CN102229953B

CN102229953B - Application of light harvesting pigment chlorophyll a/b combined protein LHCB4 in plant breeding

Info

Publication number: CN102229953B
Application number: CN2011101488112A
Authority: CN
Inventors: 张大鹏; 徐艳红; 刘蕊; 王小芳; 严璐; 刘志强
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2011-06-03
Filing date: 2011-06-03
Publication date: 2013-12-11
Anticipated expiration: 2031-06-03
Also published as: CN102229953A

Abstract

The invention discloses application of a light harvesting pigment chlorophyll a/b combined protein LHCB4 in plant breeding. The invention provides application of the LHCB4 protein to improving the stress tolerance of plants. An amino acid sequence of the LHCB4 protein is a sequence 1 in a sequence table. The improvement of the stress tolerance is stressed by hormone. The hormone is ABA (Abscisic Acid) and a nucleotide sequence of a coding gene of the LHCB4 protein is a sequence 2 in the sequence table. The improvement of the stress tolerance is shown in aspects of (1) or (2): (1) the coding gene of the LHCB4 protein is introduced into a mutant plant which is expressed and inactivated by the LHCB4 to obtain a recovered plant which is expressed by the LHCB4 protein, wherein the stress tolerance of the plant which is expressed and recovered by the LHCB4 protein is higher than that of the mutant which is expressed and inactivated by the LHCB4 protein; and (2) the stress tolerance of the mutant which is expressed and inactivated by the LHCB4 protein is higher than that of the plant expressed by the LHCB4. According to the invention, experiments prove that the process of adjusting the expression of the LHCB4 by the ABA is carried out by a signal channel involved by ABAR/CHLH (abscisic acid receptor/ magnesium-protoporphyrin IX chelatase H subunit).

Description

Catch the application in plant breeding in conjunction with albumen LHCB4 of photopigment chlorophyll a/b

Technical field

The present invention relates to biological technical field, relate in particular to a kind of photopigment chlorophyll a/b application in plant breeding in conjunction with albumen LHCB4 of catching.

Background technology

Light harvesting chlorophyll a/b is the lipophorin of photosynthetical system II (PSII) complex body in conjunction with albumen (LHCB).LHCB albumen is usually caught the optical antenna complex body with chlorophyll, xenthophylls composition complex body become photosynthetical system.These are caught the optical antenna complex body and absorb luminous energy and transmit excitation energy to PSII photoresponse center to drive photosynthetic electron transport system.The exterior antenna albumen LHCBs of PSII complex body catches the important integral part of light complex, and it is the abundantest membranin of occurring in nature content.LHCBs albumen is by less antenna complex body: LHCB4 (CP29), LHCB5 (CP26) and LHCB6 (CP24) and larger antenna complex body: tripolymer LHCB1, LHCB2 and the LHCB3 of homology and allos.

These chloroplast(id)/thylakoid proteins are by nuclear gene encoding, and it is expressed factors such as mainly being subject to environment and growth and regulates, and comprises mainly being subject to light (Silverthorne and Tobin, 1984; Sun and Tobin, 1990; Peer et al, 1996; Millar and Kay, 1996; Weatherwax et al, 1996; Yang et al, 1998; Humbeck and Krupinska, 2003; Nott et al, 2006; Staneloni et al, 2008; De Montaigu et al, 2010; Pruneda-Paz and Kay, 2010; Thines and Harmon, 2010); Active oxygen (Nott et al, 2006; Staneloni et al, 2008), chloroplast(id) reverse signal (Nott et al, 2006), diel rhythm (Paulsen and Bogorad, 1988; Strayer et al, 2000; Alabadi et al, 2001; Thain et al, 2002; Andronis et al, 2008; Pruneda-Paz et al, 2009; De Montaigu et al, 2010; Pruneda-Paz and Kay, 2010; Thines and Harmon, 2010) and regulation and control (Bartholomew et al, 1991 of bio-hormone dormin (ABA); Chang and Walling, 1991; Weatherwax et al, 1996; Staneloni et al, 2008).Plant is considered to plant to the adjusting of LHCB protein expression and regulates function of chloroplast with one of the important mechanisms of reply adverse circumstance (Nott et al, 2006; De Montaigu et al, 2010; Pruneda-Paz and Kay, 2010; Thines and Harmon, 2010).

ABA is the important biomolecule hormone of coordinate plant growth and growth, especially in (Finkelstein et al, 2002 of playing an important role aspect the plant responding adverse circumstance; Adie et al, 2007).It is believed that in the past the expression of ABA negative regulation LHCB gene, and regulated the expression of LHCB with response high light adverse circumstance (Bartholomew et al, 1991 with the light synergy; Chang andWalling, 1991; Weatherwax et al, 1996; Staneloni et al, 2008).The expression that high light is turned down LHCB may be (Weatherwax et al, 1996) that the change by ABA concentration in plant materials realizes.Yet ABA is not understood fully for the physiological significance of LHCB genetic expression.

ABA signal transduction process has obtained research widely, and people's evaluation has obtained many moietys of participating, and this is comprising being positioned at cytolemma and intracellular ABA acceptor.Plasmalemma protein GCR2 and GTG1/GTG2 are considered to be in the ABA acceptor that cell surface works.One class START albumen PYR/PYL/RCAR is considered to act on intracellular ABA acceptor, directly acts on the upstream of PP2C signal path to regulate the expression of downstream gene.The H subunit (CHLH/ABAR) of having reported magnesium porphyrin IX (Mg-ProtoIX) chelatase in Arabidopis thaliana can, in conjunction with ABA, act on ABA signal transduction process as the ABA acceptor.Recently, reported that again ABAR can suppress the expression of some ABA responsive genes in conjunction with one group of WRKY transcription factor, as ABI5.In fact, thus ABA is considered to plant by diel rhythm and is subject to a key link between ABA regulation and control response drought environment.ABAR/CHLH also plays many effects in vegetable cell: the combination of catalysis magnesium ion and porphyrin IX in the chlorophyll building-up process, and as genome uncoupling protein (GUN5) mediation plastid-core reverse signal transduction process.

ABAR/CHLH is not only the ABA acceptor, also in the chloroplast(id) reverse signal, has the expression that the GUN phenotype participates in regulating the LHCB gene.

Summary of the invention

An object of the present invention is to provide a kind of method of controlling plant trait.

The invention provides the method for controlling plant trait, is the expression that improves LHCB4 protein coding gene in the plant that sets out, and obtains having following 1) or 2) the purpose plant of feature:

1) resistance of reverse is higher than the described plant that sets out;

2) delayed growth is in the described plant that sets out.

It is following A or B that described purpose plant-growth delays in the described plant that sets out:

The sprouting of A, described purpose plant seed is later than the described plant that sets out;

The growth of B, described purpose roots of plants is later than the described plant that sets out;

The sprouting of described purpose plant seed is later than the described plant that sets out and is presented as that the germination rate of described purpose plant seed is lower than the described plant that sets out;

The growth of the root of described purpose plant seed is later than the described plant that sets out and is presented as that described purpose roots of plants length is less than the described plant that sets out;

The described raising expression of LHCB4 encoding gene in plant of setting out is to carry out under hormone is coerced;

Described hormone is specially ABA;

The nucleotides sequence of the encoding gene of described LHCB4 albumen is classified the sequence 1 in sequence table as.

Described resistance of reverse is drought-resistant property;

The described plant that sets out is monocotyledons or dicotyledons, and described monocotyledons is Arabidopis thaliana.

The described raising expression of LHCB4 encoding gene in plant of setting out realizes by import described LHCB4 encoding gene in the plant that sets out.

Described ABA coerces as in Young Plant seedling stage or plant, becoming carry out the length of time;

Described is 0.5-5 μ M in the Young Plant concentration that ABA coerces seedling stage; Describedly in the Young Plant concentration that ABA coerces seedling stage, be specially 0.5 μ M, 1 μ M, 2 μ M, 3 μ M or 5 μ M;

Describedly become concentration that the length of time, ABA coerced lower than 200 μ M plant but be not 0 μ M, be specially 20 μ M, 50 μ M, 100 μ M, 150 μ M, 200 μ M, 300 μ M.

Described drought-resistant property embodies by stomatal aperture and/or percentage of water loss;

Described root growth embodies by main root length.

The expression of described raising LHCB4 encoding gene is to activate the ABAR-WRKY40 signal path by Exogenous ABA to realize.

The ABA acceptor of the ABA activated plant that described activation ABAR-WRKY40 signal path is external source, ABA acceptor and the LHCB4 promotor that is combined with transcription factor WRKY40 are competed in conjunction with transcription factor WRKY40, discharge the promotor of LHCB4, the LHCB4 encoding gene is expressed;

The aminoacid sequence of described ABA acceptor is the sequence 2 in sequence table;

The nucleotides sequence of the encoding gene of described ABA acceptor is classified the sequence 3 in sequence table as;

The aminoacid sequence of described transcription factor WRKY40 is the sequence 4 in sequence table;

The nucleotides sequence of the encoding gene of described transcription factor WRKY40 is classified the sequence 5 in sequence table as;

The nucleotides sequence of described LHCB4 promotor is classified the sequence 6 in sequence table as.

Another object of the present invention is to provide a kind of method of controlling plant trait.

Method provided by the invention, be the expression that reduces LHCB4 protein coding gene in the purpose plant, obtains having following 1) or 2) plant of feature:

1) resistance of reverse is lower than described purpose plant;

2) growth adds faster than described purpose plant.

It is following A or B that the growth of described plant adds faster than the described plant that sets out:

A) sprouting of the seed of described plant is early than described purpose plant;

B) growth of the root of described plant is early than described purpose plant;

The sprouting of the seed of described plant is presented as that early than described purpose plant the germination rate of the seed of described plant is greater than described purpose plant;

The growth of the root of described plant is presented as that early than described purpose plant the root of described plant grows up in described purpose plant;

In described reduction purpose plant, the expression of LHCB4 protein coding gene is to carry out under hormone is coerced;

Described hormone is specially ABA;

The nucleotides sequence of the encoding gene of described LHCB4 albumen is classified the sequence 1 in sequence table as;

Described resistance of reverse is drought-resistant property;

The described plant that sets out is monocotyledons or dicotyledons, and described monocotyledons is Arabidopis thaliana;

Describedly become concentration that the length of time, ABA coerced lower than 200 μ M plant but be not 0 μ M, be specially 20 μ M, 50 μ M, 100 μ M, 150 μ M, 200 μ M, 300 μ M;

Described drought-resistant property specifically embodies by stomatal aperture and/or percentage of water loss;

Described root growth specifically embodies by main root length;

The expression of described reduction LHCB4 encoding gene is to activate the ABAR-WRKY40 signal path by Exogenous ABA to realize;

Described activation ABAR-WRKY40 signal path is specially the ABA acceptor of the ABA activated plant of external source, ABA acceptor and the LHCB4 promotor that is combined with transcription factor WRKY40 are competed in conjunction with transcription factor WRKY40, discharge the promotor of LHCB4, the LHCB4 encoding gene is expressed;

The 3rd purpose of the present invention is to provide a kind of method that improves plant stress tolerance or delay plant-growth.

Method provided by the invention, comprise the steps: with ABA, plant to be coerced, and obtains having the plant of following proterties:

1) the purpose plant stress tolerance is higher than the plant that sets out;

2) delayed growth of purpose plant is in the plant that sets out;

Plant before processing is denoted as to the plant that sets out, the plant after processing is denoted as to the purpose plant.

Describedly plant, become concentration that the length of time, ABA coerced lower than 200 μ M, be specially 20 μ M, 50 μ M, 100 μ M, 150 μ M, 200 μ M, 300 μ M;

Described feature also is embodied in following 1)-3) in any:

1), under ABA coerces, the wild-type plant resistance of reverse is higher than the mutant LHCB4 of LHCB4 protein expression inactivation;

2), under ABA coerces, the germination rate of the seed of wild-type plant is lower than the mutant LHCB4 of LHCB4 protein expression inactivation;

3), under ABA coerces, the root length of wild-type plant is lower than the mutant LHCB4 of LHCB4 protein expression inactivation.

Of the present invention experimental results show that, the expression that ABA regulates LHCB4 is the signal path participated in by ABAR/CHLH, each LHCB family member's normal expression needs the participation of ABA, and in physiological level, the ABA of high density promotes the expression of LHCB, WRKY40 directly acts on the LHCB gene as negative regulator, the signal path that LHCB mediates by ABAR-WRKY40 participates in ABA signal transduction process, a positive regulon in ABA signal transduction process, seed germination, growth of seedling and stomatal movement isophenous that response ABA regulates.

The accompanying drawing explanation

Fig. 1 is that ABA stimulates the mRNA of LHCBs and the expression of albumen

Fig. 2 is that ABA stimulates LHCB4 phenotype aspect seed germination and growth of seedling

Fig. 3 is that ABA stimulates the impact of LHCB4 on stomatal movement and drought stress

Fig. 4 is that ABA stimulates the impact of LHCB4 on germination rate, stomatal movement and drought stress

Fig. 5 is that 35S drives the LHCBs gene to express in the lhcbs mutant, recovers the phenotype of plant

Fig. 6 is that ABA stimulates lower LHCB gene to express fully

Fig. 7 transcribes the sub-WRKY40 of inhibition to be combined with LHCB family member's promotor

Fig. 8 is the expression that WRKY40 suppresses the LHCB gene

The expression that Fig. 9 is LHCB gene in the abar-3 mutant

Molecule and biochemistry detection that Figure 10 is lhcb double-mutant and lhcbcch double-mutant

The expression that Figure 11 is ROS stable state and a series of ABA responsive genes in the lhcb mutant

Figure 12 is that the reduction of LHCB4 gene expression amount makes the wrky40 mutant partly return to wild-type status to the susceptibility of ABA

The molecule that Figure 13 is mutant lhcb1-6 and biochemical identification

Figure 14 is endogenous aba content and dry matter content mensuration in different lhcb mutant

Figure 15 is that wrky40 and wrky40wrky18 mutant do not have the gun phenotype

Figure 16 is the mode chart that LHCBs albumen response physiological level concentration ABA increases

Figure 17 is the expression of LHCB4 in different tissues

Embodiment

The experimental technique used in following embodiment if no special instructions, is ordinary method.

In following embodiment, agents useful for same etc., if no special instructions, all can obtain from commercial channels.

Material used in following embodiment is as follows:

1, T-DNA insertion mutation body comprises that the lhcb1.1 (SALK-134810) of LHCB1.1 gene (At1g29920) is (hereinafter to be referred as lhcb1, LHCB1 gene in the wild-type Arabidopis thaliana is inserted and being knocked out sudden change and obtain by T-DNA, all the other genes do not change), the lhcb2.2 (SALK-005614) of LHCB2.2 gene (At2g05070) is (hereinafter to be referred as lhcb2, LHCB2 gene in the wild-type Arabidopis thaliana is inserted and being knocked out sudden change and obtain by T-DNA, all the other genes do not change), LHCB3 gene (At5g54270, LHCB3 gene in the wild-type Arabidopis thaliana is inserted and being knocked out sudden change and obtain by T-DNA, all the other genes do not have to change) lhcb3 (SALK-036200), the lhcb4.3 (SALK-032779) of LHCB4.3 gene (At2g40100) is (hereinafter to be referred as lhcb4, LHCB4 gene in the wild-type Arabidopis thaliana is inserted and being knocked out sudden change and obtain by T-DNA, all the other genes do not change), lhcb5 (the SALK-139667 of LHCB5 gene (At4g10340), LHCB5 gene in the wild-type Arabidopis thaliana is inserted and being knocked out sudden change and obtain by T-DNA, all the other genes do not change), lhcb6 (the SALK-074622 of LHCB6 gene (At1g15820), LHCB6 gene in the wild-type Arabidopis thaliana is inserted and being knocked out sudden change and obtain by T-DNA, all the other genes do not change), the seed of these mutant is all purchased from ABRC.。

2, purchased from cold spring harbor laboratory, this mutant has a Ds transposon to insert at second exon of WRKY40 (At1g80840) gene to wrky40-1 (ET5883, the environmental background of Ler, hereinafter to be referred as the wrky40 mutant).The wrky40-1 mutant is to be transformed into the Col background by backcrossing from the Ler background.

Wrky18-1 (SALK-093916), purchased from ABRC, is that the T-DNA insertion on first exon of WRKY18 (At4g31800) gene knocks out mutant, before these two mutant, has identified it is two amorphss.

3, two sudden changes

All double-mutants all pass through hybridization and the PCR evaluation obtains: the double-mutant construction process: the petal that a kind of mutant is not bloomed is removed only surplus gynoecium of its stamen with tweezers, take off the stamen in the petal of in another kind of mutant, blooming or not blooming, rub peeling off on remaining pistil stigma, make pollen fully contact column cap, this gynoecium is made marks, obtain the first familiar generation seed after the pod maturation, F1 is sowed again for seed, F2, for growth, identifies and can obtain homozygote through PCR.

4, cch mutant (Col background, the mutant of ABA acceptor) (Shen Y-Y, Wang X-F, Wu F-Q, Du S-Y, Cao Z, Shang Y, Wang X-L, Peng C-C, Yu X-C, Zhu S-Y, Fan R-C, Xu Y-H, Zhang D-P (2006) .The Mg-chelatase H subunit is an abscisic acid receptor.Nature 443:823-826, the public can obtain from Tsing-Hua University.) by doctor J.Chory, presented.

5, ABA synthesis mutant aba2 (CS156:aba2-1, Col background, ABA acceptor) is purchased from ABRC.

Plant growing condition: MS substratum 19-20 ℃ in illumination box, light intensity is 80 μ molm- ²s ^-1, while growing in soil, illumination intensity is 120 μ molm- ²s ^-1, 16h illumination.

6, the acquisition of the complementary Arabidopis thaliana of lhcbs mutant

Extract the genomic dna of the Arabidopis thaliana plant (ABRC:CS60000, hereinafter to be referred as the wild-type Arabidopis thaliana, is denoted as col) that Colombia (Col-0) ecotype is background, with following primer, carry out pcr amplification:

forward?primer：5’-GCTCTAGAATGGCTACCACCACTGCAG-3’(XbaI)

reverse?primer：5’-ACGCGTCGACCTAATTGTTAAAGGTGGCAAGG-3’(SalI)

Obtain the PCR product of 831bp, through order-checking, the nucleotides sequence of this product is classified NM_129568 (having the Nucleotide shown in sequence 1) as.

The PCR product is cut through SmaI and SalI enzyme, obtain enzyme and cut after product, this enzyme is cut to after product and pCAMBIA1300-221 carrier (the Shang Y cut through same enzyme, Yan L, Liu ZQ, Cao Z, Mei C, Xin Q, Wu FQ, Wang XF, Du SY, Jiang T, Zhang XF, Zhao R, Sun HL, Liu R, Yu YT, Zhang D-P (2010) The Mg-chelatase H subunit antagonizes a group of WRKY transcription repressors to relieve ABA-responsive genes of inhibition.Plant Cell 22:1909-1935, the public can obtain from Tsing-Hua University.) connection, obtain transformant, extract the plasmid of transformant, through order-checking, the carrier of this plasmid for obtaining between the SmaI by LHCB4 gene insertion pCAMBIA1300-221 carrier and SalI restriction enzyme site, called after pCAMBIA1300-221-LHCB4.

PCAMBIA1300-221-LHCB4 is proceeded to agrobacterium tumefaciens GV3101 bacterial strain, obtain recombinant bacterium 3, extract plasmid and send to order-checking, the plasmid that result is recombinant bacterium 3 is pCAMBIA1300-221-LHCB4, by recombinant bacterium 3 called after GV3101/pCAMBIA1300-221-LHCB4.

GV3101/pCAMBIA1300-221-LHCB4 is infected to mutant LHCB4 by the petal osmose process, obtain T0 for the complementary Arabidopis thaliana of LHCB4.

T0, for the complementary Arabidopis thaliana sowing of LHCB4, sowing, is continued to go down to posterity, until obtain T3 for the complementary Arabidopis thaliana of LHCB4, be the complementary Arabidopis thaliana of lhcbs mutant, be denoted as lhcb4 ^*.

7, chl-1 is the chlorophyll synthesis mutant, Arabidopis thaliana Biological resources center ABRC, CS3119.

Experiment in following embodiment is three repetitions, and three experiments that are that relate to quantitative test are averaged.

Embodiment 1, under the coercing of ABA, LHCB4 expresses affects plant seed germination, growth of seedling and drought-enduring

One, external source applies the impact of lower concentration ABA (being equivalent to physiological condition high density ABA) on LHCB4 mRNA and protein expression

Carry out the ABA processing in two stages of development of plants respectively: Seedling Stage with become the length of time.

Seedling Stage: common MS substratum growth wild-type Arabidopis thaliana (col) (ABRC, the CS60000) seedling of three days move to containing 0,0.5,1,3,5 or the substratum (ABA+MS substratum) of the ABA of 10uM in regrowth two weeks;

Become the length of time: move to after wild-type Arabidopsis thaliana Seedlings in soil is grown three weeks the ABA solution (the ABA aqueous solution 10ml of different concns) that sprays different concns and grow 5 hours, collect material.

The concrete detection method of mrna expression: the expression of the mRNA level of LHCB4 is carried out to the RT-PCR detection with reference to BioRad Real-Time System CFX96TM C1000 Thermal Cycler (Singapore) instrument specification sheets, the cDNA that template is plant, primer is LHCB4:forward primer:5 '-CAGCCGTACACTGAAGTCTTTGG-3 ' reverse primer:5 '-TTCTATCCATATCAACGTCGTCAAC-3 ', internal reference is β-Actin, the primer of internal reference is forward primer:5 '-GGTAACATTGTGCTCAGTGGTGG-3 ' reverse primer 5 '-AACGACCTTAATCTTCATGCTGC-3 '.

The concrete detection method of LHCB4 protein expression: by the quick-frozen in liquid nitrogen of whole plant, grinding cymbals and the grinding rod good with precooling are clayed into power material in liquid nitrogen, in the 1.5mL centrifuge tube of packing into.The Extraction buffer composition is 50mM Tris-HCl, and pH 7.5,150mMNaCl, and 1mM EDTA, 0.1% (v/v) Triton X-100,10% (v/v) glycerol, and add proteinase inhibitor.Add centrifuge tube according to Extraction buffer and the sample ratio of 4: 1, and put into rapidly the liquid nitrogen quick-frozen.Utilize the cytoclasis ultrasonic apparatus that the mixture supersound process is extremely thawed fully, and then put into the liquid nitrogen quick-frozen.The above-mentioned steps triplicate.By centrifugal 3 minutes of mixture 10000g, remove insolubles and broken cell.Supernatant liquor is transferred to new centrifuge tube stand-by, is LHCB4 albumen.

LHCB4 albumen is carried out to SDS-PAGE and the immune marking, concrete experimental procedure is with reference to paper (the Wu F-Q delivered, XinQ, Cao Z, Liu Z-Q, Du S-Y, Mei C, Zhao C-X, Wang X-F, Shang Y, Jiang T, Zhang X-F, Yan L, Zhao R, Cui Z-N, Liu R, Sun H-L, Yang X-L, Su Z, Zhang D-P (2009) .The Mg-chelatase H subunit binds abscisic acid and functions in abscisic acid signaling:New evidence in Arabidopsis.Plant Physiol 150:1940-1954), the specific antibody of LHCB4 is purchased from Agrisera (Stockholm, Sweden, website:www.agrisera.com, product No.AS01045).

Result as shown in Figure 1,

Figure 1A: in seedling: after 0 to 5 μ M ABA processes, the mrna expression level of LHCB4 raises, but 10 μ M ABA process the mrna expression level of rear LHCB4, reduces.By the wild-type seedling of three days, move on on the substratum containing different concns ABA, continued growth sampling in two weeks detects (concrete detection method is shown in that above-mentioned mrna expression detects).Every group of data are all the mean value of three independent biology repetitions.

0 μ M ABA processes, and the mRNA relative expression quantity of LHCB4 is 0.80;

0.5 μ M ABA processes, the mRNA relative expression quantity of LHCB4 is 1.02;

1 μ M ABA processes, and the mRNA relative expression quantity of LHCB4 is 0.98;

5 μ M ABA process, and the mRNA relative expression quantity of LHCB4 is 1.04;

3 μ M ABA process, and the mRNA relative expression quantity of LHCB4 is 1.10;

5 μ M ABA process, and the mRNA relative expression quantity of LHCB4 is 0.70;

10 μ M ABA process, and the mRNA relative expression quantity of LHCB4 is 0.16;

Figure 1B: become in the seedling in age: process lower than 200 μ M ABA the mrna expression level that makes LHCB4 and raise, reduce but process higher than 200 μ M ABA the mrna expression level that makes LHCB4.The plant that ABA solution sprays three week age samples detection (concrete detection method is shown in that above-mentioned mrna expression detects) after 5 hours.Every group of data are all the mean value of three independent biology repetitions.

0 μ M ABA processes, and the mRNA relative expression quantity of LHCB4 is 0.56;

20 μ M ABA process, and the mRNA relative expression quantity of LHCB4 is 0.80;

50 μ M ABA process, and the mRNA relative expression quantity of LHCB4 is 1.48;

100 μ M ABA process, and the mRNA relative expression quantity of LHCB4 is 1.20;

150 μ M ABA process, and the mRNA relative expression quantity of LHCB4 is 1.20;

200 μ M ABA process, and the mRNA relative expression quantity of LHCB4 is 1.25;

300 μ M ABA process, and the mRNA relative expression quantity of LHCB4 is 0.50.

Fig. 1 C: in seedling: after 0 to 5 μ M ABA processes, the protein expression level of LHCB4 raises, but 10 μ M ABA process the protein expression level of rear LHCB4, reduces.Material processing is with (A).Actin is the loading contrast, tests and repeats to obtain similar result for three times.

Detection method specifically is shown in that the LHCB protein expression detects.

Fig. 1 D: become in the seedling in age: process lower than 200 μ M ABA the protein expression level that makes LHCBs (LHCB1～6) and raise, reduce but process higher than 200 μ M ABA the protein expression level that makes LHCBs (LHCB1～6).Material processing is with (B).Actin is the loading contrast, tests and repeats to obtain similar result for three times.

As can be seen from the above: at Seedling Stage, the ABA concentration of 0.5 to 5 μ M promotes the mrna expression of LHCB4, expression amount decline (Figure 1A) during 10 μ M.Becoming the length of time, lower than the ABA concentration of 200 μ M, processing the mrna expression that Arabidopis thaliana promotes LHCB4, during higher than 200 μ M, the mrna expression amount of LHCB4 descends, and the ABA of 50 μ M is the optimal concentration (Figure 1B) that promotes that LHCB4 expresses.

The further impact of research ABA on the LHCB protein expression.At Seedling Stage, response and mRNA level that the LHCB protein expression is processed for ABA are in full accord, and expression amount reaches maximum (Fig. 1 C) when 1 to 3 μ M processes.Becoming the length of time, from 20 to 300 μ M all promote the expression of LHCB albumen, and along with the increase of ABA concentration, expression amount increases (Fig. 1 D), this expression from mRNA different (Figure 1B).

Experimental result shows, the expression of LHCB is subject to different adjustings transcribing from translation skill.

In a word, above data basic explanation, the lower concentration that external source applies, be equivalent to high density ABA under physiological condition, promotes rather than suppress the expression of LHCB.

Two, improve germination rate and the long variation of root of the detection of expression seed of LHCB4 encoding gene in the plant that sets out

1, seed germination experiment

Divide three parts to broadcast at MS substratum (Sigma, St.Louis, MO, USA approximately 100 seed disinfections for the treatment of measuring plants; Full-strength MS).Substratum contain 3% sucrose and 0.8% agar powder (pH 5.9) and contain 0,0.5, the ABA of 1uM.Seed vernalization 3 days under 4 ℃ of conditions, then be placed under 20 ℃ of illumination conditions and grow, and at the time interocclusal record indicated, sprouts the data of (appearance of root).

Above-mentionedly treat that measuring plants is the lhcb4 mutant, wild-type Arabidopis thaliana col, (the double-mutant construction process: the petal that lhcb4 is not bloomed is removed only surplus gynoecium of its stamen with tweezers to double-mutant lhcb4lhcb6, take off in lhcb6 the stamen in the petal of blooming or not blooming, on the lhcb4 pistil stigma, rub, make pollen fully contact column cap, this gynoecium is made marks, obtain the first familiar generation seed after the pod maturation, F1 is sowed again for seed, F2 is for growth, identify and can obtain homozygote through PCR), the cch mutant, ch1-1 (ABRC, CS3119), (the double-mutant construction process: the petal that lhcb4 is not bloomed is removed only surplus gynoecium of its stamen with tweezers to double-mutant lhcb4cch, take off in cch the stamen in the petal of blooming or not blooming, on the lhcb4 pistil stigma, rub, make pollen fully contact column cap, this gynoecium is made marks, obtain the first familiar generation seed after the pod maturation, F1 is sowed again for seed, F2 is for growth, identify and can obtain homozygote through PCR).

Each numerical value is the mean value of three independent biology repetitions.

Result as shown in Fig. 2 A-2C and Fig. 4 A-4C,

As can be seen from Figure 2A, lhcb4 is in the substratum of the ABA of 0 μ M, and 24,36,48,60,72 germination rate is respectively 25%, 90%, 100%, 100%, 100%; In the substratum of the ABA of 0.5 μ M, 24,36,48,60, the germination rate of 72h is respectively 15%, 66%, 90%, 100%, 100%; In the substratum of the ABA of 1 μ M, 24,36,48,60, the germination rate of 72h is respectively 10%, 62%, 80%, 90%, 100%;

Ch1-1 is in the substratum of the ABA of 0 μ M, and 24,36,48,60,72 germination rate is respectively 10%, 80%, 98%, 100%, 100%; In the substratum of the ABA of 0.5 μ M, 24,36,48,60,72 germination rate is respectively in the substratum of 1%, 30%, 72%, 90%, 100% ABA at 1 μ M, and 24,36,48,60,72 germination rate is respectively 0%, 20%, 43%, 75%, 84%;

Col in the substratum of the ABA of 0 μ M, 24,36,48,60, the germination rate of 72h is respectively 10%, 78%, 98%, 100%, 100%; In the substratum of the ABA of 0.5 μ M, 24,36,48,60, the germination rate of 72h is respectively in the substratum of 0%, 22%, 70%, 86%, 100% ABA at 1 μ M, 24,36,48,60, the germination rate of 72h is respectively 0%, 17%, 35%, 70%, 83%;

As can be seen from Figure 2B,

Double-mutant lhcb4 lhcb6 in the substratum of the ABA of 0 μ M, 24,36,48,60, the germination rate of 72h is respectively 10%, 90%, 100%, 100%, 100%; In the substratum of the ABA of 0.5 μ M, 24,36,48,60, the germination rate of 72h is respectively in the substratum of 8%, 50%, 78%, 92%, 100% ABA at 1 μ M, 24,36,48,60, the germination rate of 72h is respectively 5%, 38%, 56%, 82%, 90%;

Col in the substratum of the ABA of 0 μ M, 24,36,48,60, the germination rate of 72h is respectively 8%, 80%, 96%, 100%, 100%; In the substratum of the ABA of 0.5 μ M, 24,36,48,60, the germination rate of 72h is respectively in the substratum of 2%, 22%, 65%, 82%, 100% ABA at 1 μ M, 24,36,48,60, the germination rate of 72h is respectively 0%, 10%, 30%, 62%, 80%.

From Fig. 2 C, can find out,

Lhcb4 cch

24,36,48,60, the germination rate of 72h is respectively 14%, 92%, 97%, 100%, 100%,

Wild-type col is in the substratum of the ABA of 1 μ M, and 24,36,48,60,72 germination rate is respectively 10%, 78%, 88%, 100%, 100%,

Cch in the substratum of the ABA of 1 μ M, 24,36,48,60, the germination rate of 72h is respectively 19%, 90%, 98%, 100%, 100%;

Above description of test: under the stimulation of ABA, turn down LHCB4 gene expression amount (lhcb4), germination rate improves on the contrary, improve LHCB4 gene expression amount (col), germination rate reduces, and can find out, turns down the LHCB4 gene expression amount and has reduced the responding ability of seed germination to ABA.

From Fig. 4 A-4C, also can find out, the germination rate of mutant LHCB2 is higher than wild-type col.

From Fig. 4 D, can find out, mutant desensitizes for ABA aspect growth of seedling, and the double-mutant of LHCB family desensitizes for ABA aspect growth of seedling, and the double-mutant of mutant and cch desensitizes for ABA aspect growth of seedling.

Above description of test: the 1.lhcb4 mutant shows desensitization to ABA aspect seed germination; 2.lhcb4 the cch double-mutant is to ABA performance desensitization aspect seed germination, the degree of desensitization, lower than lhcb or cch single mutant, illustrates the downstream of the ABA signal transduction process that LHCB4 participates at ABAR.

2, the long experiment of root

Plant seed to be measured is directly broadcast in the MS of the ABA that contains 1uM substratum and is sowed, and 12 (E, the F) that grow after vernalization take pictures, and to observe the statistics root long, with main root length, adds up.

Treat that measuring plants is single mutant lhcb4, wild-type Arabidopis thaliana col, cch single mutant, double-mutant lhcb4 cch.Each numerical value is the average of three independent biology repetitions.

Result as shown in Fig. 2 E-2F,

From 2E, can find out, the root of lhcb4 is long is 5.8mm;

The root of Col is long is 2.0mm;

From Fig. 2 F, can find out,

Lhcb4 cch double-mutant is long at the seedling root containing on the ABA substratum of 1 μ M is 5.9mm;

The cch single mutant is long at the seedling root containing on the ABA substratum of 1 μ M is 6.3mm;

Wild-type col is long at the seedling root containing on the ABA substratum of 1 μ M is 2.0mm;

Above above-mentioned experiment can be found out, all lhcb2 mutant are showing the phenotype that ABA is desensitized aspect seed germination and growth of seedling: ABA suppresses seed germination, and ABA suppresses growth of seedling.

Three, improve the impact of the expression of LHCB4 encoding gene in the plant that sets out on resistance of reverse

1, stomatal movement experiment:

The stomatal closure system that ABA induces: the growth plant leaf to be measured of 3 weeks in soil is immersed in the damping fluid that contains 50mM KCl and 10mMMes-Tris (pH 6.15), with 200 μ molm- ²s ^-1intensity is placed 2.5h under cold light source, then adds (±)-ABA of different concns.After ABA processes 2.5h, tear and get the epidermis bar, the measurement and record of stomatal aperture.

ABA suppresses the stomatal opening system: the growth plant leaf to be measured of 3 weeks in soil is immersed in identical damping fluid, and dark processing 2.5h then adds ABA to move under cold light source and irradiates 2h, the measurement and record of stomatal aperture in damping fluid.

Treat that measuring plants is col, ch1-1 (ABRC, CS3119), cch, lhcb4 mutant.

As shown in Figure 3A, wherein induce stomatal closure is upper figure to result, and the inhibition stomatal opening is figure below.Each numerical value is the average of five independent biology repetitions.The aperture of each 60 pores of duplicate record.

From Fig. 3 A, can find out,

Col 0uMABA process 0h, 0uMABA process 2.5h, 20uMABA process 2.5h, 30uMABA process 2.5h induce stomatal closure the time pore aperture be 5.0 μ M, 5.1 μ M, 2.8 μ M, 0.3 μ M;

When Col processes the inhibition stomatal opening of 0h, 0uMABA processing 2.5h, 20uMABA processing 2.5h, 30uMABA processing 2.5h at 0uMABA, the aperture of pore is 1.4 μ M, 4.4 μ M, 2.2 μ M, 0.2 μ M;

Ch1-1 0uMABA process 0h, 0uMABA process 2.5h, 20uMABA process 2.5h, 30uMABA process 2.5h induce stomatal closure the time pore aperture be 5.3 μ M, 5.0 μ M, 2.6 μ M, 0.7 μ M;

When ch1-1 processes the inhibition stomatal opening of 0h, 0uMABA processing 2.5h, 20uMABA processing 2.5h, 30uMABA processing 2.5h at 0uMABA, the aperture of pore is 1.1 μ M, 5.4 μ M, 2.6 μ M, 0.9 μ M;

Cch 0uMABA process 0h, 0uMABA process 2.5h, 20uMABA process 2.5h, 30uMABA process 2.5h induce stomatal closure the time pore aperture be 6.2 μ M, 6.3 μ M, 5.8 μ M, 2.6 μ M;

When cch processes the inhibition stomatal opening of 0h, 0uMABA processing 2.5h, 20uMABA processing 2.5h, 30uMABA processing 2.5h at 0uMABA, the aperture of pore is 1.5 μ M, 6.7 μ M, 5.5 μ M, 2.3 μ M;

The lhcb4 mutant 0uMABA process 0h, 0uMABA process 2.5h, 20uMABA process 2.5h, 30uMABA process 2.5h induce stomatal closure the time pore aperture be 5.5 μ M, 5.4 μ M, 5.0 μ M, 3.8uM;

When the lhcb4 mutant is processed the inhibition stomatal opening of 0h, 0uMABA processing 2.5h, 20uMABA processing 2.5h, 30uMABA processing 2.5h at 0uMABA, the aperture of pore is 1.8 μ M, 5.2 μ M, 4.5 μ M, 4.0uM;

Can find out, the stomatal closure of inducing at ABA and suppressing aspect stomatal opening, the lhcb4 mutant shows the desensitization phenotype to ABA: ABA induces down, crosses expression lhcb4 (col) and compares with suppressing lhcb4 (lhcb4), and stomatal aperture becomes greatly.

2, arid experiment and percentage of water loss experiment

To until measuring plants after growth substratum immigration soil, not water and carry out drought stress (arid is rehydration after 21 days, after 2 days, adds up percentage of water loss), take pictures.Normally to water as contrast.By wild-type and different lhcb mutant, each sample is got 5 strain blades and is tested and (choose the fully-developed blade on the bolting plant not in surrounding age, be placed on pan paper, weigh once every 1h, calculate the blade percentage of water loss at every turn.Formula should be: (0h weight-1h weight)/0h weight)) rate-of-loss of coolant of excised leaf.

Treat that measuring plants is wild-type Arabidopis thaliana Col, mutant lhcb6.

Result is as Fig. 3 C-3D, and normal growth wild-type Arabidopis thaliana Col and mutant lhcb2 be without significant difference (3C), after drought stress wild-type Arabidopis thaliana Col growth better, mutant lhcb2 substantially wither (3D).

The result of percentage of water loss, as shown in Fig. 4 G, can find out,

Lhcb4 is respectively 18%, 30%, 46%, 55%, 70% in the percentage of water loss of

ABA processing

1,2,3,4,5,6h;

Col is respectively 12%, 24%, 34%, 44%, 54% in the percentage of water loss of

ABA processing

1,2,3,4,5,6h.

Research finds that the excised leaf of lhcb mutant is under the dry condition more with the equal time dehydration.This may be because these mutant are desensitizing and causing ABA aspect stomatal movement.Simultaneously, find that the lhcb mutant is normally to water under water condition growth conditions consistent with wild-type, and, under drought condition, the water retention capacity of mutant is lower than wild-type.

4, the complementary Arabidopis thaliana of lhcbs mutant is in seed germination, growth of seedling and drought-enduring research

1) seed germination, root are long

Lhcb4* represents the complementary strain (preparation method is as implied above) of LHCB4 gene.

By lbcb4* and col on the ABA substratum of 1 μ M after vernalization after 48 hours, the statistics seed germination rate.

By lbcb4* and col, on the ABA substratum of 1 μ M after vernalization after 12 days, the statistics root is long.

Result as shown in Figure 5A, col, lbcb4* on the ABA substratum of 1 μ M after vernalization the germination rate of 48 hours be respectively 32%, 24%, can find out that lhcb4 mutant phenotype to ABA desensitization aspect seed germination causes because the LHCB4 gene expression amount reduces.

Col, lbcb4* on the ABA substratum of 1 μ M after vernalization the root of 12 days long (main root length) be respectively 2.5mm, 1.8mm, can find out that lhcb4 mutant phenotype to the ABA desensitization aspect growth of seedling causes because the LHCB4 gene expression amount reduces.

2) drought-enduring (stomatal aperture)

Lbcb4* and col are induced according to the method described above to stomatal closure and suppress the stomatal opening experiment, and at the initial stomatal aperture of 0 μ M ABA 0 hour record, 0 μ M or 20 μ M ABA process after 2.5 hours measurement and record of stomatal aperture again.Each numerical value is the average of five independent biology repetitions.The aperture of each 60 pores of duplicate record.The identical expression of letter (in P<0.05 level of difference) does not have significant difference, and letter is different means that there were significant differences.

Result is as shown in 5B, and it is upper figure that ABA induces stomatal closure, and the inhibition stomatal opening is figure below,

The stomatal aperture of inducing stomatal closure that col and lbcb4* process latter 0 hour on the ABA substratum of 0 μ M is respectively 6.0 μ m, 5.6 μ m, can find out that in this experiment, wild-type is identical with the transgenic line original state.

The stomatal aperture that col and lbcb4* process the inhibition stomatal opening of latter 0 hour on the ABA substratum of 0 μ M is respectively 1.0 μ m, 0.9 μ m, can find out that in this experiment, wild-type is identical with the transgenic line original state.

The stomatal aperture of inducing stomatal closure that col and lbcb4* process latter 2.5 hours on the ABA substratum of 0 μ M is respectively 5.1 μ m, 5.1 μ m, can find out in experiment, usings water as negative control treatment wild-type and transgenic line, and its phenotype is consistent.。

The stomatal aperture that col and lbcb4* process the inhibition stomatal opening of latter 2.5 hours on the ABA substratum of 0 μ M is respectively 5.5 μ m, 6.0 μ m, can find out in this experiment, usings water as negative control treatment wild-type and transgenic line, and its phenotype is consistent.

The stomatal aperture of inducing stomatal closure that col and lbcb4* process latter 2.5 hours on the ABA substratum of 20 μ M is respectively 3.2 μ m, 2.8 μ m, can find out after ABA processes, transgenic line recovers lhcb4 mutant phenotype to the ABA desensitization aspect stomatal movement substantially, illustrates that the lhcb4 mutant causes because the LHCB4 gene expression amount reduces the phenotype of ABA desensitization.

The stomatal aperture that col and lbcb4* process the inhibition stomatal opening of latter 2.5 hours on the ABA substratum of 20 μ M is respectively 2.4 μ m, 2.2 μ m, can find out after ABA processes, transgenic line recovers lhcb4 mutant phenotype to the ABA desensitization aspect stomatal movement substantially, illustrates that the lhcb4 mutant causes because the LHCB4 gene expression amount reduces the phenotype of ABA desensitization.

The expression of embodiment 2, LHCB4 gene is regulated by the ABAR-WRKY40 signal path

One, the normal expression of LHCB4 gene needs the participation of ABA

Whether be necessary in order to detect ABA to the expression of LHCB4, utilize ABA defect mutant aba2 to be tested.

The detection of expression method of the mRNA of LHCB4: the aba2 seedling of three days moves on on the substratum that does not contain or contain (1-60 μ M) ABA, and continued growth is sampling after two weeks, carries out quantitative fluorescent PCR;

LHCB4:forward primer:5 '-CAGCCGTACACTGAAGTCTTTGG-3 ' reverse primer:5 '-TTCTATCCATATCAACGTCGTCAAC-3 ', the primer that internal reference is β-Actin, internal reference is forward primer:5 '-GGTAACATTGTGCTCAGTGGTGG-3 ' reverse primer 5 '-AACGACCTTAATCTTCATGCTGC-3 ';

Detect the expression level of LHCB4, take wild-type Arabidopis thaliana (col) as contrast.Every group of data are all the mean value that three times independently biology repeats.

Result as shown in Figure 6A, can find out, in ABA synthesis mutant aba2, (aba2-ABA) turned down in the expression of LHCB4, after ABA processes, can make the expression of the LHCB4 in ABA synthesis mutant aba2 be restored (aba2+ABA), but occur restraining effect during higher than 40 μ M when ABA concentration.

The detection method of LHCB4 protein expression: the aba2 seedling of three days moves on to and contains (0,1,3,5,10,20,40, and60 μ M) on the substratum of ABA, continued growth is sampling after two weeks, extract total protein, (antibody is that specific antibody is purchased from Agrisera (Stockholm, Sweden to carry out Western blot detection; Website:www.agrisera.com; Product No.AS01045), as shown in Figure 6B, Western blot band concentration means the albumen relative expression quantity to result.Numerical value below band represents the relative level of protein expression.Setting LHCB protein content in Col is 100, and in mutant, the LHCB protein content is quantized by comparison.Actin is the loading contrast, tests and repeats to obtain similar result for three times.

As can be seen from the above, the mRNA of LHCB4 and expressing quantity in the aba2 mutant all lower than wild-type.The processing of ABA can recover the expression level of each LHCB member in the aba2 mutant, but the ABA of higher concentration processes, (>20or>40 μ M process LHCBmRNAs;>20 μ M process the LHCB albumen except LHCB5 albumen:>40 μ M) mRNA and the expression of protein level in mutant all start descend (Fig. 6 A, 6B).These experimental results show that LHCB4 is transcribing the participation that needs ABA with the normal expression of translation skill.

It should be noted that in the aba2 mutant, LHCB4 for the response of ABA at protein level and mRNA level (Fig. 6 A, 6B) in full accord.Yet, in the aba2 mutant, the maximum concentration that stimulates LHCB to express is three times in the wild-type experimental system.

Three, the WRKY40 transcription factor is combined with LHCB family member's promotor and is suppressed its expression

Attempting a key that research directly acts on the ABAR downstream transcribes and suppresses son---and whether the WRKY40 transcription factor can directly be combined with the promoter region of LHCB and regulate the expression of LHCB.

1, by PCR and the real-time fluorescence quantitative PCR of the experiment of chromatin co-immunoprecipitation

Specifically experiment is shown in shown in following Fig. 7 A-7D,

The structure that Fig. 7 A is the LHCB2 gene promoter: Wn (W1, W2 ...) represent the order of W-box from left to right and they position with respect to initiator codon (ATG).Red line represents that CHIP analyzes the sequence fragment detected in (Fig. 7 B).Arrow represents sequence fragment used in gel retardation assay, and same fragment means with two arrows of same color, p1, p2 ... represent the number of institute's selected episode.

The promotor that Fig. 7 B is WRKY40 and LHCB2 gene is done mutually:

Concrete grammar: with the N terminal specific antibody of WRKY40 (the N end of WRKY40 be the 21-131 position Nucleotide of genbank CP002684, the nucleotides sequence of WRKY40 is classified the sequence 5 of sequence table as, aminoacid sequence is the sequence 4 in sequence table) PCR electrophoresis detection result (the Saleh A that carries out the CHIP analysis, Alvarez-Venegas R, Avramova Z (2008) An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants.Nat Protoc 3:1081-1025, Shang Y, Yan L, Liu ZQ, Cao Z, Mei C, Xin Q, Wu FQ, Wang XF, Du SY, Jiang T, Zhang XF, Zhao R, Sun HL, Liu R, Yu YT, ZhangD-P (2010) The Mg-chelatase H subunit antagonizes a group of WRKY transcription repressors to relieve ABA-responsive genes of inhibition.Plant Cell 22:1909-1935).The seedling in two week age that experiment material is the wild-type Arabidopis thaliana.In order to detect the combination activity of WRKY40 and LHCB promotor, with reference to (Mukhopadhyay A, Deplancke B, Walhout AJM, Tissenbaum HA (2008) .Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans.Nat Protoc 3:698-709; Shang Y, Yan L, Liu ZQ, Cao Z, Mei C, Xin Q, Wu FQ, Wang XF, Du SY, JiangT, Zhang XF, Zhao R, Sun HL, Liu R, Yu YT, Zhang D-P (2010) The Mg-chelatase H subunit antagonizes a group of WRKY transcription repressors to relieve ABA-responsive genes of inhibition.Plant Cell 22:1909-1935) method, 3 ' the non-translational region sequence of Actin2 of take is carried out the real-time quantitative PCR detection as internal reference contrast.

Internal reference primer Actin2:

forward?primer：5’-GGTAACATTGTGCTCAGTGGTGG-3’

reverse?primer：5’-AACGACCTTAATCTTCATGCTGC-3’

The sequence of promoter fragment indicates in (Fig. 7 A).As shown in Figure 7 B, ' Input ' swimming lane: the genomic dna of wild-type Arabidopis thaliana seedling in two week age is template to result, the PCR product of amplification; ' control ' swimming lane: the DNA that preimmune serum (deriving from antibody manufacturing company) obtains is template, the PCR product of amplification; ' LHCB-p ' swimming lane: the DNA that WRKY40N terminal specific antibody precipitation obtains is template, the PCR product of amplification (order-checking is the sequence 6 in sequence table from 5 ' end 5-201 position Nucleotide).

pLHCB4：

forward?primer：5’-CTTTCGGCGTTGACCCATC-3’

reverse?primer：5’-CATTATATTAGTTAGGGCGATTT-3’

Can find out, WRKY40 is combined with the promotor of LHCB1 gene.

The promotor that Fig. 7 C is WRKY40 and LHCB2 gene is done mutually: the quantitative fluorescent PCR that carries out the CHIP analysis with the N terminal specific antibody of WRKY40,

Result is as shown in 7C, and Actin promotor (Actin-p) (take Actin2 3 ' non-translational region sequence carry out the product of real-time quantitative PCR as internal reference contrast) contrasts for negative.

Can find out, the combination rate that the promotor of LHCB4 gene (LHCB4-p) is combined with WRKY40 is 12.0.

Every group of data are all the mean value that three times independently biology repeats.

Show, WRKY40 is combined with the promotor of LHCB gene.

Fig. 8 C is for detecting wild-type Arabidopis thaliana (Col), mutant wrky40, the mrna expression of the LHCB4 of double-mutant wrky40/18 and protein expression: real-time quantitative PCR detects wild-type Arabidopis thaliana (Col), mutant wrky40, the mRNA horizontal expression of the LHCB4 of double-mutant wrky40/18, LHCB4:forward primer:5 '-CAGCCGTACACTGAAGTCTTTGG-3 ' reverseprimer:5 '-TTCTATCCATATCAACGTCGTCAAC-3 ', internal reference is β-Actin, the primer of internal reference is forwardprimer:5 '-GGTAACATTGTGCTCAGTGGTGG-3 ' reverse primer5 '-AACGACCTTAATCTTCATGCTGC-3 '.

Immunoblotting detects the expression of protein level, Actin is that loading contrast Arabidopis thaliana total protein extracting method carries out with reference to the scheme of LHCB antibody supplier Agrisera suggestion, SDS-PAGE and immunoblotting detect with reference to Shang Y, Yan L, LiuZQ, CaoZ, Mei C, Xin Q, Wu FQ, Wang XF, Du SY, Jiang T, Zhang XF, Zhao R, Sun HL, Liu R, Yu YT, Zhang D-P (2010) The Mg-chelatase H subunit antagonizes a group of WRKYtranscription repressors to relieve ABA-responsive genes of inhibition.Plant Cell22:1909-1935.

As shown in Figure 8 C, the numerical value in square frame represents relative expression's level of albumen to the above results.Setting LHCB protein content in Col (wild-type Arabidopis thaliana) is standard, and in mutant, the LHCB protein content is quantized by comparison.Immunoblotting assay carries out three independent biology to be repeated, and obtains similar result.Every group of data of RT-PCR are the mean value of three independent biology repetitions.

Can find out, in wrky40 single mutant and wrky40wrky18 double-mutant, the expression amount of LHCB4 significantly improves, and the LHCB4 expressing quantity significantly improves in the wrky40 single mutant.Yet, in the wrky40wrky18 double-mutant, the LHCB4 expression amount does not have noticeable change.

In sum, above experimental result shows, WRKY40 and WRKY18 suppress LHCB genetic expression jointly.

The hormesis of LHCB expression is depended on to a certain extent to the function of ABAR and WRKY40 in order to study ABA, tests as follows:

Cch, wrky40, the col growth seedling of three days are moved to respectively containing continued growth on the substratum of 0,1,3,5 μ M ABA and sample after two weeks and detect LHCB4:forward primer:5 '-CAGCCGTACACTGAAGTCTTTGG-3 ' reverse primer:5 '-TTCTATCCATATCAACGTCGTCAAC-3 ', the primer that internal reference is β-Actin, internal reference is forward primer:5 '-GGTAACATTGTGCTCAGTGGTGG-3 ' reverse primer 5 '-AACGACCTTAATCTTCATGCTGC-3 ', and Actin is the loading contrast.The experiment triplicate, result is consistent.

As a result shown in Fig. 8 D and 8E,

Wherein, shown in the 8D left hand view, cch compares with the wild-type Arabidopis thaliana, and the LHCB4 of cch (' * ' marks) is in the slight rising of only having of protein level.

Shown in Fig. 8 D right part of flg, cch compares with wrky40, and the protein expression of LHCB4 is unaffected.

According to the concentration of immunoblotting band, expressing quantity is estimated, the expression amount of take in the wild-type of not carrying out the ABA processing is standard reference (in column diagram shown in red arrow).Result as shown in Fig. 8 E, 1,3, the hormesis that the ABA of 5 μ M expresses LHCB4, in cch and wrky40 mutant significantly lower than wild-type in.The mean value that data are three independent biology repetitions.

Cch LHCB4 relative expression quantity under the ABA of 0,1,3,5 μ M processes is 30,40,40,55,

Wrky40 mutant LHCB4 relative expression quantity under the ABA of 0,1,3,5 μ M processes is 340,310,310,310,

Col LHCB4 relative expression quantity under the ABA of 0,1,3,5 μ M processes is 100,400,300,150.

Three, the mutant of ABAR and WRKY40 affects the regulation and control that ABA expresses LHCB

By abar-3 mutant (the allelic simple point mutation body of ABAR, Shang Y, Yan L, LiuZQ, Cao Z, MeiC, Xin Q, Wu FQ, Wang XF, Du SY, Jiang T, Zhang XF, Zhao R, Sun HL, Liu R, Yu YT, Zhang D-P (2010) The Mg-chelatase H subunit antagonizes a group of WRKY transcription repressors to relieve ABA-responsive genes of inhibition.Plant Cell 22:1909-1935, the mutant of ABAR, the public can obtain from Tsing-Hua University.) process 2 time-of-weeks in the ABA substratum that is 0 μ M in concentration, sampling detects the expression of (detection method is the same) LHCB gene mRNA and protein level by quantitative fluorescent PCR and immunoblotting, and immunoblotting be take Actin as the loading contrast.Numerical value under protein band in square frame represents the relative expression quantity of LHCB albumen.Take the Actin band as standard.Immunoblotting three secondary pollutants are learned and are repeated, and result is consistent.Each numerical value is the mean value of three independent biology repetitions.

As shown in Figure 9, the LHCB4 gene mRNA relative expression quantity of col is 1.0 to result;

The LHCB4 gene mRNA relative expression quantity of abar-3 mutant is 0.5;

In conjunction with above-mentioned LHCB family member's protein level at lower concentration ABA (1,3 or 5 μ M) processing lower expression amount improves, but LHCB3 in cch and wrky40 mutant, the result that the ability of LHCB4 and LHCB4 response ABA descends significantly, show: the signal path that ABA mediates by ABAR-WRKY40 promotes the expression of LHCB.

Four, the double-mutant of ABAR and LHCB gene has the phenotype to the ABA desensitization, and in the cch mutant, expressing the LHCB6 gene can partly recover its phenotype of desensitization to ABA excessively

By double-mutant lhcb1 lhcb3, (the double-mutant construction process: the petal that lhcb1 is not bloomed is removed only surplus gynoecium of its stamen with tweezers for lhcb1 lhcb6, lhcb1 cch, lhcb3 cch, lhcb4 cch, lhcb6 cch, take off in lhcb6 the stamen in the petal of blooming or not blooming, rub peeling off on remaining pistil stigma, make pollen fully contact column cap, this gynoecium is made marks, obtain the first familiar generation seed after the pod maturation, F1 is sowed again for seed, F2, for growth, identifies and can obtain homozygote through PCR, the double-mutant construction process: the petal that lhcb1 is not bloomed is removed only surplus gynoecium of its stamen with tweezers, take off in lhcb6 the stamen in the petal of blooming or not blooming, rub peeling off on remaining pistil stigma, make pollen fully contact column cap, this gynoecium is made marks, obtain the first familiar generation seed after the pod maturation, F1 is sowed again for seed, F2, for growth, identifies and can obtain homozygote through PCR, the double-mutant construction process: the petal that lhcb1 is not bloomed is removed only surplus gynoecium of its stamen with tweezers, take off in cch the stamen in the petal of blooming or not blooming, on the lhcb1 pistil stigma, rub, make pollen fully contact column cap, this gynoecium is made marks, obtain the first familiar generation seed after the pod maturation, F1 is sowed again for seed, F2 is for growth, identify and can obtain homozygote through PCR) and wild-type Col extraction RNA and protein, detect the expression of (detection method is the same) LHCB gene mRNA and protein level by quantitative fluorescent PCR and immunoblotting, immunoblotting be take Actin as the loading contrast.Numerical value under protein band in square frame represents the relative expression quantity of LHCB albumen.Sampling is processed, result is as shown in figure 10: Figure 10 A is double-mutant lhcb1 lhcb3, the expression of Different L HCB gene in lhcb1 lhcb6, the expression that Figure 10 B is Different L HCB gene in double-mutant lhcb1 cch, lhcb3 cch, lhcb4 cch, lhcb6 cch.

Fluorescence quantitative PCR detection (Figure 10 A, the column diagram in 10B) is passed through in the expression of mRNA level,

Protein expression detects by immunoblotting, and Actin is the loading contrast.The relative expression quantity of the numeric representation LHCB albumen in Figure 10 A below protein band, the wild-type of take compares quantification as standard.The expression of six LHCB (LHCB1-LHCB6) all detects in cch mutant (Figure 10 B) and all double-mutants (10A, 10B).Three independent biology of immunoblotting assay repeat, and result is similar.Each numerical value of quantitative fluorescent PCR is the mean value of three independent biology repetitions.

In order to study ABAR and LHCBs gene in genetic upstream and downstream relation, with cch-lhcbs double-mutant lhcb1cch, lhcb3 cch, lhcb4 cch, and lhcb6 cch is the phenotype that object is studied its response ABA.In the cch mutant, LHCB gene expression amount significantly descend (Figure 10 B).Yet double-mutant and cch single mutant that the mutant (lhcb1, lhcb4, or lhcb6) that single LHCB lowers obtains after hybridizing with cch are compared, LHCB family gene content is not significantly lowered, and has promoted on the contrary other LHCB family members' expression.LHCB3 knocks out in the double-mutant obtained after mutant and the hybridization of cch mutant, and the LHCB3 gene is knocked, but LHCB2, LHCB4, the mRNA of LHCB6 gene is raised, and the LHCB4 protein expression is raised (Figure 10 B).These data declarations have the feedback regulation mechanism of a set of complexity to maintain the stability of LHCB gene family integral body in double-mutant.

Five, turn down or knock out the expression of the gene of the gene of the stable state of LHCB effect gene active oxygen and a series of participation ABA signal paths and response ABA

1, affect the stable state of active oxygen

As everyone knows, active oxygen (ROS) participates in ABA signal transduction process (Pei et al, 2000; Murata et al, 2001; Mustilli et al, 2002; Kwak et al, 2006; Miao et al, 2006; Zhang et al, 2009), and in vegetable cell, ROS mainly results from (Kwak et al, 2006 in chloroplast(id); Nott et al, 2006; Galvez-Valdivieso and Mullineaux, 2010).In chloroplast(id), the generation of ROS is main relevant with the electronics transmission with photoabsorption, and LHCB also in these two processes, play an important role (Jansson, 1994,1999; Galvez-Valdivieso and Mullineaux, 2010).

Therefore, detect the content of ROS in the lhcb4 mutant.

Blade is taken from soil the growth wild-type Arabidopis thaliana plant of three weeks, at 50mMKCl, in the damping fluid of 10mMMes-Tris (pH 6.15) the ABA that contains different concns, is placed in 200umol m ^-2sec ^-1pre-treatment 1h under illumination.Then make leaf dyeing (Amresco, Solon, OH, USA) by adding 0.1mg/mL NBT (being dissolved in the 100mM potassium phosphate solution) in damping fluid and vacuumizing infiltration.Then sample moves into 80% ethanol at the dark 2h. that places of room temperature, and boiling water bath is processed 10min, to remove chlorophyllous interference.

In the guard cell, the detection of ROS content utilizes H2DCF-DA (SIGMA, D6883) contaminate the blade table leather strap peeled off, method is with reference to Miao Y, Lv D, Wang P, Wang XC, Chen J, Miao C, Song CP (2006) An Arabidopsis glutathione peroxidase functions as both a redox transducer and a scavenger in abscisic acid and drought stress responses.Plant Cell 18:2749-2766.Pre-treatment epidermis bar in containing 0 (equal volume ethanol in contrast) or 5 μ M (±)-ABAMES-Tris damping fluid (with above), promote stomatal opening.Then in the damping fluid that epidermis bar immersion 50mMTris-KCl (pH 7.2) is comprised to 50mM H2DCF-DA, dark processing is 20 minutes.Utilize MES-Tris damping fluid rinsing sample, remove unnecessary dyestuff.Utilize fluorescence microscopy (Olympus, BX51, Japan) to detect the fluorescence in the epidermis bar.ROS contaminates fluorescence and clicks the parameter observation: exciting light, 488nm; Transmitted light, 525nm.

As shown in figure 11, Figure 11 A is the generation that NBT staining examine different concns ABA (wild-type col 0-50 μ M, 0-10 μ M in the lhcb6 mutant) processes ROS after wild-type and lhcbs mutant blade to experimental result.The experiment triplicate obtains similar result.

Figure 11 B is the quantification that in Figure 11 A, ROS produces.By the scanning brightness of dyeing, estimated, the dyeing concentration of ROS in wild-type is decided to be to 100%, and other are quantized by comparison.

Result is that the ROS amount of lhcb4 under 0,5,10,50 μ MABA process is 200%, 60%, 50%;

The ROS amount of col under 0,5,10,50 μ MABA process is 100%, 150%, 100%, 50%;

Figure 11 C is the epidermis bar that 5 μ M ABA process wild-type and different lhcb mutant, by H2DCF-DA, dyes and observes the ROS production in the guard cell.Test and repeat to obtain similar result three times.

Figure 11 D is the expression that real-time quantitative PCR detects a series of ABA responsive genes in the lhcbs mutant.Every group of data are all the mean value of three independent biology repetitions.

As can be seen from the above, in the lhcb mutant, the content of ROS is higher than the wild-type plant, and this has embodiment (Figure 11 A to C) in blade and guard cell.Find to process with the ABA of low concentration (1 to 5 μ M) generation that blade can promote ROS.These (Pei et al, 2000 consistent with former result of study; Murata et al, 2001; Mustilli et al, 2002; Kwak et al, 2006; Miao et al, 2006; Zhang et al, 2009); And just no longer include with the ABA processing of higher concentration (10 μ M) content (50 μ M) (Figure 11 A) that accelerating effect even can reduce ROS.Yet in the lhcb mutant, with low concentration ABA, process plant, no matter be that in complete blade (1 to 10 μ M ABA, Figure 11 A and Figure 11 B) or guard cell, the ROS content of (5 μ M ABA process, Figure 11 C) all descends.These experimental results show to turn down or knock out the LHCB gene all will affect the stable state of ROS in vegetable cell and the ROS response to ABA.

2, turn down or knock out the expression of the gene of the gene of a series of participation of LHCB effect gene ABA signal path and response ABA

Adopt fluorescent quantitation to detect the following expression that participates in ABA signal path gene and ABA responsive genes in lhcb mutant 1-6: ABF1, ABF2/AREB1, ABF3, ABF4/AREB2 (Choi et al, 2000; Uno et al, 2000), ABI1 (Leunget al, 1994; Meyer et al, 1994; Gosti et al, 1999), ABI2 (Leung et al, 1997), ABI3 (Giraudat et al, 1992), ABI4 (Finkelstein et al, 1998), ABI5 (Finkelstein and Lynch, 2000), ERD10 (Kiyosue et al, 1994), KIN1 and KIN2 (Kurkela and Borg-Franck, 1992), MYB2 and MYC2 (Abe et al, 2003), OST1 (Mustilli et al, 2002), RAB18 (Lang and Palva, 1992), RD29A (Yamaguchi-Shinozaki and Shinozaki, 1994).

Primer is as follows:

ABF1(At1g49720)：

forward?primer：5’-TCAACAACTTAGGCGGCGATAC-3’

reverse?primer：5’-GCAACCGAAGATGTAGTAGTCA-3’

ABF2(At1g45249)：

forward?primer：5’-TTGGGGAATGAGCCACCAGGAG-3’

reverse?primer：5’-GACCCAAAATCTTTCCCTACAC-3’

ABF3(At4g34000)：

forward?primer：5’-CTTTGTTGATGGTGTGAGTGAG-3’

reverse?primer：5’-GTGTTTCCACTATTACCATTGC-3’

ABF4(At3g19290)：

forward?primer：5’-AACAACTTAGGAGGTGGTGGTC-3’

reverse?primer：5’-CTTCAGGAGTTCATCCATGTTC-3’

ABI1(At4g26080)：

forward?primer：5’-AGAGTGTGCCTTTGTATGGTTTTA-3’

reverse?primer：5’-CATCCTCTCTCTACAATAGTTCGCT-3’

ABI2(At5g57050)：

forward?primer：5’-GATGGAAGATTCTGTCTCAACGATT-3’

reverse?primer：5’-GTTTCTCCTTCACTATCTCCTCCG-3’

ABI3(At3g24650)：

forward?primer：5’-TCCATTAGACAGCAGTCAAGGTTT-3’

reverse?primer：5’-GGTGTCAAAGAACTCGTTGCTATC-3’

ABI4(At2g40220)：

forward?primer：5’-GGGCAGGAACAAGGAGGAAGTG-3’

reverse?primer：5’-ACGGCGGTGGATGAGTTATTGAT-3’

ABI5(At2g36270)：

forward?primer：5’-CAATAAGAGAGGGATAGCGAACGAG-3’

reverse?primer：5’-CGTCCATTGCTGTCTCCTCCA-3’

ERD10(At1g20450)：

forward?primer：5’-TCTCTGAACCAGAGTCGTTT-3’

reverse?primer：5’-CTTCTTCTCACCGTCTTCAC-3’

KIN1(At5g15960)：

forward?primer：5’-ACCAACAAGAATGCCTTCCA-3’

reverse?primer：5’-CCGCATCCGATACACTCTTT-3’

KIN2(At5g15970)：

forward?primer：5’-ACCAACAAGAATGCCTTCCA-3’

reverse?primer：5’-ACTGCCGCATCCGATATACT-3’

MYB2(At2g47190)：

forward?primer：5’-TGCTCGTTGGAACCACATCG-3’

reverse?primer：5’-ACCACCTATTGCCCCAAAGAGA-3’

MYC2(At1g32640)：

forward?primer：5’-TCATACGACGGTTGCCAGAA-3’

reverse?primer：5’-AGCAACGTTTACAAGCTTTGATTG-3’

OST1(At4g33950)：

forward?primer：5’-TGGAGTTGCGAGATTGATGAGAG-3’

reverse?primer：5’-CCTGTGGTTGATTATCTCCCTTTTT-3’

RAB18(At5g66400)

forward?primer：5’-CAGCAGCAGTATGACGAGTA-3’

reverse?primer：5’-CAGTTCCAAAGCCTTCAGTC-3’

RD29A(At5g52310)：

forward?primer：5’-ATCACTTGGCTCCACTGTTGTTC-3’

reverse?primer：5’-ACAAAACACACATAAACATCCAAAGT-3’

Result as shown in Figure 11 D, ten important gene: ABI4 wherein, ABI5, ERD10, KIN1, KIN2, MYB2, MYC2, OST1, RAB18 and the RD29A expression in the lhcb mutant is significantly suppressed.All these repressed genes are all just to respond the gene of ABA or the positive regulon of coding ABA signal path.Three coding important transcription factor---genes of the positive regulon of ABA signal path, ABF1, ABF4 and ABI3, at lhcb2, lhcb4, also significantly suppressed in the lhcb5 mutant.ABF1 and the ABF4 expression in the lhcb6 mutant does not have considerable change, and the expression of ABI3 does not have noticeable change (Figure 11 D) in lhcb1 and lhcb3 mutant yet.Yet, these two of ABI1 and ABI2 directly act on ABA acceptor PYR/PYL/RCAR (Fujii et al, 2009) downstream, the gene of coding ABA signal path negative regulator, the expression amount in the lhcb mutant do not have noticeable change (Figure 11 D).On the contrary, gene expression amount in the lhcb mutant except lhcb5 and lhcb6 of these two the positive regulon of coding ABA signal path of ABF2 and ABF3 raises, in lhcb5 and lhcb6 mutant, ABF2 and ABF3 gene expression amount do not have considerable change (Figure 11 D).The expression amount of these ABA signal transduction process genes involveds changes the positive regulon that LHCB Gene A BA signal fundamentally is described, but this wherein has a potential complex mechanism.

Six, turn down or knock out the LHCB Gene Partial and suppress the phenotype of wrky40 mutant to the ABA sensitivity

1, the protein expression box is sprouted speed

By wrky40lhcb1, wrky40lhcb3, wrky40lhcb6 mutant, (wrky40lhcb1 double-mutant construction process: the petal that lhcb1 is not bloomed is removed only surplus gynoecium of its stamen with tweezers, take off in wrky40 the stamen in the petal of blooming or not blooming, rub peeling off on remaining pistil stigma, make pollen fully contact column cap, this gynoecium is made marks, obtain the first familiar generation seed after the pod maturation, F1 is sowed again for seed, F2, for growth, identifies and can obtain homozygote through PCR, wrky40lhcb3 double-mutant construction process: the petal that lhcb3 is not bloomed is removed only surplus gynoecium of its stamen with tweezers, take off in wrky40 the stamen in the petal of blooming or not blooming, rub peeling off on remaining pistil stigma, make pollen fully contact column cap, this gynoecium is made marks, obtain the first familiar generation seed after the pod maturation, F1 is sowed again for seed, F2, for growth, identifies and can obtain homozygote through PCR, wrky40lhcb6 double-mutant construction process: the petal that lhcb6 is not bloomed is removed only surplus gynoecium of its stamen with tweezers, take off in wrky40 the stamen in the petal of blooming or not blooming, rub peeling off on remaining pistil stigma, make pollen fully contact column cap, this gynoecium is made marks, obtain the first familiar generation seed after the pod maturation, F1 is sowed again for seed, F2 is for growth, identify and can obtain homozygote through PCR), wrky40 and wild-type Col extract RNA and protein, detect the expression of (detection method is the same) LHCB6 gene mRNA and protein level by quantitative fluorescent PCR and immunoblotting, immunoblotting be take Actin as the loading contrast.Numerical value under protein band in square frame represents the relative expression quantity of LHCB albumen, detects after (detection method is the same) vernalization the expression of the mRNA of LHCB6 and the protein expression of LHCB6 after 72 hours, and the sprouting speed of 72 hours after statistics vernalization.Take col as contrast.

Result is as shown in Figure 12 A, and the sprouting speed of wrky40lhcb1 on 0,0.5,1,3 μ MABA substratum is 100%, 80%, 72%, 45%;

The sprouting speed of wrky40lhcb3 on 0,0.5,1,3 μ MABA substratum is 100%, 85%, 65%, 50%;

The sprouting speed of wrky40lhcb6 mutant on 0,0.5,1,3 μ MABA substratum is 100%, 80%, 62%, 47%;

The sprouting speed of wrky40 on 0,0.5,1,3 μ MABA substratum is 100%, 75%, 50%, 25%;

The sprouting speed on 0,0.5,1,3 μ MABA substratum of col is 100%, 100%, 90%, 59%;

Can find out, double-mutant can significantly suppress the wrky40 mutant and suppress seed germination at ABA.

2, LHCB1, LHCB3, the LHCB6 expression amount reduces makes the wrky40 mutant weaken in the susceptibility to ABA aspect the rear growth of sprouting

Wrky40lhcb1, wrky40lhcb3, wrky40lhcb6 mutant, wrky40 seed are directly sowed and do not contained or containing the result of growth after 9 days on the substratum of (0.6 μ M) ABA.

Result is as shown in Figure 12 B, and following column diagram has shown that on the substratum that contains 0.6 μ M ABA growth is after 9 days, wild-type and mutant main root extended length;

The main root extended length of wrky40lhcb1 is 0.58cm;

The main root extended length of wrky40lhcb3 is 0.58cm;

The main root extended length of wrky40lhcb6 mutant is 0.58cm;

The main root extended length of wrky40 is 0.34cm;

The main root extended length of col is 1.00cm.

3, ABA induce stomatal closure (on) and ABA suppress aspect stomatal opening, LHCB1, LHCB3, the LHCB6 expression amount reduces does not affect the response of wrky40 mutant to ABA

Wrky40lhcb1, wrky40lhcb3, wrky40lhcb6 mutant, wrky40 seed are directly sowed in the result containing after growing 9 days on the substratum of (0.6 μ M) ABA.

Result as shown in Figure 12 C,

Wrky40lhcb1 processes 0h, 0 μ M ABA processing 2.5h at 0 μ M ABA, 20 μ M ABA processing 2.5h, 30 μ MABA process 2.5h and induce the stomatal aperture of stomatal closure to be respectively 6.0 μ m, 5.9 μ m, 2.5 μ m, 0.9 μ m;

Wrky40lhcb3 processes 0h, 0 μ M ABA processing 2.5h at 0 μ M ABA, 20 μ M ABA processing 2.5h, 30 μ MABA process 2.5h and induce the stomatal aperture of stomatal closure to be respectively 5.7 μ m, 5.9 μ m, 2.0 μ m, 1.0 μ m;

Wrky40lhcb6 processes 0h, 0 μ M ABA processing 2.5h at 0 μ M ABA, 20 μ M ABA processing 2.5h, 30 μ MABA process 2.5h and induce the stomatal aperture of stomatal closure to be respectively 6.0 μ m, 5.9 μ m, 2.5 μ m, 1.0 μ m;

Wrky40 processes 0h, 0 μ M ABA processing 2.5h at 0 μ M ABA, 20 μ M ABA processing 2.5h, 30 μ MABA process 2.5h and induce the stomatal aperture of stomatal closure to be respectively 5.9 μ m, 5.9 μ m, 2.7 μ m, 0.8 μ m;

Col processes 0h, 0 μ M ABA processing 2.5h at 0 μ M ABA, 20 μ M ABA processing 2.5h, 30 μ MABA process 2.5h and induce the stomatal aperture of stomatal closure to be respectively 6.0 μ m, 6.0 μ m, 2.3 μ m, 1.0 μ m;

The stomatal aperture that wrky40lhcb1 processes 0h, 0 μ M ABA processing 2.5h, 20 μ M ABA processing 2.5h, 30 μ MABA processing 2.5h inhibition stomatal openings at 0 μ M ABA is respectively 0.8 μ m, 6.0 μ m, 3.0 μ m, 1.0 μ m;

Wrky40lhcb3 processes 0h, 0 μ M ABA at 0 μ M ABA and processes the inhibition stomatal opening that 2.5h, 20 μ M ABA process 2.5h, 30 μ MABA processing 2.5h, and stomatal aperture is respectively 0.8 μ m, 5.8 μ m, 3.0 μ m, 1.2 μ m;

The inhibition stomatal opening stomatal aperture that wrky40lhcb6 processes 0h, 0 μ M ABA processing 2.5h, 20 μ M ABA processing 2.5h, 30 μ MABA processing 2.5h at 0 μ M ABA is respectively 0.8 μ m, 5.6 μ m, 3.0 μ m, 1.0 μ m;

The inhibition stomatal opening stomatal aperture that wrky40 processes 0h, 0 μ M ABA processing 2.5h, 20 μ M ABA processing 2.5h, 30 μ MABA processing 2.5h at 0 μ M ABA is respectively 0.6 μ m, 5.8 μ m, 2.8 μ m, 1.2 μ m;

The inhibition stomatal opening stomatal aperture that col processes 0h, 0 μ M ABA processing 2.5h, 20 μ M ABA processing 2.5h, 30 μ MABA processing 2.5h at 0 μ M ABA is respectively 0.8 μ m, 5.7 μ m, 2.9 μ m, 1.0 μ m;

The mean value that every group of data of 1-3 are three independent biology repetitions.

The results show wrky40 mutant in the past suppresses seed germination at ABA, suppresses to show the phenotype to the ABA sensitivity aspect growth of seedling, but the stomatal closure of inducing at ABA and ABA suppress not show the phenotype that responds ABA aspect stomatal opening.Lhcb1, lhcb3, lhcb6 mutant are found with the hybridization of wrky40 mutant respectively, and double-mutant can significantly suppress wrky40 mutant phenotype (Figure 12 A, 12B) for the ABA sensitivity aspect ABA inhibition seed germination and growth of seedling; But the phenotype that the stomatal closure of inducing at response ABA for the wrky40 mutant and ABA suppress aspect stomatal opening does not change (Figure 12 C).This may be that in this process, the disappearance of WRKY40 function may cause the effect of ABA signal path complexity because the ABA signal path that WRKY40 participates in is having the mechanism of a set of complexity to cause aspect the adjusting stomatal movement.Yet these data provide enough genetics evidences to show that LHCB acts on the downstream of WRKY40 transcription factor, with the LHCB gene, to be that WRKY40 transcribes the direct target gene result that suppresses son consistent.

Seven, the molecule of mutant lhcb1-6 and biochemical identification

The T-DNA of Figure 13 (A-F) LHCB1-6 inserts schematic diagram .A, lhcb1-1 (salk-134810); B, lhcb2 (salk-005614); C, lhcb3 (salk-036200); D, lhcb4 (salk-032779); E, lhcb5 (salk-139667); F, lhcb6 (salk-074622) .LP and RP identify left end primer and right-hand member primer on the genome that lhcbs uses; LBa1, T-DNA sequence left end primer; RBa1, T-DNA sequence right-hand member primer; T-DNA1 and T-DNAn represent respectively first copy and last copy (more than, the copy differential concatenation is inserted) that insert in the same site of insertion.

Figure 13 (A) is in LHCB1, and the T-DNA sequence of single copy is inserted into the promoter district of LHCB1 gene, concrete on position atg start codon ATG upstream-592nt and-523nt between, insert and cause the 70bp disappearance;

Figure 13 (B) is in LHCB2, and the T-DNA sequence is inserted into the promoter region of LHCB2 gene, concrete on position atg start codon ATG upstream-102nt and-126nt between, insert the disappearance cause 24bp;

In Figure 13 (C) LHCB3, the T-DNA sequence is inserted into first exon 1 of LHCB3 gene, and concrete on position, between atg start codon ATG downstream 120nt and 125nt, inserts the disappearance that causes 6bp;

In Figure 13 (D) LHCB4, the T-DNA sequence is inserted into the promoter district of LHCB4 gene in the mode of differential concatenation, concrete on position atg start codon ATG upstream-1263nt and-1247nt between, insert the disappearance cause 17bp;

In Figure 13 (E) LHCB5, the T-DNA sequence is inserted into the promoter district of LHCB5 gene, concrete on position atg start codon ATG upstream-483nt and-477nt between, insert the disappearance that causes 7bp;

In Figure 13 (F) LHCB6, the T-DNA sequence is inserted into the promoter district of LHCB6 gene, concrete on position atg start codon ATG upstream-391nt and-346nt between, insert the disappearance that causes 46bp.

In Figure 13 (G) RT-PCR and western blot detection mutant lhcb1～6, LHCB's transcribes and translation skill.

Immunoblotting detects take Actin as the loading contrast.The column diagram of real-time quantitative PCR, be made as standard with the expression of corresponding gene in wild-type, and the expression amount in mutant is relative value, lhcb1-1 wherein, and lhcb2, hcb4, lhcb5, lhcb6 is turned down in various degree, and lhcb3 is the gene knockout mutant.Western blot detects, and the numerical value in square frame represents the relative level of protein expression.Setting LHCB protein content in Col is 100, and in mutant, the LHCB protein content is quantized by comparison.This tests triplicate, and result is consistent.

Figure 13 (H) mutant (lhcb1～lhcb6) Determination of Chlorophyll a/b content significantly is not affected.Left figure: different mutants Determination of Chlorophyll a, chlorophyll b and total chlorophyll content.Each numerical value is the mean value of three independent biology repetitions.Right figure: the growth of seedling state of different mutants does not have the phenotype of chlorophyll disappearance.

Eight, in different lhcb mutant, endogenous aba content and dry matter content are measured

Analysis of material is the growth seedling (wild-type Arabidopis thaliana col and mutant lhcb2) of two weeks

(A) ABA level.Test kit is measured the ABA content (Sigma test kit, Plant GrowthRegulator Immmunoassay DETECTION Kit) in the lotus throne blade by ELISA.

(B) estimate dry matter content by dry weight/fresh weight.

Result as shown in figure 14, shows that ABA and the dry matter content in mutant and wild-type do not have significant difference.

Nine, wrky40 and wrky40wrky18 mutant do not have the gun phenotype

A, by wild-type Col, cch, wrky40 single mutant, wrky40wrky18 double-mutant seed are directly broadcast and are not being contained or containing 5 μ MNF (NF:Norflurazon, it is a kind of weedicide, after it is added to substratum, the development of plastid of seedling is by havoc, and plastid sends plastid-core reverse signal to nucleus; And the mutant that this process produces is not affected by NF, the phenotype that cell nuclear energy continues the coding plastogene is phenotype---the GUN phenotype with the core uncoupling) substratum on, vernalization, after three days, is put into 100 μ molm ^-2s- ¹under illumination, carry out continuous illumination in 24 hours, after 6 days, sample fluorescence quantitative PCR detection.The experiment triplicate obtains same result.

Primer is LHCB4:forward primer:5 '-CAGCCGTACACTGAAGTCTTTGG-3 ' reverse primer:5 '-TTCTATCCATATCAACGTCGTCAAC-3 ', the primer that internal reference is β-Actin, internal reference is forward primer:5 '-GGTAACATTGTGCTCAGTGGTGG-3 ' reverse primer 5 '-AACGACCTTAATCTTCATGCTGC-3 ', result, as shown in Figure 15 A, all can't detect the expression of LHCBsmRNA in wrky40 single mutant and wrkywrky18 double-mutant.

B, by wild-type Col, cch, wrky40 single mutant seed directly broadcast containing or containing on the substratum of 5 μ MNF, vernalization, after three days, is put into 100 μ molm ^-2s- ¹under illumination, carry out continuous illumination in 24 hours, sample immunoblotting after 6 days and detect.The experiment triplicate obtains same result.

Result is as shown in Figure 15 B, and with the same at wild-type, wrky40 and wrkywrky18 mutant, after NF processes cch, the expression of six genes of LHCBs all can't detect, and has shown the complicacy of GUN-type chloroplast(id) reverse signal.All these results show that wrky40 and wrky40wrky18 mutant are non-gun mutant.

As can be seen from the above, LHCBs albumen response physiological level concentration ABA and a mode chart increasing are as shown in figure 16, the WRKY40 transcription factor suppresses the expression of LHCB, ABA promotes that WRKY40 enters tenuigenin from nucleus, promote ABAR and WRKY40 to do mutually, by the expression of lowering WRKY40, remove the restraining effect to LHCBs (LHCB1/2/3/4/5/6) gene.In tenuigenin, synthetic LHCB albumen is transported to chloroplast(id) to maintain the stable state of LHCB albumen, the challenge that plant is conformed, and the stable state of LHCB albumen is essential to the foundation of ROS stable state.

Ten, the expression of LHCB4 in different tissues

Extract respectively the RNA of the different tissues of wild-type Arabidopis thaliana, reverse transcription obtains cDNA, forward primer:5 '-CAGCCGTACACTGAAGTCTTTGG-3 ' reverse the primer:5 '-TTCTATCCATATCAACGTCGTCAAC-3 ' of take is primer, internal reference: β-Actin, the primer of internal reference is forward primer:5 '-GGTAACATTGTGCTCAGTGGTGG-3 ' reverse primer5 '-AACGACCTTAATCTTCATGCTGC-3 ', fluorescence quantitative PCR detection, result is as Figure 17, LHCB4 is at the root of wild-type Arabidopis thaliana, stem, leaf, flower, mRNA relative expression quantity in angle fruit and seed is respectively 0.04, 0.40, 0.40, 0.75, 1.75, 0.00.

Claims

1. a method of controlling plant trait, be the expression that reduces LHCB4 protein coding gene in the purpose plant, obtains having following 1) or 2) plant of feature:

1) resistance of reverse is lower than described purpose plant; Described resistance of reverse is drought-resistant property;

2) growth is faster than described purpose plant;

Described purpose plant is Arabidopis thaliana.

2. method according to claim 1 is characterized in that:

Described growth faster than described purpose plant is: A) sprouting of seed ahead of time; Perhaps B) growth of root ahead of time.

3. method according to claim 2 is characterized in that:

Described hormone is ABA.

4. method according to claim 3 is characterized in that:

Described is 0.5-5 μ M in the Young Plant concentration that ABA coerces seedling stage;

Describedly plant, become concentration that the length of time, ABA coerced lower than 200 μ M.

5. method according to claim 4 is characterized in that: describedly in the Young Plant concentration that ABA coerces seedling stage, be specially 0.5 μ M, 1 μ M, 2 μ M, 3 μ M or 5 μ M; Describedly plant, become the concentration that the length of time, ABA coerced to be specially 20 μ M, 50 μ M, 100 μ M, 150 μ M or 200 μ M.

6. method according to claim 4 is characterized in that: described drought-resistant property embodies by stomatal aperture and/or percentage of water loss; The growth of described embodies by main root length.

7. method according to claim 4 is characterized in that:

The expression of described reduction LHCB4 protein coding gene is to activate the ABAR-WRKY40 signal path by Exogenous ABA to realize;

Described activation ABAR-WRKY40 signal path is specially the ABA acceptor of the ABA activated plant of external source, ABA acceptor and the LHCB4 promotor that is combined with transcription factor WRKY40 are competed in conjunction with transcription factor WRKY40, discharge the promotor of LHCB4, the LHCB4 protein coding gene is expressed;