CN117924450B - Transcription factor CIB1 and application of coding gene thereof - Google Patents
Transcription factor CIB1 and application of coding gene thereof Download PDFInfo
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Abstract
The invention discloses desmodium styracifolium transcription factor CIB1 and application of a coding gene thereof, wherein the amino acid sequence of the desmodium styracifolium transcription factor CIB1 is shown as SEQ ID NO. 2. The invention discovers that desmodium styracifolium transcription factor CIB1 can promote the hairy roots of desmodium styracifolium and liquorice to be obviously enlarged and grow obviously faster, so that the desmodium styracifolium transcription factor CIB1 and the coding gene thereof have important application prospects in promoting the growth of the hairy roots of desmodium styracifolium, liquorice and other plants and in molecular breeding for cultivating new plant species with hairy roots growing in high quality and high yield.
Description
Technical Field
The invention belongs to the technical field of biology, relates to plant genetic engineering technology, and in particular relates to application of a transcription factor CIB1 and a coding gene thereof in promoting growth of hairy roots of plants.
Background
The hairy root has the characteristics of stable genetic property, rapid growth and short growth time. In the plant field, the hairy root can realize mass production of secondary metabolites of key medicinal components of plants, so that the hairy root of the plants is a natural high-quality raw material of the medicinal components and can synthesize secondary metabolites of flavonoids, alkaloids, terpenes and the like. Up to now, more than 100 plants are available to obtain hairy roots, mainly dicotyledonous plants, with fewer monocotyledonous plants. In the field of medicinal plants, such as baikal skullcap root, tuber fleeceflower root, kuh-seng, ginseng, licorice, single body, stramonium and the like, hairy root culture systems have been established, and even hairy roots of some medicinal plants enter a mass production stage.
At present, people mainly improve the content of secondary metabolites in hairy roots by changing the external conditions of hairy root culture, such as the pH value of a culture medium, the components of the culture medium, illumination, temperature, exogenous hormones, inducers and the like. However, the methods of changing these conventional external growth environments are often limited by genetic factors of plants themselves, resulting in limited effects of increasing the secondary metabolite content in many hairy roots of plants.
Because of the complex secondary metabolism of plants, overexpression of one or more synthetases solely by hairy roots often fails to synthesize large quantities of secondary metabolites. Transcription factors can regulate the transcription of genes in multiple metabolic pathways simultaneously, so that the accumulation of metabolites is better improved. For example, transcription factor MYBs regulates tanshinone synthesis in hairy roots of red sage root, transcription factor ZIP promotes rutin synthesis in hairy roots of tartary buckwheat, and transcription factor AP2/ERF promotes Bai Langen lignin synthesis.
The basic-helix-loop-helix bHLH protein is a transcription factor superfamily, and plays an important role in regulating and controlling the growth and development of plants and stress response species. CIB1 (cryptochrome-INTERACTING BASIC-helix-loop-helix 1) is a protein which can interact with cryptoanthocyanin CRY2 in blue light specificity for the first time in plants, and CIB1 depends on CRY2 to regulate and control the initiation of flower formation.
CIB1 of Arabidopsis thaliana can be combined with a G-box (CANNTG) gene sequence with high affinity in vitro. CIL, CIL2, CIB4 and CIB5 in the CIB protein evolution branch have been reported in literature to be involved in cell elongation and growth, CIL1 is named ACE1, CIB4 is named ACE2, and CIL2 is named ACE3.ACEs form bHLH triple antagonism system with PRE1 (HLH protein for promoting growth signal) and IBH1 (HLH factor for inhibiting cell elongation) to control cell elongation. However, studies using CIB1 transcription factor are currently focused mainly on arabidopsis thaliana, and most of them are flower-forming studies of arabidopsis thaliana. However, the research on regulating and controlling plant hairy root growth by CIB1 has not been reported so far, which limits the industrialized development of plant hairy root and the development of secondary metabolite synthesis and improved plant varieties.
Disclosure of Invention
Based on the above, the invention aims to provide application of desmodium styracifolium transcription factor CIB1 and a coding gene thereof, wherein the desmodium styracifolium transcription factor CIB1 and the coding gene thereof can remarkably promote growth of hairy roots of plants.
The technical scheme for realizing the aim of the invention comprises the following steps.
In a first aspect, the invention provides application of desmodium styracifolium transcription factor CIB1 in promoting growth of plant hairy roots or molecular breeding of plant hairy roots, wherein the amino acid sequence of the desmodium styracifolium transcription factor CIB1 is shown as SEQ ID NO. 2.
In a second aspect of the invention, the application of the coding gene of desmodium styracifolium transcription factor CIB1 in promoting plant hairy root growth or plant hairy root molecular breeding is provided, wherein the nucleotide sequence of the coding gene of desmodium styracifolium transcription factor CIB1 is shown as SEQ ID NO. 1.
In a third aspect, the invention provides an application of an overexpression vector of the desmodium styracifolium transcription factor CIB1 coding gene in promoting plant hairy root growth or plant hairy root molecular breeding.
In a fourth aspect, the invention provides the application of the genetically engineered bacterium transformed with the overexpression vector of the coding gene of desmodium styracifolium transcription factor CIB1 in promoting the growth of hairy roots of plants or the molecular breeding of hairy roots of plants.
In a fifth aspect of the present invention, there is provided a method of promoting growth of hairy roots of plants comprising the steps of: the expression of desmodium styracifolium transcription factor CIB1 or the coding gene of desmodium styracifolium transcription factor CIB1 in plants is improved.
The invention has the following beneficial effects:
The inventor firstly clones and obtains the coding gene of desmodium styracifolium (Grona styracifolia (Osbeck) H.ohashi & K.ohashi) transcription factor CIB1, then constructs a vector for over-expressing the coding gene of desmodium styracifolium transcription factor CIB1, transfers the vector into agrobacterium rhizogenes K599 to obtain a genetic engineering bacterium of the over-expression vector, and then induces desmodium styracifolium and liquorice to grow hairy roots, so that the desmodium styracifolium and liquorice hairy roots are found to be obviously enlarged and obviously grow faster, and therefore, the desmodium styracifolium transcription factor CIB1 and the coding gene thereof have important application prospects in promoting the growth of the hairy roots of desmodium styracifolium, liquorice and other plants and the molecular breeding for cultivating new plant species with hairy roots high quality and high yield development, and a feasible method is provided for plant hairy root application.
Drawings
FIG. 1 is a phylogenetic diagram of desmodium styracifolium transcription factor CIB1 and other CIB family members in example 1 of the present invention.
FIG. 2 is a chart of PCR identification of desmodium styracifolium and licorice hairy roots in example 3 of the present invention.
FIG. 3 is a morphological feature of inducing desmodium styracifolium and licorice hairy roots for 1 month in example 3 of the present invention.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The experimental methods, which are not specified in the following examples, are generally carried out according to conventional conditions, such as Green and Sambrook-s.A.fourth edition, molecular cloning, instruction manual (Molecular Cloning: ALaboratory Manual), published in 2013, or according to the conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
In some embodiments of the invention, the application of desmodium styracifolium transcription factor CIB1 in promoting the growth of hairy roots of plants is disclosed, wherein the amino acid sequence of the desmodium styracifolium transcription factor CIB1 is shown as SEQ ID NO. 2.
In other embodiments of the invention, the application of desmodium styracifolium transcription factor CIB1 in plant hairy root molecular breeding is disclosed, wherein the amino acid sequence of the desmodium styracifolium transcription factor CIB1 is shown as SEQ ID NO. 2.
In other embodiments of the invention, the application of the coding gene of desmodium styracifolium transcription factor CIB1 in promoting the growth of hairy roots of plants is disclosed, wherein the nucleotide sequence of the coding gene of desmodium styracifolium transcription factor CIB1 is shown as SEQ ID NO. 1.
In other embodiments of the invention, the application of the coding gene of desmodium styracifolium transcription factor CIB1 in plant hairy root molecular breeding is disclosed, wherein the nucleotide sequence of the coding gene of desmodium styracifolium transcription factor CIB1 is shown as SEQ ID NO. 1.
SEQ ID NO:1：
ATGTTGCACTGTATGAACACGTCGGGGAATGTGGGTGGAGATATGACAGTCTTGGAAAGACAGAGAGCGAGGATGATGAAGTTGCAAGAAGACCAAGGTTACTTTTCTTCCTTCAATGGCGTTTTCTCATCTTCTCTAAATACTCATCATTCCATGATGCTAGCTGCGGATTCCGGTTCTGCCCTTGCTGAAGTAGTGGCTCATGCTCAGCATCAACCTCAGCCAACAACAGTTCATGGGTTTGATGCGGGTTCTTCACTATCCAGGACTTTTAGCTGTCCACCTACTCTCGTGGAGCCCGAGCCCGAGCCCGAGCCCAAGCCCAAGCCCACGAATTCTTCAATTGGTAAAGAGAGTTTAAAGAAGAGGAAGCATGACAAGACTCCGAATACAAAGTTTGTTGCAGAAAATGATAACAAAGACAAGAGGATCAAAGTGGGTGCAGATGATGGTGAATCCAAAATCACGGTGCAGACCAACAGCAAGAGTAACACAACAAACGCAAATAGCAACAAAAGAGAAACCTGTGCGGATAATACTTCAAATTCAAAACCTTTAGAGGTTCAAAAGTCCGATTACATTCATGTTCGTGCACGTCGTGGTCAAGCCACAGATAGCCATAGCTTAGCTGAAAGAGTTAGAAGGGAAAAAATTAGTGAGAGAATGAAGTATTTACAAGATTTAGTACCCGGTTGCAACAAAATCACTGGAAAAGCTGGAATGCTTGATGAAATCATCAATTATGTTCAATCTCTTCAGCGGCAAGTTGAGTTTCTGTCAATGAAATTGGCTTCTGTGAACCCAAGGCTTGACTTCAGTGTTGATGATCTATTTGAAAAAGAGGTGTTTCCTGCTTGTGCTACAAATTTTCCAAACATGGGAATGCCATCAGATATGACTAACGCTGCATATCTTCAGTTTAATTCACCGCATCAAATTGTTTCGTTTGGTGGATTAGATACAGTGATTAACCCTTCCGATGTGGGGCTTAAAAGGACCATAAGTGCCCCTGTAACAATGCCTGAAACATATCTTCACTCGTCTGGTTTCACACAAATGATACCT TCCTCAACATGGGAAGGTGATTTCCAAAACCTGTGCAATTTTGATTTTGATCAAGCACGAGCAACATCTTTCCCTTCTCAGCTGTTCTCAGGTCTTGTTGAAGCTAGCAATCTAAAGATGGGGATGTAG
SEQ ID NO:2：
MLHCMNTSGNVGGDMTVLERQRARMMKLQEDQGYFSSFNGVFSSSLNTHHSMMLAADSGSALAEVVAHAQHQPQPTTVHGFDAGSSLSRTFSCPPTLVEPEPEPEPKPKPTNSSIGKESLKKRKHDKTPNTKFVAENDNKDKRIKVGADDGESKITVQTNSKSNTTNANSNKRETCADNTSNSKPLEVQKSDYIHVRARRGQATDSHSLAERVRREKISERMKYLQDLVPGCNKITGKAGMLDEIINYVQSLQRQVEFLSMKLASVNPRLDFSVDDLFEKEVFPACATNFPNMGMPSDMTNAAYLQFNSPHQIVSFGGLDTVINPSDVGLKRTISAPVTMPETYLHSSGFTQMIPSSTWEGDFQNLCNFDFDQARATSFPSQLFSGLVEASNLKMGM*
In some embodiments, the plant is a leguminous plant, and/or the leguminous plant is desmodium styracifolium or licorice.
In other embodiments of the invention, the application of an over-expression vector of the coding gene of desmodium styracifolium transcription factor CIB1 in promoting plant hairy root growth or plant hairy root molecular breeding is disclosed, wherein the nucleotide sequence of the coding gene of desmodium styracifolium transcription factor CIB1 is shown as SEQ ID NO. 1.
In some of these embodiments, the over-expression vector comprises psuper promoter; or, the over-expression vector is psuper1300,1300 vector.
In other embodiments of the invention, the application of the genetically engineered bacterium transformed with the overexpression vector of the coding gene of desmodium styracifolium transcription factor CIB1 in promoting plant hairy root growth or plant hairy root molecular breeding is disclosed, wherein the nucleotide sequence of the coding gene of desmodium styracifolium transcription factor CIB1 is shown as SEQ ID NO. 1.
In some embodiments, the genetically engineered bacterium is agrobacterium rhizogenes.
In other embodiments of the present invention, a method for promoting growth of hairy roots of plants is disclosed, comprising the steps of: the expression of desmodium styracifolium transcription factor CIB1 or the coding gene of desmodium styracifolium transcription factor CIB1 in plants is improved, the amino acid sequence of desmodium styracifolium transcription factor CIB1 is shown as SEQ ID NO. 2, and the nucleotide sequence of the coding gene of desmodium styracifolium transcription factor CIB1 is shown as SEQ ID NO. 1.
In some embodiments, the plant is a medicinal plant; and/or, the medicinal plant is desmodium styracifolium or liquorice.
The invention will be described in further detail with reference to the drawings and the specific examples.
Example 1 cloning of the coding gene of desmodium styracifolium transcription factor CIB1 and construction of the over-expression vector comprises the following steps:
1. Taking whole tissue sample of rhizome and leaf of desmodium styracifolium stored at-80deg.C, grinding into powder with liquid nitrogen, and extracting RNA (product number: R4151-03C, brand: magen/Meyer group organism) with plant RNA double column kit.
2. The synthesis of the first strand of cDNA was performed according to the procedure of PRIMESCRIPT TM II 1st Strand cDNA Synthesis Kit reverse transcription kit (cat# 6210A, brand: takara/Bao Bio) instructions.
3. Based on the CDS sequence GsCIB obtained by sequencing the desmodium styracifolium genome, an upstream primer F and a downstream primer R are designed and utilizedMax DNAPolymerase Hi-Fi enzyme (cat# R045B, brand: taKaRa) amplified the cDNA sequence of GsCIB.
Upstream primer F (SEQ ID NO: 3):
CGACTCTAGAAAGCTTATGTTGCACTGTATGAACAC；
downstream primer R (SEQ ID NO: 4):
CCTCGCCCTTGCTCATGGTACCCATCCCCATCTTTAGATTG；
PCR reaction system: primestarMix. Mu.L, 2. Mu.L of upstream primer, 2. Mu.L of downstream primer, 1. Mu.L of template and 20. Mu.L of sterile water.
The reaction procedure: pre-denaturation at 98 ℃ for 5min; denaturation at 98℃for 30s, annealing at 55℃for 30s, extension at 72℃for 1min for 33 cycles; extending at 72℃for 5min.
4. The PCR product is subjected to agarose gel and then the target fragment is recovered by using a DNA gel recovery kit.
5. Over-expression vectors were constructed using FastDNAAssemblyMix (cat# E0201S, brand: CISTRO). And (3) connecting the target fragment purified in the step (4) with an over-expression vector to obtain a connection product.
Homologous recombination system: after purification, 1. Mu.L of the target fragment (100 ng/. Mu.L), 1. Mu.L of the vector (100 ng/. Mu.L) after cleavage and AssemblyMix. Mu.L were ligated at 50℃for 15 minutes.
E.coli competent DH5 alpha ice bath sample adding, ice bath standing for 5min until competent melting. Adding the ligation product to 100 mu L of escherichia coli DH5 alpha in an ice-water mixture state, flicking and uniformly mixing, and then carrying out ice bath for 20min; heat shock at 42 ℃ for 60s; standing on ice for 3min; adding 700 mu L of LB liquid medium without antibiotics; 200rpm, and culturing at 37 ℃ for 1h in a shaking way; centrifuging for 1min at 10,000Xg; the supernatant was discarded, and after 50. Mu.L of the suspended cells remained, an appropriate amount of the cell liquid was applied to LB solid medium containing 50mg/LKan, and the culture was inverted at 37℃overnight until single colonies were grown.
6. The positive clone obtained in the previous step is selected for positive clone detection and sequencing (Optimus Prinsepia Roxburghii Co.) to obtain the over-expression vector (named OE-GsCIB hereinafter) of the coding gene (the nucleotide sequence is shown as SEQ ID NO:1, the amino acid sequence is shown as SEQ ID NO: 2) of desmodium styracifolium transcription factor GsCIB.
The sequences of desmodium styracifolium transcription factor CIB1 obtained by cloning in the present invention and other CIB family members (5 CIB transcription factors, 2 CIL transcription factors) were aligned by using clustalW, and the result of the phylogenetic tree of 11 candidate proteins obtained by using the NJ distance method is shown in FIG. 1. The results indicate that among 17 members of subgroup 18 of the bHLH family, cyclocarya paliurus CIB1 was found to be similar in affinity to arabidopsis CIB 1.
Examples 2 OE-GsCIB1 transfer into Agrobacterium rhizogenes K599 competent
Agrobacterium rhizogenes K599 competent ice bath sample addition, ice bath standing for 10min until Agrobacterium competent melting. Adding OE-GsCIB1 with correct sequence to 100 mu L of Agrobacterium rhizogenes K599 in an ice-water mixture state, flicking and mixing uniformly, and then carrying out ice bath for 5min; liquid nitrogen for 5min; water bath at 37 ℃ for 5min, ice bath for 5min. Adding 700 mu L of LB liquid medium without antibiotics; 200rpm, and culturing at 28 ℃ for 3 hours in an oscillating way; centrifuging for 1min at 10,000Xg; after 50. Mu.L of suspended bacteria remained, the supernatant was discarded, and an appropriate amount of the bacterial liquid was applied to LB solid medium containing 50mg/LKan +50mg/L of Str, and the culture was inverted at 28℃overnight until single colonies were grown, and positive clone detection was performed.
Example 3 OE-GsCIB1 bacterial liquid promoting growth of desmodium styracifolium and Glycyrrhiza uralensis hairy root
The positive Agrobacterium single colony obtained in example 2 was picked up, cultured to an OD600 of 0.8, and centrifuged at 4000rpm for 10min to collect the cells. The suspension (100. Mu. Mol. L -1,MgCl2 mM, MES10 mM) was used as suspension to resuspend the bacterial suspension, the OD600 was adjusted to 0.8, and the suspension was allowed to stand at room temperature for 1h to obtain Agrobacterium resuspension.
Cutting cotyledons of the aseptic seedlings of the desmodium styracifolium and the liquorice, respectively soaking the cotyledons into the prepared agrobacterium tumefaciens heavy suspension for 1h, taking out, sucking residual bacterial liquid by filter paper, and placing the bacterial liquid on a 1/2MS solid culture medium for dark culture for 3 days. Then transferred to 1/2MS+200cefmg/L solid medium, grown under light, and medium changed every 15 days. After 1 month, positive hairy roots were transplanted to 1/2MS+200cefmg/L for further growth, and photographed for recording.
The results of PCR identification of hairy roots of desmodium styracifolium (NC represents negative control, sterile water as template, PC represents positive control, and Agrobacterium positive monoclonal as template) by rolB and rolC, respectively, are shown in FIG. 2. From the results, two bands of 750bp and 500bp were obtained, indicating that Agrobacterium rhizogenes carried desmodium transcription factor CIB1 into desmodium to produce hairy roots.
Figure 3 is a morphological feature of desmodium styracifolium and licorice hairy roots at 1 month of age. As can be seen from FIG. 3, when the transcription factor GsCIB was overexpressed, the hairy roots of desmodium styracifolium and Glycyrrhiza uralensis were significantly larger and grown faster than CK (hairy roots of desmodium styracifolium induced by empty plasmid without transcription factor CIB 1).
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (8)
1. The application of desmodium styracifolium transcription factor CIB1 in promoting growth of hairy roots of plants, wherein the amino acid sequence of the desmodium styracifolium transcription factor CIB1 is shown as SEQ ID NO. 2; the plant is herba Desmodii Styracifolii or Glycyrrhrizae radix.
2. The application of desmodium styracifolium transcription factor CIB1 in molecular breeding of plant hairy roots is provided, wherein the amino acid sequence of the desmodium styracifolium transcription factor CIB1 is shown as SEQ ID NO. 2; the plant is herba Desmodii Styracifolii or Glycyrrhrizae radix.
3. The application of the coding gene of desmodium styracifolium transcription factor CIB1 in promoting the growth of hairy roots of plants is provided, wherein the nucleotide sequence of the coding gene of desmodium styracifolium transcription factor CIB1 is shown as SEQ ID NO. 1; the plant is herba Desmodii Styracifolii or Glycyrrhrizae radix.
4. The application of the coding gene of desmodium styracifolium transcription factor CIB1 in plant hairy root molecular breeding is disclosed, wherein the nucleotide sequence of the coding gene of desmodium styracifolium transcription factor CIB1 is shown as SEQ ID NO. 1; the plant is herba Desmodii Styracifolii or Glycyrrhrizae radix.
5. The application of an over-expression vector of a coding gene of desmodium styracifolium transcription factor CIB1 in promoting plant hairy root growth or plant hairy root molecular breeding, wherein the nucleotide sequence of the coding gene of desmodium styracifolium transcription factor CIB1 is shown as SEQ ID NO. 1; the plant is herba Desmodii Styracifolii or Glycyrrhrizae radix.
6. The use of claim 5, wherein the over-expression vector comprises psuper promoter.
7. The application of the genetically engineered bacterium transformed with the overexpression vector of the coding gene of desmodium styracifolium transcription factor CIB1 in promoting the growth of plant hairy roots or the molecular breeding of the plant hairy roots is disclosed, wherein the nucleotide sequence of the coding gene of desmodium styracifolium transcription factor CIB1 is shown as SEQ ID NO. 1; the plant is desmodium styracifolium or liquorice; the original strain of the genetically engineered bacterium is agrobacterium.
8. A method for promoting growth of hairy roots of plants, comprising the steps of: improving expression of desmodium styracifolium transcription factor CIB1 or a coding gene of desmodium styracifolium transcription factor CIB1 in plants, wherein the amino acid sequence of desmodium styracifolium transcription factor CIB1 is shown as SEQ ID NO. 2, and the nucleotide sequence of the coding gene of desmodium styracifolium transcription factor CIB1 is shown as SEQ ID NO. 1; the plant is herba Desmodii Styracifolii or Glycyrrhrizae radix.
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