CN1249310A - Cationic polypeptide process for expressing antimicrobe in plant - Google Patents

Abstract

A transgenic plant expression vector containing three antimicrobe cationic polypeptides is constructed. The plant tissue is introduced to these genes via agrobacillus and in the callus culture, the callus resisting phytopathogen (bacteria and fungus) can be externally chosen. Choosing these calli can regenerate plants and directly test its antifungual and antibacterial powder. After these transgenic plants are ripened, whole or partial plant can be harvested.

Description

A kind ofly in plant, express antimicrobial cationic polypeptide technology

The invention belongs to biological technical field.Specifically, the present invention relates to antimicrobial cationic polypeptide (cationic peptide) and expression vector and promoter, transfection in plant and expression, selection of transgenic plant and regeneration, transgenic plant are to the resistance of its bacterium and fungal pathogen bacterium.

Economic plants pathogenetic bacteria and fungi cause the loss of tens million of units.There is 40% crop failure directly relevant approximately in developing country with plant pathogenetic bacteria and fungi.Plant gene engineering technology can be integrated in the Plant Genome exogenesis disease-resistant gene.Genetically modified technology has obtained success in some pest-resistant and disease-resistant plants.But in existing transgenic plant, its disease resistance is very limited, lacks broad spectrum.Express antimicrobial cationic polypeptide and can introduce the broad spectrum resistance gene and advance in the plant, increase the resistance of plant bacterium.

Cationic polypeptide comes from insect, in vertebra and invertebrates and the plant.According to its structure, be divided into alpha-helix, several big classes such as beta sheet and while tool α spiral and βZhe Die.These polypeptide have the microorganism active of wide spectrum, can resist Gram-positive and negative bacteria, parasite, even virus.Carry out the hybridization of structural modification and different cationic polypeptides by computer simulation, can create than natural more great-hearted cationic polypeptide.Express these polypeptide and not only can improve the resistance of plant, attempt using plant simultaneously, produce antimicrobial cationic polypeptide class microbiotic of new generation as factory plant.

The present invention seeks to:

(1) in plant, expresses several special cationic polypeptides, strengthen plant bacillary and resistance fungal disease;

(2) utilize plant as factory, produce microbiotic of new generation;

(3) express antibacterial peptide in the genetically modified Chinese herbal medicine,, improve and improve the property of medicine of herbal medicine to improve herbal medicine output.

The working of an invention scheme:

The invention provides antimicrobial polypeptide gene and the clone position on special expression vector thereof.In general, these carrier systems comprise the gene of cationic polypeptide or go into to the plant expression vector of various promoter controls with their precursor forms skewer.Goal gene on these carriers is changed in the plant by Agrobacterium or particle gun, genetically modified plant is at first in the callus stage, by external disease-resistant selection, and then generate plant, directly detect the resistance of whole strain plant in the body again plant-pathogenic bacterium or fungi.At last, plant-growth maturation, the whole strain plant of results or part plant such as stem tuber, piece root etc.

The selection of antimicrobial polypeptide gene

We have selected the precursor forms of 3 kinds of antimicrobial cationic polypeptides and a kind of cationic polypeptide, and are as shown in the table:

A.CEMA is in proper order:

KWKLFKKIGIGAVLKVLTTGLPALKLTK

Its Pro of b.Pro-CEMA is in proper order:

EPLQARAEVAAAPEQIAADIPEVVVSLAWDESLAPKHPGSRKN

c.Dermaseptin?B：

AMWKDVLKKIGTVALHAGKAALGAVADTISQ

d.Temporin?A：

FLPLIGRVLSGIL

The structure of plant expression vector

Shown in following description, contain the structure of the expression of plants carrying agent of antimicrobial gene:

(1)pSAI4：

Remove CaMV 35S promoter and β-glucuronidase on the PBI121, insert the HindIII-EcoRI dna fragmentation.This fragment contains 2 * 35S promoter, an AMV RNA4 translational enhancer, and CEMA gene and NOS-ter order are inserted and are cloned in pBI121.

(2)pRSPC4，pSSPC4，pPINC4：

0.65kb the promoter fragment is removed from pSAI4, inserts the HindIII-Xbal fragment.These HindIII-Xbal fragments are to come from pRSP221 (promoter that root-specific is expressed), pSSP221 (seed-specific expression promoter) and pRT210 (promoter of recovery emergency reaction) respectively.

(3) pDPC121 and pDPC221:

The former contains the pro-CEMA gene order, and on the pBI121 carrier, tool 2 * 35S promoter is used for the expression that carcinogenic Agrobacterium is intermediary's transfection.The latter contains the pro-CEMA DNA sequence on the pBI121 carrier, is used for the direct transduction of particle gun and goes into vegetable cell.

(4) pD5B1217 and pD5B2212, pTA1217, pTA2217:

Remove the Gus gene of 1.8Kb respectively from pBI121 and pBI221 with XbaI and SstI, skewer go into Dermaseptin B (DSB) gene order (pDSB1217, pDSB2212) or Temporin A gene order (pTA1217, pTA2217).

(5)pDDSB1212：

Dermaseptin B gene structure contains 2 * 35S promoter and AMV RNA4 enhanser on the pBI121 carrier.

(6)pPDSB1217，pSDSB1212，pRTA1217：

Digest pRSP121 or pSSP221 respectively with XbaI and SstI, insert from pDSB1217 (the about 120bp of DSB gene) or the next XbaI-SstI fragment of pTA1217 (Temporin A gene, about 60bp).

(7)pRDSB1217：

Remove about 0.6kb HindIII-XbaI promoter fragment on pRDSB1217, skewer is gone into from the HindIII-XbaI fragment of the next about 1kb of pRT210, contains the promoter order (Wound-inducible promoter) of emergency reaction.

(8) pDTA1217, pSTA1217 and pPTA1217:

Remove the CaMV35Spromoter of 0.8kb on the pTA1217 carrier.Go into the next 2XCaMV35SPromoter from pBI525 as skewer, an AMV RNA4 enhanser constitutes pDTA1217; The promoter of going into the seed-specific expression of the 1.16kb from pSSP121 as skewer constitutes pSTA1217 in proper order.Go into from the promoter of the next injured emergency reaction of pRT210 to constitute pPTA1217 as skewer.

(9)pSUPC4：

Remove CaMV 35S promoter and β-glucuronidase on the pBI121, constructed super promoter before CEMA.

(10)pSUPD1217：

Remove CaMV 35S promoter and β-glucuronidase on the pBI121, constructed super promoter before Dermaseptin B.

(11)pSUPT：

Remove CaMV 35S promoter and β-glucuronidase on the pBI121, constructed super promoter before Temporin A.

(12)pRSUPC4：

Remove CaMV 35S promoter and β-glucuronidase on the pBI121, the super promoter of structure reverse property is before CEMA.

(13)pRSUPD1217：

Remove CaMV 35S promoter and β-glucuronidase on the pBI121, the super promoter of structure reverse property is before Dermaseptin B.

(14)pRSUPT：

Remove CaMV 35S promoter and β-glucuronidase on the pBI121, the super promoter of structure reverse property is before Temporin A.

(15) remove CaMV 35S promoter and β-glucuronidase on the pHSUP1:pBI121, constructed super promoter before the Dermaseptin B of 6 Histidine-tagged is arranged.

(16)pHRSUP1：

Remove CaMV 35S promoter and β-glucuronidase on the pBI121, constructed super promoter before the Dermaseptin B of 6 Histidine-tagged is arranged.

(17)pTerm3：

Remove CaMV 35S promoter and β-glucuronidase on the pBI121, construct double-CaMV 35S+AMVRNA4-CEMA, double CaMV 35S+AMV RNA4-Dermaseptin B, double CaMV 35S+AMVRNA4-temporin A is on this carrier.

All said gene carriers are all by restriction endonuclease digestion, and PCR amplifies and dna sequence analysis confirms.

Agrobacterium transfection plant tissue

Utilize Agrobacterium transfection plant to be undertaken by the method that Block M.D. (1998, Theor Appl Genet 76:767-774) describes.

The plant of the anti-phytopathogen of early stage external selection

In the callus stage, in the substratum of cultured calli, directly add phytopathogen, select to resist the callus of these pathogenic bacterias again they to be induced into plant.

The antimicrobial polypeptide gene integration advances in the plant

Utilize PCR or Rt-PCR method, can detect antimicrobial gene and whether put in order in plant, and in plant, express.

The regeneration of transgenic plant

Shown in regeneration induction method of transgenic plant such as Block M.D. (1998, Theor Appl Genet 76:767-774) describe.

Embodiment

Plasmid vector pSAI4 makes up as shown in the figure.

The CEMA gene that comes from pR78h proCEMA carrier is cloned on the pBI524, finally is cloned on the pBI121 then, names in pSAI4 (Figure 1A).

5 '-terminal primer is in proper order:

CAA GGA AAA ACG GTC TAG AGC ATA TGA AAT GGA AAC contains the XbaI site.

3 '-terminal primer is in proper order:

GAA CTC GAG CAG CGA GCT CTT ACT TAG TTA GCT TC contains the BamHI site.

Figure 1B shows CecropinA, the order of melittin and CEMA, and cationic amino acid is a bold-type letter, anionic amino acid is at alphabetical underscoring.

Claims (7)

1. the present invention is the technology of a kind of transgenic plant, the carrier structure that the precursor pro-CEMA of claim three kind of antimicrobial cationic polypeptide (CEMADemaseptin B and Temporin A) and CEMA expresses in plant, and promoter.

2. the precursor forms of three kinds of cationic polypeptides as claimed in claim 1 and CEMA, its polypeptide sequence is:

A.CEMA is in proper order:

KWKLFKKIGIGAVLKVLTTGLPALKLTK

Its Pro of b.Pro-CEMA is in proper order:

EPLQARAEVAAAPEQIAADIPEVVVSLAWDESLAPKHPGSRKN

c.Dermaseptin?B：

AMWKDVLKKIGTVALHAGKAALGAVADTISQ

d.Temporin?A：

FLPLIGRVLSGIL

3. the carrier of genetic expression is pBI121 and pBI221, and its promoter has in the whole strain plant expresses or the promoter of tissue specific expression.

A. the promoter of whole strain expression of plants has:

(a)CaMA?35S?promoter

(b)2X?CaMV?35S+AMV?RNA4?translation?enhance?element

(c) super promoter (Super promoter)

(d) the super promoter of reverse property

B. the promoter of tissue specific expression has:

(a) promoter of seed specific expression

(b) promoter of root-specific expression

(c) promoter of injured emergency reaction

4. the external selection of anti-plant-pathogenic of callus: promptly callus can be selected its disease resistance callus external inoculation pathogenic bacteria or fungi.

5. the expression of above-mentioned three kinds of genes in potato, tobacco, tomato, rape, paddy rice, cotton, broccoli, radish, wheat, soybean and other various plants.

6. genetically modified plant is to plant-pathogenic bacterium and the good resistance of fungi tool.

7. utilize above-mentioned transgenic plant as plant produced a new generation antibiotic.