CN110386966A - Application of a gene sequence containing chloroplast transit peptide - Google Patents

Abstract

The invention discloses a kind of applications of gene order containing chloroplast transit peptides.By constructing the phosphatide containing chloroplast transit peptide sequence: diacylglycerol acyltransferase expression vector; conversion is into wild type Chlamydomonas reinhardtii and is overexpressed; strengthen chlamydomonas TAG synthesis capability, while reaching TAG accumulation and the synchronized purpose of micro algae growth.This transgenosis chlamydomonas is passed through normal TAP culture medium culture by the present invention, 22% and 32% is respectively increased in total fatty acid content and TAG accumulation, it realizes protein expression and oil and fat accumulation and grows synchronized target, this will have significant application prospect in microalgae bioengineering field to bioenergy important role.

Description

Application of a gene sequence containing chloroplast transit peptide

technical field

The invention belongs to the technical field of biological genetic engineering, and relates to transferring a target gene into the genome of Chlamydomonas reinhardtii, enhancing the accumulation of fatty acids in Chlamydomonas reinhardtii and synchronizing with growth, especially enhancing the accumulation of total fatty acids and TAG, and relates to a chloroplast transit peptide containing application of gene sequences.

Background technique

Microalgae have been an attractive potential source of biodiesel, and it is a good model in eukaryotes to study the synthesis of triglycerides (TAGs) through multiple pathways. In order to obtain more TAGs, microalgae are usually cultured under stress conditions, which limit the growth of algal cells, so the overall TAG yield is limited. After extensive experiments, the synchronization of TAG accumulation and algal cell growth was still not achieved. To efficiently accumulate TAG, achieving synchronization of TAG accumulation with algal cell growth is considered as one of the characteristics of an ideal strategy in the future.

In order to improve the yield of TAG, in addition to engineering methods, another method is to use molecular biology methods to modify genes related to TAG synthesis in microalgae. Now, most researchers focus on the modification of acyltransferase genes, such as glycerol triphosphate acyltransferase (GPAT, EC 2.3.1.15), lysophosphatidic acid acyltransferase (LPAAT, EC 2.3.1.51), acyl Transferases: syltransferases (DGAT, EC 2.3.1.20) and phospholipids: acylglycerol acyltransferases (PDAT, EC 2.3.1.158). Among the above acyltransferases, PDAT is the only acyltransferase that does not rely on acyl CoA, but synthesizes TAG through the acyl group of phospholipid as a donor. This conclusion is the first in castor oil by Anders and his colleagues. sub-determined (Dahlqvist A et al. 2000). Until then, DGAT was thought to be the only enzyme in the synthesis of DAG to TAG, and acyl-CoAs were thought to be indispensable for the synthesis.

After Anders et al. proposed this new idea about PDAT, more researchers focused on PDAT, studying the function of PDAT in different species in the source and further to direct TAG production, for example, Zhang et al. (Chisti Y. et al. 2007) showed that: in Arabidopsis, PDAT1 affects the normal development and growth of pollen and seeds; and Yoon et al. (Yoon K et al. 2012) pointed out that CrPDAT has broad substrate specificity and diverse lipase functions , CrPDAT can hydrolyze glycolipids, polar and neutral lipids, cholesterol lipids (similar to LCAT function), CrPDAT is more inclined to act on negatively charged phospholipids (PA/PS/PI/PG). While other sources of PDAT, including LuPDAT/ScPDAT/AtPDAT, tend to use positively charged phospholipids (PC/PE) as acyl donors. The results of this study also demonstrate the importance of CrPDAT.

By comparing the PDAT sequences of Chlamydomonas reinhardtii and other species, it was found that ScPDAT (GenBank: NM_001183185) and CrPDAT are highly similar, with a similarity of 35%. Through sequence alignment analysis, CrPDAT has 2 unique GAP regions, GAP1 (570-700) with 14 consecutive SG repeats, GAP2 (840-930) with 30 amino acid residues A and 13 G . Here, we regard ScPDAT as a mutant of CrPDAT with the GAP region removed, thus overexpressing ScPDAT in C. reinhardtii. This study will help to explore the regulatory function of PDAT in the mechanism of TAG accumulation.

Unlike cytoplasmic proteins, nuclear-encoded chloroplast proteins require the help of chloroplast transit peptide (cTP) to locate and correctly fold in the chloroplast to function. Therefore, for exogenous genes, they also need to be inserted upstream of their functional domains. Chloroplast transit peptide fragment.

SUMMARY OF THE INVENTION

The purpose of this method is a method for increasing the oil accumulation of microalgae in synchronization with growth.

To achieve the above object, the present invention provides a phospholipid diacylglycerol acyltransferase gene and an expression vector that can be localized in chloroplasts, and transforms the recombinant expression vector to obtain transgenic Chlamydomonas, which increases fatty acid accumulation and synchronizes with growth.

An application of a gene sequence containing a chloroplast transit peptide is characterized in that a target gene is inserted downstream of the gene sequence of the chloroplast transit peptide, so that the encoded protein of the target gene is located in the chloroplast.

The gene sequence containing the chloroplast transit peptide is shown in SEQ ID NO. 2 of the sequence table.

The target gene is a gene base sequence encoding the functional domain of phospholipid diacylglycerol acyltransferase.

Described target gene is SEQ ID NO.1;

Or, the gene sequence of the phospholipid diacylglycerol acyltransferase is a gene sequence whose amino acid sequence of the encoded protein has a similarity of 30% or more with the amino acid sequence encoded by the gene of SEQ ID NO.1.

Applied to increase fatty acid accumulation in Chlamydomonas and synchronize with growth.

A recombinant plasmid is characterized in that: the plasmid contains the gene sequence of the chloroplast transit peptide of claim 1 and the fragment inserted into the target gene downstream.

The gene sequence of the chloroplast transit peptide is SEQ ID NO.2;

Described target gene sequence is SEQ ID NO.1,

Or, the amino acid sequence of the encoded protein has a similarity of 30% or more with the amino acid sequence encoded by the SEQ ID NO.1 gene sequence;

A transgenic Chlamydomonas containing the recombinant plasmid.

The application of the transgenic Chlamydomonas in the process of increasing fatty acid accumulation in Chlamydomonas and synchronizing with growth.

The construction method of transgenic Chlamydomonas, the steps are as follows:

Obtained phospholipids containing chloroplast transit peptide sequence: diacylglycerol acyltransferase gene;

Construction of chloroplast-type phospholipid: diacylglycerol acyltransferase gene recombinant expression vector;

Transforming the recombinant expression vector into Chlamydomonas reinhardtii cells to obtain transgenic Chlamydomonas reinhardtii;

The transformation method adopts electroporation method, glass bead method, gene gun method, etc.;

By culturing the transgenic Chlamydomonas in normal TAP medium; measuring the growth indicators of the transgenic Chlamydomonas, including algal cell density and chlorophyll fluorescence; simultaneously measuring the protein expression and fatty acid content of exogenous genes, and comparing the wild type Various indicators of Chlamydomonas reinhardtii were obtained, and a transgenic Chlamydomonas that was synchronized with the growth and increased the oil accumulation of microalgae was obtained.

The invention overcomes the unstable factors in gene silencing technology or other physical and chemical technologies, and provides a method for improving the accumulation of fatty acids in Chlamydomonas reinhardtii and synchronizing with growth through genetic optimization and modification. A chloroplast transit peptide was fused to the front end of the exogenous gene of phospholipid:diacylglycerol acyltransferase, and transformed into Chlamydomonas reinhardtii, thereby obtaining a transgenic Chlamydomonas with improved fatty acid accumulation and growth synchronization.

The technical scheme of the present invention is as follows:

(1) Transgenic recipient algal strain and its culture

C. reinhardtii was cultured in TAP medium under the conditions of temperature of 22-25℃ and light of 50μmol photons m ^-2 s ^-1 , and wild-type C. reinhardtii CC-137 was selected as the receptor of the target gene.

(2) Construction of target gene expression vector

The recombinant expression vector pChlamy-ScPDAT was constructed based on the expression system of pChlamy plasmid and fused with ScPDAT as shown in FIG. 1 , as shown in FIG. 1 .

The ScPDAT target gene includes the ScPDAT gene (as shown in SEQ ID No. 1 in the sequence table) and the 279bp chloroplast transit peptide (cTP) fused to the front end of the gene (as shown in SEQ ID No. 2 in the sequence table), in addition to , pChlamy vector sequence also includes 673bp PUCori; 495bp Hsp70A-RbcS2 hybrid promoter; 144bp Int-1Rbc S2; 312bp β2-tubulin; 1310bp Hygromycin; 860bp Ampicilin; 51bp Pbla.

(3) Transfer the recombinant expression vector into Chlamydomonas reinhardtii, and integrate it into the Chlamydomonas nuclear genome

The recombinant plasmid was linearized by electroporation, transformed into wild-type Chlamydomonas reinhardtii CC-137, screened on TAP plate containing hygromycin resistance, and positive algal colonies were selected for single colony PCR detection. The expression of ScPDAT protein was detected by western blot method.

(4) Growth assay of transformed strains and wild strains

Under normal light and temperature, the transgenic Chlamydomonas reinhardtii were screened. Three parallels were set for each algae, and the optical density of the cells was measured once a day. At the same time, the wild-type Chlamydomonas reinhardtii cc-137 was cultivated, and the growth curve and chlorophyll were measured. Fluorescence curve; and the fatty acid and total lipid content of transgenic Chlamydomonas were measured.

The beneficial effects of the present invention are:

Compared with the prior art, the present invention has the following advantages and effects:

1. The present invention proves that the phospholipid with chloroplast transit peptide is expressed in wild-type Chlamydomonas reinhardtii: the diacylglycerol acyltransferase gene is cultivated under certain conditions, which can realize the coupled expression of exogenous proteins with growth, and promote the expression of exogenous proteins. Transgenic Chlamydomonas in which TAG accumulation and growth are synchronized.

2. Under the same conditions of the obtained transgenic Chlamydomonas and wild-type Chlamydomonas reinhardtii, the total fatty acid and TAG were increased by 22% and 32% respectively, and the expression of exogenous protein and the accumulation of fatty acid were synchronized with the growth of Chlamydomonas reinhardtii.

3. This transgenic Chlamydomonas improves fatty acid accumulation and synchronizes with growth, which will also contribute to the development of downstream industries of microalgae.

4. In the development and application of microalgae, the method of stress culture is not adopted to realize the synchronization of oil accumulation and algal cell growth, which is an important significance for the development of microalgae bioenergy.

Description of drawings

Figure 1: pChlamy-ScPDAT plasmid sequence.

Figure 2: PCR validation of transgenic Chlamydomonas Scpdat. The g lane is the PC-F/R primer verification result, and the h lane is the ScPDAT-F/R primer verification result.

Figure 3: Western blot validation of transgenic Chlamydomonas Scpdat, and growth curves of transgenic Chlamydomonas Scpdat and wild strain CC-137.

Figure 4: Chlorophyll fluorescence of transgenic Chlamydomonas Scpdat and wild strain CC-137.

Figure 5: Total fatty acid content of transgenic Chlamydomonas Scpdat and wild strain CC-137.

Figure 6: SFA, MUFA and PUFA content of transgenic Chlamydomonas Scpdat and wild strain CC-137.

Figure 7: Lipid content of transgenic Chlamydomonas Scpdat and wild strain CC-137.

Figure 8: Significantly different fatty acid content in SQDG, MGDG, DGDG, DGTS and TAG between transgenic Chlamydomonas Scpdat and wild strain CC-137.

Detailed ways

The present invention provides a transgenic Chlamydomonas by transforming and overexpressing the ScPDAT gene, which can efficiently accumulate oil. We inserted a piece of chloroplast transit peptide (cTP) upstream of the ScPDAT gene, and at the same time used the Hsp70-RBSC2 hybrid promoter to express ScPDAT in Chlamydomonas reinhardtii CC-137 to obtain a transformed algal strain named Scpdat. 137, especially for lipid accumulation and fatty acid changes. In this way, we can gain insight into the function of PDAT in lipid accumulation.

A recombinant expression vector was constructed by RF cloning technology, into which an expression vector with a resistance gene and a high-efficiency Hsp70-RBSC2 hybrid promoter was inserted, transformed into the genome of wild-type Chlamydomonas reinhardtii CC-137, and the transgenic Chlamydomonas was obtained by screening. Multiple passages demonstrate its genetic stability. The screened transgenic Chlamydomonas was cultured, and the algal cell density, chlorophyll fluorescence, fatty acid content and other indicators were measured and analyzed.

Example 1: Construction of phospholipid:diacylglycerol acyltransferase gene expression vector

The algal strain selected in this example is wild-type Chlamydomonas reinhardtii CC-137 as the receptor for transgenic operation.

The medium used when Chlamydomonas reinhardtii is cultivated is TAP medium, and the TAP medium formula is as follows:

Mother liquor 1 (salt solution): dilute 20g NH ₄ Cl, 5g MgSO ₄ ·7H ₂ O, 2.5g CaCl ₂ ·2H ₂ O to 500ml deionized water;

Mother liquor 2 (phosphate solution): dilute 10.8g K ₂ HPO ₄ and 5.6g KH ₂ PO ₄ in 500 ml of deionized water;

Mother liquor 3 (Hutner's trace metal salt solution): 50.0 g Na ₂ -EDTA·2H ₂ O, 22 g ZnSO ₄ ·7H ₂ O, 5.06 g MnCl ₂ ·4H ₂ O, 1.61 g CoCl ₂ ·6H ₂ O, 1.57 g CuSO ₄ ·5H ₂ O, 1.10 g (NH ₄ ) ₆ Mo ₇ O ₂₄ · 4H ₂ O, 4.99 g FeSO ₄ · 7H ₂ O in 500 ml deionized water; dissolve Nitrilotriacetic acid, adjust the pH to 7.0 with about 7.3 g KOH , Dissolve other salts separately, combine and adjust pH to 6.8 with _NaOH or _H2SO4

To configure 1L TAP, you need to add: 20ml of 1M Tris (sigma), 10ml of salt solution (mother solution 1), 1 ml of phosphate solution (mother solution 2), 1ml of Hutner's trace elements (mother solution 3), 1ml of glacial acetic acid, and the volume is adjusted to 1L.

The culture conditions of Chlamydomonas reinhardtii are as follows: the temperature is 22～25℃ and the illumination is 50～100μmolphotons m ^-2 s ^-1 under the condition of continuous light culture, and the growth period is 5 days.

(1) Construction of target gene expression vector

The pPICZαA-ScPDAT plasmid was obtained by artificially synthesizing the ScPDAT gene, and the 1957bp ScPDAT fragment on the pPICZαA-ScPDAT expression vector was amplified by the method of RF cloning. The specific PCR system: 50μl system, pPICZαA-ScPDAT plasmid 80～100ng, 0.4μM ScPDAT-specific primer (ScPDAT-F/R), 25μl PrimeSTAR Max DNA Polymerase (TaKaRa, Dalian, CodeNo.R045Q), reaction conditions: pre-denaturation at 94°C for 5min; then denaturation at 94°C for 30s; annealing at 55°C for 30s; Extend at 72°C for 30s; 30 cycles; the last 72min extension for 7min, and keep at 4°C after the reaction is completed to obtain the ScPDAT fragment. Then the ScPDAT fragment was used as a primer, and it was integrated into the pChlamy expression vector. The specific PCR reaction system is shown in Table 1. The reaction conditions: pre-denaturation at 98°C for 5 min; then denaturation at 98°C for 10s; annealing at 60°C for 5s; extension at 72°C for 2 min ; 30 cycles; the last 72min extension 7min, after the completion of the reaction kept at 4 ℃. The recombinant plasmid pChlamy-ScPDAT was obtained, and the obtained PCR product was transformed into E. coli DH5α competent cells, then spread on LB dishes containing ampicillin, and cultured at 37°C overnight. Single clones were picked, plasmids were extracted, and recombinant plasmids were obtained, which were verified by sequencing.

Table 1 PCR reaction system (50μl)

component Final concentration pChlamy 300pM PrimeSTAR HS DNA premixed polymerase 1.25U Primer (ScPDAT) 6nM dNTPs 200μM Buffer 1×

In the embodiment, ScPDAT-specific primer ScPDAT-F: 5'

GATTCGAGGTACCCTTCACCACGACCCCA ACAAAAGGCA 3'.

Example 2: Genetic Transformation of Chlamydomonas reinhardtii

The recombinant plasmid pChlamy-ScPDAT was extracted by conventional SDS alkaline lysis method

The specific steps of "electric transfer method" are as follows:

(1) Obtain a linearized plasmid

The plasmid was extracted using a plasmid extraction kit, the concentration of the plasmid was determined, and the plasmid was linearized with Sca I or Pvu I restriction enzymes. After the reaction, the linearized recombinant plasmid was directly recovered by alcohol precipitation.

(2) Cultivation of Chlamydomonas reinhardtii

Pick out the wild-type CC-137 single clone of C. reinhardtii from the TAP plate and put it into a sterile six-well plate for TAP culture, put it in a 24h light incubator, 28℃, 50～100μE m ^-2 s ^-1 , Chlamydomonas reinhardtii CC -137 OD ₇₅₀ = 0.6~0.7, take algal cells and dilute them to 50ml TAP medium OD ₇₅₀ = 0.05~0.06, after algal cells are cultured in a 100ml conical flask for 24h, the density of algal cells is determined to reach 0.3~0.5; if If the cell density is not reached, it needs to continue to culture; if the cell density exceeds the standard value, it needs to be re-cultured.

(3) The algal cells and the linearized recombinant plasmid are mixed and electroshocked

Take 15ml of suitable Chlamydomonas reinhardtii, centrifuge at 2500rpm for 10min, remove the supernatant, use a pipette tip to aspirate the supernatant, resuspend the algal cells with 250μl of 40mM sucrose TAP solution and add it to the electric shock cup. 2-3 μg of linearized plasmid, shake the electric shock cup gently to mix, and place it at room temperature for 5 minutes before electric shock, voltage=600V, capacitance=50μF, resistance=infinite. (No plasmid is added to the blank control, only the corresponding volume of sterile water can be added)

(4) Recovery culture

About 5 minutes after the electric shock, add 5 ml of 40 mM sucrose TAP solution to the well plate, add 125 μl of the mixture after electric shock to 2 wells of the six-well plate, rinse with 1 ml of 40 mM sucrose TAP solution, and then add them to the well plate respectively. Each 500μl was placed in a lighted incubator for 24h.

(5) Screening of coated plates

Take 2ml of algal cells in the six-well plate and put them in a 15ml centrifuge tube, centrifuge at 2500rpm for 10min, first suck out the supernatant with a pipette tip, resuspend the algal cells with 150μl of 40mM sucrose TAP solution, and spread them on a 10μg/mL hygromycin solution. The growth of algal cells was observed on the TAP plate of virgin algal cells, and the appearance of algal cells was recorded.

Example 3: Screening and identification of transgenic Chlamydomonas reinhardtii

Since the recombinant plasmid expression vector contains the hygromycin resistance gene, positive transgenic Chlamydomonas can grow on TAP plates containing hygromycin resistance. The detection of transgenic Chlamydomonas includes single colony PCR verification at the gene level and western blot verification at the protein level.

(1) PCR verification of single algal drop of transgenic Chlamydomonas Scpdat at the gene level

Pick a single algae on the plate and drop it into a PCR tube, add 10 μl of sterile water to resuspend the algal cells, cook at 98°C for 20-30min, centrifuge to take the supernatant, use PC-F/R primer first, and then use the recombinant expression vector The ScPDAT-specific primer (ScPDAT-F/R) was used to amplify the ScPDAT fragment from the algae by PCR technology, using PrimeSTAR Max DNA Polymerase, the reaction system was 25 μl, and the reaction conditions: 98 ℃ pre-denaturation for 15min; 10s; annealing at 55°C for 10s; extension at 72°C for 10s; 30 cycles; kept at 4°C after the reaction was completed. It was confirmed that the target gene fragment ScPDAT had been transferred into Chlamydomonas reinhardtii by electrophoresis bands.

In the example, the primer PC-F of the vector itself: 5' GATTCGAGGTACCCTTCACC 3'.

(2) Western blot verification of transgenic Chlamydomonas Scpdat at the protein level

Western blot was used to detect the expression of ScPDAT protein in Scpdat algal cells. Since the end of ScPDAT was tagged with histidine, His-Taq antibody was used to detect the expression of ScPDAT protein. The specific process is as follows:

a) Algal cell sample processing: 50 ml of Scpdat and CC-137 algal cells were harvested regularly every day, centrifuged at 12,000 g for 10 min in a refrigerated centrifuge, the supernatant was removed, and the algal cells were quickly frozen in liquid nitrogen, and then placed in -80 Store in the refrigerator at °C and store overnight.

b) Extraction of algal cell protein samples: Take out each algal cell sample, thaw it on ice, add 20-30 ml of absolute ethanol to each sample, and repeat this operation to remove the pigment, and then rinse with deionized water for two times. After the algal cells were obtained, 1 ml of lysis buffer was added to each sample, and protease inhibitors were added at the same time, and the cells were disrupted on a tissue cell shaker. Refrigerate and centrifuge at 12000g for 20min, suck the supernatant to obtain protein samples of algal cells, and measure the protein concentration of each sample by Coomassie brilliant blue method.

c) Protein separation by SDS-PAGE electrophoresis: uniformly load the protein of each sample, mix the protein and loading buffer (Coomassie Brilliant Blue G-250), heat and denature them, add them to a 10% SDS-PAGE gel, and conduct constant pressure Proteins were separated by electrophoresis at 90V.

d) PVDF membrane transfer and protein color development: first cut off the concentrated gel part of the SDS-PAGE gel, leave the gel at the position of the target protein and put it into the transfer solution, and at the same time put the cut PVDF membrane with the same size as the gel into methanol In the solution for 10min, take out the PVDF membrane and put it in the transfer solution, put the glue and membrane into the electroporator for electroporation. Considering the size of the ScPDAT protein, the electroporation is set to 250mA and the constant current is transferred for 1h. Blocked in milk for 1 hour, briefly rinsed twice with 1×PBST, and incubated overnight in the primary antibody solution. Here, the samples at each time point need to be incubated with His-Taq and GAPDH antibodies respectively, and the primary antibodies are both diluted 1:1000. After the primary antibody incubation, wash three times/6min with 1 × PBST, put in the secondary antibody solution and incubate for 2h. After completion, wash the membrane and put it on Tanon ^TM High-sig ECL Western Blotting Substrate for exposure and color development. This step is performed on the Multifunctional imager: FUSION-FX5-820. Western blot results are shown in Figure 3

Example 4: Cultivation of transgenic Chlamydomonas reinhardtii

(1) Growth curve and chlorophyll fluorescence measurement of transgenic Chlamydomonas

By culturing the transgenic Chlamydomonas and wild strains for 5 consecutive days, OD ₇₅₀ and F _v /F _m were measured regularly, and combined with the expression of ScPDAT protein, as shown in Figure 3, the expression of exogenous ScPDAT and its effect on growth can be seen . The ScPDAT transformants showed a delay of about one day in the initial growth stage, and the expression of ScPDAT reached the maximum at the end of the exponential phase with the growth, and then began to decline. At the same time, the changes of F _v /F _m were observed. As shown in Figure 4, the effect on the chlorophyll fluorescence kinetic parameter F _v / F _m was mainly manifested in the second day when the growth rate was the fastest, which may have interfered with the system in the chloroplast. Synthesis.

(2) Fatty acid composition and total lipid content of transgenic Chlamydomonas reinhardtii

Take about 5 mg of algal cells, add 5% sulfuric acid methanol solution, and add internal standard C17 at the same time, transesterify in an oil bath at 70 °C for 1 h, add n-hexane and deionized water, shake to take the upper layer, remove moisture and put it in In the chromatographic bottle, the data was analyzed by gas chromatography. As shown in Figure 5, the changes in the total fatty acid content of the transgenic Chlamydomonas, the transfer of ScPDAT caused the total fatty acid content of the transgenic Chlamydomonas to increase by 22%. Figure 6 shows the analysis of the content of fatty acids with different degrees of saturation. The results showed that ScPDAT mainly caused the accumulation of polyunsaturated fatty acids in transgenic Chlamydomonas.

By extracting oil from each sample and then separating it by TLC, the corresponding esters were obtained, and the esters of MGDG, DGDG, SQDG, DGTS, and TAG were quantitatively transesterified to determine the fatty acid composition and content of various esters, as shown in Figure 7. As shown in the analysis of the content of different functional esters, the transformation of _ScPDAT leads to the decrease of MGDG, an important lipid of the photosynthetic system, which is a reason for the decrease of Fv _/ Fm, but at the same time promotes the production of TAG. From the perspective of fatty acids, as shown in Figure 8, it is a component with obvious changes in fatty acid content in different lipids. The transfer of ScPDAT enhanced the transfer of lipids to TAG in chloroplasts.

Example 5: Demonstration of ScPDAT gene localization in chloroplasts

Through the research results of Yang et al. (Miao Y et al. 2017) on the characteristic fatty acid composition of chloroplasts, it is found from Figure 8 that the C16 series of fatty acids in the constructed transgenic Chlamydomonas have accumulated a lot, especially C16 in the accumulation of TAG: 4n3, C16:3n6, these are fatty acids synthesized through the chloroplast prokaryotic pathway, indicating that the introduced exogenous PDAT played a role in the chloroplast.

(a) Sequence features:

*Length: 1674bp

*Type: Nucleic acid

*Chain type: double chain

*Topology: Linear

(b) Molecular type: cDNA

(c) Assumption: No

(d) Antonym: No

(e) Original Source: Synthetic

(a) Sequence features:

*Length: 279bp

*Type: Nucleic acid

*Chain type: double chain

*Topology: Linear

(b) Molecular type: cDNA

(c) Assumption: No

(d) Antonym: No

(e) Original Source: Synthetic.

sequence listing

<110> Dalian Institute of Chemical Physics, Chinese Academy of Sciences

<120> Application of a gene sequence containing a chloroplast transit peptide

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1674

<212> DNA

<213> Artificial Sequence

<400> 1

gatagcgact tgtttgacaa ctttgtaaat tttgattcac ttaaagtgta tttggatgat 60

tggaaagatg ttctcccaca aggtataagt tcgtttattg atgatattca ggctggtaac 120

tactccacat cttctttaga tgatctcagt gaaaattttg ccgttggtaa acaactctta 180

cgtgattata atatcgaggc caaacatcct gttgtaatgg ttcctggtgt catttctacg 240

ggaattgaaa gctggggagt tattggagac gatgagtgcg atagttctgc gcattttcgt 300

aaacggctgt ggggaagttt ttacatgctg agaacaatgg ttatggataa agtttgttgg 360

ttgaaacatg taatgttaga tcctgaaaca ggtctggacc caccgaactt tacgctacgt 420

gcagcacagg gcttcgaatc aactgattat ttcatcgcag ggtattggat ttggaacaaa 480

gttttccaaa atctgggagt aattggctat gaacccaata aaatgacgag tgctgcgtat 540

gattggaggc ttgcatattt agatctagaa agacgcgata ggtactttac gaagctaaag 600

gaacaaatcg aactgtttca tcaattgagt ggtgaaaaag tttgtttaat tggacattct 660

atgggttctc agattatctt ttactttatg aaatgggtcg aggctgaagg ccctctttac 720

ggtaatggtg gtcgtggctg ggttaacgaa cacatagatt cattcattaa tgcagcaggg 780

acgcttctgg gcgctccaaa ggcagttcca gctctaatta gtggtgaaat gaaagatacc 840

attcaattaa atacgttagc catgtatggt ttggaaaagt tcttctcaag aattgagaga 900

gtaaaaatgt tacaaacgtg gggtggtata ccatcaatgc taccaaaggg agaagaggtc 960

atttgggggg atatgaagtc atcttcagag gatgcattga ataacaacac tgacacatac 1020

ggcaatttca ttcgatttga aaggaatacg agcgatgctt tcaacaaaaa tttgacaatg 1080

aaagacgcca ttaacatgac attatcgata tcacctgaat ggctccaaag aagagtacat 1140

gagcagtact cgttcggcta ttccaagaat gaagaagagt taagaaaaaa tgagctacac 1200

cacaagcact ggtcgaatcc aatggaagta ccacttccag aagctcccca catgaaaatc 1260

tattgtatat acggggtgaa caacccaact gaaagggcat atgtatataa ggaagaggat 1320

gactcctctg ctctgaattt gaccatcgac tacgaaagca agcaacctgt attcctcacc 1380

gagggggacg gaaccgttcc gctcgtggcg cattcaatgt gtcacaaatg ggcccagggt 1440

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gatcgatttg atatacgtgg tggagcaaaa agcgccgaac acgtagacat cctcggcagc 1560

gcggagttga acgattacat cttgaaaatt gcaagcggta atggcgatct cgtcgagcca 1620

cgccaattgt ctaatttgag ccagtgggtt tctcagatgc ccttcccaat gtga 1674

<210> 2

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<212> DNA

<213> Artificial Sequence

<400> 2

acgaccccaa caaaaggcaa tagcaatgct cgacaacgaa agaagggagg aaccagtgcc 60

gaggcgtccg cggcgacacc gtcaaaagcc aagcctggaa gggaccataa cgtgcacgca 120

acccccagcc actcgcactc gcactcgcac tcccagtctc agcagcaccg gcaaggaccg 180

catgctgccc aaccaaaatc ggagcggcgt ctggtgctat ggctggcggc ggcgggggtg 240

gtgctgctgc cgctggtgct gctgccgccc gcactaagc 279

Claims (10)

1. An application of a gene sequence containing a chloroplast transit peptide, characterized in that a target gene is inserted downstream of the gene sequence of the chloroplast transit peptide, so that the encoded protein of the target gene is located in the chloroplast. 2 . The application of the chloroplast transit peptide-containing gene sequence according to claim 1 , wherein the chloroplast transit peptide-containing gene sequence is shown in SEQ ID NO. 2 of the sequence table. 3 . 3 . The application of the gene sequence containing a chloroplast transit peptide according to claim 1 or 2 , wherein the target gene is a gene base sequence containing a functional domain encoding a phospholipid diacylglycerol acyltransferase. 4 . 4. the application of the gene sequence containing chloroplast transit peptide as claimed in claim 3, is characterized in that: described target gene is SEQ ID NO.1; Or, the amino acid sequence of the encoded protein has a similarity of 30% or more with the amino acid sequence encoded by the gene of SEQ ID NO.1. 5. The application according to any one of claims 1-4 containing the gene sequence of the chloroplast transit peptide, characterized in that: it is applied to increase fatty acid accumulation in Chlamydomonas and synchronize with growth. 6 . A recombinant plasmid, characterized in that: the plasmid contains the gene sequence of the chloroplast transit peptide of claim 1 and the segment of the target gene inserted downstream thereof. 7. recombinant plasmid as claimed in claim 6, is characterized in that: The gene sequence of the chloroplast transit peptide is SEQ ID NO.2; Described target gene sequence is SEQ ID NO.1, Or, the amino acid sequence of the encoded protein has a similarity of 30% or more with the amino acid sequence encoded by the gene of SEQ ID NO.1. 8. A transgenic Chlamydomonas comprising the recombinant plasmid of claim 5 or 6. 9. The application of the transgenic Chlamydomonas as claimed in claim 8 in the process of increasing fatty acid accumulation in Chlamydomonas and synchronizing with growth. 10. A construction method with the Chlamydomonas that improves fatty acid accumulation and growth synchronization, the steps are as follows: 1) construct the overexpression vector that contains the gene sequence of the described chloroplast transit peptide of claim 1 and the fragment described in the downstream insertion target gene; The gene sequence of the chloroplast transit peptide is SEQ ID NO.2; Described target gene is SEQ ID NO.1, Or, a gene whose amino acid sequence of the encoded protein has a similarity of 30% or more with the amino acid sequence encoded by the SEQ ID NO.1 gene; 2) transforming the chloroplast-type phospholipid diacylglycerol acyltransferase overexpression vector obtained in step 1) into a Chlamydomonas cell to obtain a transgenic Chlamydomonas overexpressing phospholipid diacylglycerol acyltransferase; The construction process of the expression vector is as follows: using RF cloning technology to construct a recombinant expression vector; The transformation method is as follows: the transformation method adopts electroporation method, glass bead method, and gene gun method; The Chlamydomonas is wild-type Chlamydomonas reinhardtii.