CN106119249B

CN106119249B - Identification and application of two rhizoctonia solani induced novel cis-acting elements

Info

Publication number: CN106119249B
Application number: CN201610533275.0A
Authority: CN
Inventors: 李宁; 丁新华; 储昭辉
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2016-07-07
Filing date: 2016-07-07
Publication date: 2020-02-04
Anticipated expiration: 2036-07-07
Also published as: CN106119249A

Abstract

The invention relates to the technical field of plant genetic engineering, and provides two cis-acting elements induced by rhizoctonia solani and application thereof, wherein the two elements are derived from a promoter sequence at the upstream of a banded sclerotial blight disease-resistant related gene GRMZM2G315431, and specifically, in the application, a green fluorescent protein gene and cis-series repeated fusion of the two elements are carried out to obtain recombinant DNA, and rice is transformed to obtain a new transgenic plant. The expression of the green fluorescent protein gene in the transgenic rice is regulated and controlled by the infection of rhizoctonia solani; thus, the cis-acting element of the invention can be used for constructing a plant expression vector for improving the resistance of plants to banded sclerotial blight through a plant genetic engineering technology.

Description

Identification and application of two rhizoctonia solani induced novel cis-acting elements

Technical Field

The invention relates to the technical field of plant genetic engineering, and provides two cis-acting elements for inducing rhizoctonia solani, which can be used for improving the resistance of plants to rhizoctonia solani by using a plant genetic engineering technology.

Background

Plant diseases caused by fungi, bacteria, viruses and nematodes have a great influence on crop production. Plants possess two different sets of resistance mechanisms: one is a passive defense mechanism, which refers to some resistance-related morphological structures, such as epidermis and rigid cell wall (Brady JD, front S. Format of di-isocratin and loss of isocratin in the cell walls of substrate cell-culture cells with a long functional entity or H₂O₂Plant Physiol 1997,115: 87-92.). The other is active defense, which is caused by the recognition and interaction of resistance genes with pathogenic bacteria. The elicitor coded by the pathogen avirulence gene (avr) interacts with the receptor molecule of the elicitor coded by the disease-resistant gene (R) responded by the host plant to generate disease-resistant reaction. The plant disease-resistant defense reaction generally comprises three links of signal identification, signal conduction and defense gene expression. All types of disease-resistant mechanisms in active defense rely on defensive gene expression, and the effectiveness of resistance depends on the spatiotemporal characteristics of gene expression. Due to the temporal and spatial expression characteristics of defense genes, promoters thereforHeretofore, with certain specific cis-acting elements, one could utilize defensive genes, particularly their cis-elements, to provide an advantageous tool for genetic engineering improvements. The vast majority of disease-resistant genes in plants encode proteins with nucleotide binding sites and leucine repeats (NBS-LRR), (McHale L, Tan X, Koehl P, Michelmore RW. plant NBS-LRR proteins: adaptive targets. genome Biol 2006,7: 212.). However, the promoter of such genes has been studied only rarely.

Plant gene promoters are important cis-acting elements, are DNA sequences located in the 5-terminal upstream region of structural genes, and are central to transcriptional regulation. Since the first transgenic plant appeared in 1983, the promoter is always a research hotspot of genetic engineering, and the selection of a proper and effective expression promoter lays a solid foundation for the research of the genetic engineering. The tissue-specific promoter can ensure that the expression of the exogenous gene only occurs in certain specific organs or tissue parts and often shows development regulation characteristics; the inducible promoter can make the exogenous gene respond to certain signals and only express under the stimulation of special signals. The greatest advantages of these promoters are: the method overcomes the waste caused by nonspecific continuous and efficient expression of exogenous genes started by a constitutive promoter in a receptor plant, and meets certain requirements for specific expression of the exogenous genes. Tissue-specific or inducible expression promoters have been the focus and difficulty of genetic engineering research in plants.

At present, some tissue-specific or inducible expression promoters are found, and corresponding cis-factors existing on the promoters are found, so that the bases are laid for the subsequent promoter research. The Sar8.2b gene of tobacco is induced by Salicylic Acid (SA), and several cis-acting factors including as-1 factor, GT21 and Dof binding sequences are found on its promoter. SA induces expression of the Sar8.2b gene in cis elements that may be present in the-728 to-927 bp and-197 to-351 bp regions (Song F, Goodmann RM. cloning and identification of the promoter of the tobaco Sar8.2b gene, gene incorporated in systematic acquired resistance 2002,290: 115; 124). Corn sucrose synthase I is expressed only in phloem cells (Yang NS, Russell D.maize sucrose synthase-promoter direct from phylem cell specific expression of gus gene in transgenic bacco plants. Proc Natl Acad Sci 1990,87: 4144-. The PsGNS2 promoter is specifically expressed only in seeds (Buchner P, Rochat C, Wuuilleme S, Boutin JP. characteristics of tissue-specific and developmentally regulated beta-1,3-glucanase gene in pea (Pisum sativum). Plant Mol Biol 2002,49: 171. 186.).

Corn is one of the most important food crops, and the fungal diseases and bacterial diseases of corn cause the reduction of yield and quality. The cloning of the corn disease-resistant gene and the separation of the promoter can enable people to better understand the interaction between hosts and pathogenic bacteria and lay a foundation for the improvement of genetic engineering. Therefore, how to obtain a disease-resistant plant by cloning a corn disease-resistant gene and separating a promoter becomes one of the problems to be solved urgently.

The inventor discloses a corn pathogenic bacteria inducing promoter pGRMZM2G315431 in ZL201410104914.2, the promoter can be activated by corn rhizoctonia solani YWK-196, rice bacterial blight and bacterial streak, but specific function analysis of the promoter is not made in the prior art, and deeper properties and functions cannot be revealed.

Disclosure of Invention

Aiming at the conditions of the prior art, particularly on the basis of the technology disclosed in ZL201410104914.2, the inventor of the invention obtains two cis-acting elements induced by the maize rhizoctonia solani, wherein the two elements can respond to the infection of the rhizoctonia solani and can not be activated by the rice bacterial blight bacteria and the bacterial streak bacteria, so that the two elements are the rhizoctonia solani specific induction regulation and control elements, and the regulation and control elements can be utilized to construct various plant expression vectors for improving the resistance of plants to the rhizoctonia solani through the plant genetic engineering technology.

The inventor carries out truncation on the region from-1243 to-1228 of the GRMZM2G315413 promoter in the technology disclosed in ZL201410104914.2, and determines that the two sequences from-1243 to-1239 and from-1232 to-1228 are action elements responding to the infection of rhizoctonia solani, and the nucleotide sequences are shown as SEQ ID No.1 and SEQ ID No. 2. After the gene is stably expressed in rice, the two action elements are only activated by rhizoctonia solani but not by bacterial blight bacteria and bacterial streak bacteria.

On the basis, the inventor utilizes the green fluorescent protein gene and the cis-form tandem repeated fusion of the two elements to obtain recombinant DNA, transforms rice to obtain a new transgenic plant, and experiments prove that the expression of the green fluorescent protein gene in the transgenic rice is controlled by the infection of rhizoctonia solani; as the green fluorescent protein gene is used as a visual marker gene in the field and is a commonly used marker gene in the research of promoters and regulatory elements, the function of the regulatory elements can be verified according to the marker gene, and based on the condition, the cis-acting element can be proved to be completely utilized to construct a plant expression vector for improving the resistance of plants to banded sclerotial blight through the plant genetic engineering technology.

In conclusion, the inventor provides two action elements specifically induced by rhizoctonia solani, and the functional research of the action elements is helpful for disclosing the expression regulation mechanism of the GRMZM2G315431 gene, and widens the application of the promoter and the action elements in basic research and disease-resistant genetic engineering.

Drawings

FIG. 1 is a gray scale of results of fluorescent observation and quantitative analysis of GFP induced by Rhizoctonia solani after transient expression of a truncated segment of the region-1243 to-1228 promoter of GRMZM2G315413 in tobacco;

in the figure, a is a truncated schematic diagram from-1243 to-1228; b is the GFP fluorescence detection result of each truncated segment pathogen induction activity; c is the result of quantitative analysis of the pathogen induction activity GFP of each truncated fragment;

wherein, the non-inoculated bacteria is used as a reference, and after being inoculated for 24h, the fluorescence observation and the quantitative detection are carried out, and the result shows that the-1243 to-1239 and-1232 to-1228 fragments can be activated by rhizoctonia solani, which indicates that the two sequences are rhizoctonia solani induction elements;

FIG. 2 is a gray scale graph showing the results of fluorescent observation and quantitative analysis of GFP induced by Rhizoctonia solani after transient expression of the action element of the present invention in tobacco, wherein a is the fluorescent observation result of GFP induced by pathogen-induced activity of the regulatory element; b is the result of quantitative analysis of regulatory element pathogen induction activity GFP;

wherein, the non-inoculated bacterium is used as a control, and the fluorescence observation and the quantitative detection are carried out after inoculation for 24h, the result shows that the pathogen induction activity of the GTTGA after being serially repeated twice is basically equivalent to that of the full-length GRMZM2G315431 promoter, and the induction activity of the TATTT after being serially repeated twice is equivalent to that of a single element;

FIG. 3 is a gray scale graph of GFP fluorescence observation and quantification results after rhizoctonia solani is induced in flowers 11 of rice transformed by the action element of the present invention, wherein a is a GFP fluorescence detection result after rhizoctonia solani is inoculated in transgenic rice; b is a GFP quantitative detection result after inoculating rhizoctonia solani on the transgenic rice;

selecting 3T per element₁The generation of transgenic strains, by taking non-inoculated bacteria as a reference, the pathogenic induction activity of the transgenic strains is basically equivalent to that of a full-length GRMZM2G315431 promoter after the GTTGA is repeated in series twice, and the induction activity of the transgenic strains after the TATTT is repeated in series twice is equivalent to that of a single element, so that the stable expression and the transient expression result are consistent;

FIG. 4 shows the result of quantitative detection of GFP in rice flowers 11 transformed with the working element of the present invention after induction by bacterial blight (a) and bacterial streak (b);

the result shows that no obvious fluorescent signal is detected after inoculation of the bacterial blight bacteria and the bacterial streak bacteria by taking the non-inoculated bacteria as a contrast, which indicates that the two elements can not respond to the infection of the bacterial blight bacteria and the bacterial streak bacteria;

FIG. 5 is a schematic color diagram of FIG. 1;

FIG. 6 is a schematic color diagram of FIG. 2;

fig. 7 is a color schematic of fig. 3.

Detailed Description

The present invention is further defined in the following examples, from which one skilled in the art can ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Except for special notes, the invention adopts the prior art in the field;

example 1 construction of a recombinant vector of a-1243 bp to-1228 bp different truncated sequence and GFP Gene and transient expression on native tobacco

The truncated promoter referred to in this example was derived from the GRMZM2G315413 promoter sequence and was obtained in particular according to the technique disclosed in ZL201410104914.2, in which a truncated sequence of-1243 to-1228 was obtained, and in this example further investigation and analysis of the region-1243 to-1228.

To identify the active elements of the region-1243 to-1228 in response to Rhizoctonia solani, this region was truncated again. Using 35S basic promoter as template, respectively using forward primers with the sequences shown in SEQ ID No.3, 4, 5 and 6

(5-GTTGAGGCACTTATTTCGCAAGACCCTTCCTCTATATAA-3，

5-GGCACTTATTTCGCAAGACCCTTCCTCTATATAA-3，

5-TATTTCGCAAGACCCTTCCTCTATATAA-3,

5-CGCAAGACCCTTCCTCTATATAA-3) and a reverse primer, the sequence of which is shown as SEQ ID NO.7

(5-GGGACTGACCTACCCGGG-3) amplifying the promoter fragment;

the PCR reaction procedure was as follows: pre-denaturing at 94 ℃ for 5min, denaturing at 94 ℃ for 40S, annealing at 55 ℃ for 40S, extending at 72 ℃ for 30S, reacting for 35 cycles, and then extending at 72 ℃ for 7min to obtain fragments of sequence SEQ ID No.8, 9, 10, 11, wherein SEQ ID No.8 is the sequence of-1243 to-1228 plus the 35S base promoter, SEQ ID No.9 is the sequence of-1238 to-1228 plus the 35S base promoter, SEQ ID No.10 is the sequence of-1232 to-1228 plus the 35S base promoter, and SEQ ID No.11 is the sequence of the 35S base promoter;

after the reaction is finished, carrying out 1.0% agarose gel electrophoresis detection on the PCR product, recovering and purifying a target fragment, sequencing and verifying the obtained PCR product, connecting the PCR product with a plant expression vector pCXGFP-P (containing a green fluorescent protein gene) digested by XcmI, and transforming the recombinant vector into agrobacterium GV3101 by an electrical excitation method.

The cultured Agrobacterium GV3101 and p19K were treated with 10mM MgCl₂Resuspending and OD adjusting₆₀₀1.0, the same volume is mixed and then the injection is carried outAnd (3) generating tobacco leaves, and injecting for 3-5d for transient expression detection.

Example 2 detection of the response of different truncated promoters of-1243 bp to-1228 bp to Rhizoctonia solani after transient expression on this tobacco

Shearing the lamina of the Bunsen tobaccos after 5 days of agrobacterium injection, spreading the lamina in an inoculation tray, taking off the circular bacterial block of the cultured rhizoctonia solani YWK196 by using a puncher, inversely covering the circular bacterial block on the lamina of the tobaccos, preserving moisture at 25 ℃, treating for 24 hours, and taking the lamina at the inoculation point for analysis.

The above results of fluorescence intensity of GFP in inoculated tobacco leaves are shown in FIG. 1, and after inoculation of YWK 19624 h, the fluorescence intensity was reduced by half compared with that of-1243 to-1228 after deletion of GTTGA, whereas the fluorescence intensity of deletion of GGTGAGGCACT was comparable to that of GTTGA, and the remaining sequence TATTT maintained the fluorescence intensity, indicating that both GTTGA and TATTT are involved in response to Rhizoctonia solani.

Example 3 construction of recombinant vectors of GTTGA and TATTT tandem repeats with GFP Gene and transient expression on Nicotiana benthamiana

To verify that the individual GTTGA and TATTT respond to infection by Rhizoctonia solani, these two elements were repeated in tandem twice, using a 35S-based promoter as a template and forward primers, respectively, having the sequences shown in SEQ ID Nos. 12, 13 and 6

(5-GTTGAGTTGACGCAAGACCCTTCCTCTATATAA-3，

5-TATTTTATTTCGCAAGACCCTTCCTCTATATAA-3，

(5-GGGACTGACCTACCCGGG-3) amplifying the promoter fragment; meanwhile, a maize B73 genome is taken as a template, a forward primer and a reverse primer are respectively used, the sequences of the forward primer and the reverse primer are shown as SEQ ID NO.14 (5-TTCTATGGCAAAATCAATGAAGG-3), and the sequence of the reverse primer is shown as SEQ ID NO.15 (5-TGGCGGTGACGATGGTAA-3), so that the full-length promoter fragment of GRMZM2G315431 is amplified;

the PCR reaction procedure was as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 40S, annealing at 55 ℃ for 40S, extension at 72 ℃ for 60S, reaction for 35 cycles, and post-extension at 72 ℃ for 7min to obtain fragments of sequences SEQ ID No.16, 17, 11, 18, wherein SEQ ID No.16 is the sequence of GTTGA repeat 2 times plus 35S basal promoter, SEQ ID No.17 is the sequence of TATTT repeat 2 times plus 35S basal promoter, and SEQ ID No.18 is the full-length sequence pGRM2G 315431;

after the reaction is finished, carrying out 1.0% agarose gel electrophoresis detection on the PCR product, recovering and purifying a target fragment, sequencing and verifying the obtained PCR product, connecting the PCR product with a plant expression vector pCXGFP-P digested by XcmI, and transforming the recombinant vector into agrobacterium GV3101 by an electric excitation method.

The cultured Agrobacterium GV3101 and p19K were treated with 10mM MgCl₂Resuspending and OD adjusting₆₀₀When the mixture is equal to 1.0, the benoxanil leaves are injected after equal volume mixing, and the benoxanil leaves are used for transient expression detection after 3-5 days of injection.

Example 4 testing of the response of GTTGA and TATTT tandem repeats to Rhizoctonia solani following transient expression on this tobacco

The GFP fluorescence intensity results of the tobacco leaves inoculated with the above-mentioned inoculation are shown in FIG. 2, and after YWK 19624 h, the pathogen-inducing activity of the tobacco leaves after twice-repeated GTTGA tandem (SEQ ID NO.16) was substantially equivalent to that of the full-length promoter, and the pathogen-inducing activity of the tobacco leaves after twice-repeated TATTT tandem (SEQ ID NO.17) was about half of that of the full-length promoter, indicating that GTTGA and TATTT alone could respond to the infection of Rhizoctonia solani.

Example 5 verification of tobacco transient expression results Using Stable Rice transgenes

To verify the results of tobacco transient expression in example 4, the expression vectors constructed in example 3 (SEQ ID NO.11,16-18) were introduced into Agrobacterium EHA105 by electric stimulation, and each expression vector was introduced into rice variety flower 11 by Agrobacterium-mediated genetic transformation (Lin and Zhang, optimization of the tissue culture conditions for high efficiency transformation of index rice, 2005, Plant Cell Rep.23: 540-. The obtained genetically transformed plants were named GTTGA, TATTT, pGRMZM2G31543, respectively1 and 35S mini. The invention obtains 6, 5, 9 and 7 independent transformation positive plants respectively. For GTTGA and TATTT T respectively₁And 3 generation transgenic lines are used for inoculation identification of the maize rhizoctonia solani. The results show that after inoculation of rhizoctonia solani for 24h, stronger fluorescence signals can be detected in GTTGA and TATTT transgenic plants (figure 3), and the results are consistent with tobacco transient expression results.

Meanwhile, in order to verify whether the two elements can respond to other pathogenic bacteria, the transgenic plants are inoculated and identified with the bacterial blight bacteria and the bacterial streak pathogens. As a result, no significant fluorescent signal was detected after inoculation of both GTTGA and TATTT transgenic plants with the pathogen (FIG. 4), indicating that both elements are specifically responsive to infection by Rhizoctonia solani and not to infection by bacterial blight and bacterial streak.

EXAMPLE 6 prospect of application of two acting elements

The gene is fused with the plant sheath blight disease resistance related gene (such as Os2H16, OsACS2, OsJERF1 and the like) by utilizing the pathogen induction action element with the nucleotide sequence shown as SEQ ID NO.1 and SEQ ID NO.2 to construct a transgenic plant material;

the inventor finds that the two action elements respond to the rhizoctonia solani when the rhizoctonia solani is infected on the premise of not influencing the growth and development of the plant, so that the expression of downstream disease-resistant related genes is activated, the resistance of the plant to the rhizoctonia solani is improved, and the important significance is realized on the research of the resistance of the plant to the rhizoctonia solani.

Claims

1. The application of the cis-acting element induced by the maize rhizoctonia solani in improving the resistance of the rhizoctonia solani is characterized in that: the cis-acting element nucleotide sequences are respectively shown as a sequence table SEQ ID NO.1 and a sequence table SEQ ID NO. 2.