CN106432449A

CN106432449A - Protein VPS23A related to drought tolerance of plants, encoding gene of protein VPS23A and application

Info

Publication number: CN106432449A
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: 2017-02-22
Anticipated expiration: 2036-11-04
Also published as: CN106432449B

Abstract

The invention discloses a protein VPS23A related to the drought resistance of plants, an encoding gene of the protein VPS23A and application. The protein VPS23A is named as a VPS23A protein and is a protein (a) or a protein (b). The protein (a) comprises amino acid sequences shown as sequences 1 in sequence tables; an amino acid residue or a plurality of amino acid residues of the protein (a) is substituted and/or deleted and/or added, so that the proteins (b) can be derived from the sequences 1 related to the drought resistance of the plants. The encoding gene (VPS23A gene) for encoding the VPS23A protein also belongs to the protection range of the invention. The protein VPS23A, the encoding gene and the application have the advantages that as shown by experiments, expression of the VPS23A gene in the plants can be inhibited, and accordingly the drought resistance of the plants can be obviously improved; the protein VPS23A, the encoding gene and the application have extremely important significance on crop improvement and drought-resistant crop cultivation, and the protein VPS23A and the encoding gene are suitable for popularization and application.

Description

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

Technical field

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

Background technology

Increasingly tight with ecological problems such as the change of global climate, soil desertification and salination and shortage of water resources Weight, especially arid have become as the principal element of restriction agricultural development./ 3rd of world's land area are 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 Suffer from drought cultivated area about 2,231.6 ten thousand hectares, accounting for various meteorological disasters affects 2/3rds of cultivated areas, because drought is annual 10,000,000,000 kilograms of grain of loss.With economic fast development and population increase, shortage of water resources phenomenon is increasingly serious, exacerbates The condition of a disaster of many arid and semi-arid lands.As 2014, Hebei province Cangzhou Area, because of the lasting sultry weather short of rain of high temperature, was made Become a large amount of underproduction of crops.According to its agricultural sector statistics, have in more than 780 ten thousand mu of crops nearly half be subject to drought stress, nearly 1/6 Disaster-stricken, or even have tens thousand of mu of Semen Maydiss that the consequence almost having no harvest occurs.Therefore, arid is a disaster of impact growth and development of plants Property stress.Because most of crops are very sensitive to drought stress, therefore probe into how plant tackles drought stress and find The key gene adjusting plant responding drought stress is particularly important.

In the middle of the process of plant responding drought stress it has been found that degeneration-resistant hormone abscisic acid (ABA) play extremely heavy The regulating and controlling effect wanted.In root system of plant, ABA is significantly correlated with root surrounding soil moisture.ABA can effectively slow down the moisture side of body Compel, when by drought stress, plant synthesizes a large amount of ABA to plant, and ABA passes through xylem transport and adjusts pore to aerial partss Aperture, reduces transpiration, for promoting pore to close to reduce moisture evaporation；ABA promotes moisture absorption, increases symplast way Footpath current ABA；Reduce vane extension rate, induction drought resisting specific protein synthesis, adjust guard cell's ion channel, induce phase The expression of correlation gene, thus help plant to successfully manage the threat of arid.But, the content of ABA is not increasing of can having no limits Plus, after plant adapts to drought environment, the content of ABA can decline, and is in slow ascendant trend with drought stress.This Outward, ABA can improve the activity of In Antioxidant Systems of Plants, improves the efficiency of Scavenger of ROS.If outer apply debita spissitudo ABA pair In improving, plant resistance to environment stress effect is more notable.But, costly, it can't directly apply to production to current synthetic ABA. Therefore, research and the ABA signal transduction pathway using plant, find the gene adjusting ABA signal pathway, are to cultivate drought-resistant plant Breach.

Content of the invention

It is an object of the invention to provide a kind of drought tolerance in plants associated protein VPS23A and its encoding gene and application.

The protein that the present invention provides, available from arabidopsiss, is named as VPS23A albumen, is following (a) or (b)：

A protein that () aminoacid sequence shown in sequence in sequence table 1 forms；

B () is by (a) through the replacement of one or several amino acid residues and/or disappearance and/or interpolation and and plant drought Property related protein derived from sequence 1.

In order that the VPS23A albumen in (a) is easy to purification and detection, can in by sequence table the aminoacid shown in sequence 1 The amino terminal of protein of sequence composition or carboxyl terminal connect upper label as shown in table 1.

The sequence of table 1 label

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 synthetic, also can first synthesize its encoding gene, then carry out biological expression and obtain Arrive.The encoding gene of the VPS23A albumen in above-mentioned (b) can be by lacking one in the DNA sequence shown in sequence in sequence table 2 The codon of individual or several amino acid residue, and/or carry out the missense mutation of one or several base pairs, and/or at its 5 ' end And/or 3 ' end connect the coded sequence of the label shown in table 1 and obtain.

The gene (VPS23A gene) encoding described VPS23A albumen falls within protection scope of the present invention.

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

(1) coding region as sequence table sequence 2 from 5 ' end 1001-2197 position nucleotide shown in DNA molecular；

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

(3) hybridize to the DNA sequence that (1) or (2) limits under strict conditions and encode the related protein of drought resistance DNA molecular；

(4) there is to the DNA sequence that (1) or (2) or (3) limit more than 90% homology and encode the related egg of drought resistance The DNA molecular of white matter.

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

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

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

(c1) regulate and control plant drought resistance；

(c2) regulate and control the sensitivity to degeneration-resistant hormone ABA signal for the plant.

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 cultivating transgenic plant, is the table of VPS23A gene in suppression purpose plant Reach, obtain the transgenic plant that drought resistance is higher than described purpose plant.

Described " expression of VPS23A gene in suppression purpose plant " is realized by CRISPER-Cas9 gene editing.

For described CRISPER-Cas9 gene editing sgRNA target sequence as shown in the sequence 4 of sequence table.

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

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

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

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

The present invention also protects for suppressing the material of VPS23A gene expression or being used for reducing VPS23A egg in purpose plant The application in cultivating drought-resistant plant of the material of white expression and/or activity or described sgRNA.

Plant described in any of the above is monocotyledon or dicotyledon.Described dicotyledon can be planted for Chinese lime mesh Thing.Described Chinese lime mesh plant can be crucifer.Described crucifer can be Nan Jie race plant.Described Nan Jie race plants Thing can be Arabidopsis plant.Described Arabidopsis plant concretely arabidopsiss, such as Columbia ecotype arabidopsiss.

The invention provides VPS23A albumen and its encoding gene, the expression of VPS23A gene, Ke Yixian in suppression plant Write the drought tolerance improving plant.The present invention has very important significance for crops improvement and cultivation drought-resistant crops, is suitable for In popularization and application.

Brief description

Fig. 1 is mutant vps23a and its testing result of transgenic complementary expression plant pair ABA sensitivity.

Fig. 2 is the structural representation of recombiant plasmid pCAMBIA1300-VPS23A

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

Fig. 4 is mutant vps23a drought resistance testing result.

Fig. 5 is recombinant expression carrier pYAO：The structural representation of hSpCas9-AtVPS23A-sgRNA.

Specific embodiment

Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method, if no special instructions, is conventional method.Test material used in following embodiments, if no special instructions, is certainly Routine biochemistry reagent shop is commercially available.Quantitative test in following examples, is respectively provided with three times and repeats to test, result is made even Average.

Mutant CS878714：ABRC(Arabidopsis Biological Resource Center).

Columbia ecotype arabidopsiss (Arabidopsis thaliana ecotype Columbia, Col-0)： ABRC(Arabidopsis Biological Resource Center).

PCAMBIA1300 carrier：List of references：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.

Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 strain：List of references：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 with Developmental Biology research from Chinese Academy of Sciences's heredity.

AtU6-26-sgRNA-SK plasmid：List of references：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 with Developmental Biology research from Chinese Academy of Sciences's heredity.

pYAO：HSpCas9 plasmid：List of references：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 with Developmental Biology research from Chinese Academy of Sciences's heredity.

Bsa I-HF：NEB company.

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

Table 2 MS medium component

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

By some Arabidopsis Mutants plant ordered from ABRC are carried out with ABA phenotypic screen, find 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 is in the 1st exon of VPS23A gene), all same brother of the sequence beyond VPS23A gene Rival Asia Arabidopsis thaliana ecotype, is named as mutant vps23a.

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

Arabidopsiss source VPS23A gene as shown in the sequence 2 of sequence table, wherein from 5 ' end 1001-2197 position cores Thuja acid is coding region.

Embodiment 2, the physiological feature detection of mutant vps23a

1st, by the seed of mutant vps23a and Columbia ecotype arabidopsiss (Col-0) with 10% bleaching agent solution Carry out surface sterilizing, then with aseptic water washing 3 times.

2nd, the seed after processing step 1 carries out grouping experiment：

Matched group：Aseptic seed is suspended in aqueous and bed board is on 1/2MS culture medium flat plate, by flat board at 4 DEG C Place 3 days under dark condition, the tissue culture room (16h illumination/8h is dark) then moving into 24 DEG C is cultivated one week, takes pictures and observes The growth conditions of plant.

Experimental group：Aseptic seed is suspended in aqueous and bed board is to the 1/2MS culture medium containing 0.3 or 0.5 μM of ABA On flat board, flat board is placed 3 days under 4 DEG C of dark conditions, (16h illumination/8 hour are black to then move into 24 DEG C of tissue culture room Secretly) cultivate one week, take pictures and observe the growth conditions of plant.

Result is as shown in Figure 1.Figure 1A takes pictures photo for plant cotyledon.Figure 1B is cotyledon greening ration statisticses result, wherein, 1 is the statistical result of Columbia ecotype arabidopsiss (Col-0), and 2 is the statistical result of mutant vps23a.Fig. 1 C is plant Root growth situation is taken pictures photo.Fig. 1 D is the long statistical result of root, and wherein, 1 is the system of Columbia ecotype arabidopsiss (Col-0) Meter result, 2 is the statistical result of mutant vps23a.

Cotyledon greening aspect on the ground, in matched group (1/2MS, 0 μM of ABA) is processed, mutant vps23a and brother's rival The growth conditions of sub- Arabidopsis thaliana ecotype (Col-0) are not significantly different from.In experimental group is processed, intend with Columbia ecotype Southern mustard (Col-0) is compared, and in mutant vps23a, the number of cotyledon greening is less.On the flat board containing 0.3 μM of ABA, The cotyledon greening of 6.7% mutant vps23a, and the cotyledon of 80% Colombia's Arabidopsis thaliana ecotype (Col-0) stretches simultaneously Greening；Under 0.5 μM of ABA is processed, all non-greening of cotyledon of mutant vps23a, and 64.4% Columbia ecotype is intended The cotyledon greening of southern mustard (Col-0).

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

Above experimental analysiss explanation, mutant vps23a is more sensitive for the response of ABA, in mutant vps23a The intensity of activation of ABA signal is more than Columbia ecotype arabidopsiss (Col-0).

Embodiment 3, transgenic complementary expression plant

First, the acquisition of transgenic complementary expression plant

1st, extract the total serum IgE of Columbia ecotype arabidopsiss (Col-0) and reverse transcription is cDNA, with cDNA as template, Performing PCR amplification is entered using the primer pair of VPS23A-FW and VPS23A-Rev composition, reclaims 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)

2nd, use restricted enzyme Kpn I and BamH I enzyme action pCAMBIA1300 carrier, reclaim the carrier of about 10000bp Skeleton.

3rd, the carrier framework that the pcr amplification product obtaining step 1 and step 2 obtain connects, and obtains recombiant plasmid pCAMBIA1300-AtVPS23A.According to sequencing result, structure description is carried out to recombiant plasmid pCAMBIA1300-AtVPS23A As follows：Small fragment between KpnI the and BamHI restriction enzyme site of pCAMBIA1300 carrier is replaced for sequence 2 institute of sequence table The double chain DNA molecule showing.The structural representation of recombiant plasmid pCAMBIA1300-AtVPS23A is shown in Fig. 2.

4th, recombiant plasmid pCAMBIA1300-AtVPS23A is imported in Agrobacterium tumefaciens EHA105 strain, obtain restructuring soil Earth bacillus.

5th, the restructuring edaphic bacilluss that step 4 obtains are passed through 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, results seed (seed in T0 generation, The plant that this seed grows up to is T1 for plant).

6th, the seed that step 5 harvests is placed on the 1/2MS culture medium flat plate containing 20 μ g/mL hygromycin, by flat board first 4 Place 2-4 days under DEG C dark condition, then move into 24 DEG C of tissue culture room (16h illumination/8h is dark) culture 1 week, Ran Houyi The greenhouse entering 24 DEG C is cultivated and is harvested seed (T₁The seed in generation, the plant that this seed grows up to is T₂For plant).

7th, the seed that step 6 harvests is placed on the 1/2MS culture medium flat plate containing 50 μ g/mL kanamycin and carries out resistance sieve Choosing, and harvest the T that the transfer-gen plant of single copy obtains₂(plant that this seed grows up to is T to seed for resistant plant₃Dai Zhi Strain).If a certain T₁The T obtaining for plant₂In plant, the quantity ratio about 3: 1 of resistant plant and non-resistance plant, illustrates this T₁ The transfer-gen plant being single copy for plant.

8th, the seed that step 7 harvests is placed on the 1/2MS culture medium flat plate containing 50 μ g/mL kanamycin and carries out resistance sieve Choosing.If a certain T₂The T obtaining for plant₃It is resistant plant for plant, this T is described₂Plant for the transgenic of homozygosis for plant Strain, this T₂It is homozygosis complementary expression strain for plant and its offspring.

9th, two (3.3 and 4.6) are chosen from the homozygosis that step 8 obtains complementary expression strain.Take Columbia ecotype Arabidopsiss (Col-0), mutant vps23a, the T of 3.3 strains₃For the 2 week old seedling of plant and the T of 4.6 strains₃For plant 2 Week old seedling, extracting total serum IgE reverse transcription is cDNA, with cDNA for the table of VPS23A gene in the plant to each strain for the template The amount of reaching carries out real-time fluorescence quantitative PCR detection, using Actin2 gene as reference gene, with Columbia ecotype arabidopsiss The expression of the expression of middle VPS23A gene, as 1, calculates the relative expression quantity of AtVPS23A gene in each strain other, Carry out three times repeating to test, average.

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’.

Result is shown in Fig. 3.Result shows, the expression water of VPS23A gene in homozygosis complementary expression strain (3.3 and 4.6) plant The flat expression beyond VPS23A gene in Columbia ecotype arabidopsiss, is not detected by mutant vps23a The expression of VPS23A gene.

2nd, the phenotype analytical of transgenic complementary expression plant

Method detection homozygosis complementary expression strain (3.3 and 4.6) T according to embodiment 2₃Phenotype for plant.

Result is as shown in Figure 1.Figure lA takes pictures photo for plant cotyledon.Figure 1B is cotyledon greening ration statisticses result, wherein, 3 is the statistical result of homozygosis complementary expression strain 3.3 plant, and 2 is the statistical result of homozygosis complementary expression strain 4.6 plant.Figure 1C takes pictures photo for plant-root growth situation.Fig. 1 D is the long statistical result of root, and wherein, 1 is homozygosis complementary expression strain 3.3 plant Statistical result, 2 is the statistical result of homozygosis complementary expression strain 4.6 plant.

Result shows, homozygosis complementary expression strain (3.3 and 4.6) T₃Phenotype and Columbia ecotype for plant intend south Mustard is identical, and they are in cotyledon greening and the long-living long side of root all phenotypes of energy complemented mutant body vps23a.VPS23A gene is described Disappearance be the reason lead to mutant vps23a phenotype.VPS23A gene can regulate and control the response to ABA signal for the plant.Mutation Body vps23a can be used to carry out the arid phenotype experiment of next step.

Embodiment 4, mutant vps23a Identification of Drought

2nd, aseptic seed is suspended in aqueous and bed board is on 1/2MS plate, 4 DEG C of vernalization 3 days, then 21 DEG C of cultures 11 My god, then it is transplanted to continuation culture 14 days about in soil, plant 5 in each hole.

3rd, treat that plant growing is in non-bolting period, whole leaf dish is cut off, so that pore on each blade is independently placed, with When do not rewater, carry out drought tolerance test.Osmotic treatment carried out rehydration after 21 days.

Plant phenotype observed result is as shown in Figure 4 A.Mutant vps23a and Columbia ecotype arabidopsiss (Col-0) Growth conditions be not significantly different from when there is no drought stress (according to normal growth described in step 24 weeks).In rehydration the previous day With rehydration one day after, the growth conditions of mutant vps23a plant are significantly better than Columbia ecotype arabidopsiss.One after rehydration My god, count the survival rate of each plant, find that the survival rate of mutant vps23a plant is significantly higher than Columbia ecotype and intends south Mustard (Col-0) (Fig. 4 B).

4th, take the open and flat lotus throne leaf of each plant of step 2, blade lower epidermis of tearing, be immersed in pore buffer (10mM KCl, 50 μM of CaCl₂, 10mM MES-Tris, pH 6.15) in, it is placed in high light (90 μm of ol.m^-2.s^-1) lower 3 hours；By leaf Piece shifts to process 3-5h in the pore buffer of the ABA containing 0 μM or 10 μM；Blade lower epidermis are laid on microscope slide, use Brush pen brushes off mesophyll cell, covered, and in Imager.A1 basis of microscopic observation pore, Axio software is taken pictures and counted gas Hole length-width ratio.

Statistical result is as shown in Figure 4 C.In the case of not having ABA, mutant vps23a and Columbia ecotype intend south The stomatal aperture of mustard (Col-0) is not clearly distinguished from, and under 10 μM of ABA are processed, the stomatal aperture of mutant vps23a is substantially little In Columbia ecotype arabidopsiss (Col-0).Stomatal aperture is little effectively can to reduce loss of moist, and this is mutant vps23a The major reason of drought resisting.

Result above shows, the disappearance of VPS23A gene can strengthen the sensitivity to degeneration-resistant hormone ABA signal for the plant, makes The rising of mutant plant degeneration-resistant hormone ABA level in quick response body under drought condition, closes Stoma of Leaves, reduces into one The transpiration of step, the very strong drought-resistant ability of final imparting plant.

Embodiment 5, the foundation of VPS23A afunction transgenic arabidopsis strain and identification

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

2nd, synthetic primer VPS23A-FW2 and primer VPS23A-Rev2.

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

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

3rd, the primer VPS23A-FW2 of 2-in-1 for step one-tenth and primer VPS23A-Rev2 is annealed, obtained double-stranded DNA piece Section (interference fragment).

4th, the AtU6-26-sgRNA-SK carrier that the double chain DNA fragment (interference fragment) obtaining step 3 and step 1 obtain Connect, obtain connection product.

5th, by the connection product of step 4 Nhe I and Spe I restriction enzymes double zyme cutting, obtain purpose fragment.

6th, adopt Spe I restricted enzyme to pYAO：HSpCas9 plasmid carries out enzyme action, obtains about 12,000bp's pYAO：HSpCas9 carrier.

7th, the pYAO that the purpose fragment obtaining step 5 and step 6 obtain：HSpCas9 carrier connects, and obtains recombinant 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：By pYAO：Insert shown in sequence 3 between the Spe I restriction enzyme site of hSpCas9 plasmid DNA molecular.Recombinant expression carrier pYAO：The structural representation of hSpCas9-VPS23A-sgRNA is shown in Fig. 5.

8th, the recombinant expression carrier pYAO that step 7 is obtained：HSpCas9-VPS23A-sgRNA imports Agrobacterium tumefaciens In EHA105 strain, obtain edaphic bacilluss of recombinating.

9th, the restructuring edaphic bacilluss that step 8 obtains are passed through 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 arabidopsiss (Col-0), harvests (seed in T0 generation, the plant that this seed grows up to is T to seed₁For plant).

10th, the seed that step 9 harvests is placed on the 1/2MS culture medium flat plate containing 20 μ g/mL hygromycin, flat board is first existed Place 2-4 days under 4 DEG C of dark conditions, the tissue culture room (16h illumination/8h is dark) then moving into 24 DEG C is cultivated 1 week, Ran Houyi The greenhouse entering 24 DEG C is cultivated and is harvested seed (T₁The seed in generation, the plant that this seed grows up to is T₂For plant).

11st, the seed that step 10 harvests is placed on the 1/2MS culture medium flat plate containing 50 μ g/mL kanamycin and carries out resistance Screening, and harvest the T that the transfer-gen plant of single copy obtains₂(plant that this seed grows up to is T to seed for resistant plant₃Dai Zhi Strain).If a certain T₁The T obtaining for plant₂In plant, the quantity ratio about 3: 1 of resistant plant and non-resistance plant, illustrates this T₁ The transfer-gen plant being single copy for plant.

12nd, the seed that step 11 harvests is put and carry out resistance on the dry 1/2MS culture medium flat plate containing 50 μ g/mL kanamycin Screening.If a certain T₂The T obtaining for plant₃It is resistant plant for plant, this T is described₂Plant for the transgenic of homozygosis for plant Strain, this T₂It is Transgenic wheat line for plant and its offspring.

13rd, choose one from the Transgenic wheat line that step 12 obtains, detect T₃For VPS23A gene in plant Expression (method with the 9 of embodiment 3 step one).

Through identification, in homozygosis VPS23A afunction transgenic line plant, VPS23A gene is not expressed.

2nd, turn the acquisition of empty carrier strain

1st, enzyme action is carried out to AtU6-26-sgRNA-SK plasmid using Nhe I and Spe I restricted enzyme, obtain purpose Fragment.

2nd, adopt Spe I restricted enzyme to pYAO：HSpCas9 plasmid carries out enzyme action, obtains about 12,000bp's pYAO：HSpCas9 carrier.

3rd, the pYAO that the purpose fragment obtaining step 1 and step 2 obtain：HSpCas9 carrier connects, and obtains recombinant expressed Carrier pYAO：HSpCas9-sgRNA (without interference fragment).

4th, by pYAO：HSpCas9-sgRNA carrier replaces recombinant expression carrier pYAO：HSpCas9-VPS23A-sgRNA carries Body, according to 8 operations of step one, obtains turning empty carrier strain.

3rd, Identification of Drought

Plant to be measured is：The T of homozygosis VPS23A afunction transgenic line₃For plant, the T that turns empty carrier strain₃Dai Zhi Strain, mutant vps23a, Columbia ecotype arabidopsiss (Col-0).

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

3rd, treat that plant growing is in non-bolting period, whole leaf dish is cut off, so that pore on each blade is independently placed, with When do not rewater, carry out drought tolerance test.Osmotic treatment carries out rehydration in 21 days about.

Phenotypic Observation result shows, VPS23A afunction transfer-gen plant, turn empty carrier plant, mutant vps23a and The growth conditions of Columbia ecotype arabidopsiss (Col-0) are not having drought stress (according to normal growth described in step 24 weeks) When be not significantly different from.In rehydration the previous day and rehydration one day after, VPS23A afunction transfer-gen plant and mutant The growth conditions of vps23a plant are significantly better than Columbia ecotype arabidopsiss and turn empty carrier plant.After rehydration one day, system Count the survival rate of each plant, find that the survival rate of VPS23A afunction transfer-gen plant is 60%, with mutant vps23a's Survival rate no significant difference, is significantly higher than the survival rate (23%) of Columbia ecotype arabidopsiss.Turn depositing of empty carrier plant Motility rate and the survival rate no significant difference of Columbia ecotype arabidopsiss.

Statistical result shows, in the case of there is no ABA, VPS23A afunction transfer-gen plant, turn empty carrier plant, The stomatal aperture of mutant vps23a and Columbia ecotype arabidopsiss (Col-0) is not clearly distinguished from.At 10 μM of ABA Under reason, the stomatal aperture of VPS23A afunction transfer-gen plant is 0.25, no notable with the stomatal aperture of mutant vps23a The stomatal aperture (0.42) of difference, substantially less than Columbia ecotype arabidopsiss (Col-0).The pore turning empty carrier plant is opened Degree and the stomatal aperture no significant difference of Columbia ecotype arabidopsiss.

Based on the above results, VPS23A afunction transgenic Arabidopsis plants show compared with Columbia ecotype arabidopsiss Go out stronger drought resistance.

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

<120>Drought tolerance in plants associated protein VPS23A and its encoding gene and application

<130> GNCYXMN161964

<160> 5

<210> 1

<211> 398

<212> PRT

<213>Arabidopsiss（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>Arabidopsiss（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. a kind of protein, is following (a) or (b)：

(b) by (a) through the replacement of one or several amino acid residues and/or disappearance and/or interpolation and with plant drought resistance phase The protein derived from sequence 1 closing.

2. the gene of protein described in coding claim 1.

3. gene as claimed in claim 2 it is characterised in that：Described gene is following (1) or (2) or (3) or (4)：

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

(3) hybridize to the DNA sequence that (1) or (2) limits under strict conditions and the related protein DNA of coding drought resistance divides Son；

(4) there is to the DNA sequence that (1) or (2) or (3) limit more than 90% homology and encode the related protein of drought resistance DNA molecular.

4. the recombinant expression carrier containing gene described in Claims 2 or 3, expression cassette, transgenic cell line or recombinant bacterium.

5. the application of protein described in claim 1 or gene described in Claims 2 or 3, for following (c1) and/or (c2)：

(c1) regulate and control plant drought resistance；

6. gene described in Claims 2 or 3 is cultivating the application in drought-resistant plant as target spot.

7. a kind of method cultivating transgenic plant, is the expression of gene described in Claims 2 or 3 in suppression purpose plant, obtains It is higher than the transgenic plant of described purpose plant to drought resistance.

8. a kind of cultivate transgenic plant method, be reduce purpose plant in protein expression amount described in claim 1 and/ Or activity, obtain the transgenic plant that drought resistance is higher than described purpose plant.

9. a kind of special sgRNA for CRISPER-Cas9 gene editing；The sequence of the target sequence of described sgRNA such as sequence table Shown in 4.

10. it is used for suppressing the material of gene expression described in Claims 2 or 3 or being used for reducing claim 1 institute in purpose plant State sgRNA described in the material of protein expression amount and/or activity or claim 9 application in cultivating drought-resistant plant.