CN116904635B - A gene and SNP molecular marker closely related to the content of borneol in the Acacia cinnamon plant and its primers and applications - Google Patents

Abstract

The invention belongs to the technical field of molecular biology, and specifically relates to a gene closely related to the borneol content of the Acacia sinensis plant, SNP molecular markers and their primers and applications. The gene number is Cbur03G002680, and its coding sequence is shown in SEQ ID NO.1. The SNP molecular markers are the 14th, 16th and 135th positions in the 7th exon of the gene Cbur03G002680; the nucleic acid sequence of the 7th exon is as shown in SEQ ID NO.2 Shown; the SNP molecular marker is obtained by screening the SNP site on the Cbur03G002680 gene of Cinnamomum cinnamomi. The accuracy of the obtained SNP molecular marker and its primers for detecting the borneol content of the Cinnamomum cinnamomi plant is more than 98%, and the accurate prediction rate is 82.7%, the detection level is high in accuracy and easy to repeat.

Description

A gene and SNP molecular marker closely related to the content of borneol in the Acacia cinnamon plant and its Primers and applications

Technical field

The invention belongs to the technical field of molecular biology, and specifically relates to a gene closely related to the borneol content of the Acacia sinensis plant, SNP molecular markers and their primers and applications.

Background technique

Natural borneol (Natural borneol), also known as natural borneol and plum slices, is a precious and rare medicinal material in southern medicine. It has antibacterial, anti-inflammatory, analgesic, refreshing, improving cardiovascular and cerebrovascular circulation systems, anti-cancer and cancer treatment, etc. This kind of effect is the main component of more than 60 famous and high-quality Chinese patent medicines in my country. According to statistics, the current international demand for dexborneol is 10,000 tons/year, and the domestic demand is 500 tons/year. The raw material forests of dipterocarps in Southeast Asia have been nearly exhausted due to over-exploitation. However, industrial synthetic products contain toxic isoborneol and are of poor quality and efficacy, which affects the quality and safety of Chinese patent medicines and is not conducive to the entry of my country's famous and high-quality Chinese medicine formulas and preparations into the international market. Therefore, there is a very urgent practical need to independently supply natural dexborneol.

Cinnamomum burmannii (Nees&T.Nees) Blume, commonly known as the plum tree, is a tree species of the genus Lauraceae in the Lauraceae family. It is widely distributed in southern my country, including Guangdong, Guangxi, Hunan, Jiangxi, Fujian, Guizhou and Yunnan. land. At the end of the last century, scientific researchers discovered that there is a chemical type of Chinese yinxiang that contains dextroborneol. Among them, the natural d-borneol extracted from yinxiang does not contain toxic camphor, isoborneol and carcinogens safrole and other ingredients, and is of better quality. The products with a purity of 99% and 99.99% are of high quality, priced at 10,000 yuan/kg and 80,000 yuan/kg respectively, which are particularly valuable for development and utilization. Although the occurrence rate of individual borneol-type Acacia cinnamon plants in nature is not low, resources with high borneol content (the relative content of leaf essential oil content is more than 40%) are scarce, and the scarcity of high-quality resources has seriously hindered the development of the Acacia cinnamon essential oil industry.

It is relatively clear that borneol is the key enzyme that catalyzes various reactions in the upstream pathway of monoterpene biosynthesis, mainly through the 2-methylerythritol-4-phosphate (MEP) pathway in the upstream plastid and the methyl erythritol-4-phosphate (MEP) pathway in the cytoplasm. It is synthesized through the MVA pathway, and then further synthesized by various terpenoid synthases (TPS) in the downstream pathway stages. Ma et al. (2021) first discovered a ginseng TPS gene, CbTPS1, in Cinnamomum cinnamon. They used synthetic biology technology to successfully obtain d-borneol in yeast and demonstrated the effectiveness of the synthetic enzyme in vitro. In addition, Hou et al. (2023) used the full-length transcriptome of C. cinnamomi at different leaf development stages and combined with the changing patterns of dextroborneol content to discover the gene Cbur03G002680 related to borneol synthesis and confirmed that it is homologous to CbTPS1.

Contents of the invention

In response to the above problems, the purpose of the present invention is to provide a gene, SNP molecular marker, and primers and applications that are closely related to the borneol content of the Acacia cinnamon plant.

The technical content of the present invention is as follows:

The present invention provides a gene related to the borneol content of the Cinnamomum chinensis plant. The gene number is Cbur03G002680, and its coding sequence is shown in SEQ ID NO.1.

The present invention also provides a SNP molecular marker related to the borneol content of the Cinnamomum cinnamon plant. The SNP molecular marker is the 14th position, the 16th position and the 135th position in the 7th exon of the gene Cbur03G002680. point;

The nucleic acid sequence of the 7th exon is shown in SEQ ID NO.2;

When the genotypes of the 14th site, the 16th site and the 135th site are A/A, T/T and C/C respectively, it indicates that the Cinnamomum cirrhata plant has a higher content of dexborneborneol;

When the genotypes of the 14th site, the 16th site and the 135th site are A/T, T/C and C/T respectively, it indicates that the Cinnamomum cirrhosa plant has a certain content of dextroborneol, which can as candidate materials;

When the genotypes of the 14th site, the 16th site and the 135th site are T/T, C/C and T/T respectively, it indicates that the Cinnamomum chinensis plant has a very small amount of dextroborneol or other Chemical material.

When the genotypes of the 14th site, 16th site and 135th site are respectively

The present invention also provides a primer for detecting the content of borneol in Cinnamomum bursa plant based on the SNP molecular marker, including the 14th position A/T, the 16th position T/C and the 135th position C/T. Forward primer and reverse primer;

The nucleic acid sequence of the forward primer of the 14th position A/T is shown in SEQ ID NO.3, and the nucleic acid sequence of the reverse primer is shown in SEQ ID NO.4;

The nucleic acid sequence of the forward primer of the 16th position T/C and the 135th position C/T is shown in SEQ ID NO.5, and the nucleic acid sequence of the reverse primer is shown in SEQ ID NO.6.

The present invention also provides a kit for detecting the content of borneol in Cinnamomum chinensis plant. The kit includes DNA (SEQ ID NO. 1), the above set of primers and PCR reaction reagents.

The present invention also provides the application of the above-mentioned SNP molecular markers, the primers or the kit in detecting or predicting the borneol content of the A. cadavera plant or in A. cadavera breeding.

The invention also provides a method for detecting the content of borneol in the Cinnamomum cinnamon plant, which includes the following steps:

1) Select Acacia cinnamon variety materials and extract their DNA;

2) Using the DNA obtained in step 1) as a template, use the above primers to perform PCR amplification to obtain a PCR amplification product;

3) After purifying the PCR amplification product obtained in step 2), collect amplified fragments of 69bp (site 14) and 155bp (site 16 and site 135) for testing. When the 14th site is When the genotypes of the site, site 16, and site 135 are A/A, T/T, and C/C respectively, it indicates that the C. cadavera plant has a higher content of dexborneborneol;

When the genotypes of the 14th site, the 16th site and the 135th site are A/T, T/C and C/T respectively, it indicates that the Cinnamomum cirrhosa plant has a certain content of dextroborneol, which can as candidate materials;

When the genotypes of the 14th site, the 16th site and the 135th site are T/T, C/C and T/T respectively, it indicates that the Cinnamomum chinensis plant has a very small amount of dextroborneol or other Chemical material;

Preferably, the reagents used in the PCR amplification in step 2) include 15 μL 2×Taq PCR Master Mix, 1.0 μL genomic DNA, 1 μL forward primer (10 pmol/μL), 1 μL reverse primer (10 pmol/μL), 12 μL ddH _2O ;

Preferably, the reaction program used in the PCR amplification in step 2) is pre-denaturation at 95°C for 5 minutes; 35 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds; final extension at 72°C for 5 minutes; and 1 minute at 16°C. .

The beneficial effects of the present invention are as follows:

The gene Cbur03G002680 of the present invention, which is related to the borneol content of the Cinnamomum chinensis plant, is related to the synthesis of borneol. The SNP molecular marker is obtained by screening the SNP site on the Cbur03G002680 gene of Cinnamomum bursa. The obtained SNP molecular marker and its primers are used for The accuracy rate of detecting the borneol content of the Acacia cinnamon plant is over 98%, and the accurate prediction rate is 82.7%. The detection level is high in accuracy and easy to repeat.

Screen the single nucleotide polymorphism - SNP (single nucleotide polymorphism) site on the Cbur03G002680 gene of Cinnamomum bursa in different provinces in the south, and develop accurate, rapid and high borneol content in combination with the applicant's micro-extraction results of essential oil chemical components. Molecular evaluation technology (CN202011539774.3 A method for extracting organic compounds from plant tissues containing borneol). The detection method of the present invention is suitable for detecting Yinxiang resources in the entire South China region. Therefore, it has broad spectrum and can detect different chemicals. The cost of massive screening and detection of type A. cadavera germplasm is much lower than that of conventional gas chromatography or GC-MS chromatography. The invention is an effective method to realize efficient mining of borneol-type yinxiang resources and obtain high-quality resources of Lauraceae essential oil tree species.

Description of drawings

Figure 1 is a schematic diagram of the chemical composition and distribution of different groups of Acacia incense samples collected from 7 provinces in the south;

Figure 2 is the amplification electrophoresis gel image (target sites Site 2 and Site 3) described in the Example.

Figure 3 EMMAX model diagram related to the relative content of dextroborneol.

Detailed ways

The present invention will be described in further detail below through specific implementation examples and accompanying drawings. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, those skilled in the art will Modifications to various equivalent forms of the present invention are defined by the claims appended to this application.

Unless otherwise specified, all raw materials and reagents of the present invention are those found in the conventional market.

Example 1

Screening and verification of a SNP molecular marker related to the content of borneol in Acacia cinnamon plant

1. Collection of Yin Xiang materials

From 2018 to 2021, surveys of A. cadavera population resources were carried out in Guangxi, Guizhou, Jiangxi, Hunan, Yunnan, Fujian and Guangdong, etc., and a total of 141 A. cadavera germplasm resources were collected. After micro-extraction method and GC-MS detection, the metabolite micro-extraction technology developed by the inventor (patented as CN202011539774.3, a method for extracting organic compounds from plant tissues containing borneol) was used, combined with gas chromatography-mass spectrometry. (GC/MS) system measures and detects the chemotypes and volatile metabolite contents of different A. cinnamomi germplasm resources, with a detection accuracy of 99%.

The chemotype distribution of the 141 Yinxiang samples collected is shown in Figure 1. The Yinxiang populations in Guangdong, Guangxi, Hunan, Fujian and Jiangxi are dominated by borneol type Yinxiang (the relative content of borneol is 14.8% to 49.6% ), while the cinnamaldehyde type is mostly found in Yunnan and Guizhou Yinxiang resources (the relative content of borneol is close to 0%).

2. Screening of SNPs related to the content of borneol in Acacia cinnamon plants

The 141 samples of A. cinnamomi collected were divided into three groups (grouped as needed), with 45 samples in the first screening group, 44 samples in the second verification group, and 52 samples in the third actual group.

The first screening group includes 15 high-borneol content germplasm resources (relative borneol content 30-60%), 15 medium-content resources (20-29%) and 15 low-content or other chemotype germplasm resources (0~19%), the first screening group uses second-generation sequencing technology, the specific method is as follows:

Using ultrasonic fragmentation (or enzyme digestion), the DNA is randomly broken into fragments of about 300 bp. The DNA fragments are end-repaired, A is added to the 3' end, sequencing adapters are added, purified, and PCR amplified to complete the construction of a sequencing library. After passing the quality inspection, the library will be sequenced on the Illumina platform. Library construction and sequencing were performed at Beijing Biomic Biotechnology Co., Ltd. After the sequencing data is off-machine, the raw data needs to be quality-controlled according to certain standards to remove sequences with adapters and a pair of sequences with more than 50% low-quality bases. After removing low-quality sequences and linker sequences, the software Bowtie2 in the Geneious software platform was used to compare the original data with the coding sequence (CDS) of the gene Cbur03G002680 in the assembled reference genome of C. elegans of the present invention, whose nucleic acid sequence is shown in SEQ ID NO.1 ) alignment, the parameters of the consensus sequence are set to highestsensitivity/slow, and the consensus site requires at least 90% of the aligned bases. Use the software MAFFT software (Scorematrix: 200PM/k=2) to obtain the CDS sequences of 45 samples matrix, and then use the FinderVariation/SNP module in the Geneious software platform to find the SNPs of the 45 sample sequences, and set the parameters to the minimum mutation frequency of each site at 30%.

Finally, according to the test results obtained by the above-mentioned essential oil microextraction method, it was found that 3 essential oils related to high, medium and low Acacia sinensis were found in the 7th exon part (its nucleic acid sequence is shown in SEQ ID NO.2) The SNP sites corresponding to borneol content, as shown in Table 1, appear at site 14 (Site 1), site 61 (Site 2) and site 135 (Site 3).

Table 1 Three functional sites closely related to borneol content traits

3. Functional test of SNP of borneol type Yinxiang

The present invention has no restrictions on the method of extracting DNA from the stems and leaves of P. sinensis. The kit method or the CTAB method can be used. This time, the magnetic bead method genomic DNA extraction kit (NanoMagBio) is used to extract the genomic DNA of the material to be bred. This method is used After extracting the DNA of the second group of 44 samples, first use gel electrophoresis to detect it. The detection parameters are as follows: agarose gel concentration 1%, voltage 120v, electrophoresis time: 20min, take 2μL DNA sample, add 2uL 6×Loading Buffer, electrophoresis After running, put the gel block into the gel imaging analyzer for gel imaging. The main band is required to be bright and clear, without drag, and the size of the main band should be around 10kb. Then carry out absorbance detection, take 2μL DNA sample, and use NanoDROP 8000 ultra-micro spectrophotometer to detect the nucleic acid concentration. It is required that the concentration of the DNA sample (ng/μL) ≥ 30ng/μL, and the A260/A280 value remains within the range of 1.8 to 2.0 . After the DNA extraction of 96 samples was qualified, PCR amplification of the sequence shown in SEQ ID NO. 2 was carried out using the DNA of Cinnamomum chinensis as a template, and the PCR amplification was performed with the primers to obtain the PCR amplification product.

The amplification primers used are shown in Table 2:

Table 2 PCR amplification primers

The PCR amplification system used is shown in Table 3:

Table 3 Reagent ratio information and PCR reaction conditions

In order to ensure the specificity of PCR amplification, after the PCR amplification is completed, 2 μL of the PCR product is taken for agarose gel electrophoresis detection (1% concentration), and the specificity of the amplification product of each sample is judged by the banding pattern of the PCR product, as shown in Figure 2 (a. Spotting sequence GXYF02, GXYF03, GXYF04, GXYF05, GXRA05, GXRA03, GXLC04, GX LC03; b. Marker) is shown, corresponding to the results of 8 strips from left to right in Figure 2a.

After the PCR amplification is completed, the obtained PCR amplification products are preferably purified, and amplified fragments of 69 bp and 155 bp in length are respectively collected for sequencing. The sequencing is preferably bidirectional sequencing.

When the genotypes obtained by sequencing are A/A (site 1), T/T (site 2), and C/C (site 3), it means that the selected Yinxiang variety materials have a higher content of dextroborneol. It can be used as an important resource for subsequent clonal breeding;

When the genotypes are A/T (site 1), T/C (site 2), and C/T (site 3), there are resources with a certain borneol content that can be used as candidate materials;

When the genotypes are T/T (site 1), C/C (site 2), or T/T (site 3), the borneol content is very small or is of other chemical types and can be discarded.

The above breeding method will help to quickly detect the germplasm resources of Cinnamomum chinensis with high dextrorotary borneol content, and help the development of the woody essential oil industry in southern China.

For the functional test of SNPs, GWAS analysis was further performed on all 141 selected C. cadavera samples. The EMMAX model was used to obtain a total of 1024 SNPs related to the relative content of dextroborneol. The results are shown in Figure 3. The three points circled in the middle are the three amplification target sites involved in the present invention.

4. Practical testing of high-quality resources of borneol-type Yinxiang

According to the DNA extraction method and PCR amplification method mentioned above, PCR amplification and sequencing were carried out on the third group of samples (52 samples) of Yinxiang collected from seven provinces in South China. At the same time, the above-mentioned essential oil micro-extraction method and GC-MS detection method were used to obtain the relative content in the leaves of Cinnamomum sinensis as a detection method for the molecular marker. The results are shown in the following table:

Table 4 Comparison between the evaluation of national Aminica sinensis resources using molecular markers and GC-MS evaluation

As can be seen from Table 4, among the evaluation results, 43 were completely correct, 8 were partially correct, and 1 was incorrect. It can be seen that through the SNP molecular markers and corresponding evaluation methods of the present invention, the accuracy of the results obtained is more than 98%, and it is accurate. The prediction rate is 82.7%, the detection level is high in accuracy and easy to repeat.

Claims (10)

1. A group of SNP molecular markers related to the borneol content of the Acacia cinnamon plant, characterized in that the SNP molecular markers include the nucleic acid sequence shown in SEQ ID NO. 2, which is the 7th extern of the gene Cbur03G002680 Exon; The genotypes of the 14th position A/T, the 16th position T/C and the 135th position C/T in the SEQ ID NO. 2 sequence are related to the borneol content of the Acacia chinensis plant. 2. The SNP molecular marker related to the borneol content of the Acacia cinnamon plant according to claim 1, characterized in that when the genotypes of the 14th site, the 16th site and the 135th site are A respectively. /A, T/T, C/C, it indicates that the Cinnamomum cinnamon plant has a higher content of dexborneol; When the genotypes of the 14th site, the 16th site and the 135th site are A/T, T/C and C/T respectively, it indicates that the Cinnamomum cirrhosa plant has a certain content of dextroborneol, which can as candidate materials; When the genotypes of the 14th site, the 16th site and the 135th site are T/T, C/C and T/T respectively, it indicates that the Cinnamomum chinensis plant has a very small amount of dextroborneol or other Chemical material. 3. The application of the SNP molecular marker according to claim 1 in predicting the borneol content of the A. cadavera plant or in the breeding of A. cadavera. 4. A primer for predicting the borneol content of A. cinnamomia plant based on the SNP molecular marker of claim 1, which is characterized in that it includes the 14th position A/T of the SEQ ID NO.2 sequence of claim 1, the Forward primer and reverse primer of T/C at position 16 and C/T at position 135; The nucleic acid sequence of the forward primer of the 14th position A/T is shown in SEQ ID NO.3, and the nucleic acid sequence of the reverse primer is shown in SEQ ID NO.4; The nucleic acid sequence of the forward primer of the 16th position T/C and the 135th position C/T is shown in SEQ ID NO.5, and the nucleic acid sequence of the reverse primer is shown in SEQ ID NO.6. 5. A kit for predicting the content of borneol in Cinnamomum chinensis plant, characterized in that the kit includes the primers and PCR reaction reagents of claim 4. 6. Application of the kit according to claim 5 in predicting the content of borneol in the Acacia sinensis plant. 7. Application of the kit according to claim 5 in A. cadavera breeding. 8. A method for predicting the content of borneol in Acacia cinnamon plant, which is characterized by including the following steps: 1) Select Acacia cinnamon variety materials and extract their DNA; 2) Using the DNA obtained in step 1) as a template, perform PCR amplification using the primers described in claim 4 to obtain a PCR amplification product; 3) After purifying the PCR amplification product obtained in step 2), collect the amplified fragments for testing. The amplified fragments are A/T amplification of the 14th position of the SEQ ID NO.2 sequence described in claim 1. The 69bp fragment and the 155bp fragment amplified by T/C at position 16 and C/T at position 135. 9. The method for predicting the borneol content of the Acacia cinnamon plant according to claim 8, characterized in that the reagents used in the PCR amplification in step 2) include 15 μL 2×Taq PCR Master Mix, 1.0 μL genomic DNA, 1 μL of the forward primer according to claim 4 (10 pmol/μL), 1 μL of the reverse primer according to claim 4 (10 pmol/μL), and 12 μL of ddH ₂ O. 10. The method for predicting the borneol content of the Acacia cinnamon plant according to claim 8, characterized in that the reaction program used in the PCR amplification in step 2) is pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds and annealing at 60°C. 30sec, 72℃ extension for 30sec, 35 cycles; final extension at 72℃ for 5min; 16℃ for 1min.