Protein related to regulation and control of growth and development state of litsea cubeba, biological material and application of protein and biological material

Abstract

The invention provides a protein related to regulation and control of growth and development states of litsea cubeba, a biological material and application thereof, relates to the technical field of variation or genetic engineering, and discloses a key gene LcTGA10 for regulating and controlling the growth and development states of litsea cubeba, the gene LcTGA10 is transferred into arabidopsis thaliana, the gene expression quantity of PDCD of the arabidopsis thaliana can be increased, and the gene expression quantity of PDCD of the arabidopsis thaliana can be increased. When the expression of the LcTGA10 gene is inhibited, the gene expression quantity of PDCD is reduced. PDCD is a programmed cell death regulation gene, and growth and development of litsea cubeba can be effectively promoted by inhibiting expression of specific LcTGA10 in litsea cubeba.

Description

Technical Field

[0001] The present invention relates to the technical field of mutation or genetic engineering, and particularly relates to a protein, a biological material related thereto, and their applications, which are related to regulating the growth and development state of Litsea cubeba. Background Art

[0002] The plant essential oil produced by Litsea cubeba (also known as Litsea cubeba Pers.) is an important raw material for medicine and spices, and the development of its plants can determine the yield of essential oil. However, there is a lack of a method for intervening in the development of Litsea cubeba by molecular regulation means.

[0003] In view of this, the present invention is specifically proposed. Summary of the Invention

[0004] One of the objectives of the present invention is to provide a protein to solve at least one of the technical problems existing in the prior art.

[0005] Another objective of the present invention is to provide a biological material related to the above protein.

[0006] Another objective of the present invention is to provide a product for regulating the growth and development state of Litsea cubeba.

[0007] Another objective of the present invention is to provide the application of the above product.

[0008] Another objective of the present invention is to provide a method for cultivating Litsea cubeba.

[0009] In order to achieve the above objectives of the present invention, the following technical solutions are specifically adopted: The present invention provides a protein, which is a protein of any one of the following A1), A2), or A3): A1) A protein with an amino acid sequence as shown in SEQ ID NO.2; A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues to the amino acid sequence shown in SEQ ID NO.2, having more than 90% identity with the protein shown in A1), and being related to the gender of Litsea cubeba; A3) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0010] The present invention also provides a biological material related to the above protein, which is any one of the following B1) to B5): B1) A nucleic acid molecule encoding the protein; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2).

[0011] Furthermore, the nucleic acid molecule described in B1) has the nucleotide sequence shown in SEQ ID NO.1.

[0012] The present invention also provides a product for regulating the growth and development state of Litsea cubeba, and the product contains a substance that inhibits the expression of the above-mentioned protein and / or biomaterial.

[0013] The present invention also provides the application of the above-mentioned product in promoting the growth and development of Litsea cubeba.

[0014] Furthermore, the product achieves C1)-C2) by negatively regulating PDCD the expression level. Furthermore, the product negatively regulates PDCD the expression level by inhibiting the protein from binding to the TGACG element.

[0015] In addition, the present invention also provides a method for cultivating Litsea cubeba, which includes the step of inhibiting the expression level of the above-mentioned protein or its coding gene in the target plant to obtain Litsea cubeba with good growth and development.

[0016] Furthermore, the inhibition of the expression level of the above-mentioned protein or its coding gene in the target plant is achieved by reducing or knocking out the coding gene of the above-mentioned protein.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses a key gene for regulating the growth and development state of Litsea cubeba LcTGA10 , transferring LcTGA10 gene into Arabidopsis thaliana can increase the gene expression level of PDCD in Arabidopsis thaliana, and when inhibiting the expression of LcTGA10 gene, the gene expression level of PDCD decreases. PDCD is a gene for regulating programmed cell death. By inhibiting the expression of specific LcTGA10 in Litsea cubeba, the growth and development of Litsea cubeba can be effectively promoted. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0019] Figure 1a The alignment result of the LcTGA10 homologous protein provided by the embodiment of the present invention; where black represents 100% identity, pink represents 75% identity, and bright blue represents 50% identity; Figure 1b The alignment result of the LcTGA10 homologous protein provided by the embodiment of the present invention; where black represents 100% identity, pink represents 75% identity, and bright blue represents 50% identity; Figure 2 The yeast one-hybrid and EMSA verification test results of the LcTGA10 binding LcPDCD provided by the embodiment of the present invention; Figure 3 The LcTGA10 and AtPDCD in LcTGA10 The result diagram of the expression levels in 5 Arabidopsis overexpression lines provided by the embodiment of the present invention; Figure 4 The LcTGA10 and LcPDCD in LcTGA10 The expression levels in 3 groups of transiently overexpressed male flowers of Litsea cubeba provided by the embodiment of the present invention. Specific Embodiments

[0020] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meanings and scopes of the terms should be clear. However, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or extrinsic definition. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms is non-restrictive.

[0021] In general, the nomenclature and the techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art or as described herein. The nomenclature, as well as the laboratory procedures and techniques used in connection with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well known and commonly used in the art.

[0022] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0023] According to a first aspect of the present invention, there is provided a protein, which is found in Litsea cubeba and named TGA10, and is a protein of any one of A1), A2) or A3) as follows: A1) a protein having an amino acid sequence as shown in SEQ ID NO.2; A2) a protein having more than 90% identity with the protein shown in A1) and related to the gender of Litsea cubeba, obtained by substitution and / or deletion and / or addition of amino acid residues to the amino acid sequence shown in SEQ ID NO.2; A3) a fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0024] In the above protein, the sequence shown in SEQ ID NO.2 consists of 453 amino acid residues.

[0025] The above protein can be artificially synthesized, or its coding gene can be synthesized first and then expressed biologically.

[0026] In the above protein, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein by using DNA in vitro recombination technology, so as to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0027] For the above-mentioned protein, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined by using homology search sites on the Internet, such as the BLAST web page on the NCBI home page website.

[0028] For the above-mentioned protein, the identity of more than 90% can be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0029] According to the second aspect of the present invention, the present invention also provides biomaterials related to the above-mentioned protein, which are any one of the following B1) to B5): B1) A nucleic acid molecule encoding the above-mentioned protein; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2).

[0030] Among them, the nucleic acid molecule described in B1) has the nucleotide sequence shown in SEQ ID NO.1.

[0031] According to the third aspect of the present invention, a product for regulating the growth and development state of Litsea cubeba is provided, and the product contains a substance that inhibits the expression of the above-mentioned protein and / or biomaterial.

[0032] According to the fourth aspect of the present invention, the application of the above-mentioned product in promoting the growth and development of Litsea cubeba is provided.

[0033] In some preferred embodiments, the product promotes the growth and development of Litsea cubeba by negatively regulating PDCD the expression level.

[0034] More preferably, the product negatively regulates PDCD the expression level by inhibiting the protein from binding to the TGACG element.

[0035] According to the fifth aspect of the present invention, a method for cultivating Litsea cubeba is also provided, and the method includes the step of inhibiting the expression level of the above-mentioned protein or its coding gene in the target plant to obtain Litsea cubeba with good growth and development.

[0036] Among them, the expression level of the protein or its coding gene in the plant to be inhibited is achieved by reducing or knocking out the coding gene of the protein.

[0037] It should be noted that the plants described in the present invention include Litsea cubeba and Arabidopsis thaliana.

[0038] The present invention will be further described below through examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0039] Experimental materials: Tissues of various parts of Litsea cubeba used in the experiment were collected from the forest farm of the Research Institute of Subtropical Forestry, Chinese Academy of Forestry. Arabidopsis thaliana and tobacco seedlings were planted in the greenhouse of the Research Institute of Subtropical Forestry. The light and temperature cycle conditions were 14 hours of light per day at 25 °C and 10 hours of darkness at 22 °C. Fresh plant tissue samples for DNA or RNA extraction experiments were picked and quickly frozen with liquid nitrogen, and then stored in a -80 °C refrigerator for later use.

[0040] Example 1 Cloning and sequence alignment of the TGA10 gene in Litsea cubeba According to the instruction manual, RNA was extracted from the female flowers of Litsea cubeba using TRIzol reagent, and the single-stranded cDNA synthesized by reverse transcription of RNA was used as a template. The design of the amplification primers referred to the LcTGA10 transcript sequence in the Litsea cubeba transcriptome database, and the primer sequences used are shown in Table 1. The complete coding sequence fragment of the LcTGA10 amplified gene was sequenced to obtain accurate sequence information, which was calibrated with the information in the transcriptome database. The full protein sequence of LcTGA10 was used to align with the homologous protein sequences of other species.

[0041] The results showed that the complete coding DNA sequence (CDS) (SEQ ID NO.1) was cloned from the Litsea cubeba transcriptome data. LcTGA10 The coding sequence size of LcTGA10 is 1362 bp, encoding 453 amino acids (SEQ ID NO.2), and about 70% is conserved, belonging to the plant TGA family (Figure 1).

[0042] Table 1 CDS cloning primers for LcTGA10

[0043] Example 2 LcTGA10 can bind to the LcPDCD promoter element of the Yeast one-hybrid assay: The complete coding sequence fragment of the LcTGA10 gene was cloned into the pGADT7 vector, and the TGACG element and its mutant element were cloned into the pAbAi vector respectively. The constructed pGADT7 vector was digested with restriction enzymes BstBITransform yeast after digestion with restriction enzymes. Inoculate the transformed yeast on an SD / -Ura / -Leu agar plate containing 600 ng / mL AbA and culture at 30 °C for 3 d.

[0044] EMSA: Amplify the LcTGA10 full coding sequence from the cDNA of Litsea cubeba. Then ligate it to the pET-32a vector containing a His tag. To successfully induce the target protein, transform the recombinant vector into BL21 Escherichia coli cells and mix the E. coli with 0.2 mM isopropyl-β-D-thiogalactoside (IPTG) overnight at 16 °C. Perform EMSA using the LightShift™ Chemiluminescent EMSA Kit (Thermo Fisher Scientific, USA). Mix the purified LcTGA10-His protein with the biotin-labeled probe for 30 minutes. Separate the mixed solution of the reaction system on a PAGE gel at 180 V. Then transfer it to a nylon membrane and perform UV cross-linking. Detect the biotin-labeled probe using an antibiotin antibody.

[0045] The results show that the TGA10 transcription factor plays an important role in regulating floral organ development by binding to the TGACG element in the promoter of the target gene. A TGACG element was found in the LcPDCD gene promoter fragment of Litsea cubeba, and yeast one-hybrid experiments showed that LcTGA10 can bind to the LcPDCD TGACG element in the promoter. EMSA experiments showed that LcTGA10 can bind to the TGACG element probe and cause a mobility shift. In addition, as the number of probes without bioluminescence labeling of the TGACG element gradually increases, the ability of LcTGA10 to bind to the TGACG element probe gradually decreases ( Figure 2 ). The above two experiments show that: LcTGA10 can directly bind to the LcPDCD TGACG element in the gene promoter to regulate the LcPDCD gene expression level.

[0046] Example 3 Overexpression of LcTGA10 in the model plant Arabidopsis thaliana can increase the AtPDCD expression level Heterologously overexpress the LcTGA10 gene of Litsea cubeba in Arabidopsis thaliana, and a total of 5 transgenic lines were obtained. Among them, the expression level was the highest in line 2 (OE2) ( Figure 3 ). By measuring the expression levels of LcTGA10 and AtPDCD in different transgenic lines, the results showed that the AtPDCD gene expression level also increased with LcTGA10and increase with the increase in expression level ( Figure 3 , Table 2).

[0047] Table 2 Primers used for qRT-PCR

[0048] Example 4 Overexpression in the Litsea cubeba plant LcTGA10 can also improve LcPDCD expression level Furthermore, a transient transformation experiment was conducted on the male flower organs of Litsea cubeba. The Agrobacterium tumefaciens strain LBA4404 containing LcTGA10 was co-injected with the pCAMBIA1300S vector into male flowers of Litsea cubeba with a diameter of 3 mm for transient overexpression. After 3 days, the expression levels of LcTGA10 and LcPDCD genes were detected. The results showed that compared with the control (CK), the expression level of LcPDCD in transient overexpressed male flowers (TOE1, TOE2, and TOE3) also showed an obvious upward trend with the increase in the LcTGA10 gene expression level ( Figure 4 ). Combining the overexpression results of LcTGA10 in Arabidopsis thaliana with this experimental result, it was proved that LcTGA10 can effectively and positively regulate the expression level of the key gene for degradation PDCD .

[0049] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention, and are not intended to limit it; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A protein, characterized in that, The protein is a protein of any one of the following A1), A2) or A3): A1) A protein having an amino acid sequence as shown in SEQ ID NO.2; A2) A protein which is obtained by substituting and / or deleting and / or adding amino acid residues to the amino acid sequence shown in SEQ ID NO.2, has an identity of more than 90% with the protein shown in A1), and is related to the gender of Litsea cubeba; A3) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of A1) or A2).

2. A biological material related to the protein described in claim 1, which is any one of the following B1) to B5): B1) A nucleic acid molecule encoding the protein described in claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2).

3. The biomaterial according to claim 2, wherein The nucleic acid molecule described in B1) has a nucleotide sequence as shown in SEQ ID NO.

1.

4. A product for regulating the growth and development state of Litsea cubeba, characterized in that, The product contains a substance that inhibits the expression of the protein described in claim 1, and / or the biological material described in claim 2 or 3.

5. Use of the product described in claim 4 in promoting the growth and development of Litsea cubeba.

6. The application according to claim 5, wherein The product promotes the growth and development of Litsea cubeba by negatively regulating PDCD the expression level.

7. The application according to claim 6, characterized in that, The product negatively regulates the expression level by inhibiting the binding of proteins to the TGACG element. PDCD ​ 8. A method for cultivating Litsea cubeba, characterized in that, The method includes the step of obtaining Litsea cubeba by inhibiting the expression level of the protein described in claim 1 or its encoding gene in a target plant.

9. The method according to claim 8, wherein The inhibition of the expression level of the protein described in claim 1 or its encoding gene in the target plant is achieved by reducing or knocking out the encoding gene of the protein described in claim 1.

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