CN116200523B - Identification method and application of plant tissue specific promoter - Google Patents
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Abstract
The invention relates to the technical field of biology, in particular to an identification method and application of a plant tissue specific promoter. Based on the characteristic that cis-regulatory element loci show chromatin opening, the gene promoter is identified efficiently by searching open chromatin DHS in a near-gene region upstream of a gene; and a large number of screening and identification show that when the gene has the tissue specific high expression under certain conditions, the stem tissue specific promoter can be effectively separated; taking sugarcane as an example, the method can be used for efficiently identifying the specific promoter of the sugarcane stem, and the detection rate can reach 60%. The invention can rapidly and accurately identify the tissue specific promoter, is beneficial to the development of excellent new variety cultivation of sugarcane and other crops through excellent gene utilization; meanwhile, the method can be popularized to different crops, and has wide application potential and commercial product development value.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an identification method and application of a plant tissue specific promoter.
Background
In genetic engineering breeding, one transforms an excellent gene into a target crop, improves stress resistance or yield traits of the target species by efficient expression control of the exogenous excellent gene, and cultivates excellent new germplasm and new varieties. In the current genetic engineering breeding, in order to realize high expression of excellent genes, constitutive promoters such as 35S are often adopted. The promoter has wide applicability and can realize the high-efficiency expression of different genes in various tissues of different species. However, the use of such constitutive promoters, while achieving improvement of related traits, often brings about 'side effects', i.e. affecting other agronomic traits, which makes it difficult to achieve true production applications of the transformed new material. For example, transformation of a trehalose synthesis-related gene TPPF which can improve drought resistance of plants can effectively improve drought resistance of plants, but plant development is seriously affected, growth is slow, biomass is obviously reduced compared with wild type, and the plant is difficult to apply in production. This is due mainly to the fact that the superior trait results in specific spatiotemporal expression from one or more genes, whereas non-specific expression of genes inevitably affects other gene expression and ultimately the related trait.
In addition, exogenous promoters often have some disadvantages in genetic transformation applications, such as low transformation efficiency, easy loss or methylation inactivation after long passage time, and difficulty in achieving endogenous tissue space-time specific expression requirements, because the exogenous promoters do not belong to the host itself. Thus, screening for endogenous tissue-specific promoters has become an urgent need and key factor for genetic engineering research and effective use. The conventional tissue specific promoter method at present mainly adopts upstream sequences of different expressed genes to perform cloning transformation verification one by one, and blindness exists, and time and labor are wasted. The tissue specific promoter identification method provided by the invention has the characteristics of accuracy, high efficiency and wide applicability, and therefore, has better application potential.
Disclosure of Invention
The invention aims to solve the difficult problem of identifying an endogenous tissue specific promoter in genetic engineering research and breeding utilization, and provides an identification method of a plant tissue specific promoter and application thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a method for identifying a plant tissue specific promoter, comprising the steps of:
step 1, carrying out genome-wide identification of open chromatin sites (DHS) of different tissues; taking sugarcane stem tissue specific promoter identification as an example, respectively carrying out stem and leaf tissue DHS whole genome identification work, and drawing DHS sites of the whole genome;
step 2, respectively carrying out transcriptome analysis of different tissues, taking sugarcane as an example, obtaining transcriptome data of stems and leaves, determining expressed genes, selecting a 1-5kb interval upstream of the active expressed genes, and screening DHS positioned in the interval;
step 3, analyzing the expression of genes in each tissue, screening the DHS which is specific to the target tissue and has high expression on the upstream of the genes, and taking the DHS as a candidate target tissue specific promoter;
and 4, performing transient transformation function verification on the DHS nucleotide sequence represented by the candidate target tissue specific promoter, and determining the tissue specific promoter function.
Preferably, in order to identify stem specific promoters, as in sugarcane, a gene is selected for stem tissue expression greater than 30FPKM (Fragments Per Kilobase of exon model per Million mapped fragments), which gene should be expressed in leaf tissue at less than 3FPKM, and stem/leaf expression at an FPKM ratio greater than 30. A stem-specific DHS in the region 1-5kb upstream of this type of gene was used as a candidate stem-specific promoter.
The invention also provides application of the tissue specific promoter obtained by the identification method in plants.
Preferably, the plant is sugarcane, 6 stem tissue specific promoters are identified in the sugarcane, and the sequences of the promoters are shown in sequence tables 1-6.
Preferably, 6 stem tissue specific promoters identified in sugarcane are verified by a transient transformation system to specifically perform promoter functions in stem tissue cells.
Compared with the prior art, the invention has the advantages that:
1. the invention adopts open chromatin locus to screen the promoter, and can accurately position non-coding functional areas including the promoter, so that compared with the conventional single gene upstream DNA fragment one by one verification mode, the method is quick and has high accuracy; meanwhile, the whole genome layer analysis is adopted, so that the method can simultaneously identify a large number of tissue specific promoters and has the characteristic of high flux.
2. A large number of analyses show that, in sugarcane, up to 60% of candidate specific promoter sequences screened by adopting the specific expression gene screening parameter provided by the invention show stem specific promoter characteristics, and the parameter condition has better applicability.
3. The invention effectively solves the development problem of endogenous tissue specific promoters in the long-term puzzled gene engineering research and utilization; meanwhile, the method can be popularized to different crops, and has wide application potential and commercial product development value.
Drawings
FIG. 1 is a diagram of genomic level identification open chromatin sites according to the invention;
the graph shows the identification result of the chromosome 3 development chromatin locus of the tropical sugarcane species. A is the result of mapping the high throughput sequencing data, and B is a partial magnified image. The identified DHS is indicated by shading, wherein leaf and stem specific DHS are indicated by filled and open arrows, respectively, with the remainder being DHS common to both tissues.
FIG. 2 is a diagram showing an example of the identification of a stem-specific promoter of the present invention;
the sugarcane stalk gene Soffic.03G0023920-3C is shown to be expressed in a high degree in the stalk specificity, namely FPKM values in the stalk and the leaf are 45 and 0.42 respectively, and a stalk-specific DHS site is identified in 5kb upstream of the stalk gene as a candidate stalk-specific promoter.
FIG. 3 is a graph showing the results of transient transformation verification of a stem specific promoter (DHS#3) of the present invention;
among them, panels A and C are vectors containing a conventional 35S promoter and linked to GFP reporter gene, and a large amount of GFP signal was seen in the protoplast transformation results of the stem (A) and leaf (C), and the cloning vector was used as a positive control; b and D are negative controls, and the vector is a 35S enhanced linked GFP reporter gene, and the absence of a promoter cannot initiate the expression of the reporter gene, so that the expression of the reporter gene cannot be observed in protoplasts of transformed stems (B) or leaves (D). The candidate DHS to be verified (DHS # 3) site was connected between GFP and 35S enhancer, and detection of the reporter gene expressed in protoplasts of stem (E) but not in protoplasts of leaf (F) indicated that DHS #3 to be tested had stem specific promoter function.
Detailed Description
The following technical solutions in the embodiments of the present invention will be clearly and completely described with reference to the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present invention, and thus the protection scope of the present invention is more clearly defined. The described embodiments of the present invention are intended to be only a few, but not all embodiments of the present invention, and all other embodiments that may be made by one of ordinary skill in the art without inventive faculty are intended to be within the scope of the present invention.
Example 1: genome-wide identification of open chromatin site (DHS)
1. Taking 3-5g of fresh sugarcane stem and leaf tissues, grinding into powder in liquid nitrogen, and transferring to a 50ml centrifuge tube;
2. adding precooled cell nucleus extract NIB (10 mM Tris-HCl,80mM KCl,10mM EDTA,1mM spermidine, 1mM arginin, 0.15% mercaptoethanol, 0.5M sucrose,pH9.5) into the centrifuge tube, mixing thoroughly, and filtering with nylon cloth;
3. the filtered liquid was centrifuged at 1200g for 10min at 4℃and the pellet was resuspended in 10ml of nuclear wash buffer NWB (NIB solution added to 0.5% Triton X-100);
4. re-suspending at 4 ℃, centrifuging for 5min at 1200g, re-suspending the pellet with 10ml of Nuclear Wash Buffer (NWB);
5. adding 5ml of a cell nucleus enzymolysis buffer NDB (10 mM Tris-HCl,10mM NaCl,3mM MgCl2,pH 7.4) into the precipitate for resuspension, centrifuging at 4 ℃ for 5min by 1200g, and discarding the supernatant;
6. re-suspending the nuclear pellet with 800-1000 μl of nuclear enzymolysis buffer NDB, and sub-packaging into 6 1.5ml centrifuge tubes, each tube 80 μl;
7. the cell nucleus suspension is placed in a metal bath at 37 ℃, 0.5U DNase I is added, and the cell nucleus suspension is incubated for 10min at 37 ℃;
8. after the incubation is finished, 80 μl of 50mM EDTA is added into each centrifuge tube to terminate the enzymolysis reaction, and the mixture is placed on ice after being inverted and fully mixed;
9. the incubated sample is subjected to conventional phenol chloroform extraction to obtain a pure DNA sample;
10. taking 5 mu l of the DNA sample, and carrying out electrophoresis on 1% agarose gel to detect the enzyme digestion effect;
11. performing fragment screening on the DNA sample meeting the enzyme digestion effect by using AMPure beads to remove large fragments;
12. library construction and high-throughput sequencing are carried out on the recovered DNA samples by using a library construction kit;
13. the identification work of DHSs was performed using Popera (https:// github. Com/forrestzhang/Popera), and stem and leaf tissue whole gene DHS sites were obtained as shown in FIG. 1.
Example 2: tissue specific and highly expressed gene identification
1. Transcriptome sequencing was performed using the same tissue samples identified by DHS in example 1, and the methods of transcriptome sequencing and analysis were conventional.
2. Analysis of Gene expression in various tissues, such as sugarcane, in order to identify stem-specific promoters, stem tissue was screened for expression of greater than 30FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) genes, which should be expressed in leaf tissue at less than 3FPKM and stem/leaf expression at a FPKM ratio greater than 30 (FIG. 2).
3. In combination with DHS data, searches were performed in the 1-5kb region upstream of the above identified stem-specific high-expression genes to identify DHS nearest to the gene and stem-specific, and as a candidate stem-specific promoter (FIG. 2).
Example 3: transient transformation verification of tissue specific promoter protoplasts
1. The construction of a promoter verification vector from each candidate promoter sequence to be verified is disclosed in the literature "Han J, wang P, wang Q, lin Q, chen Z, yu G, miao C, dao Y, wu R, schnatable JC, tang H, wang K (2020) Genome-wide characterization of DNase I-hypersensitive sites and cold response regulatory landscapes in grams. The Plant Cell 32:2457-2473". Promoter verification vectors are available from university of south general.
2. Protoplast preparation was performed using The same tissue as in The DHS assay of example 1, and The protoplast preparation method is disclosed in The literature "Wang Q, yu G, chen Z, han J, hu Y, wang K (2021) Optimization of protoplast isolation, transformation and its application in sugarcane (Saccharum spontaneum L). The Crop Journal 9:133-142", and is not repeated.
3. The candidate vectors were transformed into stem and leaf protoplasts, respectively, and the stem-specific promoters were obtained by screening candidate promoters expressed only in the stem protoplasts by observation (FIG. 3). Among them, panels A and C are vectors containing a conventional 35S promoter and linked to GFP reporter gene, and a large amount of GFP signal was seen in the protoplast transformation results of the stem (A) and leaf (C), and the cloning vector was used as a positive control; b and D are negative controls, and the vector is a 35S enhanced linked GFP reporter gene, and the absence of a promoter cannot initiate the expression of the reporter gene, so that the expression of the reporter gene cannot be observed in protoplasts of transformed stems (B) or leaves (D). The candidate DHS to be verified (DHS # 3) site was connected between GFP and 35S enhancer, and detection of the reporter gene expressed in protoplasts of stem (E) but not in protoplasts of leaf (F) indicated that DHS #3 to be tested had stem specific promoter function.
In conclusion, the invention can rapidly and accurately identify the tissue specific promoter, is beneficial to the development of excellent new variety cultivation of sugarcane and other crops through excellent gene utilization; meanwhile, the method can be popularized to different crops, and has wide application potential and commercial product development value.
The description and practice of the invention disclosed herein will be readily apparent to those skilled in the art, and may be modified and adapted in several ways without departing from the principles of the invention. Accordingly, modifications or improvements may be made without departing from the spirit of the invention and are also to be considered within the scope of the invention.
Claims (1)
1. A sugarcane stalk-specific promoter, characterized in that: the nucleotide sequence of the sugarcane stem specific promoter is shown as SEQ ID NO. 3.
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