Gene editing mediated paddy rice transcription factor target gene screening technology

Abstract

The invention provides a gene editing mediated rice transcription factor target gene screening technology, which mainly constructs a novel transcription factor target gene screening vector through base editing enzymes ABE7.10 and BE3, and obtains a corresponding plant sample material and a protoplast sample material through transgene stable transformation and protoplast instantaneous transformation. The preliminary analysis and screening of downstream target sites of the functional transcription factors are completed by virtue of DNA whole genome sequencing results of the materials. The method is short in time consumption and simple and convenient in steps, can greatly overcome the defects that other transcription factor target gene screening technologies are long in time consumption, tedious in method and the like, can be applied to research on functions of transcription factors and the like in the future, and provides rapid and simple technical support for genetic engineering research in the future.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to a rice transcription factor target gene screening technology mediated by gene editing. Background Art

[0002] Transcription factors are important participants in the growth and development of rice, so the study of transcription factors is an important part of the study of rice nutritional physiology or stress physiology. Transcription factors have one or more binding regions in the genome, which can bind to the adjacent DNA regions of downstream genes to upregulate or downregulate the expression of related genes. A full understanding of the regulatory network in which transcription factors are located and their downstream regulatory regions will help us more clearly understand the mechanism of action of different transcription factors in rice, so as to use the mechanism of action of these transcription factors to obtain high-yield ideal rice plant types.

[0003] With the continuous deepening of the research on the function of transcription factors, many researchers have developed a variety of analytical techniques related to rice transcription factor target genes. Common transcription factor target gene screening techniques include chromatin immunoprecipitation technology, DamID chromatin analysis technology, gel migration or electrophoretic mobility shift assay (EMSA), yeast single hybrid assay, and bioinformatics and other related technologies; most of these technologies are based on the interaction between proteins and DNA and use the second-generation sequencing technology to complete the screening or identification of related transcription factor target genes. However, these existing transcription factor target gene screening technologies have different disadvantages such as complex principles, cumbersome processes, and long experiments, which affect the progress of research on related transcription factor functions.

[0004] Therefore, it is necessary to provide a new gene editing technology-mediated rice transcription factor target gene screening technology to solve the above technical problems. Summary of the invention

[0005] The main purpose of the present invention is to provide a gene editing-mediated rice transcription factor target gene screening technology, aiming to solve the technical problems of the current transcription factor target gene screening technology, which has complex principles, cumbersome processes and long experiments.

[0006] To achieve the above purpose, the present invention provides a gene editing-mediated rice transcription factor target gene screening technology, the technical solution is:

[0007] Step 1: Use two base editing enzymes ABE7.10 and BE3 to construct transcription factor target gene screening systems and obtain corresponding vector plasmids;

[0008] Step 2: Under laboratory conditions, corresponding plant sample materials and protoplast sample materials are obtained by genetic modification technology and protoplast transient transformation technology respectively;

[0009] Step 3: Send the plant sample materials and protoplast sample materials obtained in step 2 to relevant biological companies to extract DNA for DNA resequencing;

[0010] Step 4: Conduct preliminary analysis and screening of downstream target genes of transcription factors based on DNA resequencing results.

[0011] Preferably, in step one, two screening systems are constructed using two base editing enzymes ABE7.10 and BE3 respectively, the pOX rice overexpression vector is selected, and two restriction sites PstⅠ and HindⅢ are selected downstream of its promoter Ubi, and ABE7.10 or BE3 is inserted to obtain pOX-ABE7.10 and pOX-BE3 vector plasmids, and then the rice transcription factor is inserted at the HindⅢ restriction site to obtain the final transcription factor target gene screening vector plasmid.

[0012] Preferably, in the protoplast transformation in step 2, due to the particularity of the screening system, equal amounts of GFP plasmid and the constructed vector plasmid are used to co-transfect protoplasts. If the GFP plasmid is successfully transfected and emits green fluorescence, it is considered that the constructed vector plasmid is also successfully transferred into the protoplasts to a large extent.

[0013] Preferably, in step four, based on the characteristics that adenine base editor ABE7.10 can effectively and specifically convert the target A·T to G·C in a programmable manner and that cytosine base editor BE3 can convert the base pair C·G on the target locus to T·A, the regions where the corresponding base pair changes appear in the resequencing results are extracted, thereby performing a preliminary screening of possible binding regions of transcription factors on the DNA genome.

[0014] The 1,4-D degrading bacteria C. metallidurans ZM02 provided by the present invention is derived from activated sludge of a sewage treatment plant and is obtained through artificial domestication, enrichment and separation. The strain is a Gram-negative bacterium.

[0015] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0016] 1. The present invention adopts two base editing enzymes, and constructs a rice transcription factor target gene screening system according to their action principles and characteristics. The preliminary screening results of the downstream target genes of the transcription factors can be obtained through the steps of protoplast transient transformation, DNA extraction and DNA resequencing, and DNA resequencing result analysis, which is time-saving;

[0017] 2. The present invention can obtain preliminary screening results of downstream target genes of transcription factors only through a series of basic molecular biology techniques and DNA whole genome sequencing, and the method is simple;

[0018] 3. The present invention is mainly applicable to the screening and analysis of downstream target genes of transcription factors in plants such as rice. If this technology is modified, it may be applied to other fields and has great development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the vector construction of the present invention;

[0020] Among them: ①, base editing enzyme; ②, rice transcription factor. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with the accompanying drawings and implementation modes.

[0022] The present invention provides Figure 1 The rice transcription factor target gene screening technology shown in the figure selects the pOX rice overexpression vector, selects two restriction sites PstⅠ and HindⅢ in the downstream of its promoter Ubi, inserts them into ABE7.10 or BE3, and obtains pOX-ABE7.10 and pOX-BE3 vector plasmids. Then, the rice transcription factor is inserted into the HindⅢ restriction site to obtain the final transcription factor target gene screening vector plasmid.

[0023] Step 1: Construction of screening system: Figure 1 Six vector plasmids pOX-ABE7.10, pOX-ABE7.10-SPL9, pOX-ABE7.10-SPL14, pOX-BE3, pOX-BE3-SPL9 and pOX-BE3-SPL14 were constructed using two base editing enzymes ABE7.10 and BE3, respectively.

[0024] Step 2: Under laboratory conditions, corresponding plant sample materials and protoplast sample materials are obtained by genetic modification technology and protoplast transient transformation technology respectively;

[0025] Step 3: Send the materials obtained in step 2 to relevant biological companies to extract DNA for DNA resequencing;

[0026] Step 4: Preliminary analysis and screening of downstream target genes of transcription factors based on DNA resequencing results (these two samples were missing due to the failure of sequencing of pOX-ABE7.10-SPL14 plant samples and protoplast samples):

[0027] 1. Based on the specificity of the two base editing enzymes, the sample mutation types under the action of the editing enzymes were compared to preliminarily verify whether the editors had specific editing effects. As shown in Table 1, the mutation frequency of the sample A·T→G·C under the action of the editing enzyme ABE7.10 was greater than the mutation frequency of C·G→T·A, and the mutation frequency of the sample C·G→T·A under the action of the editing enzyme BE3 was greater than the mutation frequency of the sample A·T→G·C, indicating that the two editing enzymes do work and have a certain gene editing specificity in the whole DNA genome, and the technology is feasible.

[0028] Table 1 is a comparison of base pair mutation frequencies in different samples

[0029]

[0030]

[0031] 2. 19 genes in the plant sample pOX-BE3-SPL14 can be found in the reported target genes of OsSPL14, while 81 genes in the protoplast sample pOX-BE3-SPL14 can be found in the reported target genes of OsSPL14. This result proves that the technology of using the editing enzyme BE3 to screen the downstream target genes of transcription factors is feasible. At the same time, due to the absence of pOX-ABE7.10-SPL14 plant samples and protoplast samples, another method was used to verify the technical feasibility of the editing enzyme ABE7.10 to screen the downstream target genes of transcription factors. The analysis found that 889 of the possible target genes of OsSPL9 analyzed from the plant sample pOX-ABE7.10-SPL9 overlapped with the results analyzed from the plant sample pOX-BE3-SPL9, and 276 of the possible target genes of OsSPL9 analyzed from the protoplast sample pOX-ABE7.10-SPL9 overlapped with the results analyzed from the protoplast sample pOX-BE3-SPL9. This verifies that the results obtained by screening the editing enzyme ABE7.10 have a certain degree of credibility, which proves that the technology is feasible.

[0032] 3. Through the plant sample pOX-BE3-SPL9 and the protoplast sample pOX-BE3-SPL9, we obtained a total of 2714 possible downstream target genes of OsSPL9, and by analyzing the plant sample pOX-ABE7.10-SPL9 and the protoplast sample pOX-ABE7.10-SPL9, we obtained 5400 possible downstream target genes of OsSPL9.

[0033] As shown above, this gene editing technology-mediated rice transcription factor target gene screening technology is considered to be simple, time-saving, and a feasible biotechnology.

[0034] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A gene editing-mediated rice transcription factor target gene screening technology, It is characterized in that The following steps are involved: Step 1: Use two base editing enzymes ABE7.10 and BE3 to construct transcription factor target gene screening systems and obtain corresponding vector plasmids; Step 2: Under laboratory conditions, corresponding plant sample materials and protoplast sample materials are obtained by genetic modification technology and protoplast transient transformation technology respectively; Step 3: sending the plant sample materials and protoplast sample materials obtained in step 2 to relevant biological companies to extract DNA for DNA resequencing; Step 4: Conduct preliminary analysis and screening of downstream target genes of transcription factors based on DNA resequencing results.

2. The rice transcription factor target gene screening technology mediated by gene editing technology according to claim 1, It is characterized in that In step one, two screening systems were constructed using two base editing enzymes ABE7.10 and BE3 respectively. The pOX rice overexpression vector was selected, and two restriction sites PstⅠ and HindⅢ were selected downstream of its promoter Ubi and inserted into ABE7.10 or BE3 to obtain pOX-ABE7.10 and pOX-BE3 vector plasmids. Then, the rice transcription factor was inserted at the HindⅢ restriction site to obtain the final transcription factor target gene screening vector plasmid.

3. The rice transcription factor target gene screening technology mediated by gene editing technology according to claim 1, It is characterized in that In the protoplast transformation in step 2, due to the particularity of the screening system, equal amounts of GFP plasmid and the constructed vector plasmid are used to co-transfect protoplasts. If the GFP plasmid is successfully transfected and emits green fluorescence, it is considered that the vector plasmid we constructed has also been successfully transferred into the protoplasts to a large extent.

4. The rice transcription factor target gene screening technology mediated by gene editing technology according to claim 1, It is characterized in that In step four, based on the characteristics that adenine base editor ABE7.10 can effectively and specifically convert the target A·T to G·C in a programmable manner and that cytosine base editor BE3 can convert the base pair C·G on the target locus to T·A, the regions where the corresponding base pair changes appear in the resequencing results are extracted, thereby performing a preliminary screening of possible binding regions of transcription factors on the DNA genome.

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