CN106432449B

CN106432449B - Drought tolerance in plants GAP-associated protein GAP VPS23A and its encoding gene and application

Info

Publication number: CN106432449B
Application number: CN201610973639.7A
Authority: CN
Inventors: 谢旗; 于菲菲
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2016-11-04
Filing date: 2016-11-04
Publication date: 2019-07-26
Anticipated expiration: 2036-11-04
Also published as: CN106432449A

Abstract

The invention discloses a kind of drought tolerance in plants GAP-associated protein GAP VPS23A and its encoding gene and applications.Protein provided by the invention is named as VPS23A albumen, is following (a) or (b): (a) protein that the amino acid sequence shown in sequence 1 in sequence table forms；(b) by (a) by the substitution and/or deletion and/or addition of one or several amino acid residues and the protein as derived from sequence 1 relevant to plant drought resistance.The gene (VPS23A gene) for encoding the VPS23A albumen also belongs to protection scope of the present invention.The present invention is experimentally confirmed, and is inhibited the expression of VPS23A gene in plant, can be significantly improved the drought tolerance of plant.The present invention improves crops and cultivates drought-resistant crops and has very important significance, and is suitable for promoting and applying.

Description

Drought tolerance in plants GAP-associated protein GAP VPS23A and its encoding gene and application

Technical field

The present invention relates to a kind of drought tolerance in plants GAP-associated protein GAP VPS23A and its encoding gene and applications.

Background technique

As the ecological problems such as the variation of global climate, soil desertification and salination and shortage of water resources are increasingly tight Weight, especially arid have become the principal element for restricting agricultural development.The one third of world's land area is all in arid With semiarid state, cause economic loss very big, arid is one of natural hazards risk the most serious.China is every year on average It suffers from drought cultivated area about 2,231.6 ten thousand hectares, accounting for about various meteorological disasters influences 2/3rds of cultivated areas, because drought is annual 10,000,000,000 kilograms of grain of loss.With economical fast development and population increase, shortage of water resources phenomenon gets worse, exacerbates The condition of a disaster of many arid and semi-arid lands.Such as 2014, Hebei province Cangzhou Area was made because of the lasting sultry weather short of rain of high temperature At a large amount of underproduction of crops.It is counted according to its agricultural sector, has nearly half in more than 780 ten thousand mu of crops by drought stress, nearly 1/6 It is disaster-stricken, or even there are tens of thousands of mu of corns the consequence almost having no harvest occur.Therefore, arid is to influence a disaster of plant growth and development Property stress.Since most of crops are very sensitive to drought stress, probe into how plant copes with drought stress and find The key gene for adjusting plant responding drought stress is particularly important.

In the process of plant responding drought stress, it has been found that degeneration-resistant hormone abscisic acid (ABA) plays extremely heavy The regulating and controlling effect wanted.ABA and root surrounding soil moisture content are significant related in root system of plant.ABA can effectively slow down the moisture side of body Compel, for plant when by drought stress, plant synthesizes a large amount of ABA, and ABA adjusts stomata by xylem transport to aerial part Aperture reduces transpiration, for promoting stomata to close to reduce moisture evaporation；ABA promotes moisture absorption, increases symplast way Diameter water flow ABA；Vane extension rate is reduced, induction drought resisting specific protein synthesis adjusts guard cell's ion channel, induces phase The expression of correlation gene, so that plant be helped to successfully manage arid threat.But the content of ABA is not the increasing that can have no limits Add, after plant adapts to drought environment, the content of ABA can be declined, and as drought stress is in slow ascendant trend.This Outside, the activity of In Antioxidant Systems of Plants can be improved in ABA, improves the efficiency of Scavenger of ROS.If applying ABA pairs of debita spissitudo outside In improving, plant resistance to environment stress effect is more significant.But artificial synthesized ABA is costly at present, it can't directly apply to production. Therefore, the ABA signal transduction pathway for studying and utilizing plant, finds the gene for adjusting ABA signal pathway, is to cultivate drought-resistant plant Breach.

Summary of the invention

The object of the present invention is to provide a kind of drought tolerance in plants GAP-associated protein GAP VPS23A and its encoding gene and applications.

Protein provided by the invention is obtained from arabidopsis, is named as VPS23A albumen, is following (a) or (b):

(a) protein that the amino acid sequence shown in sequence 1 in sequence table forms；

(b) by (a) by the substitution and/or deletion and/or addition of one or several amino acid residues and and plant drought The relevant protein as derived from sequence 1 of property.

In order to make VPS23A albumen in (a) convenient for purifying and detection, can in as sequence table amino acid shown in sequence 1 The amino terminal or carboxyl terminal of the protein of sequence composition connect upper label as shown in Table 1.

The sequence of 1 label of table

Label	Residue	Sequence
			Poly-Arg	5-6 (usually 5)	RRRRR
Poly-His	2-10 (usually 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

VPS23A albumen in above-mentioned (b) can be artificial synthesized, can also first synthesize its encoding gene, then carries out biological expression and obtain It arrives.The encoding gene of VPS23A albumen in above-mentioned (b) can be by will lack one in DNA sequence dna shown in sequence 2 in sequence table The codon of a or several amino acid residues, and/or the missense mutation of one or several base-pairs is carried out, and/or at its 5 ' end And/or 3 ' end connect the coded sequence of label shown in table 1 and obtain.

The gene (VPS23A gene) for encoding the VPS23A albumen also belongs to protection scope of the present invention.

The gene is following (1) or (2) or (3) or (4):

(1) DNA molecular shown in the nucleotide of 5 ' end 1001-2197 of sequence 2 of code area such as sequence table；

(2) DNA molecular shown in sequence 2 in sequence table；

(3) the DNA sequence dna hybridization that limits under strict conditions to (1) or (2) and the relevant protein of drought resistance is encoded DNA molecular；

(4) there is 90% or more homology and the relevant egg of coding drought resistance to the DNA sequence dna that (1) or (2) or (3) limits The DNA molecular of white matter.

Above-mentioned stringent condition can be for 0.1 × SSPE (or 0.1 × SSC), the solution of 0.1%SDS be miscellaneous in DNA or RNA It hands over and hybridizes at 65 DEG C in experiment and wash film.

Recombinant expression carrier, expression cassette, transgenic cell line or recombinant bacterium containing the VPS23A gene belong to this The protection scope of invention.

The present invention also protects the application of VPS23A albumen or VPS23A gene, for as follows (c1) and/or (c2):

(c1) regulate and control plant drought resistance；

(c2) sensibility of the regulation plant to degeneration-resistant hormone ABA signal.

The present invention also protects VPS23A gene cultivating the application in drought-resistant plant as target spot.

The present invention also protects a kind of method for cultivating genetically modified plants, is the table for inhibiting VPS23A gene in purpose plant It reaches, obtains the genetically modified plants that drought resistance is higher than the purpose plant.

" expression for inhibiting VPS23A gene in purpose plant " is realized by CRISPER-Cas9 gene editing.

The target sequence of sgRNA for the CRISPER-Cas9 gene editing is as shown in the sequence 4 of sequence table.

For the target sequence binding region in the sgRNA of the CRISPER-Cas9 gene editing as shown in sequence 5.

Recombinant vector for the CRISPER-Cas9 gene editing is concretely by the Spe of pYAO:hSpCas9 plasmid DNA molecular shown in sequence 3 is inserted between I restriction enzyme site.

The present invention also protects a kind of method for cultivating genetically modified plants, is the expression for reducing VPS23A albumen in purpose plant Amount and/or activity obtain the genetically modified plants that drought resistance is higher than the purpose plant.

The present invention also protects a kind of special sgRNA for CRISPER-Cas9 gene editing；The target sequence of the sgRNA As shown in the sequence 4 of sequence table.

The present invention protects substance for inhibiting VPS23A gene expression or also for reducing VPS23A egg in purpose plant White expression quantity and/or active substance or the sgRNA are cultivating the application in drought-resistant plant.

Any description above plant is monocotyledon or dicotyledon.The dicotyledon can be planted for Chinese lime mesh Object.The Chinese lime mesh plant can be crucifer.The crucifer can be Nan Jie race plant.The Nan Jie race plants Object can be Arabidopsis plant.The Arabidopsis plant concretely arabidopsis, such as Columbia ecotype arabidopsis.

The present invention provides VPS23A albumen and its encoding genes, inhibit the expression of VPS23A gene in plant, Ke Yixian Write the drought tolerance for improving plant.The present invention improves crops and cultivates drought-resistant crops and has very important significance, and is suitble to In popularization and application.

Detailed description of the invention

Fig. 1 is the testing result that plant pair ABA sensibility is expressed in mutant vps23a and its transgenosis complementation.

Fig. 2 is the structural schematic diagram of recombinant plasmid pCAMBIA1300-VPS23A

Fig. 3 is relative expression levels of the VPS23A gene in each plant.

Fig. 4 is mutant vps23a drought resistance testing result.

Fig. 5 is the structural schematic diagram of recombinant expression carrier pYAO:hSpCas9-AtVPS23A-sgRNA.

Specific embodiment

Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even Mean value.

Mutant CS878714:ABRC (Arabidopsis Biological Resource Center).

Columbia ecotype arabidopsis (Arabidopsis thaliana ecotype Columbia, Col-0): ABRC(Arabidopsis Biological Resource Center)。

PCAMBIA1300 carrier: bibliography: Yiyue Zhang, Chengwei Yang, Yin Li, Nuoyan Zheng, Hao Chen, Qingzhen Zhao, Ting Gao, Huishan Guo and Qi Xie (2007) SDIR1 Is a RING Finger E3 Ligase That Positively Regulates Stress-Responsive Abscisic Acid Signaling in Arabidopsis.Plant Cell.19 (6): 1912-1929.；The public can be from Chinese science Institute's heredity is obtained with Developmental Biology research.

EHA105 plants of Agrobacterium tumefaciens (Agrobacterium tumefaciens): bibliography: Yiyue Zhang, Chengwei Yang, Yin Li, Nuoyan Zheng, Hao Chen, Qingzhen Zhao, Ting Gao, Huishan Guo and Qi Xie(2007)SDIR1Is a RING Finger E3 Ligase That Positively Regulates Stress-Responsive Abscisic Acid Signaling in Arabidopsis.Plant Cell.19 (6): 1912-1929；The public can be obtained from Inst. of Genetics and Development Biology, CAS's Developmental Biology research.

AtU6-26-sgRNA-SK plasmid: bibliography: Liuhua Yan, Shaowei Wei, Yaorong Wu, Ruolan Hu, Hongju Li, Weicai Yang, Qi Xie (2015) High Efficiency Genome Editing In Arabidopsis Using Yao Promoter-Driven CRISPR/Cas9 System.Mol.Plant8 (12): 1820-1823；The public can be obtained from Inst. of Genetics and Development Biology, CAS's Developmental Biology research.

PYAO:hSpCas9 plasmid: bibliography: Liuhua Yan, Shaowei Wei, Yaorong Wu, Ruolan Hu, Hongju Li, Weicai Yang, Qi Xie (2015) High Efficiency Genome Editing In Arabidopsis Using Yao Promoter-Driven CRISPR/Cas9 System.Mol.Plant 8 (12): 1820-1823；The public can be obtained from Inst. of Genetics and Development Biology, CAS's Developmental Biology research.

Bsa I-HF:NEB company.

The ingredient of MS culture medium is shown in Table 2.

2 MS medium component of table

The acquisition of embodiment 1, VPS23A albumen and its encoding gene

By carrying out ABA phenotypic screen to several Arabidopsis Mutants plant ordered from ABRC, it is found that an ABA is super quick The mutant CS878714 of sense, through genome sequencing, mutant CS878714 is the deletion mutant (T- of VPS23A gene DNA insertion mutation body, insertion point are in the 1st exon of VPS23A gene), the same brother of sequence other than VPS23A gene Rival Asia Arabidopsis thaliana ecotype, is named as mutant vps23a.

The VPS23A albumen in arabidopsis source is as shown in the sequence 1 of sequence table.

The VPS23A gene in arabidopsis source is as shown in the sequence 2 of sequence table, wherein from the core of 5 ' end 1001-2197 Thuja acid is code area.

Embodiment 2, the detection of the physiological characteristic of mutant vps23a

1, by the seed of mutant vps23a and Columbia ecotype arabidopsis (Col-0) in 10% bleaching agent solution Surface sterilizing is carried out, then with sterile water washing 3 times.

2, by step 1, treated that seed is grouped experiment:

Control group: aseptic seed is suspended in aqueous solution and on bed board to 1/2MS culture medium flat plate, by plate at 4 DEG C It is placed 3 days under dark condition, then moves into 24 DEG C of tissue culture room (16h illumination/8h is dark) culture one week, taken pictures and observe The growth conditions of plant.

Experimental group: by aseptic seed suspend in aqueous solution and bed board arrive containing 0.3 or 0.5 μM of ABA 1/2MS culture medium On plate, plate is placed 3 days under 4 DEG C of dark conditions, then moving into 24 DEG C of tissue culture room, (illumination/8 hour 16h are black It secretly) cultivates one week, takes pictures and observe the growth conditions of plant.

As a result as shown in Figure 1.Figure 1A is that plant cotyledon is taken pictures photo.Figure 1B is cotyledon greening ration statistics result, wherein 1 is the statistical result of Columbia ecotype arabidopsis (Col-0), and 2 be the statistical result of mutant vps23a.Fig. 1 C is plant Root growth situation is taken pictures photo.Fig. 1 D is root long statistical result, wherein 1 is the system of Columbia ecotype arabidopsis (Col-0) Meter is as a result, 2 be the statistical result of mutant vps23a.

On the ground in terms of cotyledon greening, in control group (1/2MS, 0 μM of ABA) processing, mutant vps23a and brother's rival The growth conditions of sub- Arabidopsis thaliana ecotype (Col-0) are not significantly different.It is quasi- with Columbia ecotype in experimental group processing Southern mustard (Col-0) is compared, and the number of cotyledon greening is less in mutant vps23a.On the plate containing 0.3 μM of ABA, The cotyledon greening of 6.7% mutant vps23a, and the stretching, extension of the cotyledon of 80% Colombia's Arabidopsis thaliana ecotype (Col-0) is simultaneously Greening；In the case where 0.5 μM of ABA is handled, the non-greening of the cotyledon of mutant vps23a, and 64.4% Columbia ecotype is quasi- The cotyledon greening of southern mustard (Col-0).

In terms of the root growth of underground, in control group (1/2MS, 0 μM of ABA) processing, mutant vps23a and Colombia The growth conditions of Arabidopsis thaliana ecotype (Col-0) are not significantly different.In experimental group, the root long of mutant vps23a is shorter than brother Rival Asia Arabidopsis thaliana ecotype (Col-0).On the plate containing 0.3 μM of ABA, the root long of mutant vps23a is about brother's human relations Than the 50% of sub- Arabidopsis thaliana ecotype (Col-0) root long；In the case where 0.5 μM of ABA is handled, the root long of mutant vps23a is about brother The 30% of rival Asia Arabidopsis thaliana ecotype (Col-0) root long.

The above experimental analysis explanation, mutant vps23a is more sensitive for the response of ABA, in mutant vps23a The intensity of activation of ABA signal is greater than Columbia ecotype arabidopsis (Col-0).

Plant is expressed in embodiment 3, transgenosis complementation

One, the acquisition of transgenosis complementation expression plant

1, the total serum IgE and reverse transcription for extracting Columbia ecotype arabidopsis (Col-0) are cDNA, using cDNA as template, PCR amplification is carried out using the primer pair that VPS23A-FW and VPS23A-Rev is formed, recycles pcr amplification product.

VPS23A-FW:5 '-GGGTACCCTCTCGTCGTTGGAGTTG-3 ' (underscore is Kpn I restriction enzyme site)

VPS23A-Rev:5 '-CGGATCCAGCCGATGTTTATTACCT-3 ' (underscore is BamH I restriction enzyme site)

2, with restriction enzyme Kpn I and BamH I digestion pCAMBIA1300 carrier, the carrier of about 10000bp is recycled Skeleton.

3, the pcr amplification product that step 1 obtains is connected with the carrier framework that step 2 obtains, obtains recombinant plasmid pCAMBIA1300-AtVPS23A.According to sequencing result, structure description is carried out to recombinant plasmid pCAMBIA1300-AtVPS23A It is as follows: the small fragment between KpnI the and BamHI restriction enzyme site of pCAMBIA1300 carrier is replaced into 2 institute of sequence for sequence table The double chain DNA molecule shown.The structural schematic diagram of recombinant plasmid pCAMBIA1300-AtVPS23A is shown in Fig. 2.

4, recombinant plasmid pCAMBIA1300-AtVPS23A is imported in Agrobacterium tumefaciens EHA105 plants, obtains recombination soil Earth bacillus.

5, the recombination agrobacterium for obtaining step 4 by plant vacuum-infiltration (method reference literature: BentAF, Clough S J.Agrobacterium.Germ-Line Transformation:Transformation of Arabidopsis, without Tissue Culture [M] //Plant Molecular Biology Manual.Springer Netherlands, 1998:17-30) untransformed mutants vps23a, harvest seed (seed in T0 generation, The plant that the seed grows up to is T1 for plant).

6, the seed for harvesting step 5 is placed on the 1/2MS culture medium flat plate containing 20 μ g/mL hygromycin, by plate first 4 It is placed 2-4 days under DEG C dark condition, then moves into 24 DEG C of tissue culture room (16h illumination/8h is dark) culture 1 week, then moved Enter 24 DEG C of greenhouse to be cultivated and harvest seed (T₁The seed in generation, the plant which grows up to are T₂For plant).

7, the seed for harvesting step 6 is placed in progress resistance sieve on the 1/2MS culture medium flat plate containing 50 μ g/mL kanamycins Choosing, and harvest the T that the transgenic plant singly copied obtains₂For resistant plant seed (plant that the seed grows up to be T₃Dai Zhi Strain).If a certain T₁The T obtained for plant₂The quantity of resistant plant and non-resistance plant illustrates the T than about 3: 1 in plant₁ It is the transgenic plant singly copied for plant.

8, the seed for harvesting step 7 is placed in progress resistance sieve on the 1/2MS culture medium flat plate containing 50 μ g/mL kanamycins Choosing.If a certain T₂The T obtained for plant₃It is resistant plant for plant, illustrates the T₂It is that homozygous transgenosis is planted for plant Strain, the T₂It is homozygous complementary expression strain for plant and its offspring.

9, two (3.3 and 4.6) are chosen from the complementary expression strain of homozygosis that step 8 obtains.Take Columbia ecotype Arabidopsis (Col-0), mutant vps23a, 3.3 strains T₃For the T of the 2 week old seedling and 4.6 strains of plant₃For the 2 of plant Week old seedling, it is template to the table of VPS23A gene in the plant of each strain using cDNA that extracting total serum IgE and reverse transcription, which is cDNA, Real-time fluorescence quantitative PCR detection is carried out up to amount, using Actin2 gene as reference gene, with Columbia ecotype arabidopsis The expression quantity of the expression quantity of middle VPS23A gene calculates the relative expression quantity of AtVPS23A gene in other each strains as 1, Three repeated experiments are carried out, are averaged.

Primer sequence for identifying VPS23A gene is as follows:

QRT-VPS23A-Fw:5 '-CACTTGAACAACAATTACAGA-3 ':

QRT-VPS23A-Rev:5 '-AAGCATTATCCACATCCAA-3 '.

Primer sequence for identifying Actin2 gene is as follows:

QRT-Actin2-FW:5 '-GGTAACATTGTGCTCAGTGGTGG-3 '；

QRT-Actin2-Rev:5 '-AACGACCTTAATCTTCATGCTGC-3 '.

As a result see Fig. 3.The result shows that the homozygous complementary expression water for expressing VPS23A gene in strain (3.3 and 4.6) plant The expression for having exceeded VPS23A gene in Columbia ecotype arabidopsis is put down, is not detected in mutant vps23a The expression of VPS23A gene.

Two, the phenotypic analysis of transgenosis complementation expression plant

According to homozygous complementary expression strain (the 3.3 and 4.6) T of the method detection of embodiment 2₃For the phenotype of plant.

As a result as shown in Figure 1.Figure lA is that plant cotyledon is taken pictures photo.Figure 1B is cotyledon greening ration statistics result, wherein 3 be the homozygous complementary statistical result for expressing 3.3 plant of strain, and 2 be the statistical result of homozygous complementary expression 4.6 plant of strain.Figure 1C is that plant-root growth situation is taken pictures photo.Fig. 1 D is root long statistical result, wherein 1 is homozygous complementary expression 3.3 plant of strain Statistical result, 2 for homozygous complementary expression 4.6 plant of strain statistical result.

The result shows that homozygous complementary expression strain (3.3 and 4.6) T₃The quasi- south of phenotype and Columbia ecotype for plant Mustard is identical, the phenotype of their equal energy complemented mutant body vps23a in terms of cotyledon greening and root long growth.Illustrate VPS23A gene Missing be the reason of leading to mutant vps23a phenotype.VPS23A gene can regulate and control response of the plant to ABA signal.Mutation Body vps23a can be used to carry out the arid phenotype experiment of next step.

Embodiment 4, mutant vps23a Identification of Drought

2, by aseptic seed suspend in aqueous solution and bed board to 1/2MS plate on, 4 DEG C vernalization 3 days, then 21 DEG C culture 11 It, then be transplanted in soil and continue culture 14 days or so, 5 are planted in each hole.

3, it is in non-bolting period to plant growth, entire leaf dish is cut off, places stomata on each blade independently, together When do not rewater, carry out drought tolerance test.Rehydration is carried out after Osmotic treatment 21 days.

It is as shown in Figure 4 A that plant phenotype observes result.Mutant vps23a and Columbia ecotype arabidopsis (Col-0) Growth conditions be not significantly different at no drought stress (according to normal growth 4 weeks described in step 2).On the day before rehydration One day after with rehydration, the growth conditions of mutant vps23a plant are significantly better than Columbia ecotype arabidopsis.Rehydration is latter It, counts the survival rate of each plant, and the survival rate of discovery mutant vps23a plant is significantly higher than the quasi- south of Columbia ecotype Mustard (Col-0) (Fig. 4 B).

4, the open and flat lotus throne leaf of each plant of step 2 is taken, blade lower epidermis of tearing is immersed in stomata buffer (10mM KCl, 50 μM of CaCl₂, 10mM MES-Tris, pH 6.15) in, it is placed in strong light (90 μm of ol.m^-2.s^-1) lower 3 hours；By leaf Piece shifts in the stomata buffer of the ABA containing 0 μM or 10 μM and handles 3-5h；Blade lower epidermis is laid on glass slide, is used Writing brush brushes off mesophyll cell, and covered, in Imager.A1 microscopically observation stomata, Axio software takes pictures and counts gas Hole length-width ratio.

Statistical result is as shown in Figure 4 C.In the case where no ABA, mutant vps23a and the quasi- south of Columbia ecotype The stomatal aperture of mustard (Col-0) does not have significant difference, and in the case where 10 μM of ABA are handled, the stomatal aperture of mutant vps23a is obviously small In Columbia ecotype arabidopsis (Col-0).Stomatal aperture is small can to effectively reduce loss of moist, this is mutant vps23a The major reason of drought resisting.

The above result shows that the missing of VPS23A gene can enhance plant to the sensibility of degeneration-resistant hormone ABA signal, make The raising of mutant plant degeneration-resistant hormone ABA level in quick response body under drought condition is closed Stoma of Leaves, is reduced into one The transpiration of step, it is final to assign the very strong drought-resistant ability of plant.

The foundation and identification of embodiment 5, VPS23A afunction transgenic arabidopsis strain

1, digestion is carried out to AtU6-26-sgRNA-SK plasmid using Bsa I-HF, obtains the AtU6-26- of about 4000bp SgRNA-SK carrier.

2, artificial synthesized primer VPS23A-FW2 and primer VPS23A-Rev2.

VPS23A-FW2:5 '-ATTG(underscore is carrier AtU6-26-sgRNA- to GGACGGCGTCGAGAATAAAG-3 ' The complementary series of cohesive end sequence of the SK after Bsa I digestion)；

VPS23A-Rev2:5 '-AAAC(underscore is carrier AtU6-26-sgRNA- to CTTTATTCTCGACGCCGTCC-3 ' The complementary series of cohesive end sequence of the SK after Bsa I digestion).

3, primer VPS23A-FW2 and primer VPS23A-Rev2 that step 2 synthesizes are annealed, obtains double-stranded DNA piece Section (interference fragment).

4, by the double chain DNA fragment (interference fragment) that step 3 obtains and the AtU6-26-sgRNA-SK carrier that step 1 obtains Connection, obtains connection product.

5, by the connection product of step 4 Nhe I and Spe I restriction enzymes double zyme cutting, target fragment is obtained.

6, digestion is carried out to pYAO:hSpCas9 plasmid using Spe I restriction enzyme, obtains about 12,000bp's PYAO:hSpCas9 carrier.

7, the target fragment that step 5 obtains is connect with the pYAO:hSpCas9 carrier that step 6 obtains, is recombinantly expressed Carrier pYAO:hSpCas9-VPS23A-sgRNA.According to sequencing result, to recombinant expression carrier pCAMBIA1300-Cas9- VPS23A carries out structure and is described as follows: will insert shown in sequence 3 between the Spe I restriction enzyme site of pYAO:hSpCas9 plasmid DNA molecular.The structural schematic diagram of recombinant expression carrier pYAO:hSpCas9-VPS23A-sgRNA is shown in Fig. 5.

8, the recombinant expression carrier pYAO:hSpCas9-VPS23A-sgRNA for obtaining step 7 imports Agrobacterium tumefaciens In EHA105 plants, recombination agrobacterium is obtained.

9, the recombination agrobacterium for obtaining step 8 by plant vacuum-infiltration (method reference literature: Bent AF, Clough S J.Agrobacterium, Germ-Line Transformation:Transformation of Arabidopsis, without Tissue Culture [M] //Plant Molecular Biology Manual.Springer Netherlands, 1998:17-30.) conversion Columbia ecotype arabidopsis (Col-0), harvest (seed in T0 generation, the plant which grows up to are T to seed₁For plant).

10, the seed for harvesting step 9 is placed on the 1/2MS culture medium flat plate containing 20 μ g/mL hygromycin, and plate is first existed It is placed 2-4 days under 4 DEG C of dark conditions, then moves into 24 DEG C of tissue culture room (16h illumination/8h is dark) culture 1 week, then moved Enter 24 DEG C of greenhouse to be cultivated and harvest seed (T₁The seed in generation, the plant which grows up to are T₂For plant).

11, the seed for harvesting step 10 is placed on the 1/2MS culture medium flat plate containing 50 μ g/mL kanamycins and carries out resistance Screening, and harvest the T that the transgenic plant singly copied obtains₂For resistant plant seed (plant that the seed grows up to be T₃Dai Zhi Strain).If a certain T₁The T obtained for plant₂The quantity of resistant plant and non-resistance plant illustrates the T than about 3: 1 in plant₁ It is the transgenic plant singly copied for plant.

12, the seed for harvesting step 11 is set and carries out resistance on the dry 1/2MS culture medium flat plate containing 50 μ g/mL kanamycins Screening.If a certain T₂The T obtained for plant₃It is resistant plant for plant, illustrates the T₂It is that homozygous transgenosis is planted for plant Strain, the T₂It is Transgenic wheat line for plant and its offspring.

13, one is chosen from the Transgenic wheat line that step 12 obtains, and detects T₃For VPS23A gene in plant Expression quantity (method with 3 step 1 of embodiment 9).

By identification, VPS23A gene is not expressed in homozygous VPS23A afunction transgenic line plant.

Two, turn the acquisition of empty carrier strain

1, digestion is carried out to AtU6-26-sgRNA-SK plasmid using Nhe I and Spe I restriction enzyme, obtains purpose Segment.

2, digestion is carried out to pYAO:hSpCas9 plasmid using Spe I restriction enzyme, obtains about 12,000bp's PYAO:hSpCas9 carrier.

3, the target fragment that step 1 obtains is connect with the pYAO:hSpCas9 carrier that step 2 obtains, is recombinantly expressed Carrier pYAO:hSpCas9-sgRNA (is free of interference fragment).

4, pYAO:hSpCas9-sgRNA carrier is carried instead of recombinant expression carrier pYAO:hSpCas9-VPS23A-sgRNA Body obtains turning empty carrier strain according to 8 operations of step 1.

Three, Identification of Drought

Plant to be measured are as follows: the T of homozygous VPS23A afunction transgenic line₃For plant, turn the T of empty carrier strain₃Dai Zhi Strain, mutant vps23a, Columbia ecotype arabidopsis (Col-0).

1, by the seed of plant to be measured with surface sterilizing is carried out in 10% bleaching agent solution, then with sterile water washing 3 times.

3, it is in non-bolting period to plant growth, entire leaf dish is cut off, places stomata on each blade independently, together When do not rewater, carry out drought tolerance test.Osmotic treatment 21 days or so progress rehydrations.

Phenotypic Observation the result shows that, VPS23A afunction transgenic plant, turn empty carrier plant, mutant vps23a and The growth conditions of Columbia ecotype arabidopsis (Col-0) are in no drought stress (according to normal growth 4 weeks described in step 2) When be not significantly different.On the day before rehydration and rehydration one day after, VPS23A afunction transgenic plant and mutant The growth conditions of vps23a plant are significantly better than Columbia ecotype arabidopsis and turn empty carrier plant.After rehydration one day, system The survival rate of each plant is counted, the survival rate of discovery VPS23A afunction transgenic plant is 60%, with mutant vps23a's Survival rate is significantly higher than the survival rate (23%) of Columbia ecotype arabidopsis without significant difference.Turn depositing for empty carrier plant The survival rate of motility rate and Columbia ecotype arabidopsis is without significant difference.

Statistical result shows in the case where no ABA, VPS23A afunction transgenic plant, turn empty carrier plant, The stomatal aperture of mutant vps23a and Columbia ecotype arabidopsis (Col-0) does not have significant difference.At 10 μM of ABA Under reason, the stomatal aperture of VPS23A afunction transgenic plant is 0.25, and the stomatal aperture with mutant vps23a is without significant Difference, the substantially less than stomatal aperture (0.42) of Columbia ecotype arabidopsis (Col-0).The stomata for turning empty carrier plant is opened The stomatal aperture of degree and Columbia ecotype arabidopsis is without significant difference.

Based on the above results, VPS23A afunction transgenic Arabidopsis plants are showed compared with Columbia ecotype arabidopsis Stronger drought resistance out.

<110>Inst. of Genetics and Development Biology, CAS

<120>drought tolerance in plants GAP-associated protein GAP VPS23A and its encoding gene and application

<130> GNCYXMN161964

<160> 5

<210> 1

<211> 398

<212> PRT

<213>arabidopsis (Arabidopsis thaliana)

<400> 1

Met Val Pro Pro Pro Ser Asn Pro Gln Gln Val Gln Gln Phe Leu Ser

1 5 10 15

Ser Ala Leu Ser Gln Arg Gly Pro Ser Ser Val Pro Tyr Glu Glu Ser

20 25 30

Asn Lys Trp Leu Ile Arg Gln His Leu Leu Asn Leu Ile Ser Ser Tyr

35 40 45

Pro Ser Leu Glu Pro Lys Thr Ala Ser Phe Met His Asn Asp Gly Arg

50 55 60

Ser Val Asn Leu Leu Gln Ala Asp Gly Thr Ile Pro Met Pro Phe His

65 70 75 80

Gly Val Thr Tyr Asn Ile Pro Val Ile Ile Trp Leu Leu Glu Ser Tyr

85 90 95

Pro Arg His Pro Pro Cys Val Tyr Val Asn Pro Thr Ala Asp Met Ile

100 105 110

Ile Lys Arg Pro His Ala His Val Thr Pro Ser Gly Leu Val Ser Leu

115 120 125

Pro Tyr Leu Gln Asn Trp Val Tyr Pro Ser Ser Asn Leu Val Asp Leu

130 135 140

Val Ser Asp Leu Ser Ala Ala Phe Ala Arg Asp Pro Pro Leu Tyr Ser

145 150 155 160

Arg Arg Arg Pro Gln Pro Pro Pro Pro Ser Pro Pro Thr Val Tyr Asp

165 170 175

Ser Ser Leu Ser Arg Pro Pro Ser Ala Asp Gln Ser Leu Pro Arg Pro

180 185 190

Phe Pro Pro Ser Pro Tyr Gly Gly Gly Val Ser Arg Val Gln Val Gln

195 200 205

His Val His His Gln Gln Gln Ser Asp Asp Ala Ala Glu Val Phe Lys

210 215 220

Arg Asn Ala Ile Asn Lys Met Val Glu Met Val His Ser Asp Leu Val

225 230 235 240

Ser Met Arg Arg Ala Arg Glu Ala Glu Ala Glu Glu Leu Leu Ser Leu

245 250 255

Gln Ala Gly Leu Lys Arg Arg Glu Asp Glu Leu Asn Ile Gly Leu Lys

260 265 270

Glu Met Val Glu Glu Lys Glu Thr Leu Glu Gln Gln Leu Gln Ile Ile

275 280 285

Ser Met Asn Thr Asp Ile Leu Asp Ser Trp Val Arg Glu Asn Gln Gly

290 295 300

Lys Thr Lys Asn Leu Val Asp Leu Asp Val Asp Asn Ala Phe Glu Cys

305 310 315 320

Gly Asp Thr Leu Ser Lys Gln Met Leu Glu Cys Thr Ala Leu Asp Leu

325 330 335

Ala Ile Glu Asp Ala Ile Tyr Ser Leu Asp Lys Ser Phe Gln Asp Gly

340 345 350

Val Val Pro Phe Asp Gln Tyr Leu Arg Asn Val Arg Leu Leu Ser Arg

355 360 365

Glu Gln Phe Phe His Arg Ala Thr Gly Ser Lys Val Arg Ala Ala Gln

370 375 380

Met Glu Val Gln Val Ala Ala Ile Ala Gly Arg Leu His Ser

385 390 395

<210> 2

<211> 2805

<212> DNA

<213>arabidopsis (Arabidopsis thaliana)

<400> 2

ctctcgtcgt tggagttggc gtccagtgga caccgtttag ttattactcc gatccaagac 60

cgaataacta ctacgccgat cctccgccga taagatacta ctcagataat ccagcggata 120

ttctccagtt atgcgtgggt aatcgatgtc tcatcatcca gttaggttac tgtgaccaag 180

tacctaacaa cctccgcagt ttcttagcag atccagaaac cacgttcgtc ggtgtctgga 240

atggtcaaga tgcaggaaag cttgctcggt gttgccacca gttggagatc ggagaacttc 300

tggacataag gcggtacgtg actgattcgt ggggaaggag catgaggcgt tcttcgtttg 360

aagagattgt tgaggaatgt atgggctatc aaggagtgat gctagatccg gagataagca 420

tgagcgattg gaccgcttac gatctagacc ttgatcagat tcttcaggcg tcactagatg 480

cttacgtttg ccatcagctt ggtgtttgga ctcgtctctg ggaagtttga aaacaagtat 540

caaaatgaat aaattgctac ttggattttt ctgtcttttg ttgacaagtg acttgaatct 600

tctttttgtt ttgttatgag aaaaaaattg tggtttgaat cttttcgttt atttttgttg 660

tcaagtggtt tgaatcttct tattgttaaa gttatgagaa aaaaactgtt ttgtttttca 720

ctcgttactt acatgagtaa ggtgaataaa actttgatca caaactcgct ttatgttttg 780

ggtgttaaac tttggacttt ggaacatgtc cactccttac aaatttttat ggagtaaacc 840

ggattaaaca ctatccaaaa caacactgaa gggtaaaaga gtaaatagct aataaagacg 900

cgtataataa cattacttct tcttcttaaa tcgaatctcc tccccagttg tcgtcgtcat 960

cttcaatttg gacttccaac gccgacaccg tttaactccg atggttcccc cgccgtctaa 1020

tccgcagcag gttcagcagt tcctctcctc tgccctctcc cagcgcggcc catcttcagt 1080

cccctacgaa gagtccaaca agtggttgat ccggcaacat ctacttaacc taatctcttc 1140

ttacccttcc ttagagccca aaacggcatc gtttatgcac aacgatggtc gctccgtcaa 1200

cctccttcaa gcagatggta cgattccgat gccttttcat ggagtcacct ataacatacc 1260

tgtgattatc tggctcctcg agtcatatcc tcgtcatcct ccttgcgtct atgtgaatcc 1320

caccgctgat atgatcatca agcgacctca cgcacatgtc actccttctg gtctcgtttc 1380

tcttccgtac cttcagaatt gggtctaccc tagctccaat ctcgtagatc tcgtctccga 1440

tctcagcgct gcttttgctc gtgatccgcc tctttattct cgacgccgtc ctcagccacc 1500

gccaccgtct cctcctacgg tatacgattc gtctctgtca cgacctcctt cggctgatca 1560

gtcattgcct agaccgttcc cgccatcacc ttacggcgga ggagtaagta gggtgcaagt 1620

gcagcatgtt caccaccagc agcaatctga tgatgcggcg gaggttttca agagaaatgc 1680

gattaataag atggtggaga tggttcatag cgatttggtt tcgatgagga gagccagaga 1740

agctgaagca gaggagctgc tgagcttgca agctgggctg aagagaagag aggatgagct 1800

taatataggg ttgaaagaga tggttgagga gaaagaaaca cttgaacaac aattacagat 1860

tatctccatg aacactgata ttctagactc gtgggttaga gagaaccaag gcaaaaccaa 1920

gaatttagtt gatttggatg tggataatgc ttttgaatgt ggtgacacac tctctaagca 1980

gatgttagag tgtactgctt tggatttagc cattgaagat gctatttatt ccttggataa 2040

gtcgtttcaa gatggtgttg ttccctttga tcagtatttg aggaatgtga ggttgttgtc 2100

gagagaacag ttcttccacc gagccacggg ttctaaagtc agggcggcac aaatggaggt 2160

tcaggttgca gccatcgcag gtaggttaca ttcatgaatg aaattcagtg tttttgtttg 2220

cgggatactg tgtcacacaa tgctggttta tatagtctga gattagcatc tttgaggagt 2280

atttctgtgg ctatgcgtgt gatgatggtt cctctttaag attgaattaa atgtctatag 2340

actatatata cacgtacact gttgtgcaca tacatatttg aatggatcag ggcctgagaa 2400

tcgtctcctt ttgtgatcta tgactacttt tttacatgtt ggggatcttt gtgataaaca 2460

gtattattat ctaatgaatc aatcaattgg tttgagaatg ttagtatctt tgttttggat 2520

attggaacag ttttgatgac tcttctttct tctccattaa aaaaatgttg ctttagttta 2580

ttcaccatgc aaaaggaaac taaaaccttg tcgaattgaa cccattagct ccaccatcaa 2640

tgatgaaacc ttgtcaagtc ttcttatcag caactgcaac atccttacgc cttgttttgc 2700

ttcatcagtg tgcctttgaa tctttcgtag tcttggataa aaccagctcc ttccttgcaa 2760

ctccctagcc tccttccttg aggagttagg taataaacat cggct 2805

<210> 3

<211> 635

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 3

aagcttcgtt gaacaacgga aactcgactt gccttccgca caatacatca tttcttctta 60

gctttttttc ttcttcttcg ttcatacagt ttttttttgt ttatcagctt acattttctt 120

gaaccgtagc tttcgttttc ttctttttaa ctttccattc ggagtttttg tatcttgttt 180

catagtttgt cccaggatta gaatgattag gcatcgaacc ttcaagaatt tgattgaata 240

aaacatcttc attcttaaga tatgaagata atcttcaaaa ggcccctggg aatctgaaag 300

aagagaagca ggcccattta tatgggaaag aacaatagta tttcttatat aggcccattt 360

aagttgaaaa caatcttcaa aagtcccaca tcgcttagat aagaaaacga agctgagttt 420

atatacagct agagtcgaag tagtgattgg gacggcgtcg agaataaagg ttttagagct 480

agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc 540

ggtgcttttt ttgccattct tttcaagctc cattgtcaaa ttttcggggg gttttgaagt 600

cgcctatctg aggttagtct ctctgcatct gatca 635

<210> 4

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 4

ctttattctc gacgccgtcc 20

<210> 5

<211> 20

<212> RNA

<213>artificial sequence

<220>

<223>

<400> 5

ggacggcguc gagaauaaag 20

Claims

1. in sequence table protein shown in sequence 1 or and its encoding gene application, for following (c1) and/or (c2):

(c1) regulate and control plant drought resistance；

2. application according to claim 1, it is characterised in that: the encoding gene is following (1) or (2):

(2) DNA molecular shown in sequence 2 in sequence table.

3. the gene of protein shown in sequence 1 is cultivating the application in drought-resistant plant as target spot in polynucleotide.

4. application according to claim 3, it is characterised in that: the gene is following (1) or (2):

(2) DNA molecular shown in sequence 2 in sequence table.

5. a kind of method for cultivating genetically modified plants is the coding for inhibiting protein shown in sequence 1 in sequence table in purpose plant The expression of gene obtains the genetically modified plants that drought resistance is higher than the purpose plant.

6. according to the method described in claim 5, it is characterized by: the encoding gene is following (1) or (2):

(2) DNA molecular shown in sequence 2 in sequence table.

7. a kind of method for cultivating genetically modified plants is to reduce a kind of expression quantity and/or activity of protein in purpose plant, obtains It is higher than the genetically modified plants of the purpose plant to drought resistance；

The protein is as shown in sequence 1 in sequence table.

8. for the substance that inhibits the encoding gene of protein shown in sequence 1 in sequence table to express or for reducing in purpose plant The expression quantity of protein shown in sequence 1 and/or active substance are cultivating the application in drought-resistant plant in sequence table；

The substance is recombinant expression carrier pYAO:hSpCas9-VPS23A-sgRNA；

The recombinant expression carrier pYAO:hSpCas9-VPS23A-sgRNA is by the Spe I in pYAO:hSpCas9 plasmid Insert what DNA molecular shown in sequence 3 constructed between restriction enzyme site.

9. application according to claim 8, it is characterised in that: the encoding gene is following (1) or (2):

(2) DNA molecular shown in sequence 2 in sequence table.