Modunaliella pralinae salt stress response lncRNAs and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to modunaliella pralinae salt stress response lncRNAs and application thereof. In order to realize intraspecific and interspecific rapid and accurate identification of high-salt-tolerance modunaliella pralinae and efficient screening of dunaliella functional gene GGPS cDNA core sequences, the invention provides the salt stress response lncRNAs of the modunaliella pralinae, and the nucleotide sequences are sequentially shown as SEQ ID NO.1-6; the nucleotide sequences of the target sequences of the Modunaliella pralinae salt stress response lncRNAs LNC000866 and LNC000981 are as shown in SEQ ID NO.7 to SEQ ID NO.8, and the nucleotide sequences of the target sequences are as shown in SEQ ID NO.7 to SEQ ID NO.8; the PCR reaction specific primer pair is used for amplifying the lncRNAs and GGPS cDNA core sequences of LNC000866 and LNC000981 target functional genes, and the nucleotide sequences of the PCR reaction specific primer pair are shown as SEQ ID NO.9-20 in sequence.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to Dunaliella primigensis salt stress-responsive lncRNAs and applications thereof. Background Art

[0002] Salt stress is a very important abiotic stress factor, which has a significant impact on the research of growth and development, metabolic regulation, signal transduction, and functional gene mining of plants, especially algae. At present, the systematic research and large-scale application of plant salt stress response at home and abroad are mainly concentrated on higher plants. As a kind of primitive stress-resistant lower plants, algae have relatively few studies on salt stress response and application. Using unique halophilic microalgae as experimental materials, screening and identifying regulatory factors or key genes closely related to salt stress response, and analyzing their unique molecular mechanism of salt stress response is very necessary for the study of salt tolerance mechanism of lower algae and its application in the field of plants.

[0003] Dunaliella primogeniture belongs to the genus Dunaliella in the phylum Chlorophyta. Its cell morphology, physiological and biochemical characteristics are very similar to those of other Dunaliella species, making it difficult to distinguish using traditional algal species identification methods, such as morphological or physiological techniques. Using evolutionarily conserved genes such as rbcL and cox2-3 to construct phylogenetic trees between different algal species is currently a relatively quick method for molecular identification of Dunaliella species. However, this method often has the disadvantage that the nucleotide sequence differences of the conserved genes themselves are small, making it impossible to quickly and accurately distinguish different strains (species) within the same genus. Sometimes, it is necessary to combine new conserved genes or morphological and physiological analysis methods for comprehensive identification. In addition, although Dunaliella primogeniture has a broad spectrum of salt tolerance (generally referring to sodium salts here), the NaCl tolerance varies between different strains (and within the species), which is often related to factors such as its natural growth environment, genetic variation, and human activities. Traditional methods for screening highly salt-tolerant Dunaliella species primarily rely on physiological experiments across varying salt concentration gradients. However, these methods are often limited by long cultivation and monitoring cycles (approximately 20 days), stringent external control conditions (which must be maintained consistently to prevent bacterial contamination), degradation of the original algae, and the potential for errors in physiological assays. More importantly, Dunaliella is an important model algae for salt stress research, and the identification of uniquely salt-tolerant strains within this genus is crucial for the subsequent screening and cloning of functional genes associated with salt tolerance. The development of relevant technologies is urgently needed to accurately identify salt-tolerant Dunaliella strains while rapidly mapping functional gene sequences related to salt stress response and accelerating the acquisition of key functional gene information. Therefore, it is imperative to establish a set of rapid, sensitive, accurate, and inexpensive new technologies and methods for interspecific identification of Dunaliella primordium, intraspecific identification of highly salt-tolerant strains, and rapid acquisition of unique functional gene information, as well as the development of downstream biopharmaceutical products (such as biochemical and molecular biology reaction kits) based on these technologies.

[0004] Currently, research on Dunaliella primogeniture, both domestically and internationally, focuses on optimizing production processes, cloning key genes, systematic evolutionary analysis, and the manufacture of downstream functional products. However, there is limited research on the identification of non-coding RNAs in Dunaliella primogeniture and their use in screening and identifying specialized strains, as well as cloning functional genes.

[0005] LncRNA is a type of non-coding RNA with a length greater than 200 nt, which can be divided into sense lncRNA, antisenselncRNA, intronic lncRNA, and intergenic lncRNA. LncRNA does not have protein coding capabilities, but it plays an important regulatory role in plants and animals. Studies have confirmed that lncRNA is involved in regulating gene expression at the post-transcriptional level in multiple aspects such as transcription, translation, growth and development, and response to biotic or abiotic stresses. In higher plants, lncRNA has been found to regulate the expression of salt-tolerance genes. Therefore, the present invention intends to identify and analyze lncRNAs and key target genes related to salt stress response in Dunaliella primordium. Using specific sequences as targets, it can be used for precise molecular identification of the algae and rapid mining of its characteristic functional genes. The content of the present invention has important reference value for the protection and utilization of this characteristic salt algae resource and the market application of its high-value-added products. Summary of the Invention

[0006] In response to the above problems, the present invention provides Dunaliella primigensis salt stress-responsive lncRNAs and applications thereof.

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

[0008] The present invention provides salt stress-responsive lncRNAs of Dunaliella purinata, including: LNC_000228, LNC_000862, LNC_000732, LNC_000643, LNC_000866, and LNC_000981, wherein LNC_000228 and LNC_000862 are intergenic lncRNAs (intergenic region lncRNAs), LNC_000732 and LNC_000643 are incRNAs lncRNAs (intron lncRNAs), LNC_000866 is antisense lncRNAs (antisense lncRNAs), and LNC_000981 is senselncRNA (sense lncRNA); the nucleotide sequences of the above lncRNAs are shown in SEQ ID NOs. 1 to 6, respectively.

[0009] The present invention also provides target sequences of the target genes GGPS of LNC_000866 and LNC_000981 in the salt stress-responsive lncRNAs of Dunaliella primordium, namely LNC_000866_target and LNC_000981_target, and the nucleotide sequences are shown in SEQ ID NOs. 7 to 8.

[0010] The present invention also provides a PCR reaction specific primer pair for amplifying LNC_000228, LNC_000862, LNC_000732, and LNC_000643 among the above-mentioned lncRNAs, comprising:

[0011] dpr-LNC_000228-F / dpr-LNC_000228-R;

[0012] dpr-LNC_000862-F / dpr-LNC_000862-R;

[0013] These two pairs of specific primers for PCR reaction can be used for intraspecific identification of highly salt-tolerant Primodophyllum strains;

[0014] dpr-LNC_000732-F / dpr-LNC_000732-R;

[0015] dpr-LNC_000643-F / dpr-LNC_000643-R;

[0016] These two pairs of PCR-specific primers can be used to differentiate and identify Dunaliella primordium and other species of Dunaliella.

[0017] The nucleotide sequences of the four pairs of PCR reaction specific primers are shown in SEQ ID NOs. 9 to 16, respectively.

[0018] The present invention also provides a PCR reaction specific primer pair for amplifying the above-mentioned GGPS target sequence, comprising:

[0019] dpr-GGPS_target-F1 / dpr-GGPS_target-R1;

[0020] dpr-GGPS_target-F2 / dpr-GGPS_target-R2;

[0021] These two pairs of PCR-specific primers can be used for molecular screening of the core sequence of the GGPS cDNA of the functional gene of Dunaliella, namely the geranylgeranyl pyrophosphate synthase gene (GGPS, which is one of the key enzymes in the synthesis of carotenoids, a high-value-added product of Dunaliella);

[0022] The nucleotide sequences of these two pairs of PCR reaction specific primers are shown in SEQ ID NOs. 17 to 20, respectively.

[0023] Dunaliella 18S rRNA-F / 18S rRNA-R are upstream and downstream amplification primers of the internal reference gene 18S rRNA used in the fluorescent quantitative PCR reaction of Dunaliella primordium lncRNAs, and their nucleotide sequences are shown in SEQ ID NOs. 21 and 22.

[0024] A method for screening highly salt-tolerant strains of Dunaliella primidii based on salt stress response-related lncRNAs (LNC_000228, LNC_000862) and their primer information, comprising the following steps:

[0025] Step 1, salt stress treatment of different Primolecta algae strains; specifically, 7 Primolecta algae strains collected from different regions were selected (each strain was divided into 3 experimental groups + 1 control group), namely D.primolecta_CN4 (collected from inland salt lake), D.primolecta_CN5 (collected from inland salt lake), D.primolecta_CN9 (collected from salt water wetland), D.primolecta_Q1 (collected from coastal coast), D.primolecta_Q2 (collected from coastal coast), D.primolecta_Q3 (collected from near Sea area), D. primolecta_Dpr-C40 (purchased from the UTEX algae seed bank), were cultured statically in DM culture medium at room temperature of 25°C and a light intensity of 10,000 lux (light-dark ratio of 12:12) until the 20th day (stable growth period); NaCl was added to the experimental groups to a final concentration of 1.0 mol / L (low salt stress group), 2.0 mol / L (medium salt stress group), and 3.0 mol / L (high salt stress group), respectively. In the control group, no additional NaCl was added except for the normal physiological salinity of 0.05 mol / L NaCl in the DM culture medium. Each stress was for 48 h.

[0026] Step 2, extracting and reverse-transcribe total RNA from each group of Dunaliella purinata strains treated in step 1; specifically comprising the following steps:

[0027] Step 2.1: Place 400-500 mg of sterile algae strains from each group into a pre-chilled mortar, add 6-9 mL of liquid nitrogen, grind thoroughly for 15 seconds, and then transfer to a sterile EP tube.

[0028] In step 2.2, add 1.2 mL of Trizol solution to the EP tube, followed by 250 μL of a 24:1 chloroform / isoamyl alcohol mixture. Shake vigorously for 15 seconds, then centrifuge at 12,000 rpm at 4°C for 6 minutes. Transfer the supernatant to another sterile EP tube.

[0029] In step 2.3, extract once with an equal volume of the supernatant with saturated phenol / chloroform / isoamyl alcohol in a volume ratio of 25:24:1 and once with chloroform / isoamyl alcohol in a volume ratio of 24:1;

[0030] In step 2.4, the extracted total RNA precipitate was precipitated with 2.5 volumes of 4°C pre-cooled isopropanol for 30 min, washed 2-3 times with 75% ethanol, and then dried in a sterile environment.

[0031] In step 2.5, dissolve the dried RNA from step 2.4 in 50 μL of DEPC-treated RNase-free ultrapure water.

[0032] In step 2.6, analyze RNA by electrophoresis on 1.5% agarose gel and test RNA quality by UV-visible spectrophotometry. Acceptable RNA, i.e., RNA with clear 28S rRNA and 18S rRNA bands and a 28S:18S ratio > 1 and an OD260 / OD280 ratio of 1.9 to 2.0 by UV-visible spectrophotometry, should be diluted to 30 ng / μL with DEPC-treated ultrapure water.

[0033] Step 2.7: Use a commercially available conventional plant reverse transcription kit to reverse transcribe the RNA of each group of Dunaliella purinata into cDNA. The reverse transcription reaction system is as follows: 2 μL total RNA, 0.5 μL RT primer, All-in-One First-Strand cDNA Synthesis SuperMix for PCR (4 μL) was added with RNase-free water to a volume of 20 μL. Reverse transcription conditions were as follows: 25°C for 5 min; 40°C for 15 min; 85°C for 5 min; and 4°C for 5 min.

[0034] Step 3: Fluorescence quantitative PCR amplification of lncRNAs (LNC_000228, LNC_000862) in response to salt stress in different strains of Dunaliella primordium. The amplification system was as follows: 2 μL of the reverse transcription product from step 2, 0.5 μL of each upstream and downstream primer, 10 μL of 2× Rotor-Gene SYBR Green PCR Master Mix, and 7 μL of ultrapure water. The reverse transcription conditions were: 95°C for 5 min, 95°C for 15 s, 60°C for 30 s, and 72°C for 20 s; 40 cycles, with Dunaliella 18S rRNA as the internal reference, and Realtime melting curve detection.

[0035] The fluorescent quantitative PCR primers include dpr-LNC_000228-F, dpr-LNC_000228-R, dpr-LNC_000862-F, and dpr-LNC_000862-R, whose nucleotide sequences are shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively; the nucleotide sequences of the dpr-18S rRNA-F and dpr-18S rRNA-R primers are shown in SEQ ID NO.21 and SEQ ID NO.22, respectively.

[0036] Step 4, use 2 -△△CT The relative expression levels of lncRNAs (LNC_000228, LNC_000862) were calculated, and the high-salt-tolerant Primodophyllum strains were screened according to the relative expression levels, i.e., log2 (Fold change) value > 3 (q value < 0.05).

[0037] The application of the above-mentioned lncRNAs responsive to salt stress of Dunaliella purinata and their PCR reaction primer pair sequences can be used to develop a PCR reaction kit for rapidly screening high-salt-tolerant Dunaliella purinata strains, including the dpr-LNC_000228, dpr-LNC_000862, dpr-18S rRNA and dpr-lncRNAs primer pairs, a fluorescent quantitative PCR reaction system, a reaction procedure, an operating guide, other general reagents, and a description of the identification standards for high-salt-tolerant strains.

[0038] A molecular identification method for the algae strain based on lncRNAs (LNC_000732, LNC_000643) related to salt stress response of the algae and their primer information, comprising the following steps:

[0039] Step 1: Several algae species of the genus Dunaliella of different strains (including one identified strain of Dunaliella primordium) are routinely cultured in DM medium, and about 150 to 200 mg of algal cell slurry is respectively enriched on the 20th day of culture;

[0040] Step 2: The enriched algal cell slurries were placed in sterile mortars, ground with liquid nitrogen for 15 seconds, and gDNAs of each algal strain were extracted using the CTAB method. The specific steps are as follows:

[0041] (1) Add 600 μL of preheated CTAB extract (about 60°C), mix well, and incubate at 60°C for 50 min;

[0042] (3) Add 600 μL Tris saturated phenol solution, mix thoroughly, and centrifuge at 12,000 rpm for 2 min at room temperature;

[0043] (4) After taking the supernatant, add 600 μL of phenol / chloroform / isoamyl alcohol mixture (volume ratio 25:24:1) respectively, mix well, and centrifuge at 12000 rpm for 2 min at room temperature;

[0044] (5) After collecting the supernatant, add 1 / 10 volume of acetic acid (3 mol / L) and 250 mL of 4°C pre-cooled anhydrous ethanol, and let it stand at -20°C for 40 min;

[0045] (6) Centrifuge at 12000 rpm and 4°C for 6 min, discard the supernatant, add 1 mL of 4°C pre-cooled 70% ethanol to the precipitate, wash 2-3 times, and vacuum for 10 min;

[0046] Step 3: Wash the gDNAs of each algae strain with 75% ethanol 2-3 times, dry them in a sterile environment, and dissolve them in a sterile EP tube containing 50 μL ultrapure water or TE solution.

[0047] Step 4. PCR amplification was performed on the LNC_000732 and LNC_000643 fragments of each algal strain. The PCR amplification system was as follows: 1 μL gDNA, 1 μL upstream and downstream primers, 10 μL 2× Plant Master Mix, 5 μL Buffer P2, and 7 μL ddH2O. The PCR amplification program was as follows: 94°C for 3 min; 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, for 35 cycles; 72°C for 5 min, and stored at 4°C for later use. The PCR amplification primers were dpr-LNC_000732-F, dpr-LNC_000732-R, and dpr-LNC_000643-F, and dpr-LNC_000643-R, and their nucleotide sequences were shown in SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, and SEQ ID NO. 16, respectively.

[0048] Step 5: The amplified products were sent to a biological company for sequencing. The sequencing results were analyzed using bioinformatics software such as MEGA2.0. The step size was set to 1000 and the calculation was repeated 100 times to construct the maximum likelihood method (ML) phylogenetic tree of the test algal strains LNC_000732 and LNC_000643 respectively.

[0049] Step 6: Through the ML phylogenetic tree analysis of LNC_000732 and LNC_000643, the algae species with the closest phylogenetic relationship to the identified Primodulinae (i.e., the evolutionary distance between the two algae strains is between 0.99 and 1.00, and the probability value is between 98% and 100%) is identified as the Primodulinae species.

[0050] A method for rapidly identifying the GGPS cDNA core sequence of the Dunaliella geranylgeranyl pyrophosphate synthase gene based on information on salt stress response-related lncRNAs (LNC_000866, LNC_000981) and their targets in Dunaliella primordium comprises the following steps:

[0051] Step 1: The identified Dunaliella primidiocarpa species were cultured in a conventional DM medium. On the 20th day of culture, NaCl was added to a final concentration of 3.0 mol / L and subjected to salt stress for 48 h.

[0052] Step 2: After 48 hours of salt stress, the algal cells were enriched in a pre-cooled mortar and rapidly ground with liquid nitrogen for 15 seconds. Total RNA was extracted from the algal strain according to the above-mentioned total RNA extraction steps.

[0053] Step 3, referring to the above reverse transcription step, reverse transcribe the total RNA in step 2 into cDNA;

[0054] Step 4: Based on the possible cis or trans interaction mechanism between lncRNA and mRNA confirmed by research, bioinformatics prediction of mRNA targeted by dpr-lncRNAs was performed.

[0055] (1) For cis-action, the present invention predicts target mRNAs based on the following principles: the dpr-lncRNA is located about 50 kb upstream and downstream of the mRNA, and the expression correlation between the two is not less than 0.9. The target sequence with a P value of <0.01 is determined to be the target mRNA of the lncRNA. (2) For trans-action, the present invention predicts target mRNAs based on the following principles: the target mRNA of the lncRNA is predicted based on the expression levels of the lncRNA and the protein-coding gene, using the Pearson correlation coefficient method and the cor function to calculate the correlation between the two. The higher the correlation coefficient, the clearer the targeting relationship between the two. The target mRNA with a correlation coefficient close to 1.00 is determined to be the target mRNA of the lncRNA.

[0056] According to the above method, the six dpr-lncRNAs identified in the present invention predicted a total of 10 target mRNAs, including 8 cis-acting target mRNAs and 2 trans-acting target mRNAs. Furthermore, the present invention found that only two of the cis-acting target mRNAs had clear functional annotations in bioinformatics databases such as NCBI and EMBL, and both were Geranylgeranylpyrophosphate synthase, corresponding to LNC_000866 and LNC_000981, respectively. The target mRNAs of the remaining dpr-lncRNAs had no clear functional annotation information.

[0057] Step 5: BLASTn sequence alignment of the target mRNAs of LNC_000866 and LNC_000981 was performed. After deleting the differential sequences, the shared target sequence (sequence similarity of 99%) was extracted. Using this as a template, specific nested PCR primer pairs were designed, namely, dpr-GGPS_target-F1 / dpr-GGPS_target-R1 (first round amplification) and dpr-GGPS_target-F2 / dpr-GGPS_target-R2 (second round amplification). The nucleotide sequences are shown in SEQ ID NOs. 17, 18, 19, and 20.

[0058] Step 6: Using the cDNA product from step 3 as a template, perform the first round of PCR amplification. The amplification system is as follows: 2 μL of cDNA, 1 μL each of dpr-GGPS_target-F1 and dpr-GGPS_target-R1, 2× PCR mix (containing dNTPs, Mg 2+ , Buffer (6 μL), Taq enzyme (3U) 0.3 μL, and ddH2O to make up the total volume to 20 μL; the first-round PCR amplification program was: 95°C for 5 min; 95°C for 45 s, 55°C for 40 s, 72°C for 45 s, 35 cycles; 72°C for 5 min; PCR products were electrophoresed on a 1.2% agarose gel; lanes with diffuse bands were considered positive for the first-round PCR and could proceed to the second-round PCR amplification;

[0059] Step 7: The second round of PCR amplification was performed using the PCR product from step 6 as a template. The amplification system was as follows: 1.8 μL of positive PCR product, 1 μL each of dpr-GGPS_target-F2 / dpr-GGPS_target-R2, 2× PCR mix (containing dNTPs, Mg 2+ Buffer) 5 μL, Taq enzyme (3U) 0.2 μL, ddH2O to make up the total volume to 20 μL; the second round of PCR amplification program was: 95°C for 4 min; 95°C for 30 s, 55°C for 40 s, 72°C for 50 s, 37 cycles; 72°C for 5 min;

[0060] Step 8: The PCR product from step 7 was electrophoresed on a 1.2% agarose gel. The positive bands between 750 and 1000 bp were cut, dissolved, purified, and sent to a biotechnology company for sequencing.

[0061] In step 9, the sequencing results were compared with the known GGPS cDNA sequence of Dunaliella salina (gene ID: MN172172) by BLASTn. The matching rate between the sequences was higher than 99%, and the second-round PCR amplified sequence fragment was identified as the core fragment of the Dunaliella GGPS gene cDNA.

[0062] Compared with the prior art, the present invention has the following advantages:

[0063] 1. The present invention discovers and provides new lncRNAs (LNC_000228, LNC_000862, LNC_000732, LNC_000643, LNC_000866, and LNC_000981) that are significantly associated with the salt tolerance of Dunaliella primogeniture. Experiments have shown that LNC_000228 and LNC_000862, whose expression is significantly upregulated, and their amplification primer information can be used for molecular identification of highly salt-tolerant strains within the Dunaliella primogeniture species; LNC_000732 and LNC_000643 and their amplification primer information can be used for interspecific identification of Dunaliella primogeniture within the genus Dunaliella; and the target sequences of LNC_000866 and LNC_000981 and their primer information can be used for amplification of the cDNA core fragment of the GGPS gene in the genus Dunaliella. The present invention has important guiding significance for in-depth research on the salt stress regulation mechanism of Dunaliella primordium and molecular breeding of characteristic salt-tolerant Dunaliella strains.

[0064] 2. The detection method of the present invention can complete the molecular identification of the test Dunaliella strain within 4 to 5 hours using fewer primer combinations. Compared with traditional identification methods for highly salt-tolerant strains and algae species, it has the advantages of accuracy, efficiency, sensitivity, low cost and convenient operation.

[0065] 3. The present invention also designs specific primers based on the target sequences (LNC_000866_target, LNC_000981_target) of lncRNAs (LNC_000866, LNC_000981) related to salt stress in Dunaliella primordium. Through experiments, it was found that the specific primer pairs based on their target sequences can be used to rapidly amplify the cDNA core sequence of the functional gene GGPS in Dunaliella. This discovery can greatly improve the molecular cloning efficiency of the GGPS gene in Dunaliella genus, laying the foundation for the full-length amplification and market application of this functional gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 This is the macroscopic morphology of Dunaliella purinosa on the 20th day of indoor cultivation (CO: control group, LS: low salt stress group, MS: medium salt stress group, HS: high salt stress group);

[0067] Figure 2 This is the single-cell microscopic morphology of Dunaliella purinosa cultured indoors on the 20th day (magnification 20× under an optical microscope, CO: control group, LS: low-salt stress group, MS: medium-salt stress group, HS: high-salt stress group);

[0068] Figure 3 This is a statistical chart of lncRNAs screening in Dunaliella purinosa (the horizontal axis is the screening step, and the vertical axis is the number of transcripts after screening in the corresponding step);

[0069] Figure 4 Analysis of lncRNA sequence characteristics in Dunaliella purinata (a. Classification of dpr-lncRNAs; b. Comparison of lengths of dpr-lncRNAs and dpr-mRNAs; c. Comparison of exon numbers between dpr-lncRNAs and dpr-mRNAs; d. Comparison of open reading frames (ORFs) between dpr-lncRNAs and dpr-mRNAs);

[0070] Figure 5 The localization of lncRNAs in response to salt stress in Dunaliella primordium on the chromosome of Dunaliella primordium (black lines represent the positions of related lncRNAs, and the length of the lines represents their expression levels);

[0071] Figure 6 is the relative expression level of Dunaliella primordica LNC_000228 in different samples (with Dunaliella 18SrRNA as the internal reference);

[0072] Figure 7 is the relative expression level of Dunaliella primordica LNC_000862 in different samples (with Dunaliella 18SrRNA as the internal reference);

[0073] Figure 8 This is the cell number statistics of Dunaliella purinosa under high salt stress (NaCl concentration is 3.0 mol / L);

[0074] Figure 9 The agarose gel electrophoresis amplification map of the LNC_000732 core transcript of the tested algae strain (the arrow points to the amplified fragment of the transcript, about 280 bp);

[0075] Figure 10 The agarose gel electrophoresis amplification map of the LNC_000643 core transcript of the tested algae strain (the arrow points to the amplified fragment of the transcript, about 220 bp);

[0076] Figure 11 This is the maximum likelihood tree of LNC_000732 for the tested algae strain (each node from left to right is the evolutionary distance and the percentage of genetic similarity);

[0077] Figure 12This is the maximum likelihood tree of LNC_000643 of the tested algae strain (each node from left to right is the evolutionary distance and the percentage of genetic similarity);

[0078] Figure 13 This is the nested PCR amplification pattern of the GGPS gene cDNA core sequence of two strains of Dunaliella primidii (first round);

[0079] Figure 14 This is the nested PCR amplification pattern of the cDNA core sequence of the GGPS gene of two strains of Dunaliella primidii (second round);

[0080] Figure 15 This is the NJ evolutionary tree of the GGPS gene cDNA core sequence of Dunaliella primordium, Dunaliella salina, and Chlorella vulgaris (each node from left to right is the evolutionary distance and genetic similarity percentage). DETAILED DESCRIPTION

[0081] The following is a specific and detailed description of the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention and the accompanying drawings. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the principles of the present invention, and these should also be considered to fall within the scope of protection of the present invention.

[0082] Example 1

[0083] Screening of lncRNAs related to salt stress response in Dunaliella primordium

[0084] 1. Algae material cultivation and salt stress treatment

[0085] A highly salt-tolerant strain of Dunaliella primolecta (D. primolecta) (number: Dpr-C40, which can tolerate 2.8-3.0 mol / L NaCl) was purchased from the UTEX algae seed bank. After sterilization with hypochlorous acid, the algae was cultured in a constant-temperature incubator with a temperature of 25°C and a light intensity of 10,000 lux (light-dark ratio of 12:12). The culture was statically cultured under DM culture medium until the 20th day (generally reaching the mature or stable stage of Dunaliella). The algae solution was evenly divided into 4 groups (3 biological replicates per group), namely the CO group (control group, the original NaCl concentration of the culture medium was 0.05 mol / L), the LS group (low salt stress group, NaCl was added to a final concentration of 1.0 mol / L), the MS group (medium salt stress group, NaCl was added to a final concentration of 2.0 mol / L), and the HS group (high salt stress group, NaCl was added to a final concentration of 3.0 mol / L). After the addition of NaCl, the three experimental groups were each stressed for 48 h (see Figure 1-2 ).

[0086] The DM culture medium adopts the following formula (Table 1-2).

[0087] Table 1 DM medium formula

[0088]

[0089] Table 2 A5 formulation in DM medium

[0090]

[0091] 2. Total RNA extraction, lncRNA library construction and high-throughput sequencing

[0092] According to the instructions of the commercially available TRIzol Kit (including but not limited to this kit), total RNA was extracted from the experimental group (LS / MS / HS group, 3 biological replicates each) and the control group (CO group, 3 biological replicates). After collecting the total RNA, the Ribo-zero TM rRNA Removal Kit (including but not limited to this kit) operation guide, respectively, to remove rRNA, using Agilent 2100 Bioanalyzer and 2.0 Fluorometer (including but not limited to this product) to detect the quality of total RNA. Ultra TM Following the instructions for the II Directional RNA Library Prep Kit (including but not limited to this kit), 3 μg of total RNA was collected from each of the experimental and control groups to construct the lncRNA library. The constructed library was subjected to high-throughput RNA-seq sequencing on the BGISEQ-500 sequencing platform at BGI. The statistical analysis results of the sequencing in this example are shown in Table 3.

[0093] Table 3 Statistical analysis of lncRNA sequencing of Dunaliella purinata

[0094]

[0095] 3. Screening of lncRNAs in Dunaliella primordium

[0096] Bioinformatics screening of lncRNAs of Dunaliella purinata was performed according to the process in Table 4.

[0097] Table 4. Screening process of Dunaliella purinata lncRNAs

[0098]

[0099] Through the above process, a total of 1006 Dunaliella primordica lncRNAs (dpr-lncRNAs) were screened in this example, and the screening results are shown in Figure 3 The results of sequence feature analysis are shown in Figure 4 .

[0100] 4. Screening of dpr-lncRNAs related to salt stress response

[0101] The CPC, PhyloCSF method and Pfam database were used for analysis and screening, and the intersection screening was used to obtain candidate salt stress-related dpr-lncRNAs. The differentially expressed dpr-lncRNAs under different salinity stress were screened from the candidate novel lncRNAs of HS vs.CO, MS vs.CO, and LS vs.CO, which were dpr-lncRNAs related to salt stress response. The differential expression analysis (FPKM quantitative analysis) of dpr-lncRNAs was performed using Cuffdiff software, and the differential expression analysis criteria were |log2(Fold change)| value ≥ 1 and q < 0.05. In this example, a total of 6 salt stress response-related dpr-lncRNAs were screened, and their nucleotide sequences are shown in SEQ ID NO.1 to 6, their differential expression is shown in Table 5, and their location on the chromosome of Dunaliella is shown in Table 5. Figure 5 and Table 5.

[0102] Table 5 Relative expression analysis of lncRNAs in response to salt stress in Dunaliella purinata

[0103]

[0104] Example 2

[0105] Molecular identification of highly salt-tolerant strains using salt-stress-responsive lncRNAs in Dunaliella primordium

[0106] Based on the two lncRNA sequences (LNC_000228 and LNC_000862) of Dunaliella primordium that were screened in Example 1, a specific primer pair was designed. The Tm value was set at 56-65°C, the GC percentage was set at 40-55%, and the amplified region length was set at 200-250 bp. The relative expression level was expressed as mean ± standard deviation and analyzed by ANOVA. The nucleotide sequence information of the specific amplification primers used in this example is shown in SEQ ID NOs. 9-12. The specific implementation steps are as follows:

[0107] 1. Total RNA extraction and reverse transcription of the test Dunaliella purinata strain

[0108] Seven samples of Dunaliella primolecta strains identified in the present invention (including D. primolecta_CN4, D. primolecta_CN5, D. primolecta_CN9, D. primolecta_Q1, D. primolecta_Q2, D. primolecta_Q3, and the known highly salt-tolerant strain D. primolecta_dpr-C40) were selected and cultured as described in Example 1. Around day 20, the culture broth of each sample was equally divided into two aliquots: one serving as a control group, i.e., no NaCl was added in addition to the basal physiological salinity (0.05 mol / L NaCl); and the other serving as a stress group, i.e., a 3.0 mol / L NaCl stress group, subjected to 48 h of stress. Total RNA from each experimental group was prepared according to the procedures of a commercially available Qiagen RNeasy Mini kit (including but not limited to this product). The above RNAs were reverse transcribed into cDNAs respectively with reference to the commercially available TransScript II All-in-One First-Strand cDNA Synthesis SuperMix for PCR (including but not limited to this product), and reverse transcription was performed according to the following reaction system and reaction procedure (see Table 6).

[0109] Table 6 Reverse transcription experimental system and reaction procedure

[0110]

[0111] 2. Real-time PCR amplification of lncRNAs responsive to salt stress in the tested algae

[0112] Refer to commercially available Prepare Premix×EX TaqTMII using the operating manual (including but not limited to this product), follow the real-time PCR reaction system and procedure in Table 7, use the above-mentioned cDNAs as templates and Dunaliella 18S rRNA as an internal reference to amplify LNC_000228 and LNC_000862, respectively, and perform real-time melting curve analysis.

[0113] Table 7 Real-time PCR reaction system and procedure

[0114]

[0115] After the reaction is completed, use 2 -△△CT The relative expression levels of LNC_000228 and LNC_000862 were calculated, that is, samples with log2 (Fold change) values greater than 3 (q value less than 0.05) can be identified as highly salt-tolerant Primoduliella strains. Figure 6 、 Figure 7 It can be seen that the order of the relative expression levels of Real time PCR in the salt stress group of 7 Dunaliella primolecta LNC_000228 is: D.primolecta_dpr-C40>D.primolecta_CN9>D.primolecta_Q2>D.primolecta_Q3>D.primolecta_Q1>D.primolecta_CN4>D.primolecta_CN5, and the order of the relative expression levels of Real time PCR in the salt stress group of 7 Dunaliella primolecta LNC_000862 is: D.primolecta_dpr-C40>D.primolecta_CN9>D.primolecta_CN5>D.primolecta_Q2>D.primolecta_Q1>D.primolecta_Q3>D.primolecta_CN4. It can be seen that under high salt stress conditions, the up-regulated expression of the two lncRNAs in the algae strain D.primolecta_CN9 was very significant (P value < 0.01), and the relative expression levels were comparable to those of the identified highly salt-tolerant D.primolecta_dpr-C40 (△FPKM value LNC_000228 =1.51±0.02, ΔFPKM value LNC_000862 =1.49±0.05), which can be identified as a highly salt-tolerant strain of Dunaliella purinata.

[0116] 3. Result Verification

[0117] Dunaliella cell biomass, to some extent, reflects the algal strain's tolerance to salt stress. Microscopic observation of algal cells from the experimental group in this example (cultured to day 20 and then subjected to 3.0 mol / L NaCl stress for 48 h, for a total of 22 days) was performed. 1.0 mL of algal solution was collected and the cells counted using a cell counter. Algal cell density was measured under different salinity stresses. Each count was repeated five times.

[0118] Depend on Figure 8 It can be seen that after 48 hours of salt stress (i.e., on the 22nd day), the cell density of the seven strains of Dunaliella primolecta was in the following order: D.primolecta_dpr-C40>D.primolecta_CN9>D.primolecta_CN4>D.primolecta_CN5>D.primolecta_Q1>D.primolecta_Q3>D.primolecta_Q2. The cell density of the highly salt-tolerant algae strain D.primolecta_dpr-C40 was comparable to that of the tested algae strain D.primolecta_CN9, i.e., 细胞数The average value is (0.04±0.002)×10 6 mL -1 ; and much higher than other algae strains, that is, the average is higher than other algae strains (2.01±0.05)×10 6 mL -1 The above results indicate that the tested algae strain D.primolecta_CN9 is a highly salt-tolerant strain in this example. This conclusion is consistent with the Real-time PCR detection conclusion, demonstrating the feasibility and accuracy of the identification method.

[0119] 4. Experimental time, accuracy and cost analysis of identification of highly salt-tolerant Dunaliella purinata

[0120] Based on the time required to successfully complete the required experimental steps in this embodiment, the accuracy of the results, the costs involved in purchasing instruments and equipment, reagents and consumables, etc., through market research, the results of the comparison between the traditional highly salt-tolerant algae identification method and the identification method of the present invention are summarized as follows (Table 8):

[0121] Table 8 Comparison between traditional high salt-tolerant algae identification technology and the technology of the present invention

[0122]

[0123] As can be seen from the above table, the identification method of the present invention is superior to traditional identification methods in terms of identification time, accuracy of identification results, and identification cost for highly salt-tolerant Dunaliella purinata species. This also indirectly demonstrates the advancement and practicality of the lncRNAs and corresponding primer sequences provided by the present invention.

[0124] Example 3

[0125] Interspecific identification of Dunaliella primordium using salt stress-responsive lncRNAs

[0126] Based on the two lncRNA sequences (LNC_000732 and LNC_000643) screened in Example 1, a specific primer pair was designed, with a Tm value of 55-68°C, a GC percentage of 40-55%, and an amplification region length of 200-350 bp. The nucleotide sequence information of the specific amplification primers used in this example is shown in SEQ ID NOs. 13-16. The specific implementation steps are as follows:

[0127] 1. Cultivation of test algae and extraction of genomic DNA

[0128] DM culture medium was prepared according to the method of Example 1, and 7 test Chlorophyta strains (including unknown Chlorophyta A, unknown Chlorophyta B, unknown Chlorophyta C, unknown Chlorophyta D, unknown Chlorophyta E, unknown Chlorophyta F, and known Primoduliella dpr-C40) were cultured indoors under light. On the 20th day of culture, approximately 100 to 200 mg of algal cells were enriched. After grinding with liquid nitrogen for 15 seconds, genomic DNA (gDNA) of each algal strain was extracted according to the operating instructions of a commercially available Plant gDNA Extract Kit (including but not limited to this product).

[0129] 2. PCR amplification of salt stress-responsive lncRNAs transcripts

[0130] The core fragments of LNC_000732 and LNC_000643 transcripts of each algae strain were PCR amplified. The PCR amplification system and reaction procedures are shown in Table 9:

[0131] Table 9 PCR amplification system and procedure for Dpr-lncRNAs transcripts

[0132]

[0133] 3. Identification of Dunaliella primordium species

[0134] The amplified products were subjected to agarose gel electrophoresis (concentration 1.2%). Figure 9 、 Figure 10 It can be seen that except for the unknown green algae D and the known Dunaliella primidioides dpr-C40, which have obvious amplification bands between 200 and 300 bp (the size range of the core fragments of the LNC_000732 and LNC_000643 transcripts) (pointed by the arrow), the other algae strains have no obvious amplification products at this location, and the unknown green algae D can be identified as Dunaliella primidioides.

[0135] 4. Result Verification

[0136] The PCR amplification products were sent to a biotechnology company for sequencing. The sequencing results were assembled using DNAstar software and verified by BLASTn. Nucleotide sequence alignment was performed using Clustal X 2.0 software, and ambiguous segments were deleted. Maximum likelihood estimation (based on the Tamura-Nei model to estimate substitution patterns and ratios) of the LNC_000732 and LNC_000643 transcript sequences was performed using the Models module of MEGA 2.0 software. Genetic distance was analyzed using the Distance module (pairwise calculation mode was selected, with the model set to No. of differences and p-distance). The step size was set to 1000, and the calculation was repeated 100 times. Maximum likelihood method (ML) phylogenetic trees were constructed for LNC_000732 and LNC_000643, respectively.

[0137] Depend on Figure 11 、 Figure 12 As can be seen, the unknown green algae D and the dpr-C40 strain of Dunaliella purinata were clustered separately, and the evolutionary distance between the two strains was between 0.99 and 1.00, with a genetic similarity of 100, indicating that the unknown green algae D is Dunaliella purinata. This result is consistent with the PCR test results of this example, confirming the effectiveness of the lncRNAs and corresponding primer sequences provided by this invention.

[0138] 5. Time and cost analysis of interspecific identification of Dunaliella primidiocarpa

[0139] Based on the time required to successfully complete the experimental procedures of this embodiment, the costs of instruments, reagents, consumables, and electrical energy, and through market research, a comparison between the traditional algae species identification method and the method of the present invention is summarized as follows (Table 10):

[0140] Table 10 Comparison between traditional algae species identification methods and the identification method of the present invention

[0141]

[0142]

[0143] As can be seen from the above table, the identification method of the present invention is superior to traditional identification methods in terms of identification time, accuracy of identification results, and identification cost of Dunaliella purinata species, which also indirectly demonstrates the advanced nature and practicality of the lncRNAs and corresponding primer sequences provided by the present invention.

[0144] Example 4

[0145] Molecular screening of the GGPS cDNA core sequence of the functional gene in Dunaliella primordium based on salt stress-responsive lncRNAs and their target sequences

[0146] Using the two strains of Dunaliella primolecta (D. primolecta_CN4 and D. primolecta_Q2) from Example 1 as the research subjects, and using the common target sequences of the lncRNAs (LNC_000866 and LNC_000981) identified in Example 1 as templates, nested PCR primers were designed with Tm values set at 55-68°C and GC percentages set at 40-55%. The nucleotide sequences of the nested PCR primers used in this example are shown in SEQ ID NOs. 17-20. The specific implementation steps are as follows:

[0147] 1. Cultivation of test algae, total RNA extraction and cDNA amplification

[0148] Following the method of Example 1, two algal species, D. primolecta_CN4 and D. primolecta_Q, were cultured in DM medium under constant temperature and illumination. On day 20, NaCl was added to 3.0 mol / L, and salt stress was applied for 48 hours. Total RNA from each strain was extracted according to the instructions of the commercially available Qiagen RNeasy Mini kit (Beijing Tiangen), including but not limited to this product. The total RNA was then reverse-transcribed into cDNAs according to the instructions of the commercially available HiScript II 1st Strand cDNA Synthesis Kit (Nanjing Novezan), including but not limited to this product.

[0149] 2. Nested PCR amplification

[0150] Using the Dunaliella cDNAs as templates, and primers dpr-GGPS_target-F1 / dpr-GGPS_target-R1 (first round amplification) and dpr-GGPS_target-F2 / dpr-GGPS_target-R2 (second round amplification), amplification was performed according to the following nested PCR system and reaction procedures (Table 11).

[0151] Table 11 Dunaliella GGPS cDNA core sequence amplification system and procedure

[0152]

[0153]

[0154] 3. Screening of Dunaliella GGPS core sequences

[0155] The two PCR amplification products were run on 1.2% agarose gels. Positive bands between 750 and 1000 bp (the range of the amplified fragments) were excised, dissolved, purified, and sent to a biotechnology company for sequencing. The sequencing results were then compared with the known Dunaliella salina GGPS cDNA sequence (GenBank gene ID: MN172172) using BLASTn. The alignment rates for the amplified sequences of D. primolecta_CN4 and D. primolecta_Q2 were 99.5% and 99.8%, respectively, confirming that the PCR-amplified sequences from these two strains were core fragments of the Dunaliella GGPS cDNA.

[0156] Depend on Figure 13 、 Figure 14 It can be seen that the first-round PCR amplification lanes of the two algae strains were diffuse, indicating that multiple non-specific amplification bands were produced in the first round; the second-round PCR amplification lanes showed obvious bands at 750-1000bp (the range where the target fragment is located), indicating that the GGPS cDNA specific core sequence has been successfully cloned.

[0157] 4. Result Verification

[0158] The GGPS cDNA core fragment sequences of the above two algae strains were compared with the GGPS cDNA core fragment of Dunaliella salina and the GGPS cDNA core fragment of an outgroup (Chlorella vulgaris, belonging to the genus Chlorella of the phylum Chlorophyta) by BLASTn alignment. A phylogenetic tree was constructed using Clustal W and MEGA 5.0 software, and the neighbor-joining (NJ) method was selected as the tree construction method.

[0159] Depend on Figure 15 As can be seen, the GGPS cDNA core sequences of D.primolecta_CN4, D.primolecta_Q2, and D.salina clustered together, while Chlorella clustered alone. This demonstrates that the GGPS sequences of the first three share high homology and belong to the Dunaliella GGPS core sequence. The core sequence of this gene in Chlorella shares low homology and does not belong to the Dunaliella GGPS cDNA core sequence. The method used in this example can greatly improve the success rate of researchers cloning GGPS genes from other strains of the genus Dunaliella, avoid false positives in molecular cloning experiments, and lay an information foundation for the research of GGPS genes in algal genetic engineering and the in-depth application of this gene's function.

[0160] 5. Analysis of Experimental Time, Accuracy, and Cost for Screening the Core Sequence of Dunaliella GGPS Gene cDNA

[0161] Based on the time required to successfully complete the required experimental steps in this example, the accuracy of the results, the costs involved in purchasing instruments and equipment, reagents and consumables, etc., and through market research, a comparison between the traditional algae gene molecular cloning technology method and the technical method of the present invention is summarized as follows (Table 12):

[0162] Table 12 Comparison between traditional algae gene molecular cloning methods and the method of the present invention

[0163]

[0164] As can be seen from the table above, the method of the present invention is superior to traditional methods in terms of successful gene sequence cloning time, sequence result accuracy, and experimental cost. This also indirectly demonstrates the advanced nature and practicality of the lncRNAs and corresponding primer sequences provided by the present invention.

[0165] The above embodiments are preferred implementations of the present invention, but the implementation of the present invention is not limited to the above embodiments. For ordinary technicians in this field, several modifications and improvements can be made without departing from the principles of the present invention, which are all included in the scope of protection of the present invention.

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

Claims

1. Dunaliella primordium salt stress responsive lncRNAs, characterized by: include: LNC_000228, LNC_000862, LNC_000732, LNC_000643, LNC_000866, and LNC_000981, whose nucleotide sequences are shown in SEQ ID NOs. 1 to 6 respectively.

2. The GGPS target sequence of LNC_000866 and LNC_000981 in the salt stress-responsive lncRNAs of Dunaliella primordium according to claim 1, characterized in that The nucleotide sequences are shown in SEQ ID NOs. 7 to 8, namely, LNC_000866_target and LNC_000981_target.

3. A PCR reaction specific primer pair for amplifying LNC_000228, LNC_000862, LNC_000732, and LNC_000643 in the salt stress-responsive lncRNAs of Dunaliella primordium according to claim 1, characterized in that: include: dpr-LNC_000228-F / dpr-LNC_000228-R; dpr-LNC_000862-F / dpr-LNC_000862-R; dpr-LNC_000732-F / dpr-LNC_000732-R; dpr-LNC_000643-F / dpr-LNC_000643-R; The nucleotide sequences of the primer pairs are shown in SEQ ID NOs. 9 to 16, respectively.

4. A PCR reaction specific primer pair for amplifying the target GGPS target sequence according to claim 2, characterized in that: include: dpr-GGPS_target-F1 / dpr-GGPS_target-R1; dpr-GGPS_target-F2 / dpr-GGPS_target-R2; The nucleotide sequences of the primer pairs are shown in SEQ ID NOs. 17 to 20.

5. The use of the specific primers for PCR reaction according to claim 3, characterized in that: The dpr-LNC_000228-F / dpr-LNC_000228-R and dpr-LNC_000862-F / dpr-LNC_000862-R can be used for intraspecific identification of highly salt-tolerant Dunaliella purinata strains; the dpr-LNC_000732-F / dpr-LNC_000732-R and dpr-LNC_000643-F / dpr-LNC_000643-R can be used for interspecific identification of Dunaliella purinata and other Dunaliella species.

6. The use of the specific primers for PCR reaction according to claim 4, characterized in that: The dpr-GG PS_target-F1 / dpr-GGPS_target-R1 and dpr-GGPS_target-F2 / dpr-GGPS_target-R2 can be used for molecular screening and cloning of the GGPS cDNA core sequence, a functional gene of Dunaliella.

7. A real-time PCR reaction kit for rapid screening of highly salt-tolerant Dunaliella purinata strains, characterized in that: The method comprises the PCR reaction specific primer pair for amplifying LNC_000228 and LNC_000862 in the lncRNAs responsive to salt stress of Dunaliella primordium as described in claim 3.

8. A PCR reaction kit for accurately identifying Dunaliella primidioides strains, characterized in that: The method comprises the PCR reaction specific primer pair for amplifying LNC_000732 and LNC_000643 in the lncRNAs responsive to salt stress of Dunaliella primordium as described in claim 3.

9. A nested PCR reaction kit for efficiently cloning the core sequence of the GGPS cDNA of the functional gene of Dunaliella primordium strain, characterized in that: The invention comprises the PCR reaction specific primer pair for amplifying the target GGPS cDNA core sequence as claimed in claim 4.

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