CN108517333A

CN108517333A - Application of the rice Os BBTI4 protein gene on improving rice anti-rice blast

Info

Publication number: CN108517333A
Application number: CN201810336811.7A
Authority: CN
Inventors: 宁约瑟; 张重阳
Original assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Priority date: 2018-04-16
Filing date: 2018-04-16
Publication date: 2018-09-11
Anticipated expiration: 2038-04-16
Also published as: CN108517333B

Abstract

The invention discloses applications of the rice trypsin inhibitor protein gene OsBBTI4 on improving rice anti-rice blast, the cDNA nucleotides sequences of the rice trypsin inhibitor protein gene OsBBTI4 are classified as the degenerate sequence shown in SEQ ID No.1 or for SEQ ID No.1.Transgenic experiments prove the resistance that the overexpression of the gene improves rice to Pyricularia oryzae.Therefore it can be used as target gene and import plant, disease resistance of plant is improved, to carry out plant species improvement.The trypsin inhibitor being transferred to also by the hydrolysis of protease inhibition, protects other albumen not to be degraded, effectively improves plant defense ability.

Description

Application of the rice Os BBTI4 protein gene on improving rice anti-rice blast

Technical field

The invention belongs to genetic engineering fields, and in particular to a kind of rice Os BBTI4 protein gene is improving Rice Resistance rice It is applied in seasonal febrile diseases.

Background technology

Trypsin inhibitor (Trypsin inhibitor, TI) is prevalent in the animal, plant and micro- life of nature In object.Currently, studying more trypsin inhibitor is mainly derived from the main grain of the mankind, such as pulse family and Qiao Benke, It is also had been reported that in Cruciferae.And in the animal kingdom, the microorganisms such as blood, pancreas and the saccharomycete of animal, streptomyces In also have the presence of trypsin inhibitor.

Protease inhibitors can be divided into serine, cysteine, aspartic acid, 4 major class of metal protease inhibitors.And pancreas Protease inhibitors belongs to serpin (serine proteinase inhibitor, Serpin) family.Its Middle research the most extensive is two class of Kunitz types inhibitor and Bowman-Birk types inhibitor, is Ku Nizi respectively Trypsin inhibitor (Kunitz type Trypsin inhibitor, abbreviation KTI) and Bao Man-problem about Bellco haemodialysis trypsase suppression Preparation (Bowman-Birk type Trypsin inhibitor, abbreviation BBTI).

Bowman-Birk originates from Kunitz, and is replaced it.Bowman-Birk trypsin inhibitor (BBTI) acquisition is detached from soybean in nineteen forty-six.The molecular mass of the bowman-Birk trypsin inhibitor is about 8kD is rich in cysteine, including 2 reaction sites and 7 disulfide bond.2 reaction sites inhibit trypsase and pancreas solidifying respectively Cyclic structure is presented in the single-side structural of galactase, reaction site.One cyclic structure is that trypsase calmodulin binding domain CaM contains 4 two Sulfide linkage, can inhibit the activity of trypsase, and the combined area that another cyclic structure is chymotrypsin, there are 3 disulfide bond, Inhibition can be combined with chymotrypsin, and it is active.Since intramolecular is rich in the polar interaction between disulfide bond and subunit, The thermal stability and ph stability of these BBTI is preferable.

Extensive research based on trypsin inhibitor structure and biochemical function, and people were it is also noted that pancreas in recent years The biological function of protease inhibitors, including：Antimycotic, antiviral and antitumaous effect, and carried out the phase of this respect extensively Close research.

In anti-virus aspect, Evandro etc. is extracted kunitz trypsin inhibitor from beans, which can To inhibit to be proliferated HIV-1 the activity of vital HIV-1 RT, so that this trypsin inhibitor is provided with Inhibit the possibility of HIV-1 proliferation.Ye etc. has found that the Bowman-Birk types inhibitor from broad bean similarly has and inhibits HIV- The effect of 1 reverse transcriptase activity.These researchs all illustrate that trypsase inhibits and with the possibility for inhibiting virus multiplication.

At antimycotic aspect, trypsin inhibitor is mainly widely studied in corn.The study found that in corn, Corn trypsin inhibitor can inhibit the growth of Aspergillus flavus and the sprouting of spore, be referred to as a kind of antifungal protein.And And think that this inhibitory activity may inhibit aflatoxins generation related with host itself.Also it has been reported that, is deposited when there is pathogen When, which can inhibit the activity of the amylase of pathogen, to make disease fungus that can not draw nourishment from, Cause pathogen that can not survive, to inhibit the normal growth of pathogen.Therefore, trypsin inhibitor takes part in plant really Disease-resistant sexual approach.And after the processing of hormone jasmonic or injury processing, trypsin inhibitor has apparent induced expression, These researchs all demonstrate resistance signal's approach that trypsin inhibitor takes part in plant really, and are deposited with JA signal transductions In reciprocation.

Therefore, we are such as antiviral it is not difficult to find that the trypsin inhibitor in plant has the function of various biological, Antimycotic and pest-resistant biological function.Meanwhile trypsin inhibitor has regulated and controled Genes For Plant Tolerance caused by a variety of pathogen infections Sick signal path.

But the trypsin inhibitor research in rice is very few, the trypsin inhibitor in rice is to disease fungus Biological function research also have no any report.

Invention content

The technical problems to be solved by the invention are：A kind of purposes that rice Os BBTI4 protein gene is new is provided.

The technical scheme is that：Rice trypsin inhibitor protein gene OsBBTI4 is improving Rice Resistance rice blast The cDNA nucleotides sequences of application in disease, the rice trypsin inhibitor protein gene OsBBTI4 are classified as SEQ ID No.1 Degenerate sequence shown or for SEQ ID No.1.

Polypeptide the answering on improving rice anti-rice blast of rice trypsin inhibitor protein gene OsBBTI4 codings With the amino acid sequence of the polypeptide is as shown in SEQ ID No.2.

A method of rice anti-rice blast being improved, by the OsBBTI4 progress gram of rice trypsin inhibitor protein gene It is grand and build plant expression vector, which is transferred to Agrobacterium, is then seeded to rice, selection is anti-with blast resisting The overexpression strain and its offspring, the cDNA nucleotides sequences of rice trypsin inhibitor protein gene OsBBTI4 of property are classified as SEQ Degenerate sequence shown in ID No.1 or for SEQ ID No.1.

Further, in a kind of method of raising rice anti-rice blast described above, expression vector pCXUN.

Further, in a kind of method of raising rice anti-rice blast described above, Agrobacterium EHA105.

The disease resistance evaluation of progress rice after qRT-PCR verifications is carried out to the transfer-gen plant of acquisition.Punching is vaccinated with life Transgenic rice plant when long to 5-8 weeks big, the 10-14 days investigation state of an illness.It is consistent the result shows that, transfer-gen plant with compare Compared to the apparent resistance shown to Pyricularia oryzae.

Compared with prior art, the invention has the advantages that：

Transgenic experiments prove the resistance that the overexpression of the gene improves rice to Pyricularia oryzae.Therefore it can be used as mesh Gene transfered plant, improve disease resistance of plant, to carry out plant species improvement.The trypsin inhibitor being transferred to also passes through suppression The hydrolysis of protease processed protects other albumen not to be degraded, effectively improves plant defense ability.

Description of the drawings

Fig. 1 is pCXUN expression vector collection of illustrative plates；

Fig. 2 is the expression analysis of OsBBTI4 in transgenic paddy rice.WT indicate wild type, OsBBTI4-OX-7-6 and OsBBTI4-OX-16-2 indicates two overexpression strains；

Fig. 3 is that OsBBTI4-OX transfer-gen plants enhance the disease resistance to rice blast fungus biological strain RB22；

Fig. 4 is that morbidity lesion area substantially reduces after OsBBTI4-OX transfer-gen plants are inoculated with；

Fig. 5, which is the fungal biomass in OsBBTI4-OX transfer-gen plant scabs region, to be significantly reduced.

Specific implementation mode

Embodiment 1

1) extraction of total serum IgE

Rice japonica rice variety Nipponbare is selected, waits for that rice seedling is grown to two weeks or so, takes blade liquid nitrogen frozen immediately, is preserved In -80 DEG C of refrigerators.Partial blade is taken, is ground with mortar, the 1.5ml EP pipes for filling Trizol lysates is transferred to, fully vibrates Afterwards, extracted total RNA, electroresis appraisal total serum IgE quality.

2) structure of the clone and plant expression vector of rice trypsin inhibitor protein gene OsBBTI4

Design both ends primer：

P1：5-ATGAGCAACACCACCATGGC-3 (SEQIDNo.3),

P2：5-CTAGTTCTCCGCTCGGGGTT-3(SEQ ID No.4)

The total serum IgE reverse transcription that step 1) is obtained synthesizes the first chains of cDNA, is carried out with high-fidelity Pfu enzymes as template PCR amplification, PCR programs are as follows：94 DEG C of pre-degeneration 5min, 94 DEG C of denaturation 45s, 60 DEG C of renaturation 1min, 72 DEG C of extension 1min, 35 After cycle, 72 DEG C of extension 5min recycle target fragment after 72 DEG C of ends heat preservation 10min of last Taq enzyme add A.Meanwhile with limitation Property restriction endonuclease XcmI digestion pCXUN carriers, then recycle digestion after carrier segments, OsBBTI4 is further cloned into double base Expression vector pCXUN (Fig. 1), sequencing identification ensure that code area reading frame is correct in expression vector.

3) acquisition of transfer-gen plant

The expression vector pCXUN-OsBBTI4 that step 2) obtains is transferred to agrobacterium strains EHA105, is further transferred to water Rice japonica rice variety Nipponbare carries out the transfer-gen plant of acquisition in the disease resistance evaluation (figure of progress rice after qRT-PCR verifications 2)。

Embodiment 2

OsBBTI4-OX transgenic line Disease Resistance Identifications：

Punching inoculation：When transgenic paddy rice is grown to 5-8 weeks leaf age, selects down two leaves and carry out rice blast fungus punching inoculation, so After rice is placed in growth chamber dark culturing 24 hours afterwards, it is transferred to illumination in 12 hours, it is raw under 12 hours dark conditions It is 10-14 days long；Spot expansion situation is observed, the state of an illness is investigated, counts lesion area and biomass, is had compared with the control apparent anti- Property transgenic rice plant be obtain blast resisting transfer-gen plant.

As a result, it has been found that compared with wild type (WT), OsBBTI4 overexpression transfer-gen plants enhance small to rice blast fungus physiology The disease resistance (Fig. 3) of kind RB22, meanwhile, count the opposite lesion area (table 1) of transgenic line and wild type incidence of leaf With opposite fungal biomass (table 2), morbidity lesion area is only after being embodied in OsBBTI4 overexpression transfer-gen plant inoculations 50% or so (Fig. 4) of control, the fungal biomass in scab region fall to 20% of control or so (Fig. 5).

The opposite lesion area of 1 transgenic line of table and wild type incidence of leaf

The relative biomass of table 2 transgenic line and wild type incidence of leaf

The specific implementation mode of the application above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously Cannot the limitation to the application protection domain therefore be interpreted as.It should be pointed out that for those of ordinary skill in the art For, under the premise of not departing from technical scheme design, various modifications and improvements can be made, these belong to this The protection domain of application.

Sequence table

<110>Applicant's title:Plant Protection institute, Chinese Academy of Agricultral Sciences

<120>Rice Os BBTI4 protein gene is applied on improving rice anti-rice blast

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 756

<212> DNA

<213>Rice (Oryza sativa)

<400> 1

atgagcaaca ccaccatggc tatttccacc atccttctct tcctcctcgc cggcctcgtc 60

gccgcccacg gcgacggcga caccatgatc cgtctcccaa gcgacggcgc cgaagcacca 120

ccacgcccgc ccaaaccctg ggactgctgc gacaacatcg agatgtcccc gctcgagatc 180

ttcccgccgc tgtaccgctg caacgacgag gtgaagcagt gctccgccgc ctgcaaggag 240

tgcgtggagg cgcccggcga cttcccccgc ggcgccttcg tgtgccgcga ctggtactcg 300

acggtggacc cgggccacat gtgcacggcg ccggatcagc cgacgacgaa gaggccgtgg 360

aagtgctgtg acagcatcgt gcagctgccg cagaggatct tcccgccgtt ctggcgctgc 420

gacgacgagc tggagcccgg caagtgcacc gccgcgtgca agtcgtgcag ggaggcgccg 480

gggccgttcc cggggccgct catctgcgag gacgtctact ggggcgccga cccgggcccc 540

ttgtgcacgc cgcggccatg ggggaaatgc tgcgacaagg ccttctgcaa caagatgaac 600

ccgccgacct gccgctgcat ggacgaggtg aacaagtgcg ccgccgcgtg caaggattgc 660

cagcgtgtgg agtcgtcaga gccgcctcgc tacgtctgca aggaccgctt caccggccag 720

cccgggccca tgtgcaaacc ccgagcggag aactag 756

<210> 2

<211> 251

<212> PRT

<213>Rice (Oryza sativa)

<400> 2

Met Ser Asn Thr Thr Met Ala Ile Ser Thr Ile Leu Leu Phe Leu Leu

1 5 10 15

Ala Gly Leu Val Ala Ala His Gly Asp Gly Asp Thr Met Ile Arg Leu

20 25 30

Pro Ser Asp Gly Ala Glu Ala Pro Pro Arg Pro Pro Lys Pro Trp Asp

35 40 45

Cys Cys Asp Asn Ile Glu Met Ser Pro Leu Glu Ile Phe Pro Pro Leu

50 55 60

Tyr Arg Cys Asn Asp Glu Val Lys Gln Cys Ser Ala Ala Cys Lys Glu

65 70 75 80

Cys Val Glu Ala Pro Gly Asp Phe Pro Arg Gly Ala Phe Val Cys Arg

85 90 95

Asp Trp Tyr Ser Thr Val Asp Pro Gly His Met Cys Thr Ala Pro Asp

100 105 110

Gln Pro Thr Thr Lys Arg Pro Trp Lys Cys Cys Asp Ser Ile Val Gln

115 120 125

Leu Pro Gln Arg Ile Phe Pro Pro Phe Trp Arg Cys Asp Asp Glu Leu

130 135 140

Glu Pro Gly Lys Cys Thr Ala Ala Cys Lys Ser Cys Arg Glu Ala Pro

145 150 155 160

Gly Pro Phe Pro Gly Pro Leu Ile Cys Glu Asp Val Tyr Trp Gly Ala

165 170 175

Asp Pro Gly Pro Leu Cys Thr Pro Arg Pro Trp Gly Lys Cys Cys Asp

180 185 190

Lys Ala Phe Cys Asn Lys Met Asn Pro Pro Thr Cys Arg Cys Met Asp

195 200 205

Glu Val Asn Lys Cys Ala Ala Ala Cys Lys Asp Cys Gln Arg Val Glu

210 215 220

Ser Ser Glu Pro Pro Arg Tyr Val Cys Lys Asp Arg Phe Thr Gly Gln

225 230 235 240

Pro Gly Pro Met Cys Lys Pro Arg Ala Glu Asn

245 250

<210> 3

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 3

atgagcaaca ccaccatggc 20

<210> 4

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

ctagttctcc gctcggggtt 20

Claims

1. applications of the rice trypsin inhibitor protein gene OsBBTI4 on improving rice anti-rice blast, the rice pancreas The cDNA nucleotides sequences of protease inhibitor protein gene OsBBTI4 are classified as shown in SEQ ID No.1 or for SEQ ID No.1's Degenerate sequence.

2. application of the polypeptide of rice trypsin inhibitor protein gene OsBBTI4 codings on improving rice anti-rice blast, The amino acid sequence of the polypeptide is as shown in SEQ ID No.2.

3. a kind of method improving rice anti-rice blast, which is characterized in that by rice trypsin inhibitor protein gene OsBBTI4 is cloned and is built plant expression vector, which is transferred to Agrobacterium, is then seeded to rice, selection Overexpression strain with blast resisting resistance and its offspring, the rice trypsin inhibitor protein gene OsBBTI4's CDNA nucleotides sequences are classified as the degenerate sequence shown in SEQ ID No.1 or for SEQ ID No.1.

4. a kind of method improving rice anti-rice blast according to claim 3, which is characterized in that expression vector is pCXUN。

5. a kind of method improving rice anti-rice blast according to claim 3, which is characterized in that Agrobacterium is EHA105。