CN105273070B

CN105273070B - Rubber tree dead skin GAP-associated protein GAP HbMC2 and its encoding gene and application

Info

Publication number: CN105273070B
Application number: CN201510770686.7A
Authority: CN
Inventors: 刘辉; 邓治; 李德军
Original assignee: Rubber Research Institute Chinese Academy Tropical Agricultural Sciences
Current assignee: Rubber Research Institute Chinese Academy Tropical Agricultural Sciences
Priority date: 2015-11-12
Filing date: 2015-11-12
Publication date: 2018-05-15
Anticipated expiration: 2035-11-12
Also published as: CN105273070A

Abstract

The invention discloses rubber tree dead skin GAP-associated protein GAP HbMC2 and its encoding gene and application.Rubber tree dead skin GAP-associated protein GAP HbMC2 provided by the present invention is following A1) or A2)：A1) amino acid sequence is the protein shown in sequence 1 in sequence table；A2) in the amino acid sequence of sequence 1 by substitution and/or missing and/or add that one or several amino acid residues obtain have identical function as A1) derived from protein.It is demonstrated experimentally that HbMC2 encoding genes are overexpressed the death that can promote cell under oxidative stress, resistance of the biology to oxidative stress is reduced.The important target closely related, HbMC2 and its encoding gene can be prevented as rubber tree dead skin occurs with rubber tree dead skin for the expression of HbMC2 encoding genes.

Description

Rubber tree dead skin GAP-associated protein GAP HbMC2 and its encoding gene and application

Technical field

The present invention relates to rubber tree dead skin correlation GAP-associated protein GAP HbMC2 and its encoding gene and two in biological technical field Application of the person in regulating cell death, oxidative stress resistance and rubber tree dead skin occur.

Background technology

Para rubber tree (Hevea brasiliensis Muell.Arg.) is because good, economical with rubber content height, quality Long lifespan, easily harvesting etc. particular advantages and as natural rubber main source.China is the big natural rubber consumption of the first in the world State, annual consumption are up to 3,700,000 tons.Since China belongs to non-traditional Zhi Jiao areas, it is limited to plant glue region, China year gum yield less than 800000 tons, the degree of self-sufficiency is less than 22%.In the case where plant puddle ground area is limited, to ensure natural rubber supply capacity, just must The Rubber Yield of unit area must be greatly improved.In the factor for influencing per unit area yield, dead skin is one of major limitation sex factor.Mesh Before, each Zhi Jiao states plantation dead skin rate in the world is between 20%~50%, annual output loss up to 15%~20% (Venkatachalam etc., 2010).In view of dead skin seriously affects caoutchouc industry, dead skin must be solved by improving per unit area yield Problem, and it is to solve the problems, such as the premise of dead skin to illustrate dead skin mechanism, carries out rubber tree dead skin mechanism and prevention and control research With important theory and application value.

The bark of Para rubber tree is outwards differentiated to form by vascular cambium, thin with phloem ray, bast thin-walled The structure of the elementary organizations such as born of the same parents, screen casing, companion cell, bast fiber.As most important lactiferous plant, the bark of rubber tree has one Production glue tissue-latex dust of kind specialization.In natural rubber production, people by cutting the latex dust in rubber tree bark, collect by The latex of secant discharge, the raw material as extracting natural rubber.Rubber tree dead skin (Tapping Panel Dryness, TPD) Symptom reduces for secant dumping or stops to arrange completely.Rubber tapping is a kind of inevitable mechanical wounding for rubber tree.Rubber tree The origin cause of formation of dead skin is sufficiently complex, it spontaneous can both be produced, and can also be induced and produced by various internal and external factors, intensity mistake of tapping rubber Greatly, generation that is long-term and can excessively causing rubber tree dead skin using ethrel stimulation dumping, research finds living in dead skin rubber tree Property oxygen level rise.Researcher was once explored and had been studied from pathology, physiology, heredity, soil and biochemistry etc., but it is sent out Life system is still unclear.At present, dead skin is considered as that a kind of physiology caused, complicated by excessively rubber tapping and the stimulation of strong ethene is comprehensive Close disease (Fan Siwei and Yang Shaoqiong, 1995).In recent years, as molecular biology and related gene engineering technology are in rubber tree physiology Application in Biochemical Research, the research to rubber tree dead skin mechanism achieve preliminary progress, thus it is speculated that active oxygen metabolism, general Element-proteasome pathway, programmed cell death (Programed Cell Death, PCD), jasmonic biosynthesis and rubber The gene expression such as biosynthesis pathway change is related with rubber tree dead skin generation.Chen etc. (2003) is identified in rubber tree bark And dead skin related gene HbMyb1 is cloned, which is probably the negative regulatory factor of programmed cell death, and proposes " rubber tree Dead skin is a kind of programmed cell death phenomenon " viewpoint.Peng etc. (2011) further study showed that：With healthy rubber tree phase Than dead skin rubber tree duct cell has the characteristic feature of programmed cell death, and overexpression HbMyb1 can press down in tobacco The cell death of adverse circumstance induction processed.Venkatachalam etc. (2007), Li et al. (2010；2012) utilized with deep green grade (2012) Inhibition subtractive hybridizes and biochip technology divides gene expression difference between dead skin and healthy latex of panama rubber tree or bark Analysis, find largely with the relevant gene of programmed cell death approach differential expression between, it was demonstrated that it is cellular programming dead Die and play an important role in rubber tree dead skin generating process.Important gene is in rubber tree in research programmed cell death approach Effect during dead skin occurs will be helpful to the parsing for the molecular mechanism that dead skin occurs, and can be provided for rubber tree dead skin prevention and control important Target gene.

During zooblast programmed cell death, Caspase (cysteine-dependent Aspartate-specific proteases, caspase) play key effect (Lam and Zhang, 2012).Research is aobvious Show, some plants can produce the material of similar caspase activity, and this enzymatic activity regulates and controls programmed cell death mistake Journey.Nevertheless, the protein homologous with caspase is not found in plant, however, it was found that there is one kind to contain caspase The half Guang asparagus fern albumen (caspase-like protein) of class of domain (caspase domain), is known as metacaspase (MC).Metacaspases is present in cyanobacteria, fungi, yeast and higher plant (Jiang etc., 2010).According to caspase- The sequence similarity and sequential structure of like functional areas, metacaspases are segmented into 2 types, i.e. I types and II types (Uren Deng 2000).Except contain about 150 amino acid similar to caspase large subunits conservative region and existing for C-terminal Outside the second conservative region similar to caspase small subunits, the I type metacaspase albumen n ends from plant and fungi Prodomain contains 1 specific amino acid die body, and (motif, is typically the repetition die body (proline- of Pro-rich Rich repeat motif) or zinc finger die body (zinc finger motif).This zinc fingers and plant hypersensitivity Relevant albumen LSD-1 is similar.II types metacaspase does not have prodomain in plant, but sub- single in p20 and p10 two A fragment for being about 200 amino acid residues (Ma Cong and hole Balakrishnan, 2012) is inserted between position.

Although metacaspase does not show class caspase activity, they are still in plant programmed cell death During play a role (Zhang and Lam, 2011).Hoeberichts etc. (2003) is in the microbial tomato leaf of Botrytis cinerea Observe that II type metacaspase gene LeMCA1 expression quantity is significantly raised, illustrates that metacaspase take part in hypersensitivity The hypersensitivity of plant disease-resistant.In tobacco, overexpression metacaspase genes NbMC1 can strengthen tobacco to destroying anthrax The resistance (Hao etc., 2007) of bacterium.It is thin that the downward of European spruce II types metacaspase can suppress its embry ogenesis period suspensor The programmed cell death process (Suarez etc., 2004) of born of the same parents.9 metacaspase genes are co-existed in arabidopsis gene group, Wherein, two I type metacaspase genes (AtMC1 and AtMC2) have been shown to regulating cell programmed cell death process, AtMC1 is positive regulatory factor, and AtMC2 is negative regulatory factor (Coll etc., 2010).Watanabe and Lam (2011) are proved In biology and the arabidopsis cell programmed cell death of abiotic stress induction, AtMC4 plays the effect of positive regulatory factor.It is small In wheat the homologous gene TaMC4 of the gene also be proved strip rust bacteria induction programmed cell death process (Wang etc., 2012).TMC8 is in UVC and H for arabidopsis II type metacaspase Gene As₂O₂Expressed during the programmed cell death of mediation Up-regulation, the missing of the gene can weaken cell death, thus it is speculated that the arabidopsis cell sequencing that AtMC8 take part in Oxdative stress induction is dead Die process (He etc., 2008).Ahmad etc. (2012) it has also been found that corn II type metacaspase genes expression and activity by smelly Oxdative stress and the induction of aging, the protein of metacaspase mediations hydrolyze what is induced in blade reply ozone stress and age Play an important role in aging.Arabidopsis metacaspase Gene As tMC9 is then special to express in the xylem of development, participates in The dead regulation and control (Bollhoner etc., 2013) of tracheid.These results of study show, metacaspase gene family members Key effect is played during the programmed cell death of development, biology or abiotic stress induction.

Up to the present, still reported without the research in relation to rubber tree metacaspase family genes.It is cellular programming dead Die and play an important role in rubber tree dead skin generation, and metacaspase family genes rise in programmed cell death Important regulating and controlling effect.Metacaspase family members may take part in rubber tree dead skin and regulate and control.

The content of the invention

The technical problems to be solved by the invention be how the death of regulating cell, and how to suppress or prevent rubber tree Dead skin.

In order to solve the above technical problems, present invention firstly provides a kind of and relevant protein of rubber tree dead skin, its name Referred to as HbMC2, from the Para rubber tree (Hevea brasiliensis Muell.Arg.) of Euphorbiaceae Hevea, is Following A1) or A2)：

A1) amino acid sequence is the protein of sequence 1 in sequence table；

A2 it is) residual by substituting and/or lacking and/or add one or several amino acid in the amino acid sequence of sequence 1 Base obtain have identical function as A1) derived from protein；

Wherein, sequence 1 is made of 361 amino acid.

In order to make A1) in protein easy to purifying, the amino terminal of protein that can be in sequence table shown in sequence 1 or The upper label as shown in Table 1 of carboxyl terminal connection.

The sequence of 1. label of table

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

Above-mentioned A2) in HbMC2 can be artificial synthesized, also can first synthesize its encoding gene, then carry out biological expression and obtain.On State A2) in the encoding gene of HbMC2 can be by the way that one or several ammonia will be lacked in the DNA sequence dna shown in sequence in sequence table 2 The codon of base acid residue, and/or the missense mutation of one or several base-pairs is carried out, and/or connect at its 5 ' end and/or 3 ' ends The coded sequence of label shown in upper table 1 obtains.

In order to solve the above technical problems, present invention also offers with the above-mentioned relevant biomaterial of HbMC2 albumen, the life Any of thing material is following B1) to B12)：

B1 the nucleic acid molecules of HbMC2) are encoded；

B2 B1) is contained) expression cassettes of the nucleic acid molecules；

B3 B1) is contained) recombinant vectors of the nucleic acid molecules；

B4 B2) is contained) recombinant vector of the expression cassette；

B5 B1) is contained) recombinant microorganisms of the nucleic acid molecules；

B6 B2) is contained) recombinant microorganism of the expression cassette；

B7 B3) is contained) recombinant microorganism of the recombinant vector；

B8 B4) is contained) recombinant microorganism of the recombinant vector；

B9 B1) is contained) the transgenic plant cells systems of the nucleic acid molecules；

B10 B2) is contained) the transgenic plant cells system of the expression cassette；

B11 B3) is contained) the transgenic plant cells system of the recombinant vector；

B12 B4) is contained) the transgenic plant cells system of the recombinant vector.

In above-mentioned biomaterial, B1) nucleic acid molecules can be following b1) b2) or b3) or gene b4)：

B1) nucleotide sequence is the cDNA molecules of 98-1183 or DNA molecular of sequence 2 in sequence table；

B2) nucleotide sequence is the cDNA molecules or DNA molecular of sequence 2 in sequence table；

B3) and b1) or b2) nucleotide sequence that limits has more than 90% homogeneity, and the cDNA molecules of coding HbMC2 Or genomic DNA molecule；

B4) under strict conditions with b1) or b2) limit nucleotide sequence hybridization, and encode HbMC2 cDNA molecules or Genomic DNA molecule.

Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules also may be used To be RNA, such as mRNA or hnRNA.

Wherein, sequence 2 is made of 1267 nucleotide, and 98-1183 are code area, shown in coded sequence 1 HbMC2。

Those of ordinary skill in the art can easily use known method, such as the side of orthogenesis and point mutation Method, is mutated the nucleotide sequence of the coding HbMC2 of the present invention.Those have and the present invention point by manually modified From the obtained nucleotide sequence 90% of coding HbMC2 or the nucleotide of higher homogeneity, as long as encoding HbMC2 and having HbMC2 functions, are the nucleotide sequence derived from the present invention and are equal to the sequence of the present invention.

Term " homogeneity " used herein refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes and this hair The nucleotide sequence of the protein of amino acid sequence composition shown in bright coding HbMC2 has 90% or a higher, or 95% or The nucleotide sequence of higher homogeneity.Homogeneity can with the naked eye or computer software is evaluated.Using computer software, two Homogeneity between a or multiple sequences can use percentage (%) to represent, it can be same between correlated series for evaluating Property.

Above-mentioned stringent condition is in 2 × SSC, the solution of 0.1%SDS, hybridizes at 68 DEG C and washes film 2 times, every time 5min, and in 0.5 × SSC, the solution of 0.1%SDS, hybridize at 68 DEG C and wash film 2 times, each 15min；Or, 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS solution in, hybridize under the conditions of 65 DEG C and wash film.

Above-mentioned more than 90% homogeneity, can be 90% or more than 95% homogeneity.

In above-mentioned biomaterial, B2) described in the nucleic acid molecules containing coding HbMC2 expression cassette (HbMC2 gene expressions Box), it is the DNA for referring to express HbMC2 in host cell, which not only may include the startup for starting HbMC2 genetic transcriptions Son, may also include the terminator for terminating HbMC2 genetic transcriptions.Further, the expression cassette may also include enhancer sequence.It can use Include but not limited in the promoter of the present invention：Constitutive promoter, is organized, the promoter that organ and development are special, and induction Type promoter.The example of promoter includes but not limited to：The constitutive promoter 35S of cauliflower mosaic virus；From tomato Wound-inducible promoter, leucine aminopeptidase (" LAP ", Chao et al. (1999) Plant Physiol 120：979- 992)；Chemical inducible promoter from tobacco, pathogenesis correlation 1 (PR1) (by salicylic acid and BTH (diazosulfide- 7- carbothioic acid S-methyl esters) induction)；Tomato protease inhibitors II promoters (PIN2) or LAP promoters (available jasmine Ketone acid methyl esters induces)；Heat-shock promoters (United States Patent (USP) 5,187,267)；Tetracycline inducible promoter (United States Patent (USP) 5, 057,422)；Seed specific promoters, such as Millet Seed specificity promoter pF128 (CN101063139B (Chinese patents 200710099169.7)), the special promoter of seed storage protein matter (for example, phaseolin, napin, oleosin and big (Beachy et al. (1985) EMBO is J.4 for the promoter of beans beta conglycin：3047-3053)).They can be used alone Or it is used in combination with other plant promoters.All references cited herein is cited in full text.Suitable tanscription termination Son includes but not limited to：Agrobacterium nopaline syntase terminator (NOS terminator), cauliflower mosaic virus CaMV35S are terminated Son, tml terminators, pea rbcS E9 terminators and nopaline and octopine synthase terminator (see, e.g.：Odell Et al. (1985) Nature 313:810；Rosenberg et al. (1987) Gene, 56:125；Guerineau et al. (1991) Mol.Gen.Genet,262:141；Proudfoot(1991)Cell,64:671；Sanfacon et al. Genes Dev., 5: 141；Mogen et al. (1990) Plant Cell, 2:1261；Munroe et al. (1990) Gene, 91:151；Ballad et al. (1989)Nucleic Acids Res.17:7891；Joshi et al. (1987) Nucleic Acid Res., 15:9627).

The recombinant vector of the HbMC2 expression casettes can be contained with existing expression vector establishment.The plant expression Carrier includes double base agrobacterium vector and the carrier available for plant micropellet bombardment etc..As pAHC25, pBin438, PCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or PCAMBIA1391-Xb (CAMBIA companies) etc..The plant expression vector can also include 3 ' end non-translational regions of foreign gene Domain, i.e., comprising polyadenylation signals and the DNA fragmentation of any other participation mRNA processing or gene expression.The polyadenylic acid letter Number bootable polyadenylic acid is added to 3 ' ends of mRNA precursor, as Agrobacterium crown gall nodule induces (Ti) plasmid gene (such as nopaline Synthase gene Nos), plant gene (such as soybean storage protein genes) 3 ' end transcription non-translational region be respectively provided with similar functions. During using gene constructed plant expression vector of the invention, enhancer, including translational enhancer or transcriptional enhancer also can be used, These enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., but must be read with coded sequence Frame is identical, to ensure the correct translation of whole sequence.The source of the translation control signal and initiation codon be it is extensive, It can be natural or synthesis.Translation initiation region can come from transcription initiation region or structural gene.In order to just In transgenic plant cells or plant are identified and screened, plant expression vector used can be processed, can as added The coding expressed in plant can produce the enzyme of color change or gene (gus gene, the luciferase genes of luminophor Deng), the marker gene of antibiotic (as assigned the nptII genes to kanamycins and associated antibiotic resistance, assigned to herbicide The bar genes of phosphinothricin resistance, assign the hph genes to antibiotic hygromycin resistance, and assign to methotrexate resistance Dhfr genes, assign the EPSPS genes to glyphosate) or (such as anti-herbicide base such as anti-chemical reagent marker gene Cause), provide metabolism mannose ability mannose-6-phosphate isomerase gene., can not from the security consideration of genetically modified plants Add any selected marker, transformed plant is directly screened with adverse circumstance.

In above-mentioned biomaterial, the carrier can be plasmid, sticking grain, bacteriophage or viral vector.The plasmid can be ferment Female expression vector pYES2.

In above-mentioned biomaterial, the microorganism can be yeast, bacterium, algae or fungi, such as Escherichia coli.The yeast can For Wine brewing yeast strain INVScl.

In above-mentioned biomaterial, the transgenic plant cells system does not include propagating materials.

In an embodiment of the invention, the encoding gene of HbMC2 passes through the restructuring containing HbMC2 expression casettes Recombinant microorganism is obtained in vector introduction Wine brewing yeast strain INVScl.The recombinant vector is by Yeast expression carrier pYES2 BamH I and Not I recognition sites between sequence replace with DNA in sequence table shown in the 98-1183 nucleotide of sequence 2 Molecule, obtained recombinant vector.HbMC2 shown in the recombinant vector expressed sequence 1.The recombinant microorganisms express sequence 1 Shown HbMC2.

In order to solve the above technical problems, the present invention provides HbMC2 or its relevant biomaterial in following C1)-C5) appoint Application in one：

C1) regulating cell is dead；

C2 biological oxidation stress resistance) is regulated and controled；

C3 rubber tree dead skin) is regulated and controled；

C4) the rubber tree kind that selection and breeding dead skin incidence reduces；

C5 the prevention product of rubber tree dead skin) is prepared.

In above application, the dead skin can be the dead skin that occurs under natural conditions, or the dead skin that incitant induces； The cell death can be the cell death of cell death incitant induction.The cell death incitant and the dead skin Incitant can be injury, hydrogen peroxide or ethephon (CEPHA),2-(chloroethyl) phosphonic acid.Described injury can be mechanical wounding and/or injure naturally.The oxygen Change the oxidative stress that stress can be hydrogen peroxide-induced.

In above application, the cell can be microbial cell or plant cell.The microbial cell can be that yeast is thin Born of the same parents；The plant cell can be the cell of rubber tree bark.

In order to solve the above technical problems, present invention also offers HbMC2 or B1) nucleic acid molecules are preventing as target Application in rubber tree dead skin.

HbMC2 or B1 provided by the present invention) application of the nucleic acid molecules as target in rubber tree dead skin is prevented For following any purposes：

I1 application of the material of HbMC2 expression in rubber tree dead skin is prevented) is suppressed；

I2 application of the material of the transcription of HbMC2 encoding genes in rubber tree dead skin is prevented) is suppressed；

I3 application of the material of the expression of HbMC2 in rubber tree dead skin is prevented) is reduced；

I4 application of the material of the transcriptional level of HbMC2 encoding genes in rubber tree dead skin is prevented) is reduced；

I5 application of the material of HbMC2 inactivations in rubber tree dead skin is prevented) is made.

In above application, the dead skin can be the dead skin that occurs under natural conditions, or dead skin incitant induces Dead skin；The dead skin incitant can be injury, hydrogen peroxide or ethephon (CEPHA),2-(chloroethyl) phosphonic acid.Described injury can be mechanical wounding and/or from So injury.

In above application, the HbMC2 encoding genes can be B1) nucleic acid molecules.

There is following D1)-D5) at least one of material prepare prevent rubber tree dead skin product in application Belong to the scope of protection of the invention：

D1 the expression of HbMC2) is suppressed；

D2 B1) is suppressed) expression of the nucleic acid molecules；

D3 the expression of HbMC2) is reduced；

D4 B1) is reduced) expression of the nucleic acid molecules；

D5) HbMC2 is inactivated.

In order to solve the above technical problems, present invention also offers following E1)-E4) in any method：

E1 the method for) cultivating the genetically modified organism that oxidative stress resistance reduces, includes the following steps：Led into receptor biological Enter B1) nucleic acid molecules, obtain genetically modified organism；The genetically modified organism is compared with the receptor biological to oxidative stress Resistance reduces；

E2 the method that oxidative stress resistance improves genetically modified organism) is cultivated, is included the following steps：Knock out in purpose biology B1) nucleic acid molecules or suppress B1 in the purpose biology) expression of the nucleic acid molecules, obtain genetically modified organism；It is described The resistance that genetically modified organism compares oxidative stress with the purpose biofacies improves；

E3 the method that genetically modified organism occurs for anti-dead skin) is cultivated, is included the following steps：Knock out B1 in purpose biology) it is described Nucleic acid molecules suppress B1 in the purpose biology) expression of the nucleic acid molecules, obtain genetically modified organism；The transgenosis life Dead skin incidence in thing colony is less than the dead skin incidence in the purpose biocenose；

E4 the method that genetically modified organism occurs for non-anti- dead skin) is cultivated, is included the following steps：B1 is imported into receptor biological) The nucleic acid molecules, obtain genetically modified organism；Dead skin incidence in the genetically modified organism colony is higher than purpose biology Dead skin incidence in colony.

In the above method, B1) nucleic acid molecules can be modified first as follows, then import in the receptor biological, with up to To more preferable expression effect：

1) modified and optimized according to actual needs, so that gene efficient expression；For example, can be according to recipient plant institute partially The codon of love, changes its codon to meet while the amino acid sequence of HbMC2 encoding genes of the present invention is kept Plant-preference；In optimization process, it is desirable that certain G/C content is kept in the coded sequence after optimization, to be best implemented with The high level expression of quiding gene in plant, wherein G/C content can be 35%, more than 45%, more than 50% or more than about 60%；

2) gene order of neighbouring initial methionine is modified, so that translation effectively starting；For example, using in plant The effective sequence known is modified；

3) promoter with the expression of various plants is connected, in favor of its expression in plant；The promoter may include Composing type, induction type, sequential adjusting, growth adjustment, Chemical Regulation, tissue are preferably and tissue-specific promoter；Promoter Selection will be needed and changed with expression time and space, and also depend on target kind；Such as the specificity of tissue or organ Promoter is expressed, acceptor as needed is depending on what period of development；Although demonstrate many from dicotyledon Promoter can act in monocotyledon, and vice versa, but it is desirable to select dicot promoters are used for Expression in dicotyledon, monocotyledonous promoter are used for the expression in monocotyledon；

4) it is connected with suitable transcription terminator, the expression efficiency of gene of the present invention can also be improved；Such as from The tml of CaMV, from the E9 of rbcS；Any known available terminator to work in plant can be with the present invention Gene is attached；

5) enhancer sequence is introduced, such as intron sequences (such as from Adhl and bronzel) and viral leader sequence (such as from TMV, MCMV and AMV).

In the above method, the genetically modified plants are interpreted as not only including obtain the genetic transformation recipient plant the Generation genetically modified plants, also including its filial generation.For genetically modified plants, the gene can be bred in the species, it is also possible to often Other kinds that the gene transfer is entered same species by breeding technique are advised, particularly including in commercial variety.The transgenosis is planted Thing includes seed, callus, intact plant and cell.

In the above method, the HbMC2 encoding genes can be B1) nucleic acid molecules.

In the above method, the purpose is biological or the receptor biological can be microorganism and plant.The microorganism is specific Can be yeast, the plant concretely rubber tree.

In the above method, the dead skin can be the dead skin that occurs under natural conditions, or dead skin incitant induces Dead skin.The dead skin incitant can be injury, hydrogen peroxide or ethephon (CEPHA),2-(chloroethyl) phosphonic acid.Described injury can be mechanical wounding and/or from So injury.The oxidative stress can be the oxidative stress of hydrogen peroxide-induced.

It is demonstrated experimentally that the HbMC2 and its encoding gene of the present invention can reduce the oxidative stress resistance of biology, promote oxidation The death of the lower cell of stress：Without H₂O₂During processing, the recombination yeast INVSc1 (pYES2- containing HbMC2 encoding genes HbMC2 lifes of the recombination yeast INVSc1 (pYES2) in liquid and solid inducing culture) and without HbMC2 encoding genes Long no notable difference.The recombination yeast INVSc1 (pYES2-HbMC2) of equivalent and INVSc1 (pYES2) are inoculated into 3mM H₂O₂Solid inducing culture on, 30 DEG C culture 3 days after, find H₂O₂Under Stress treatment, recombination yeast INVSc1 (pYES2- HbMC2 bacterial plaque number) will be considerably less than INVSc1 (pYES2).By the recombination yeast INVSc1 (pYES2-HbMC2) of equivalent and INVSc1 (pYES2) is inoculated into 3mM H₂O₂Liquid inducing culture in, 30 DEG C, 200rpm shaken cultivations, in different time points Measure bacterium solution OD₆₀₀Value.The result shows that H₂O₂After handling 24h, the OD of INVSc1 (pYES2-HbMC2)₆₀₀Value is significantly less than INVSc1 (pYES2), and as processing time extends, this gap is gradually increased, when handling 48h, INVSc1 (pYES2) bacterium The OD of liquid₆₀₀Value is about 10 times of INVSc1 (pYES2-HbMC2).Further study show that high concentration H₂O₂(10mM) handles 6h Afterwards, the cell mortality of recombination yeast INVSc1 (pYES2-HbMC2) is apparently higher than INVSc1 (pYES2).Result above shows, HbMC2 genes are expressed in yeast can suppress growths of the recombination yeast INVSc1 (pYES2-HbMC2) under oxidative stress, improve Cell mortality under oxidative stress, reduces resistance of the recombination yeast to oxidative stress.

Quantitative fluorescent PCR analysis shows, expression of the HbMC2 genes in dead skin rubber tree bark are higher than healthy rubber Tree, and the expression of HbMC2 genes is injured by dead skin inducible factor, hydrogen peroxide and ethephon-induction, shows the table of HbMC2 genes It is closely related up to occurring with rubber tree dead skin.The Function Identification of HbMC2 genes is shown using yeast expression system, HbMC2 bases Because of positive regulation and control H₂O₂The cell death of mediation.Result above shows that rubber tree HbMC2 genes play important work in dead skin generation With the up-regulation of the gene expression can cause cell death, and then trigger the generation of dead skin.This not only enriches rubber tree dead skin hair Raw molecular mechanism, the also effect for further research apoptosis in rubber tree dead skin generation are laid a good foundation. HbMC2 and its encoding gene can be also expected to by regulating and controlling HbMC2 and its coding base as the important target of rubber tree dead skin prevention and control The expression of cause prevents rubber tree dead skin, including:(1) antisense expression, RNAi and clpp gene of the structure for HbMC2 encoding genes Except carrier, and it is transformed into rubber tree, is screened by suppressing the expression of HbMC2 genes or knocking out the cultivation of HbMC2 genes The transgenosis rubber tree reduced to dead skin incidence；(2) rubber tree dead skin prevention is developed as target using HbMC2 or its encoding gene Medicine.

Brief description of the drawings

Fig. 1 is rubber tree HbMC2 albumen conserved domain prediction results.

Fig. 2 is the systematic evolution tree of rubber tree HbMC2 and other plant metacaspase albumen.

Fig. 3 is differential expression analysis of the rubber tree HbMC2 genes in health and dead skin latex of panama rubber tree and bark.

Wherein " it is notable that * * " represent that the expression quantity of the HbMC2 genes in 0.01 level has between health tree and Dry bark tree Difference.

Fig. 4 is influence of the injury processing to HbMC2 gene expressions in latex.

Fig. 5 is H₂O₂The expression of HbMC2 genes changes in latex after processing.Wherein, " * " and " * * " represent respectively processing with Control (0h) has significant difference in 0.05 and 0.01 level.

Fig. 6 is the expression change of HbMC2 genes in latex after ethephon (CEPHA),2-(chloroethyl) phosphonic acid is handled.

Wherein " * " and " * * " represent that processing with compareing (0h) has significant difference in 0.05 and 0.01 level respectively.

Fig. 7 is the detection of expression of HbMC2 genes in different induction time point recombination yeasts.

Wherein 1,2 and 3 are respectively to compare HbMC2 genes spy when recombination yeast INVSc1 (pYES2) induces 24,36 and 48h The testing result of different primer；4th, 5 and 6 be respectively HbMC2 when recombination yeast INVSc1 (pYES2-HbMC2) induces 24,36 and 48h The testing result of gene specific primer.

Fig. 8 is the growth differences of recombination yeast INVSc1 (pYES2-HbMC2) and INVSc1 (pYES2) under oxidative stress. Wherein HbMC2 represents recombination yeast INVSc1 (pYES2-HbMC2)；PYES2 is control INVSc1 (pYES2)；A figures are control group (i.e. without H₂O₂Oxidative stress processing)；B figures are oxidative stress (3mM H₂O₂) processing.

Fig. 9 is the bacterial concentration of recombination yeast INVSc1 (pYES2-HbMC2) and INVSc1 (pYES2) under oxidative stress (OD₆₀₀Value) comparison in difference.Wherein HbMC2-CK and pYES2-CK is respectively recombination yeast INVSc1 (pYES2-HbMC2) and right According to INVSc1 (pYES2) in normal condition (without H₂O₂Oxidative stress processing) under growth curve；HbMC2-3 and pYES2-3 points Not Wei recombination yeast INVSc1 (pYES2-HbMC2) and control INVSc1 (pYES2) in oxidative stress (3mM H₂O₂) processing under Growth curve.

After Figure 10 handles 6h for oxidative stress, recombination yeast INVSc1 (pYES2-HbMC2) and INVSc1 (pYES2's) deposits Situation living.Wherein HbMC2 represents recombination yeast INVSc1 (pYES2-HbMC2)；PYES2 represents control INVSc1 (pYES2).A Figure is control group (without H₂O₂The recombinant bacterium of Stress treatment)；B figures are 10mM H₂O₂Handle the recombinant bacterium after 6h.

Embodiment

The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining The bright present invention, the scope being not intended to be limiting of the invention.

Experimental method in following embodiments, is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples, is commercially available unless otherwise specified.

It is (yellow that Para rubber tree (Hevea brasiliensis Muell.Arg.) heat in following embodiments grinds 7-33-97 Magnificent grandson, Liang Maohuan, Wu Yuntong, Li Deshun, what grinds the selection and breeding of 7-33-97 into scale popularization level rubber tree improved seeds heat in prestige Tropical crops journal, 1994,02:1-6.), it is Rubber Institute, Chinese Academy of Agricultural Science's product.

YPD fluid nutrient mediums in following embodiments are the aseptic liquid nutrient medium being made of solute and solvent, solute and Its concentration is 1% (mass percent concentration) yeast extract, 2% (mass percent concentration) peptone and 2% (mass percent is dense Degree) glucose, solvent is water.

SC-Ura in following embodiments^-Fluid nutrient medium for lack urinary ammonia acid sterilised yeast minimal medium, 1L SC-Ura^-Fluid nutrient medium is that (yeast nitrogen base, YNB, are free of the basic nitrogen source of addition yeast into deionized water Amino acid) 6.7g, glucose 20g, adenine, arginine, cysteine, leucine, lysine, threonine and tryptophan are each 0.1g, aspartic acid, histidine, isoleucine, methionine, phenylalanine, proline, serine, tyrosine and valine Each 0.05g, and it is settled to the culture medium that 1L is obtained.1L SC-Ura^-Solid medium is to be added into above-mentioned fluid nutrient medium The culture medium that 20g agar powders obtain.

Liquid inducing culture in following embodiments is the SC-Ura using 2% galactolipin as carbon source^-Fluid nutrient medium, 1L Liquid inducing culture is that the basic nitrogen source of yeast (yeast nitrogen base, YNB, without amino are added into deionized water Acid) 6.7g, galactolipin 20g, adenine, arginine, cysteine, leucine, lysine, threonine and each 0.1g of tryptophan, Aspartic acid, histidine, isoleucine, methionine, phenylalanine, proline, serine, tyrosine and valine are each 0.05g, and it is settled to the culture medium that 1L is obtained.1L solids inducing culture is to add 20g agar powders to above-mentioned inducing culture Obtained culture medium.

0mM H in following embodiments₂O₂Solid inducing culture be above-mentioned solid inducing culture；2mM H₂O₂ Solid inducing culture to add H into solid inducing culture₂O₂, obtained H₂O₂Concentration be 2mM solid induction training Support base；2.5mM H₂O₂Solid inducing culture to add H into solid inducing culture₂O₂, obtained H₂O₂Concentration be The solid inducing culture of 2.5mM；3mM H₂O₂Solid inducing culture to add H into solid inducing culture₂O₂, obtain The H arrived₂O₂Concentration be 3mM solid inducing culture.

2mM H in following embodiments₂O₂Liquid inducing culture to add H into liquid inducing culture₂O₂, obtain The H arrived₂O₂Concentration be 2mM liquid inducing culture；2.5mM H₂O₂Liquid inducing culture be to liquid Fiber differentiation H is added in base₂O₂, obtained H₂O₂Concentration be 2.5mM liquid inducing culture；3mM H₂O₂Liquid inducing culture be H is added into liquid inducing culture₂O₂, obtained H₂O₂Concentration be 3mM liquid inducing culture.

0mM H in following embodiments₂O₂Aqueous solution is sterile distilled water.5mM and 10mM H₂O₂Aqueous solution is respectively H is added into sterile distilled water₂O₂, obtained H₂O₂Concentration be respectively 5mM and 10mM aqueous solution.

The acquisition and sequence analysis of 1. rubber tree dead skin GAP-associated protein GAP HbMC2 of embodiment and its encoding gene

Applicant utilizes the gene expression difference between RNA-Seq technical Analysis health and dead skin rubber tree bark, finds One EST differential expressions between health and dead skin rubber tree bark, the high expression in dead skin rubber tree bark.Then applicant is with this Est sequence is probe, to Para rubber tree EST (Expressed Sequence Tags), TSA (Transcriptome Shotgun Assembly) and genome database carry out it is homologous compare analysis, by electronic splicing and bioinformatic analysis, A gene order for including complete coding region, gene code metacaspase albumen are obtained from Para rubber tree, therefore is incited somebody to action It is named as HbMC2, is HbMC2 genes by the unnamed gene for encoding the albumen.

The accuracy of HbMC2 gene orders is obtained for verification, (Beijing hundred is safe using generic plant total RNA extraction reagent box Gram Bioisystech Co., Ltd) extraction Para rubber tree heat grinds 7-33-97 bark total serum IgEs, removed using DNase I in RNA DNA, with reference to RevertAid^TMFirst Strand cDNA Synthesis Kit (Fermentas companies) specification synthesis the One chain cDNA.Primers (forward primer sequence by inference：5′-TGATTTTCCAGTCCTGATCCTGA-3′；Reversely Primer sequence：5 '-ACAGCCAGTCAGTTGCAAAC-3 '), using bark cDNA as template, pass through PCR amplification target gene.PCR Reaction system is：2 μ L of cDNA templates,5 μ L, 2.5mM dNTPs of FastPfu Buffer, 2 μ L, just, Reverse primer (10 μm of ol/L) each 0.5 μ L,FastPfu DNA Polymerase (2.5U/ μ L) 0.5 μ L, add ddH₂O to 25 μ L of cumulative volume.Response procedures are：95 DEG C of pre-degeneration 3min；95 DEG C of denaturation 20s, 55 DEG C of annealing 20s, 72 DEG C extend 1min 30s, set 35 circulations altogether；72 DEG C of extension 10min.

1% agarose gel electrophoresis detection is carried out to PCR product, the results showed that the band of about 1200bp or so is obtained, with Target is consistent.Band is cut, the fragment is recycled with Ago-Gel QIAquick Gel Extraction Kit (OMEGA companies).Then will purifying DNA fragmentation be connected to carrier(the full formula gold biotechnology in Beijing is limited by-Blunt Simple Cloning Vector Company), escherichia coli DH5a competent cell is converted, picking positive colony, serves the sequencing of Hai Shenggong bioengineering Co., Ltd.

Sequencing result shows, the long 1267bp of nucleotide sequence of the HbMC2 gene cDNAs of acquisition, nucleotide sequence such as sequence In table shown in sequence 2.The coded sequence of HbMC2 genes is as shown in 98-1183 nucleotide of sequence 2 in sequence table, coding Protein HbMC2 shown in sequence 1.Wherein, sequence 2 is the cDNA sequence of HbMC2 genes.

Prediction HbMC2 molecular weight of albumen is 39.55kD, and theoretical isoelectric point is 6.59.Utilize NCBI conserved structure numeric field datas Storehouse (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) HbMC2 amino acid is guarded Structure domain analysis, it is found that the albumen contains LSD1 Zinc finger domains and caspase domains (Peptidase-C14) (Fig. 1), Show that HbMC2 albumen belongs to I type metacaspase family proteins.HbMC2 amino acid sequences are carried out on NCBI websites Blastp homogeneity compares analysis, and choose with homologous HbMC2 other plant metacaspase albumen using ClustalX and MEGA6.0 Software on Drawing chadograms, the results showed that：HbMC2 and Jatropha curcas JcMC1, apple MdMC1, tomato SlMC1, potato StMC1-like and grape VvMC1 Characterizations are nearer, positioned at same sub- branch (Fig. 2).In arabidopsis with HbMC2 albumen relationships Relation it is nearest be AtMC1.

The expression pattern analysis of 2. rubber tree dead skin related gene HbMC2 of embodiment

(1) HbMC2 genes high expression in dead skin rubber tree

Applicant grinds the bark and latex cDNA of 7-33-97 health and dead skin rubber tree as mould using Para rubber tree heat respectively Plate carries out real-time fluorescence quantitative PCR, and experiment in triplicate, repeats comprising the following steps that for experiment every time：

To grind the total serum IgE extracted in 7-33-97 health and dead skin rubber tree bark and latex from rubber tree heat respectively The cDNA of reverse transcription is template, and with gene specific primer, (forward primer is 5 '-CTCCTAATTCCTCTTACGCGGG-3 ', reversely Primer is 5 '-TTCTTCCTACCGTGCGCATT-3 ') carry out real-time fluorescence quantitative PCR analysis.Real-time fluorescence quantitative PCR exists Bio-Rad CFX96 fluorescence quantitative PCR instruments carry out, and a parallel test sets 3 repetitions.Reaction system：Premix Ex Taq^TM(2 ×) 10 μ L, forward and reverse each 2 μ L of 1 μ L, cDNA template of primer, mends ddH₂O to 20 μ L of cumulative volume.Reaction condition：95℃ 5min；95℃10s；58℃30s；Melting curve analysis is carried out after 40 circulations, to determine the specificity of primer.With paragutta 18S rRNA genes are set as reference gene, the forward and reverse primer sequence of reference gene is respectively 5 '- GCTCGAAGACGATCAGATACC-3 ' and 5 '-TTCAGCCTTGCGACCATAC-3 '.The calculating of gene relative expression quantity uses Formula 2^–ΔΔCt.Data processing uses 12.0 softwares of SigmaPlot with mapping, and relative expression quantity result is averaged for 3 repetitions Value ± SD, significance difference analysis use Student ' s t test.

HbMC2 genes express that the results are shown in Figure 3 in dead skin rubber tree, compared with healthy rubber tree, HbMC2 genes Significant changes, but table of the HbMC2 genes in dead skin rubber tree bark do not occur for the expression quantity in dead skin latex of panama rubber tree Up to amount significantly rise.It is closely related to show that the gene occurs with rubber tree dead skin, which may just regulate and control rubber tree dead skin hair Raw, i.e., HbMC2 gene expression amounts raise the generation that may result in rubber tree dead skin in rubber tree bark.

(2) HbMC2 gene expressions are by the injury of rubber tree dead skin incitant, hydrogen peroxide (H₂O₂) and ethephon (CEPHA),2-(chloroethyl) phosphonic acid processing Induction

In natural rubber production, people (are commonly called as using the latex in special cutter (glue knife) cutting rubber tree bark Rubber tapping), latex is flowed out from scarfing, the raw material as extracting natural rubber.Rubber tapping is a kind of inevitable for rubber tree Mechanical wounding.Research shows, rubber tapping intensity is excessive, long-term and excessive can cause rubber tree dead skin using ethrel stimulation dumping Generation.Meanwhile research finds that reactive oxygen species raise in dead skin rubber tree.Correlation factor pair occurs for further research dead skin The influence of HbMC2 gene expressions, applicant analyze injury, H₂O₂And glue after the processing of the rubber tree dead skin such as ethephon (CEPHA),2-(chloroethyl) phosphonic acid incitant The expression change of HbMC2 genes, experiment in triplicate, repeat comprising the following steps that for experiment every time in breast：

Choose tree enclose reached open the standard of cutting do not open cut Para rubber tree heat grind 7-33-97 injured respectively, H₂O₂And Ethephon (CEPHA),2-(chloroethyl) phosphonic acid processing.Injury processing is with reference to (Tang et al., 2010, The sucrose transporter such as Tang HbSUT3plays an active role in sucrose loading to laticifer and rubber productivity in exploited trees of Hevea brasiliensis(para rubber tree),Plant Cell Environ.,33(10):Method 1708-1720), H₂O₂Processing with reference to Zhu etc. (Zhu et al., 2010, HbMT2, an ethephon-induced metallothionein gene from Hevea brasiliensis responds to H₂O₂stress,Plant Physiol.Biochem.,48:Method 710-715), ethephon (CEPHA),2-(chloroethyl) phosphonic acid processing is with reference to Hao and Wu (Hao B.Z.,Wu J.L.,2000,Laticifer differentiation in Hevea brasiliensis:induction by exogenous jasmonic acid and linolenic acid.Ann.Bot.,85:Method 37-43), not make The normal plant of processing compares.Often handle per 3 repetitions of time point, often repeatedly 5 rubber trees, the rubber tree of every kind of processing are equal For different rubber trees.Injury and H₂O₂Processing handle 0,6,24 and 48h collection latex, ethephon (CEPHA),2-(chloroethyl) phosphonic acid processing processing 0,4,8, 24 and 48h gathers latex, liquid nitrogen cryopreservation after sample collection.

According to the fluorescence quantifying PCR method analysis injury in step (1), H₂O₂And the HbMC2 genes after ethephon (CEPHA),2-(chloroethyl) phosphonic acid processing Expression.The result shows that injury, H₂O₂And ethephon (CEPHA),2-(chloroethyl) phosphonic acid processing can raise the expression of HbMC2 genes in latex.Injury, H₂O₂ And ethephon (CEPHA),2-(chloroethyl) phosphonic acid processing can raise the expression of HbMC2 genes in latex.After injury processing, expression of the HbMC2 genes in latex Slightly rise, but difference is not notable (Fig. 4) compared with before processing.Under oxidative stress, the expression of HbMC2 genes is in first rising in latex Trend is reduced after height.When handling 24h, the relative expression quantity highest of HbMC2 genes, is significantly higher than the tables of before processing HbMC2 genes Up to level, for 3.0 times of before processing.Hereafter, the expression of HbMC2 genes declines, but the still significantly high before processing (figure of expression 5).After ethephon (CEPHA),2-(chloroethyl) phosphonic acid processing, expression of the HbMC2 genes in latex is in downward trend after first raising, when handling 8h, the gene Expression reaches maximum, is 2.3 times of before processing, is significantly higher than the expression of before processing HbMC2 genes, hereafter, The expression of HbMC2 genes declines, and when handling 48h, the expression of the gene substantially returns to before processing expression (Fig. 6).More than The result shows that the expression of HbMC2 genes is by H₂O₂With the induction of the dead skin incitant such as ethephon (CEPHA),2-(chloroethyl) phosphonic acid, this is further demonstrated that, HbMC2 The expression of gene occurs closely related with rubber tree dead skin.

Embodiment 3.HbMC2 genes can reduce the oxidative stress resistance of transgenic yeast, induce the dead of transgenic yeast Die

First, the structure of recombination yeast

(1) structure of Yeast expression carrier

The primer pair of amplification coding area (5 ' the 98th to 1183 nucleotide in end) is designed according to sequence in sequence table 2, it is positive and negative BamH I and Not I restriction enzyme sites and protection base are introduced respectively to the end of primer 5 '.Forward primer：5′- GCGGATCCATGTTCATGCTCGTCGACTG-3 ' (being the identification sequence of BamH I at underscore), reverse primer：5′- TAGCGGCCGCTCAGAAAGAAAAAGGTCTGG-3 ' (being the identification sequence of Not I at underscore).Using bark cDNA as mould Plate, passes through PCR amplification target gene.PCR reaction systems are：2 μ L of cDNA templates,FastPfu Buffer5 μ L, 2.5mM dNTPs 2 μ L, each 0.5 μ L of forward and reverse primer (10 μm of ol/L),FastPfu 0.5 μ L of DNA Polymerase (2.5U/L), add ddH₂O to 25 μ L of cumulative volume.Response procedures are：95 DEG C of pre-degeneration 3min；95 DEG C denaturation 20s, 55 DEG C annealing 20s, 72 DEG C extension 1min 10s, altogether set 35 circulation；72 DEG C of extension 10min.

PCR product is connected to after agarose gel electrophoresis, recovery purifying-Blunt Simple Cloning Vector (Beijing Quanshijin Biotechnology Co., Ltd), converts escherichia coli DH5a competent cell, picking positive colony, send Shanghai Sheng Gong bioengineering Co., Ltd is sequenced.Using the correct clone of plasmid extraction kit (OMEGA companies) extracting sequencing and Yeast expression carrier pYES2 (Invitrogen companies) empty carrier plasmid, with restriction enzyme BamH I and Not I (Fermentas companies) carries out double digestion.Double digestion system：1 μ L, Not I (10U/ μ L) of BamH I (10U/L) 1 μ L, 10 × 6 μ L of Tango Buffer, 8 μ L of Plasmid DNA, mend ddH₂O to 30 μ L.37 DEG C of water-bath 3h.Digestion products carry out 1.0% agarose After gel electrophoresis, gel extraction target stripe.The target gene of recycling and pYES2 carrier frameworks are connected using T4DNA ligases Connect, linked system：6 μ L, pYES2 carrier framework of target gene recovery product, 2 μ L, T4DNA ligase, 11 μ of μ L, 10 × Buffe L；16 DEG C of connections are overnight.Connection product converts escherichia coli DH5a competent cell, then containing ampicillin (100mg/L) LB solid plates on 37 DEG C of overnight incubations.Picking monoclonal shakes bacterium, utilizes pYES2 vector primers (T7：5′- TAATACGACTCACTATAGGG-3 ') gene mentation specific reverse primer to bacterium colony carry out positive-selecting, positive colony further into Row sequencing identification.Sequencing result is shown between BamH I and Not the I recognition sites of recombinant plasmid pYES2-HbMC2 containing orderly The 98th to 1183 nucleotide sequence in row 2, shows that the recombinant expression carrier of structure is correct.The recombinant vector is named as PYES2-HbMC2, pYES2-HbMC2 express amino acid sequence are the protein HbMC2 of sequence 1 in sequence table.

(2) structure of recombination yeast

1. the structure of recombination yeast

Wine brewing yeast strain INVScl (Invitrogen companies) is urinary ammonia acid auxotroph (Ura^-) bacterial strain, SC-Ura^-On culture medium, which can hardly grow and breed.And contain URA3 bases on Yeast expression carrier pYES2 Cause, and the expression of the gene can make yeast transformant in SC-Ura^-Normal growth on culture medium.Therefore, SC-Ura can be used^- Culture medium screens positive yeast transformant.PYES2 and recombinant expression carrier pYES2-HbMC2 plasmids are extracted, and is passed through They are transformed into competent yeast bacterial strain INVSc1 by Li-acetate method respectively, and transformed yeast bacterium solution is coated in SC-Ura^-Solid On culture medium, compareed with unconverted yeast, 30 DEG C are inverted culture, after 2 days in addition to unconverted yeast is not completely long, conversion The yeast of two kinds of plasmids can grow and have bacterium colony to grow, and illustrate yeast conversion success.Picking yeast single bacterium colony, is incubated overnight After abolish cell membrane, and pass through the further identification that bacterium solution PCR method carries out positive transformant.Specific construction step is such as Under：

1) picking yeast INVSc1 monoclonals are added in 10mL YPD fluid nutrient mediums, 30 DEG C, and 200rpm shakes training overnight.

2) OD of yeast liquid is detected₆₀₀Value, the yeast liquid being incubated overnight is added in 50mL YPD fluid nutrient mediums, dilute Release to OD₆₀₀For 0.4,30 DEG C, 200rpm continues to shake training 2-4h.

3) 4 DEG C, 2500rpm centrifugation 5min, abandon supernatant and collect thalline, thalline is resuspended with 40mL l × TE buffer solutions.

4) 4 DEG C, 2500rpm centrifuges 5min again, abandons supernatant and collects thalline, and bacterium is resuspended with 2mL l × LiAc/0.5 × TE Body.

5) the resuspension thalline of acquisition is dispensed into 1.5mL centrifuge tubes, often 100 μ L of pipe.

6) yeast cells of packing is placed in incubation at room temperature 10min.

7) in each transformation system (100 μ L), 1 μ g pYES2-HbMC2,100 μ g denaturated salmon essence DNA are added, are mixed It is even.

8) 700 μ L l × LiAc/40%PEG-3350/l × TE is added, is mixed.

9) the mixed liquor 30min in 30 DEG C of incubation steps 1.8.

10) DMSO of 88 μ L, after mixing, 42 DEG C of thermal shock 7min are added.

11) 4 DEG C of 5000rpm centrifugation lmin, supernatant discarding.

12) thalline is resuspended with lmL l × TE buffer solutions, 4 DEG C, 5000rpm centrifuges lmin, supernatant discarding.

13) thalline is resuspended with the buffer solution of 100 μ L l × TE, and is applied to SC-Ura^-On solid medium, 30 DEG C of inversions Culture 2 days.From SC-Ura^-Random picking INVSc1 (pYES2-HbMC2) monoclonal 3 on solid medium, 30 DEG C, 200rpm Shaken cultivation is stayed overnight.Overnight culture is taken, boiling water bath 5min, places 5min smudge cellses, be repeated several times, with clasmatosis on ice Liquid centrifugal concentrating sample carries out bacterium solution PCR positive detections as template.Positive colony is the restructuring ferment containing pYES2-HbMC2 Mother, is named as INVSc1 (pYES2-HbMC2).

According to the method described above, pYES2-HbMC2 is replaced with into pYES2, other steps are constant, obtain containing pYES2's Recombination yeast, is named as INVSc1 (pYES2).

2. the detection that HbMC2 is expressed in recombination yeast

INVSc1 (pYES2-HbMC2) and INVSc1 (pYES2) (being used as negative control) is preserved bacterium solution to be inoculated in respectively SC-Ura^-In fluid nutrient medium, 30 DEG C, 200rpm shaken cultivation 24h, measure its OD₆₀₀Value.A certain amount of culture is taken, 4000r/min, centrifuges 1min, abandons supernatant, thalline is resuspended with liquid inducing culture, and adjust OD₆₀₀=0.2,50mL is respectively taken, 30 DEG C, 200rpm Fiber differentiations, when Fiber differentiation 24,36 and 48h, respectively take 10ml bacterium solutions, low-speed centrifugal collects thalline, uses Yeast total serum IgE rapid extraction kit (Sangon Biotech (Shanghai) Co., Ltd.) extracts total serum IgE, using DNase I DNA in RNA is removed, with reference to RevertAid^TMFirst Strand cDNA Synthesis Kit (Fermentas companies) are said Bright book synthesizes the first chain cDNA.First with saccharomyces cerevisiae Actin gene (GeneBank accession number：L00026.1) will be per PCR The content tune of template cDNA is consistent in reaction, then recycles HbMC2 gene specific primers to carry out PCR amplification to template.PCR is anti- The system is answered to be：Template 2-3 μ L, Buffer 2.5 μ L, dNTPs 2 μ L, forward and reverse each 0.5 μ L of primer, 0.5 μ L of Taq enzyme, are mended ddH₂O to 25 μ L of cumulative volume.Response procedures are：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C extend 1min, sets 30 circulations altogether；72 DEG C of extension 10min.

The results show that in Fiber differentiation 24,36 and 48h, turn empty carrier negative control INVSc1 (pYES2) and be not detected by HbMC2 gene expressions, and the expression of HbMC2 genes can be detected in recombinant bacterium INVSc1 (pYES2-HbMC2), wherein luring When leading culture 36h, HbMC2 gene expression amounts highest (Fig. 7).Result above shows that rubber tree HbMC2 genes can be in recombination yeast Expression in INVSc1 (pYES2-HbMC2).

The oxidative stress Resistance detecting of two, recombination yeasts INVSc1 (pYES2-HbMC2)

1. recombination yeast INVSc1 (pYES2-HbMC2) and INVSc1 (pYES2) are in H containing various concentrations₂O₂Solid induction training The growth differences supported on base compare

The INVSc1 (pYES2-HbMC2) of step 1 is inoculated in SC-Ura^-In fluid nutrient medium, 30 DEG C, 200rpm shakes Culture 24h is swung, to OD₆₀₀It is worth for 2.0 or so, 4000r/min, centrifuges 1min, abandon supernatant, is resuspended with liquid inducing culture Thalline, and adjust OD₆₀₀=0.2,10mL is taken, the induced expression culture 36h under 30 DEG C, 200rpm, measures its OD₆₀₀Value, and will Its concentration is adjusted to OD₆₀₀=1, obtain INVSc1 (pYES2-HbMC2) nutrient solution.Then INVSc1 (pYES2-HbMC2) is trained Nutrient solution is respectively with 5 times of inducing culture dilution, 5²Again, 5³Times and 5⁴Again, the INVSc1 (pYES2- of 5 times of dilution are respectively obtained HbMC2) nutrient solution, dilution 5²INVSc1 (pYES2-HbMC2) nutrient solution again, dilution 5³INVSc1 (pYES2-HbMC2) again Nutrient solution and dilution 5⁴INVSc1 (pYES2-HbMC2) nutrient solution again.

According to the method described above, INVSc1 (pYES2-HbMC2) being replaced with into INVSc1 (pYES2), other steps are constant, Respectively obtain INVSc1 (pYES2) nutrient solution, dilute 5 times of INVSc1 (pYES2) nutrient solution, dilution 5²INVSc1 again (pYES2) nutrient solution, dilution 5³INVSc1 (pYES2) nutrient solutions again and dilution 5⁴INVSc1 (pYES2) nutrient solution again.

H₂O₂Processing：INVSc1 (pYES2-HbMC2) nutrient solution is distinguished into point sample in 0mM H₂O₂Solid Fiber differentiation Base and 3mMH₂O₂Solid inducing culture on, each 5 μ L nutrient solutions of culture medium flat plate point, often handle 4 repetitions.30 DEG C of cultures After 3 days, untreated INVSc1 (pYES2-HbMC2) and 3mM H is respectively obtained₂O₂The INVSc1 (pYES2-HbMC2) of processing.

According to above-mentioned H₂O₂Processing method, INVSc1 (pYES2-HbMC2) nutrient solution is replaced with into 5 times of dilution INVSc1 (pYES2-HbMC2) nutrient solution, respectively obtains untreated INVSc1 (pYES2-HbMC2) and dilution 5 after 5 times of dilution 3mM H after times₂O₂The INVSc1 (pYES2-HbMC2) of processing.

According to above-mentioned H₂O₂Processing method, INVSc1 (pYES2-HbMC2) nutrient solution is replaced with into dilution 5²Times INVSc1 (pYES2-HbMC2) nutrient solution, respectively obtains dilution 5²Untreated INVSc1 (pYES2-HbMC2) and dilution after times 5²3mM H after times₂O₂The INVSc1 (pYES2-HbMC) of processing.

According to above-mentioned H₂O₂Processing method, INVSc1 (pYES2-HbMC2) nutrient solution is replaced with into dilution 5³Times INVSc1 (pYES2-HbMC2) nutrient solution, respectively obtains dilution 5³Untreated INVSc1 (pYES2-HbMC2) and dilution after times 5³3mM H after times₂O₂The INVSc1 (pYES2-HbMC2) of processing.

According to above-mentioned H₂O₂Processing method, INVSc1 (pYES2-HbMC2) nutrient solution is replaced with into dilution 5⁴Times INVSc1 (pYES2-HbMC2) nutrient solution, respectively obtains dilution 5⁴Untreated INVSc1 (pYES2-HbMC2) and dilution after times 5⁴3mM H after times₂O₂The INVSc1 (pYES2-HbMC2) of processing.

According to above-mentioned H₂O₂Processing method, INVSc1 (pYES2-HbMC2) nutrient solution is replaced with into INVSc1 (pYES2) Nutrient solution, respectively obtains untreated INVSc1 (pYES2) and 3mM H₂O₂The INVSc1 (pYES2) of processing.

According to above-mentioned H₂O₂Processing method, INVSc1 (pYES2-HbMC2) nutrient solution is replaced with into 5 times of dilution INVSc1 (pYES2) nutrient solution, 3mM H after respectively obtaining untreated INVSc1 (pYES2) after diluting 5 times and diluting 5 times₂O₂ The INVSc1 (pYES2) of processing.

According to above-mentioned H₂O₂Processing method, INVSc1 (pYES2-HbMC2) nutrient solution is replaced with into dilution 5²Times INVSc1 (pYES2) nutrient solution, respectively obtains dilution 5²Untreated INVSc1 (pYES2) and dilution 5 after times²3mM after times H₂O₂The INVSc1 (pYES2) of processing.

According to above-mentioned H₂O₂Processing method, INVSc1 (pYES2-HbMC2) nutrient solution is replaced with into dilution 5³Times INVSc1 (pYES2) nutrient solution, respectively obtains dilution 5³Untreated INVSc1 (pYES2) and dilution 5 after times³3mM after times H₂O₂The INVSc1 (pYES2) of processing.

According to above-mentioned H₂O₂Processing method, INVSc1 (pYES2-HbMC2) nutrient solution is replaced with into dilution 5⁴Times INVSc1 (pYES2) nutrient solution, respectively obtains dilution 5⁴Untreated INVSc1 (pYES2) and dilution 5 after times⁴3mM after times H₂O₂The INVSc1 (pYES2) of processing.

The growth of recombination yeast after any of the above processing is as shown in figure 8, without H₂O₂Handle in control group, INVSc1 (pYES2-HbMC2) and the bacterial plaque number of INVSc1 (pYES2) does not have notable difference (A in Fig. 8), but in 3mM H₂O₂Stress treatment Under, the bacterial plaque number of INVSc1 (pYES2-HbMC2) will be considerably less than INVSc1 (pYES2) (B in Fig. 8), show overexpression The recombination yeast of HbMC2 genes is more sensitive to oxidative stress, and HbMC2 and its high expression of encoding gene can suppress thin under oxidative stress The growth of born of the same parents.

2. recombination yeast INVSc1 (pYES2-HbMC2) and INVSc1 (pYES2) are in various concentrations H₂O₂Liquid Fiber differentiation Growth differences in base compare is inoculated in SC-Ura by the INVSc1 (pYES2-HbMC2) of step 1^-In fluid nutrient medium, 30 DEG C, 200rpm shaken cultivation 24h, to OD₆₀₀It is worth for 2.0 or so, 4000r/min, centrifuges 1min, abandon supernatant, induced with liquid Thalline is resuspended in culture medium, and adjusts OD₆₀₀=0.2, untreated INVSc1 (pYES2-HbMC2) nutrient solution is obtained, experiment repeats Three times.

According to the method described above, liquid inducing culture is replaced with into 3mM H₂O₂Liquid inducing culture, other steps are equal It is constant, obtain 3mM H₂O₂INVSc1 (pYES2-HbMC2) nutrient solution of processing.

According to the method described above, INVSc1 (pYES2-HbMC2) being replaced with into INVSc1 (pYES2), other steps are constant, Obtain untreated INVSc1 (pYES2) nutrient solution.

According to the method described above, INVSc1 (pYES2-HbMC2) is replaced with into INVSc1 (pYES2), and liquid is induced and is trained Foster base replaces with 3mM H₂O₂Liquid inducing culture, other steps are constant, obtain 3mM H₂O₂The INVSc1 of processing (pYES2) nutrient solution.

Above-mentioned each nutrient solution is respectively taken into 10mL, 30 DEG C, Fiber differentiation under 200rpm, OD is measured every 12h₆₀₀Value, it is continuous to survey To culture 48h, as a result as shown in Fig. 9 and table 2.

The results show that without H₂O₂Under treatment conditions, INVSc1 (pYES2-HbMC2) and INVSc1 (pYES2) bacterium solution be not There is no difference substantially with incubation time point concentration；3mM H₂O₂Before processing 24h, INVSc1 (pYES2-HbMC2) and INVSc1 (pYES2) OD of bacterium solution₆₀₀It is worth no notable difference, but after processing 24h, the OD of INVSc1 (pYES2-HbMC2)₆₀₀Value is obvious small In INVSc1 (pYES2), and as processing time extends, this gap is gradually increased, when handling 48h, INVSc1 (pYES2) The OD of bacterium solution₆₀₀Value is about 10 times of INVSc1 (pYES2-HbMC2).This further confirms the recombination yeast pair for turning HbMC2 genes Oxidative stress is more sensitive, and the high expression of the oxidative stress resistance of HbMC2 negative regulations biology, i.e. HbMC2 genes can reduce biology The high expression of oxidative stress resistance, HbMC2 and its encoding gene can suppress the growth of cell under oxidative stress.

Table 2.H₂O₂Processing different time points, recombination yeast INVSc1 (pYES2-HbMC2) and INVSc1's (pYES2) OD₆₀₀Value

3. high concentration H₂O₂Recombinant yeast INVSc1 (pYES2-HbMC2) and INVSc1 (pYES2's) deposits after Stress treatment Difference living

The INVSc1 (pYES2-HbMC2) of step 1 is inoculated in SC-Ura^-In fluid nutrient medium, 30 DEG C, 200rpm shakes Culture 24h is swung, to OD₆₀₀It is worth and abandons supernatant for 2.0 or so, 4000r/min, centrifugation 1min, thalline is resuspended with inducing culture, And adjust OD₆₀₀=0.2,10mL is taken, the Fiber differentiation 36h under 30 DEG C, 200rpm, takes 5mLINVSc1 (pYES2-HbMC2) to train Nutrient solution two is managed, 4000r/min, is centrifuged 3min, is abandoned supernatant, thalline is resuspended in 0mM H respectively₂O₂Aqueous solution (i.e. sterile distillation Water) and 10mM H₂O₂In aqueous solution so that OD₆₀₀=1.0.After mixing, 2mL is respectively taken in 10mL centrifuge tubes, 30 DEG C, 160rpm Lower incubation 6h, respectively obtains untreated INVSc1 (pYES2-HbMC2) bacterium solutions and 10mM H₂O₂INVSc1 (the pYES2- of processing HbMC2) bacterium solution.The bacterium solution of different disposal is carried out to 5 times of gradient dilution with sterile distilled water respectively, respectively obtains 5 times of dilution Untreated INVSc1 (pYES2-HbMC2) bacterium solution, dilution 5²Untreated INVSc1 (pYES2-HbMC2) bacterium solution again, Dilution 5³Untreated INVSc1 (pYES2-HbMC2) bacterium solution again, the 10mM H for diluting 5 times₂O₂The INVSc1 of processing (pYES2-HbMC2) bacterium solution, dilution 5²10mM H again₂O₂INVSc1 (pYES2-HbMC2) bacterium solutions of processing and dilution 5³Times 10mM H₂O₂INVSc1 (pYES2-HbMC2) bacterium solution of processing.By two kinds of undiluted bacterium solutions and the bacterium of different extension rates Liquid respectively takes 5 μ L to distinguish point sample in SC-Ura^-On solid medium, 4 repetitions are often handled.30 DEG C are cultivated 3 days, are respectively obtained and are not located The INVSc1 (pYES2-HbMC2) of reason, the INVSc1 (pYES2-HbMC2) of untreated 5 times of dilution, untreated dilution 5²Times INVSc1 (pYES2-HbMC2), it is untreated dilution 5³INVSc1 (pYES2-HbMC2), 10mM H again₂O₂Processing INVSc1(pYES2-HbMC2)、10mM H₂O₂INVSc1 (pYES2-HbMC2), the 10mM H of 5 times of the dilution of processing₂O₂Processing Dilution 5²INVSc1 (pYES2-HbMC2), 10mM H again₂O₂The dilution 5 of processing³INVSc1 (pYES2-HbMC2) again.

According to the method described above, INVSc1 (pYES2-HbMC2) being replaced with into INVSc1 (pYES2), other steps are constant, Respectively obtain untreated INVSc1 (pYES2), the INVSc1 (pYES2) of untreated 5 times of dilution, untreated dilution 5²Times INVSc1 (pYES2), it is untreated dilution 5³INVSc1 (pYES2), 10mM H again₂O₂The INVSc1 (pYES2) of processing, 10mM H₂O₂INVSc1 (pYES2), the 10mM H of 5 times of the dilution of processing₂O₂The dilution 5 of processing²INVSc1 (pYES2) again, 10mM H₂O₂The dilution 5 of processing³INVSc1 (pYES2) again.

The growing state of above-mentioned each yeast is as shown in Figure 10,10mM H₂O₂After handling 6h, under identical extension rate The bacterial plaque number of INVSc1 (pYES2-HbMC2) is considerably less than INVSc1 (pYES2), through dilution 5³INVSc1 (pYES2- after times HbMC2) grown without bacterial plaque, and INVSc1 (pYES2) still has bacterial plaque growth.Show under oxidative stress, INVSc1 (pYES2- HbMC2) bacterium colony death toll is raised compared with INVSc1 (pYES2), and result above shows, under oxidative stress, the high expression of HbMC2 can promote The death of yeast cells under into oxidative stress, and then show the phenotype more sensitive to oxidative stress.

Claims

1. a kind of and relevant protein of rubber tree dead skin, is the protein that amino acid sequence is sequence 1 in sequence table.

Any of 2. it is following B1 with the relevant biomaterial of protein described in claim 1) to B8)：

B1 the nucleic acid molecules of protein described in claim 1) are encoded；

B2 B1) is contained) expression cassettes of the nucleic acid molecules；

B3 B1) is contained) recombinant vectors of the nucleic acid molecules；

B4 B2) is contained) recombinant vector of the expression cassette；

B6 B2) is contained) recombinant microorganism of the expression cassette；

B7 B3) is contained) recombinant microorganism of the recombinant vector；

B8 B4) is contained) recombinant microorganism of the recombinant vector.

3. biomaterial according to claim 2, it is characterised in that：B1) nucleic acid molecules are following b1) or b2) Gene：

B2) nucleotide sequence is the cDNA molecules or DNA molecular of sequence 2 in sequence table.

4. biomaterial is in following C3 described in protein described in claim 1 or Claims 2 or 3)-C5) in it is any should With：

C3 rubber tree dead skin) is regulated and controled；

C5 the prevention product of rubber tree dead skin) is prepared.

5. application according to claim 4, it is characterised in that：The dead skin is the dead skin or dead skin occurred under natural conditions The dead skin that incitant induces.

6. application according to claim 5, it is characterised in that：The dead skin incitant is injury, hydrogen peroxide or second Alkene profit.

7. biomaterial is in regulating and controlling microbial cell death or oxidation described in protein described in claim 1 or Claims 2 or 3 Application in stress resistance.

8. application according to claim 7, it is characterised in that：The microbial cell is yeast cells.

9. the application according to claim 7 or 8, it is characterised in that：The cell death lures for cell death incitant The cell death led；The oxidative stress is the oxidative stress of hydrogen peroxide-induced.

10. application according to claim 9, it is characterised in that：The cell death incitant is injury, hydrogen peroxide Or ethephon (CEPHA),2-(chloroethyl) phosphonic acid.

11. the answering in rubber tree dead skin is prevented as target of gene described in protein described in claim 1 or claim 3 With.

12. application according to claim 11, it is characterised in that：The dead skin is the dead skin or dead occurred under natural conditions The dead skin that skin incitant induces.

13. application according to claim 12, it is characterised in that：The dead skin incitant for injury, hydrogen peroxide or Ethephon (CEPHA),2-(chloroethyl) phosphonic acid.

14. have the function of following D1), D2) and material at least one of D5) prepare prevent in rubber tree dead skin product should With：

D1 the expression of protein described in claim 1) is suppressed；

D2 B1 in claim 3) is suppressed) expression of the nucleic acid molecules；

D5 protein inactivation described in claim 1) is made.

15. application according to claim 14, it is characterised in that：The dead skin is the dead skin or dead occurred under natural conditions The dead skin that skin incitant induces.

16. application according to claim 15, it is characterised in that：The dead skin incitant for injury, hydrogen peroxide or Ethephon (CEPHA),2-(chloroethyl) phosphonic acid.

17. cultivating the method for the genetically modified organism that oxidative stress resistance reduces, include the following steps：Power is imported into receptor biological B1 during profit requires 3) nucleic acid molecules, obtain genetically modified organism；The genetically modified organism is compared with the receptor biological to oxygen Changing the resistance of stress reduces；The receptor biological is microorganism.

18. according to the method for claim 17, it is characterised in that：The microorganism is yeast.

19. the method according to claim 17 or 18, it is characterised in that：The oxidative stress is the oxygen of hydrogen peroxide-induced Change stress.