CN114350666B - Isolation and application of promoter Pssi of stem, stem tip and small ear Jiang Biaoda - Google Patents

Abstract

The invention provides isolation and application of a stem, a stem tip and a small ear Jiang Biaoda promoter Pssi. According to the invention, a stem tip and a small ear Jiang Biaoda promoter Pssi are isolated and cloned, the promoter activity is identified through expression analysis and tissue staining, and the Pssi is further utilized to control the expression of Cas9 nuclease, so that a CRISPR/Cas9 mediated homology directed repair screening-free marker system is developed. The invention shows that in transgenic plants, the Pssi promoter can drive the Cas9 nuclease to be highly expressed in stems, stem tips and spikelets, and to be low-expressed or not-expressed in organs such as callus, roots, leaves and the like; the system can efficiently remove the screening marker gene in monocotyledonous plants, and can remove the screening marker gene in T ₁ A large number of plants with homozygous deletion of the marker gene can be obtained in the generation, the efficiency of complete deletion of the screening marker is 55.6%, and a large number of plants without the screening marker gene can be obtained in the generation of the transgenic plant.

Description

Isolation and application of promoter Pssi of stem, stem tip and small ear Jiang Biaoda

Technical Field

The invention belongs to the technical field of biotechnology and plant genetic engineering. More particularly, it relates to the isolation of a stem, stem tip and spike Jiang Biaoda promoter Pssi and uses thereof.

Background

Meristem refers to an undifferentiated cell mass in a plant that produces new tissue or organs, wherein all above-ground tissue of the plant is produced by shoot tip meristem (shoot apical meristem, SAM) and is the portion located at about 0.5mm from the shoot tip. The periphery of SAM can produce collateral tissue (including leaves and flowers) that is regulated primarily by different concentrations of auxin distribution (Ali, et al 2020.Molecular and Hormonal Regulation of Leaf Morphogenesis in arabidopsis. International journal of molecular sciences.21, 5132.). Genome editing in shoot apex meristematic cells can be efficiently transferred to reproductive organs such as spikelets in rice by cell division and stably inherited to the next generation.

Exogenous genes are introduced into plants by using plant genetic engineering and transgenic methods, thereby setting a foundation for functional genome research and crop genetic engineering breeding. For efficient screening of transgenic positive plantlets, marker genes encoding antibiotics or herbicide resistance are widely used in genetic transformation of plants. However, after the transgenic positive plants are obtained, the constitutive expression resistance marker genes can cause toxicity to plant growth, and also cause public safety concerns. Therefore, the establishment of a time-saving and efficient plant recombinant expression vector system without the screening mark has very important effect and significance for improving the transgenic safety of plants and promoting the commercialization process of products. The method for obtaining the plant without the screening marker gene mainly comprises the following steps: co-transformation, transposon systems (Ac/Ds), homologous recombination and site-specific recombination systems (Cre/loxP, FLP/FRT and R/RS). Among the most widely used are site-specific recombination systems, which typically require the use of heat shock, chemical induction or tissue-specific promoters to drive expression of the recombinase. Although screening marker-free plants are reported based on the above methods, most of them are only suitable for sexual plants or require time-consuming and laborious breeding screening processes. In addition, the specific recombination site (e.g., 34-bp loxP site) left in the plant genome after recombination remains a marker for transgene (Thakore, et al 2015.Chapter 3-Genome Engineering for Therapeutic applications. Transformation Gene Therapy to the clinical.27-43.).

Homologous-directed repair (HDR) is an important repair method of double-strand breaks (DSBs) in plants, which uses a Homology arm DNA fragment with Homology to the sequence of the break site at both ends as a template for repair. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated nuclease Cas 9) system, which is widely used at present, has been demonstrated to be a powerful gene editing tool that can efficiently induce DSBs at specific genomic sites, thereby activating the HDR pathway of the body. CRISPR/Cas9 mediated HDR has been successfully applied to deletion, knock-in and substitution of genomic fragments in multiple species. Compared to non-homologous end joining (non-homologous end joining, NHEJ), HDR can provide more precise and seamless DNA joining, with the potential to develop a screenless marker system.

Regarding a screening-marker-free system, the prior art discloses a chemical induction deletion expression vector of a transgenic plant without a selection marker, wherein the whole induction deletion system and a selection marker gene expression frame in the vector system are arranged between two loxP/FRT deletion sites, two MAR sequences which are arranged in the same direction are introduced outside the deletion sites, the whole exogenous gene expression frame can be inserted into the expression vector to enhance the stability of exogenous gene expression, and after the optimized estrogen-induced deletion vector is transformed into rice, the exogenous gene can be normally expressed before the selection marker gene is deleted. The research also discloses a specific promoter for flowering of rice and a screening marker-free transformation vector thereof, wherein the recombinant expression vector contains a plant expression vector of a recombinant Cre/loxP system of the promoter, and can control the specific expression of Cre recombinase coding genes in pistils and stamens, and the defects of low efficiency, time and labor waste when the screening markers are deleted by using the plant reproductive tissue specific promoter and the Cre/loxP system in the prior art are overcome, and the method can only be applied to plants which are propagated sexually by using the plant reproductive tissue promoter. At present, no research on rice stem tip and spike promoters is seen. Therefore, the novel and more effective stem tip tissue and small ear strong promoters are searched and separated, and the CRISPR/Cas9 mediated HDR plant screening-free marker expression system is controlled to delete marker genes in stems, stem tips and small ears efficiently, so that the method has practical significance for improving the safety of transgenic plants and saving the time for screening the screening-free marker plants.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the problems, and provides a stem, a stem tip and a small ear Jiang Biaoda promoter Pssi and a CRISPR/Cas 9-mediated HDR-based efficient deletion system comprising the same.

The first object of the present invention is to provide a stem, stem tip and spike Jiang Biaoda promoter Pssi.

A second object of the invention is to provide the use of the stem, stem tip and spike Jiang Biaoda promoter Pssi.

A third object of the present invention is to provide a gene expression cassette which is strongly expressed in stems, stem tips and spikelets.

A fourth object of the present invention is to provide a gene expression vector which is strongly expressed in stems, stem tips and spikelets.

It is a fifth object of the present invention to provide a plant screening marker-free system.

It is a sixth object of the present invention to provide the use of said screenless marker system.

The above object of the present invention is achieved by the following technical scheme:

in order to overcome the defects of low efficiency, time and labor waste when the screening mark deletion is performed by utilizing a plant reproductive tissue specific promoter and a Cre/loxP system in the prior art, a system for performing efficient screening mark deletion based on CRISPR/Cas9 mediated HDR is developed, and a stem, a stem tip and a small ear Jiang Biaoda promoter Pssi are used for driving Cas9 genes to cut targets at two sides of the screening mark, so that HDR is induced and marker gene fragments are deleted.

The present invention first provides a stem, stem tip and spike Jiang Biaoda promoter Pssi, which is a DNA molecule having the following characteristics: the nucleotide sequence of the polypeptide is shown as SEQ ID NO. 1; or a DNA molecule which hybridizes under stringent conditions to the DNA sequence defined in SEQ ID No. 1; or DNA molecules which have more than 90 percent of similarity with the DNA sequence defined by SEQ ID NO. 1 and can regulate and control the strong expression of the target genes in stems, stem tips and spikelets.

Preferably, the stem, tip and spike Jiang Biaoda promoter Pssi is a promoter of rice OsSRABB (loc_os 11g 05290) capable of driving high expression of downstream genes in rice stem, tip and spike, but not or low expression in other tissues and organs such as callus, root, leaf sheath.

The invention provides an application of a gene expression cassette for strongly expressing the stem, the stem tip and the small ear Jiang Biaoda promoter Pssi in the construction of the stem, the stem tip and the small ear, or an expression vector for strongly expressing the stem, the stem tip and the small ear, or a system without screening markers in the construction of plants.

The invention provides a gene expression cassette which is expressed in stems, stem tips and spikelets strongly, and contains a promoter Pssi of the stems, the stem tips and the spikelets Jiang Biaoda.

Preferably, the expression cassette is a Cas9 expression cassette driven by Pssi.

The invention provides a gene expression vector which is expressed in stems, stem tips and spikelets strongly, or a gene expression cassette which contains the gene expression vector and is expressed in stems, stem tips and spikelets strongly.

The invention provides a plant screening-free marking system.

Preferably, the transformation vector of the selectable marker-free system is a recombinant expression vector for CRISPR/Cas 9-mediated homology-directed repair for plant transformation.

Preferably, the T-DNA region of the recombinant expression vector comprises two homologous arm sequences that are homologous in orientation; a Cas9 nuclease expression cassette, a marker gene expression cassette, a guide RNA expression cassette and a corresponding target site sequence are contained between the two homologous arm sequences; the Cas9 nuclease expression cassette employs the stem, stem tip, and spike Jiang Biaoda promoter Pssi of claim 1 to drive Cas9 nuclease-encoding gene expression.

Preferably, the nucleic acid sequences of the two homologous arms are shown in SEQ ID NO. 2-3 in sequence.

Preferably, the CRISPR/Cas9 mediated HDR screening marker-free system is arranged in a T-DNA region and outside a homologous arm sequence, and further comprises a multiple cloning site or an expression cassette of an exogenous target gene, and can be used for genetic transformation of the exogenous gene.

Preferably, the nucleic acid sequence of the multiple cloning site is shown in SEQ ID NO. 6.

The invention also provides application of the screening marker-free system in culturing screening marker-free transgenic plants.

Preferably, the CRISPR/Cas9 mediated HDR screenless marker system can be used for monocots or dicots to develop screenless transgenic plants.

More preferably, the monocotyledonous plant comprises rice, maize, wheat, sorghum, barley, oats, etc., preferably rice.

The invention has the following beneficial effects:

the invention provides a stem, a stem tip and a small ear Jiang Biaoda promoter Pssi separation and identification method and application thereof in a plant screening marker-free system. According to the invention, a stem tip and a small ear promoter Pssi are isolated and cloned, the promoter activity is identified through expression analysis and tissue staining, and the Pssi is further utilized to control the expression of Cas9 nuclease, so that a CRISPR/Cas9 mediated Homology Directed Repair (HDR) screening-free marker system is developed. In transgenic plants, the Pssi promoter can drive the Cas9 nuclease to be highly expressed in stems, stem tips and spikelets, but not expressed or lowly expressed in organs such as calli, roots, leaves and the like, so that transgenic plants can be obtained under the condition of not influencing screening of transgenic resistant calli, cells for deleting marker genes are generated in the stems, the stem tips and the spikelets, the cells are differentiated and developed into reproductive organs, and finally transgenic plants without screening markers are quickly and efficiently screened in transgenic plant offspring. The system can efficiently remove the screening marker gene in monocotyledonous plants, and can remove the screening marker gene in T ₁ A large number (55.6%) of homozygous deleted plants of the marker gene can be obtained in the generation, and a large number of plants without the screening marker gene can be obtained in the offspring of the transgenic plant.

Drawings

FIG. 1 is a schematic diagram showing the expression pattern of OsSRABB (a: the relative expression level of OsSRABB compared with OsUFC1 is shown in a cartoon chart (data derived from Rice eFPBrowser; b, c: the expression level of OsSRABB in various tissues at different developmental stages is expressed by a standardized signal (log 2) and an original signal (Cy 3) in a heat chart (data derived from Rice XPro) and a histogram (data derived from CREP; light color indicates a low transcription level; dark color indicates a high transcription level; NA, cannot be detected);

FIG. 2 shows the expression patterns of endogenous OsSRABB of rice in various tissues of different development periods of rice (China 11) (wherein, the later "V" indicates collection in vegetative growth phase; the later "R" indicates collection in reproductive growth phase; the OsUFC1 gene of rice is used as an internal reference);

FIG. 3 is a schematic representation of predicted hormone-induced expression associated cis-elements in the Pssi promoter (wherein, the transcription associated cis-elements are underlined; the hormone-induced expression associated cis-elements are underlined; the 5' UTR region is underlined);

FIG. 4 is a schematic diagram of the recombinant expression vector pYLPssi of a plant CRISPR/Cas 9-mediated HDR containing the Pssi promoter, wherein the Cas9 structure and selection marker deletion schematic (pYLPssi: cas9 vector T-DNA region contains a GUS expression cassette split into two parts, "GU" and "US", each containing a 1027bp homology arm "U"; a pair of target site sequences TS1 and TS2 are inserted between immediately adjacent homology arms, a HPT expression cassette, two guide RNA expression cassettes carrying target sequences (T1 and T2) and a Cas9 expression cassette driven by Pssi are contained inside the target sequences, and when Pssi drives Cas9 gene expression in the shoot tip and spike, sequences between TS1 and TS2 are cut, the homology arm "U" is induced and deleted, and finally plants completely free of selection markers are obtained in the offspring;

FIG. 5 is a schematic diagram of homology arms of "GU" and "US" fragments (a: "GU" white ground color is "G", gray ground color is "U"; "US" gray ground color is "U", white ground color is "S");

FIG. 6 is a schematic representation of target sequences TS1 and TS2 (wherein, italics is PAM);

FIG. 7 is a schematic representation of a multiple cloning site fragment of the cleavage site (wherein the underlined PstI/Fse I/HindIII/SacI/Asc I/Swa I/Sbf I/Pac I/Mlu I/Pme I sites, respectively);

FIG. 8 shows pYLPssi:: T of Cas9 ₀ Screening marker deletion in transformants (a: T of Cas9 with the GU-F, T35S-R and US-R primer pair pYLPssi: ₀ detecting the deletion condition of the marker gene of the transformant, wherein plants with heterozygous deletion can be amplified into 1094bp of small band (amplified by GU-F and US-R) and 1277bp of large band (amplified by GU-F and T35S-R); plants whose screening markers are not deleted can only be amplified to form a 1277bp large band; CK (CK) ⁺ The vector is pCAMBIA1305 carrying GUS gene; CK (CK) ^- Is a pYLPssi:: cas9 vector; m is marker; b: t for pYLPssi::: cas9 ₀ Deletion statistics of transformants);

FIG. 9 shows pYLPssi:: T of Cas9 ₀ Analysis of the expression Activity of Cas9 and GUS in transformants (a: pYLPssi:: T of Cas9 ₀ qRT-PCR quantitative analysis of Cas9 and GUS in transformants, wherein the latter "V" represents collection in vegetative growth phase; the latter "R" represents collection in reproductive phase; the rice OsUFC1 gene is used as an internal reference; b: PYLPssi:: T of Cas9 ₀ GUS staining analysis in various tissues of transformants at different developmental stages. Wherein wild type middle flower 11 served as a negative control; scale 0.5 cm);

FIG. 10 shows pYLPssi:: T of Cas9 ₁ Screening marker deletion in transformants (a: T of Cas9 with the GU-F, T35S-R and US-R primer pair pYLPssi: ₁ detecting the deletion condition of the marker gene of the transformant, wherein plants with heterozygous deletion can be amplified into 1094bp of small band (amplified by GU-F and US-R) and 1277bp of large band (amplified by GU-F and T35S-R); plants with homozygous deletions (indicated by asterisks) can only be amplified with a band of 1094 bp; plants whose screening markers are not deleted can only be amplified to form a 1277bp large band; CK (CK) ⁺ The vector is pCAMBIA1305 carrying GUS gene; CK (CK) ^- Is a pYLPssi:: cas9 vector; m is marker; b: t for pYLPssi::: cas9 ₁ Deletion statistics of transformants);

FIG. 11 shows pYLPssi:: T of Cas9 ₁ Sequencing of the recombinant products amplified from GU-F and US-R in the transformants.

Detailed Description

The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

The following table shows the primers and sequences used in the examples of the present invention.

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EXAMPLE 1 cloning and analysis of the Rice Stem, stem tip and spike promoter Pssi

In the study of the invention, in order to isolate and clone Rice stems, stem tips and spikelets Jiang Biaoda promoters, a protein coding gene OsSRABB (LOC_Os11g 05290) containing an alpha/beta barrel domain of stress reaction in Rice is found to be highly expressed in Rice stems, stem tip meristems and spikelets by analyzing public databases of Rice eFP Browser (https:// bar.utoronto.ca /), rice XPro (http:// ricxpro.dna.afrc.go.jp /) and CREP (http:// crep.ncpgr.cn /), and is not expressed in calli, and a schematic diagram showing the relative expression level of OsSRABB compared with OsUFC1 is shown in a cartoon diagram; and in the heat map and the histogram, the expression level of OsSRABB in various tissues at different development stages shows that the OsSRABB can be a stem, a stem tip (containing meristem) and a spikelet strong expression gene by using a standardized signal (log 2) and a raw signal (Cy 3).

To further confirm the expression pattern of ossrobb, calli, roots, stems, leaves, leaf sheaths, stem tips, spikelets, glumes, anthers and pistils of rice (chinese 11) plants at different developmental stages were extracted and reverse transcribed into cDNA. The expression of OsSRABB gene (SRABB RT-F and SRABB RT-R primers) was detected by qRT-PCR, and rice OsUFC1 was used as an internal reference gene (UFC 1 RT-F and UFC1 RT-R primers).

qRT-PCR reaction procedure: pre-denaturation at 95 ℃ for 30s; denaturation at 95℃for 5s, annealing at 58℃for 20s, extension at 72℃for 15s, reading at 72℃for 15s, 40 cycles; dissolution profile from 55 ℃ to 90 ℃.

qRT-PCR reaction system: SYBR Green RT-PCR Mix 10. Mu.L, F-and R-terminal primers (10. Mu. Mol/L) each 0.4. Mu.L, cDNA template 0.2. Mu.L, ddH ₂ O was made up to 20. Mu.L.

As a result, as shown in FIG. 2, the OsSRABB gene was highly expressed in the stems, the stem tips and the spikelets, and not expressed or weakly expressed in other tissues. Through sequence analysis, the upstream of the start codon ATG of the OsSRABB gene is found to be the 3' end of another gene, so that a 858bp upstream sequence is selected as a promoter Pssi (stem-, shoot tip-and inflorescence-strong promoter) of the OsSRABB gene, and the sequence of the promoter Pssi is shown as SEQ ID NO. 1. Analysis of the SEQ ID NO:1 sequence by promoter cis-element prediction website PLACE (https:// www.dna.affrc.go.jp/PLACE /), revealed the presence of multiple elements of hormone-induced expression in Pssi, as shown in FIG. 3, which is a schematic diagram of the related cis-elements of the predicted hormone-induced expression in the Pssi promoter.

Example 2 construction of plant CRISPR/Cas 9-mediated HDR recombinant expression vector containing Pssi promoter

In order to establish a time-saving and efficient plant screening marker deletion system, the invention constructs a system carrier pYLPssi for screening marker deletion based on CRISPR/Cas9 mediated HDR, wherein Cas9 is shown in figure 4 and mainly comprises the following 6 elements:

(1) One is composed of P _35S An isolated GUS expression cassette driven by a constitutive promoter. Wherein, GUS gene is divided into two parts, namely, GU whose nucleic acid sequence is shown in SEQ ID NO. 2 (amplified by the primers I-1-FU1, I-1-R1 and I-1-R2) and US whose nucleic acid sequence is shown in SEQ ID NO. 3 (amplified by the primers III-FU3 and III-RU 3); both "GU" and "US" contain a 1027bp DNA homology arm "U", both homology arms being shown in FIG. 5, which can be reconstituted into a complete GUS expression cassette by HDR and induce deletion of fragments between the two homology arm sequences.

(2) The two sets of optimally designed target sequences TS1 and TS2 are shown in FIG. 6, and the nucleic acid sequences are shown in SEQ ID NO. 4 and SEQ ID NO. 5 (which are not present in the plant genome). Immediately inside the "GU" and "US" fragments, amplified by the I-2-F1, I-2-F2 and I-2-R primers (amplifying the target sites immediately adjacent to "GU"), and by the II-FU2, II-RU3-2 and II-RU3-3 primers (amplifying the target sites immediately adjacent to "US"), respectively.

(3) One is composed of P _35S Constitutive promoter-driven HPT resistance expression cassette (vs. pYLCRISPR/Cas9P _ubi The HPT expression cassette sequences in H are identical, reference: ma et al 2015.A Robust CRISPR/Cas9 System forConvenent, high-Efficiency Multiplex Genome Editing in Monocot and Dicot plants. Molecular Plant 8, 1274-1284.) was amplified from I-2-F2 and I-2-R primers together with TS1, TS2 and used as a screen for resistant calli.

(4) Two guide RNA expression cassettes with target sequences of interest (identical to the sequences of the gRNA expression cassettes in pYLgRNA-mOsU3 and pYLgRNA-mOsU6a, ref: ma et al 2015.A Robust CRISPR/Cas9 System for Convenient, high-Efficiency Multiplex Genome Editing in Monocot and Dicot plants. Molecular Plant 8, 1274-1284.). Wherein the nuclear RNA promoter OsU drives expression of T1-sgRNA (amplified from U-F, RU3T1, fgT1 and gRNA-R primers, target sequence corresponding to TS 1); the nuclear RNA promoter OsU a drives expression of T2-sgRNA (amplified from U-F, RU6aT2, fgT2 and gRNA-R primers, target sequence corresponding to TS 2). The assembly method is described in the prior art (Ma, et al 2015.A Robust CRISPR/Cas9 System for Convenient, high-Efficiency Multiplex Genome Editing in Monocot and Dicot plants. Molecular plant.8, 1274-1284.). The assembled OsU 3:T1-sgRNA and OsU a:T2-sgRNA were fusion ligated with the Up-GA-T4, gR-GA-T4, up-GA-T5 and gR-GA-T1 primers.

(5) A Cas9 expression cassette driven by Pssi (vs. pYLCRISPR/Cas9P _ubi Cas9 expression cassette sequence in H is identical, reference: ma et al 2015.A Robust CRISPR/Cas9 System for Convenient, high-Efficiency Multiplex Genome Editing in Monocot and Dicot plants.molecular Plant 8,1274-1284. Wherein the Pssi and Cas9 expression cassettes are amplified from the II-FU2, II-RU3-1, II-RU3-2 and II-RU3-3 primers and ligated by overlapping PCR.

(6) A PstI/Fse I/HindIII/Sac I/Asc I/Swa I/Sbf I/Pac I/Mlu I/Pme I multiple cloning site fragment comprising multiple unique cleavage sites is shown in FIG. 7, the nucleic acid sequence of which is shown in SEQ ID NO. 6. The multiple cloning site fragment was synthesized directly and ligated outside of the "US" fragment.

The above elements are sequentially arranged on the pCAMBIA1300 vector framework, and pYLPssi::: cas9 is obtained after sequencing correctly. When the Cas9 nuclease cleaves the TS1 and TS2 targets, the exposed homology arm "U" will undergo HDR, resulting in deletion of the spacer fragment and reconstitution of a complete GUS reporter gene expression cassette.

Example 3 obtaining and identification of transgenic plants without selectable markers

Cas9 vector constructed in example 2 was transformed into rice (China 11) callus using Agrobacterium-mediated transformation of rice callus. The genetic transformation of rice was carried out by reference to methods in the prior art (Nishimura, et al 2006.A protocol for Agrobacterium-mediated transformation in rice protocols.1, 2796-2802.).

Obtaining transgenic T ₀ After the plants, the genomic DNA was extracted by SDS method as a template and detected by using three primer combinations of GU-F, T S-R and US-R. Wherein, the T35S-R is positioned at the inner side of the T35S terminator of the HPT expression cassette, and amplified with GU-F to obtain a product which does not undergo recombination, and the size is 1277bp; GU-F and US-R are located outside the target sites TS1 and TS2, are about 12.1kb apart when no recombination deletion occurs, cannot be amplified under 1min extension conditions, and can amplify a 1098bp fragment after recombination only when deletion occurs.

Amplification procedure used: pre-deforming for 4min at 94 ℃; denaturation at 94℃for 30s, annealing at 60℃for 30s, extension at 72℃for 1min for 30 cycles; finally, the extension is carried out for 3min at 72 ℃.

PCR system: PCR Mix 10. Mu.L, 0.3. Mu.L each of F-and R-terminal primers (10. Mu. Mol/L), 0.5. Mu.L of genomic DNA template, ddH ₂ O was made up to 20. Mu.L.

As a result, as shown in FIG. 8, in FIG. 8a, the primer pair pYLPssi was used to:: T of Cas9 with GU-F, T S-R and US-R ₀ Detecting the deletion condition of the marker gene of the transformant, and amplifying a 1094bp small band (amplified by GU-F and US-R) and a 1277bp large band (amplified by GU-F and T35S-R) of the heterozygous deleted plant; plants whose screening markers are not deleted can only be amplified to form a 1277bp large band; CK (CK) ⁺ The vector is pCAMBIA1305 carrying GUS gene; CK (CK) ^- Is a pYLPssi:: cas9 vector; m is marker, T ₀ Heterozygous deletion of the selection marker occurred mostly in the plants, with a deletion rate of 73.3% (11/15) in FIG. 8 b.

Selecting the T of Cas9 ₀ Callus, root and stem of transformant at different development stagesThe total RNA was extracted and reverse transcribed into cDNA, leaf sheath, glume, anther and pistil. The expression of Cas9 gene (Cas 9 RT-F/Cas9 RT-R primer) and GUS gene (GUS RT-F/GUS RT-R primer) was detected by qRT-PCR, and rice OsUFC1 was used as an internal reference gene (UFC 1 RT-F/UFC1 RT-R primer), and qRT-PCR detection conditions were the same as in example 1. The method of staining GUS tissue is described in the prior art (Jefferson, et al 1987.GUS fusion: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. Embo j.6, 3901-7.).

The result is shown in FIG. 9 as pYLPssi:: T of Cas9 ₀ In the transformed strain, the Cas9 and the GUS genes of the transformed strain are both expressed in stem tissues in high levels in fig. 9a, but the Cas9 expression level decreases with the advancement of the plant development period, while the GUS expression level increases with the advancement of the plant development period. The above results indicate that the selection markers in the stem, stem tip and spikelet cells are continuously deleted as the plant is differentiated and grown, and the number of intact GUS genes after recombination is continuously increased. In FIG. 9b, GUS tissue staining showed that strong GUS activity was detected in stem tissue during reproductive growth, whereas GUS expression was also observed in glumes and anthers, indicating that some of the stem, shoot tip and spikelet cells had undergone deletion of the selection marker and transferred to the reproductive organ by cell division.

T for pYLPssi::: cas9 ₁ The transformants were further grown, and after the genomic DNA had been extracted by SDS method, the transformants were detected by using a combination of three primers, GU-F, T S-R and US-R. The result is shown in FIG. 10, T in FIG. 10a ₁ A considerable number of plants which had only amplified a 1094bp short recombinant product appeared. Statistical findings, at pYLPssi:: T of Cas9 ₁ In the transformant, the plant with the homozygous deletion of the screening mark (only one 1094bp short band is amplified) accounts for 55.6 percent (25/45); plants with heterozygous deletion of the screening mark (amplified into a 1094bp short band and a 1277bp large band) account for 26.7% (12/45); as shown in FIG. 10b, the total deletion rate of the selectable markers was 82.2% (37/45).

The 1094bp short recombinant product was subjected to agarose gel electrophoresis and recovered, and the deleted interface sequence of the fragment was determined by sequencing analysis (sequencing with GU-F and US-R). As a result, the fragment contained the GUS gene seamlessly joined, as shown in FIG. 11.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Sequence listing

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tctatgatga tgatgatagt tacagaaccg acgaactagt ctgtacccga tcaacaccga 240

gacccgtggc gtcttcgacc tcaatggcgt ctggaacttc aagctggact acgggaaagg 300

actggaagag aagtggtacg aaagcaagct gaccgacact attagtatgg ccgtcccaag 360

cagttacaat gacattggcg tgaccaagga aatccgcaac catatcggat atgtctggta 420

cgaacgtgag ttcacggtgc cggcctatct gaaggatcag cgtatcgtgc tccgcttcgg 480

ctctgcaact cacaaagcaa ttgtctatgt caatggtgag ctggtcgtgg agcacaaggg 540

cggattcctg ccattcgaag cggaaatcaa caactcgctg cgtgatggca tgaatcgcgt 600

caccgtcgcc gtggacaaca tcctcgacga tagcaccctc ccggtggggc tgtacagcga 660

gcgccacgaa gagggcctcg gaaaagtcat tcgtaacaag ccgaacttcg acttcttcaa 720

ctatgcaggc ctgcaccgtc cggtgaaaat ctacacgacc ccgtttacgt acgtcgagga 780

catctcggtt gtgaccgact tcaatggccc aaccgggact gtgacctata cggtggactt 840

tcaaggcaaa gccgagaccg tgaaagtgtc ggtcgtggat gaggaaggca aagtggtcgc 900

aagcaccgag ggcctgagcg gtaacgtgga gattccgaat gtcatcctct gggaaccact 960

gaacacgtat ctctaccaga tcaaagtgga actggtgaac gacggactga ccatcgatgt 1020

ctatgaagag ccgttcggcg tgcggaccgt ggaagtcaac gacggcaagt tcctcatcaa 1080

caacaaaccg ttctacttca agggctttgg caaacatgag gacactccta tcaacggccg 1140

tggctttaac gaagcgagca atgtgatgga tttcaatatc ctcaaatgga tcggcgccaa 1200

cagcttccgg accgcacact atccgtactc tgaagagttg atgcgtcttg cggatcgcga 1260

gggtctggtc gtgatcgacg agactccggc agttggcgtg cacctcaact tcatggccac 1320

cacgggactc ggcgaaggca gcgagcgcgt cagtacctgg gagaagattc ggacgtttga 1380

gcaccatcaa gacgttctcc gtgaactggt gtctcgtgac aagaaccatc caagcgtcgt 1440

gatgtggagc atcgccaacg aggcggcgac tgaggaagag ggcgcgtacg agtacttcaa 1500

gccgttggtg gagctgacca aggaactcga cccacagaag cgtccggtca cgatcgtgct 1560

gtttgtgatg gctaccccgg agacggacaa agtcgccgaa ctgattgacg tcatcgcgct 1620

caa 1623

<210> 3

<211> 1457

<212> DNA

<213> homology arm "US" (SIPOSEQUENCELISTERING 1.0)

<400> 3

gcgtcaccgt cgccgtggac aacatcctcg acgatagcac cctcccggtg gggctgtaca 60

gcgagcgcca cgaagagggc ctcggaaaag tcattcgtaa caagccgaac ttcgacttct 120

tcaactatgc aggcctgcac cgtccggtga aaatctacac gaccccgttt acgtacgtcg 180

aggacatctc ggttgtgacc gacttcaatg gcccaaccgg gactgtgacc tatacggtgg 240

actttcaagg caaagccgag accgtgaaag tgtcggtcgt ggatgaggaa ggcaaagtgg 300

tcgcaagcac cgagggcctg agcggtaacg tggagattcc gaatgtcatc ctctgggaac 360

cactgaacac gtatctctac cagatcaaag tggaactggt gaacgacgga ctgaccatcg 420

atgtctatga agagccgttc ggcgtgcgga ccgtggaagt caacgacggc aagttcctca 480

tcaacaacaa accgttctac ttcaagggct ttggcaaaca tgaggacact cctatcaacg 540

gccgtggctt taacgaagcg agcaatgtga tggatttcaa tatcctcaaa tggatcggcg 600

ccaacagctt ccggaccgca cactatccgt actctgaaga gttgatgcgt cttgcggatc 660

gcgagggtct ggtcgtgatc gacgagactc cggcagttgg cgtgcacctc aacttcatgg 720

ccaccacggg actcggcgaa ggcagcgagc gcgtcagtac ctgggagaag attcggacgt 780

ttgagcacca tcaagacgtt ctccgtgaac tggtgtctcg tgacaagaac catccaagcg 840

tcgtgatgtg gagcatcgcc aacgaggcgg cgactgagga agagggcgcg tacgagtact 900

tcaagccgtt ggtggagctg accaaggaac tcgacccaca gaagcgtccg gtcacgatcg 960

tgctgtttgt gatggctacc ccggagacgg acaaagtcgc cgaactgatt gacgtcatcg 1020

cgctcaatcg ctataacgga tggtacttcg atggcggtga tctcgaagcg gccaaagtcc 1080

atctccgcca ggaatttcac gcgtggaaca agcgttgccc aggaaagccg atcatgatca 1140

ctgagtacgg cgcagacacc gttgcgggct ttcacgacat tgatccagtg atgttcaccg 1200

aggaatatca agtcgagtac taccaggcga accacgtcgt gttcgatgag tttgagaact 1260

tcgtgggtga gcaagcgtgg aacttcgcgg acttcgcgac ctctcagggc gtgatgcgcg 1320

tccaaggaaa caagaagggc gtgttcactc gtgaccgcaa gccgaagctc gccgcgcacg 1380

tctttcgcga gcgctggacc aacattccag atttcggcta caagaacgct agccatcacc 1440

atcaccatca cgtgtga 1457

<210> 4

<211> 23

<212> DNA

<213> TS1( SIPOSequenceListing 1.0)

<400> 4

actgtgactc caatcccagg tgg 23

<210> 5

<211> 23

<212> DNA

<213> TS2( SIPOSequenceListing 1.0)

<400> 5

gtgatagtgc cactcacaag agg 23

<210> 6

<211> 105

<212> DNA

<213> multiple cloning site fragment (SIPOSEQUENCELISTERING 1.0)

<400> 6

aattaactgc agatccaggc cggccataag ctttgagctc taaggcgcgc ctatttaaat 60

acctgcaggt ttaattaaga acgcgttcag tttaaactta aatta 105

Claims (9)

1. Promoter P of stem, stem tip and small spike Jiang BiaodassiCharacterized in that the promoter PssiIs a DNA molecule having the following characteristics: the nucleotide sequence is shown as SEQ ID NO. 1.

2. The stem, stem tip and spike Jiang Biaoda promoter P of claim 1ssiThe application of the gene expression cassette in constructing the stem, the stem tip and the small ear or the expression vector in constructing the stem, the stem tip and the small ear.

3. A gene expression cassette which is strongly expressed in stems, stem tips and spikelets, comprising the promoter P of Jiang Biaoda of the stems, stem tips and spikelets of claim 1ssi。

4. A gene expression vector which is strongly expressed in stems, stem tips and spikelets, characterized by comprising the gene expression cassette according to claim 3.

5. The gene expression vector of claim 4, wherein the gene expression vector is a recombinant expression vector for CRISPR/Cas 9-mediated homology-directed repair for plant transformation.

6. The gene expression vector of claim 5, wherein the recombinant expression vector comprises a T-DNA region comprising two homologous arm sequences in the same orientation; containing two homologous arm sequences between themCas9A nuclease expression cassette, a marker gene expression cassette, a guide RNA expression cassette and a corresponding target site sequence; the saidCas9The nuclease expression cassette employs the stem, stem tip and spike Jiang Biaoda promoter P of claim 1ssiTo driveCas9Nuclease encodes gene expression.

7. The gene expression vector of claim 6, wherein the recombinant expression vector further comprises multiple cloning sites or an expression cassette for an exogenous gene of interest within the T-DNA region of the recombinant expression vector outside the homology arm sequence.

8. The stem, stem tip and spike Jiang Biaoda promoter P of claim 1ssi、Use of the gene expression cassette of claim 3 or the gene expression vector of claim 4 for breeding transgenic plants without selectable markers.

9. The use according to claim 8, wherein said growing transgenic plant without a selectable marker is a monocot.

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