CN112553204B - Rice stem borer harm inducing promoter pOsISA2 and application thereof in cultivation of intelligent anti-borer rice - Google Patents

Abstract

The invention discloses a rice chilo suppressalis harm inducing promoter pOsISA2, a gene OsISA2 and application thereof in cultivation of rice with chilo suppressalis resistance; the promoter pOsISA2 at least comprises a nucleotide sequence shown as SEQ ID NO.1, or the nucleotide sequence is shown as SEQ ID NO. 2; the gene OsISA2 is shown in SEQ ID NO. 3. The invention finds a rice chilo suppressalis harm inducing promoter pOsISA2 and a gene OsISA2, finds new application of the promoter and the gene in cultivation of rice resistant to chilo suppressalis, and provides important gene resources for cultivation of rice varieties resistant to chilo suppressalis.

Description

Rice stem borer harm inducing promoter pOsISA2 and application thereof in cultivation of intelligent anti-borer rice

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a rice stem borer harm inducing promoter pOsISA2 and application thereof in cultivating intelligent rice resistant to stem borers.

Background

Insect damage is one of the most important factors causing the reduction of yield of rice, and data statistics show that the reduction of yield caused by insect damage is remarkable every year. At present, chemical pesticides are mainly used for preventing and controlling pests in agricultural production. The use of chemical pesticides in large quantities not only increases the production cost, but also causes environmental pollution and even threatens human health. It is urgently needed to cultivate insect-resistant rice varieties so as to improve the insect resistance of rice. However, the cultivation of new insect-resistant varieties by conventional rice breeding techniques not only requires a long time, but also for the most important pests of rice, such as chilo suppressalis, tryporyza incertulas, cnaphalocrocis medinalis and the like, no effective resistant germplasm resources are found in rice at present, so that the improvement of insect resistance of rice by conventional breeding is still unavailable at present. The most direct method is to introduce exogenous insect-resistant gene into receptor rice by using transgenic technology to create new insect-resistant variety. To date, many useful insect-resistant genes have been identified and cloned in plants, animals and even microorganisms, some of which have been transformed into rice to obtain transgenic insect-resistant lines, and some of which have been field tested to show good resistance to the above-mentioned pests.

From the development of transgenic insect-resistant rice, the expression of insect-resistant genes is driven by using constitutive expression promoters such as 35sCaMV, Actin I and Ubiquitin from the starting stage, and a plurality of new transgenic rice lines showing good resistance to borers are obtained. With the development of transgenic technology and detection means, it has been found that the use of constitutive expression promoters exposes some problems in the course of the test, such as excessive consumption of materials and energy in plant cells, alteration of agronomic traits caused by increased metabolic burden of rice, and excessive public concerns about food safety. The main reason for these problems is that the constitutive expression promoter is used to drive the target gene, and the expression of the target gene cannot be regulated and controlled effectively in time and space, but can be expressed continuously and constantly in various tissues and organs of the transgenic recipient plant. In addition, when the same constitutive expression promoter is repeatedly used to drive the expression of two or more exogenous genes, transgene silencing or cosuppression phenomena may be caused, which brings great trouble to multigene transformation. Therefore, with the development of plant genetic engineering, there is a need to find more effective tissue-and organ-specific expression promoters or inducible expression promoters to replace constitutive expression promoters, so as to better control the expression of target genes in target tissues or organs in time and space.

An inducible promoter is a promoter that can greatly increase the transcription level of a target gene when stimulated by some physical or chemical signal. Among these, there are mainly hormone-inducible promoters, such as SAUR15(Small Autoxin-up RNA15) gene, which are up-regulated by Auxin and brassinolide induction (Walcher, C.L., Nemhauser, J.L., double promoter element required for an Auxin response [ J ]. Plant physiology,2012,158 (1), pp 273-282.); secondly, there is a non-biological adversity stress induced promoter, the rice Wsi18 gene can be significantly induced by NaCl, ABA and drought stress to be up-regulated and expressed, but has no obvious response to low temperature stress. The promoter is isolated and cloned, and the background activity of the promoter in Transgenic rice is found to be very low, but the promoter can be induced to be increased by the above-mentioned adversity stress (Yi, N, Oh, S.J., Kim, Y.S., etc., Analysis of the Wsi18, a stress-induced promoter which is active in the genetic gain of the genetic rice [ J ]. Transgenic research,2011,20(1), pp 153-. Also, a biological stress-inducible promoter such as PmTNL1 gene promoter can be specifically and efficiently induced by white rust (Liu, J. -J., Ekramoddoulla, A.K., Genomic organization, induced expression and promoter activity of a resistance gene analyzer (PmTNL1) in western white pine [ J ]. plant,2011, 233(5), pp 1041-. The promoter fragment specifically induced and expressed by Chilo suppressalis feeding is separated and cloned to the Chilo suppressalis feeding by Hua et al (Hua, h., Lu, q., Cai, m., etc.), which has a very good prospect in insect resistance transgenic research.

However, the research on pest damage inducible promoters is still less, while the research on chilo suppressalis damage inducible promoters in rice is less, and further intensive research is needed to be carried out to excavate more chilo suppressalis damage inducible promoters and genes.

Disclosure of Invention

The invention provides a new rice chilo suppressalis harm inducing promoter pOsISA2, a gene OsISA2 and a new application thereof in cultivating rice resistant to rice borers, and provides a basis for cultivating intelligent rice resistant to rice borers.

The specific technical scheme is as follows:

the invention provides a rice stem borer harm inducing promoter pOsISA2, which at least comprises a nucleotide sequence shown in SEQ ID No. 1.

Further, the nucleotide sequence of the rice stem borer harm inducible promoter pOsISA2 is shown in SEQ ID No. 2.

The promoter pOsISA2 is a predicted promoter of terpene synthase family gene (LOC _ Os04g27670), and has a total length of 2161bp, wherein the last 1160bp region is predicted by bioinformatics to contain a promoter core element, namely a nucleotide sequence shown in SE Q ID No. 1. The website is predicted to contain core elements for starting transcription such as TATA box and CAAT box and predict a defense and stress response element GTTTTCTTAC through promoters such as New PLACE (https:// www.dna.affrc.go.jp/PLACE/a section ═ newspace) and plantaCARE (http:// bioinformatics. psb. element. be/webtools/plantarte/html /). The promoter can quickly respond to Chilo suppressalis harm and start transcription of downstream genes.

According to the transcriptome sequencing data of rice before and after inoculation of Chilo suppressalis, the gene OsISA2 has extremely low mRNA transcription level in each tissue and part before the treatment of Chilo suppressalis damage, and can hardly be detected; after the chilo suppressalis is damaged, the transcription level in tissues damaged by stalks and the like is sharply up-regulated, and the up-regulation multiple is over 75 times; the gene is shown to be capable of rapidly and efficiently responding to the harm of chilo suppressalis, and the promoter of the gene is shown to be a chilo suppressalis harm inducing promoter. Therefore, the promoter and the insect-resistant element are cloned from japonica rice variety Nipponbare to form an insect-resistant module and are introduced into the chassis crop rice, so that the intelligent expression of the insect-resistant gene can be realized. The specific expression is that the insect-resistant module does not work when the pests are not harmed, no insect-resistant protein is expressed, so that the growth and development of rice are not influenced, and the insect-resistant protein is quickly and efficiently expressed when the chilo suppressalis is harmed to resist the damage of the pests. And finally, no production and accumulation of the insect-resistant protein at the edible part is ensured. The invention also discloses a cloning method of the promoter and a construction method of a corresponding expression vector thereof, and a genetic transformation method of rice mediated by agrobacterium. The promoter has strong application value in cultivating novel intelligent insect-resistant rice.

The invention also provides application of the rice stem borer harm inducible promoter pOsISA2 in culturing intelligent rice resistant to stem borers.

The borers include striped rice borers, tryporyza incertulas, rice leaf rollers and other lepidoptera pests.

Further, the application comprises the following steps:

(1) constructing a recombinant vector containing the rice stem borer harm inducing promoter pOsISA 2;

(2) transferring the recombinant vector into an agrobacterium-infected cell to construct a genetic engineering bacterium containing the recombinant vector in the step (1);

(3) and (3) performing mediated transformation on the rice callus by the genetically engineered bacteria in the step (2) to obtain a transgenic positive rice plant.

Further, in the step (1), the original expression vector of the recombinant vector is pSB130, and the recombinant vector also comprises an insect-resistant gene.

Further, in the step (2), the agrobacterium is EHA 105.

The invention also provides application of the rice gene OsISA2 as a chilo suppressalis harm inducing gene in cultivating rice resistant to chilo suppressalis, wherein the nucleotide sequence of the gene OsISA2 is shown as SEQ ID No. 3.

The borers include striped rice borers, tryporyza incertulas, rice leaf rollers and other lepidoptera pests.

Further, the application comprises the following steps:

(A) constructing a recombinant vector containing a rice gene OsISA2, wherein the nucleotide sequence of the gene OsISA2 is shown as SEQ ID No. 3;

(B) transferring the recombinant vector into an agrobacterium-infected cell to construct a genetic engineering bacterium containing the recombinant vector in the step (A);

(C) and (C) performing mediated transformation on the rice callus by using the genetic engineering bacteria in the step (B) to obtain a transgenic positive rice plant.

Further, in the step (A), the original expression vector of the recombinant vector is pSB130, and the recombinant vector also comprises a rice constitutive initiation type promoter pActin I.

Further, in the step (B), the Agrobacterium is EHA 105.

The invention also provides a recombinant vector which comprises the rice stem borer harm inducing promoter pOsISA 2; or the rice gene OsISA2 with the nucleotide sequence shown in SEQ ID NO. 3.

Further, the recombinant vector also comprises an insect-resistant gene; further, the insect-resistant gene is a Bt gene.

The invention also provides a transformant which comprises the rice stem borer harm inducing promoter pOsISA 2; or comprises a rice gene OsISA2 with the nucleotide sequence shown as SEQ ID NO. 3.

Further, the transformant also comprises an insect-resistant gene; further, the insect-resistant gene is a Bt gene.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention finds a rice stem borer harm inducing promoter pOsISA2 and a gene OsISA2, finds new application of the promoter and the gene in cultivation of rice resistant to stem borers, and provides important gene resources for cultivation of intelligent rice varieties resistant to stem borers.

(2) The invention provides a method for cultivating intelligent anti-borer rice by utilizing a transgenic technology, and obtains an intelligent anti-borer rice material.

Drawings

FIG. 1 is a schematic diagram of the general flow of the present invention for constructing intelligent insect-resistant rice.

FIG. 2 shows the relative expression amounts of the OsISA2 gene in example 1 in various tissues of japonica rice Nippon, and the data is derived from a rice gene annotation database (http:// rice. plant. biology. msu. edu /).

FIG. 3 shows the average relative expression level of transcripts of OsISA2 gene in example 1 when it is endangered by Chilo suppressalis.

Wherein, HPL2 is a positive control, and the promoter is a chilo suppressalis harm inducible type; the abscissa 1-4 represents pre-treatment (0h), treatment 24h,48h and 72h, respectively; the ordinate represents the relative expression amount of the transcript; a total of 4 biological replicates were set up.

FIG. 4 is a schematic diagram of the pSB130-rbcS-TP-Bt expression vector of example 1.

FIG. 5 is a schematic diagram of the pOsISA2-Bt expression vector of example 1.

FIG. 6 is the molecular detection of cry1Ab/cry1Ac genes of interest in the transgenic-resistant healing lesion of example 1;

wherein, M: DL 2000 molecular weight markers; +: a positive control; -: negative control; 1-21: callus after transformation; the arrows indicate the target bands.

FIG. 7 shows the results of protein detection in the stem Cry1Ab/Cry1Ac of example 2;

wherein, IC2-1, IC2-6, IC2-8 and IC2-12 are 4 independent transformants; ZY3(TT51 derived line) as positive control, NT as non-transgenic negative control; 4 of IC2-1-T, etc. were the results after treatment with artificial inoculation of Chilo suppressalis, while 4 of IC2-1, etc. were the corresponding untreated control samples.

FIG. 8 shows the results of detection of the Cry1Ab/Cry1Ac proteins in the seeds of example 4;

wherein, IC2-1, IC2-6, IC2-8 and IC2-12 are 4 independent transformants; ZY3(TT51 derived line) as positive control; NT is a non-transgenic negative control.

FIG. 9 is a schematic diagram of the pOsISA2-OE expression vector in example 5.

FIG. 10 shows the average relative expression level of transcripts of OsISA2 gene in example 5 when it was overexpressed.

Wherein NT is a negative control; the abscissa represents 3 independent transformants; the ordinate represents the relative expression amount of the transcript; a total of 3 biological replicates were set up.

Detailed Description

The present invention will be further described with reference to the following specific examples, which are only illustrative of the present invention, but the scope of the present invention is not limited thereto.

Example 1

1. Discovery of chilo suppressalis harm inducing gene and promoter

Transcriptome sequencing data of rice stalks before (0h) and after (24h, 48h and 72h) chilo suppressalis damage in the tillering stage are respectively taken, and a target gene which can quickly respond to chilo suppressalis damage and is hardly expressed when no chilo suppressalis damage is caused is screened by using a bioinformatics technology.

The gene has the following characteristics: before the chilo suppressalis damage treatment, the gene transcription level is low, and the limitation is that FPKM is less than or equal to 20; after the chilo suppressalis harm treatment, the expression level is sharply up-regulated, and the up-regulation multiple is preferably more than or equal to 50 times; and requires extremely low FPKM values of various tissues and sites in a rice gene annotation database (http:// rice. plant biology. msu. edu /), particularly requires a value of 0 in endosperm.

A terpene synthase gene (LOC _ Os04g27670) was selected according to the above requirements and named OsISA 2(Induced by strained materials oligonucleotides attached 2) (FIG. 2). The stem graft transcriptome sequencing data show that OsISA2 is sharply up-regulated and expressed after being damaged by chilo suppressalis (figure 3). Therefore, the promoter of the chilo suppressalis has the function of quickly and efficiently responding to the chilo suppressalis damage. According to a Nipponbare sequencing sequence published by a rice gene annotation database (http:// rice. plant biology. msu. edu /), the size of about 2Kb before ATG or 5' UTR is used as a predicted promoter region. In the patent, 2161bp before the 5' UTR of the initiation codon of the OsISA2 gene is selected as a promoter and is named as pOsISA 2.

2. Construction of Intelligent insect-resistant expression Module

According to the sequence information provided by a rice gene annotation website (http:// rice. plant biology. msu. edu /) and the sequence information of the expression vector pSB130-rbcS-TP-Bt (figure 4), refer to a one-step rapid cloning kit Hieff

Plus One Step Cloning Kit Specification design of Cloning primers containing kozak sequence (pOsISA 2-F: 5'-TAAAACGACGGCCAGTGCCCCTTTGATGTTTTGTGTTGTAG-3', pOsISA 2-R: 5 ' -GCAGTTGTTGTCCAT)GGTGGCCAGAACACAGTACTCCTTCTAG-3’)。

High quality of wild type riceQuantitative DNA as a template, and the sequence 2161bp and Kozak of pOsISA2 (GCCACC) are amplified by using KOD FX high-fidelity enzymeATGGAnd does not contain underlined partial bases) and homologous sequences at both sides of the vector framework, and a total of 2197bp DNA fragment is connected to the vector framework pSB130-Em-Bt after the rbcS-TP fragment is cut by the pSB130-rbcS-TP-Bt, and HindIII and SalI cutting sites at the homologous connection positions are not reserved.

The specific experimental method is as follows:

2.1 cloning of the target fragment

KOD FX reaction System (40. mu.L):

the reaction program is 94 ℃ for 3min, 98 ℃ for 10s, 68 ℃ for 3min, amplification is carried out for 32 cycles, and finally extension is carried out for 7min at 68 ℃. The above products were separated by electrophoresis on a 1% agarose gel.

2.2 recovery of target fragments

The Zymoclean Gel DNA Recovery Kit is used for recovering the target fragment Gel, and the specific steps are as follows: cutting off the target band on the agarose gel by using a blade, and putting the cut target band into a 1.5mL centrifuge tube; adding 3 times volume of ADB buffer into the centrifuge tube, namely adding 300 mu L of ADB buffer into 100 mu L (mg) of glue, and then placing the gel in a metal bath at 55 ℃ for 5-10 minutes to completely melt the gel; transferring the agarose solution to an adsorption column sleeved on a collecting pipe, centrifuging at 12000rpm for 30sec, and discarding effluent; adding 200 mu L of Wash Buffer into the adsorption column, centrifuging at 12000rpm for 1 minute, and discarding the effluent; washing twice repeatedly, and centrifuging for 2 min; the adsorption column is sleeved on a new 1.5mL centrifuge tube, 6-20 μ L of precipitation Buffer is added, and the DNA is eluted after standing for 1min and then centrifuging for 1 min.

2.3 vector linearization

The pSB130-rbcS-TP-Bt vector was double digested with Hind III and SalI from TaKaRa, according to the following specific steps:

after incubation for 1 hour at 37 ℃, the carrier skeleton pSB130-Em-Bt with larger fragments is recovered through electrophoresis.

2.4 mesh vector construction

The recovered PCR product and the linearization vector are quickly cloned by a one-step method to obtain a kit Hieff

The Plus One Step Cloning Kit was used to construct the expression vector as follows:

mixing, centrifuging, reacting at 50 ℃ for 20 minutes in a PCR instrument, cooling on ice for 5 minutes, and transforming escherichia coli DH5 alpha, wherein the method comprises the following steps:

taking out DH5 alpha competence from a refrigerator at minus 80 ℃, putting on ice for melting, adding the ligation product, and gently and uniformly mixing; performing ice bath for 30min, performing heat shock for 45s in a water bath kettle at 42 ℃, and cooling for 2min on ice; adding 500 μ L LB liquid culture medium, placing on 37 deg.C shaking table, and recovering at 200rpm for 1 h; centrifuging at 5000rpm for 3min to precipitate thallus, sucking 400 μ L of supernatant, and blowing and beating the remaining supernatant and thallus; uniformly coating the bacterial liquid on LB solid culture medium containing the clarithromycin, and carrying out inverted culture in a constant-temperature incubator at 37 ℃ for overnight (12-16 h); selecting 3 monoclonals, inoculating the monoclonals into LB liquid culture medium containing the clarithromycin, placing the monoclonals on a shaking table at 37 ℃ and 250rpm until the solution is turbid, detecting positive clones through PCR of the bacterial solution, and sending the positive clones to sequencing identification. And (4) storing bacterial liquid and plasmids for later use for a sample with correct sequencing.

The expression vector with correct sequencing is named as pOsISA 2-Bt. The expression vector map is shown in FIG. 5. So far, the intelligent insect-resistant expression module is successfully constructed.

3. Agrobacterium-mediated genetic transformation of rice

3.1 expression vector transformation of Agrobacterium EHA105

The pOsISA2-Bt vector is used for transforming agrobacterium by an electric shock transformation method, and the used agrobacterium strain is EHA 105. The specific procedure is as follows: adding 0.5 mu L of plasmid into a 1.5mL centrifuge tube containing 50 mu L of agrobacterium EHA105 electric shock competent cells, sucking and uniformly mixing by using a pipette, and then transferring into an electrode cup; after electric shock, quickly adding 1mL of LB liquid culture medium, sucking, uniformly mixing, moving into the previous 1.5mL centrifuge tube, and carrying out shake culture on a constant temperature shaking instrument at 28 ℃ for 1 h; after the bacterial liquid is recovered, sucking 100 mu L of bacterial liquid, uniformly coating the bacterial liquid on the surface of an LB solid screening culture medium (containing 50mg/L kanamycin and 25mg/L rifampicin), and inversely placing a culture dish in an incubator at 28 ℃ for culturing for 2 days; after the positive clone was confirmed by colony PCR, the positive clone was shaken to preserve the bacterial liquid (250. mu.L of 80% sterile glycerol was added to 1mL of bacterial liquid). Storing at-80 deg.C for use.

3.2 Agrobacterium-mediated genetic transformation of Rice

Rice transformation is carried out according to the method steps reported by Chenhao (Chenhao. "Gene switch" system development and the application thereof in breeding endosperm zero-expression type "green" insect-resistant rice [ D ]. Zhejiang: Zhejiang university, 2016.). The specific procedure is as follows:

taking out and storing the agrobacterium liquid at minus 80 ℃, sucking 200 mu L of the agrobacterium liquid, uniformly coating the liquid on the surface of an LB solid culture medium containing 25mg/L of rifampicin and 50mg/L of kanamycin, and culturing the liquid at 28 ℃ for overnight; then selecting a single colony from the culture medium for amplification culture, wherein the liquid culture medium is as described above; then, 200-300. mu.L of fresh bacterial liquid was aspirated into 20mL of LB liquid medium containing 25mg/L rifampicin and 50mg/L kanamycin, and cultured at 28 ℃ for 16-18 hours with shaking (220 rpm). Centrifuging sufficient bacterial liquid at 4000rpm for 15min, and discarding the supernatant of the LB culture medium; resuspend Agrobacterium with 20mL of 0.1M MgSO4 solution (resuspend by gentle pipetting), centrifuge at 4000rpm for 10-15min, discard the MgSO containing the antibiotic ₄ Supernatant fluid; adding 5mL of AA-AS infection culture medium containing 200 mu M Acetosyringone (AS) to re-suspend the agrobacterium tumefaciens, adding a proper amount of AA-AS infection culture medium to adjust the OD600 value of the bacterium liquid to be between 0.2 and 0.8 finally; and (4) subpackaging the bacteria liquid by using a sterile 50mL centrifuge tube, and keeping the bacteria liquid in 20-25mL tube for later use.

Transferring the embryonic callus of the Xishui 134 which is pre-cultured for about 7 days from the subculture dish to an empty culture dish covered with sterile filter paper, air-drying for about 10-15min on a super-clean workbench, and slowly turning over a sterilized stainless steel spoon for callus to be fully dried; after the bacterial liquid is dried, transferring the bacterial liquid into a 50mL centrifugal tube containing the bacterial liquid, slightly shaking (not too violent) at room temperature for about 40min, and standing the centrifugal tube on an ultra-clean workbench for 10 min; discarding the bacterial liquid, and placing the embryogenic callus on sterile filter paper to dry for about 15 min; then, the infected calli were transferred to co-culture medium (Table 1) containing AS (200. mu.M) with the surface covered with sterile filter paper and cultured in the dark at 28 ℃ for 50-55 h; selecting embryogenic callus with surface agrobacterium not growing in large quantity or being not polluted, transferring to an antibacterial culture medium (table 2) containing 500mg/L of cephalosporins, and performing antibacterial culture in a dark room at 28 ℃ for 3-4 days; transferring the callus after bacteriostatic culture to a screening culture medium containing 500mg/L of cefuroxime and 75mg/L of hygromycin, and culturing in a dark room at 28 ℃; in the first week, the agrobacterium contamination condition is checked every day, if the contamination can not be controlled, the screening culture medium needs to be replaced in time, callus with good growth state is selected every half month and subcultured on the fresh screening culture medium, the concentration of the cefamycin in the culture medium is adjusted according to the self contamination degree of the agrobacterium, and the concentration of the cefamycin in the culture medium can be reduced by half in the third or fourth subculture screening under the general condition.

A total of 21 independent transformants were obtained following the procedure described above.

TABLE 1 AA Medium formulation

TABLE 2 CC Medium formulation

3.3 PCR identification of transgenic resistant calli

3.3.1 extraction of resistant callus genomic DNA

Weighing 0.1g of transgenic resistant callus from each independent transformant after subculture, and placing the transgenic resistant callus in a sterilized 1.5mL centrifuge tube filled with grinding beads; adding 500 μ L of 1.5 × CTAB extractive solution, grinding on sample grinder to homogenate, and placing in 56 deg.C water bath for 20-30min (taking out during water bath, repeatedly reversing, and mixing for 2 times); then adding 500 μ L chloroform, reversing the above materials for several times, mixing, and centrifuging at room temperature for 10min (8000 rpm); sucking 400 μ L of supernatant into a new centrifugal tube, adding 800 μ L of anhydrous ethanol, mixing, and standing at-30 deg.C for about 30 min; centrifuging at 12000rpm at room temperature for 5min, and removing supernatant; soaking and washing the DNA precipitate with 75% ethanol, removing the ethanol, and drying at room temperature; add 100. mu.L of sterile water and dissolve overnight for use.

3.3.2 molecular detection of cry1Ab/cry1Ac in transgenic resistance healing wounds

Molecular detection of cry1Ab/cry1Ac in transgenic-resistant healing wounds was performed using conventional PCR techniques. The detection primers used were: Bt-F: 5'-TGGTTCTGCCCAAGGTATCG-3' and Bt-R: 5'-AACGGTTC CGCTCTTTCTGT-3', the size of the amplified target fragment is about 369bp, the PCR reaction system is the same as the conventional system, and the used PCR reaction program is 94 ℃ denaturation for 5 min; denaturation at 95 ℃ for 15sec, annealing at 55 ℃ for 30sec and extension at 72 ℃ for 30sec (32 cycles); then the mixture is extended for 10min at 72 ℃ and stored at 12 ℃ at low temperature. The obtained PCR amplification product is separated and identified by 1 percent agarose gel electrophoresis.

The PCR detection result shows that: about 76.2% of 16 of the 21 transgenic resistant cured strains selected detected the cry1Ab/cry1Ac gene (FIG. 6). Indicating that the gene of interest has integrated into the recipient plant, while the negative individuals indicate that the resistant calli may have integrated only the Hpt gene and can be grown on selection medium without integrating the gene of interest.

Therefore, the above-mentioned negative materials are discarded, and only the positive materials are regenerated and redifferentiated.

3.3.3 differentiation of cry1Ab/cry1Ac Positive calli

Differentiation of cry1Ab/cry1Ac positive calli was performed according to methods reported by Chenhao (Chenhao. "Gene switch" system development and its application in breeding endosperm zero expression type "green" insect-resistant rice [ D ]. Zhejiang: Zhejiang university, 2016.). The specific experimental procedure is as follows: resistant calli with positive target genes cry1Ab/cry1Ac are transferred to an N6 differentiation medium (N6 minimal medium +2mg/L Kinetin +1mg/L NAA + 4% Gelrite), pre-differentiated in a dark room at 28 ℃ for 7-9 days, then transferred to a fresh differentiation medium, and differentiated into green seedlings in a light room at 25 ℃ (generally, the green point of differentiation can be seen after 7-14 days, and the green seedlings can be differentiated into green seedlings after 3 weeks). The obtained green seedling is cleaned and adhered to the culture medium on the root system, directly (the root bud is differentiated simultaneously) or after the root is strengthened by a rooting culture medium (the bud is differentiated first), the green seedling is transferred into a Yoshida culture solution for transitional culture, and after the growth state is good and stable, the green seedling is transferred to a greenhouse until the green seedling is mature.

Example 2 qualitative detection of insect-resistant protein in transformants such as Positive transgenic plant IC2-1

Cry1Ab/Cry1Ac protein in 4 transformants such as a positive transgenic plant IC2-1 and the like is detected and analyzed by Cry1Ab/Cry1Ac test paper strips of Shanghai Youlong company. Protein extraction and test paper detection in the rice heading period stalks are carried out according to the steps described in the product specification with slight modification. And respectively selecting stalks damaged by chilo suppressalis (artificial inoculation for 48 h) and stalks not damaged by chilo suppressalis to perform a comparative experiment, and detecting the 4 transformants to verify the function of the intelligent insect-resistant module.

The method comprises the following specific steps: transgenic rice stalks with the length of about 2-3cm are respectively put into a mortar, and liquid nitrogen is added for full grinding. About 0.2g of the powder is added into a 1.5mL centrifugal tube filled with a 0.5mL kit self-contained protein extraction Buffer in advance, and the mixture is vortexed, shaken and mixed uniformly and then centrifuged at 12000rpm for 30 sec. 0.3mL of the supernatant was transferred to another sterile 1.5mL centrifuge tube. Then, the test paper is put in the test paper, and the color development condition of the test paper is observed for about 3 min.

The results show that the protein samples of the positive transgenic plants can detect the target bands after being damaged by chilo suppressalis, but can not detect the target bands in the corresponding controls (figure 7), and the results show that the Cry1Ab/Cry1Ac protein is not expressed when being not damaged by chilo suppressalis in the receptor genome, and is expressed only after being damaged by chilo suppressalis. It is inferred that the intelligent insect-resistant module can work normally according to the expected design.

Example 3 identification of insect resistance of To-generation individuals of transformants such as IC2-1

And 4 independent transformants such as the IC2-1 transformant are selected for artificial inoculation identification, and the artificial in vitro stalk method is adopted for the inoculation identification.

The in vitro stalk method comprises the following specific steps: taking 2 main ear rice seedlings in heading period, wiping the rice seedlings, cutting the 2 rice seedlings into 2 stems of 5cm containing nodes and leaf sheaths, and pressing small filter paper sheets soaked by benzimidazole preservative solution of 0.1g/L on two ends of the stems. Then, 10 pieces of ant borers (chilo suppressalis) are respectively inoculated into each root stalk, 2 pieces of the root stalks are displaced to the inner diameter of a small flat-bottom glass tube (9.5cm multiplied by 1.5cm), and the degreased cotton is tightly plugged at the tube opening. Larval mortality and live weight were examined or determined, respectively, from day 7 after the initial inoculation. Meanwhile, a small filter paper piece which is soaked by 0.1g/L of benzimidazole preservative solution is added at the two ends of the stem on the 3 rd day to ensure that the stem is moisturized. On day 7, the rice plants were picked up and the number of larvae surviving was recorded and the live insect mass per glass tube was weighed if necessary.

The test result proves that 4 independent transformants such as IC2-1 and the like show high resistance to the chilo suppressalis of the artificial inoculation. As shown in the attached table 3, the mortality rate of the inoculated chilo suppressalis on the control is 10%, which is very significantly lower than that of the chilo suppressalis of 4 transformants, wherein the mortality rate in IC2-6 is as high as 85%. Shows that: the 4 transformants can intelligently respond to the harm of the chilo suppressalis and directionally express the insect-resistant protein to achieve the insect-resistant effect. Finally, the death rate difference of the chilo suppressalis is extremely obvious after the transgenic new material and the parent are inoculated with the insects.

TABLE 3

Example 4 qualitative detection of Cry1Ab/Cry1Ac proteins in To generation seeds of transformants such as IC2-1

Cry1Ab/Cry1Ac protein in T0 generation seeds of 4 transformants such as IC2-1 and the like is detected and analyzed by Cry1Ab/Cry1Ac test paper strips of Shanghai Youlong company. Protein extraction and test paper detection in rice seeds are carried out according to the steps described in the product specification with slight modification. About 50 glumes of seeds were individually selected for detection.

The method comprises the following specific steps: about 50 transgenic rice seeds are respectively taken and placed in a sample grinding box filled with steel balls with the diameter of 1.5cm, and the sample grinding box is placed on a sample grinding machine to grind the seeds into powder. Adding about 0.2g of the powder into a 1.5mL centrifuge tube preloaded with a 0.5mL kit self-contained protein extraction Buffer, and centrifuging at 12000rpm for 30sec after vortex and uniform mixing. 0.3mL of the supernatant was transferred to another sterile 1.5mL centrifuge tube. Then, the test paper is put in the test paper, and the color development condition of the test paper is observed for about 3 min.

The results show that the target protein can not be detected in the protein samples of the seeds of the transformants such as IC2-1 and the like, and the results are consistent with the wild type control (figure 8), and the results show that the Cry1Ab/Cry1Ac protein is not expressed in the seeds. Thus, the creation of novel intelligent insect-resistant rice germplasm represented by transformants such as IC2-1 was successfully demonstrated.

Example 5 acquisition of OsISA2 Gene overexpression Material and functional verification

OsISA2 gene (LOC _ Os04g27670) was searched for by KEYWORDs in the genomic database of phytozome rice (https:// phytozome.jgi.doe.gov/pz/portal.html #. s earch show ═ KEYWORD & method ═ Org _ Ostiva) and its genomic sequence (including exons, introns, and 5 'and 3' UTR sequences) (SEQ ID No.3) was obtained, OsISA2 gene was cloned using the experimental method in example 1 and constructed in the same way on expression vector pLB (development of "Gene switch" system and its use in breeding of zero endosperm expressing "green" insect-resistant rice [ D ]. Zhejiang university, 2016.) to excise LNL-Bt fragment.

The cloning primer of the gene is (ISA 2-OE-F: 5'-GCTTTTTTGTAGGTAGACTGCAGGTTCT GGTTCATCTATATATTAGAG-3', ISA 2-OE-R: 5'-CGATCGGGGAAATTCGTCGACTT TTGGTGGAGCGCAACCATTG-3'), and the skeleton of the expression vector is linearized by adopting Pst I and Sal I enzyme digestion. The construction was carried out in the same manner as in example 1, and the constructed expression vector was named pOsISA2-OE (FIG. 9).

Similarly, the rice is transformed into Xiushui 134 rice variety by using an agrobacterium infection method, and the expression level of OsISA2 gene in T0 generation positive seedlings is detected. The transcriptional level of this gene was increased by about 156.03 to 555.14 fold in 3 independent transformants, respectively, compared to the non-transgenic control (FIG. 10). According to the characteristic of the rapid up-regulation of the expression quantity of OsISA2 after inoculation of chilo suppressalis in the foregoing, the fact that the resistance to chilo suppressalis is possibly enhanced to a certain extent when the gene is over-expressed is inferred. Therefore, the gene resource can be used for cultivating a new insect-resistant rice material.

Finally, it is also noted that the above-mentioned list is only a few specific embodiments of the present invention. In addition, the present invention is not limited to the above embodiments, and may be modified to some extent. Accordingly, all modifications directly derivable or suggested by the person skilled in the art from the present disclosure should be considered within the scope of the present invention.

Sequence listing

<110> Zhejiang university

<120> rice stem borer harm inducing promoter pOsISA2 and application thereof in cultivation of intelligent anti-borer rice

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1160

<212> DNA

<213> Rice (Oryza sativa L.)

<400> 1

tagggttttt gtaagataac aacagttcaa aacatatttt tcggtgcatc gccaacccta 60

ttattagttt tgacgagttc ttgtttttaa catggtagag agctcttaca taagggactt 120

atttggcgaa tagttgatgg aaattcagtt gatgtaatca aggataactg ggacaagagg 180

aacagccttc tcaaaccact tggttttatc aatgctttac tattcatcat tctatagtta 240

aaattttaca gttttcttta aaaaaataaa ctgcactaaa gcttttattg agatggatgg 300

tcaactatta tttcaaaact caacaacttt tcgcacatgg aaggagtact acataattat 360

gttgcaaata aaggggatac acagctacat atgcatagat taatcttaaa aacactcaat 420

aaaatcataa tatatatata tatatatata tatagactgc gtgtccttgt tcaaaacact 480

aaggcctcct ttaatggttg agctagtgca ctagcaaaaa gtctaccttt ttaatagatt 540

gtctcatagc acattgttca aaatgctttg ggttccacaa tccctctcac atttttttag 600

gagcttagct cttgaagaag agatagtgca ttctctctcc ttatcttctc tctcttccac 660

atcatacaaa aactgatgtg gagatttatg agctagctga ctcacccttg ttggaggagg 720

cctaagtact accaacctgg agggagtacc gtgttttttt ttaaaaaaaa actgcactaa 780

accatttagt aaaacaaatg gccaactatt attgcaaagc tcaacaactt tgtacatatg 840

gaggagtaca taactttcaa aacggaggga gtgggtacgc aatttctccc tctaggtaga 900

taatacttac cattttggat aaaactgata tcaaacttta aaatctttaa ctaagaataa 960

tttctaaaat atttcactta taagtattga gaccatatgt atagatttgt cttaaaaagc 1020

acttcaataa attcatatat tcgttgatat tactatatat atcataatat aaaatagttg 1080

tcatagttat tttttgaaca tcatgcgtat ccttatccaa aaggataagt actccaatct 1140

agaaggagta ctgtgttctg 1160

<210> 2

<211> 2161

<212> DNA

<213> Rice (Oryza sativa L.)

<400> 2

cctttgatgt tttgtgttgt agtttactat ctcagtttca tattataact cattttgact 60

tttttagtat aatttctttt aaacttgata agctttagaa aaatgtaaca atattttaag 120

cacaaaacaa atatattatc aaaatatatt taatatttct ttcaattaaa ctaatttggt 180

attgtagatg ttgctaaatt tttctataaa cttagtccaa attaaaaaaa attaaaaaga 240

agtcaaggcg acttataata tgaaaataag gtgggttcta acaaaaaata ggaaattgtt 300

tttcaaaatc tcacaatgtg taggggaaaa tatggtcaca ccgaaaagtg tgttacaatc 360

taacagtgtg taataacata ttaagaaaga atcaacacac atagctaaga gaattcattc 420

tttgttattt cttgccagta ggttgtagtg ctatttcatg atattcgttc ataatgttta 480

ttatttggta taaaaaatgt atttaacact tattgcaccc tcgttataaa agtgtttatt 540

ttttacatat tccacgtatt agtccaatac cttttactac cataatactc ttactttttc 600

aaatcccaat gcaattattc cttgctaagg aagtaaatgt ttttttgttc cttgttagga 660

ggttgtagtg ctattgcctg ataaatgttg cttagtacct ccacaatgtt tgtcgcttac 720

tacaccctcg tctgaatgca atatactatc tcctaaacag tgcttgtcaa tggcaaaatt 780

tggcaatttc cattagtcaa ggccaaatca aaggtagagt tctaagctga ggaagtcatg 840

ttagaattgg actcagttag tttctttttg ctacatatta gctgattaga gtttgagttg 900

gagttgtata agtagtttct tgtttaggat agagtgctaa gtcgatccga tcttagtata 960

accagactta gttttgccta tgggtataat atgtacacac atagggtttt tgtaagataa 1020

caacagttca aaacatattt ttcggtgcat cgccaaccct attattagtt ttgacgagtt 1080

cttgttttta acatggtaga gagctcttac ataagggact tatttggcga atagttgatg 1140

gaaattcagt tgatgtaatc aaggataact gggacaagag gaacagcctt ctcaaaccac 1200

ttggttttat caatgcttta ctattcatca ttctatagtt aaaattttac agttttcttt 1260

aaaaaaataa actgcactaa agcttttatt gagatggatg gtcaactatt atttcaaaac 1320

tcaacaactt ttcgcacatg gaaggagtac tacataatta tgttgcaaat aaaggggata 1380

cacagctaca tatgcataga ttaatcttaa aaacactcaa taaaatcata atatatatat 1440

atatatatat atatagactg cgtgtccttg ttcaaaacac taaggcctcc tttaatggtt 1500

gagctagtgc actagcaaaa agtctacctt tttaatagat tgtctcatag cacattgttc 1560

aaaatgcttt gggttccaca atccctctca cattttttta ggagcttagc tcttgaagaa 1620

gagatagtgc attctctctc cttatcttct ctctcttcca catcatacaa aaactgatgt 1680

ggagatttat gagctagctg actcaccctt gttggaggag gcctaagtac taccaacctg 1740

gagggagtac cgtgtttttt tttaaaaaaa aactgcacta aaccatttag taaaacaaat 1800

ggccaactat tattgcaaag ctcaacaact ttgtacatat ggaggagtac ataactttca 1860

aaacggaggg agtgggtacg caatttctcc ctctaggtag ataatactta ccattttgga 1920

taaaactgat atcaaacttt aaaatcttta actaagaata atttctaaaa tatttcactt 1980

ataagtattg agaccatatg tatagatttg tcttaaaaag cacttcaata aattcatata 2040

ttcgttgata ttactatata tatcataata taaaatagtt gtcatagtta ttttttgaac 2100

atcatgcgta tccttatcca aaaggataag tactccaatc tagaaggagt actgtgttct 2160

g 2161

<210> 3

<211> 4083

<212> DNA

<213> Paddy rice (Oryza sativa L.)

<400> 3

gttctggttc atctatatat tagaggcatg gtgctatgca ttcgcattca taaatatcaa 60

gccaatcggc aatcgctgag atttactagt acttactgta cctgcataaa tacataaaca 120

cataccatat tatggcctcg agtgatactc ctacccatga ggaggcactc agttttgagc 180

cctcgatatg gggcgacttc tttatcaact acgaaccaca accattgcag gcatcaccac 240

tactcattcc tatgtgtaca tctgcccttc taattttaat taatgccatg ctaactagct 300

aatagctagc taattcgatg gatctatgct ataatttcat gcagagatca gagaggtgga 360

tgcaggagag ggctgagaag ctgagagggc aaatccagac actatttggg acctgccacg 420

acatgtcggc gaggatgaac ttagtggatt ccgtccaaca tctcggaatt gatcacctct 480

tccaagagga gatagaagat gctctaacaa gcatccatgg cagtgaattc agaagctcta 540

gtctctatga gattgctctt cggtttcgct tgcttaggga acatgggttt tgggtgtctc 600

caggtatcca aatgcatcca tccacctaat atatagataa atagtttatt attccgagat 660

taggaatttt tgtcttcgag atttgctgaa gcttactttt gttcgtttgc gacattatag 720

aaatttttta gtctaaaacc atacgacaca tgcaaaataa caaaacacag aaaatgttca 780

aaacacggga aatgtactaa tttgatgtca ggtacagcaa aaaagcgact gaaaaattat 840

gggggaaact aatcatgtta cgttacggat agaataagtc cagccattac tgtgttcaat 900

gacaaatatt ttttgtgacg cagatgcatt caataaattt aagggagatg atggcaagtt 960

tcgaaatgat atagcgaatg acccaaaggg gctactaagt ttatacaacg ctgcacacct 1020

cctcattcat ggcgagccag aacttgaaga agcaatctca tttgcacgaa aacaccttga 1080

attgatgagt caagacagtg ttctcaaccc tccattagct gagcaagtca agcgtgcact 1140

tagcctacca ctgccaagga ctttcaagag agttgagact atttgctaca tgtcggagta 1200

cgaacgagag gtgggaaata ttccacttct tcttgaactt gcgaagctag attttaacct 1260

cctgcaacat atccacttgg aggaactcaa agcaatttct gagtatgcct atctatcaat 1320

ctctttgaga attctccact ggataaataa tatgcggctc tgtgcatcgt agatataacg 1380

gctatgtgca tccattatct aaaaatataa ttaatatgtt ttttaataat aatggaatga 1440

actaaataac ttaattatta tgtgcaactg aattaacagt atcgatgtgt ttttgcaaat 1500

aaattattat gatagcatga atatattgtc agtagtaatg caggttatta tataatctct 1560

gtttttttta taatacctgt cattttagcg ttgaatttta tttgaagtaa atccacatcc 1620

attattttgg tcttatcaca tatccaatac ccacaagcta gccgttaaaa taaaacttag 1680

caccttaatt gtgaatataa ttaatggtct tgtcacactt ttgaaataat taatttcaga 1740

tggtggaaag atctttatgg atatatggag ctacgttaca ttagggatcg tacgatagag 1800

gcatacgctt ggtcctacat gatgttctac gaagaagact ttgcattcac cagaatgttt 1860

gttgccaaaa taattgcact ggatactgtg atggatgata cgtatgatgc tcatgccacc 1920

gttgaggaat gccggcagct aaacacagcc atacaaaggt tggataagtt cagtttgttt 1980

tattgataac acatatatga taagtgaaga caaacagtaa tatcttagcc tacaaatatt 2040

caaaataaga tatccaaatt tgatatggag catatcaaat tgacattact ccctaatata 2100

tatttaggga tcgagtaggg aatgttcaaa tgacttatat ttatggatca ggaggaatat 2160

tagatttgtc ttgaaaaata ctttcatgca gttatatttt aaatacattt atgtatggat 2220

ggcatatgat tagaaaaatt aatgattaaa actatgttct gaagttcgtg tcaatatttt 2280

aattcataag aaagaaatat tggatggaac atagtgacaa ataatgtatc ctagcatggc 2340

taattctcat gcaaaactaa ttgcagatgg gacaaaagcg ctatttctat tttgccggag 2400

tacctgaaaa agttctatat taaattgcta atcaactttg aggagtttga gcaccaagtg 2460

tccgacaacg aaaaatacaa ggttacttac accaaacaag aggtagaagc atccaaatat 2520

ctctatggga aattttttgt tattaccact ataagttata ttcttattta gcgacaacca 2580

actaaatttt gaggtagcgc gtggcaaaaa aaaaaaggaa acttgatttt gggtgagaaa 2640

agcgaaatta catacttttc aagtgtcaac ataaaactaa tttttgcaca tatatgttcg 2700

ggtattatta tgttgtagtt attaattttg ttttatttgc ttatagtttc aaaggcagtc 2760

tacttattac ctacaagaag ctgaatggtc ctaccagaaa cacaagccaa gctttaaaga 2820

tcaggtggtt ttgtccacca aatcctcggc cgtgcaatta gtgtgtgtgg ctgctatgat 2880

tggctggggt aatgcagtga cgacagaagc attcgagtgg gcagctagtg gtaatgatgc 2940

agtcatagca tgcgcaaaga ttggacgttt tatgaatgat attgctgcat ttaaggtaca 3000

tatatagtag tattactctc actgttctat tcggcttctg gattgtgaaa tcaatactag 3060

ctttatccca taatatgcat agccaaaata taacctcata atacgggaca gactgagtag 3120

tctgtgatta atagacattt ttgtcattgc cttttctaat caatatgtcc atcaaaattg 3180

taaaaagtaa caatatgaaa ttatttttta caataagtct actaatgtca ttttcgcttg 3240

acaagtctta ataactgtaa atgtattaat ttttcatgat tttgtggctc gtctacgtat 3300

attaaaaaca tcatctattt aagaacggaa taagtaaaaa aaactctttt caccaagtag 3360

ttaaatataa ttctttttca cattttcata tgcattatac acatgcagtg tggaaagaac 3420

aagggagatg ttgcgagctc cgtagagtgt tacatgaatg agaatagggt cacaagcgag 3480

gtcgcctttg caaagattga ttcactggtc gaagatgaat ggagaaccac gaaccagacc 3540

cgccttgagc atggtacact gctgcccatg gtgcagcgag ttgtgaactt caccgtttct 3600

atggtactct tctacgatga caggaatgat gcctacacat ttgccacact tcttagggag 3660

attatagagt ctctcttcgt gaggcctgct cccatctaga ccttaatggt taactttcta 3720

gccaatgtgg caatccatct gcatgtatac ttgtattgaa tatgcttttg gcttctgtta 3780

ttcatgttaa caataaactt gtgtgctaag ctacttaatc tgcatgtgtt tcattttgag 3840

tctgtatgcg tgagcccacg atctatgtgg atagccatcg cgaggagtca gttgtgaggg 3900

catccgaggc tagctcgctg tccgcgtcgt gcgccgaatt caaagacacg tcgtgtggct 3960

gtggggatgg acgcagctag atcacggcgg cagaatcggt cctggtggtt gtatttccag 4020

tttttgagtt gcttttttta aggaagtaaa cagaaaagca gcaatggttg cgctccacca 4080

aaa 4083

<210> 4

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

taaaacgacg gccagtgccc ctttgatgtt ttgtgttgta g 41

<210> 5

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gcagttgttg tccatggtgg ccagaacaca gtactccttc tag 43

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tggttctgcc caaggtatcg 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aacggttccg ctctttctgt 20

<210> 8

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gcttttttgt aggtagactg caggttctgg ttcatctata tattagag 48

<210> 9

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cgatcgggga aattcgtcga cttttggtgg agcgcaacca ttg 43

Claims (7)

1. A rice stem borer harm inducing promoter pOsISA2 is characterized in that the nucleotide sequence is shown as SEQ ID No. 2.

2. The application of the rice stem borer harm-inducing promoter pOsISA2 as claimed in claim 1 in culturing intelligent rice resistant to stem borers.

3. The use according to claim 2, comprising the steps of:

(1) constructing a recombinant vector containing the rice stem borer harm inducing promoter pOsISA2 of claim 1;

(2) transferring the recombinant vector into an agrobacterium-infected cell to construct a genetic engineering bacterium containing the recombinant vector in the step (1);

(3) and (3) carrying out mediated transformation on the rice callus by the genetically engineered bacteria in the step (2) to obtain a transgenic positive rice plant.

4. A recombinant vector comprising the rice stem borer hazard-inducing promoter pOsISA2 according to claim 1.

5. The recombinant vector of claim 4, further comprising an insect-resistant gene.

6. A transformant comprising the rice chilo suppressalis hazard-inducing promoter pOsISA2 according to claim 1.

7. The transformant according to claim 6, further comprising an insect-resistant gene.

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