CN101792747A - Soybean AOC and coding gene and application thereof - Google Patents

Abstract

The invention discloses allene oxide cyclase (AOC), an important catalytic enzyme in the generation path of glycine max(L.)Merr. jasmonic acid and a coding gene and application thereof, belonging to the biotechnology field. The soybean AOC is a protein with the amino acid sequence in SEQ ID NO.2 in a sequence table. The coding gene is the DNA sequence of SEQ ID NO.1 of a GmAOC1 gene. The soybean AOC and the coding gene can be used for cultivating insect resistant plant varieties, especially soybean varieties. When the transgenic tobacco leaves and the wild leaves are simultaneously fed to prodenia litura, the loss rate of the transgenic tobacco leaves is significantly lower than that of the wild leaves, showing that the GmAOC1 gene plays an important role in improving the insect resistance of the plants.

Description

A kind of soybean AOC fermentoid and encoding gene and application

Technical field

The present invention relates to a kind of soybean AOC fermentoid and encoding gene and application, belong to plant genetic engineering field.Be specifically related to and pest-resistant coherent signal material jasmonic route of synthesis key enzyme allene oxide synthase reductase enzyme (Allene oxide cyclase, AOC) and encoding gene and application, and relate to derive from soybean with the synthetic relevant AOC fermentoid GmAOC of jasmonic and encoding gene and its application in cultivation zoophobous kind.

Background technology

Insect pest is one of principal element that causes crop production reduction, and according to the World Food Programme (FAO) statistics, in worldwide, the annual crop loss that causes because of insect pest accounts for 14% of total yield, loses hundreds billion of approximately dollars.As a kind of important food and cash crop, various pests seriously can influence its yield and quality to soybean [Glycine max (L.) Merr.].Use chemical insecticide so far, promptly increased agricultural cost, cause many pest resistance to insecticide to strengthen rapidly again, the while environment, food chain and water resources also are subjected to severe contamination.

At present, using gene engineering technique carries out breeding and has become one of important method that improves the crop insect-resistance.Studies show that jasmonic can activated plant in the body with pest-resistant relevant defensin gene, make plant produce (Ryan and Pearce, 1998) such as proteinase inhibitor, secondary compound and release volatile matters.Allene oxidecyclase (AOC) is jasmonic (EC 5.3.99.6) route of synthesis key enzyme, and it can be catalyzed into unstable substrate first stable pentacyclic compound of jasmonic approach.The AOC fermentoid extensively is present in the plant, mainly clones this gene from Arabidopis thaliana, clover, paddy rice etc. at present.

Improve the soybean insect-resistance, reduce the influence of insect pest, have actively and great realistic significance improving agricultural performance and improving the human life quality to soybean yields and quality.

Summary of the invention

Technical problem

The purpose of this invention is to provide a kind of soybean AOC fermentoid and encoding gene and application.

Technical scheme

Soybean AOC fermentoid provided by the present invention, name is called GmAOC1, derives from Glycine soybean [Glycinemax (L.)], is the protein with the described aminoacid sequence of SEQ ID NO.2 in the sequence table.

Above-mentioned soybean AOC zymoid encoding gene, its cDNA gene has the dna sequence dna of GmAOC1 genes of SEQ IDNO.1 in the sequence table; Wherein, the GmAOC1 genes of SEQ ID NO.1 in the sequence table is by deoxynucleotide, and this sequence is the reading frame of GmAOC1 gene, and coding has the protein of the amino acid residue sequence of SEQ ID NO.2 in the sequence table.

The expression vector that contains GmAOC1 genes of SEQ ID NO.1 of the present invention is meant pMDC83-GmAOC1 plant overexpression carrier, and the host bacterium is meant the agrobacterium tumefaciens bacterial strain EHA105 that the GmAOC1 gene is changed over to.

The primer of amplification GmAOC1 gene:

GmAOC1 ORF forward primer: 5 ' ATGGCTTCCATGGGCTCTC-3 '

GmAOC1 ORF reverse primer: 5 ' GTTGGTGAAGTTTGGCAAA-3 ';

Above-mentioned soybean GmAOC1 enzyme and encoding gene GmAOC1 gene thereof can be applied in cultivating zoophobous.

Beneficial effect

The tobacco of overexpression GmAOC1 is compared with wild-type tobacco, and its insect-resistance significantly improves, and illustrates that the GmAOC1 gene is playing an important role aspect the raising plant resistance to insect.

GmAOC1 of the present invention is to cultivating pest-resistant farm crop, and it is particularly significant to the influence of soybean yields to the influence of crop yield to reduce insect pest.

Utilize plant expression vector, the encoding gene importing vegetable cell with GmAOC1 of the present invention can obtain the insect-resistant transgenic plant.

When using GmAOC1 to make up plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or inducible promoter.For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, as adding selected marker's (gus gene, luciferase genes etc.) that can in plant, express or antibiotic marker thing (gentamicin marker, kantlex marker etc.) with resistance.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

Carry GmAOC1 of the present invention plant expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and the plant transformed tissue cultivating is become plant.By the plant transformed host both can be monocotyledonss such as paddy rice, wheat, corn, also can be dicotyledonss such as soybean, cucumber, tomato, willow, turfgrass, clover.

Description of drawings

Fig. 1 is that the GmAOC1 gene is at soybean chromosome position structural representation

Fig. 2 is the part-structure synoptic diagram that contains the plant expression vector of GmAOC1

Fig. 3 is the blade that changes GmAOC1 tobacco strain system and the wild-type tobacco prodenia litura result schematic diagram of feeding simultaneously

First row: transgene tobacco blade, second row: wild-type tobacco blade

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment.

Method therefor is ordinary method if no special instructions among the following embodiment.

One, the cDNA of soybean GmAOC1 and encoding gene thereof clone and evaluation soybean (Glycine max (L.) Merr.) the little green soya bean of material Calusena lansium (juanjuan Wu, et al.Constitutive overexpression ofAOS-l ike gene from soybean enhanced tolerance to insect attack intransgenic tobacco.Biotechnology letters, 2008), material is planted in the solarium, conventional field management.

AOC gene with evaluation in the plant clover of having reported (Medicago truncatula): MtAOC1 (AJ308489) and MtAOC2 (AJ866733), homology according to aminoacid sequence between species, TIGR database and NCBI (WGS) database of search soybean, through splicing, extend the back and obtain a height homologous sequence.Its sequence 5 ' end is all imperfect.Designed Auele Specific Primer (GSP) and the nested primer (NGSP) of 5 ' RACE respectively, the order-checking of amplification rear clone, splicing obtains comprising the sequence of complete ORF.

The design special primer is that template is cloned checking with the cDNA of soybean gene group and soybean tender leaf respectively.The primer that comprises complete ORF sequence with the Peimer3 programdesign:

AOC ORF forward primer: 5 ' ATGGCATCATCCTCATCAA-3 '

AOC ORF reverse primer: 5 ' GTCAGTGAAGCCAGCAATA-3 '; Use the RT-PCR method, cDNA is a template amplification GmAOC1 gene with the total RNA counter-rotating of soybean synthetic.Get the little green soya bean blade of Calusena lansium, place liquid nitrogen to grind, RNA extracts and carries out according to TIANGEN total RNA extraction reagent box RNA Plant Extraction kit DP417.CDNA first chain is synthetic according to the TaKaRa reagent cDNA of the company first chain synthetic agent box TaKaR a PrimeScript ^TM1st strand cDNA Synthesis KitD6110A specifically sees operation instructions for details.With the cDNA fragment that obtains is template, carries out pcr amplification reaction with 1 pair of primer respectively.25 μ l μ l PCR reaction systems are: 1 μ l, one chain cDNA (0.05 μ g), 1 μ l primer (10 μ M), 2.5 μ l, 10 * PCR damping fluid, 2.5 μ l Mg ²⁺, 4 μ l dNTP (10mM) and 1.25U LA TaqDNA polysaccharase, supply 25 μ l with ultrapure water.Be reflected on the BIO-RAD PTC-200 type PCR instrument and carry out, its program is 95 ℃ of sex change 5min; 94 ℃ of 30sec again, 56 ℃ of 50sec, 72 ℃ of 1min, totally 30 circulations; 72 ℃ are extended 10min then; 4 ℃ of preservations.The PCR product reclaims after link PMD19-T carrier (TaKaRa), transformed into escherichia coli DH5 α, the screening of blue hickie, shake bacterium, order-checking, sequential analysis, the result shows that the PCR product has the nucleotide sequence of SEQ ID NO.1 in the sequence table, called after GmAOC1 gene.ORF sequence and genome sequence comparison back are found that as shown in Figure 1: the GmAOC1 gene contains two exons, an intron.

Two, the Function Identification of GmAOC1 gene coded protein

Utilize the Gateway of Invitrogen company

Technology with Clonase ^TMThe II test kit, GmAOC1 gene forward is inserted into expression vector pMDC83 (Sokolov et al (2005) A redox-regulat ed chloroplast protein phosphatase binds to starch diurnally andfunctions in its accumulation, PNAS, 103:9732-9737), obtain pMDC83-GmAOC1 plant overexpression carrier, Fig. 2 has shown the pMDC83-GmAOC1 partial sequence.Respectively pMDC83-GmAOC1 is changed over to agrobacterium tumefaciens bacterial strain EHA105 (Avsian-Kretchmer et al with freeze-thaw method, TheSalt-Stress Signal Transd uction Pathway That Activates the gpx1 PromoterIs Mediated byIntracell ular H2O2, Diff erent from the Pathway Inducedby Extracellular H2O2,2004, Plant Physiology, 135:1685-1696) in.PMDC83-GmAOC1 is by Agrobacterium EHA105 mediated transformation tobacco, and the PCR detected result shows acquisition 56 positive plants.With the blade of transgene tobacco strain system and the wild-type tobacco prodenia litura (purchasing) of feeding simultaneously in the academy of agricultural sciences, Jiangsu Province, the result is as shown in Figure 3: the loss amount of transgene tobacco blade significantly is less than the loss amount of wild-type tobacco blade, illustrate that transgenic line compares insect-resistance with wild-type tobacco and significantly improve, overexpression GmAOC1 gene can improve the insect-resistance of transgene tobacco.

Sequence table

＜110〉Agricultural University Of Nanjing

＜120〉a kind of soybean AOC proteinoid and encoding gene and application

＜130〉specification sheets

<160>4

<170>PatentIn?version?3.1

<210>1

<211>765

<212>DNA

＜213〉Glycine soybean (Glycine max L.)

<220>

＜221〉GmAOC1 gene reading frame (ORF)

<222>(1)..(765)

<223>

<400>1

atggcttcca?tgggctctct?gaagatgatt?tcgtccctca?aactctcccg?ttcaagttgt 60

tctatctctc?cccttcaaac?ccaaaagcaa?gtaggttcaa?gtctctttca?atccttccca 120

accaaaactt?taaaattctc?agctacccct?caagtatcta?catccagaag?aagtaccaac 180

aagaccacta?ccactgcatt?cttcttcaat?aaccaaaagc?agcatcaaga?ttcctcacag 240

ccagccaaag?ttcaagaact?ctttgtctac?gagatcaacg?aacgcgaccg?aggaagtcct 300

gcatacctta?ggctaagcca?gaagccagtt?aactctctag?gagacttagt?gccattcagc 360

aacaagatat?actctggaga?cttgcaaaag?agactaggga?taactgcagg?cttgtgtgtg 420

ctcatccagc?atgagcctga?gaaaaagggt?gatagatatg?aggccattta?cagcttctac 480

tttggaaact?atggccacat?atcagtgcaa?ggagcctatc?tcacattcca?agacacatat 540

ctggcagtta?caggaggctc?tggaatcttt?gaaggtgctt?ctggacaagt?gaagcttcac 600

caacttgtgt?tccctttcaa?gctgttctac?accttctatt?tgaagggtgt?tcctgatttg 660

cctcctgaac?tgcttgggaa?acctgttgaa?ccttcaccaa?gtgttgagcc?ttctcctgct 720

gctatggcta?ccgagcctca?tgcctgtttg?ccaaacttca?ccaac 780

<210>2

<211>255

<212>PRT

＜213〉Glycine soybean (Glycine max L.)

<220>

＜221〉GmAOC1 protein sequence

<222>(1)..(255)

<223>

<400>2

Met?Ala?Ser?Met?Gly?Ser?Leu?Lys?Met?Ile?Ser?Ser?Leu?Lys?Leu?Ser

1 5 10 15

Arg?Ser?Ser?Cys?Ser?Ile?Ser?Pro?Leu?Gln?Thr?Gln?Lys?Gln?Val?Gly

20 25 30

Ser?Ser?Leu?Phe?Gln?Ser?Phe?Pro?Thr?Lys?Thr?Leu?Lys?Phe?Ser?Ala

35 40 45

Thr?Pro?Gln?Val?Ser?Thr?Ser?Arg?Arg?Ser?Thr?Asn?Lys?Thr?Thr?Thr

50 55 60

Thr?Ala?Phe?Phe?Phe?Asn?Asn?Gln?Lys?Gln?His?Gln?Asp?Ser?Ser?Gln

65 70 75 80

Pro?Ala?Lys?Val?Gln?Glu?Leu?Phe?Val?Tyr?Glu?Ile?Asn?Glu?Arg?Asp

85 90 95

Arg?Gly?Ser?Pro?Ala?Tyr?Leu?Arg?Leu?Ser?Gln?Lys?Pro?Val?Asn?Ser

100 105 110

Leu?Gly?Asp?Leu?Val?Pro?Phe?Ser?Asn?lys?Ile?Tyr?Ser?Gly?Asp?Leu

115 120 125

Gln?Lys?Arg?Leu?Gly?Ile?Thr?Ala?Gly?Leu?Cys?Val?Leu?Ile?Gln?His

130 135 140

Glu?Pro?Glu?Lys?Lys?Gly?Asp?Arg?Tyr?Glu?Ala?Ile?Tyr?Ser?Phe?Tyr

145 150 155 160

Phe?Gly?Asn?Tyr?Gly?His?Ile?Ser?Val?Gln?Gly?Ala?Tyr?Leu?Thr?Phe

165 170 175

Gln?Asp?Thr?Tyr?Leu?Ala?Val?Thr?Gly?Gly?Ser?Gly?Ile?Phe?Glu?Gly

180 185 190

Ala?Ser?Gly?Gln?Val?Lys?Leu?His?Gln?Leu?Val?Phe?Pro?Phe?Lys?Leu

195 200 205

Phe?Tyr?Thr?Phe?Tyr?Leu?Lys?Gly?Val?Pro?Asp?Leu?Pro?Pro?Glu?Leu

210 215 220

Leu?Gly?Lys?Pro?Val?Glu?Pro?Ser?Pro?Ser?Val?Glu?Pro?Ser?Pro?Ala

225 230 235 240

Ala?Met?Ala?Thr?Glu?pro?His?Ala?Cys?Leu?Pro?Asn?Phe?Thr?Asn

245 250 255

<210>3

<211>19

<212>DNA

＜213〉synthetic

<220>

＜221〉GmAOC1 ORF forward primer

<222>(1)..(19)

<223>

<400>3

atggcttccatgggctctc 19

<210>4

<211>19

<212>DNA

＜213〉synthetic

<220>

＜221〉GmAOC1 ORF reverse primer

<222>(1)..(19)

<223>

<400>4

gttggtgaagtttggcaaa 19

Claims (10)

1. soybean jasmonic route of synthesis key enzyme allene oxide synthase reductase enzyme, called after GmAOC enzyme is the protein with the described aminoacid sequence of SEQ ID NO.2 in the sequence table.

2. coding claim 1 described soybean AOC zymoid gene.

3. encoding gene according to claim 2 is soybean AOC zymoid cDNA gene, has the dna sequence dna of GmAOC1 genes of SEQ ID NO.1 in the sequence table.

4. contain claim 2 or 3 described expression carrier.

5. expression vector according to claim 4 is meant pMDC83-GmAOC1 plant overexpression carrier.

6. the host bacterium that contains claim 2 or 3 described genes.

7. host bacterium according to claim 6 is meant the agrobacterium tumefaciens bacterial strain EHA105 that the GmAOC1 gene is changed over to.

8. the primer of amplification claim 2 or 3 described genes is characterized in that,

GmAOC1 ORF forward primer: 5 ' ATGGCTTCCATGGGCTCTC-3 '

GmAOC1 ORF reverse primer: 5 ' GTTGGTGAAGTTTGGCAAA-3 '.

9. the application of the described soybean AOC of claim 1 fermentoid in cultivating zoophobous.

10. claim 2 or the 3 described soybean AOC class enzyme coding genes application in cultivating zoophobous.

Images