Method for identifying exchanged mRNA based on interaction between rootstock and scion of Camellia oleifera

Abstract

The present invention discloses a method for identifying exchange mRNA based on the interaction between oil-tea camellia rootstock and scion, comprising the following steps: S1: third-generation full-length transcriptome sequencing; S2: second-generation transcriptome sequencing; S3: adding suffixes _Co and _Cc to the transcript ID of two third-generation full-length transcription data, merging them into a set as a reference genome, using second-generation transcriptome samples to perform reference comparison with the merged third-generation full-length transcription data; S4: verification of two-way exchange of mRNA between rootstock and scion. The present invention can accurately identify mRNA exchange, analyze key gene pathways, has a multi-level verification mechanism, optimizes breeding strategies, enriches plant grafting theory, and has strong versatility and practicality.

Description

Technical Field

[0001] The invention relates to the technical field of oil-tea camellia planting, and in particular to a method for identifying exchanged mRNA based on interaction between oil-tea camellia rootstock and scion. Background Art

[0002] Grafting, as the main means of propagating improved plant varieties, can not only promote plant renewal, but also improve plant yield and resistance. In recent years, more and more studies have shown that grafting is not only a physical combination process of plant bodies, but also an interaction of genetic material between the scion and the rootstock, especially the long-distance transport of messenger RNA (mRNA). As a molecule encoding proteins, mRNA plays an important role in plant growth and development, metabolic regulation and stress response. During the grafting process, the transfer of mRNA between the rootstock and the scion may play a key role in regulating the growth and development, phenotypic variation and resistance of the grafted plants. Therefore, exploring the transfer mechanism of mRNA between the rootstock and the scion during the grafting process of Camellia oleifera is of great significance for revealing the phenotypic differences caused by grafting and the regulatory mechanisms behind them.

[0003] Existing studies have shown that the grafting process causes the exchange of genetic information between the rootstock and the scion. For example, mRNA can be transferred from the rootstock to the scion through long-distance transport in the phloem, regulating the growth and development of the scion and causing phenotypic variation in the scion. At the same time, plant hormones, secondary metabolites and epigenetic regulation are also involved in the variation process of grafted plants. However, there is currently a lack of systematic research on the specific mechanism of mRNA transfer during grafting, the signal molecules involved, and the impact on plant phenotypes.

[0004] Existing studies have used high-throughput RNA sequencing technology (such as RNA-Seq) to analyze the distribution and expression of mRNA in plants. By sampling and sequencing different tissues of grafted plants (such as phloem and junction), the phenomenon of mRNA transfer between the rootstock and scion can be captured. Secondly, some studies have reported that fluorescent labeling and in situ hybridization technology (FISH) can locate specific mRNA in cells or tissue sections, which can intuitively show the distribution and transfer of mRNA between the rootstock and scion; using specific fluorescent probes, the dynamic transfer process of mRNA can be observed in real time in grafted plants. In addition, by detecting the abundance changes of specific mRNAs through real-time quantitative PCR (qRT-PCR), the expression levels of certain target mRNAs can be accurately quantified, which is suitable for small-scale verification of candidate mRNAs screened by RNA-Seq or other methods.

[0005] However, existing schemes are mostly focused on the study of specific plants, and most of them are observations of mRNA expression levels in stockstocks and scions. These existing schemes can only detect pre-selected specific mRNAs and cannot be used for global mRNA screening; current technologies mostly focus on the expression and distribution of mRNA, but lack a comprehensive explanation of the mechanism of mRNA cross-tissue transfer. Summary of the invention

[0006] The purpose of the present invention is to solve the shortcomings existing in the prior art, and a method for identifying exchange mRNA based on the interaction between oil-tea rootstocks and scions is proposed. On the basis of the existing technology, the mRNA transfer law between oil-tea rootstocks and scions is further studied through grafting experiments, dynamic observations and analysis combined with third-generation full-length transcriptome and second-generation transcriptome sequencing, and the transfer and regulation mechanism of mRNA in the grafting process and its influence on the phenotypic differences of oil-tea are explored, which provides a theoretical basis and technical support for oil-tea grafting breeding; secondly, after a series of experiments such as gene cloning, Agrobacterium transformation, and genetic transformation are carried out on the transferred mRNA, a grafting experiment is carried out again on transgenic tobacco materials and wild-type tobacco, and it is found that the transferred mRNA can also undergo two-way communication, which once again verifies that the technical method is scientific and feasible.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The method for identifying exchanged mRNA based on the interaction between Camellia oleifera rootstock and scion includes the following specific steps:

[0009] S1: Iso-seq analysis: The scion and rootstock samples in the Co / Co combination 90 days after grafting were mixed, and the scion and rootstock samples in the Cc / Cc combination 90 days after grafting were mixed, and then Iso-seq analysis was performed separately. The two groups were combined and used as the reference genome for RNA-seq;

[0010] S2: RNA-seq analysis: Leaf samples and root samples of the four grafting combinations were collected 90 days after grafting, and each sample had three biological replicates, and RNA-seq analysis was performed separately;

[0011] S3: Identification of bidirectional exchange of mRNA between rootstock and scion: The two third-generation full-length transcript data in S1 were merged into one set as the reference genome after adding the suffixes _Co and _Cc after the transcript ID, and the second-generation transcriptome samples in S2 were used for reference comparison with the merged three-generation full-length transcript data;

[0012] S3a: First screening: Screening the gene sets whose CcCc_root sample count is 0 and CoCc_root sample count is not 0, that is, the genes that may be the unidirectional downward communication of Co;

[0013] S3b: Second screening: screening the gene set with the “_Co” suffix, i.e., genes that were not originally present in the Cc root system and were confirmed to be transferred unidirectionally downward from Co;

[0014] S3c: The third screening: exclude the gene sets with count values ​​not 0 and suffix _Co in the scion samples of Cc / Cc (CcCc_apex); then exclude the gene sets with count values ​​0 in the scion samples of CoCo (CoCo_apex) and rootstock samples (CoCo_root);

[0015] S4: Verification of bidirectional exchange of mRNA between stock and scion:

[0016] S4a: A series of experiments will be conducted on the two key genes screened for plant hormone signal transduction, auxin biosynthesis and other pathways, including gene cloning, vector construction, Agrobacterium transformation, and tobacco overexpression genetic transformation;

[0017] S4b: Xiangyan No. 7 was used as the genetic transformation material. After the genetic transformation was completed, the transgenic tobacco was grafted with wild-type tobacco and grafted with each other. Four experimental combinations were set up, namely, wild-type tobacco grafted with the same species, transgenic tobacco as scion grafted with wild-type tobacco, transgenic tobacco as rootstock grafted with wild-type tobacco, and transgenic tobacco grafted with the same species;

[0018] S4c: After the rootstock and scion are healed, RT-PCR is performed on the leaves to detect whether the key gene exchange has been transferred. Specific primers are used to amplify the marker gene to prove whether the transgenic process is successful;

[0019] S4d: qRT-PCR was then performed to determine the expression levels of key genes, and GFP-tagged protein was used as the antibody protein to detect whether the protein transcribed by the target gene linked to GFP was translated, thereby verifying whether the target gene was functional.

[0020] As a further technical solution of the present invention, in S1, Iso-seq is the third-generation full-length transcriptome sequencing.

[0021] As a further technical solution of the present invention, in S2, RNA-seq is the second-generation transcriptome sequencing.

[0022] As a further technical solution of the present invention, in S3, the operations of S3a-S3c are taken as an example of the root sample (CoCc_root) of the grafting combination Co / Cc.

[0023] As a further technical solution of the present invention, in the S4a, the two key genes are the GH3 gene and the UGT gene.

[0024] As a further technical solution of the present invention, in the S4c, the marker gene is such as the GFP gene.

[0025] The beneficial effects of the present invention are:

[0026] 1. Accurate identification of mRNA exchange: Through the comprehensive analysis of the third-generation full-length transcriptome and the second-generation transcriptome, the mRNA exchange between the rootstock and the scion during the grafting process can be accurately identified. This method improves the accuracy of identifying gene transfer and provides important data support for understanding plant interactions.

[0027] 2. Multi-level verification mechanism: Combining transgenic experiments to verify the exchange of mRNA ensures that the identified mRNA transfer is not a coincidence, but a transfer process with biological significance. Through different combinations of grafting experiments, the function and communication effect of the gene can be systematically evaluated.

[0028] 3. Analysis of key gene pathways: Focusing on the analysis of key genes such as plant hormone signal transduction and auxin biosynthesis, the regulatory mechanism of communication between rootstock and scion was revealed, which provided potential molecular targets for subsequent breeding and improvement, and helped to improve the resistance and yield of Camellia oleifera.

[0029] 4. Optimize breeding strategies: By identifying and understanding the mRNA communication and its regulation between the rootstock and scion, we can provide a scientific basis for the grafting breeding of Camellia oleifera, optimize the breeding strategy, and improve the breeding efficiency of excellent varieties.

[0030] 5. Enrich the theory of plant grafting: This method provides a new research perspective for the phenomenon of gene exchange in plant grafting, helps promote the advancement of plant grafting theory, and further expands research in the field of plant breeding.

[0031] 6. Wide application: Although the focus is on Camellia oleifera, the principles and techniques of this method can be extended to the grafting research of other plant species, and it has strong versatility and practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Flow chart of the third-generation full-length and second-generation transcriptome analysis methods for experimental samples;

[0033] Figure 2 Flow chart of the first and second screening for the identification of bidirectional exchange of mRNA between rootstock and scion;

[0034] Figure 3 Flow chart of the third screening for the identification of bidirectional exchange of mRNA between rootstock and scion;

[0035] Figure 4 This is a flow chart of the method for identifying exchanged mRNA based on the interaction between Camellia oleifera rootstock and scion proposed in the present invention. DETAILED DESCRIPTION

[0036] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further explained below in conjunction with specific implementation methods.

[0037] Scion-rootstock interaction refers to the interaction between the rootstock and the scion. This interaction can determine the overall performance of the grafted plant by affecting the growth, development, yield, disease resistance, etc. of the scion. The rootstock absorbs water and nutrients through its roots and passes them to the scion, which also affects the hormone level and metabolic process of the scion, while the scion determines the upper morphological characteristics of the plant and the quality of the fruit. The physiological, genetic and environmental interactions between the rootstock and the scion may produce different grafting effects, which is the key to the rootstock-scion interaction.

[0038] Interspecific grafting: refers to the grafting of plants of different species, grafting the scion of one plant onto the rootstock of another plant; interspecific grafting is usually used to study physiological interactions and genetic exchanges between plants, as well as to improve plant resistance, yield or adaptability.

[0039] Genetic transformation: refers to the process of introducing exogenous genes into the genome of a target organism so that the organism acquires new genetic characteristics; in plant science, genetic transformation is often used to improve plant traits, such as improving disease resistance, stress resistance, yield and quality.

[0040] mRNA communication (or mRNA transfer) refers to the phenomenon of transferring mRNA molecules between different cells or tissues in plants or animals through material transport pathways. In plants, mRNA communication is an important signal transmission mechanism between plant cells. It regulates gene expression between different organs and tissues through the vascular system or apoplast pathway, thereby coordinating the plant's growth and development, nutritional metabolism, and ability to respond to environmental changes.

[0041] Third-generation full-length transcriptome sequencing technology (also known as third-generation sequencing or third-generation single-molecule real-time sequencing technology, mainly including PacBio and Nanopore technologies): is a technology that directly reads the complete mRNA molecular sequence, which can provide a new means for studying the exchange and transfer of mRNA between rootstock and scion during plant grafting; it has the advantages of obtaining full-length transcript information, discovering new transcripts, detecting transcript diversity, studying complex genomic regions and studying non-coding RNA; using it as a reference genome for comparison with second-generation transcriptome data can efficiently and scientifically obtain mRNA information.

[0042] Next-generation transcriptome sequencing (also called high-throughput RNA sequencing or RNA-seq) is a technology widely used in gene expression analysis that can study mRNA in samples through large-scale parallel sequencing; second-generation sequencing technologies include Illumina, BGI and other platforms, which can generate high-precision and high-throughput data.

[0043] Example 1

[0044] Please see attached Figure 1 -Attached Figure 4 The method for identifying exchange mRNA based on the interaction between Camellia oleifera rootstock and scion includes the following specific steps:

[0045] S1: Iso-seq (third-generation full-length transcriptome sequencing) analysis: The scion and rootstock samples in the Co / Co combination 90 days after grafting were mixed, and the scion and rootstock samples in the Cc / Cc combination 90 days after grafting were mixed, and then Iso-seq analysis was performed separately. The two groups were combined as the reference genome for RNA-seq, such as Figure 1 As shown;

[0046] S2: RNA-seq (next-generation transcriptome sequencing) analysis: Leaf samples and root samples of the four grafting combinations were collected 90 days after grafting, and each sample had three biological replicates, and RNA-seq analysis was performed separately, such as Figure 1 As shown;

[0047] S3: Identification of bidirectional exchange of mRNA between rootstock and scion: The two third-generation full-length transcript data in S1 were merged into one set as the reference genome after adding the suffixes _Co and _Cc after the transcript ID, and the second-generation transcriptome samples in S2 were used for reference comparison with the merged three-generation full-length transcript data;

[0048] Take the root sample (CoCc_root) of the grafted combination Co / Cc as an example;

[0049] S3a: First screening: Screening the gene sets whose CcCc_root sample count is 0 and CoCc_root sample count is not 0, that is, the genes that may be the one-way downward communication of Co, such as Figure 2 As shown;

[0050] S3b: Second screening: Screening the gene set with the suffix “_Co”, i.e., genes that were not originally present in the Cc root system and were confirmed to be transferred unidirectionally downward from Co, e.g. Figure 2 As shown;

[0051] S3c: The third screening: exclude the gene sets with non-0 count values ​​and suffix _Co in the scion samples of Cc / Cc (CcCc_apex); then exclude the gene sets with count values ​​of 0 in the scion samples of CoCo (CoCo_apex) and rootstock samples (CoCo_root), such as Figure 3 As shown;

[0052] S4: Verification of bidirectional exchange of mRNA between stock and scion:

[0053] S4a: A series of experiments will be conducted on the two key genes (GH3 gene and UGT gene) involved in plant hormone signal transduction, auxin biosynthesis and other pathways, including gene cloning, vector construction, Agrobacterium transformation, and tobacco overexpression genetic transformation;

[0054] S4b: Xiangyan No. 7 was used as the genetic transformation material. After the genetic transformation was completed, the transgenic tobacco was grafted with wild-type tobacco and grafted with each other. Four experimental combinations were set up, namely, wild-type tobacco grafted with the same species, transgenic tobacco as scion grafted with wild-type tobacco, transgenic tobacco as rootstock grafted with wild-type tobacco, and transgenic tobacco grafted with the same species;

[0055] S4c: After the rootstock and scion are healed, RT-PCR is performed on the leaves to detect whether key gene transfer has occurred. Specific primers are used to amplify the marker gene (such as GFP gene) to prove whether the transgenic process is successful;

[0056] S4d: qRT-PCR was then performed to determine the expression levels of key genes, and GFP-tagged protein was used as the antibody protein to detect whether the protein transcribed by the target gene linked to GFP was translated, thereby verifying whether the target gene was functional.

[0057] The verification results showed that after the transgenic Xiang tobacco and wild-type safflower tobacco were grafted together and grafted together, DNA detection, fluorescence quantification, and protein identification were performed on the samples of wild-type safflower tobacco in the heterogeneous grafting treatment. It was found that gene exchange was detected in the wild-type safflower tobacco samples, that is, the GH3 and UGT genes can be transferred bidirectionally through the grafting junction between the rootstock and the scion. In summary, through the grafting experiment of the transgenic Xiang tobacco with GH3 and UGT genes and the wild-type safflower tobacco, it was found that the GH3 and UGT genes can be transported bidirectionally through the grafting junction, which verified the results of this study.

[0058] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects:

[0059] Method integration: Combined third-generation full-length transcriptome sequencing (Iso-seq) and second-generation transcriptome sequencing (RNA-seq) to comprehensively analyze the mRNA exchange between rootstock and scion during the grafting process of Camellia oleifera.

[0060] Identification of bidirectional exchange of mRNA: Through specific screening steps, the unidirectional and bidirectional exchange of mRNA between the rootstock and scion was successfully identified and verified, and the genes were clarified.

[0061] Experimental verification: Grafting experiments were conducted using transgenic tobacco models to verify the actual existence of mRNA exchange, enhancing the reliability of the research results.

[0062] Example 2

[0063] The method for identifying exchanged mRNA based on the interaction between Camellia oleifera rootstock and scion includes the following specific steps:

[0064] S1: Same as Example 1;

[0065] S2: Same as Example 1;

[0066] S3: Same as in Example 1;

[0067] S4: Verification of bidirectional mRNA communication between rootstock and scion: Use gene editing technology to directly modify specific communication genes (GH3 gene and UGT gene) and observe their functions and communication effects in grafting.

[0068] The advantage of this method is that it can directly verify the function of a specific gene and can better exclude the interference of other variables.

[0069] Example 3

[0070] The method for identifying exchange mRNA based on the interaction between Camellia oleifera rootstock and scion includes the following specific steps: performing genome-wide association analysis (GWAS) on different varieties or individuals to identify genetic variations related to mRNA exchange.

[0071] Advantages of this method: It can find the potential genetic basis and help understand the genetic mechanism of mRNA communication.

[0072] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples. Under the concept of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0073] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for identifying exchanged mRNA based on the interaction between Camellia oleifera rootstock and scion, characterized in that: The specific steps include: S1: Iso-seq analysis: The scion and rootstock samples in the Co / Co combination 90 days after grafting were mixed, and the scion and rootstock samples in the Cc / Cc combination 90 days after grafting were mixed, and then Iso-seq analysis was performed separately. The two groups were combined and used as the reference genome for RNA-seq; S2: RNA-seq analysis: Leaf samples and root samples of the four grafting combinations were collected 90 days after grafting, and each sample had three biological replicates, and RNA-seq analysis was performed separately; S3: Identification of bidirectional exchange of mRNA between rootstock and scion: The two third-generation full-length transcript data in S1 were merged into one set as the reference genome after adding the suffixes _Co and _Cc after the transcript ID, and the second-generation transcriptome samples in S2 were used for reference comparison with the merged three-generation full-length transcript data; S3a: First screening: Screening the gene sets whose CcCc_root sample count is 0 and CoCc_root sample count is not 0, that is, the genes that may be the unidirectional downward communication of Co; S3b: Second screening: screening the gene set with the "_Co" suffix, that is, the genes that were not originally present in the Cc root system and were confirmed to be transferred unidirectionally downward from Co; S3c: The third screening: exclude the gene sets with count values ​​not 0 and suffix _Co in the scion samples of Cc / Cc (CcCc_apex); then exclude the gene sets with count values ​​0 in the scion samples of CoCo (CoCo_apex) and rootstock samples (CoCo_root); S4: Verification of bidirectional exchange of mRNA between stock and scion: S4a: A series of experiments including gene cloning, vector construction, Agrobacterium transformation, and tobacco overexpression genetic transformation were performed on the two key genes screened for involvement in plant hormone signal transduction and auxin biosynthesis pathway. In S4a, the two key genes are GH3 gene and UGT gene; S4b: Xiangyan No. 7 was used as the genetic transformation material. After the genetic transformation was completed, the transgenic tobacco was grafted with wild-type tobacco and grafted with each other. Four experimental combinations were set up, namely, wild-type tobacco grafted with the same species, transgenic tobacco as scion grafted with wild-type tobacco, transgenic tobacco as rootstock grafted with wild-type tobacco, and transgenic tobacco grafted with the same species; S4c: After the healing, RT-PCR is performed on the leaves of the rootstock and scion to detect whether the key gene exchange has been transferred, and specific primers are used to amplify the marker gene to prove whether the genetic modification process is successful. In S4c, the marker gene is GFP gene; S4d: qRT-PCR was then performed to determine the expression levels of key genes, and GFP-tagged protein was used as the antibody protein to detect whether the protein transcribed by the key gene linked to GFP was translated, so as to verify whether the key gene was functional.

2. The method for identifying exchanged mRNA based on the interaction between Camellia oleifera rootstock and scion according to claim 1, characterized in that: In S1, Iso-seq is the third-generation full-length transcriptome sequencing.

3. The method for identifying exchanged mRNA based on the interaction between Camellia oleifera rootstock and scion according to claim 1, characterized in that: In S2, RNA-seq refers to the second-generation transcriptome sequencing.

Images