CN117625616A - Long-chain non-coding RNA and application thereof in regulating and controlling ginkgo flavonoid content - Google Patents

Abstract

The invention discloses a long-chain non-coding RNA and application thereof in regulating and controlling ginkgo flavonoid content, wherein the long-chain non-coding RNA comprises any one or more of RNA LncNAT30, RNA LncNAT31, RNA LncNAT3 and RNA LncNAT11, and nucleotide sequences of the long-chain non-coding RNA are respectively shown by SEQ NO. 1-4. The non-coding RNA provided by the invention is used for regulating and controlling the synthesis of ginkgo flavonoids after being overexpressed in ginkgo, and the flavonoid content is obviously reduced after the long-chain non-coding RNA is overexpressed, which shows that the non-coding RNA is a key lncRNA for regulating and controlling the synthesis of ginkgo flavonoids, the content of ginkgo flavonoids is regulated and controlled through the overexpression or knockout of the non-coding RNA, and meanwhile, the content of flavonoids can be obviously improved through silencing the non-coding RNA.

Description

Long-chain non-coding RNA and application thereof in regulating and controlling ginkgo flavonoid content

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to long-chain non-coding RNA and application thereof in regulating and controlling ginkgo flavonoid content.

Background

Ginkgo biloba (Gingo bioloba l.) is an important economic tree species, a rare tree species for the life and the wiggle in ginkgo biloba, which is a special product in China and is called as a plant activated stone. Integrates the functions of eating, medical use, material use, health care and ecology, and has high economic, ecological, social and humane values. The She kernel and exodermis of Ginkgo biloba both contain medicinal ingredients. The ginkgo leaf contains abundant secondary metabolites such as flavonoids, terpene lactones, and other active substances, and ginkgo leaf extracts (Ginkgo biloba Extract, gbE) are raw materials of various medicines, and currently, the international content of related medicines of ginkgo leaf extracts is more than 30. Flavonoid compounds are the main active ingredient of GbE, more than 40 flavonoid compounds have been isolated from ginkgo biloba, and ginkgo biloba extract contains approximately 6% terpene lactones and 24% flavonoid glycosides. Ginkgo flavonoids and terpene lactones are the primary secondary metabolites of ginkgo biloba, and are often used to treat cancer, cardiovascular diseases and neurodegenerative diseases.

Long non-coding RNAs (lncRNAs) are a ubiquitous transcript of length exceeding 200nt, and have little or no protein-encoding capability, but are functional. lncRNA is involved in a variety of fundamental biological processes as a key regulatory molecule at the transcriptional, posttranscriptional and epigenetic levels. lncRNA has been found to play an important role in plant growth, development and stress tolerance.

At present, the research on the lncRNA is generally focused in the fields of animals and medicine, and the basic strategies of the research mainly comprise screening and identifying the lncRNA, expressing and positioning the lncRNA and researching the functions and mechanisms of the lncRNA. With the wide application of chain-specific pooling, the development of lncRNA predictive algorithms has been followed to screen and identify a large number of potential lncRNAs in Arabidopsis, mulberry, cabbage, cotton, and other plants. In recent years, the rapid development of the third generation sequencing technology featuring single molecule sequencing can realize the direct sequencing of RNA and the detection of modification on RNA, and a large number of lncRNAs are identified from species such as Arabidopsis thaliana, rice, australian cotton and the like; then, verifying the expression level and the positioning of the lncRNA screened by high-throughput sequencing by using a mature technology, wherein the techniques comprise Northern imprinting, real-time fluorescence quantitative PCR (qRT-PCR) and the like; finally, the biological functions of the candidate lncRNA can be further revealed through a high-efficiency stable plant genetic transformation system. The basic strategy mainly comprises functional availability studies and functional deficiency studies. The functional acquisition research is mainly realized by introducing an over-expression vector, and the functional deletion research is mainly performed by adopting CRISPR/Cas9 gene knockout, RNAi interference and other methods.

The exploration in the lncRNA of plants is still in the primary stage, the research is mainly focused on plants such as arabidopsis, tomatoes, rice and the like, the research on the plants involved in the growth and development of the plants is mainly focused on auxin transportation and signal transduction, and meanwhile, the lncRNA plays an important role in plant response to abiotic stress. lncRNADRIR (drought induced RNA) is activated after drought, salt stress and abscisic acid treatment, and regulates and controls the expression of ABA signal transduction, water transport and other genes relevant to abiotic stress in plants. There is a large distance from the complete explanation of the mechanism of action and biological function of lncRNA in plants, and few studies have been reported on woody plants lncRNA, especially in gymnosperms.

Disclosure of Invention

The invention aims to: aiming at the defects existing in the prior art, the invention provides long-chain non-coding RNA and application thereof in regulating and controlling the content of ginkgo flavonoids, wherein the long-chain non-coding RNA comprises one or more of lncRNA LncNAT30, lncRNA LncNAT31, lncRNALncNAT3 and lncRNALncNAT11, and the content of the apricot flavonoids can be effectively controlled by regulating and controlling the expression of one or more of the lncRNA.

The invention also provides application of the long-chain non-coding RNA for negatively regulating/inhibiting the content of ginkgo flavonoids.

The technical scheme is as follows: in order to achieve the above purpose, the long-chain non-coding RNA for negatively regulating/inhibiting the flavonoid content of ginkgo provided by the invention comprises one or more of lncRNA LncNAT30, lncRNA LncNAT31, lncRNA LncNAT3 and lncRNA LncNAT11, and the nucleotide sequences of the 4 long-chain non-coding RNA are respectively shown in SEQ NO. 1-4.

The invention relates to an overexpression vector of ginkgo long-chain non-coding RNA, wherein the vector contains one or more of lncRNA LncNAT30, lncRNA LncNAT31, lncRNA LncNAT3 and lncRNA LncNAT11.

Preferably, the over-expression vector assembles a constitutive strong expression promoter CAMV35S at the 5' end of the long-chain non-coding RNA of ginkgo, which can enable the long-chain non-coding RNA of ginkgo to be expressed in the ginkgo body with high efficiency; a strong terminator NOS-ter is assembled at the 3' end of the long-chain non-coding RNA of ginkgo, and can effectively terminate transcription of the long-chain non-coding RNA of ginkgo.

Wherein, the over-expression vector is assembled with an NPT II gene expression cassette, and can be used as a screening marker of transgenic ginkgo, and kanamycin can be used for screening the transgenic ginkgo.

Wherein, the over-expression vector is assembled with LB and RB sequences, which promotes the integration of the gene expression frame and the screening marker gene NPTII assembled between the LB and RB sequences into the chromosome of ginkgo receptor cells.

Host cells of the over-expression vectors of the invention.

Preferably, the host cell is an agrobacterium-based strain.

The application of the long-chain non-coding RNA for negatively regulating/inhibiting the content of ginkgo flavonoids in regulating and controlling the synthesis of the ginkgo flavonoids is provided.

Wherein the application is: the transgenic gingko callus with the long-chain non-coding RNA is transferred into gingko callus, and the flavonoid content of the transgenic gingko callus with the long-chain non-coding RNA in the overexpression mode is obviously reduced.

Wherein the application is: the long-chain non-coding RNA is transferred into ginkgo callus, and the flavonoid content of the transgenic ginkgo callus is obviously increased by knocking out or silencing the long-chain non-coding RNA.

The invention takes ginkgo leaves as materials, and clones four new lncRNAs LncNAT30, lncNAT31, lncNAT3 and LncNAT11. Meanwhile, the gene is constructed to AN overexpression vector pRI 101-AN (TaKaRa, japan) through enzyme digestion connection, and a 35S: lncNAT30, 35S: lncNAT31, 35S: lncNAT3 and 35S: lncNAT11 vector is obtained through the construction of a homologous recombination technology. The gene is positioned behind a promoter CaMV35S, and under the drive of the promoter CaMV35S, lncNAT30, lncNAT31, lncNAT3 and LncNAT11 can be efficiently expressed in ginkgo callus, so that the synthesis of flavonoids is regulated and controlled.

Generally, no other genes are arranged on the complementary strand of the genes, 4 novel lncRNA LncNAT30, lncNAT31, lncNAT3 and LncNAT11 genes are cloned in ginkgo for the first time, the 4 genes are positioned on the complementary strand of the protein coding genes and are partially or completely complementary to the protein coding genes, and the 4 genes are found to inhibit the synthesis of ginkgo flavonoids for the first time. After the 4 genes are overexpressed in ginkgo, the flavonoid content can be obviously reduced, which shows that the 4 genes are key lncRNA for regulating and controlling the synthesis of ginkgo flavonoid. Through over-expression or knocking out or silencing of any one or more of lncRNA LncNAT30, lncRNA LncNAT31, lncRNA LncNAT3 and lncRNA LncNAT11, ginkgo with different flavone contents can be cultivated according to requirements, so that the method has important application value in the process of ginkgo molecular breeding. The research result provides theoretical basis for improving synthesis and accumulation of ginkgo flavonoids by adopting gene regulation technology improvement, and provides reference for selecting high-quality seed sources and later planting popularization in the future ginkgo industry production.

The beneficial effects are that: compared with the prior art, the invention has the following advantages:

the invention clones four new lncRNA-LncNAT 30, lncNAT31, lncNAT3, lncNAT11 genes for the first time from ginkgo, and the transgenic ginkgo callus of any one or more of LncNAT30, lncNAT31, lncNAT3 or LncNAT11 genes is subjected to over-expression or knocked out or silenced, so that the flavonoid content can be regulated and controlled, the LncNAT30, lncNAT31, lncNAT3 and LncNAT11 are key lncRNA for regulating and controlling the synthesis of ginkgo flavonoids, the overexpression of LncNAT30, lncNAT31, lncNAT3 or LncNAT11 can be regulated and controlled, the overexpression of LncNAT3 or LncNAT11 can inhibit the synthesis of flavonoids, belongs to negative regulation factors of the synthesis of ginkgo flavonoids, and provides precious reference value for improving the flavonoids by carrying out fixed-point knockout on genes through CRISPR/Cas9 gene editing technology in the future, so that the regulation and control of the expression of LncNAT30, lncNAT31, lncNAT3 or LncNAT11 has important application values in the aspects of improving the medicinal quality of ginkgo leaves and the like; meanwhile, the invention also constructs an over-expression vector and host cell containing LncNAT30, lncNAT31, lncNAT3 or LncNAT11, and regulates and controls the content of ginkgo flavonoids through the expression quantity of LncNAT30, lncNAT31, lncNAT3 or LncNAT11, and further the invention can obviously improve the synthesis of flavonoids by knocking out or silencing any one or more of LncRNA LncNAT30, lncRNA LncNAT31, lncRNA LncNAT3 and LncRNA LncNAT11.

Drawings

FIG. 1 is a clone electrophoresis diagram of LncNAT30 and LncNAT 31;

FIG. 2 is a clone electrophoresis pattern of LncNAT 3;

FIG. 3 is a clone electrophoresis pattern of LncNAT 11;

FIG. 4 is a schematic diagram of the structures of constructed plant expression vectors 35S:: lncNAT30 (A), 35S:: lncNAT31 (B), 35S:: lncNAT3 (C) and 35S:: lncNAT11 (D).

FIG. 5 shows the expression level detection (A) and total flavone content detection (B) of LncNAT30 transgenic ginkgo callus, and the significant difference is represented between different letters;

FIG. 6 shows the expression level detection (A) and total flavone content detection (B) of LncNAT31 transgenic ginkgo callus, and the significant difference is represented between different letters;

FIG. 7 shows the expression level detection (A) and total flavone content detection (B) of LncNAT3 transgenic gingko callus ^* P<0.05， ^** P<0.01， ^*** P<0.001)

FIG. 8 shows the expression level detection (A) and total flavone content detection (B) of LncNAT11 transgenic ginkgo callus ^* P<0.05， ^** P<0.01， ^*** P<0.001)。

FIG. 9 shows the expression level detection (A) and total flavone content detection (B) of transgenic ginkgo with silencing LncNAT11 ^* P<0.05， ^** P<0.01， ^*** P<0.001)。

Detailed Description

The invention is further described below with reference to the drawings and examples.

Example 1

Cloning LncNAT30, lncNAT31, lncNAT3 and LncNAT11

(1) Based on ginkgo lncRNA-seq data, 4 lncRNA were obtained by screening, and primers were designed manually using Primer Premier 5.0 software. Wherein the forward primer (F primer) is: 5'-TCGTCAGCTGGTAGTCGG-3', the reverse primer (R primer) is: 5'-AGCAGATTGTAGTCCAGAATGCC-3', which was designated LncNAT30 (FIG. 1) by PCR amplification; wherein the forward primer (F primer) is: 5'-CTCCCTCACAACCTGTTACTGC-3', the reverse primer (R primer) is: 5'-CGAATGCATCGTGAAGATGG-3' the sequence was designated LncNAT31 (FIG. 1) by PCR amplification. Wherein the forward primer (F primer) is: 5'-ATAACCCATTATTGATTGATTGAGAAA-3', the reverse primer (R primer) is: 5'-TTCTTCCCTTCCCTCACGAA-3' the sequence was designated LncNAT3 (FIG. 2) by PCR amplification. Wherein the forward primer (F primer) is: 5'-GCCTCCAGGGGAGGGGAA-3', the reverse primer (R primer) is: 5'-CCAAGATCTCGAGTAAGCCAGAAA-3' the sequence was designated LncNAT11 (FIG. 3) by PCR amplification.

(2) PCR amplification was performed using the high-fidelity enzyme PrimeSTAR Max (Takara, japan), and the PCR system was as follows:

the mixed solution is gently mixed, and is placed in a common PCR reaction instrument after instantaneous low-speed centrifugation, and the following procedures are set:

running glue: taking out the gene amplification product in the PCR instrument, detecting a proper amount of product on 1% agarose gel by using an electrophoresis instrument, taking out the product after about 25min and observing by using an imaging system to obtain a target fragment, wherein the cloning electrophoresis patterns of LncNAT30, lncNAT31, lncNAT3 and LncNAT11 are shown in figures 1-3.

(3) Ligation of purified fragments with cloning vector

The gel recovery product was ligated into the cloning vector as described in pEASY-Blunt Zero Cloning Kit (full gold, china) protocol, the specific system being as follows:

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

(4) Coli transformation

Referring to Trans1-T1 Phage Resistant Chemically Competent Cell product instruction (full gold, china), the connected product is mixed with competent cells, and after ice bath, heat shock and resuscitation, a proper amount of the mixture is coated on an LB plate, the plate is inverted, and the mixture is cultured at 37 ℃ overnight.

(5) Positive clone screening and sequencing analysis

Selecting single colony from the screening culture plate, inoculating the single colony into LB liquid culture medium, shaking at 37 ℃ and 250rmp overnight; PCR detection of recombinant transformants was performed directly with the overnight cultured broth as template.

The reaction system:

the reaction procedure:

sequencing and identifying clone Yingjun biotechnology company (Shanghai) with positive bacterial liquid PCR detection, and measuring that LncNAT30 has a sequence of 468bp and is shown as SEQ ID NO. 1; the LncNAT31 has a sequence of 262bp and is shown as SEQ ID NO. 2; the measured sequence of LncNAT3 is 1100bp, and the sequence is shown as SEQ ID NO. 3; the detected LncNAT11 has a sequence of 227bp and is shown as SEQ ID NO. 4.

Example 2

Construction of plant expression vectors of LncNAT30, lncNAT31, lncNAT3 and LncNAT11

(1) In the experiment, the sequence of pRI 101-AN vector (TaKaRa, japan, the vector has a promoter CAMV35S, a strong terminator NOS-ter, AN NPT II gene expression cassette, LB and RB sequences) LncNAT30, lncNAT31, lncNAT3 and LncNAT11 is subjected to enzyme digestion reaction experiments by using TaKaRa QuickCut restriction enzyme (TaKaRa, japan), and the specific reaction system is as follows:

and mixing the solutions in the system, performing instantaneous centrifugation, performing heat preservation for 30min in a water bath at 37 ℃, ending the enzyme digestion reaction, observing enzyme digestion strips by agarose gel electrophoresis, and then respectively cutting and recycling the target genes and the carrier fragments for subsequent carrier connection reaction.

(2) Referring to the instructions of TaKaRa T4 DNA Ligase (TaKaRa, japan), the expression vector recovered after the double cleavage reaction was ligated with the target DNA fragment product as follows:

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

Through PCR detection, the construction success of the overexpression vectors of LncNAT30, lncNAT31, lncNAT3 and LncNAT11 is confirmed, the expression vectors are named as 35S, lncNAT30, 35S, lncNAT31, 35S, lncNAT3 and 35S, lncNAT11 are shown in figures 4A-D, the constructed expression vectors are assembled with a constitutive strong expression promoter CaMV35S at the 5 'end of LncNAT30, lncNAT31, lncNAT3 and LncNAT11, a terminator NOS is assembled at the 3' end, NPT II gene expression boxes are assembled on the expression vectors and serve as screening markers of transgenic ginkgo, and LB and RB sequences are assembled on the expression vectors, so that the gene expression frameworks assembled between the LB and RB sequences and the screening marker genes NPT II are integrated into ginkgo receptor cell chromosomes.

(3) Transformation of Agrobacterium

Referring to the operating instruction of GV3101/EHA105 Chemically Competent Cell product (full gold, china), 35S of LncNAT30, 35S of LncNAT31, 35S of LncNAT3 and 35S of LncNAT11 expression vector plasmids are mixed with EHA105 agrobacterium competent cells, and then the mixture is added into a culture medium for shake culture after standing for 5min, liquid nitrogen for 5min, water bath at 37 ℃ for 5min and ice bath for 5min. And (3) a proper amount of the culture medium is coated on an LB plate, and the culture medium is cultured in an inverted mode at the temperature of 28 ℃. Selecting a monoclonal on a flat plate, adding an appropriate amount of LB liquid culture medium, culturing for 48 hours, and sequencing bacterial liquid to obtain the agrobacterium containing 35S: lncNAT30, 35S: lncNAT31, 35S: lncNAT3 and 35S: lncNAT11 vectors respectively.

Example 3

Genetic transformation of LncNAT30, lncNAT31, lncNAT3 and LncNAT11

1. Ginkgo callus transformation

(1) The agrobacteria containing 35S:: lncNAT30, 35S: lncNAT31, 35S:: lncNAT3 and 35S:: lncNAT11 vectors were obtained in example 2, respectively, and applied on LB plates. After culturing, the agrobacterium monoclonal on the LB plate is selected and inoculated into LB liquid culture medium for culturing at 28 ℃ for 16h to OD ₆₀₀ 0.5-0.6;

(2) Placing the bacterial liquid into a centrifuge tube, centrifuging at 18 ℃ and at 350 rpm for 15min, and removing supernatant;

(3) Adding a heavy suspension (100 mL of MS liquid culture medium contains 100 mu M acetosyringone) into the centrifuge tube to heavy suspension the bottom thalli, and standing at room temperature for 2 hours;

(4) Placing ginkgo callus small pieces with consistent sizes into agrobacterium tumefaciens heavy suspension, standing at room temperature, soaking for 15min, then gently clamping by forceps, and sucking the heavy suspension liquid on the surface by using sterile filter paper;

(5) The infected calli were placed in callus medium (MS+4.0mg.L ^-1 NAA+2.0mg·L ^-1 Kt+100 μm acetosyringone), culturing in darkness at 25deg.C for 3d, taking out, quick freezing in liquid nitrogen, and storing in ultralow temperature refrigerator for subsequent flavonoid content measurement.

2. Detection of transgenic Material and determination of flavonoid content

Using PrimeScript ^TM Reverse Transcriptase Reagent Kit (TaKaRa, japan) were subjected to real-time quantitative PCR to examine the expression of LncNAT30, lncNAT31, lncNAT3 and LncNAT11 at the RNA level, and the expression levels of LncNAT30 (FIG. 5A), lncNAT31 (FIG. 6A), lncNAT3 (FIG. 7A) and LncNAT11 (FIG. 8A) in the transgenic ginkgo callus obtained in step 3 were significantly increased, indicating that LncNAT30, lncNAT31, lncNAT3 and LncNAT11 had been successfully transferred into ginkgo callus. The flavonoid content of the ginkgo callus without the transgenes (WT, 35S:: lncNAT30, 35S:: lncNAT31, 35S:: lncNAT3 and 35S:: agrobacterium infection of LncNAT11 vector, other culture conditions were the same) and the transgenic (the flavonoid content of the ginkgo callus with the LncNAT30, 35S:: lncNAT31, 35S:: lncNAT3 and 35S:: agrobacterium infection of LncNAT11 vector) were all significantly reduced by using a plant flavonoid extraction kit (Suzhou Ke Ming biotechnology Co., china) according to the present invention. These results indicate that LncNAT30, lncNAT31, lncNAT3 and LncNAT11 are all effective in inhibiting flavonoid synthesis, but they are not limited to the same extent, so that the objective of obtaining different flavonoid contents can be achieved by using these 4 lncnrnas alone or in combination.

Example 4

The sequence of LncNAT11 cloned in example 1 was ligated to pTRV2 vector (conventional commercially available VIGS vector) in the same manner as in example 2 to construct TRV2: lncNAT11. The constructed vector TRV2:: lncNAT11 was transferred into Agrobacterium according to the method of example 2 to obtain Agrobacterium containing the TRV2:: lncNAT11 vector. Cutting germinated seeds of Ginkgo biloba with knife, soaking in Agrobacterium solution containing TRV 2:LncNAT 11 vector, OD ₆₀₀ And (3) vacuumizing for 20min to enable the bacterial liquid to fully infect seeds, and planting the infected seeds in a flowerpot, wherein the bacterial liquid is 0.5-0.6. Leaves were taken after the seedlings were grown for about 20 days, and the transgenic materials were subjected to expression level detection and flavonoid content detection in the same manner as in example 3. TRV 2-LncNAT 11 expression level in LncNAT11 transgenic ginkgo callus was significantly reduced (FIG. 9A), and flavonoid content was significantly increased (FIG. 9B). Whereas LncNAT30, lncNAT31, lncNAT3 can all achieve the above effect through silencing. These results indicate that long non-coding RNAs can effectively regulate flavonoid synthesis.

Claims (10)

1. The long-chain non-coding RNA for negatively regulating/inhibiting the content of ginkgo flavonoids is characterized by comprising one or more of lncRNA LncNAT30, lncRNA LncNAT31, lncRNA LncNAT3 and lncRNA LncNAT11, wherein the nucleotide sequences of the 4 long-chain non-coding RNA are respectively shown in SEQ NO. 1-4.

2. An overexpression vector containing long-chain non-coding RNA for negatively regulating/inhibiting the flavonoid content of ginkgo biloba according to claim 1, which is characterized in that the vector contains one or more of lncRNA LncNAT30, lncRNA LncNAT31, lncRNA LncNAT3 and lncRNA LncNAT11.

3. The overexpression vector according to claim 2, characterized in that it assembles a constitutive strong expression promoter CAMV35S at the 5' end of long non-coding RNA.

4. The overexpression vector according to claim 2, characterized in that it is assembled with a strong terminator NOS-ter at the 3' -end of the long non-coding RNA.

5. The overexpression vector according to claim 2, characterized in that it is assembled with an NPT ii gene expression cassette as a screening marker for transgenic ginkgo.

6. The overexpression vector according to claim 2, characterized in that it is assembled with LB and RB sequences, promoting the integration of the gene expression framework and the selectable marker gene nptii assembled in between into the chromosome of ginkgo receptor cells.

7. A host cell comprising the over-expression vector according to claim 2, characterized in that the host cell is preferably an agrobacterium-based starting strain.

8. Use of long non-coding RNA of negative control/inhibition of ginkgo flavonoid content according to claim 1 for controlling ginkgo flavonoid synthesis.

9. The use according to claim 8, characterized in that the use is: the transgenic gingko callus with the long-chain non-coding RNA is transferred into gingko callus, and the flavonoid content of the transgenic gingko callus with the long-chain non-coding RNA in the overexpression mode is obviously reduced.

10. The use according to claim 8, characterized in that the use is: the long-chain non-coding RNA is transferred into ginkgo callus, and the flavonoid content of the transgenic ginkgo callus is obviously increased by knocking out or silencing the long-chain non-coding RNA.