CN115851737B - Fir constitutive promoter Cula and application thereof - Google Patents
Fir constitutive promoter Cula and application thereof Download PDFInfo
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Abstract
The invention provides a fir constitutive promoter Cula and application thereof. The nucleotide sequence of the fir constitutive promoter Cula is shown as SEQ ID NO.1, and the nucleotide sequence of the complement is shown as SEQ ID NO. 2. The Cula promoter is more active in fir protoplasts than the traditional promoters CaM35S, zmubi and CmYLCV, and may be more suitable for fir genetic engineering. The Cula11 promoter has stronger activity in roots and leaves of poplar than the currently-used promoter CaM 35S. In rice, the Cula promoter has stronger expression activity in roots and leaves than promoters CaM35S, zmubi, actin1, CMYLCV and the like widely applied to rice genetic engineering.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a fir constitutive promoter Cula and application thereof, wherein the Cula promoter can efficiently drive the expression of a target gene in fir protoplasts and has strong activity in over-expressing poplar, rice roots and leaves.
Background
China fir (Cunninghamia lanceolata (lamb.) Hook) is a tree of the genus Cunninghamia of the family Cunninghamiae, and is mainly distributed in 17 provinces in the southern subtropical zone of China. Molecular breeding of fir by genetic engineering is a current research hotspot and difficulty. Promoters are essential elements for gene overexpression. Wherein, the constitutive strong promoter can promote gene expression in all tissues, organs and development stages, and has strong expression target genes without space-time specificity in all tissues. In practice, constitutive promoters are expressed in most tissues under different conditions, usually derived from plant virus or plant housekeeping genes. However, promoters currently applicable to molecular breeding of fir, particularly strong constitutive promoters isolated from fir, have been rarely reported.
In the field of dicotyledonous genetic engineering, the CaMV35S promoter from cauliflower mosaic virus is one of the most widely used promoters for basic research and transgenic plant development. The expression level of the promoter in different tissues of many plants is relatively high. This strong expression in different plant tissues is caused by the additive effects of multiple tissue-specific elements. The CmYLCV promoter isolated from yellow leaf curl virus is also a more commonly used constitutive expression promoter and has been shown to be highly active in calli, meristematic and vegetative and reproductive tissues of arabidopsis, tobacco, tomato, maize and rice. In addition to its extremely narrow host selection range, the CmYLCV promoter is a powerful tool for regulating transgene expression in a variety of plant species. At present, the CaMV35S promoter and CmYLCV promoter are representative of constitutive promoters and are mainly applied to the field of genetic engineering of dicotyledonous plants.
In monocots such as rice, the activity of the CaM35S promoter is relatively low. Thus, a series of constitutive promoters derived from monocots have been identified separately and applied to genetic engineering of monocots. Promoters which are commonly used at present are ZmUBI promoter derived from corn, act1, osTubA1, osCc1, RUBQ1 and the like derived from rice. These promoters are strongly expressed in monocotyledonous plants such as rice, maize, wheat, bamboo, etc., and play an important role in molecular breeding of monocotyledonous plants. ZmUBI promoter is most widely used and is strongly expressed in almost all cell types, young tissues, but is poorly expressed in older tissues.
When in transgenic breeding or gene function research, the constitutive promoter can make the exogenous gene express in plant in high efficiency, continuously and stably for a long time, and is favorable for the exogenous gene to exert its function. Promoters from other species often undergo gene silencing when used for heterologous expression, and in addition, different promoters, particularly endogenous promoters, are required to avoid gene suppression or silencing by driving multiple genes with the same type of promoter at the same time. Over the past few decades, although there have been many choices for promoters applied to monocotyledonous and dicotyledonous plants, promoters isolated from gymnosperms have been rarely reported. Few gymnosperms have been reported to be transgenic, and most of them use CaM35S as a promoter, which cannot meet the genetic engineering requirements of gymnosperms such as fir. Therefore, the isolation and identification of the strong constitutive promoter of the Chinese fir endogenous has important significance for transgenic breeding of gymnosperms such as Chinese fir and the like.
Disclosure of Invention
The invention aims to provide a promoter Cula11 for driving exogenous genes to express in fir, and application of the promoter, in particular application in dicotyledonous plants represented by poplar and monocotyledonous plants represented by rice.
In order to achieve the above purpose, the invention adopts the following technical scheme:
A constitutive expression promoter Cula is derived from fir, its nucleotide sequence is shown in SEQ ID NO.1, and its complement nucleotide sequence is shown in SEQ ID NO. 2.
A recombinant vector comprising the fir constitutive promoter Cula as described above.
A recombinant strain comprising the recombinant vector described above.
Preferably, the recombinant strain is agrobacterium tumefaciens.
The application of the constitutive expression promoter Cula to the cultivation of transgenic plants.
Preferably, the plant is fir, rice or poplar.
Preferably, the application is specifically: and connecting the constitutive expression promoter Cula11 to the sequence upstream of the gene to be expressed of the plant binary expression vector PCAMBIA to obtain a recombinant vector, and transforming the product of the recombinant vector into plant cells, tissues or organs for cultivation.
The invention has the remarkable advantages that:
Compared with the traditional promoters CaM35S, zmubi and CmYLCV, the Cula promoter provided by the invention has stronger activity in fir protoplasts. The Cula11 promoter has stronger activity in roots and leaves of poplar than the currently-used promoter CaM 35S. In rice, the Cula promoter has stronger expression activity in roots and leaves than promoters CaM35S, zmUBI, actin1, CMYLCV and the like widely applied to rice genetic engineering.
Drawings
Fig. 1: plasmid map of recombinant vector pCambia 1301-Cula. .
Fig. 2: cula11-eGFP plasmid map.
Fig. 3: the Cula promoter was expressed in China fir protoplast (A), and the fluorescence intensity of expression was measured (B).
Fig. 4: activity analysis of Cula A promoter in transgenic Rice.
Fig. 5: activity analysis of Cula A promoter in transgenic poplar.
Detailed Description
The invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are for illustration of the invention only and are not intended to limit the scope of the invention in any way.
The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials and reagent materials used in the examples below are all commercially available products unless otherwise specified.
EXAMPLE 1 cloning of fir constitutive expression promoter Cula11
According to the sequence of the three-generation full-field transcription group of fir of the subject group, an amplification primer is designed according to the upstream sequence of the gene Cula of fir, and an in-fusion homologous recombination site of the primer is designed according to the characteristics of the selected vector and target gene. The designed primer sequences were as follows:
forward primer: 5'-ggccagtgccaagctctcgaAGGCGATGTGTCAACTAGTGAGT-3' the process of the preparation of the pharmaceutical composition,
Reverse primer:
5’-cctcgcccttgctcaccatgGTTGATGATCACGAATAACACTAGAAATT-3’。
the fir genome DNA is used as a template, a forward primer and a reverse primer are used for amplifying the promoter fragments, and a PCR reaction system and an amplification program are as follows:
PCR reaction system:
amplification procedure ：94 ℃, 3 min→(94 ℃, 30 s→68 ℃, 30 s→68 ℃, 1 min 20 s)×2 cycles→(94 ℃, 30s→67 ℃, 30 s→68 ℃, 1 min 20 s)×2 cycles→(94 ℃, 30s→66 ℃, 30 s→68 ℃, 1min20 s)×2 cycles……(94 ℃, 30 s→57 ℃, 30 s→68 ℃, 1 min 20 s) ×2 cycles→(94 ℃, 30s→56 ℃, 30 s→68 ℃, 1 min 20 s)×15 cycles→68 ℃, 5 min→12 ℃, 10 min.
And (3) performing agarose gel electrophoresis on the amplified fragments, wherein the electrophoresis conditions are as follows: electrophoresis was performed at a voltage of 140V in a 1.5% agarose gel, at 20 min. Gel with the same size as the target strip is cut off for glue recovery, then the gel is connected to the upstream of GUS gene of a plant binary expression vector pCambia1301 (purchased from Camcia company) by an infusion method to obtain a recombinant vector pCambia1301-Cula (the plasmid map is shown in figure 1), the recombinant vector pCambia1301-Cula is transformed into competent cells of escherichia coli JM109 according to a heat shock method, positive clones are obtained through colony PCR screening, and then monoclonal shaking liquid extract plasmids are selected and sequenced for verification. The correct clone is verified to be the fir constitutive expression promoter Cula11 to be obtained, the nucleic acid sequence of which is shown as SEQ ID NO.1, and the nucleotide sequence of the complement of which is shown as SEQ ID NO. 2.
Example 2 expression of Green fluorescent protein (eGFP) reporter Gene Using promoter Cula11
The plasmid pCambia1301-Cula containing Cula promoter sequence is used as a template, a primer containing the specificity of the promoter of the XhoI cleavage site Cula is utilized, a Cula promoter sequence fragment is obtained through PCR, the Cula promoter is cloned to the upstream of the sequence of the modified 35S: NLS-eGFP vector eGFP gene through the XhoI cleavage site, the CaMV35S promoter in the original 35S: NLS-eGFP vector is replaced, the Cula promoter drives the final vector Cula-eGFP of the eGFP reporter gene (the plasmid map is shown in figure 2), the final vector is transferred into agrobacterium tumefaciens EHA105 through a freeze thawing method after sequencing verification, bacterial liquid PCR detection positive cloning is carried out, the positive cloning is picked up and introduced into fir protoplast, observation is carried out under a fluorescence microscope, and the activity of the promoter is analyzed.
Meanwhile, the ZmUbi promoter and the CmYLCV promoter are cloned to the upstream of the sequence of the eGFP gene of the modified 35S: NLS-eGFP vector respectively, the CaMV35S promoter in the original 35S: NLS-eGFP vector is replaced, the final vector ZmUbi-eGFP of the eGFP reporter gene driven by the ZmUbi promoter and the final vector CmYLCV-eGFP of the eGFP reporter gene driven by the CmYLCV promoter are formed, the sequence is verified, the obtained product is transferred into agrobacterium tumefaciens EHA105 by a freeze thawing method, bacterial liquid PCR detection is carried out, positive clones are picked up and introduced into fir protoplasts, observation is carried out under a fluorescent microscope, photographing is carried out, and the promoter activity is analyzed.
Wherein the ZmUbi promoter is disclosed in the literature 【Christensen A H , Quail P H . Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants[J]. Transgenic Research, 1996, 5(3):213-218.】.
Wherein said CmYLCV promoter has been disclosed in the literature [ A Multipurpose Toolkit to Enable Advanced Genome ENGINEERING IN PLANTS [ J ]. THE PLANT CELL, 2017 ].
Wherein the engineered 35S: NLS-eGFP vector is disclosed in the literature 【Liu K H ,Niu Y ,KonishiM , et al. Discovery of nitrate-CPK-NLP signalling in central nutrient-growth networks[J]. Nature, 2017, 545(7654):311-316.】.
The result shows (figure 3) that Cula11 promoter presents stronger fluorescence intensity in fir protoplast, which shows that Cula11 promoter has activity for driving gene expression in fir; by comparison of fluorescence intensities, we found that Cula promoter was 4.2, 3.2 and 1.6 times more active in fir protoplasts than CaMV35S, zmUbi, cmYLCV, respectively.
Example 3 expression of GUS Gene in Rice and poplar Using promoter Cula11
The pCambia1301-Cula plasmid containing Cula promoter sequence is transferred into agrobacterium tumefaciens EHA105 by freeze thawing method, bacterial liquid PCR is carried out to detect positive clone, and the positive clone is selected for genetic transformation of rice. Removing shell of rice seed, sterilizing, sucking surface water with sterile filter paper, inoculating onto callus induction medium, and culturing at 28deg.C. Transforming rice callus by agrobacterium-mediated method, screening to obtain resistant callus, inoculating into differentiation medium, and inoculating into rooting medium after green bud grows. When the young seedling is stronger, cutting the leaf, extracting its genome DNA by using plant genome DNA extraction kit, then amplifying GUS gene partial sequence to make PCR verification, making GUS histochemical staining analysis on leaf and root system tissue of positive plant, observing under the stereoscopic microscope, photographing and analyzing promoter activity.
The pCambia1301-Cula plasmid containing Cula promoter sequence is transferred into agrobacterium tumefaciens EHA105 by freeze thawing method, bacterial liquid PCR is carried out to detect positive clone, and the positive clone is selected for genetic transformation of rice. Taking poplar aseptic seedling leaves as explants, placing the poplar aseptic seedling leaves into agrobacterium tumefaciens EHA105 bacterial liquid containing pCambia1301-Cula11 plasmids to be infected for 15-20 min, taking out the explants after infection, placing the explants on aseptic filter paper to suck the bacterial liquid on the surfaces of the explants, placing the explants on a co-culture medium, and co-culturing the explants under the dark condition of 25 ℃ for 3 d; then taking out the leaves, placing the leaves in sterile water for washing 5-6 times, transferring the leaves into sterile water containing 250 mg/L of carboxybenzyl, putting the leaves into a shaking table for shaking 10 min at the temperature of 28 ℃, and then taking out the leaves and sucking the surface moisture by sterile filter paper; then spreading the leaves on a culture medium for screening, transferring the leaves into a new culture medium every 20d for subculture until adventitious buds emerge from the edges of the leaves, and then inoculating the leaves into a rooting culture medium. When the young seedling is stronger, cutting the leaf, extracting its genome DNA by using plant genome DNA extraction kit, then amplifying GUS gene partial sequence to make PCR verification, making GUS histochemical staining analysis on leaf and root system tissue of positive plant, observing under the stereoscopic microscope, photographing and analyzing promoter activity.
Meanwhile, the promoters of CaM35S, zmubi, actin and CMYLCV are respectively connected to the upstream of GUS gene of a plant binary expression vector pCambia1301, a recombinant vector is transformed into competent cells of escherichia coli JM109 according to a heat shock method, positive clones are obtained through colony PCR screening, then, a monoclonal shaking liquid extract plasmid is selected, the correct plasmid is verified to be transferred into agrobacterium tumefaciens EHA105 by a freeze thawing method, bacterial liquid PCR detection of the positive clones is carried out, and the positive clones are selected for genetic transformation of rice and poplar.
GUS staining results show (figures 4-5), cula promoter can regulate GUS gene expression in all tissues on the ground and underground of rice and poplar; meanwhile, the Cula promoter has stronger activity in roots and leaves of poplar than the currently-used promoter CaM 35S; in rice, cula a promoter has stronger expression activity in roots and leaves than the promoters CaM35S, zmubi, actin and CMYLCV widely used in rice genetic engineering.
Claims (6)
1. A fir constitutive promoter Cula, characterized in that: the nucleotide sequence of the fir constitutive promoter Cula is shown as SEQ ID NO. 1.
2. The fir constitutive promoter Cula according to claim 1, characterized in that: the nucleotide sequence of the complement of the fir constitutive promoter Cula is shown as SEQ ID NO. 2.
3. A recombinant vector, characterized in that: the recombinant vector comprises the fir constitutive promoter Cula of claim 1.
4. A recombinant strain, characterized in that: the recombinant strain contains the recombinant vector of claim 3.
5. The recombinant strain according to claim 4, wherein: the recombinant strain is agrobacterium tumefaciens.
6. Use of a fir constitutive promoter Cula according to claim 1 for the cultivation of transgenic plants, characterized in that: the plant is fir, rice or poplar.
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