Application of SmOPR3 gene in creation of male sterile line of salvia miltiorrhiza

Abstract

The invention relates to the technical field of gene engineering, in particular to application of a SmOPR3 gene in creation of a male sterile line of salvia miltiorrhiza. The invention provides application of a SmOPR3 gene in creation of a male sterile line of salvia miltiorrhiza. The nucleotide sequence of the SmOPR3 gene is shown as SEQ ID NO: 1. Experimental results show that three regenerated salvia miltiorrhiza bunge are obtained in this conversion, and results of PCR amplification and Sanger sequencing on a SmOPR3 mutation region show that the three salvia miltiorrhiza bunge strains are effectively edited. The subcloning analysis confirms that the three materials successfully realize accurate and synchronous editing of two targets, the editing form of one salvia miltiorrhiza (no.1) realizes allele knockout, and the deletion of SmOPR3 directly leads to maldevelopment of stamens of salvia miltiorrhiza, cannot normally generate pollen and finally leads to the phenotype of male sterility.

Description

Technical Field

[0001] The invention relates to the technical field of gene engineering, and in particular to the application of SmOPR3 gene in the creation of male sterile lines of Salvia miltiorrhiza. Background Art

[0002] Salvia miltiorrhiza is a plant of the Lamiaceae family that is widely used to treat cardiovascular diseases. Its active ingredients, such as tanshinone and salvianolic acid, have significant pharmacological effects. As demand increases, research on the cultivation and breeding of Salvia miltiorrhiza is becoming increasingly important.

[0003] Male sterility is usually caused by nuclear genes, cytoplasmic genes or the interaction between the two, which manifests as abnormal pollen development or loss of function. The creation of male sterile lines of Salvia miltiorrhiza requires a deep understanding of its sterility mechanism. Male sterile lines play a key role in hybrid breeding, which can simplify the hybridization process, improve seed purity and reduce production costs. By creating male sterile lines of Salvia miltiorrhiza, the development of hybrid breeding of Salvia miltiorrhiza can be promoted, and the yield and quality can be improved. Since Salvia miltiorrhiza is a monoecious plant, its breeding system is self-compatible and facultative outcrossing. Therefore, emasculation is required to exclude self-pollination so that it can better serve as the mother parent for hybrid breeding.

[0004] The creation methods of male sterile lines of Salvia miltiorrhiza include: Natural variation screening: Screening sterile plants from natural populations. Mutation breeding: Using physical or chemical mutagens to induce sterile mutations. Genetic engineering: Directed knockout or modification of genes related to pollen development through gene editing technology (such as CRISPR / Cas9). Cell engineering: Using the characteristics of cytoplasmic male sterility (CMS) to introduce sterile cytoplasm through distant hybridization or protoplast fusion.

[0005] There are significant advantages in creating male sterile lines of Salvia miltiorrhiza by targeted knockout or modification of genes related to pollen development through gene editing technology (such as CRISPR / Cas9). Compared with traditional mutagenesis methods, gene editing technology can quickly and efficiently achieve the knockout or modification of target genes, significantly shortening the breeding cycle. Without the introduction of exogenous DNA, the obtained male sterile lines are usually not classified as genetically modified organisms (GMOs) and are more easily accepted by the market. Gene editing technology can create stable male sterile lines in a short period of time. The obtained sterile lines can be directly used for hybrid breeding without the need for additional emasculation steps, significantly improving breeding efficiency.

[0006] Therefore, how to quickly obtain a male sterile line of Salvia miltiorrhiza through gene editing is an urgent problem that technicians in this field need to solve. Summary of the invention

[0007] The purpose of the present invention is to provide application of SmOPR3 gene in establishing male sterile lines of Salvia miltiorrhiza.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides application of SmOPR3 gene in establishing male sterile line of Salvia miltiorrhiza. The nucleotide sequence of the SmOPR3 gene is shown in SEQ ID NO:1.

[0010] SEQ ID NO: 1:

[0011]

[0012] Preferably, the SmOPR3 gene of Salvia miltiorrhiza is knocked out to obtain a male sterile line of Salvia miltiorrhiza.

[0013] The present invention also provides a method for creating a male sterile line of Salvia miltiorrhiza, wherein the SmOPR3 gene of Salvia miltiorrhiza is knocked out to cause mutation, thereby causing the protein encoded by the SmOPR3 gene to lose activity; the nucleotide sequence of the SmOPR3 gene is shown in SEQ ID NO: 1.

[0014] Preferably, the gene knockout method is CRISPR / Cas9.

[0015] The present invention also provides the application of the male sterile line of Salvia miltiorrhiza created by the method in Salvia miltiorrhiza breeding.

[0016] Preferably, the breeding is hybrid breeding.

[0017] Preferably, the male sterile line of Salvia miltiorrhiza created by the method described is used as the female parent for hybrid breeding.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The male sterile type of Salvia miltiorrhiza is considered to be dominant heterozygous sterility (Msms) in natural nuclear male sterility (GMS). Attempts to reveal the mechanism of male sterility in Salvia miltiorrhiza have been made through genome, transcriptome, proteome and other analyses, but no clear major effect gene has been found through forward genetics.

[0020] The present invention obtains three regenerated plants through gene editing and transformation, namely no.1-3. PCR amplification and Sanger sequencing showed that the SmOPR3 mutation region was effectively edited in all three plants. Subclone analysis confirmed that all three materials can accurately knock out all two target sites at the same time. Among them, Smopr1 and SmOPR3 showed biallelic mutations, that is, each target site on the two chromosomes had mutations, but the mutation forms were different. Smopr2 is a biallelic homozygous mutation, with a large fragment deletion of 46 bases between sgRNA1 and sgRNA2. Further analysis found that both forms of mutations led to frameshifting of SmOPR3 and complete loss of function.

[0021] We observed no significant difference between the SmOPR3 mutant and the wild type in the aboveground part, but the red color of the roots was significantly lighter than that of the wild type, and red is the hallmark of tanshinone. During the flowering stage, there were significant differences in the stamens between the mutant and the wild type, similar to the male sterility phenotype of Arabidopsis, with insufficient elongation of the stamen filaments, shrunken pollen sacs of the anthers, and the anthers did not dehiscence, and no pollen grains were observed. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0023] Figure 1 Empty vector for multi-target gene editing.

[0024] Figure 2 :SmOPR3 gene editing vector.

[0025] Figure 3 :The editing form of SmRPS5A-SmOPR3 vector transformed into Salvia miltiorrhiza plants.

[0026] Figure 4 :Phenotypes and expression levels of JAs biosynthesis genes in WT and SmOPR3 plants; A. JAs biosynthesis pathway; B. Stamens of WT and SmOPR3 plants; C. Changes in the expression levels of key enzyme genes for JAs synthesis in leaves and roots of WT and SmOPR3 plants at different time periods. DETAILED DESCRIPTION

[0027] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0028] Example 1

[0029] 1 Culture conditions

[0030] The seeds of Salvia miltiorrhiza Bge. were obtained from Shangluo City, Shaanxi Province. Salvia miltiorrhiza was cultivated in an artificial climate chamber with a temperature of 25°C, a light intensity of 10000 lx, and a 16 h light / 8 h dark period.

[0031] 2 Strains and vectors

[0032] Agrobacterium: GV3101 Chemically Competent Cell (Shanghai Weidi Biotechnology Co., Ltd.).

[0033] 3 Reagents

[0034] HSDNA Polymerase (Code No. R010A, TaKaRa); GeneJET PCR purification kit (Code No. K0702, Thermo Scientific); restriction endonucleases BamHI-HF and Spe I-HF were purchased from NEB, USA; Seamless Cloning and Assembly Kit was purchased from Beijing TransGen Biotech Co., Ltd.; Escherichia coli cloning strain Trans T1 (Code No. CD501) was purchased from Beijing TransGen Biotech Co., Ltd.; plasmid extraction kit PlasmidMini Kit (Code No. D69402, Omega Company).

[0035] 4 Experimental methods

[0036] 4.1 Construction of multi-target gene knockout vector

[0037] The vector construction method refers to ZL 202410212456.8 (a promoter, gene editing vector and application for improving the gene editing efficiency of CRISPR / Cas9 system)

[0038] (1) Use restriction endonucleases PmeI and KpnI to remove the gene editing empty vector (such as Figure 1 Double enzyme digestion was performed to obtain the Cas9-Kan linearized editing vector; Figure 1 Multi-target gene editing empty vector

[0039] (2) Construct the SmOPRsgRNA1-sgRNA2 target site into a linearized vector, such as Figure 2 :SmOPR3 gene editing vector

[0040] (3) The above gene editing vectors were transformed into Escherichia coli T1 and sequenced for identification.

[0041] 4.2 Identification of editing forms of Salvia miltiorrhiza plants

[0042] Screen suitable combinations, infect Salvia miltiorrhiza with Agrobacterium tumefaciens GV3101, regenerate and screen, obtain edited plants, identify positive edits by PCR sequencing, and further evaluate the editing form by monoclonal sequencing (for related methods, refer to PCR sequencing and identification methods, refer to ZL 202410212456.8).

[0043] 4.3 Determination of gene expression

[0044] Using Illumina TruSeq TM RNA Sample Prep Kit was used to construct cDNA library of plant RNA, and sequencing analysis was performed using Illumina HiSeq 4000 System. Referenced splicing and annotation were performed based on the Salvia miltiorrhiza genome. Five knockout strains and five wild strains with consistent growth conditions were selected to extract total RNA from roots and leaves, and reverse transcribed into cDNA. Primer Premier 5 was used to design qPCR primers for key enzyme genes in the JAs and tanshinone biosynthesis pathway (for related methods, refer to the literature "Transcriptome and yeast two-hybrid sequencing shed light on the crosstalk between light and jasmonate signaling inregulating tanshinone biosynthesis").

[0045] The amplification system and reaction procedure refer to the TB Premix Ex Taq TM II (TliRNaseH Plus) instruction manual. Reaction system: 2×SYBR qPCR Mix 5μL, cDNA 1μL, Forward Primer 0.2μL, Reverse Primer 0.2μL, ddH2O 3.6μL. PCR reaction conditions: pre-denaturation 95℃, 30s, then 40 cycles: 95℃ 5s, 60℃ 34s, 3 technical replicates per group, by comparing the relative difference of each sample To analyze the level of gene expression. The internal reference gene is Danshen Actin.

[0046] 5 Experimental results

[0047] 5.1 Obtaining SmOPR3 gene knockout plants

[0048] Three regenerated Salvia miltiorrhizae were obtained in this transformation. The results of PCR amplification and Sanger sequencing of the SmOPR3 mutation region showed that all three Salvia miltiorrhizae achieved effective editing. Subcloning analysis confirmed that the three materials successfully achieved precise and synchronous editing of two targets, and the editing form of one Salvia miltiorrhizae (no.1) achieved allele knockout, see Figure 3 , as the main material for subsequent experiments. Figure 3The edited form of SmRPS5A-SmOPR3 vector transformed into Salvia miltiorrhiza plants.

[0049] 5.2 SmOPR3 deficiency causes male sterility

[0050] During the flowering stage, there were obvious differences in the stamens between the SmOPR3 mutant and the wild type, which were similar to the male sterility phenotype of Arabidopsis thaliana, with both showing insufficient elongation of the filaments of the stamens, shriveled pollen sacs of the anthers, and the anthers not opening, and no pollen grains were observed.

[0051] The results showed that the loss of SmOPR3 directly led to the poor development of stamens in Salvia miltiorrhiza and the inability to produce pollen normally, which ultimately led to the phenotype of male sterility ( Figure 4 B). Figure 4 Phenotypes and expression levels of JAs biosynthesis genes in WT and SmOPR3 plants; A. JAs biosynthesis pathway; B. Stamens of WT and SmOPR3 plants; C. Changes in the expression levels of key enzyme genes for JAs biosynthesis in leaves and roots of WT and SmOPR3 plants at different time periods.

[0052] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of SmOPR3 gene in the creation of male sterile lines of Salvia miltiorrhiza, characterized in that: The nucleotide sequence of the SmOPR3 gene is shown in SEQ ID NO:

1.

2. The use of the SmOPR3 gene according to claim 1 in the creation of a male sterile line of Salvia miltiorrhiza, characterized in that: The SmOPR3 gene of Salvia miltiorrhiza was knocked out to obtain a male sterile line of Salvia miltiorrhiza.

3. A method for creating a male sterile line of Salvia miltiorrhiza, characterized in that: The SmOPR3 gene of Salvia miltiorrhiza is knocked out to cause a mutation, thereby causing the activity of the protein encoded by the SmOPR3 gene to be lost; the nucleotide sequence of the SmOPR3 gene is shown in SEQ ID NO:

1.

4. The method according to claim 3, characterized in that: The gene knockout method is CRISPR / Cas9 editing method.

5. Application of the male sterile line of Salvia miltiorrhiza created by the method according to claim 3 or 4 in Salvia miltiorrhiza breeding.

6. The use according to claim 5, characterized in that: The breeding is hybrid breeding.

7. The use according to claim 6, characterized in that: The male sterile line of Salvia miltiorrhiza created by the method described in claim 3 or 4 is used as the female parent for hybrid breeding.

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