CN108517322A - A kind of pinellia pedatisecta Schott trypsin inhibitor gene, the albumen of its coding and pest-resistant application - Google Patents

Abstract

The invention discloses a kind of pinellia pedatisecta Schott trypsin inhibitor gene, the albumen of its coding and pest-resistant applications.The nucleotides sequence list of gene such as SEQ ID NO：Shown in 1.When cultivating resistance to insect-resistant transgenic plants, structure contains SEQ ID NO first：Then the recombinant expression carrier of gene shown in 1 utilizes gained recombinant expression carrier to build transformant, recycle gained transformant to convert purpose plant, screen positive plant, screens to obtain the homozygous genetically modified plants with insect resistace compared with normal plants by three generations.Inventor is proved that the pest-resistant characteristic of the gene pairs plant of the present invention has by a large amount of scientific researches and is obviously improved effect, it proves that the insect resistace of itself and plant has to be closely connected, the genetically modified plants with high insect resistace have been obtained based on this and using existing gene engineering method, there is great practical significance to reducing pesticide control aphid, improving crop-planting efficiency and planting yield.

Description

A kind of pinellia pedatisecta Schott trypsin inhibitor gene, albumen of its coding and pest-resistant Using

Technical field

The present invention relates to gene engineering technology field, especially a kind of systems with pinellia pedatisecta Schott protease inhibitor gene Standby, coding albumen and its application in pest-resistant correlation.

Background technology

Aphid is to breed most fast insect, and damage to crops is extensive on earth, and because having developed a variety of self-protections Defense mechanism is difficult prevention, and some types can secrete plant hormone so that plant forms goiter and tumor nest；Some secretion coating of wax matter Being covered in body surface enables drug be difficult to penetrate into；Some strong mustard oil smells of release frighten natural enemy away.Aphid is still agriculturally many sick Harmful communications media, cotten aphid secretion are the important anaphylactogens of human airway, serious to destroy exhausting environment.Pinellia pedatisecta Schott （Pinellia pedatisecta）It is a kind of Chinese herbal medicine of the natural pest-resistant characteristic of tool.Chemical composition mainly include alkaloids and Fatty acid and Pleurotus Ostreatus, protein etc., total protein component is to the piercing and suckings mouth such as grain aphid, bird-cherry aphid, cotten aphid and black peach aphid Device pest has killing activity.It is improved on crops using the method improvement of biotechnology using this characteristic to resist aphid Property.Domestic and international present Research is summarized as follows with development trend.

(1) the aphid-resistant gene cloned at present is few.Resource resistance to aphids is present in wild species or nearly source kind more, in 3500 soybean 11 anti-sources are only screened in germplasm；2 anti-sources are screened in 1200 lettuce germplasm；In 40000 wheat resources, only 300 A performance resistance.The resistance of plants against insects generally can be divided into 3 types, i.e. anti-repelling properties, antibiosis and tolerance to insects, moderate resistance Property effect it is most apparent, including controlled by multiple genes and single aobvious（It is hidden）Gene controls, if wheat is to Diuraphis noxia（Diuraphis noxiaMordvilko resistance), soybean are to soybean aphid（Aphis glycinesMatsumura）Resistance be single aobvious gene Control；But it is had also discovered for acyrthosiphum pisim in wheat, barley（Acythosiphon pisumHarris）, corn leaf aphids （Rhopalosiphum maidisFitch）Recessive aphid-resistant gene, quantitative character control resistance it is more.Theoretically only have The resistance of single aobvious gene control is conducive to through gene cloning and applies transformations on other species, and more resources are suitable for this The distant hybridization or conventional breeding of crop.With the development of Research of Plant Genomics and functional genomics, some aphid-resistant gene quilts It clones and, such as：Wild-type tomatoMi-1Gene can anti-root of Beijing euphorbia Macrosiphus spp（Macrosiphum euphorbiae）, anotherVat Gene can anti-muskmelon cotten aphid（Aphis gossypiiGlover）, this kind of aphid-resistant gene is generally related with plant hypersensitivity, i.e., Corresponding plant can identify the signal invaded by aphid, and the side such as callose, lignin is deposited to synthesize aldehydes matter, on cell wall Formula is to aphids resistance.Though have aphid-resistant gene to be cloned, because there are many type of aphid, a kind of Aphid Species mistake that gene can resist Few, pest-resistant far from crop is met needs.

(2) the advantage of pinellia pedatisecta Schott resource resistance to aphids.Pinellia pedatisecta Schott is a kind of natural highly resistance aphid, the medium-height grass without peculiar taste Medicine, people are initially separated the anti-insect activity substance of the tuber of pinellia from the nineties, start to think that main function is by phytolectin shape At, successively cloned from pinellia pedatisecta SchottPPA-1、PTA-1Etc. multiple genes, in terms of aphids resistance there is certain meaning to answer With value.In addition, Fan Handong in 2010 et al. identifies a kind of new anti-aphid active material, i.e. plant from tuber of pinellia stem tuber total protein Trypsin inhibitor（Pinellia peatisectaTrypsin inhibitor, hereinafter referred to as PPTI albumen orPPTIBase Cause）The aphids resistance albumen of type：When its concentration reaches 0.5%, the corrected mortality of aphid reaches 100% after 5 days.It is prior It is to calculate the substance through SDS-PAGE, reversed-phase high performance liquid chromatography for the pure protein without sugar, mass spectral analysis shows its opposite point Son amount is 20596.09.PPTI albumen has the following advantages compared with other type anti-insect activity albumen：First, it is pure protein Matter effect as a result, compared to rely on sugar combine lectin family, gene be easier build conversion carrier, appliable plant model It encloses wide；Secondly, protease inhibitors generally has broader spectrum of insect resistace, therefore speculates that PPTI can be resistant to a variety of aphids； Third, insect are not likely to produce the tolerance to protein inhibitor.We are further study show that can at pinellia pedatisecta Schott blade position This albuminoid is expressed by mechanical damage and disease pest intrusion induction high concentration.

(3) the pest-resistant and its characteristic of protease inhibitors.Previous scholars have many researchs to this, such as：Up to Kedong etc., Du Cowpea is used in the system researches such as GuoqiangCpTIGenetic transformation apple has 2 to reach the resistance of bollworm in 60 transformation plants 100%；Wang Haohua etc. has studied insect pest inducing poplar trypsaseKTIGene overexpression situation, specifies willowKTIGene The functional diversity of family and its disease-resistant pest-resistant effect.Xu Pingli etc. is cloned into a BBI type protease inhibitors from wheatTaUESGene, and prove that it takes part in salt and drought stress response.Have now been found that three categories protease presses down in plant Preparation:The PPTI albumen of serine, coloured glaze base and metal protease inhibitors, the present patent application belongs to the first kind and serine egg White enzyme inhibitor, serpin also at least it is found that there is 6 families, but only few members have it is relatively strong anti- Sexual function, in addition to it is above it is several other than, also arc beans, potato protease inhibitors etc., but the protease having found inhibits Agent fights adverse circumstance type and function and effect difference is multifarious, and existing virose protease inhibitor gene collocation composing type starts The genetically modified plants of son, protease inhibitors expression quantity is still relatively low, typically up to less than the lethal dose of aphid.

To sum up, from the point of view of domestic and international present Research, there are problems to include for anti-aphid basic research：First, crop is to aphid Resistant gene research is also seldom, the monoclonal antibody natural disposition gene of wide spectrum can be used seldom；Second is that the plant lectin procatarxis of wide spectrum contains mostly Sugar chain, and be difficult the correct sugar chain of expression with the method for genetic engineering；The composing type third, existing protease inhibitor gene is arranged in pairs or groups The genetically modified plants of promoter develop the protease inhibitors of effective non-environmental pollution control aphid typically up to less than lethal dose Gene has a very important significance.

Invention content

The technical problem to be solved by the present invention is to overcome deficiencies in the prior art, provide a kind of pinellia pedatisecta Schott trypsase Inhibitor gene, the albumen of its coding and pest-resistant application.

In order to solve the above technical problems, the technical solution used in the present invention is as follows.

A kind of and relevant gene of plant resistance to insect, nucleotides sequence list such as SEQ ID NO：Shown in 1.

The invention also includes the recombinant expression carriers containing said gene.

The invention also includes the Matrix attachment region transformant containing said gene.

The invention also includes the amplimers pair of said gene.

The invention also includes the above-mentioned albumen for stating gene code.

The invention also includes application of the above-mentioned said gene in cultivating resistance to insect-resistant transgenic plants.Structure contains first SEQ ID NO：Then the recombinant expression carrier of gene shown in 1 utilizes gained recombinant expression carrier to build transformant, recycles institute Transformant converts purpose plant, screen positive plant, screening to obtain homozygous having with normal plants compared with by three generations resists The genetically modified plants of worm property.The purpose plant is tobacco.The insect resistace is primarily referred to as the resistance for Homoptera insect.

It is using advantageous effect caused by above-mentioned technical proposal：Inventor proves the present invention by a large amount of scientific researches The pest-resistant characteristic of gene pairs plant have and be obviously improved effect, it was demonstrated that the insect resistace of itself and plant is with being closely connected, base In this, the genetically modified plants with high insect resistace are obtained using existing gene engineering method, to reducing pesticide control aphid, carrying High crop plantation efficiency and plantation yield have great practical significance.The detailed advantageous effect of the present invention is described below.

(1), by 3 end RACE and 5 end DNA walk shifting technology obtain an overall length pinellia pedatisecta Schott trypsase inhibit because Sub- PPTI gene orders.642 bp of PPTI full length genes encodes 214 amino acid, and molecular weight is 23 kD, and pI values are 5.13； Signal peptide is made of 19 amino acid residues of N-terminal, has STI（Soybean proteinase inhibitor）Structural domain is a kind of tryptose Enzyme inhibition factor.Protein transmembrane domain prediction result：Inside lines never intersect always with outside lines, therefore, it is considered that the albumen Without membrane-spanning domain.Albumen hydrophilic and hydrophobic on-line analysis：Peptide chain main part is distributed in hydrophobic region mostly, and only small part is distributed In hydrophilic area, which belongs to fat-soluble albumen.

(2), the functional verification of transformation of tobacco is carried out to the gene.Different transfer-gen plant insect resistaces is widely different, from connecing After worm from the point of view of the 15th day result, it is 7 that 10 kinds of transgene tobaccos are descending to the population restraint of cigarette aphid, 1,3,5,9,8,2,6, 4、10；Wherein No. 7 and No. 1 have respectively reached 100% and 92.02%, the inhibiting rate of No. 3 plant to the population inhibiting rate highest of cigarette aphid Also more a height of 78.85%, No. 5 inhibiting rates are also respectively 58.84%, remaining transfer-gen plant does not show resistance, this may Caused by causing transgenosis not expressed with transgene silencing.Especially No. 7 transfer-gen plants cigarette aphid after being inoculated with 7 days is all dead It dies, is demonstrated by lethal effect, illustrate that the gene that we detach clone is a gene to aphid with good resistance.

(3), plant trypsin inhibitor has the following advantages compared with other type anti-insect activity albumen：First, it is True protein effect as a result, compared to rely on sugar combine lectin family, the gene cloned be easier build conversion Carrier, that is, it is easy application；Secondly, protease inhibitors generally has broader spectrum of insect resistace；Third, insect are not likely to produce To the tolerance of protein inhibitor, further study show that can be lured by mechanical damage and disease pest intrusion at pinellia pedatisecta Schott blade position It leads high concentration and expresses this albuminoid.

To sum up, we utilize itself research advantage, separation clone's pinellia pedatisecta Schott protease inhibitor（Pinellia Peatisecta trypsin inhibitor, hereinafter referred to as PPTI）, carry out aphids resistance functional analysis and utilization ways research；And It is combined with the urgency of plant aphids prevention, formulates new material and new method, the physiological inheritting mistake of the research protease suppression factor Journey, this method have important theory and realistic meaning to agricultural production and enhancement of environment.

Description of the drawings

The measurement of Fig. 1, pinellia pedatisecta Schott cDNA library clone's size.

3 ' terminal amplification of Fig. 2, PPTI gene cDNA.

Fig. 3, first time chromosome walking electrophoretogram：M:1Kb Plus DNA ladder 1B-4B:Experimental group 5B:It is negative Compare 6B-8B:For positive control.

Fig. 4, second of chromosome walking electrophoretogram：M:1Kb Plus DNA ladder 1B-4B:Experimental group 5B:It is negative Compare 6B-8B:For positive control.

The PCR amplification of Fig. 5, PPTI gene：It is followed successively by from left to right：Marker DM2000；2-3：PPTI amplified fragments； 4：Negative control water.

Fig. 6, eukaryotic expression product bioinformatic analysis：Encode 214 amino acid, predicted molecular weight 23kD, pI value For 5.13 (Fig. 6 a)；Signal peptide forms (Fig. 6 b) by 19 amino acid residues of N-terminal, has STI (soybean proteinase inhibitor) knots Structure domain (Fig. 6 c)；Protein transmembrane domain prediction result such as Fig. 6 d show that inside lines never intersect always with outside lines, therefore recognize Do not have membrane-spanning domain for the albumen；Albumen hydrophilic and hydrophobic on-line analysis such as Fig. 6 e show that peptide chain main part is distributed in hydrophobic mostly Area, only small part are distributed in hydrophilic area, thus it is speculated that the albumen belongs to fat-soluble albumen.

Fig. 7, turn the inoculation aphid experiment of PPTI genetic tobaccos：A left side is transgene tobacco, and the right side is control.

The inhibiting effect that Fig. 8, different transgene tobaccos increase aphid insect density.

Specific implementation mode

The present invention is described in detail in following embodiment.Various raw materials used in the present invention and items of equipment are conventional city Product is sold, can be bought and be directly obtained by market.Quantitative test in following embodiment, is respectively provided with three repeated experiments, Results are averaged.

The sequence information for searching protease inhibitors is sequenced by cDNA library for embodiment 1.

The stem tuber of pinellia pedatisecta Schott comes from industrial crops institute of Hebei Prov. Academy of Agricultural ＆. Forest Sciences biotechnology center.Taq DNA polymerizations Enzyme, reverse transcriptase（M-MLV）, ligase, primer synthesis, Oligo d (T), chromosome walking kit（ Genome Walking Kit）Etc. being precious bioengineering（Dalian）Co., Ltd produces, and Ago-Gel DNA QIAquick Gel Extraction Kits are by Tiangeng Biochemical technology（Beijing）Co., Ltd provides.First the stem tuber of pinellia pedatisecta Schott and the overall length of blade are constructed with CTAB- soil soap methods CDNA library clone insert major part size is between 500 to 3000 bp（Fig. 1）.

Random 6000 cloning and sequencings of picking are sequenced using M13 as sequencing primer, are analyzed the data of sequencing, are passed through U.S. Blast software on-line analysis sequences on state's world gene pool carry out sequence analysis analysis to sequence, and stressing to search has egg The cDNA sequence information of white enzyme inhibitor.According to the information design gene-specific primer of acquisition, using dT18 as downstream primer into Row RT-PCR amplifications.Using 3 '-Full RACE Core Set Ver.2.0 kits of TaKaRa amplification PPTI gene cDNAs ' End overall length（Fig. 2）.

The 3RACE and chromosome walking primer of 1. PPTI of table

Primer	Sequence	Explanation
			PPTIF1	5' CTGGTGTCCTGCAGTAGTAA 3'（SEQ ID NO：4）	3 ' the peripheries RACE primers
PPTIF2	5' TCTAGCGTTTGAGTTCACCC 3'（SEQ ID NO：5）	Primer is enclosed in 3 ' RACE
			PPTIGSP1	5' GTTTGTGAAGGTGAAGGAAG 3'（SEQ ID NO：6）	Step moves peripheral primer
PPTIGSP2	5' GAGGATGCTGCTTCGTCGTC 3'（SEQ ID NO：7）	Step encloses primer in moving
			PPTI- F	5'-TCAACCATGGAGTTTATCCTG- 3'（SEQ ID NO：8）	Full length sequence
PPTI-R	5'-ATC CCT TCC TTC ACC TTC AC-3'（SEQ ID NO：9）	Full length sequence

By target fragment purifying (being carried out according to TIANgel MiDi purification Kit operating procedures) and it is inserted into PMD19-T carriers are selected 3 clones and are sequenced (by Hua Da genetic testing sequence) at random.

Embodiment 2, CTAB methods extract pinellia pedatisecta Schott leaves genomic DNA.

(1) 100mg blades are weighed, it is in powdered to be put into liquid nitrogen grinding rapidly, is transferred in 2mL centrifuge tubes, is added 65 DEG C in advance The beta -mercaptoethanol of the extracting solution 1mL and 2% (V/V) of heat, 65 DEG C of warm bath 30min, therebetween mixing 2-3 times.

(2) it is cooled to room temperature, isometric chloroform is added:Isoamyl alcohol (24:1) mixing stands 10min, centrifuge (room temperature, 10000rpm,10min).It repeats to extract primary.

(3) it sucts clearly in new 2mL centrifuge tubes, the isopropanol mixing of 2/3 volume precooling, -20 DEG C of standings is added 15min is centrifuged (room temperature, 12000rpm, 10min), removes supernatant.

(4) it is washed twice with the ethyl alcohol of 1mL70%, it is micro- dry at room temperature, it is dissolved and is precipitated with 100 μ LTE buffer solutions (PH8.0).

(5) 10 μ L RNaseA, 37 DEG C of warm bath 30min are added.

(6) isometric chloroform is added:Isoamyl alcohol (24：1) mixing stands 10min, centrifuge (room temperature, 12000rpm, 10min).It repeats to extract primary.

(7) absolute ethyl alcohol of 3M NaAc (pH5.2) and 2 times of volumes of 1/10 volume, -20 DEG C of standing 1h, centrifugation (4 is added DEG C, 12000rpm, 10min) abandon supernatant.

(8) it is washed twice, is precipitated with the sterile water dissolutions of 100 μ L after dry, -80 DEG C save backup with 70% ethyl alcohol.

(9) 5 μ L is taken to carry out 1.0% plain agar sugar gel electrophoresis.

Embodiment 3 carries out DNA clone using cDNA known array design primers.

Gene-specific primer PPTIGSP1 is designed according to PPTI gene cDNA known arrays, utilizes TaKaRa GenomeWalker Universal Kit kits expand to obtain segment Promoter-1.By target fragment purifying (according to TIANgel MiDi purification Kit operating procedures carry out) and pMD19-T carriers are inserted into, 3 clones are selected at random It is sequenced (by Hua Da genetic testing sequence).Gene-specific primer PPTIGSP2 is designed according to Promoter-1, is utilized TaKaRa GenomeWalker Universal Kit kits expand to obtain segment Promoter-2.Target fragment is purified (being carried out according to TIANgel MiDi purification Kit operating procedures) is simultaneously inserted into pMD19-T carriers, selects 3 at random A clone is sequenced the Promoter-1 (by Hua Da genetic testing sequence) and sees that Fig. 3, Promoter-2 are shown in Fig. 4.

According to 3 end sequences of PPTI genes and the sequence of upstream, it is spliced into a complete gene order, is then designed The PPTI gene orders of primer amplification overall length.PCR is carried out by template and PPTI gene primers of the first chains of cDNA of synthesis.By mesh Fragment purification (being carried out according to TIANgen MiDi purification Kit operating procedures) and be inserted into pMD19-T carriers, with Machine selects 3 clones and is sequenced and (measures sequence by Shanghai bioengineering), sees Fig. 5.Find that has an albumen by analysis The Partial cDNA Sequence of enzyme inhibition factor structural domain, such as SEQ ID NO：Shown in 2.Hold non-volume in the gene end codon downstream 3 ' Code region sequence, such as SEQ ID NO：Shown in 3.

Embodiment 4,3 ' RACE expand PPTI gene cDNA end sequences

Utilize 3 '-Full RACE Core Set Ver.2.0 kits of TaKaRa amplification PPTI gene cDNAs ' end overall length.

(1), RT-PCR reaction systems：

Total RNA 1μg 5.5μL

3′RACE adaptor 1μL

Following reaction reagent is added in 70 DEG C of denaturation 10min, cooled on ice 3min

5 x M-MLVBuffer 2μL

dNTP(10mM each) 1μL

RNase inhibitor 0.25μL

M-MLV(RNase H^-) 0.25μL

42 DEG C of extension 60min, synthesize the first chains of cDNA.

(2), Outer PCR reaction systems and Outer PCR reaction conditions：

2 μ L of the first chains of cDNA, 94 DEG C of 3min

PPTIF1(10μM) 2μL 94℃ 30s

3′RACE Outer Primer 2μL 54℃ 30s

1×cDNA Dilution Buffer II 8μL 72℃ 3min

10 x Buffer 5μL 72℃ 10min

1 μ L of Taq enzyme

H₂O to 50 μ L.

(3), Inner PCR reaction systems and Inner PCR reaction conditions：

Outer PCR 1μL 94℃ 3min

PPTIF2(10μM) 2μL 94℃ 30s（30 cycles）

3′RACE Inner Primer 2μL 55℃ 30s（30 cycles）

dNTPs(2.5mM each) 4μL 72℃ 1min（30 cycles）

10 x Buffer 5μL 72℃ 10min

1 μ L of Taq enzyme

H₂O to 50 μ L

10 μ L Inner PCR products are taken to carry out 1.0% plain agar sugar gel electrophoresis.

(4), coupled reaction system：

Outer PCR products recycle 4.5 μ L of segment

0.5 μ L of pMD19-T carriers

Solution I 5μL

16 DEG C of connection 12h.

Embodiment 5,DraI、EcoRV、StuI、PvuTetra- kinds of digestion with restriction enzyme genomic DNAs of II.

Endonuclease reaction system：

80 μ L of genomic DNA

8 μ L of restriction enzyme

10 x Buffer 10μL

H₂O to 100 μ L.

37 DEG C of digestion 12h, each digestion products take 5 μ L, carry out 1.0% plain agar sugar gel electrophoresis, detect digestion situation.

Embodiment 6, purifying DNA fragment.

(1) each group digestion products are transferred to respectively in 1.5mL centrifuge tubes, add sterile water to 500 μ L.

(2) isometric Tris saturated phenols are added, mixing centrifuges (room temperature, 12000rpm, 10min), draws upper strata aqueous phase Into new 1.5ml centrifuge tubes.

(3) isometric chloroform is added, mixing centrifuges (room temperature, 12000rpm, 10min), draws upper strata aqueous phase to newly In 2ml centrifuge tubes.

(4) absolute ethyl alcohol of the 3M NaAc (pH5.2) and the precooling of 2 times of volumes of 1/10 volume is added, -20 DEG C stand 1h, from The heart (4 DEG C, 12000rpm, 10min), abandons supernatant.

(5) it is washed twice with the ethyl alcohol of 70% precooling, is precipitated with the sterile water dissolutions of 20 μ L after dry, 1 μ L is taken to carry out electrophoresis inspection It surveys.

Embodiment 7,5 ' RACE amplification PPTI genetic fragments

(1), the genomic DNA fragment purified is connect with connector.Coupled reaction system and connection reaction condition：

4 μ L of genomic DNA fragment, 16 DEG C of 12h of purifying

1.9 μ L of GenomeWalker connectors, 70 DEG C of 5min

0.5 μ L of T4 ligases

10 x Buffer 1.6μL。

After reaction plus sterile water is to 80 μ L, is used as the template of Primary PCR.

⑵、Primary PCR：

PCR reaction systems and PCR reaction conditions：

2 μ L of template, 94 DEG C of 5min

PPTIGSP1 (25μM) 1μL 94℃ 25s（7 cycles）

AP1 (25μM) 1μL 72℃ 3min（7 cycles）

10 x Buffer 5μL 94℃ 25s（32 cycles）

dNTPs(2.5mM each) 4μL 62℃ 3min（32 cycles）

0.5 μ L of Taq enzyme, 62 DEG C of 7min

H₂O to 50 μ L.

Primary PCR reaction products are diluted 20 times, are used as the template of Secondary PCR.

⑶、Secondary PCR.PCR reaction systems and reaction condition：

2 μ L of template, 94 DEG C of 5min

PPTIGSP2 (25μM) 1μL 94℃ 25s（5 cycles）

AP2 (25μM) 1μL 72℃ 3min（5 cycles）

10 x Buffer 5μL 94℃ 25s（35 cycles）

dNTPs(2.5mM each) 4μL 67℃ 3min（35 cycles）

0.5 μ L of Taq enzyme, 67 DEG C of 7min

H2O to 50 μ L.

PCR reaction systems and PCR reaction conditions：

2 μ L of template, 94 DEG C of 5min

2GSP2 (25μM) 1μL 94℃ 25s（5 cycles）

AP2 (25μM) 1μL 72℃ 3min（5 cycles）

10 x Buffer 5μL 94℃ 25s（35 cycles）

dNTPs(2.5mM each) 4μL 57℃ 3min（35 cycles）

0.5 μ L of Taq enzyme, 57 DEG C of 7min

H2O to 50 μ L.

10 μ L Secondary PCR products are taken to carry out 1.0% plain agar sugar gel electrophoresis.

Coupled reaction system：

Secondary PCR products recycle 4.5 μ L of segment

0.5 μ L of pMD19-T carriers

Solution I 5μL

16 DEG C of connection 12h.

Embodiment 8,PPTIFull length gene is cloned and bioinformatic analysis.

According toPPTI3 end sequences of gene and the sequence of upstream are spliced into a complete gene order, then design Primer amplification overall lengthPPTIGene order.Using the first chains of cDNA of synthesis as template andPPTIGene primer carries out PCR.By mesh Fragment purification (being carried out according to TIANgen MiDi purification Kit operating procedures) and be inserted into pMD19-T carriers, with Machine selects 3 clones and is sequenced and (measures sequence by Shanghai bioengineering).

PCR reaction systems and reaction condition：

1 μ L of the first chains of cDNA, 94 DEG C of 5min

PPTIF1(25μM) 1μL 94℃ 30s（35 cycles）

PPTIR1(25μM) 1μL 54℃ 30s（35 cycles）

10 x Buffer 2.5μL 72℃ 1min（35 cycles）

dNTPs(2.5mM each) 2μL 72℃ 10min

0.5 μ L of Taq enzyme

H₂O to 25 μ L.

10 μ LPCR products are taken to carry out 1.0% plain agar sugar gel electrophoresis.

Coupled reaction system：

PPTIGene recycles 4.5 μ L of segment

0.5 μ L of pMD19-T carriers

Solution I 5μL

16 DEG C of connection 12h.

Cloned sequence analysis shows, PPTI full length genes 642bp (SEQ ID NO：1), encode 214 amino acid (referring to Fig. 6 a), predicted molecular weight 23kD, pI value is 5.13；Signal peptide forms (Fig. 6 b) by 19 amino acid residues of N-terminal, has STI (soybean proteinase inhibitor) structural domain (Fig. 6 c) is a kind of trypsin ihhibitor.Protein transmembrane domain prediction knot Fruit such as Fig. 6 d show：Inside lines never intersect always with outside lines, therefore, it is considered that the albumen does not have membrane-spanning domain.Albumen parent Hydrophobicity on-line analysis such as Fig. 6 e show：Peptide chain main part is distributed in hydrophobic region mostly, and only small part is distributed in hydrophilic area, Speculate that the albumen belongs to fat-soluble albumen.

Embodiment 10, genetic fragment conversion bacillus coli DH 5 alpha and positive colony detection-bacterium colony PCR.

The conversion of bacillus coli DH 5 alpha is as follows.

(1) 1 competent cell (100 μ L) is taken out from -80 DEG C of refrigerators, is melted on ice.

(2) it takes 10 μ L connection products to be added in the competent cell just melted, flicks mixing, ice bath 30min.

(3) 42 DEG C of heat shock 30s, are immediately placed in 2min on ice.

(4) 500 μ L LB liquid mediums are added, 37 DEG C, 200rpm cultivates 1h.

(5) 4000rpm room temperatures centrifuge 2min, part supernatant are removed, by remaining bacterium solution mixing.

(6) bacterium solution is applied on LB+Amp solid mediums with spreading rod, 37 DEG C of inversions are incubated overnight.

Picking partial white single bacterium colony is respectively put into added with the small of 10 μ L ultra-pure waters on the solid medium being incubated overnight In Ep pipes, vortex concussion is dissolved in the water up to bacterium colony, as pcr template, is expanded, and amplification condition is expanded with back Condition, electrophoresis detection.

The verification of embodiment 11, the function of PPTI genes on tobacco.

The plant expression vector pCAMBIA1301-PPTI for being built into target gene turns PPTI with leaf disk method transformation of tobacco Gene difference strain tobacco shows the rejection ability to aphid, and the aphid on transgene tobacco is gradual with the increase of number of days It reduces, at the 15th day, aphid number was almost nil.Aphid inhibiting rate is calculated, finds to turn PI when being inoculated with aphid the 15th day The tobacco of gene is 83.4% to the average inhibition of aphid.At the same time, the anti-aphid ability of the isogenic different plants of phase inversion exists Different, this may be due in different transformed plants PPTI expression quantity it is different caused by, need further study turn The relationship of the expression quantity and aphids resistance level of the PPTI albumen of genetic tobacco（Fig. 7）.This Preliminary Study determines PPTI genes There is good resistance to aphid, this is the protease inhibitor gene of the aphids resistance obtained in pinellia pedatisecta Schott for the first time, Basis is identified further to carry out pest-resistant research using the gene.Test operation process is as follows.

Using conventional plant binary expression vector, preferred plant binary expression vector pBI121 (is commonly used in plant transgene Expression vector is purchased from Chinese Academy of Agricultural Sciences crop research institute Zhao Kaijun researcher in the present invention) and bacterial strain EHA105, it utilizes Cloning and sequencingPPTIGene order, by being incited somebody to action after Xba I and Sma I double digestionsPPTIGene is subcloned into the corresponding of pBI121 On site, it is built into 35S：PPTI+GUS:The plant expression vector pBI121-PPTI of NOS structures, is transformed into agrobacterium strains In EHA105.Anti- kanamycins is obtained using leaf disk method transformation of tobacco according to the method for Wang Guanlin etc.（nptII）The conversion of label Regeneration plant (Wang Guanlin etc., 2002).

By 1% pBI121-PPTI of the agarose electrophoresis through Xba I and Sma I double digestion plasmids, digestion generates two Segment showsPPTIGene is inserted into correct reading frame in pBI121.Conventional leaf disk method transformation of tobacco obtains anti- Kanamycins（nptII）The transgenic tobacco plant of label.

The young leaflet tablet of A transfer-gen plants PCR detection clip transfer-gen plant and nontransgenic plants, using CTAB methods Genomic DNA is extracted, with appropriate sterile dual distillation aqueous suspension, -20 DEG C of preservations after drying at room temperature.The DNA profiling amount of extraction 50ng/ μ L are adjusted to, takes 1 μ L to do template and carries out PCR amplification, 94 DEG C of 3 min；94 DEG C of 30 S, 57 DEG C of 30 S, 72 DEG C of 1min, 35 cycles；72℃ 10 min.The agarose electrophoresis that PCR product runs 1% observes result.The result shows that it is whole to obtain PPTI genes Close the transfer-gen plant of tobacco plant genome.

The Semiquatitative RT-PCR assay of B transfer-gen plants is detected using tobacco young leaflet tablet as test material, according to Promega public affairs The Trizol RNA extracts kits specifications of department extract total serum IgE to the positive plant detected with PCR, carry out sxemiquantitative RT- Whether PCR, verification trans-exogenous PPTI genes express in rna transcription level.10 transgenosis single plants are selected to be detected at random, The result shows that PPTI genes are expressed in mRNA level in-site, but it is different transfer-gen plant expression differs in vivo Sample.

The aphids resistance identification black peach aphid (Myzus persicae) of C transfer-gen plants picks up from Hebei Prov. Academy of Agricultural ＆. Forest Sciences's plant protection Abiogenous population on tobacco in institute greenhouse chooses bouncing, physiological status in being raised in greenhouse after a period of stabilisation Consistent aptery adult aphid is for examination.The black peach aphid of indoor raising is inoculated into the transgene tobacco and non-transgenic cigarette of 810 leaf ages respectively On the blade of careless plant middle level, 10 transfer-gen plant strains are randomly selected, per 3 plants of tobacco seedlings of strain, aptery adult aphid 20 is connect per young plant Head is placed under identical conditions（23~25 DEG C）Raising, the next day investigation record Aphed population in each processing tobacco seedlings, up to 15 days.It presses According to Yuan Zhengqiang etc. method using transgene tobacco to the population inhibiting rate of aphid as index（Yuan is just strong etc., and 2001）, to evaluate Virulence effect of each transgene tobacco to aphid：Population inhibiting rate（%）=（Compare strain work aphid number-treatment region work aphid number）/ control strain Aphid living number × 100.

Qualification result shows that different transfer-gen plant insect resistaces is widely different, after connecing worm from the point of view of the 15th day result, 10 Kind transgene tobacco descending to the population restraint of cigarette aphid is 7,1,3,5,9,8,2,6,4,10 (Fig. 8)；Wherein No. 7 and 1 Number 100% and 92.02% respectively reached to the population inhibiting rate highest of cigarette aphid, the inhibiting rate of No. 3 plant also more a height of 78.85%, No. 5 inhibiting rates are also respectively 58.84%, remaining transfer-gen plant does not show resistance, this may cause with transgene silencing Caused by transgenosis is not expressed.Especially No. 7 transfer-gen plants cigarette aphid after being inoculated with 7 days is all dead, is demonstrated by lethal effect, Illustrate that the gene that we detach clone is a gene to aphid with good resistance.

The description that above-described embodiment is recorded only is proposed as the enforceable technical solution of the present invention, not as to its technical side The single restrictive condition of case itself.

Sequence table

<110>Hebei Prov. Academy of Agricultural ＆. Forest Sciences

<120>A kind of and relevant gene of plant resistance to insect, albumen of its coding and its application

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 642

<212> DNA

<213>Tobacco (nicotiana tabacum)

<400> 1

atggagttta tcctgctcct tgtgtcttcc ctcctcctca ccgcccgagc cgccgccgcc 60

gcctccaatc ccatcctcga caacgacggc aacgagctcc gacgtggcca actctactat 120

gcgatgtccg tgaagaggcc cggtggcggc ctgacgctgg cgccccccgc caacgcggcg 180

cggtgccctc tcaacgtggc ccaggcgccc ttcaacgact attccggccg cccgctggcc 240

ttcttcccgg agaacgccga cgacgacacc gtgcgagagg gaagtacact aaacatcatg 300

ttcccggagc cgacggagtg cgcccagtcc accgtgtgga ctctcgacag ggagaccggc 360

ctcgtgacca ccggcggaac cgcgtcgtcg gcggtcggcc cctactacag ccggttcgcc 420

atacgcaagg ccgaggatgc tgcttcgtcg tcccagcgcg atcgctacca gatccaggtt 480

tgcccctgca gctccggcgt gcggcggcct tcctgcagga tgggctgtct cggcagtctg 540

ggtttgagcg agggagagga gaacgtcatg ctcaacatca acaacgagcg ccctcacacc 600

gtcatgtttg tggaggtgaa ggaagggatc gctgccagca ta 642

<210> 2

<211> 560

<212> DNA

<213>Tobacco (nicotiana tabacum)

<400> 2

tgcagctggc cattacggcc ggggccgacg acgacaccgt gcgagaggga agtacactaa 60

acatcatgtt cccggagccg acggagtgcg cccagtccac cgtgtggact ctcgacaggg 120

agaccggcct cgtgaccacc ggcggaaccg cgtcgtcggc ggtcggcccc tactacagcc 180

ggttcgccat acgcaaggcc gaggatgctg cttcgtcgtc ccagcgcgat cgctaccaga 240

tccaggtttg cccctgcagc tccggcgtgc ggcggccttc ctgcaggatg ggctgtctcg 300

gcagtctggg tttgagcgag ggagaggaga acgtcatgct caacatcaac aacgagcgcc 360

ctcacaccgt catgtttgtg aaggtgaagg aagggatcgc tgccagcata tagaggagcc 420

gttgatcgat cggccagtac ttgctagtcc catgttagta ctacgtacgt actttgtatc 480

gtccaccaaa taagctaggg ttcctataat agggaacgat cgagctgcca tggacagctc 540

ctctagcgtt tgagttcacc 560

<210> 3

<211> 270

<212> DNA

<213>Tobacco (nicotiana tabacum)

<400> 3

tagaggagcc gttgatcgat cggccagtac ttgctagtcc catgttagta ctacgtacgt 60

actttgtatc gtccaccaaa taagctaggg ttcctataat agggaacgat cgagctgcca 120

tggacagctc ctctagcgtt tgagttcacc cagctggtgt cctgcagtag taactacagg 180

acatggtgtg ctgtgtagta gttaagcttg tcttctactt aagtataata agtggcgacg 240

tgcatggttc tctcgcaaaa aaaaaaaaaa 270

<210> 4

<211> 20

<212> DNA

<213>Artificial sequence (artificial sequence)

<400> 4

ctggtgtcct gcagtagtaa 20

<210> 5

<211> 20

<212> DNA

<213>Artificial sequence (artificial sequence)

<400> 5

tctagcgttt gagttcaccc 20

<210> 6

<211> 20

<212> DNA

<213>Artificial sequence (artificial sequence)

<400> 6

gtttgtgaag gtgaaggaag 20

<210> 7

<211> 20

<212> DNA

<213>Artificial sequence (artificial sequence)

<400> 7

gaggatgctg cttcgtcgtc 20

<210> 8

<211> 21

<212> DNA

<213>Artificial sequence (artificial sequence)

<400> 8

tcaaccatgg agtttatcct g 21

<210> 9

<211> 20

<212> DNA

<213>Artificial sequence (artificial sequence)

<400> 9

atcccttcct tcaccttcac 20

Claims (9)

1. a kind of pinellia pedatisecta Schott trypsin inhibitor gene, it is characterised in that：Its nucleotides sequence list such as SEQ ID NO：1 institute Show.

2. the recombinant expression carrier containing gene described in claim 1.

3. the nuclear transformants containing gene described in claim 1.

4. expanding the primer pair of gene described in claim 1.

5. the albumen of gene code described in claim 1.

6. application of the gene described in claim 1 in cultivating resistance to insect-resistant transgenic plants.

7. application according to claim 6, it is characterised in that：Structure contains SEQ ID NO first：The weight of gene shown in 1 Then group expression vector utilizes gained recombinant expression carrier to build transformant, gained transformant is recycled to convert purpose plant, sieve Positive plant is selected, screens to obtain the homozygous genetically modified plants with insect resistace compared with normal plants by three generations.

8. the application described according to claim 6 or 7, it is characterised in that：The purpose plant is tobacco.

9. the application described according to claim 6 or 7, it is characterised in that：The insect resistace master refers to being directed to Homoptera insect Resistance.