor family GRAS of SCR congener family, it is the crucial regulatory factor of control asymmetric cell division, move to after SHR expresses in stelar tissue in the adjacent cells with SCR and take place to interact and be limited in the nucleus, in case be limited in the nuclear and just can't move to okioplast, then by a series of downstream gene (SCR of regulation and control, MGP, JKD) expression and to make this confluent monolayer cells specialization be the endodermis cell, the mutant root of these two gene function disappearances is long significantly to shorten, lack endodermis and Casparian strip, and Casparian strip is the control root system of plant absorbs moisture from soil important structure, the absorption that the thickening of Casparian strip can be limited salt ion especially plant to the absorption of moisture, and then the transpiration rate of restriction plant, improve the water use efficiency of plant.

The present invention passes through transgenic technology, in plants such as goal gene SHR importing acceptor Arabidopis thaliana, under the situation that does not influence its normal growth growth, improve the drought resistance of plant, not only controlled the rate of water absorption of root but also reduced the transpiration of moisture, important exemplary role had been played in the technological innovation of high-tech agricultural.

Summary of the invention

The objective of the invention is to: provide the new plant normal growth such as Arabidopis thaliana that do not influence to grow method and the application thereof that can improve its drought resistance again.

The object of the present invention is achieved like this: a kind of novel method that improves plant drought resistance, implement step by step;

Step 1SHR-1 overexpression vector construction and Agrobacterium-mediated Transformation

1) extracting the whole strain RNA of Arabidopis thaliana, after reverse transcription becomes cDNA, is primer with SHR-F:5'ATGGATACTCTCTTTAGACTAG3' and SHR-R:5'TTACGTTGGCCGCCACGCACTAG3', and PCR obtains the gene complete fragment of SHR-1;

2) with Ligation high test kit it is connected in the pBluescript KS intermediate carrier sequence verification;

3) choose the forward clone, utilize two restriction enzyme sites of BamHI and SalI that the SHR-1 gene fragment is downcut from the KS carrier, and be connected among the plant expression vector pCAMBIA1301;

4) making up pCAMBIA1301-SHR-1, to change Agrobacterium GV3101 over to standby, namely chooses the Arabidopis thaliana seedling of just having bloomed, and every the 3-5d transfection once, transfection 3-5 time is standby continuously;

Step 2 Arabidopis thaliana arid is handled and percentage of water loss is measured

The Arabidopis thaliana seedling that sprouts 5d is transferred in the soil, begun to stop to water behind the cultivation 21-25d, and continue to cultivate 21d, observe its phenotype; Get the non-transgenic wild-type and the SHR-1 that cultivate 28d and cross expression strain blade, time point weighs up fresh weight respectively, namely gets percentage of water loss;

Step 3RT-PCR and Real-Time pcr analysis

1) utilize Trizol reagent to extract plant RNA and utilize ReverTra Ace, reverse transcription becomes cDNA;

2) RT-PCR the primer: SHR-F:5'ATGGATACTCTCTTTAGACTAG3'; SHR-R:5'TTACGTTGGCCGCCACGCACTAG3'; Atactin2F:5'GGAAGGATCTGTACGGTAAC3'; Atactin:5'GGACCTGCCTCATCATACT3';

3) reaction process: 95 ℃ of 1min, 95 ℃ of 15s, 58 ℃ of 30s, 72 ℃ of 15s; Totally 30 circulations; 72 ℃, extension 5min; Real-Time PCR operates according to SYBR Green Realtime PCR MasterMix specification sheets;

The genetic transformation of step 4 paddy rice

1) inducing of paddy rice mature embryo callus: the ZH11 rice paddy seed is shelled, with 70% alcohol immersion 1min, soak 20min with 20% chlorine bleach liquor again, with sterilized water washing 3-5 time, sowing under 26-28 ℃, was secretly cultivated 28-30d after aseptic filter paper blotted soaking solution on NB callus of induce substratum, the callus particle is seeded on the new NB callus of induce substratum, and it is standby secretly to cultivate 4d;

2) transform the Agrobacterium will contain the p1301proD1-SHR-1 plasmid the day before yesterday and be inoculated in the LB substratum, it is 0.6-0.8 that 28 ℃, 200rpm concussion are cultured to OD66o;

3) callus is transferred in the aseptic triangular flask, poured into Agrobacterium bacterium liquid, namely allow bacterium liquid submergence callus; Room temperature is placed 20min, removes bacterium liquid, callus is transferred to drawn unnecessary bacterium liquid on the aseptic filter paper, forwards NB then to and is total on the substratum, 20-25 ℃, cultivates 2-3d altogether in the dark;

4) callus after will cultivating is altogether transferred in the aseptic triangular flask, earlier with sterilized water washing 2-3 time, again with the sterilized water washing 20min that contains the 500ml/L Pyocianil; After aseptic filter paper blots excessive moisture, callus is transferred to the screening of carrying out transformant on the NB screening culture medium, screening 42-65d;

5) kanamycin-resistant callus tissue after will screening changes on the pre-differentiation substratum and cultivates 7-20d, goes on the NB division culture medium again and cultivates, and condition is 26 ℃, and 16h illumination/8h is dark; The resistance regeneration plant that differentiates changes strengthening seedling and rooting on the root media over to; 21-28d, the regeneration resistant plant that will take root is transplanted to the greenhouse;

Step 5PEG simulating drought is handled paddy rice

Wild-type and transgenic paddy rice seedling behind the sprouting 7-10d are planted respectively, normally watered and cultivate 14d, use the 200ml20%PEG aqueous solution then every day, continuous pouring 12-15d observes its phenotype.

Described method, Arabidopis thaliana growth conditions are 23 ± 1 ℃, 14h illumination/10h dark; Paddy rice ZH11 growth conditions is 28 ± 1 ℃, 16h illumination/8h dark.

Principle of the present invention and the mechanism of action: Casparian strip is the control root system of plant absorbs moisture from soil important structure, the absorption that the thickening of Casparian strip can be limited salt ion especially plant to the absorption of moisture, by the Casparian strip in the constitutive expression SHR gene render transgenic roots of plants in plant why after, thereby under the prerequisite of true plant normal growth water supply, the absorption rate of control plant from soil, the transpiration rate of restriction plant, improve the water use efficiency of plant, improve the drought-resistant ability of plant.

The present invention passes through transgenic technology, in plants such as goal gene SHR importing acceptor Arabidopis thaliana, under the situation that does not influence its normal growth growth, improve the drought resistance of plant, not only control the rate of water absorption of root but also reduced the transpiration of moisture, important exemplary role has been played in the technological innovation of high-tech agricultural, shown technical progress.

Description of drawings

The present invention is described further by reference to the accompanying drawings.

Accompanying drawing 1 is the expression pattern synoptic diagram of SHR-1;

As shown in the figure: moisture absorption (root) and transportation organ (stem) the middle expression of SHR-1 gene in each organ of Arabidopis thaliana (tissue).

Accompanying drawing 2 is handled the expression of inducing SHR-1 for salt and arid;

As shown in the figure: the expression of SHR-1 in root.

Accompanying drawing 3 is handled the expression of inducing SHR-1 for salt and arid;

As shown in the figure: SHR-1 is the expression in the plant (seedling) on the ground.

Accompanying drawing 4 is the RT-PCR of the mistake express transgenic plant of SHR-1;

As shown in the figure: WT is wild-type, LB1, and LB7, LB8, LB9 and LB12 are 5 independently transgenic lines, are SHR-1 on it, it is ACTIN2 down.

Accompanying drawing 5 is expressed the rate-of-loss of coolant that has reduced transfer-gen plant for crossing of SHR-1;

As shown in the figure: WT is the wild-type contrast, and LB1 and LB7 are two independently transgenic lines.

Accompanying drawing 6 is PCR and the RT-PCR of the mistake express transgenic paddy rice of SHR-1;

As shown in the figure: A: the PCR of transgenic paddy rice identifies: M, DNA marker; P, plasmid is over against photograph; N, negative contrast; WT, the wild-type paddy rice; 1-12, different transgenic lines; B: SHR-1 expression of gene in the transgenic paddy rice.

Accompanying drawing 7 is the biomass of transgenic paddy rice;

Shown in the accompanying drawing: allos is crossed expression SHR-1 gene in paddy rice, wherein the drought resisting difference of Mo Ni transgenic paddy rice and wild-type; WT, the wild-type paddy rice; LB1, LB3, LB5 are three transgenic lines.

Embodiment

Be described further below in conjunction with the invention of embodiment.

Embodiment

One, experiment material

Testing used vegetable material comprises:

Arabidopis thaliana (Arabidopsis thaliana) Columbia(Col-0) and the Ler ecotype (1);

(2) the Arabidopis thaliana growth conditions is 23 ± 1 ℃, 14h illumination/10h dark;

(3) paddy rice ZH11: growth conditions is 28 ± 1 ℃, 16h illumination/8h dark.

Experiment bacterial strain uses therefor: (1) coli strain: DH5 α; DE3; Golden plus(2) agrobacterium strains GV3101(is used for the Arabidopis thaliana conversion); EHA105(is used for rice conversion).

Two, experimental technique

(1) SHR-1 overexpression vector construction and Agrobacterium-mediated Transformation

Extracting the whole strain RNA of Arabidopis thaliana, after reverse transcription becomes cDNA, is primer with SHR-F:5'ATGGATACTCTCTTTAGACTAG3' and SHR-R:5'TTACGTTGGCCGCCACGCACTAG3', and PCR obtains the gene complete fragment of SHR-1; With Ligation high test kit it being connected into pBluescript KS(EcoR V cuts) in the intermediate carrier, order-checking is verified; Choose the forward clone, utilize two restriction enzyme sites of BamHI and SalI that the SHR-1 gene fragment is downcut from the KS carrier, and be connected among the plant expression vector pCAMBIA1301; It is standby that the pCAMBIA1301-SHR-1 that builds changes Agrobacterium GV3101 over to; Wherein Agrobacterium-mediated Transformation is used rifle head titration column cap method, chooses the Arabidopis thaliana seedling of just having bloomed, every the 5d transfection once, and transfection 5 times (Clough and Bent, 1998) continuously.

The used enzyme of gene fragment is bought from Japan (Shanghai) TAKARA company.

(2) the Arabidopis thaliana arid is handled and percentage of water loss mensuration

The Arabidopis thaliana seedling of sprouting 5d begins to stop to water after transferring to and cultivating 21d under the normal growth condition in the soil, and continues to cultivate 21d, observes the phenotype under the drought condition; In order to carry out the mensuration of percentage of water loss, the wild-type and the SHR-1 that cultivate 28d under the same growth conditions are crossed expression strain system, respectively cut 10 blades, according to weighing up fresh weight the pitch time that designs respectively, the fresh weight of each time point is percentage of water loss with the fresh weight of start time point ratio; Experiment is carried out 3 biology and is repeated.

(3) RT-PCR and Real-Time pcr analysis

Utilize Trizol reagent (TAKARA, Japan) extract plant RNA and utilize ReverTra Ace (TOYOBO, Japan) reverse transcription becomes cDNA; The RT-PCR the primer is: SHR-F(5'ATGGATACTCTCTTTAGACTAG3') and SHR-R(5'TTACGTTGGCCGCCACGCACTAG3');

Atactin2F(5'GGAAGGATCTGTACGGTAAC3') and Atactin(5'GGACCTGCCTCATCATACT3');

Reaction process is: 95 ℃ of 1min; 95 ℃ of 15s, 58 ℃ of 30s, 72 ℃ of 15s, 30 circulations; 72 ℃, 5min, Real-Time PCR operates according to SYBR Green Realtime PCR Master Mix (TOYOBO) specification sheets.

(4) genetic transformation of paddy rice

1) inducing of paddy rice mature embryo callus: the seed of rice varieties ZH11 shells, and with 70% alcohol immersion 1min, 20%(v/v) chlorine bleach liquor constantly rocks during soaking 20min() after, fully wash 5 times with sterilized water; Be seeded in after aseptic filter paper blots on the NB callus of induce substratum, 26-28 ℃ dark cultivate 28d after, it is standby that the callus particle is inoculated on the new NB callus of induce substratum the dark 4d of cultivation;

2) transform the Agrobacterium will contain the p1301proD1-SHR-1 plasmid the day before yesterday be inoculated in the LB substratum (contain Kan50ug/ml, Rif25ug/ml) in, 28 ℃, 200rpm, it is 0.8 that concussion is cultivated to OD66o;

3) callus is transferred in the aseptic triangular flask, poured into cultured Agrobacterium bacterium liquid (allow bacterium liquid submergence callus);

4) room temperature is placed 20min, during rock 4-5 time gently;

5) remove bacterium liquid and callus transferred on the aseptic filter paper draw unnecessary bacterium liquid, forward NB then to altogether on the substratum, cultivate 3d altogether for 25 ℃ in the dark;

6) callus after will cultivating is altogether transferred in the aseptic triangular flask, earlier with sterilized water washing 3 times, again with the sterilized water washing 20min that contains the 500ml/L Pyocianil;

7) after aseptic filter paper blots excessive moisture, callus is transferred to the screening of carrying out transformant on the NB screening culture medium (containing Ticarcillin/Clavulanate Acid 200mg/L and 50mg/L Totomycin), 7d is one-period, screens 3 cycles;

8) after the kanamycin-resistant callus tissue after will screening changes over to and cultivates 7d on the pre-differentiation substratum, go to NB division culture medium (containing BAP2mg/L and NAA0.5mg/L) again and go up and cultivate, condition is 26 ℃, and 16h illumination/8h is dark;

9) the resistance regeneration plant that differentiates changes root media (containing 1/2MS+NAA0.5mg/L) over to and goes up strengthening seedling and rooting;

10) behind about 21d, the regeneration resistant plant that will take root is transplanted to the greenhouse.

Used microbiotic is bought in Japan (Shanghai) TAKARA company.

(5) the PEG simulating drought is handled paddy rice

Wild-type behind the sprouting 7d is planted in same Culture basin (different transgenic lines are planted in different Culture basins) respectively with the transgenic paddy rice seedling, normally water and cultivate 14d, every day is with 200ml20%PEG aqueous solution continuous pouring 13d then, during observe phenotype and in time take pictures.

The present invention improves the experiment show of resistance:

(1) separation of SHR-1 gene and expression pattern analysis

In TAIR10 database (http://www.arabidopsis.org/), carry out sequence alignment, find cDNA sequence and the aminoacid sequence of SHR-1, the Real-time experimental result shows that SHR-1 expression amount in the moisture absorption of Arabidopis thaliana and transportation organ root and stem is higher, and expression amount relatively low (Fig. 1) at its hetero-organization such as leaf and in spending.

(2) the SHR-1 gene is induced by salt and drought stress

The Real-time experimental result shows that SHR-1 being expressed in Arabidopis thaliana is subjected to after the coercing of salt and arid certain rise being arranged, the situation of being induced of this gene has been carried out the different time gradient takes a sample, experimental data shows that being expressed in of SHR-1 coerced and just obviously increases after handling half an hour, illustrates that SHR-1 is one and is subjected to the gene (Fig. 2,3) that the adverse circumstance factor is induced.

(3) express the drought resistance that the SHR-1 gene can improve transgenic arabidopsis excessively

The SHR-1 gene that 35S is started changes Arabidopis thaliana over to, obtain more than 20 transgenic line altogether, wherein 10 strain systems receive that T1 is for seed, select wherein 5 (LB1 of strain system, LB7, LB8, LB9 and LB12) carry out RT-PCR and detect, show all to cross in these 5 strain systems and expressed SHR-1, the Arabidopis thaliana wild-type in 4 weeks of will in soil, growing and 3 weeks of transgenic line control water that SHR-1 crosses expression (35S:SHR-1), observe them to the tolerance of arid, the result shows, control water during 3 weeks most of wild-type lose the water meter type, and the dehydration situation of crossing the transgenic line express SHR-1 is significantly better than wild-type (Fig. 4), blade rate-of-loss of coolant measurement result shows, crosses the rate-of-loss of coolant of expressing strain system and will be considerably slower than wild-type, and these results showed that expressing SHR-1 can improve the drought resistance (Fig. 5) of transgenic plant.

(4) in paddy rice, express the drought resistance that SHR-1 can improve transgenic paddy rice excessively

The SHR-1 that 35S is driven gene constructed on the pCAMBIA1301 carrier rice transformation ZH11, obtain more than 10 transgenic line altogether, through PCR, GUS dyeing and RT-PCR have identified integration and the excessively expression of SHR-1 gene in transgenic paddy rice, with (the LB1 of three strains systems of the transgenic paddy rice after 2 weeks of sprouting, LB3 and LB5) T1 plant in same Culture basin for seedling and wild-type, under the phytotron condition (28 ℃, 16h illumination/8 hour dark) continues to cultivate for 1 week, handle the experiment of (watering 20% the PEG aqueous solution 200ml/ basin every day) simulating drought with PEG then, experimental result shows, SHR-1 crosses the express transgenic strain and ties up to PEG processing back growth conditions significantly better than wild-type, shows drought resistance (Fig. 6) preferably.

(5) arid of Arabidopis thaliana experiment, wild-type are all dead, and genetically modified survival rate is 100%, and the paddy rice result is the biological quantitative statistics (Fig. 7) of fresh weight.

Claims (2)

1. a novel method that improves plant drought resistance is characterized in that: implement step by step;

Step 1SHR-1 overexpression vector construction and Agrobacterium-mediated Transformation

1) extracting the whole strain RNA of Arabidopis thaliana, after reverse transcription becomes cDNA, is primer with SHR-F:5'ATGGATACTCTCTTTAGACTAG3' and SHR-R:5'TTACGTTGGCCGCCACGCACTAG3', and PCR obtains the gene complete fragment of SHR-1;

2) with Ligation high test kit it is connected in the pBluescript KS intermediate carrier sequence verification;

3) choose the forward clone, utilize two restriction enzyme sites of BamHI and SalI that the SHR-1 gene fragment is downcut from the KS carrier, and be connected among the plant expression vector pCAMBIA1301;

4) making up pCAMBIA1301-SHR-1, to change Agrobacterium GV3101 over to standby, namely chooses the Arabidopis thaliana seedling of just having bloomed, and every the 3-5d transfection once, transfection 3-5 time is standby continuously;

Step 2 Arabidopis thaliana arid is handled and percentage of water loss is measured

The Arabidopis thaliana seedling that sprouts 5d is transferred in the soil, begun to stop to water behind the cultivation 21-25d, and continue to cultivate 21d, observe its phenotype; Get the non-transgenic wild-type and the SHR-1 that cultivate 28d and cross expression strain blade, time point weighs up fresh weight respectively, namely gets percentage of water loss;

Step 3RT-PCR and Real-Time pcr analysis

1) utilize Trizol reagent to extract plant RNA and utilize ReverTra Ace, reverse transcription becomes cDNA;

2) RT-PCR the primer: SHR-F:5'ATGGATACTCTCTTTAGACTAG3'; SHR-R:5'TTACGTTGGCCGCCACGCACTAG3'; Atactin2F:5'GGAAGGATCTGTACGGTAAC3'; Atactin:5'GGACCTGCCTCATCATACT3';

3) reaction process: 95 ℃ of 1min, 95 ℃ of 15s, 58 ℃ of 30s, 72 ℃ of 15s; Totally 30 circulations; 72 ℃, extension 5min; Real-Time PCR operates according to SYBR Green Realtime PCR MasterMix specification sheets;

The genetic transformation of step 4 paddy rice

1) inducing of paddy rice mature embryo callus: the ZH11 rice paddy seed is shelled, with 70% alcohol immersion 1min, soak 20min with 20% chlorine bleach liquor again, with sterilized water washing 3-5 time, sowing under 26-28 ℃, was secretly cultivated 28-30d after aseptic filter paper blotted soaking solution on NB callus of induce substratum, the callus particle is seeded on the new NB callus of induce substratum, and it is standby secretly to cultivate 4d;

2) transform the Agrobacterium will contain the p1301proD1-SHR-1 plasmid the day before yesterday and be inoculated in the LB substratum, it is 0.6-0.8 that 28 ℃, 200rpm concussion are cultured to OD66o;

3) callus is transferred in the aseptic triangular flask, poured into Agrobacterium bacterium liquid, namely allow bacterium liquid submergence callus; Room temperature is placed 20min, removes bacterium liquid, callus is transferred to drawn unnecessary bacterium liquid on the aseptic filter paper, forwards NB then to and is total on the substratum, 20-25 ℃, cultivates 2-3d altogether in the dark;

4) callus after will cultivating is altogether transferred in the aseptic triangular flask, earlier with sterilized water washing 2-3 time, again with the sterilized water washing 20min that contains the 500ml/L Pyocianil; After aseptic filter paper blots excessive moisture, callus is transferred to the screening of carrying out transformant on the NB screening culture medium, screening 42-65d;

5) kanamycin-resistant callus tissue after will screening changes on the pre-differentiation substratum and cultivates 7-20d, goes on the NB division culture medium again and cultivates, and condition is 26 ℃, and 16h illumination/8h is dark; The resistance regeneration plant that differentiates changes strengthening seedling and rooting on the root media over to; 21-28d, the regeneration resistant plant that will take root is transplanted to the greenhouse;

Step 5PEG simulating drought is handled paddy rice

Wild-type and transgenic paddy rice seedling behind the sprouting 7-10d are planted respectively, normally watered and cultivate 14d, use the 200ml20%PEG aqueous solution then every day, continuous pouring 12-15d observes its phenotype.

2. according to the described method of claim 1, it is characterized in that: the Arabidopis thaliana growth conditions is 23 ± 1 ℃, 14h illumination/10h dark; Paddy rice ZH11 growth conditions is 28 ± 1 ℃, 16h illumination/8h dark.

Images