Gene promoter active fragment screening method based on cross-overlap method and application

Abstract

The invention discloses a gene promoter active fragment screening method based on a cross-overlap method and application, and belongs to the technical field of biology. The screening method comprises the following steps: segmenting a target gene promoter sequence, and setting an overlapping fragment between two adjacent segments to obtain a gene promoter active fragment containing the overlapping fragment in the segments. According to the invention, the active fragment of the pennisetum americanum PgGNM2 promoter is screened for the first time, the fragment is used for a CRISPR / Cas9 system, so that the gene editing efficiency can be improved by more than 40%, and when the fragment is used for a yeast one-hybrid bait carrier, the false positive rate can be reduced to less than 5%. The screening method retains the integrity of cis-acting elements, is suitable for monocotyledonous / dicotyledonous plants, and provides an efficient tool for molecular breeding of crops.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for screening gene promoter active fragments based on a cross-overlap method and its application. Background Art

[0002] A gene promoter is a specific DNA sequence located upstream of a gene. It plays a key role in regulating gene expression, mainly in the following aspects: (1) Providing a transcription initiation signal: providing a recognition and binding site for RNA polymerase. When RNA polymerase binds to the promoter, it initiates the gene transcription process and synthesizes RNA using DNA as a template. Without a promoter, RNA polymerase cannot accurately find the transcription start site, and gene transcription cannot proceed normally; (2) Determining the temporal and spatial specificity of gene expression: Different genes have different expression patterns in different tissues, cells, and developmental stages, which is largely determined by the promoter. The promoter contains a variety of cis-acting elements that can interact with proteins such as transcription factors in the cell. In specific cell types or developmental stages, there are some specific transcription factors in the cell that can recognize and bind to the promoter of the corresponding gene, thereby activating or inhibiting gene transcription.

[0003] Regulation of gene expression intensity is a core issue in molecular biology research. As key regulatory elements for gene transcription initiation, promoters' sequence characteristics and functional properties directly influence gene expression levels. The regulatory functions of promoters are primarily manifested in the following aspects: First, specific cis-acting elements (such as the TATA box and CAAT box) within the promoter sequence and their spatial arrangement determine their binding capacity for RNA polymerase and transcription factors. Differences in this binding efficiency can lead to significant variations in transcriptional activity. Highly active promoters typically contain multiple conserved transcription factor binding sites, enabling recruitment of more transcription machinery and forming a stable transcription initiation complex, thereby achieving efficient gene expression. In contrast, low-activity promoters, due to insufficient number or affinity of binding sites, can only maintain basal levels of transcription. Second, epigenetic modifications such as DNA methylation and histone acetylation dynamically alter the chromatin accessibility of promoters. DNA methylation generally inhibits transcription factor binding, while histone acetylation promotes chromatin relaxation. Together, these modifications constitute a sophisticated epigenetic regulatory network. In molecular breeding practice, the isolation and identification of highly active promoters has multiple application values: on the one hand, these strong promoters can be used to construct efficient expression vectors, significantly increasing the expression level of exogenous genes; on the other hand, in the CRISPR / Cas9 gene editing system, the use of endogenous strong promoters to drive Cas9 protein expression can significantly improve editing efficiency.

[0004] For important forage crops like Pennisetum truncatum, establishing an endogenous promoter library not only optimizes genetic transformation systems but also provides tools for studying the species' unique gene regulation mechanisms. This, in turn, accelerates variety improvement by precisely regulating the expression levels of genes associated with key agronomic traits. Therefore, identifying active fragments of a gene promoter is crucial for improving gene editing technology platforms and enabling molecular design breeding for this species. Summary of the Invention

[0005] The present invention aims to provide a method and application for screening gene promoter active fragments based on the cross-overlap method to address the aforementioned problems in the prior art. The transcriptionally active fragments obtained through this screening method can be used to construct efficient "bait" vectors for yeast one-hybrid library screening, reducing false positives. They can also be used in the CRISPR / Cas9 editing system to improve gene editing efficiency.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for screening gene promoter active fragments, comprising segmenting a target gene promoter sequence, setting overlapping fragments between two adjacent segments, and obtaining gene promoter active fragments containing overlapping fragments in the segmentations.

[0008] Furthermore, the step also includes: cloning the fragment into a vector containing a reporter gene, introducing it into a host cell to detect activity, and screening to obtain the gene promoter active fragment.

[0009] Furthermore, the reporter gene includes LUC.

[0010] Furthermore, the vector includes pGreenII 0800.

[0011] Furthermore, the host cells include tobacco leaves transformed by Agrobacterium.

[0012] The present invention also provides a high-activity fragment obtained by screening according to the above screening method, and the nucleotide sequence of the high-activity fragment is shown in SEQ ID NO.5.

[0013] The present invention also provides the use of the above-mentioned high-activity fragment in driving the expression of target genes.

[0014] Furthermore, the application includes constructing a CRISPR / Cas9 system vector.

[0015] The present invention also provides a low-activity fragment obtained by screening according to the above screening method, wherein the low-activity fragment comprises any one or more of the nucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.6.

[0016] The present invention also provides the use of the low-activity fragment in constructing a yeast one-hybrid bait vector.

[0017] High-activity fragments were defined as having significantly higher activity than that of the empty vector (p<0.05), and low-activity fragments were defined as having significantly lower activity than that of the empty vector (p<0.05).

[0018] The present invention discloses the following technical effects:

[0019] The present invention splits the promoter by the cross-overlap method, retaining the integrity of the cis-acting elements to the greatest extent. After verification by the LUC reporter system, it was found for the first time that the P4 fragment in the PgGNM2 promoter of Pennisetum americanum has significantly high activity (fluorescence intensity is 3.2 times that of the control), while the P1 / P2 / P5 fragments show an inhibitory effect (activity is lower than that of the empty vector). The highly active fragments can be directly used in the CRISPR / Cas9 system to increase the editing efficiency of Pennisetum americanum by more than 40%; the yeast single-hybrid bait vector constructed with the low-activity fragment reduces the false positive rate to less than 5%. This method has strong universality and is suitable for the functional analysis of monocotyledonous and dicotyledonous plant promoters, providing an efficient tool for crop molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is a schematic diagram of the overlapping segments of the Pennisetum americanum PgGNM2 promoter;

[0022] Figure 2 Schematic diagram of the carrier structure used in Example 1;

[0023] Figure 3 This is a graph showing the fluorescence detection analysis of LUC driven by different PgGNM2 promoter fragments; 1-6 represent empty vector, promoter fragment 1 (P1), promoter fragment 2 (P2), promoter fragment 3 (P3), promoter fragment 4 (P4), and promoter fragment 5 (P5), respectively;

[0024] Figure 4is the ratio of the LUC fluorescence signal to the REN fluorescence signal of different PgGNM2 promoter fragments. Different letters indicate that there is significance of p < 0.05 between different fragments. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified.

[0031] The pGreenII 0800 vector is described in the non-patent document "Hellens, RP, Edwards, EA, Leyland, NR, Bean, S., & Mullineaux, PM (2000). pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plan T molecular biology, 42 (6)", which is publicly available from the applicant to repeat this experiment.

[0032] The competent Agrobacterium was GV3101 (pSoup-p19), a product of Shanghai Weidi Biotechnology Co., Ltd.

[0033] The preparation method of LB solid medium is as follows (taking 1 L as an example): 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of agar powder, distilled water to 1 L, adjust the pH to 7.2 with 5 mol / L NaOH, and sterilize at 121°C for 30 min.

[0034] The concentration of antibiotic kana sulfate (Kana) is 50 mg / L, and the concentration of antibiotic rifampicin (Rif) is 50 mg / L).

[0035] The infection solution formula is: 10mM MgCl2, 10mM MES (pH5.7), 100μmAS.

[0036] Example 1

[0037] The present invention takes Pennisetum serrata as an example. The upstream 2000bp of PgGNM2 obtained from the Pennisetum serrata website is used as the promoter sequence. The nucleotide sequence is shown in SEQ ID NO.1. Then the promoter sequence is segmented into 500bp segments, and 150bp of each adjacent segment is taken as the overlapping fragment (see Figure 1 The nucleotide sequence of the PgGNM2 promoter of Pennisetum americanum is shown in SEQ ID NO.1, the nucleotide sequence of P1 is shown in SEQ ID NO.2, the nucleotide sequence of P2 is shown in SEQ ID NO.3, the nucleotide sequence of P3 is shown in SEQ ID NO.4, the nucleotide sequence of P4 is shown in SEQ ID NO.5, and the nucleotide sequence of P5 is shown in SEQ ID NO.6 (the nucleotide sequences of the PgGNM2 promoter and P1-P5 are shown in Table 1).

[0038] 1. Obtaining Pennisetum DNA Template

[0039] The Pennisetum truncatum materials used were grown in the sixth teaching laboratory building of the Chengdu campus of Sichuan Agricultural University. Fresh samples were taken and stored in liquid nitrogen to extract the Pennisetum truncatum DNA.

[0040] 2. Using the DNA obtained in step 1 as a template, a primer pair consisting of the upstream primer SEQ ID. P6_F (SEQ ID NO. 7) and the downstream primer SEQ ID. P6_R (SEQ ID NO. 8) was amplified (instantaneous centrifugation to mix evenly, and the PCR reaction used the Novezan P520 high-fidelity enzyme PCR program). The amplification system and program are detailed in Tables 2 and 3, and then the PCR product was recovered.

[0041] 3. Using the PCR product recovered in step 2 as a template, amplification was performed using a primer pair consisting of upstream primers SEQ ID. P1-F (SEQ ID NO. 9), SEQ ID. P2-F (SEQ ID NO. 11), SEQ ID. P3-F (SEQ ID NO. 13), SEQ ID. P4-F (SEQ ID NO. 15), or SEQ ID. P5-F (SEQ ID NO. 17), and downstream primers SEQ ID. P1-R (SEQ ID NO. 10), SEQ ID. P2-R (SEQ ID NO. 12), SEQ ID. P3-R (SEQ ID NO. 14), SEQ ID. P4-R (SEQ ID NO. 16), or SEQ ID. P5-R (SEQ ID NO. 18). The nucleotide sequences of the above primers are detailed in Table 1.

[0042] Table 1 Sequences of PgGNM2 promoter, P1-P5 and primers

[0043]

[0044]

[0045]

[0046]

[0047] Table 2 PCR reaction system

[0048] Reaction components Dosage 2×TaqMasterMix 10 μL Forward primer 1 μL Reverse primer 1 μL <![CDATA[ddH2O]]> 7μL DNA 1 μL total 20 μL

[0049] Table 3 PCR amplification program

[0050]

[0051]

[0052] 4 μL of PCR product was taken and detected by 1% agarose gel electrophoresis. The target band of the reaction product of the five promoter fragments (P1-P5) of American Pennisetum was 500 bp. Figure 2 The DNA was extracted from the gel using a standard agarose gel DNA recovery kit produced by Meiji Company. The method was referred to the kit instructions to obtain specific amplified fragments. After sequencing, the following amplified fragments were obtained:

[0053] The nucleotide sequence of the fragment obtained by amplifying the Pennisetum sutchuenensis DNA using SEQ ID. P6-F and SEQ ID. P6-R is shown in positions 1-2000 of SEQ ID PgGNMP2000, referred to as fragment pgGNMP2000 (SEQ ID NO. 1). The nucleotide sequence of the fragment obtained by amplifying the Pennisetum sutchuenensis DNA fragment pgGNMP2000 using SEQ ID. P1-F and SEQ ID. P1-R is shown in P1 (SEQ ID NO. 2). The nucleotide sequence of the fragment obtained by amplifying the Pennisetum sutchuenensis DNA fragment pgGNMP2000 using SEQ ID. P2-F and SEQ ID. P2-R is shown in P2 (SEQ ID NO. 3). The nucleotide sequence of the fragment obtained by amplifying the Pennisetum sutchuenensis DNA fragment pgGNMP2000 using SEQ ID. P3-F and SEQ ID. P3-R is shown in P3 (SEQ ID NO. 4). The nucleotide sequence of the Pennisetum truncatum DNA fragment pgGNMP2000 obtained by amplifying the fragments pgGNMP2000 with SEQ ID. P4-F and SEQ ID. P4-R is shown as P4 (SEQ ID NO. 5). The nucleotide sequence of the Pennisetum truncatum DNA fragment pgGNMP2000 obtained by amplifying the fragments pgGNMP2000 with SEQ ID. P5-F and SEQ ID. P5-R is shown as P5 (SEQ ID NO. 6).

[0054] 4. Construction of Pennisetum recombinant expression vector

[0055] The vector was simultaneously digested with restriction endonucleases SalI and BamHI produced by Thermo Fisher Scientific, and the fragments were recovered after digestion to obtain digested pGreenII 0800.

[0056] 4.1 Using a one-step recombination kit produced by Novozymes, fragment P1 was recombined into pGreenII 0800 after restriction digestion, following the kit instructions to obtain a ligation product. 5 μL of the ligation product was transformed into Escherichia coli DH5a and cultured on solid LB medium containing kanamycin (Kana, concentration: 50 pg / L) for 12 h. Single colonies were selected and cultured in liquid LB medium containing Kana (concentration: 50 pg / L). After positive clones were identified by PCR in the culture medium, plasmids were extracted and double-enzyme digestion was performed. Successful plasmids were sent to Sangon Biotechnology for sequencing. Once sequencing was correct, the successfully constructed expression vector pGreenII 0800-P1 was obtained. The structure of the expression vector pGreenII 0800-P1 is described as follows: the small fragment between the restriction endonuclease SalI and BamHI recognition sequences of pGreenII0800 is replaced with SEQ ID NO. 2, while the other sequences of the vector pGreenII 0800 remain unchanged, to obtain the expression vector pGreenII 0800-P1. The promoter P1 in this expression vector drives the expression of the LUC gene, see Figure 4 .

[0057] 4.2 Using a one-step recombination kit produced by Novozymes, fragment P2 was recombined into pGreenII 0800 after restriction digestion, following the kit instructions to obtain a ligation product. 5 μL of the ligation product was transformed into Escherichia coli DH5a and cultured on solid LB medium containing kanamycin (Kana, concentration: 50 pg / L) for 12 h. Single colonies were selected and cultured in liquid LB medium containing Kana (concentration: 50 pg / L). After positive clones were identified by PCR in the culture medium, plasmids were extracted and double-enzyme digestion was performed. Successful plasmids were sent to Sangon Biotechnology for sequencing. Once sequencing was correct, the successfully constructed expression vector pGreenII 0800-P2 was obtained. The structure of the expression vector pGreenII 0800-P2 is described as follows: the small fragment between the restriction endonuclease SalI and BamHI recognition sequences of pGreenII0800 is replaced with SEQ ID NO. 3, while the other sequences of the vector pGreenII 0800 remain unchanged. The resulting expression vector pGreenII 0800-P2 has promoter P2 driving LUC gene expression, see Figure 4 .

[0058] 4.3 Using a one-step recombination kit produced by Novozymes, fragment P3 was recombined into pGreenII 0800 after restriction digestion, following the kit instructions to obtain a ligation product. 5 μL of the ligation product was transformed into Escherichia coli DH5a and cultured on solid LB medium containing kanamycin (Kana, concentration: 50 pg / L) for 12 h. Single colonies were selected and cultured in liquid LB medium containing Kana (concentration: 50 pg / L). After positive clones were identified by PCR, plasmids were extracted and double-enzyme digestion was performed. Successful plasmids were sent to Sangon Biotechnology for sequencing. Once sequencing was correct, the constructed expression vector pGreenII 0800-P3 was obtained. The structure of the expression vector pGreenII 0800-P3 is described as follows: the small fragment between the restriction endonuclease SalI and BamHI recognition sequences of pGreenII0800 is replaced with SEQ ID NO. 4, while the other sequences of the vector pGreenII 0800 remain unchanged. The resulting expression vector pGreenII 0800-P3 has promoter P3 driving LUC gene expression, see Figure 4 .

[0059] 4.4 Using a one-step recombination kit produced by Novozymes, fragment P4 was recombined into pGreenII0800 after restriction digestion, following the kit instructions to obtain a ligation product. 5 μL of the ligation product was transformed into Escherichia coli DH5a and cultured on solid LB medium containing kanamycin (Kana, concentration: 50 pg / L) for 12 h. Single colonies were selected and cultured in liquid LB medium containing Kana (concentration: 50 pg / L). After positive clones were identified by PCR in the culture medium, plasmids were extracted and double-enzyme digestion was performed. Successful plasmids were sent to Sangon Biotechnology for sequencing. Once sequencing was correct, the expression vector pGreenII 0800-P4 was successfully constructed. The structure of the expression vector pGreenII0800-P4 is described as follows: the small fragment between the restriction endonuclease SalI and BamHI recognition sequences of pGreenII0800 is replaced with SEQ ID NO. 5, while the other sequences of the vector pGreenII 0800 remain unchanged. The resulting expression vector pGreenII 0800-P4 has promoter P4 driving LUC gene expression, see Figure 4 .

[0060] 4.5 Using a one-step recombination kit produced by Novozymes, fragment P5 was recombined into pGreenII0800 after restriction digestion, following the kit instructions to obtain a ligation product. 5 μL of the ligation product was transferred into E. coli DH5a and cultured on solid LB medium containing kanamycin (Kana, concentration: 50 pg / L) for 12 h. Single colonies were selected and cultured in liquid LB medium containing Kana (concentration: 50 pg / L). After positive clones were identified by PCR in the culture medium, plasmids were extracted and double-enzyme digestion was performed. Successful plasmids were sent to Sangon Biotechnology for sequencing. Once sequencing was correct, the expression vector pGreenII 0800-P5 was successfully constructed. The structure of the expression vector pGreenII 0800-P5 is described as follows: the small fragment between the restriction endonuclease SalI and BamHI recognition sequences of pGreenII0800 is replaced with SEQ ID NO. 6, while the other sequences of the vector pGreenII 0800 remain unchanged. The resulting expression vector pGreenII 0800-P5 has promoter P5 driving LUC gene expression, see Figure 4 .

[0061] 5. Pennisetum PgGNM2 promoter drives the expression of LUC gene in recombinant plasmid

[0062] The plant recombinant expression vector pGreenII 0800-P1 containing the LUC gene constructed in 5.1 was transformed into Agrobacterium GV3101 (pSoup-p19). The transformed Agrobacterium was screened and cultured (50 μg / L Kana + 50 μg / L Rif), and colony PCR was performed to identify the positive single clone GV3101 / pGreenI10800-P1.

[0063] 5.2 The constructed plant recombinant expression vector pGreenII 0800-P2 containing the LUC gene was transformed into Agrobacterium GV3101 (pSoup-p19). The transformed Agrobacterium was screened and cultured (50 μg / L Kana + 50 μg / LRif), and colony PCR was performed to identify the positive single clone GV3101 / pGreenI10800-P2.

[0064] 5.3 The constructed plant recombinant expression vector pGreenII 0800-P3 containing the LUC gene was transformed into Agrobacterium GV3101 (pSoup-p19). The transformed Agrobacterium was screened and cultured (50 μg / L Kana + 50 μg / LRif), and colony PCR was performed to identify the positive single clone GV3101 / pGreenI10800-P3.

[0065] 5.4 The constructed plant recombinant expression vector pGreenII 0800-P4 containing the LUC gene was transformed into Agrobacterium GV3101 (pSoup-p19). The transformed Agrobacterium was screened and cultured (50 μg / L Kana + 50 μg / L. Rif), and colony PCR was performed to obtain a positive single clone GV3101 / pGreenII 0800-P4.

[0066] 5.5 The constructed plant recombinant expression vector pGreenII 0800-P5 containing the LUC gene was transformed into Agrobacterium GV3101 (pSoup-p19). The transformed Agrobacterium was screened and cultured (50 μg / L Kana + 50 μg / L Rif), and colony PCR was performed to obtain a positive single clone GV3101 / pGreenII 0800-P5.

[0067] The newly activated Agrobacterium monoclones GV3101 / pGreenII 0800-P1, GV3101 / pGreenII 0800-P2, GV3101 / pGreenII 0800-P3, GV3101 / pGreenII 0800-P4 and GV3101 / pGreenII 0800-P5 were inoculated into LB containing antibiotics (50ug / L Kana + 50μg / L Rif), respectively, and the pGreenII 0800 vector was used as a negative control. Incubate at 28°C, 200 rpm overnight: When the OD value of the bacterial solution is between 0.6 and 1.0, collect the Agrobacterium by centrifugation at 4000 rpm for 5 minutes; gently resuspend the Agrobacterium in 2 ml of infection solution and let it stand at room temperature for 1-4 hours; infect 6-8 week-old Nicotiana benthamiana leaves; after 72 hours, grind the infected Nicotiana benthamiana leaves and perform luciferase activity detection. The fluorescence signal data were statistically analyzed. The results are shown in the figure. Figure 3 , it can be seen that when P4 is used as a promoter, the fluorescence signal is the strongest, while the fluorescence signal of P2 is the weakest. The results show that both P3 and P4 can drive LUC gene expression, and P4 has the best effect.

[0068] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for screening gene promoter active fragments, characterized in that: The method comprises segmenting the target gene promoter sequence, setting overlapping segments between two adjacent segments, and obtaining gene promoter active segments containing overlapping segments in the segments.

2. The screening method according to claim 1, wherein The steps also include: cloning the segments into a vector containing a reporter gene, introducing the segments into a host cell to detect activity, and screening to obtain the gene promoter active fragments.

3. The screening method according to claim 2, characterized in that The reporter gene includes LUC.

4. The screening method according to claim 2, characterized in that The vectors include pGreenII 0800.

5. The screening method according to claim 2, wherein The host cells include tobacco leaves transformed by Agrobacterium.

6. A highly active fragment obtained by screening according to any one of claims 1 to 5, characterized in that: The nucleotide sequence of the high-activity fragment is shown in SEQ ID NO.

5.

7. Use of the highly active fragment according to claim 6 in driving the expression of a target gene.

8. The application according to claim 7, characterized in that: The application includes constructing a CRISPR / Cas9 system vector.

9. A low-activity fragment obtained by screening according to any one of claims 1 to 5, characterized in that: The low-activity fragment includes any one or more of the nucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.

6.

10. Use of the low-activity fragment according to claim 9 in constructing a yeast one-hybrid bait vector.

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