CN104099334B - MicroRNAs gene and improve chlorella content of fatty acid application - Google Patents

Abstract

The invention discloses a microRNAs gene and its application in increasing the fatty acid content of green algae. The invention uses microRNAs and their target genes as tools to regulate the activity of key enzymes in green algae metabolism (PEPC enzymes) and control the accumulation of carbon sources in algae cells, so that the green algae cells can accumulate more fatty acids.

Description

microRNAs gene and its application in improving fatty acid content of green algae

Technical field:

The invention belongs to the field of biotechnology, and relates to a microRNAs gene and its application in increasing the fatty acid content of green algae. By using the principle of regulating microRNAs to regulate its target genes, the expression level of microRNAs in green algae cells is induced by light, temperature, etc., The ultimate technology to increase the fatty acid content of Chlorella cells.

Background technique:

At present, the world's energy mainly depends on non-renewable resources such as oil, coal and natural gas, and their depletion has become the main factor affecting the development of the global economy. At the same time, the use of these petrochemical energy sources will be exhausted sooner or later, and the development of new energy sources has become an inevitable trend of economic development. Biodiesel (fatty acid methyl ester) has been obtained for its easy biodegradability, renewable and similar power and combustion characteristics to petrochemical diesel. sure. Microalgae, as a new type of raw material for biodiesel production, has become a hot spot in bioenergy research due to its characteristics of large biomass, fast growth, and high oil content, and has good development potential. Based on microRNAs regulation technology, increasing the accumulation of lipids in microalgal cells by regulating the key enzyme genes or transcription factors of fatty acid metabolism is currently the most potential metabolic regulation approach.

Phosphoenolpyruvate carboxylase (PEPC) is present in all photosynthetic organisms and most non-photosynthetic bacteria and protozoa. In microalgae, 3-phosphoglyceraldehyde formed by photosynthetic fixation of CO ₂ can not only enter the synthesis pathway of glucose and amino acids, but also form pyruvate through glycolysis and then enter lipid metabolism through acetyl-CoA or participate in various Complete Krebs cycle. PEPC can catalyze the carboxylation reaction of _CO2 with phosphoenolpyruvate (PEP) to finally generate oxaloacetate (OAA) and inorganic phosphoric acid, while PEP can be converted into acetyl CoA, which is the main raw material for fatty acid synthesis. PEPC directs the carbon source to the tricarboxylic acid cycle in the carbon metabolism pathway, so that the carbon source flows to the direction of non-lipid accumulation and is not easy to accumulate oil. Studies have shown that the activity of PEPC has an important influence on the synthesis of lipids. In the late 1980s, Japanese scholar Dr. Sugi found that the protein content in soybean grains was closely related to the enzyme activity of PEPC. According to reports, PEPC in C3 plants may play an important role in promoting the replenishment of intermediate metabolites in the TCA cycle and coordinating the metabolism of carbon and nitrogen, that is, when the demand for the carbon skeleton used in amino acid synthesis increases, the tricarboxylic The regeneration of oxaloacetate in the acid cycle coordinates the metabolism of C and N, and provides the carbon skeleton required for amino acid synthesis [GonzalezM.C, Sanehez R, Cejudo FJAbioticstresses affeeting water balance induce Phosphoenolpyruvate carboxylase expression in roots of wheat seedlings J.Planta., 2003, 216(6):985-992.]. Chen Jinqing put forward the "substrate competition" hypothesis, thinking that the synthesis of grain oil and protein requires the same substrate - pyruvate, there is substrate competition between the two, and the equilibrium point depends on the key enzymes of the metabolism of two types of substances, PEPC and acetyl-CoA carboxylate Relative activity of ACCase. PEPC catalyzes the synthesis of oxaloacetate from pyruvate to enter protein metabolism; ACCase catalyzes the synthesis of acetyl-CoA from pyruvate to enter fatty acid metabolism. Accordingly, Chen Jinqing et al. proposed to use antisense PEPC technology to inhibit the gene expression of the key protein synthesis enzyme PEPC in rapeseed to increase the supply of fatty acid biosynthesis substrate PEP. The highest content reached 49.54% [Chen Jinqing, Lang Chunxiu, Hu Zhanghua, etc. Antisense pepc gene regulates the ratio of protein/fat content in rapeseed J. Journal of Agricultural Biotechnology, 1999, 7(4): 316-32]. Hou Lijun et al. transformed Escherichia coli with the antisense pepcA gene of cyanobacteria. Compared with wild-type cells, the PEPC enzyme activity in E. coli transfected with antisense pepcA segment was reduced to 30.2% of wild bacteria, protein synthesis was reduced by 23.6%, and lipid synthesis was reduced. Increased by 46.9%: while the PEPC enzyme activity of Escherichia coli with the positive pepcA fragment was 2.38 times that of wild bacteria, protein synthesis increased by 14.5%, lipid synthesis decreased by 49.6%, and the content of octadecanoic acid in the transgenic bacteria increased significantly [Hou Lijun , Shi Dingji, Cai Zefu, etc. Regulation of lipid synthesis in Escherichia coli by cyanobacteria sense and antisense pepcA gene introduction J. China Biotechnology Journal, 2008, (5): 52-58].

Chlamydomonas reinhardtii contains two forms of PEPC coding genes CrPpc1 and CrPpc2, and it is speculated that PEPC1 is a homotetramer and PEPC2 is a complex [Eric R.Moellering, Yexin Ouyang. The two divergentPEP-carboxylase catalytic subunits in the green microalga Chlamydomonas reinhardtii respond reversibly to inorganic-N supply and co-exist in the high-molecular-mass, hetero-oligomeric Class-2 PEPC complex. E.R. Moellering et al./FEBS Letters., 581(2007):4871–4876.]. Can green algae accumulate more fatty acids by regulating the activity of PEPC enzyme? This patent technology is aimed at the two subtypes of PEPC enzymes (PEPC1 and PEPC2) in Chlamydomonas reinhardtii, and designs suitable microRNAs to participate in the regulation of the activity of PEPC enzymes. Invented a technology based on microRNAs to regulate the accumulation of fatty acids in green algae.

Invention content:

The invention provides a technique for regulating the synthesis and metabolism of fatty acids in green algae based on microRNAs. Construct transgenic algae that can regulate the expression of microRNAs by means of light intensity or temperature, and use microRNAs to inhibit their target genes as a means to regulate the synthesis of fatty acids in green algae. Inhibition of PEPC enzyme activity in Chlamydomonas reinhardtii by microRNAs enables green algae cells to accumulate more fatty acids. Concrete content of the present invention is as follows:

One aspect of the present invention relates to a Chlamydomonas reinhardtii microRNAs gene, characterized in that its sequence is SEQ ID NO.1 or SEQ ID NO.2.

The present invention also relates to the application of the above-mentioned microRNAs in increasing the fatty acid content of green algae.

Another aspect of the present invention also relates to a method for the synthesis of fatty acids in green algae based on the regulation of microRNAs genes of Chlamydomonas reinhardtii, to construct transgenic algae that can regulate the expression of microRNAs by means of light intensity or temperature, etc., and to use microRNAs and their target genes as tools to regulate green algae. The synthesis and metabolism of algae protein increases the synthesis of fatty acids in algae cells, thereby increasing the fatty acid content in green algae cells.

In a preferred embodiment of the present invention, microRNAs inhibit the target gene in the protein synthesis pathway of green algae—phosphoenolpyruvate carboxylase (PEPC) gene to increase the fatty acid content.

In a preferred embodiment of the present invention, it is characterized in that constructing transgenic algae capable of regulating the expression of microRNAs by means such as light intensity or temperature, the specific method is as follows:

Screening and cultivation of transgenic recipient algae: using green algae medium such as TAP and BBM, aerated eukaryotic green algae (such as Chlamydomonas reinhardtii, etc.) under the condition of light of 90-200 μE/m ² ·s;

According to the sequence determined by the method of claim 3, artificially synthesize microRNAs that regulate target genes;

Construction of green algae expression vectors: Insert microRNAs into green algae expression vectors with specific promoters (strong light induction or heat shock induction, etc.);

Genetic transformation of expression vectors and screening of transgenic algae: the expression vectors with microRNAs genes are introduced into the genome of green algae for expression, and the genetic transformation is carried out by "bead milling method", gene gun method or electric shock transformation method. Through resistance plate screening or auxotrophic screening, positive algae colonies were obtained, and molecular detection and expression product analysis were carried out to determine the transgenic algae with microRNA, and the transgenic algae could be induced by strong light or high temperature.

Description of drawings:

Figure 1: Comparison of fatty acid content between transformants regulated by amicroRNAs-cre-MIRpepc1 and the control group (transformants B and C represent transformants that have been transferred to microRNAs that regulate PEPC1 gene expression);

Figure 2: Comparison of the fatty acid content of transformants regulated by amicroRNAs-cre-MIRpepc2 and the control group (transformants H, V, and Z represent transformants transferred with microRNAs that regulate PEPC2 gene expression).

Specific examples:

Chlamydomonas reinhardtii is the representative species of green algae.

Example 1

(1) Selection and cultivation of transgenic recipient algal strains

Cell wall-defective Chlamydomonas reinhardtii (Chlamydomonas reinhardtii, cc-849, purchased from the Chlamydomonas Genetic Center of Duck University, USA) and cytochrome b-deficient Chlamydomonas reinhardtii (cc-2654, purchased from the Chlamydomonas Genetic Center of Duck University, USA) were selected as Transgenic recipient strains. TAP medium was used as the medium for Chlamydomonas reinhardtii, and its formulation and ingredients were as follows: 2.42g Tris, 25mL 4×Beijerinck salts (16g NH ₄ Cl, 2g CaCl ₂ .2H ₂ O, 4g MgSO ₄ .7H ₂ O dissolved Dilute to 1L in water, 1mL1M(K)PO ₄ , 1mL Trace trace element mixture (11.4g H ₃ BO ₃ , 5.6gMnCl ₂ .4H ₂ O, 22g ZnSO ₄ .7H ₂ O, 4.99 _g FeSO ₄ .7H ₂ O, 1.61g CoCl ₂ .6H ₂ O, 1.57g CuSO ₄ .5H ₂ O, 1.1g (NH ₄ ) ₆ Mo ₇ O ₂₄ .4H ₂ O, 50g Na ₂ EDTA, dissolved in water, 20% KOH Adjust the pH to 6.5-6.8, dilute to 1L), dissolve in 975mL of water, adjust the pH to 6.95-7.05 with glacial acetic acid, and dilute to 1L.

Cultivation conditions of Chlamydomonas reinhardtii: continuous culture at 22-25°C, 90 μE/m ² ·s light conditions, algal cell concentration in the logarithmic growth phase is 1-2×10 ⁶ cells/mL.

(2) Synthesis of artificial microRNAs from Chlamydomonas reinhardtii

The microRNAs sequences that can inhibit the expression of the PEPC enzyme gene were synthesized by the gene company, respectively amicroRNAs-cre-MIRpepc1 and amicroRNAs-cre-MIRpepc2, and their sequence information is as follows:

>amicroRNAs-cre-MIRpepc1 (SEQ ID NO.1)

GCTAGC GCGGGGCCCUGACACCACUGCGGCCGCuagcgaccaaacaauccaauaCCGCGCCUGGACCCGAGGGAGGACCCCUCGGGACCCGGUACGUCGUAUUGGAUUGUUUGGUCGCUAGGUCGCGGUGGGGUCAGGUCCUUCCGC CACGTG

>amicroRNAs-cre-MIRpepc2 (SEQ ID NO.2)

GCTAGC GCGGGGCCCUGACACCCACUGCGGCCGCgugccgaaaauguuugguaaCCGCGCCUGGACCCCGAGGGAGGACCCCUCGGGACCCGGUACGUCGUUAACCAAACAUUUUCGGCACGGUCGCGGUGGGGUCAGGUCCUUCCGC CACGTG

(3) Construction of amicroRNAs-cre-MIRpepc1, 2 gene Chlamydomonas nuclear genome expression vector

The regulated amicroRNAs-cre-MIRpepc1 and amicroRNAs-cre-MIRpepc2 genes were obtained by Nhe Ⅰ and Pmac Ⅰ double enzyme digestion. Vector pH105 [Wu JX et al. Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii. Chinese Journal of Oceanology and Limnology, 2008(26): 242-247] was cloned with HSP70A-RBCS2 promoter sequence and RBCS2 The terminator sequence carries multiple cloning restriction sites such as Pmac Ⅰ, Nhe Ⅰ, Xba Ⅰ and Sal Ⅰ in the middle, which can be inserted into foreign genes and expressed in the Chlamydomonas nuclear genome. The vector pSP124 ((purchased from the Chlamydomonas Center of Duck University, USA))【Lambreras v, Stevens DR, Purton S. Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron. Plant J.1998; 14(4):441-447】contains The expression cassette RBCS2::ble::RBCS2 can make transformed host cells acquire phleomycin and Zeomycin resistance, and is used as a selection marker for Chlamydomonas transformation in the present invention.

The amicroRNAs-cre-MIRpepc1 and amicroRNAs-cre-MIRpepc2 genes were inserted into the pH105 vector digested with PmacI and NheI, respectively, to obtain pH105-amicroRNA. pH105-amicroRNA was digested by EcoR Ⅰ to obtain the expression frameworks expressing amicroRNA genes (HSP70A-RBCS2::amicroRNAs-cre-MIRpepc1::RBCS2 and HSP70A-RBCS2::amicroRNAs-cre-MIRpepc2::RBCS2) were inserted into the EcoR Ⅰ site of pSP124 to obtain Chlamydomonas expression vectors pH105-amicroRNAs-cre-MIRpepc1 and pH105-amicroRNAs-cre-MIRpepc2 that can regulate pepc1 and pepc2 genes.

(4) Construction of amicroRNAs-cre-MIRpepc1, 2 gene Chlamydomonas chloroplast expression vector

Plasmid p423 (purchased from the Chlamydomonas Center of Duck University, USA) carries the aadA gene expression cassette (5'atpA::aadA::3'rbcL), and the aadA gene is used as a selection marker for genetic transformation of Chlamydomonas, endowing host cells with spectinomycin resistance . Using the genomic DNA of Chlamydomonas reinhardtii cc-124 (purchased from the Chlamydomonas Genetic Center of Duck University, USA) as a template, primers Prch1 and Prch2 were designed to amplify the 5'chlB gene (490bp) as the right-end homologous sequence of Chlamydomonas genetic transformation. Primers Prch3 and Prch4 were designed to amplify the 3'chlB gene (630bp) as the left homologous sequence of Chlamydomonas genetic transformation.

The primers for amplifying the 5'chlB gene are:

Prch1: 5'- CCTGAATTCCTGCCATTGGTGTCCATTGC -3'

EcoR Ⅰ

Prch2: 5'-GGA GATATC CATTTCCAGGCTGATAGTG-3'

EcoR Ⅰ

PCR program: Denaturation at 94°C for 1 min, renaturation at 56°C for 1 min, extension at 72°C for 1 min, 30 cycles.

The primers for amplifying the 3'chlB gene are:

Prch3: 5'-GC GGTACC GCCATTGTATGGTCTCCAG-3'

Kpn I

Prch3: 5'-CG CTGCAG CGGGTACATTAGTAGCGGTG-3'

Pst I

PCR program: Denaturation at 94°C for 1 min, renaturation at 56°C for 1 min, extension at 72°C for 1 min, 30 cycles.

The obtained 5'chlB gene and p423 plasmid were respectively digested and amplified with EcoR Ⅰ, ligated and constructed to obtain the p423c plasmid. The obtained 3'chlB gene and p423c plasmid were digested and amplified with Kpn Ⅰ and Pst Ⅰ, respectively, and then ligated and constructed to obtain the Chlamydomonas chloroplast expression vector p423chl.

The aadA gene can be excised by Nco Ⅰ and Pst Ⅰ double enzyme digestion, and the exogenous target gene amicroRNAs-cre-MIRpepc1 or amicroRNAs-cre-MIRpepc2 can be inserted to construct the amicroRNAs-cre-MIRpepc1 and amicroRNAs-cre-MIRpepc2 gene expression cassettes ( 5'atpA::amicroRNAs-cre-MIRpepc1::3'rbcL and 5'atpA::amicroRNAs-cre-MIRpepc2::3'rbcL). The amicroRNAs-cre-MIRpepc1 and amicroRNAs-cre-MIRpepc2 gene expression cassettes were obtained by double digestion with EcoR V and Sma I, and ligated with the p423chl plasmid that had been double digested with EcoR V, and the positively connected plasmids were screened by Xho I digestion , construct the PamicroRNAs-cre-MIRpepc1-chl and PamicroRNAs-cre-MIRpepc2-chl plasmids containing aadA gene expression cassette and amicroRNAs-cre-MIRpepc1 or amicroRNAs-cre-MIRpepc2 gene expression cassette. The Chlamydomonas chloroplast expression vectors PamicroRNAs-cre-MIRpepc1-chl and PamicroRNAs-cre-MIRpepc2 carry the chlB gene exon 5' and 3' end sequences, which can be used as homologous transformation fragments during the genetic transformation of Chlamydomonas The amicroRNAs-cre-MIRpepc1 or amicroRNAs-cre-MIRpepc2 gene is site-directedly integrated into the position between the 5' and 3' end sequences of the chlB gene of the chloroplast DNA.

(5) Genetic transformation of amicroRNAs-cre-MIRpepc1 and amicroRNAs-cre-MIRpepc2 genes in Chlamydomonas reinhardtii cells

(1) "Bead milling method" genetic transformation

use Plasmid Purification Kit extracted recombinant plasmids pH105-amicroRNAs-cre-MIRpepc1 and pH105-amicroRNAs-cre-MIRpepc2 respectively. The specific steps of the "bead milling method" are as follows: (1) Cultivate the cell wall-deficient Chlamydomonas reinhardtii cc-849 (purchased from the Chlamydomonas Center of Duck University, USA) to the logarithmic phase in continuous light and TAP medium, and the number of cells is about 1-2×10 ⁶ cells/ml. Centrifuge at 5000rmp for 5min at room temperature to collect the algal cells; discard the supernatant; (2) Resuspend the algal cell pellet with sterilized fresh TAP culture medium, and adjust the cell concentration to 2×10 ⁸ cells/ml; (3) Pipette 270 μl of algal cells Put the suspension into a 1.5ml EP tube (with sterilized alloy tin beads inside), add 1 μg enzyme-cut linear pH105-amicroRNAs-cre-MIRpepc1 and pH105-amicroRNAs-cre-MIRpepc2 and 30μl 50% PEG6000 to the EP tube At the same time, a sample group without PEG6000 was established; in addition, a control group without DNA was also established; (4) the mixture of Chlamydomonas cells/alloy tin beads/exogenous DNA was shaken at the highest speed for 25s on the oscillator; (5) Transfer the mixture to a centrifuge tube containing 10ml of fresh TAP medium, and culture overnight (22-25°C) on a 100rpm shaker under low light to recover the cells; (6) Centrifuge at 3000rmp for 5min at room temperature to collect the cells. Discard the supernatant; carefully resuspend the cells with 500 μl of fresh TAP medium, add 3.5ml of 0.5% TAP medium, mix well and pour it on a Zeomycin TAP solid plate containing 10 μg/ml, and place it in a light incubator at 22-25°C Cultivate it upside down for about 3 weeks, until green monoclones grow on the plate.

(2) "Gene gun" genetic transformation

"Gene gun" genetic transformation was performed using a gene gun (Bio-Rad) in a sterile ultra-clean bench. The specific steps are as follows: (1) Chlamydomonas reinhardtii cc-2654 (purchased from the Chlamydomonas Center of Duck University, USA) was cultured in TAP medium until the logarithmic growth phase (1-2×10 ⁶ cells/ml), centrifuged at 5000rmp room temperature for 5min , collect the algal cells; discard the supernatant; resuspend the algal cell pellet with sterilized fresh TAP culture medium, and adjust the cell concentration to 2×10 ⁸ cells/mL; pipette 300 μL of the algal cell suspension and spread it on the center of the TAP solid plate ( Diameter ~ 3cm); Incubate under light at 22-25°C (~90μE/m ² /s) for 24h, until a layer of cells is formed on the surface of the plate. (2) Take 100μL gold powder suspension (60mg/mL), add 5μg plasmid (PamicroRNAs-cre-MIRpepc1-chl or PamicroRNAs-cre-MIRpepc2-chl) (100μg/μL), 100μl 2.5M CaCl ₂ and 40μL 0.1M For spermidine, vortex for 2-3 minutes, let stand for 1 minute, centrifuge at 8,000 rpm for 2 seconds at room temperature, discard the supernatant; add 280 μL of 70% ethanol to wash, discard the supernatant; add 280 μL absolute ethanol to wash, discard the supernatant Clear; add 100 μL of absolute ethanol and gently resuspend. (3) Take 10 μL of DNA gold powder coating solution for bombardment; the bombardment parameters are as follows: split membrane helium pressure 1100 psi, vacuum degree 25 inches Hg, bombardment distance 9 cm, gold powder particle diameter 100 nm; The plates were placed in a light incubator at 22-25°C, and incubated in light for 8 hours. Wash the Chlamydomonas cells with 1 mL of fresh TAP culture solution, spread them on an antibiotic screening plate (transfer of PamicroRNAs-cre-MIRpepc1-chl or PamicroRNAs-cre-MIRpepc2-chl plasmid) and culture for about 3 weeks, until green single cells grow on the plate. clone.

(6) Screening and identification of transgenic microRNAs gene algae

After Chlamydomonas reinhardtii was transformed and plated, a single colony grew about a month or so. Select the transformant and inoculate it on the blank plate TAP medium. After the monoclonal grows out, inoculate it into the liquid medium. After the growth reaches the logarithmic phase, extract the genomic DNA, and regulate the amicroRNAs-cre-MIRpepc1 and amicroRNAs-cre -MIRpepc2 designed primers for genome PCR experiments.

After recovering and purifying the PCR products, they were sent to Shanghai Sangong for sequencing. The transformants confirmed by molecular biology were subcultured in the laboratory for several months, and the stability of the transformants was further confirmed according to the above method.

(7) Regulation and detection of fatty acid production of transgenic algae

Inoculate the transgenic algae liquid into the TPA liquid medium, and cultivate to the logarithmic phase after inoculation. The algae liquid cultured to the logarithmic phase is subjected to 0.5 hours of heat shock to restore growth, and the algal cells are collected by centrifugation. After drying, fatty acids are extracted and methylated. The extracted fatty acid methyl ester samples were injected into a GC-MS gas phase-mass spectrometer for detection.

In this study, GC-MS gas chromatography-mass spectrometry was used to analyze the extracted fatty acid samples. Firstly, it is necessary to detect the fatty acid standard. The standard used in this experiment is a mixed standard of 37 fatty acid methyl esters purchased from Shanghai Anpu Company. Before detecting fatty acid samples, it is necessary to set up an appropriate program so that the 37 fatty acid methyl esters in the mixed standard can be separated better, so that all peaks are single peaks and separated at the baseline.

After determining the detection process and method, the control group and transformants were detected. By referring to the peak time and mass spectrum results of 37 kinds of fatty acid methyl ester mixed standards, it was detected that the fatty acids of Chlamydomonas reinhardtii cc849 were mainly composed of C16:0 (methyl palmitate), C16:1, C18:0 (stearic acid), C18: 1T (oleic acid), C18:2T (linoleic acid) and C18:3n3 (alpha-linolenic acid). The peak times of the transformants and the control group were basically the same, indicating that the fatty acid components were consistent. In the research, treatment methods such as heat shock and strong light recovery were used for detection. Under different treatment conditions, the fatty acid content of the transformant and the control group were different accordingly.

The method of heat shock was used in the experiment, 40° heat shock for 30 minutes, after heat shock, the light recovery growth was 24 hours, and the algae cells were collected by centrifugation at 5000rpm for 5 minutes. After lyophilization, total lipid samples were extracted and methylated. Samples were detected using GC-MS. From Fig. 1 and Fig. 2, it can be seen that the fatty acid content of the transformants has a larger improvement, compared with the control group (using * to represent in the figure; * represents having significant difference compared with the control group; ** represents the control group There is a very significant difference compared with the group), and the highest improvement is nearly 20%. Artificial microRNAs have the expected regulatory effect on algal cells, and this study has laid a solid foundation for the industrial development of microalgae to prepare biodiesel in the future.

The above description is a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (2)

1. Chlamydomonas reinhardtii microRNAs gene, it is characterised in that its sequence is SEQ ID NO.1 or SEQ ID NO.2.

2. the application in improving Chlamydomonas reinhardtii content of fatty acid of the microRNAs gene described in claim 1.