CN102731638B - Hevea brasiliensis latex small G-protein Rop family protein and coding gene thereof - Google Patents

Abstract

The invention discloses a small G-protein Rop family protein, a coding gene and an application of the hevea brasiliensis latex small G-protein Rop family protein. The protein is a protein having one of the following amino acid residue sequences: 1) protein composed of an amino acid residue sequence of SEQ ID No.1 in a sequence table; 2) SEQ ID No.1 derived protein obtained by subjecting the amino acid residue sequence of SEQ ID No.1 in the sequence table to substitution and/or deletion and/or addition of one or more amino acid residues and having GTP (guanosine triphosphate) binding function. The protein has GTP binding activity, plays a certain role in basic disease-resistant mechanism and stress resistance of plants, and has the characteristics of control for secondary xylem and phloem development of woods. The protein can improve the resistance of transgenic crop or woods against adverse situation, so as to improve the yield.

Description

A kind of latex of panama rubber tree small G-protein Rop family member's albumen and encoding gene thereof

Technical field

The present invention relates to the application of a kind of latex of panama rubber tree small G-protein Rop family member albumen HbRop3 and encoding gene thereof.

Background technology

Small G-protein is the monomeric protein in conjunction with guanylic acid (GTP), effect (the Zhenbiao Yang that in eukaryotic cell, has molecular switch, Small GTPases:Versatile Signaling Switches in Plants.The Plant Cell, 2002.14, p.S375-S388; V Vernoud, V., et al., Analysis of the small GTPase gene superfamily of Arabidopsis.Plant physiology, 2003.131 (3): p.1191-1208; Nibaua, C., Wu, HM.and Cheung, AY., RAC/ROP GTPases: ' hubs ' for signal integration and diversification in plants.Trends in Plant Science, 11 (6): p.309-315).Many studies confirm that, plant small G-protein Rop subfamily member is the peculiar gene of a class that forms in the plant evolution, multiple physiological activity in their wide participation vegetable cells, tissue, cell polarity, cell growth, differentiation and death, the cell walls that comprises actin cytoskeleton forms, the regulation and control of the interior calcium ion concn of tenuigenin, O ₂ ^-And H ₂O ₂Generate, the proteolytic degradation system that ubiquitin relies on coerces or the signal transduction of hormone induction etc.Along with carrying out in a deep going way of scientific effort, it is found that the function of small G-protein Rop family gene is more diversified, in different plants, show the dependency with different economic biology characteristics.For example, in cotton, closely related with the growth of cotton fibre; In leguminous plants, relevant with formation with the intrusion of root nodule bacterium; In eucalyptus, participate in (the Foucart such as genesis and development of eucalyptus secondary xylem, C., et al., Overexpression of EgROP1, a Eucalyptus vascular-expressed Rac-like small GTPase, affects secondary xylem formation in Arabidopsis thaliana.New Phytol, 2009.183 (4): p.1014-29).In rubber tree, up to the present, do not see any relevant rubber latex small G-protein Rop family gene clone's relevant report so.

Natural rubber (cis-1,4-polyisoprene, rubber hydrocarbon) is a kind of important industrial raw material and indispensable strategic materials, plays an important role in development of world economy.At present, Para rubber tree is the main source of natural rubber.The latex dust of Para rubber tree is the place of natural rubber biosynthesizing and storage, also is a kind of defence structure of rubber tree.The natural rubber biosynthesizing is the intracellular main metabolic activity of latex dust, because rubber hydrocarbon accounts for more than 90% of its tenuigenin (latex) dry weight.The latex regenerative process requires the latex dust cell to have very strong metabolic activity, particularly energy regeneration metabolism, protein synthesis and transportation.Based on the analysis to plant small G-protein Rop family gene function, we infer small G-protein Rop member, the Rop member in the latex especially, and important function also is bound to have in rubber tree.Report oxygen production such as nearest Li Dejun (2010) and removing are probably in the important disease of rubber tree--dead skin disease (tapping panel dryness, TPD) has vital role in the generation, and small G-protein Rop member participates in the generation of ROS, therefore participates in regulating and control of dead skin disease probably.(Li, D., et al., Identification and characterization of genes associated with tapping panel dryness from Hevea brasiliensis latex using suppression subtractive hybridization.BMC Plant Biol, 2010.10:p.140), they have just reported Rop member's partial sequence, do not clone this member's gene, also not more further investigations.But their research can prove the exactness that we infer.

Summary of the invention

The purpose of this invention is to provide a kind of small G-protein Rop family member's albumen and encoding gene and application.

Small G-protein Rop family member albumen provided by the present invention derives from the Para rubber tree (Hevea brasiliensis) of Euphorbiaceae genus hevea, and name is called HbRop3, is following 1) or 2) protein:

1) protein of being formed by the aminoacid sequence shown in the sequence in the sequence table 1;

2) with the amino acid residue sequence of sequence in the sequence table 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and with albumen shown in the sequence 1 have identical activity by 1) protein of deriving.

Sequence 1 in the sequence table is made up of 198 amino-acid residues.

For the ease of the HbRop3 purifying of sequence 1 coding, label as shown in table 1 in N-terminal that can the protein that the aminoacid sequence shown in the sequence 1 is formed in by sequence table or the C-terminal connection.

The sequence of table 1 label

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

But above-mentioned HbRop3 synthetic also can synthesize its encoding gene earlier, carries out the biology expression again and obtains.The encoding gene of above-mentioned HbRop3 can be by lacking sequence in the sequence table 2 codon of one or several amino-acid residue in the dna sequence dna shown in the 5 ' terminal 94-690 bit base, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

The encoding gene of described small G-protein Rop family member albumen (HbRop3) also belongs to protection scope of the present invention.

The encoding gene of described small G-protein Rop family member albumen (HbRop3) is following 1) or 2) or 3) or 4) dna molecular:

1) dna molecular shown in the sequence 2 in the sequence table;

2) in the sequence table sequence 2 from the dna molecular shown in the Nucleotide of 5 ' terminal 94-690 position;

3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the described small G-protein Rop family member's that encodes dna molecular;

4) with sequence table in the nucleotide sequence of sequence 2 have nucleotide sequence homology and that proteins encoded has the GTP combined function more than 80%.

Sequence 2 in the sequence table is made up of 1009 Nucleotide, open reading frame (the ORF that to comprise a length be 597 Nucleotide, 5 ＇ from sequence 2 hold 94-690 position nucleotide sequence), 5 '-UTR(of 93nt holds 1-93 position nucleotide sequence from 5 ＇ of sequence 2) and 3 '-UTR(of 319nt hold 691-1009 position nucleotide sequence from 5 ＇ of sequence 2), the albumen that length is 198 amino acid (sequence 1 in the sequence table) of encoding

Described stringent condition can be at 0.1 * SSPE (or in the solution of 0.1 * SSC), 0.1%SDS, hybridizes under 65 ℃ and washes film.

The recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain described small G-protein Rop family member protein coding gene all belong to protection scope of the present invention.

HbRop3 of the present invention has GTP in conjunction with activity, has the effect of molecular switch, can the resistance regulation and control of wide participation plant basis.Have certain effect for understanding the plant disease-resistant mechanism in basis and improving plant stress resistance tool, have the feature that regulation and control forest secondary xylem and phloem are grown, in genetically modified crops or the forest, can improve it to the resistance of adverse circumstance, thereby improve output.

Description of drawings

Fig. 1 is the phylogenetic analysis of HbRop3; HbRop3 and 16 homogenic functional clustering analysis charts, wherein, the meaning of two letters of beginning is: At (Arabidopis thaliana); Os (paddy rice); Gh (upland cotton); Hv (barley); Nt (tobacco); Zm (corn); Hb (rubber tree); All the other letters are the gene title.

Fig. 2 is HbRop3 and 17 functional domain analyses from disease-resistant Rop albumen in other plant; Wherein, the meaning of two letters of beginning is: At (Arabidopis thaliana); Os (paddy rice); Gh (upland cotton); Hv (barley); Nt (tobacco); Zm (corn); Hb (rubber tree); All the other letters are protein name.

Fig. 3 is the expression of HbRop3 gene in different dead skin degree rubber trees

Fig. 4 is the tissue specific expression analysis of HbRop3 gene

Fig. 5 is the influence of rubber tapping to HbRop3 genetic expression

Fig. 6 is the influence of ethrel processing to HbRop3 genetic expression

Fig. 7 is that the short period of time mechanical wounding is to the influence of HbRop3 genetic expression

Fig. 8 for long-time mechanical wounding to the rubber tree secondary latex dust induce and to the influence of HbRop3 genetic expression

Fig. 9 is that the methyl jasmonic is to the influence of HbRop3 genetic expression

Figure 10 is the influence of phytokinin (6-BA) HbRop3 genetic expression

Embodiment

Method described in the following embodiment if no special instructions, is ordinary method.

Acquisition and the compliance test result thereof of embodiment 1, small G-protein Rop family member albumen HbRop3 encoding gene

We have cloned the full-length cDNA of small G-protein Rop family member protein coding gene HbRop3 in the latex of panama rubber tree first, and have carried out sequential analysis and functional study.

1. the acquisition of small G-protein Rop family member protein coding gene HbRop3

We have set up latex of panama rubber tree est sequence database, find the est sequence of small G-protein Rop member encoding gene HbRop3, according to the Gentyx software analysis, have obtained to have the cDNA sequence of complete reading frame.Therefore we only to need the design primer to verify again just passable, concrete steps are as follows:

＜1〉HbRop3cDNA full-length clone

Respectively design a primer (3-F(positive-sense strand primer): 5 '-TCATTGCTCTGTCTATGTTGCTCA-3 ' at 5 ' end and 3 ' end respectively according to est sequence; 5-1009R(antisense strand primer) 5 '-AGTTACAGGCCGTAAATAATACAAG-3 ').Grinding 7-33-97(Rubber Institute, Chinese Academy of Agricultural Science with Para rubber tree heat cultivates, Rubber Institute, Chinese Academy of Agricultural Science has seedling to sell for a long time) the first chain cDNA be template, use Taq plus polymerase to carry out pcr amplification, the primer final concentration is 0.4 μ mol/L, and amplification program is: 94 ℃ of pre-sex change 3min; 94 ℃ of sex change 1min, 58 ℃ of annealing 1min, 72 ℃ are extended 1.5min, totally 30 circulations; 72 ℃ are extended 10min.The fragment cloning that this PCR is obtained checks order to the pMD18-T carrier, shows that through order-checking obtaining clip size is the cDNA full length sequence of 1009bp.The fragment of this acquisition is small G-protein Rop member's of the present invention encoding gene HbRop3 gene, this fragment has the nucleotide sequence of sequence 2 in the sequence table, sequence 2 total lengths are 1009 Nucleotide (nt) in the sequence table, open reading frame (the ORF that to comprise a length be 597 Nucleotide, 5 ＇ from sequence 2 hold 94-690 position nucleotide sequence), 5 ' of 93nt-UTR(holds 1-93 position nucleotide sequence from 5 ＇ of sequence 2) and 3 '-UTR(of 319nt hold 691-1009 position nucleotide sequence from 5 ＇ of sequence 2), the length of encoding is 198 amino acid (sequence 1 in the sequence table), molecular weight is the albumen of 21.89KDa, being Rop albumen member HbRop3, is HbRop3 with this small G-protein Rop unnamed gene.The fragment cloning that above-mentioned PCR is obtained is to the pMD18-T recombinant vectors called after pMD18-HbRop3 of the Nucleotide that contains sequence 2 of pMD18-T carrier acquisition.

The pertinent literature report disease-resistant according to small G-protein gene involved in plant, the amino acid that we select from the Rop family member HbRop3 in representative 16 disease-resistant relevant Rop genes and 1 elastomer gum Ruzhong in other model plant or the important cash crop (Arabidopis thaliana, tobacco, paddy rice, barley, corn and cotton) compares cluster analysis, understands the system function evolution status of HbRop3.It is right to utilize Clustal X (version 1.81) software that these 17 small G-protein Rop member gene orders are carried out multiple ratio, and uses TreeView[Win32] (version1.6.6) constructing system evolutionary tree (Fig. 1).From cluster result (Fig. 1) as can be seen, the HbRop3 gene of cloning and HvRacB are in the same place with the OsRacB cluster.HvRacB and OsRacB are proved to be the negative regulation function (Agrawal that has in the disease-resistant signal transduction path of barley powdery mildew and paddy rice blight, G.K., H.Iwahashi and R.Rakwal, Small GTPase ' Rop ': molecular switch for plant defense responses.FEBS Lett, 2003.546 (2-3): p.173-80; Jung, Y.H., et al., Functional characterization of OsRacB GTPase--a potentially negative regulator of basal disease resistance in rice.Plant Physiol Biochem, 2006.44 (1): p.68-77).The result of this preliminary cluster analysis has further proved our supposition, and Rop member one fixes on has important function in the latex of panama rubber tree.Because in rubber tree, people think that always latex of panama rubber tree has the physiological function of defence, and just molecular biology mechanism is not clear.

Simultaneously, we have also analyzed aminoacid sequence and the selected homogenic amino acid whose multiple comparison result of elastomer gum Ruzhong HbRop3 coded by said gene.The result shows: the HbRop3 gene of cloning has all conservative structural domains of plant Rop gene and various C-terminal film signal for locating.And according to the variation of C-terminal, can be divided into 3 groups " CXX ", " CXXS " and " CXXL " (Fig. 2), this illustrates that also we have been cloned into complete gene order.

Utilize different online software (Predotar, Target P and PSORT) analyzed the Subcellular Localization of this rubber tree small G-protein Rop member HbRop3, the result shows that it may be positioned at except chloroplast(id) and extramitochondrial other organoid, has only the PSORT prediction to be positioned in the nucleus.Simultaneously, also comparative analysis the OsRacB of paddy rice, PSORT prediction is positioned at plasma membrane, nucleus or plastosome, and the HvRacB of software PSORT prediction barley is positioned in nucleus or the plastosome.Yet predicting the outcome of other two softwares is that these three albumen all are to be positioned at except in chloroplast(id) and extramitochondrial other organoid (table 2).As seen predicting the outcome of software can only be played the effect of reference, and concrete Subcellular Localization also needs concrete detection.

The prediction of table 2.HbRop3 Subcellular Localization

2.HbRop3 expression of gene research

＜1〉the expression analysis checking of HbRop3 gene in the rubber tree of different dead skin disease occurrence degrees

Dry sick (the TPD of tapped part of rubber tree, tapping panel dryness) is the comprehensive physiological disease that influences rubber tree output maximum at present, its molecular mechanism just begins one's study recently, according to HbRop3 functional clustering analytical results, infer that the gene in this latex might participate in the resistance of this disease is regulated and control.Therefore, we select for use heat to grind rubber tree (dead skin degree＜30% of 3 groups of different dead skin degree in the 7-33-97 strain; 30%＜dead skin degree＜60%; 60%＜dead skin degree ﹤ 90%) and four groups of trees of the health tree etc. around it be experiment material, detect the regulation and control of replying whether this gene participates in TPD.The calculating of TPD occurrence degree is that basis is arrheaed the scant length of glue and the ratio of secant total length, and namely TPD=arrheas the length/secant total length (%) of glue secant.This expression of gene analysis revealed, this gene may participate in these advancing of disease regulation and control.Because this gene is up-regulated along with the increase of TPD occurring degree, as seen it is relevant (Fig. 3) with this advancing of disease.

Checking is expressed by the function of organization of＜2HbRop3 gene

With Para rubber tree heat grind 7-33-97 blade, bark and latex RNA at random the cDNA of reverse transcription be template, carry out real-time fluorescence quantitative PCR with the HbRop3 gene specific primer, according to each self-corresponding Qr value (Qr=ECt (YLS8)-Ct (HbRop3), E-constant 10; The cycle number that fluorescent signal in each reaction tubes of Ct-experiences when arriving the thresholding of setting) size multiply by identical multiple simultaneously, namely obtains relative expression's abundance of different treatment.The result shows: HbRop3 expresses in latex at most, and the expression relative abundance in seed and bark lower (Fig. 4) shows that it has tangible tissue expression specificity, and may be the Rop encoding gene of performance critical function in the latex dust cell.

＜2〉rubber tapping influences the HbRop3 expression of gene

Rubber tapping can stimulate latex production, and to cut in the rubber tree this effect more obvious not opening.It is low not open the latex dust metabolic activity that cuts in the tree stem portion bark, and along with the increase of rubber tapping number of times, the latex dust metabolic activity strengthens, and final latex output has also increased.Not opening this specific character of cutting tree makes it become the ideal material identifying and study the latex regeneration associated genes.

With do not open cut latex RNA that Para rubber tree heat grinds different cuttves of 7-33-97 time at random the cDNA of reverse transcription be template, with HbRop3 gene specific primer (rRop3-F:TTATAGATTACCCTGGTGCAGTG(positive-sense strand primer, the 479-501 position Nucleotide of sequence 2 in the corresponding sequence table), rRop3-R:AGAACGTGACAAACTCGCAAC(antisense strand primer, the 846-866 position Nucleotide of sequence 2 in the corresponding sequence table)) carry out real-time fluorescence quantitative PCR, internal control gene is to select for use the gene mitogenic protein YLS8(GENBANK accession number that is fit to the latex of panama rubber tree analysis to be: HQ323250), its special real-time quantitative primer be (rYLS8-F:CCTCGTCGTCATCCGATTC, rYLS8-R:CAGGCACCTCAGTGATGTC).Shown in result such as Fig. 5 (do not cut in the rubber tree opening, cut continuously preceding 8 influences of cutting time HbRop3 genetic expression along with opening), the result shows that rubber tapping influences the HbRop3 expression of gene---significantly reduce the HbRop3 expression of gene.

＜3〉mechanical wounding is handled the gummy Ruzhong HbRop3 expression of gene influence of tapping rubber

Because rubber tapping is a kind of inevitable mechanical wounding to rubber tree, does mechanical wounding influence the HbRop3 expression of gene so? we with mechanical wounding but do not outflow the treatment process of latex, detect this expression of gene situation.The result shows: mechanical wounding is handled can obviously reduce the HbRop3 expression of gene, illustrate that it also is reason (Fig. 7 that rubber tapping influences the downward modulation expression of HbRop3 that injury is handled, ordinate zou is as HbRop3 gene relative expression quantity, be 1.0 with 0 hour expression amount, the ratio of the expression amount of all the other mechanical wounding time expression amounts and 0 hour is relative expression quantity).

＜4〉HORMONE TREATMENT influences the HbRop3 expression of gene

There is research to think, small G-protein Rop member protein genetic expression that some plant hormones are adjustable and its activity of regulation and control, and this regulating and controlling effect is brought into play central role (Moshkov in some hormone regulating and controlling growth and development of plants, I.E., et al., Ethylene regulates monomeric GTP-binding protein gene expression and activity in Arabidopsis.Plant Physiol, 2003.131 (4): p.1705-17; Zheng, Z.L., et al., Plasma membrane-associated ROP10 small GTPase is a specific negative regulator of abscisic acid responses in Arabidopsis.Plant Cell, 2002.14 (11): p.2787-97).In rubber tree produced, using ethrel (ethene sustained release dosage) at secant or face can stimulate latex output, has become the indispensable integral part of modern tapping system.

Seven kind of plant hormones or growth regulator (ethrel, jasmonic and phytokinin 6-benzyl purine 6-BA, growth hormone, dormin, Plant hormones regulators,gibberellins and Whitfield's ointment) are chosen in this research, be applied to respectively 1cm face place, rubber tree secant and secant top stimulate rubber tree (0h, 3h, 12h, 24h).The real-time fluorescence quantitative PCR result shows (interpretation of result is the same): in the seven kind of plant hormones of measuring (or growth regulator), except ethrel (ET) is reduced (Fig. 6) the HbRop3 expression of gene a little, other HORMONE TREATMENT is to the little (phytokinin (CTK of HbRop3 expression of gene influence, 6-BA) (Figure 10), jasmonic (JA) (Fig. 9), other four kinds of HORMONE TREATMENT are to HbRop3 expression of gene influence little (figure is slightly).Among Fig. 6, Fig. 9, Figure 10, ordinate zou is HbRop3 gene relative expression quantity, is 1.0 with the expression amount of handling 0 hour, and the ratio of the expression amount of all the other treatment time expression amounts and 0 hour is relative expression quantity.

＜5〉mechanical wounding is handled and is induced in the rubber tree secondary latex dust regenerative process in seedling stage HbRop3 expression of gene mutation analysis

According to reference and actual verification, injury is handled can induce the generation (Tian Weimin of secondary latex dust, Shi Minjing, Yu Fengyi, Wu Jilin, Hao Bingzhong, Cui Keming: mechanical wounding and execute jasmonic outward to the local effect of the secondary latex dust of Para rubber tree differentiation and relation (English) the .Acta Botanica Sinica 2003 (11) that distributes with jasmonic: 1366-1372 thereof; Rubber Institute, Chinese Academy of Agricultural Science's patent: predict the method .2008 of differentiation ability of rubber tree secondary milk tube a kind of seedling stage; J-L, W and H-B, Z, Laticifer Differentiation in Hevea brasiliensis:Induction by Exogenous Jasmonic Acid and Linolenic Acid.Annals of Botany, 2000.85 (1): p.37).In this experiment, (the variety certification numbering: state examines hotwork 2001001, and Rubber Institute, Chinese Academy of Agricultural Science cultivates to utilize Para rubber tree heat to grind the 8-79 strain; Rubber Institute, Chinese Academy of Agricultural Science has seedling to sell for a long time) annual branch, induce the formation of secondary latex dust with injuring the way of handling.5 groups of branches are chosen in this experiment respectively, and per 3 strains are one group.Choose the branch that the first fluffy leaf is the comparison unanimity of bronze phase or pale green phase, the stem section on the second fluffy leaf is carried out scratch and is handled.Every strain branch is in the position of symmetry, 4 the 1 * 1cm of 1cm scratch of respectively being separated by ²Processing area, totally 8 processing.Be collected in the bark of scraping the injury after 0 day, 3 days, 5 days, 7 days, the 13 day time that injury handles respectively, extract the RNA of bark, intragentic dynamic expression changes in the same period of secondary latex dust differentiation along with wounding handles the back to investigate in these barks the HbRop3 gene; And get the part bark and do the I-Br processing, follow the tracks of the situation of inducing of rubber tree secondary latex dust.The result shows that the HbRop3 gene was injury processing 3 days, 5 days, in 7 day time expression level with handle before compare remarkable downward modulation, but its expression all increases progressively along with the time, expression level at the 13rd day significantly increases, surpassed the expression level (Fig. 8 before handling, induce the atomization of secondary latex dust along with physical abuse, the HbRop3 expression of gene is analyzed, ordinate zou is as HbRop3 gene relative expression quantity, the expression amount of handling contrast with scratch not is 1.0, and all the other mechanical wounding time expression amounts are relative expression quantity with the ratio that the expression amount of contrast is handled in scratch not).Facts have proved, after annotinous branch injury is handled the 5th day or the 7th day, secondary latex dust is induced out successively.Therefore can think that these expression of gene express enhancing with the generation of secondary latex dust.

According to the influence of hormones such as the cambial active receiving growth hormone of plant, phytokinin, Plant hormones regulators,gibberellins and ethrel as can be known of report document, but nearest result of study shows the radially differentiation (Nieminen that has only phytokinin directly to influence cambial cell, K., et al., Cytokinin Signaling Regulates Cambial Development in Poplar.Proceedings of the National Academy of Sciences of the United States of America, 2008.105 (50): p.20032-37).The secondary latex dust of rubber tree is that cambial cell outwards is differentiated to form along with changes of seasons has rhythm in the bark, be the methyl jasmonic to the secondary latex dust maximum plant hormone that makes a difference, but the methyl jasmonic is handled the division (J-L that can not accelerate cambial cell, W and H-B, Z, Laticifer Differentiation in Hevea brasiliensis:Induction by Exogenous Jasmonic Acid and Linolenic Acid.Annals of Botany, 2000.85 (1): p.37.), concrete molecular mechanism also nobody was studied, although the experiment at calendar year 2001 Liu Huifang etc. has proved that phytokinin also has coordinated regulation effect (Liu Huifang to the differentiation of secondary latex dust, Wu Jilin and Hao Bingzhong, jasmonic and other hormone are to the synergy of Para rubber tree latex dust differentiation. tropical crops journal, 2001 (3)).

From detect the HbRop3 gene be subjected to hormones such as JA, 6-BA, SA and GA influence result of study as can be seen, these four kinds of HORMONE TREATMENT are to HbRop3 expression of gene influence little (Fig. 9 and 10).The HbRop3 gene be by what mode participate in the rubber tree secondary latex dust formation mechanism or remain further to be inquired into.

3. the activation analysis of prokaryotic expression and recombinant protein

Utilizing Liu's pET28a(art gold etc. the prokaryotic expression carrier of two latex invertase genes of rubber tree makes up and expression condition is optimized tropical crops journal .2010. the 7th phase) made up HbRop3 Prokaryotic Expression carrier, concrete grammar is as follows:

＜1〉contains the acquisition of Para rubber tree HbRop3 gene coding region recombinant vectors

By the coding region restriction enzyme digestion sites of Genetyx software analysis HbRop3 gene, determine the restriction enzyme (BamHI and HindIII) of two HbRop5 gene coding regions modification usefulness.Design HbRop3 gene coding region primer (5-N:C

ATGAGTGCTTCAAGGTTCATCAAGTG(positive-sense strand primer, what italic marked is the BamHI restriction enzyme site), 5-S:C

TCACAATATGGAGCAGGCCTTTTGAG(antisense strand primer, what italic marked is the HindIII restriction enzyme site), be template with pMD18-HbRop3, carry out pcr amplification, amplification program is: 95 ℃ of pre-sex change 3min; 94 ℃ of sex change 1min, 56 ℃ of annealing 1min, 72 ℃ are extended 1.5min, totally 30 circulations; 72 ℃ are extended 10min.The 94-690 position nucleotide sequence that this primer has sequence 2 in the sequence table to the fragment that increases.

Amplification obtains the product of 597bp, and order-checking shows that this fragments sequence is the 94-690 position nucleotide sequence of sequence 2 in the sequence table.This fragment used with pMD18-T be connected, obtain the pMD-HbRop3 recombinant plasmid, confirm the exactness of extension increasing sequence through order-checking.In 37 ℃ of water-baths, with BamHI and the two recombinant plasmids of cutting of HindIII, electrophoresis reclaims the purpose band about 597bp.This purpose fragment is inserted between the BamHI and HindIII restriction endonuclease sites of pET28a plasmid (the good and bio tech ltd in Shanghai), obtain recombinant vectors, recombinant vectors is carried out the enzyme evaluation of cutting and check order, evaluation is shown the recombinant vectors called after pET28a-HbRop3 of the correct 94-690 position nucleotide sequence that contains sequence 2 in the ordered list.

＜2〉expression of HbRop3 of the present invention and its functional verification

Among the recombinant vectors pET28a-HbRop3 importing E.coli BL21 (DE3) with acquisition (Lu Hai etc. the research of express recombinant protein in the e. coli bl21 (DE3). the 23rd the 6th phase of volume of the journal .2001 of Beijing Forestry University), obtain recombinant expressed bacterium, will identify that correct reorganization bacterium is cultured to OD in the LB substratum that contains 50 μ g/ml kantlex ₆₀₀=0.4～0.6, adding IPTG is 1mM to final concentration, 37 ℃ of following inducing culture, and 2h, 4h or 7h are contrast with the same reorganization bacterium of the culture medium culturing that do not add IPTG, centrifugal collection thalline, tropina carries out the 12%SDS-PAGE electrophoresis detection.The result shows, induces down this HbRop3 gene to realize efficient heterogenous expression at IPTG, and albumen has expression in supernatant liquor and inclusion body, and apparent molecular weight and the theoretical molecular of expressing protein are close, about 23.89kDa, (figure slightly).

We utilize the method for His label affinity purification to obtain having the albumen HbRop3 of GTP activity.(concrete grammar is with reference to the TALON of Clontech company behind ni-sepharose purification for protein expressioning product

The resin product description), use ³²P-mark GTP carries out GTP in conjunction with activation analysis, concrete grammar reference (Yunxia Qin, Feng Shi, Chaorong Tang ^*.Molecular characterization and expression analysis of cDNAs encoding four Rab and two Arf GTPases in the latex of Hevea brasiliensis.Plant Physiology and Biochemistry2011,49p:729-37) carry out:

1) target protein of purifying is put to nitrocellulose filter (0.45 μ m).After film dries, at first use prehybridization solution (50mM Tris-HCl, 5mM MgCl ₂, the 0.3%(concentration expressed in percentage by volume) and Tween20,0.5mM EDTA, 5m MDTT, 0.3%(concentration expressed in percentage by volume) BSA, pH7.5; ), 30 ℃ of prehybridization 30min.Change prehybridization solution, add 0.9 μ l(α- ³²P) GTP(3000 μ CimM) (contain 10 approximately ^-9M GTP), 30 ℃ of hybridization 30min.At last wash film three times for 30 ℃ with the prehybridization solution that does not contain BSA, each 5～10min suitably dries the back radioautograph.

2) target protein of purifying is put to nitrocellulose filter (0.45 μ m), carried out GTP, ATP competition according to the method described above in conjunction with test, test operation is the same.Wherein (α- ³²P) the GTP final concentration is 10 ^-9M, adding final concentration in the GTP competition experiments respectively again is 10 ^-8, 10 ^-7The heterotope mark GTP of M, namely 10 times, 10 ²Competition GTP doubly; It is 10 that the ATP competition experiments then adds final concentration respectively again ^-7, 10 ^-6The heterotope mark ATP of M, namely 10 ²Doubly, 10 ³Competition ATP doubly.Our qualitative experiment result shows that expressed recombinant protein HbRop3 has special GTP in conjunction with activity, because under the experiment condition of the nonisotopically labelled competitive GTP that has 10 times of amounts, HbRop3 obviously reduces the binding capacity of isotope-labeled GTP; The nonisotopically labelled competitive GTP of 100 times of amounts almost completely suppresses HbRop3 to the combination of isotope-labeled GTP; On the contrary, the nonisotopically labelled ATP of 100 times of amounts and 1000 times of amounts at all can not influence HbRop3 to the combination of isotope-labeled GTP, and visible HbRop3 is specifically in conjunction with GTP, and debond ATP.Therefore our functional gtp binding protein of this HbRop3 genes encoding of cloning.

4. conclusion

We have cloned the full-length cDNA of a small G-protein Rop family member gene HbRop3 first from the elastomer gum Ruzhong; It has typical structural domain feature in the plant Rop family gene; The expression analysis result shows tentatively that also it can have multi-functional (stress resistance that participates in the latex dust cell is replied, latex regenerative process and the generation etc. that promotes the secondary latex dust of rubber tree stem) in the elastomer gum Ruzhong.

This gene can be used as the important target gene of rubber tree transgenic breeding, be expected to rationally regulate the metabolism of latex dust by regulating and control this expression of gene, enhancing is to the resistance of diseases such as TPD, coordinate nourishing and growing and rubber production of rubber tree, thus the excavation rubber tree production potential of maximum potential.In addition, this gene can be used as the important function of gene resource, also may be applied in the high yield of other timber beyond the rubber tree or adversity gene engineering.

Claims (7)

1. protein, the protein of being formed by the amino acid residue sequence of the SEQ ID № .1 in the sequence table.

2. protein according to claim 1, it is characterized in that: described protein is to have GTP in conjunction with the protein of activity.

3. the encoding gene of claim 1 or 2 described protein.

4. encoding gene according to claim 3 is characterized in that: the nucleotide sequence of described encoding gene following 1) or 2) shown in:

1) SEQ ID № in the sequence table: 2 nucleotide sequence;

2) SEQ ID № in the sequence table: 2 from the nucleotide sequence shown in the Nucleotide of 5 ' terminal 94-690 position.

5. the recombinant expression vector that contains claim 3 or 4 described encoding genes.

6. the transgenosis reorganization bacterium that contains claim 3 or 4 described encoding genes.

7. the described protein of claim 1 is in conjunction with the application in the guanylic acid.

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