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CN113150091A - CsHD2 protein and gene for promoting plant lateral bud growth and application thereof - Google Patents

CsHD2 protein and gene for promoting plant lateral bud growth and application thereof Download PDF

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CN113150091A
CN113150091A CN202110190565.0A CN202110190565A CN113150091A CN 113150091 A CN113150091 A CN 113150091A CN 202110190565 A CN202110190565 A CN 202110190565A CN 113150091 A CN113150091 A CN 113150091A
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张金智
胡春根
曾仁芳
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Huazhong Agricultural University
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Abstract

The invention relates to the technical field of plant genetic engineering, in particular to CsHD2 protein and gene for promoting the growth of plant lateral buds and application thereof. The CsHD2 gene is separated from sweet orange for the first time, an overexpression vector of the gene is constructed, the overexpression vector is respectively introduced into tobacco and lemon through an agrobacterium-mediated genetic transformation method, and finally the cloned CsHD2 gene has the functions of promoting lateral shoot lateral bud development, prolonging the vegetative growth stage of plants and postponing the flowering phase through phenotype analysis of transgenic plants. The invention analyzes the mechanism of the CsHD2 gene in regulating the development of the citrus apical tissue, proves that the gene can promote the development of axillary buds and lateral branches of citrus, is beneficial to realizing the growth and development of citrus branch tips and the regulation and improvement of plant type structures in actual production, and is beneficial to achieving the purposes of increasing both production and income by utilizing a genetic engineering means.

Description

CsHD2 protein and gene for promoting plant lateral bud growth and application thereof
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to CsHD2 protein and gene for promoting the growth of plant lateral buds and application thereof.
Background
Homeobox genes (Homeobox genes) are a gene family highly conserved in animals and plants, and are first found in Drosophila melanogaster, and play an important role in the processes of biological morphogenesis, cell differentiation and the like. In the plant kingdom, homeobox genes comprise five major families, and the common characteristic of these gene families is that all have a conserved domain (HD) capable of binding with specific DNA sequences. The Arabidopsis KNOX (KNOTTED1-like homeobox genes) gene family is one of five large families of homologous heterotype box genes, the family has eight gene members, and can be divided into two subfamilies of Class I and Class II according to the amino acid difference of a conserved structural domain, and the two subfamilies have similar and relatively conserved structural domains. Besides a certain difference in structure, the expression patterns of the two subfamily genes in plants are also different, and in addition, the regulation and control functions in the plant development process are also different. The KNOX family Class I subfamily, which includes four members STM, knot 1, knot 2 and knot 6, is mainly expressed specifically in plant meristems and is involved in regulation of organ development and morphological changes, while four members of Class II are expressed in all tissues of plants and are currently less studied, and the function of most of these subfamilies in plants is not yet clear.
In the woody plant citrus, a KNOX family gene with a relatively conserved sequence also exists, and the function of the family member CsHD2 gene is found to be closely related to the morphological development of the citrus, so far, the research on the family gene in the citrus is almost blank, and the molecular regulation function of the family member in the aspects of growth and development and the like is not clear.
Disclosure of Invention
The invention aims to provide application of CsHD2 protein in promoting the growth of plant lateral buds, wherein the amino acid sequence of the CsHD2 protein is shown as SEQ ID NO. 1.
Further, the application of the CsHD2 gene coding the CsHD2 protein in promoting the growth of plant lateral buds, wherein the amino acid sequence of the CsHD2 protein is shown as SEQ ID NO: 1.
Further, the CsHD2 gene is a DNA molecule described in any one of the following (1) to (2):
(1) the nucleotide sequence of the DNA molecule is shown as SEQ ID NO. 2;
(2) a DNA molecule which is hybridized with the DNA sequence defined in (1) under strict conditions and encodes CsHD2 protein.
Further, an expression vector containing the CsHD2 gene or application of agrobacterium tumefaciens in promoting the growth of plant lateral buds, wherein the nucleotide sequence of the CsHD2 gene is shown as SEQ ID NO. 2.
Further, the vector is a PBI121 expression vector.
Further, the plants are tobacco and lemon.
The invention also aims to provide the application of the expression vector or the agrobacterium containing the CsHD2 gene in promoting the growth of the lateral bud of the plant.
Further, the plants are tobacco and lemon.
The invention also aims to provide a method for promoting the growth of plant lateral buds, which comprises the step of transferring the CsHD2 gene related to the promotion of the growth of plant lateral buds into a plant body to obtain a CsHD2 transgenic plant.
The CsHD2 gene in the KNOX family gene is separated from sweet orange for the first time, an overexpression vector is constructed through the gene, the gene is respectively introduced into tobacco and lemon through agrobacterium-mediated genetic transformation, and the obtained transgenic plant is subjected to phenotype observation, so that the cloned CsHD2 gene has the functions of promoting the development of lateral shoot lateral buds, prolonging the vegetative growth stage of the plant and postponing the flowering phase. A method for promoting lateral bud growth using the CsHD2 gene is provided.
The invention analyzes the mechanism of the CsHD2 gene in regulating the citrus top development, proves that the gene can promote the development of the lateral shoot and the lateral bud of the citrus, is beneficial to realizing the regulation and the improvement of the growth and the development of the citrus branch tip in the actual production, and is beneficial to achieving the purposes of increasing the yield and the income by utilizing a gene engineering means.
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FIG. 1(a) shows the phenotypic results of wild type tobacco seedlings and tobacco seedlings into which the CsHD2 gene has been transferred; (b) the phenotype results of the wild tobacco and the tobacco seedlings which are transferred with the CsHD2 gene are shown.
FIG. 2 shows the results of the phenotype of normal lemon and lemon with the CsHD2 gene transferred.
Detailed Description
The present invention is further described in detail below with reference to specific examples so that those skilled in the art can more clearly understand the present invention.
The following examples are given for the purpose of illustration only and are not intended to limit the scope of the invention. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without any creative effort belong to the protection scope of the present invention.
Unless otherwise specified, the test reagents and materials used in the examples of the present invention are commercially available.
Unless otherwise specified, the technical means used in the examples of the present invention are conventional means well known to those skilled in the art.
The inventor clones a new gene CsHD2 from sweet orange based on a plant gene cloning technology, the gene ID is LOC18050945, the nucleotide sequence is shown as SEQ ID NO. 2, the new gene comprises 4 exons and 3 introns, the open reading frame is 1146bp, 381 amino acids are coded, and the amino acid sequence is shown as SEQ ID NO. 1. The isoelectric point of the protein coded by the gene is 5.96, and the predicted molecular weight is 42.988 kDa.
The amino acid sequence of the CsHD2 protein is shown below:
MDGGSNSTSCMNMMAFGDNSNGLYCPMMMMPLMSSFTHQHHRHDHHHDEHHQHHLDADSNTLFLPLAAAAAATTTINNIVQNQNRNSSSGSSLILEDNTTAINTINNSSARCYFMETNYGSGASSSVKAKIMAHPHYHRLLAAYANCQKVGAPPEVVARLEEVCASAASMGSGGSSCIGQDPALDQFMEAYCEMLTKYEQELSKPFKEAMSFLQKIESQFKSLSISSPNSASSEAIDRNGSSEEDFDVNIDFIDPQAEDQELKGQLLRRYSGCLSSLKQEFMKKRKKGKLPKEARQQLLDWWSKHYKWPYPSESQKLALAESTGLDSKQINNWFINQRKRHWKPSEDMQFMVMDATQPQYYMDSTVMGNPFPMDLSPAPLL
the nucleotide sequence of the CsHD2 gene is shown as follows:
ATGGACGGTGGTTCTAATAGCACTTCTTGTATGAATATGATGGCTTTTGGAGACAACAGTAATGGACTATATTGTCCTATGATGATGATGCCTCTCATGTCTTCTTTCACTCATCAACATCATCGTCATGATCATCATCATGATGAACATCATCAACATCATCTTGATGCAGACTCAAATACCCTATTTCTTCCCCTTGCTGCTGCTGCTGCTGCTACTACTACTATCAACAATATTGTTCAAAATCAAAACCGCAATAGCAGTAGTGGCTCGTCTCTGATTCTCGAAGATAACACCACCGCCATCAATACTATTAACAACTCCTCAGCTCGGTGTTATTTCATGGAGACCAACTATGGCAGCGGCGCTTCTTCTTCTGTCAAAGCTAAGATAATGGCTCATCCTCACTATCACCGTCTCTTGGCTGCCTATGCCAATTGTCAGAAGGTTGGGGCACCACCTGAAGTGGTGGCAAGACTAGAAGAAGTTTGCGCATCTGCGGCCTCGATGGGTTCCGGCGGCTCGAGCTGTATCGGTCAAGATCCAGCTCTTGATCAGTTTATGGAAGCTTACTGTGAGATGCTGACCAAATATGAACAAGAGCTCTCTAAACCCTTCAAGGAAGCCATGTCTTTCCTACaAAAGATCGAGTCTCAATTTAAGTCCCTCTCTATTTCCTCTCCAAATTCTGCTTCTAGCGAGGCTATTGATAGGAATGGTTCGTCTGAAGAGGATTTCGATGTGAATATTGACTTCATAGATCCCCAAGCTGAAGATCAGGAGCTGAAAGGTCAGCTTCTGCGCAGGTATAGCGGATGTTTAAGTAGTCTCAAGCAGGAGTTCATGAAGAAGAGAAAGAAAGGGAAGCTGCCTAAAGAAGCCAGGCAACAGTTGTTGGATTGGTGGAGCAAACACTACAAATGGCCTTACCCATCGGAGTCACAGAAGCTGGCCCTTGCAGAATCAACAGGTCTGGATTCAAAGCAAATAAACAACTGGTTTATTAATCAAAGGAAGAGACACTGGAAGCCTTCCGAAGATATGCAGTTTATGGTGATGGATGCTACCCAACCTCAATATTACATGGACAGTACTGTTATGGGTAATCCCTTTCCAATGGATCTCTCACCTGCACCGCTGCTCTGA
the product obtained by reverse transcription of orange tissue RNA is used as a PCR amplification template, and the full-length sequence of the coding region of the gene is obtained by cloning by utilizing the PCR technology. The gene provided by the invention is constructed into an over-expression vector, and is respectively introduced into tobacco and lemon through agrobacterium-mediated genetic transformation, and phenotype observation of obtained transgenic plants shows that the CsHD2 gene cloned by the invention has the functions of promoting the development of lateral shoot lateral buds and influencing the development of citrus plant types.
Example 1 cloning of CsHD2 Gene
The embodiment provides a method for cloning a CsHD2 gene, which comprises the following specific steps:
s1, extraction of orange RNA
Collecting the top tissue of the sweet orange by using an ice box or liquid nitrogen, and immediately storing the collected tissue in a refrigerator at the temperature of minus 80 ℃ for later use. The whole process of RNA extraction needs to be carried out in an ultra-clean bench, and the reagent adopts Trizol kit of Takara company, and the main steps are as follows:
a. 1mL of RNAioso Plus reagent was placed in a 2mL RNAase free centrifuge tube and placed on ice.
b. Liquid nitrogen sample grinding: and (3) placing a proper amount of sample in a mortar, pouring liquid nitrogen into the mortar, and quickly grinding the mixture into powder.
c. Taking a proper amount of ground sample, placing the ground sample in an RNAioso Plus solution, standing at room temperature, adding 1/5 volumes of chloroform, shaking, uniformly mixing, standing at room temperature for 5min, and centrifuging at 12000r/min at 4 ℃ for 10 min.
d. Optional steps are as follows: the supernatant was transferred to a new 2mL RNAase free centrifuge tube and step c was repeated once by adding an equal volume of chloroform.
e. Transferring the supernatant (about 600 μ L) into a new 1.5mL RNAase free centrifuge tube, adding equal volume of isopropanol, shaking, mixing, standing at room temperature for 10min, and centrifuging at 12000r/min at 4 deg.C for 10 min.
f. The supernatant was discarded and 500. mu.L of pre-cooled 75% ethanol (in DEPC water) was added to the suspension for precipitation and the salts were eluted.
g. Alcohol was discarded and the precipitate was dried and dissolved by adding the appropriate amount of RNase Free ddH 2O.
h. 2.5 mul RNA solution is taken for electrophoresis detection of the quality, and the rest RNA can be directly used for reverse transcription or stored at minus 80 ℃ for standby.
S2, reverse transcription reaction
The reagent for reverse transcription reaction is PrimeScript of reverse transcription kit of Takara companyTMRT reagent Kit with gDNA Eraser (cat No. RR047A), all procedures were performed in an ultraclean bench. The reaction steps are as follows:
a. reaction process for removing genome DNA
The reaction mixture was prepared as follows, and carefully performed on ice using a RNase-free PCR tube.
TABLE 1 genome removal reaction System
Figure BDA0002943990250000061
Reacting at 42 deg.C for 2min or standing at room temperature for 5-10min, and performing the next step.
b. Reverse transcription reaction
TABLE 2 reverse transcription reaction System
Figure BDA0002943990250000062
c. Reacting the prepared system at 37 ℃ for 15-20min, then reacting at 85 ℃ for 5s, and storing at 4 ℃ for later use, wherein the prepared system is stored in a refrigerator at-80 ℃ for a long time.
S3, cloning of Gene
The experiment mainly uses the product obtained by the reverse transcription of the RNA of the sweet orange tissue as a PCR amplification template, and adopts high-fidelity DNA polymerase of Nanjing Novozam company
Figure BDA0002943990250000063
Max Super-Fidelity DNA Polymerase kit for PCR amplification reaction.
Wherein, the amplification primers are as follows: 5'-GATAGACAGACTGCCGTTTC-3', respectively;
5’-AGAACACCTGCTGAATTTGC-3’。
the system is as follows:
TABLE 3 Gene amplification System
Figure BDA0002943990250000071
TABLE 4 Gene amplification PCR program
Figure BDA0002943990250000072
And after the reaction is finished, taking out the PCR product, and carrying out electrophoresis detection or storing the PCR product in a refrigerator at 4 ℃ for a short time.
S4, vector construction
All reagents used for vector construction adopt a one-step cloning method of Nanjing NuoZan Biotechnology limited
Figure BDA0002943990250000073
The kit II is constructed by fusing a target gene and a carrier through recombination reaction, and the specific experimental operation steps are as follows:
a. firstly, carrying out plasmid extraction on escherichia coli (a strain is DH5 alpha) carrying a PBI121 vector, carrying out double enzyme digestion on the obtained vector plasmid by using restriction enzymes BamHI and SacI under the condition of enzyme digestion at 37 ℃ for 30 minutes, designing a recombinant primer to carry out PCR reaction, adding recombinant recognition sites 5'-ACGGGGGACTCTAGAGGATCC-3' and 5'-CGATCGGGGAAATTCGAGCTC-3' to two ends of a target fragment, measuring the concentration of the vector and the target fragment after the vector and the target fragment are prepared, and adjusting the proportion.
b. The ligation reaction of the vector and the target fragment is carried out at 25 ℃ or 37 ℃ according to requirements, and the specific ligation reaction system is as follows:
TABLE 5 ligation reaction System
Figure BDA0002943990250000081
S5 transformation of Agrobacterium
After the vector is successfully constructed, the plasmid is introduced into an agrobacterium-competent cell for agrobacterium transformation, and the specific operation steps are as follows:
a. taking out Agrobacterium GV3101 competence from-80 deg.C refrigerator, freeze thawing on ice, adding 3-4 μ L plasmid, quick freezing in liquid nitrogen for 1min, warm bathing in 37 deg.C thermostat for 5min, and placing on ice for 5 min.
b. Adding 600 plus 800 mu L of liquid non-resistant LB culture medium, shaking the bacteria for 2-3h under the condition of 28 ℃ and 220r/min, and recovering the competent cells.
c. Centrifuging at high speed for 1min, removing supernatant, spreading the bacterial solution on solid LB + Rif solid culture medium with corresponding resistance, and culturing at 28 deg.C for 2-3 days.
d. After the long spots, monoclonal colonies are picked for PCR detection, and positive colonies can be used for plant transformation.
The agrobacteria grown in this example were used for the dip-staining of examples 2 and 3.
Example 2 transformation of tobacco
The tobacco transformation process comprises the following steps:
a. one day in advance, Agrobacterium solution was prepared and cultured Overnight (OD) in a shaker at 28 ℃600About 1.2), the next day, the cultured cell suspension was centrifuged to collect cells, the cells were suspended in a non-antibiotic MS liquid medium, and OD was added600The value is adjusted to 0.8-1.0 and the dye liquor is used for standby.
b. A healthy and normal wild tobacco leaf is taken, a main vein is cut off, the leaf is cut into small blocks with the length of about 1cm, the back faces upwards, the leaf is placed in a staining solution to suspend for about 10min, and the leaf is continuously shaken in the process.
c. Taking out the leaves with sterile forceps, placing on sterile filter paper, blotting to remove residual liquid, spreading in a dish containing blank solid MS culture medium, sealing the dish, and culturing in dark for three days.
d. Shaking and cleaning with cephalosporin water (concentration 400mg/L) for 3 times (each time for about 5 min), and shaking and cleaning with clear water twice (each time for about 10 min).
e. Sucking residual liquid with filter paper, placing the liquid on a differentiation culture medium (MS +6-BA + NAA + Cef) with corresponding antibiotics with the back side facing upwards, culturing in an aseptic illumination culture room for about 30 days to perform bud cutting and rooting culture (the culture medium for rooting culture is MS + Cef with corresponding antibiotics), and if the buds do not grow all the time, replacing the culture medium every 20 days to prevent pollution until the seedlings grow out.
f. Seedlings were transplanted to soil for cultivation and observed for phenotype. The results are shown in FIG. 1. FIG. 1(a) shows phenotypic results of wild type tobacco seedlings (labeled control in the figure) and 2 tobacco seedlings transformed with CsHD2 gene (labeled 35CsHD2, # 1 and # 2 in the figure); FIG. 1(b) shows the phenotypic results of wild-type tobacco seedlings (labeled control in the figure) and tobacco seedlings into which the CsHD2 gene was transferred (labeled 35CsHD2 in the figure).
As can be seen from FIG. 1(a), the CsHD2 transgenic tobacco exhibited mainly phenotypes such as limited apical growth and mass growth of lateral buds, compared with the wild type. As can be seen from the figure 1(b), after the tobacco enters reproductive growth, the wild tobacco normally blooms, while the transgenic tobacco prolongs the vegetative growth stage of plants, delays the flowering phase, obviously increases lateral buds and has strong differentiation capability.
Example 3 transformation of lemon
The genetic transformation procedure for lemon was as follows:
a. 2 days before transformation, streak activation is carried out on LB solid culture medium by using an inoculating loop, the culture is carried out in an incubator at 28 ℃ until thalli grow out, and the monoclone can be picked up to ensure the activity and then streak activation is carried out once again), the thalli is scraped by using a sterile gun head or other tools and is put into 50mL of MT suspension culture medium containing 20mg/L AS (50 mu LAS mother liquor is added into each 50 mL), and shake culture is carried out for 2 hours at 28 ℃. Measure its OD600Adjusting the value to be in the range of 0.6-0.8, and then carrying out the next conversion process. The mother solution concentration of AS is 20mg/mL, and the preparation method comprises the following steps: weighing 2g of powder, dissolving, metering to 100mL of DMSO, subpackaging the dissolved liquid into a 1.5 or 2.0mL centrifuge tube, and storing at-20 ℃.
b. Taking out healthy fresh lemon seeds from fruits, soaking in 1mol/L NaOH solution for 15min to remove pectin, sterilizing with 2% NaClO in a super clean bench for about 20min, and washing with sterile water for 3-5 times, wherein the sterilized seeds can be stored in a refrigerator at 4 deg.C for half a year. When seeds are peeled, the inner and outer seed coats of the seeds are removed by using sterile forceps in a superclean bench, the seeds are sowed in an MT solid culture medium, and the seeds are cultured in a tissue culture room for 20 to 40 days in a shading mode and are cultured in the light for about one week, so that the seeds can be used for genetic transformation.
c. Taking the epicotyl of a well-grown and strong lemon seedling, cutting into stem sections with the length of about 1cm in a super clean workbench by using a sterile scalpel, inclining the cut as far as possible to increase the cut area, temporarily storing the cut stem sections in a sterile triangular flask, paying attention to the fact that a small amount of MT suspension culture medium is put in the flask for moisture preservation, pouring all agrobacterium infection liquid into the triangular flask after all stem sections are cut, infecting for 15-20min, and continuously shaking.
d. After the infection process is finished, absorbing bacteria liquid remained on the surface of the stem section by using sterile filter paper, transferring the bacteria liquid to a co-culture medium (MT + AS) with a layer of sterile filter paper paved on the surface, culturing for 3 days under a dark condition, washing for 3-5 times by using sterile filter paper, absorbing water by using the sterile filter paper, placing the bacteria liquid in a screening culture medium, culturing for 30-50 days in a culture chamber in a shading mode, and then, transferring to a normal illumination condition for culturing. When the regenerated bud grows to about 0.5cm, the regenerated bud is cut off and transferred to a germination culture medium for continuous growth, and when the regenerated bud grows to about 3cm, the regenerated bud can be transferred to a rooting culture medium for growth. The culture medium needs to be replaced by subculture every 30-40 days in the culture process; and the phenotype is observed, and the transgenic lemon grows very slowly, so the test adopts a grafting method to propagate the transgenic seedling, and the result is shown in figure 2. In the figure, non-transgenic lemon plants are marked as control, and transgenic lemon plants are marked as 35CsHD2, 1#, 2# and 3 #. The CsHD2 transgenic lemon plant mainly shows dwarfing, stronger lateral bud differentiation capability and increased lateral bud number.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (7)

  1. The application of the CsHD2 protein in promoting the growth of plant lateral buds is characterized in that the amino acid sequence of the CsHD2 protein is shown as SEQ ID NO. 1.
  2. 2. The application of the CsHD2 gene for encoding the CsHD2 protein in promoting the growth of plant lateral buds is characterized in that the amino acid sequence of the CsHD2 protein is shown as SEQ ID NO: 1.
  3. 3. The use of the CsHD2 gene encoding a CsHD2 protein for promoting the growth of lateral buds of a plant according to claim 2, wherein the CsHD2 gene is a DNA molecule according to any one of the following (1) to (2):
    (1) the nucleotide sequence of the DNA molecule is shown as SEQ ID NO. 2;
    (2) a DNA molecule which is hybridized with the DNA sequence defined in (1) under strict conditions and encodes CsHD2 protein.
  4. 4. The application of the expression vector containing the CsHD2 gene in promoting the growth of plant lateral buds is characterized in that the nucleotide sequence of the CsHD2 gene is shown as SEQ ID NO. 2.
  5. 5. The application of the expression vector containing the CsHD2 gene in promoting the growth of plant lateral buds as claimed in claim 4, wherein the expression vector is a PBI121 expression vector.
  6. 6. The use of the expression vector containing CsHD2 gene in promoting the growth of plant lateral buds as claimed in claim 4, wherein the plants are tobacco and lemon.
  7. 7. A method for promoting the growth of plant lateral buds is characterized by comprising the step of transferring a CsHD2 gene related to the promotion of the growth of plant lateral buds into a plant body to obtain a transgenic plant of the CsHD2 gene.
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Cited By (1)

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Publication number Priority date Publication date Assignee Title
CN114990132A (en) * 2022-05-11 2022-09-02 华中农业大学 Application of citrus CcLFY gene in regulation and control of shoot development

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