阅读说明：本技术 银杏长链非编码RNA Lnc2L和Lnc2S及其载体和应用 (Ginkgo biloba long-chain non-coding RNA Lnc2L and Lnc2S, and vector and application thereof ) 是由 刘思安 操萌 孟钊龙 王莉 于 2021-08-04 设计创作，主要内容包括：本发明公开了银杏长链非编码RNA Lnc2L和Lnc2S及其载体和应用,所述银杏长链非编码RNA Lnc2L或者Lnc2S,其核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。本发明提供的非编码RNA在银杏中过量表达后,可以用于调控银杏类黄酮合成,过量表达长链非编码RNA Lnc2L后类黄酮含量显著增加；而过量表达长链非编码RNA Lnc2S后类黄酮含量显著降低。这表明Lnc2L和Lnc2S是调控银杏类黄酮合成的关键lncRNA,通过Lnc2L和Lnc2S的过量表达或敲除来调控银杏类黄酮的含量,能够按照需求培育出类黄酮含量高的银杏,因此在银杏分子育种过程中具有重要的应用价值。(The invention discloses a gingko long-chain non-coding RNA Lnc2L and Lnc2S, a vector and an application thereof, wherein the nucleotide sequences of the gingko long-chain non-coding RNA Lnc2L or Lnc2S are respectively shown as SEQ ID No.1 and SEQ ID No. 2. After the non-coding RNA provided by the invention is over-expressed in ginkgo, the non-coding RNA can be used for regulating and controlling the synthesis of ginkgo flavonoids, and the flavonoid content is obviously increased after long-chain non-coding RNA Lnc2L is over-expressed; and after the long-chain non-coding RNA Lnc2S is over-expressed, the flavonoid content is obviously reduced. This shows that Lnc2L and Lnc2S are key lncRNA for regulating and controlling ginkgo flavonoid synthesis, the content of ginkgo flavonoids is regulated and controlled through overexpression or knockout of Lnc2L and Lnc2S, and ginkgo with high flavonoid content can be cultured according to requirements, so that the ginkgo molecular breeding method has important application value in the ginkgo molecular breeding process.)

1. The nucleotide sequences of the long-chain non-coding RNA Lnc2L or Lnc2S of ginkgo are respectively shown as SEQ NO.1 and SEQ NO. 2.

2. An overexpression vector comprising the long non-coding RNA Lnc2L or Lnc2S of Ginkgo biloba of claim 1.

3. The overexpression vector according to claim 2, wherein the overexpression vector is assembled to form a strong expression promoter CAMV35S at the 5' end of the long non-coding RNA Lnc2L or Lnc2S of ginkgo biloba.

4. The overexpression vector of claim 2, wherein the overexpression vector is characterized in that the strong terminator NOS-ter is assembled at the 3' end of the long non-coding RNA Lnc2L or Lnc2S of Ginkgo biloba.

5. The overexpression vector according to claim 2, wherein the overexpression vector is assembled with nptii gene expression cassette as a selection marker of transgenic ginkgo biloba.

6. The overexpression vector according to claim 2, wherein said overexpression vector is assembled with LB and RB sequences, which facilitate the integration of the gene expression cassette and the selectable marker gene nptiii assembled therebetween into the chromosome of the receptor cells of ginkgo biloba.

7. A host cell comprising the over-expression vector of claim 2.

8. The host cell according to claim 7, wherein the host cell is preferably an Agrobacterium-derived strain.

9. The use of the long non-coding RNA Lnc2L or Lnc2S of Ginkgo biloba of claim 1 for modulating flavonoid synthesis.

10. The application according to claim 9, wherein the application is: the long-chain non-coding RNA Lnc2L or Lnc2S is transferred into the gingko callus, the flavonoid content of the transgenic gingko callus over-expressing the long-chain non-coding RNA Lnc2L is obviously increased, and the flavonoid content of the transgenic gingko callus over-expressing the long-chain non-coding RNA Lnc2S is obviously reduced.

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to two novel ginkgo long-chain non-coding RNAs Lnc2L and Lnc2S, and a vector and application thereof.

Background

Ginkgo biloba (Gingo biloba L.) is an important economic tree type, and the folium ginkgo, the kernel ginkgo and the testa ginkgo contain medicinal components, which are called as 'activated stones which are precious for the whole body'. Ginkgo biloba has been used as a medicine for over 600 years, and its efficacy is first described in Shen nong's herbal Jing. Ginkgo leaves contain abundant secondary metabolites, such as active substances like flavonoid, terpene lactone, polyprenol, etc., and are widely used in a large number of medicines, health products and foods. Ginkgo biloba leaf Extract (GbE) is a raw material of various medicines, and has certain effects of preventing and treating early-stage Alzheimer disease, cardiovascular diseases and the like. At present, more than 30 related medicines of ginkgo leaf extracts are internationally available. Flavonoid compounds are the main active ingredient of GbE, and more than 40 kinds of flavonoid compounds have been isolated from ginkgo biloba, and the flavonoid compounds widely studied in recent years are mainly flavonol, anthocyanin and procyanidin. The flavonoid compounds are more than 9000 types, widely exist in organs such as leaves and roots of plants and participate in the growth and development of the plants and the regulation and control of adverse reactions. The flavonoid compound has important pharmacological action, plays an important role in preventing and treating cardiovascular sclerosis, resisting oxidation, resisting aging, resisting tumors and the like, and is widely applied to the fields of health care and medical treatment.

Long non-coding RNAs (lncRNAs) are ubiquitous transcripts over 200nt in length that have little or no protein coding ability but are functional. lncRNA is involved in a variety of basic biological processes at the transcriptional, post-transcriptional and epigenetic levels as key regulatory molecules. lncRNA has now been found to play an important role in plant growth, development and stress tolerance.

In plants, some important lncRNAs such as OsPI, TPS11, IPS1, COLDAIR, LDMAR and the like play important roles in various basic processes such as vernalization, male sterility, nodule formation, photomorphogenesis and phosphate (Pi) uptake. ENOD40 is the first plant lncRNA predicted to exert a riboregulatory effect in the nodulation of alfalfa, soybean. However, woody plants have been less studied about lncrnas than herbaceous plants.

With the development of high throughput sequencing technology, many lncRNAs have been found in plants. There are approximately 40000 potential lncrnas in arabidopsis, 27065 in rice and 20163 in maize. The research reports about plant lncRNA are more, but most of the research reports are concentrated on model plants such as arabidopsis thaliana and rice, and the like, and participate in regulating and controlling growth, development, signal transduction, morphogenesis, stress response and other life processes of the plants, the research reports about woody plant lncRNA, particularly gymnosperms are relatively less, for example, the reports about ginkgo are fresh, and meanwhile, no related report about lncRNA for regulating and controlling synthesis of ginkgo flavonoids is provided at present.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the defects in the prior art, the invention aims to provide two key lncRNA Lnc2L and Lnc2S for regulating and controlling the synthesis of ginkgo flavonoids, and the content of the ginkgo flavonoids can be controlled by regulating and controlling the expression of the two lncRNA.

The invention also provides a vector for regulating expression of key lncRNA Lnc2L and Lnc2S synthesized by ginkgo flavonoids and application thereof.

The technical scheme is as follows: in order to achieve the purpose, the two novel ginkgo long-chain non-coding RNAs Lnc2L or Lnc2S have nucleotide sequences shown as SEQ NO.1 and SEQ NO.2 respectively.

The invention relates to an overexpression vector containing gingko long-chain non-coding RNA Lnc2L or Lnc 2S.

Wherein the overexpression vector is assembled at the 5' end of the gingko long-chain non-coding RNA Lnc2L or Lnc2S to form a strong expression promoter CAMV35S which can enable Lnc2L or Lnc2S to be efficiently expressed in gingko bodies.

Wherein, the overexpression vector is assembled with a strong terminator NOS-ter at the 3' end of the gingko long-chain non-coding RNA Lnc2L or Lnc2S, and the strong terminator NOS-ter can effectively terminate the transcription of Lnc2L and Lnc 2S.

Wherein, the over-expression vector is assembled with an NPT II gene expression box, and can be used as a screening marker of transgenic ginkgo to screen the transgenic ginkgo by using kanamycin.

Wherein, the overexpression vector is assembled with LB and RB sequences, and facilitates the integration of a gene expression frame and a screening marker gene NPT II assembled between the LB and RB sequences into the chromosome of the gingko receptor cell.

Host cells containing the overexpression vectors of the invention.

Wherein, the host cell takes agrobacterium as an original strain.

The application of the gingko long-chain non-coding RNA Lnc2L or Lnc2S in the regulation of flavonoid synthesis is disclosed.

Preferably, the application is: the long-chain non-coding RNA Lnc2L or Lnc2S is transferred into the gingko callus, the flavonoid content of the transgenic gingko callus over-expressing the long-chain non-coding RNA Lnc2L is obviously increased, and the flavonoid content of the transgenic gingko callus over-expressing the long-chain non-coding RNA Lnc2S is obviously reduced.

The invention takes ginkgo leaves as a material and clones two new lncRNA Lnc2L and Lnc 2S. Meanwhile, the gene is constructed into AN overexpression vector pRI 101-AN (TaKaRa, Japan) by enzyme digestion and ligation, and 35S:: Lnc2L and 35S:: Lnc2S vectors are constructed by homologous recombination technology. After the gene is positioned behind a promoter CaMV35S, under the drive of a promoter CaMV35S, Lnc2L and Lnc2S can be efficiently expressed in ginkgo callus, so that the synthesis of flavonoids is regulated.

The invention clones two new genes lncRNA Lnc2L and Lnc2S in ginkgo for the first time, discovers that lncRNA Lnc2L and Lnc2S can regulate and control synthetic genes lncRNA Lnc2L and Lnc2S of ginkgo flavonoids for the first time are two ginkgo long-chain non-coding RNAs coded by the same gene, two brand-new ginkgo long-chain non-coding RNAs Lnc2L and Lnc2S have a difference of 98bp, and the rest sequences are completely consistent. The Lnc2L provided by the invention can be used for regulating and controlling the synthesis of ginkgo flavonoids after being over-expressed in ginkgo, which indicates that Lnc2L and Lnc2S are key lncRNA for regulating and controlling the synthesis of ginkgo flavonoids, the content of ginkgo flavonoids is regulated and controlled by over-expression or knockout of Lnc2L and Lnc2S, and ginkgo with high flavonoid content can be cultured according to requirements, so that the method has important application value in the molecular breeding process of ginkgo. The research result provides a theoretical basis for improving the synthesis and accumulation of ginkgo flavonoids by adopting a gene regulation technology, and provides a reference for selecting high-quality seed sources and planting and popularizing in the later period in the future ginkgo industrial production.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the invention clones two new lncRNA-Lnc 2L and Lnc2S genes from ginkgo for the first time, and the flavone content of transgenic ginkgo callus over-expressing Lnc2L or Lnc2S is obviously increased or reduced by transferring Lnc2L and Lnc2S into ginkgo callus, which shows that Lnc2L and Lnc2S are key lncRNA for regulating and controlling ginkgo flavone synthesis, and the regulation and control of the expression of Lnc2L and Lnc2S can control the synthesis of flavonoids, so that the regulation and control of the expression of Lnc2L and Lnc2S has important application value in the aspects of improving the medicinal quality of ginkgo leaves and the like; meanwhile, the invention also constructs an overexpression vector and a host cell containing Lnc2L or Lnc 2S. The content of ginkgo flavonoids is regulated and controlled through the expression levels of Lnc2L and Lnc2S, and ginkgo with high flavonoid content can be cultured as required, so that the ginkgo molecular breeding method has important application value in the ginkgo molecular breeding process.

Drawings

FIG. 1 is a clone electrophoretogram of Lnc2L and Lnc 2S;

FIG. 2 is a sequence alignment of Lnc2L and Lnc 2S;

FIG. 3 is a schematic structural diagram of constructed plant expression vectors 35S:: Lnc2L (a) and 35S:: Lnc2S (b);

FIG. 4 shows the expression level measurements of the Lnc2L and Lnc2S transgenic ginkgo calli (P <0.01, P < 0.001);

fig. 5 shows the measurement of flavonoid content of the calli of Lnc2L and Lnc2S transgenic ginkgo (P < 0.001).

Detailed Description

The invention is further illustrated by the following figures and examples.

Example 1

Clones Lnc2L and Lnc2S

(1) Based on ginkgo lncRNA-seq data, an lncRNA is obtained by screening, and primers are artificially designed on the lncRNA by using Primer Premier 5.0 software. Wherein the forward primer (primer F) is: 5'-GTATTCGTTTCCTCATAAACCAGG-3', the reverse primer (R primer) is: 5'-TTTCAATTGGCAGGGATAATATA-3' are provided. Two bands appeared during PCR amplification, probably due to variable splicing, and the long sequence was named Lnc2L and the short sequence was named Lnc2S (FIG. 1).

(2) PCR amplification using the high fidelity enzyme PrimeSTAR Max (Takara, Japan) was performed as follows:

and (3) gently and uniformly mixing the mixed solution, placing the mixture into a common PCR reactor after instantaneous low-speed centrifugation, and setting the following procedures:

glue running: and taking out the gene amplification product in the PCR instrument, detecting the appropriate product on 1% agarose gel by using an electrophoresis instrument, taking out the product after about 25min, and observing the product by using an imaging system to obtain the target fragment.

(3) Ligation of purified fragments to cloning vectors

The gel recovery product was ligated to the Cloning vector according to the pEASY-Blunt Zero Cloning Kit (all gold, China) protocol as follows:

the solution in the system was mixed in a microtube and reacted at room temperature for 5 min. After the reaction was completed, the reaction mixture was placed on ice for further use.

(4) Transformation of E.coli

The ligated product was mixed with Competent cells according to Trans1-T1 Phage resist chemical company Cell product Specification (all-purpose gold, China), and after ice bath, heat shock, resuscitation, an appropriate amount was applied to LB plate, the plate was inverted, and cultured overnight at 37 ℃.

(5) Positive clone screening and sequencing analysis

Selecting single colony from the screening culture plate, inoculating the single colony in LB liquid culture medium, shaking the colony at 37 ℃ and 250rmp overnight; and directly carrying out PCR detection on the recombinant transformant by taking the overnight cultured bacterial liquid as a template.

Reaction system:

reaction procedure:

the clone with positive bacteria liquid PCR detection is sent to Yingjun biotechnology company (Shanghai) for sequencing identification, the sequences of Lnc2L and Lnc2S are respectively 676bp and 578bp, the sequences are shown as SEQ ID NO.1 and SEQ ID NO.2, when the sequences are compared, the result shows that Lnc2L is 98bp longer than Lnc2S, and other sequences are completely consistent (figure 2).

Example 2

Construction of plant expression vectors of Lnc2L and Lnc2S

(1) In the experiment, TaKaRa Quickcut restriction enzyme (TaKaRa, Japan) is adopted to carry out enzyme digestion reaction experiments on a pRI 101-AN vector (TaKaRa, Japan, the vector is provided with a promoter CAMV35S, a strong terminator NOS-ter, AN NPT II gene expression box, LB and RB sequences) and Lnc2L and Lnc2S sequences respectively, and the specific reaction systems are as follows:

mixing all solutions in the system, then carrying out instantaneous centrifugation, preserving the temperature in a water bath kettle at 37 ℃ for 30min, then finishing the enzyme digestion reaction, observing an enzyme digestion strip by agarose gel electrophoresis, and then respectively cutting and recovering the target gene and the vector fragment for subsequent vector ligation reaction.

(2) The expression vector recovered after the double digestion reaction and the target DNA fragment product are connected with each other by referring to TaKaRa T4 DNA Ligase (TaKaRa, Japan) operating instructions, and the system is as follows:

the solutions in the system were mixed in a microtube and reacted in a metal bath at 16 ℃ for 5-6 h.

PCR detection confirms that the overexpression vectors of Lnc2L and Lnc2S are successfully constructed, namely 35S:: Lnc2L and 35S:: Lnc2S, as shown in FIGS. 3a and 3b, the constructed expression vectors are provided with a constitutive strong expression promoter CaMV35S at the 5 'end and a terminator NOS at the 3' end of Lnc2L and Lnc2S, NPT II gene expression cassettes are arranged on the expression vectors to serve as screening markers of transgenic ginkgo, and LB and RB sequences are assembled on the expression vectors to promote the gene expression frames and the screening marker gene NPT II assembled between the gene expression frames and the screening marker gene NPT II to be integrated into the chromosome of the ginkgo receptor cells.

(3) Transformation of Agrobacterium

Referring to the GV3101/EHA105 chemical company Cell product (gold, China) operating instructions, the 35S constructed in step (2), Lnc2L and 35S, Lnc2S expression vector plasmid and EHA105 Agrobacterium tumefaciens Competent cells are mixed, and after standing for 5min, liquid nitrogen for 5min, water bath at 37 ℃ for 5min and ice bath for 5min in sequence, the mixture is added into a culture medium for shaking culture. Coating a proper amount of the suspension on an LB flat plate, and performing inverted culture in an incubator at 28 ℃. The single clone on the plate is picked up, added with a proper amount of LB liquid culture medium, cultured for 48h, and the bacterial liquid is sequenced to respectively obtain agrobacterium containing 35S:: Lnc2L and 35S:: Lnc2S vectors.

Example 3

Genetic transformation of Lnc2L and Lnc2S

1. Ginkgo callus transformation

(1) Agrobacterium containing 35S:, Lnc2L and Lnc2S vectors obtained in example 2 were spread on LB plates, respectively. After the culture, the agrobacterium tumefaciens monoclonal on an LB plate is selected and inoculated into an LB liquid culture medium, and the culture is carried out for 16h at the temperature of 28 ℃ to OD₆₀₀0.5-0.6;

(2) putting the bacterial liquid into a centrifugal tube, centrifuging at 18 ℃ and 3500rpm for 15min, and removing supernatant;

(3) adding a resuspension (100mL of MS liquid culture medium containing 100 mu M acetosyringone) into the centrifuge tube to resuspend the bottom thalli, and standing at room temperature for 2 h;

(4) placing the small ginkgo callus blocks with the same size into the agrobacterium heavy suspension, standing and soaking at room temperature for 15min, lightly clamping out the small ginkgo callus blocks by using forceps, and sucking the heavy suspension liquid on the surface by using sterile filter paper;

(5) placing the infected callus in callus culture medium (MS +4.0 mg. L)^-1NAA+2.0mg·L^-1KT +100 mu M acetosyringone), culturing in dark at 25 ℃ for 3d, taking out, putting into liquid nitrogen, quickly freezing, storing in an ultra-low temperature refrigerator, and applying to subsequent flavonoid content determination.

2. Detection of transgenic material and determination of flavonoid content

The expression of Lnc2L and Lnc2S at the RNA level was detected by real-time quantitative PCR using PrimeScript reverse Transcriptase Reagent Kit (TaKaRa, Japan), and the expression level of Lnc2L and Lnc2S in the transgenic ginkgo callus obtained in step 3 was significantly increased (FIG. 4), indicating that Lnc2L and Lnc2S were successfully transferred into the ginkgo callus. The flavonoid content of the transgenic ginkgo callus over-expressing the long-chain non-coding RNA Lnc2L was significantly increased (by 14.5%) as measured by a plant flavonoid extraction kit (China, Coxim Biotechnology Co., Suzhou, China) on non-transgenic (CK, not subjected to Agrobacterium infection with the 35S:: Lnc2L and 35S:: Lnc2S vector of the present invention, other culture conditions were the same) and transgenic (infected with Agrobacterium with the 35S:: Lnc2L and 35S:: Lnc2S vector) ginkgo callus (FIG. 5), whereas the flavonoid content of the transgenic ginkgo callus over-expressing the long-chain non-coding RNA Lnc2S was significantly decreased (by 39.8%) (FIG. 5). These results indicate that Lnc2L effectively promotes flavonoid synthesis, Lnc2S effectively inhibits flavonoid synthesis, and the combination of the two can effectively regulate flavonoid synthesis.

Sequence listing

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