A strong endogenous promoter of lemna and a vector and application thereof

Abstract

The application provides a Lemna australis endogenous strong promoter, a recombinant vector containing the Lemna australis endogenous strong promoter, and application of the Lemna australis endogenous strong promoter and the recombinant vector in starting expression of a target gene in monocotyledonous plants and dicotyledonous plants. The Lemna australis endogenous strong promoter provided by the application has high activity in monocotyledonous plants and dicotyledonous plants, can maintain high activity under conditions of different concentrations of screening agents, and basically does not affect expression of adjacent genes under conditions of different concentrations of screening agents. The application can solve problems of the 35S promoter widely used in the prior art, such as difficulty in further improving activity, low activity in monocotyledonous plants, and influence on expression of adjacent genes.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and biotechnology, and relates to a strong endogenous promoter of duckweed, its vector, and its applications. Background Technology

[0002] Promoters are among the most critical regulatory elements controlling gene expression, enabling precise temporal and spatial regulation of gene expression. Widely used in plant science and plant biotechnology, promoters are an essential tool for studying the function of specific genes, improving crop yield, enhancing plant stress resistance, and regulating the expression of exogenous proteins.

[0003] Many plants have been widely used in bioreactors for expressing exogenous proteins. Compared with other expression systems, plant bioreactors have advantages such as low production cost and ease of scaling up. Currently, the earliest and most widely used promoter in plant bioreactors is the 35S promoter from cauliflower mosaic virus. For example, this promoter has been used for the expression of Japanese encephalitis virus envelope protein in rice, CTB-InsB3 in tobacco, and human β-amyloid protein in potato. The 35S promoter is also widely used to improve crop yield, for example, for plasma membrane H... + Overexpression of ATPase / MFAP1 / NAC23 increased rice yield; overexpression of FTO increased yields of both rice and potatoes; and H... + Overexpression of pyrophosphatase IbVP1 increases starch metabolism and yield in sweet potato. Furthermore, the 35S promoter plays a crucial role in plant stress resistance; for example, overexpression of VvCEB1opt increases salt tolerance in Arabidopsis thaliana, and overexpression of IbPSS1 enhances salt tolerance in transgenic sweet potato. However, studies have shown that the 35S promoter has some limitations, such as low activity in monocotyledonous plants, affecting the expression of adjacent genes, and potentially causing gene silencing. Therefore, scientists are increasingly focusing on the study of endogenous strong promoters in plants, such as the rice actin-1 and maize Ubi-1 promoters. Although they possess high activity, they have only been applied in monocotyledonous plants, and sometimes exhibit unstable promoter activity; for example, the activity level of the Ubi-1 promoter changes with plant development. Meanwhile, experience with the 35S promoter and the maize Ubi-1 promoter over the past few years indicates that their performance is difficult to further improve. Therefore, there is an urgent need to find new endogenous strong promoters in both monocots and dicots to increase the production of heterologous proteins in plant bioreactors, enhance plant stress resistance, and improve crop yield.

[0004] Duckweed is the world's smallest monocotyledonous flowering plant, comprising 5 genera and 36 species. Duckweed is characterized by its rapid growth, lack of competition for land with food crops, and high protein content, making it widely used as a bioreactor for producing exogenous proteins. While duckweed has been used as a bioreactor to express various exogenous proteins, a common feature of existing studies is that they all utilize the 35S promoter for expression; no reports have yet emerged regarding strong endogenous promoters in duckweed. Summary of the Invention

[0005] To address the problems of the difficulty in further improving the activity of existing 35S promoters, their low activity in monocotyledonous plants, and their impact on the expression of adjacent genes, this invention provides a strong endogenous promoter of duckweed and its vector to enrich the types of promoters and improve their activity. This invention also provides the application of the duckweed endogenous promoter and vector in promoting the expression of target genes in monocotyledonous and dicotyledonous plants.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is as follows:

[0007] A strong endogenous promoter for duckweed, derived from the monocotyledonous plant duckweed, has a nucleotide sequence selected from either sequence (a) or sequence (b) below.

[0008] The sequence (a) is shown in SEQ ID NO.1 of the sequence listing; the sequence (b) is a nucleotide sequence that has more than 90% homology with the sequence (a) and has the strong promoter function.

[0009] Further, sequence (b) is a nucleotide sequence having more than 95% homology with sequence (a) and having the strong promoter function. Even further, sequence (b) is a nucleotide sequence having more than 98% homology with sequence (a) and having the strong promoter function.

[0010] The present invention also provides a recombinant vector containing the aforementioned endogenous strong promoter of *Lemna minor*. The recombinant vector is formed by recombining the *Lemna minor* endogenous strong promoter into a vector, and the vector is a plant expression vector well known in the art. Furthermore, the recombinant vector also contains a target gene.

[0011] This invention experimentally demonstrates that the endogenous strong promoter of duckweed provided by this invention exhibits high activity in both monocotyledonous plants (e.g., duckweed) and dicotyledonous plants (e.g., tobacco). Furthermore, the endogenous strong promoter of duckweed provided by this invention maintains high activity under different concentrations of screening agents (e.g., at a concentration of screening agent G418 of 50–500 mg / L). This means that the endogenous strong promoter of duckweed described in this invention can be screened and regenerated under high concentrations of screening agents, and using high concentrations of screening agents for screening and regeneration can improve the positive rate of transgenes. This invention also experimentally demonstrates that the endogenous strong promoter of duckweed provided by this invention has virtually no impact on the expression of adjacent genes under different concentrations of screening agents.

[0012] Based on the above experimental results, this invention provides the application of the aforementioned duckweed endogenous strong promoter in initiating target gene expression in monocotyledonous and dicotyledonous plants. In application, the duckweed endogenous strong promoter is inserted before the target gene to initiate its expression. Specifically, the aforementioned duckweed endogenous strong promoter can be used to increase the yield of heterologous proteins expressed in plant bioreactors, enhance plant stress resistance, and increase crop yield.

[0013] Based on the above experimental results, this invention also provides the application of the above recombinant vector in initiating the expression of target genes in monocotyledonous and dicotyledonous plants.

[0014] Compared with the prior art, the technical solution of the present invention has the following beneficial technical effects:

[0015] This invention provides a strong endogenous promoter for duckweed and a recombinant vector containing this promoter. Taking the expression of the egfp gene driven by the provided strong endogenous promoter in tobacco leaves and duckweed as examples, experiments demonstrate that the strong endogenous promoter provided by this invention exhibits higher activity in tobacco and duckweed than the widely used 35S promoter in existing technologies. This invention addresses the deficiency of low activity of the widely used 35S promoter in monocotyledonous plants.

[0016] This invention also experimentally demonstrates that, under different concentrations of screening agents, the duckweed endogenous promoter provided by this invention exhibits higher activity than the widely used 35S promoter at both the RNA and protein levels. This means that the duckweed endogenous strong promoter described in this invention can be used for screening and regeneration under high concentrations of screening agents, and using high concentrations of screening agents for screening and regeneration can improve the positive rate of transgenes.

[0017] The present invention also experimentally demonstrates that, under different concentrations of screening agents, the endogenous strong promoter of duckweed provided by the present invention does not interfere with the expression of adjacent genes, thus solving the problem that the 35S promoter, which is widely used in the prior art, interferes with the expression of adjacent genes.

[0018] The above experimental results demonstrate that the duckweed endogenous strong promoter and recombinant vector provided by this invention can be used to initiate the expression of target genes in monocotyledonous and dicotyledonous plants. For example, the duckweed endogenous strong promoter and recombinant vector can be used to increase the exogenous protein yield in plant bioreactors, increase plant stress resistance, and increase crop yield, providing new options for solving or improving the problems and deficiencies in these fields. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the construction of the 35S:eGFP vector and the LpSUT2:eGFP vector in Comparative Example 1 and Example 1.

[0020] Figure 2 The results show the transient expression of the promoters in Comparative Example 2 and Example 2 in tobacco leaves.

[0021] Figure 3 This is the result of stable expression of the promoters in Comparative Example 3 and Example 3 in duckweed.

[0022] Figure 4 The mRNA expression levels of the egfp gene driven by the promoters in Comparative Example 4 and Example 4 under different concentrations of the screening agent G418 are shown.

[0023] Figure 5 The results show the fluorescence intensity of eGFP protein driven by the promoters in Comparative Example 4 and Example 4 under different concentrations of the screening agent G418.

[0024] Figure 6 These are the transcriptome sequencing results of 35S:eGFP positive transgenic duckweed and LpSUT2:eGFP positive transgenic duckweed. Detailed Implementation

[0025] The following examples further illustrate the endogenous strong promoter of duckweed and its vector provided by the present invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods in this technical field. Unless otherwise specified, the materials, reagents, and instruments used in the following examples are commercially available, and the duckweed is preserved in the germplasm resource bank of the Chengdu Institute of Biology, Chinese Academy of Sciences.

[0026] Comparative Example 1: Construction of a 35S promoter vector

[0027] In this comparative example, the pCambia2301:GUS vector was modified into the pCambia2301:eGFP vector, denoted as 35S:eGFP, as follows: Figure 1 As shown in the image above, the T-DNA fragment contains two 35S promoters in opposite directions, which drive the expression of the egfp gene and the nptII gene, respectively.

[0028] Example 1: Construction of an endogenous promoter vector for duckweed

[0029] Fresh duckweed samples were collected, and genomic DNA was extracted using the CTAB method. The specific method is as follows: Fresh duckweed samples were placed in a 1.5 mL EP tube and ground into powder under liquid nitrogen. 500 μL of preheated CTAB extraction buffer (preheated to 65°C) was added, and the mixture was gently stirred for 5-10 seconds. The tube was then incubated in a 65°C water bath for 30 minutes, with the tube gently inverted every 5-10 minutes to ensure thorough mixing. After cooling, 500 μL of trichloroethane and isoamyl alcohol (24:1 volume ratio) were added in a fume hood. The mixture was gently inverted and mixed, and centrifuged at 13500 × g at 25°C for 10 minutes. After centrifugation, the tubes separated into layers. The supernatant was transferred to a new EP tube, and an equal volume of anhydrous ethanol was added. The tube was incubated at -20°C for 1 hour, and then centrifuged at 13500 × g at 25°C for 10 minutes. The precipitate was washed 3-4 times with 200 μL of 70% ethanol. The EP tube was then opened and allowed to dry. 50 μL of CTAB buffer was added. DNA was dissolved in ddH2O, and the concentration of genomic DNA was measured.

[0030] The endogenous promoter LpSUT2 (Sucrose transporter 2 of Landoltiapunctata 0202) was selected for experiments. Using genomic DNA as a template, a 2000bp promoter fragment upstream of the LpSUT2 gene (nucleotide sequence shown in SEQ ID NO.1 of the sequence listing) was amplified by PCR. The amplified fragment was then recombined into the expression vector pCambia2301:eGFP, replacing the 35S promoter driving egfp gene expression to construct the LpSUT2:eGFP vector, denoted as LpSUT2:eGFP. Figure 1 The figure below shows the results. The restriction endonucleases used for linearizing the expression vector were pvuII and pstI, and the recombinant primers used were LpSUT2-F: 5'-GATTCATTAATGCAGCTGAGCTCCCTCTCCTTCTTCTCCT-3' and LpSUT2-R: 5'-GCCAAGCTTATGGCACTGCAGCATCTCCAATTCAGCCTCC-3'.

[0031] Comparative Example 2: Transient expression of the egfp gene driven by the 35S promoter in leaves of the dicotyledonous plant tobacco.

[0032] The recombinant plasmid 35S:eGFP constructed in Comparative Example 1 was transformed into Agrobacterium GV3101. Subsequently, Agrobacterium GV3101 containing the recombinant plasmid was used to infect tobacco leaves for transient expression. Two days later, the fluorescence intensity of the green fluorescent protein eGFP was observed and photographed using a laser scanning confocal microscope.

[0033] The steps for infecting tobacco leaves with Agrobacterium are as follows: Take 20 μL of Agrobacterium GV3101 stored at -80℃ and inoculate it into fresh LB broth containing the responsive antibiotic. Incubate overnight at 28℃ and 200 rpm. Transfer the culture to an Erlenmeyer flask containing LB broth at a volume ratio of 1:50 and incubate at 28℃ and 200 rpm for approximately 6 hours until OD (Organic Dry Index) is reached. 600 1. Take 1 mL of bacterial solution and centrifuge at 4000 rpm for 10 min to collect bacterial cells. Resuspend the bacterial cells in 1 mL of Agrobacterium infection solution containing acetylsuccinone AS. Place the solution at room temperature and in the dark for 2 h. Use a sterile syringe to draw up the bacterial solution and infect tobacco leaves. Place the infected tobacco leaves in the dark overnight. The next morning, transfer them to a 16 h:8 h light condition for 2 days. Observe the fluorescence intensity using a laser scanning confocal microscope.

[0034] Example 2: Transient expression of the egfp gene driven by the endogenous promoter of duckweed in leaves of the dicotyledonous plant tobacco.

[0035] The recombinant plasmid LpSUT2:eGFP constructed in Example 1 was transformed into Agrobacterium GV3101. Subsequently, the Agrobacterium GV3101 containing the recombinant plasmid was used to transiently infect tobacco leaves. Two days later, the fluorescence intensity of the green fluorescent protein eGFP was observed and photographed using a laser scanning confocal microscope. In this example, the steps for infecting tobacco leaves with Agrobacterium were the same as in Comparative Example 2.

[0036] The results of transient expression of the promoters in Comparative Example 2 and Example 2 in tobacco leaves are as follows: Figure 2 As shown, by Figure 2 It can be seen that in the dicotyledonous plant tobacco, the fluorescence intensity of green fluorescent protein eGFP expressed by the endogenous promoter LpSUT2 of duckweed is higher than that of green fluorescent protein eGFP expressed by the currently widely used 35S promoter.

[0037] Comparative Example 3: The egfp gene driven by the 35S promoter is stably expressed in the monocotyledonous plant duckweed.

[0038] The recombinant plasmid 35S:eGFP constructed in Comparative Example 1 was transformed into Agrobacterium GV3101. Then, Agrobacterium GV3101 containing the recombinant plasmid was used to infect duckweed callus tissue. After 3 days of co-culture, the callus tissue was transferred to selection medium containing 100 mg / L G418. After 60 days of selection, the callus tissue was transferred to fresh regeneration medium containing 100 mg / L G418. After 30 days of regeneration, leaves appeared. At this point, single leaves were picked and cultured in fresh medium for further expansion. A suitable amount of samples from the regenerated lines were collected, and genomic DNA was extracted. PCR was used to detect whether the target fragment was integrated into the duckweed genome. After obtaining positive transgenes, the fluorescence intensity of the green fluorescent protein eGFP in the transgenic duckweed was observed using a laser scanning confocal microscope.

[0039] The steps for infecting duckweed callus with Agrobacterium are as follows: 20 μL of Agrobacterium GV3101 stored at -80℃ was inoculated into fresh LB medium containing a responsive antibiotic and cultured overnight at 28℃ and 200 rpm. The next day, the culture was transferred to an Erlenmeyer flask containing LB medium at a volume ratio of 1:50 and cultured at 28℃ and 200 rpm for approximately 6 hours until OD (dose retardation). 600 1. The bacterial culture was placed in a 50 mL sterile tube and centrifuged at 4000 rpm for 15 min at 25°C (room temperature). After centrifugation, the bacterial cells were resuspended in 25 mL of resuspension buffer (prepared with 0.6 M mannitol solution on MS basal medium, without sucrose, and with 100 μM acetylsyringone AS, pH = 5.6). Subsequent experiments were performed in a clean bench. The resuspended Agrobacterium was placed in the dark for 1 hour to stimulate the callus tissue for a period of time after adding AS, thereby improving its infection efficiency. Growth samples were picked from the plate. Healthy callus tissue was placed in Agrobacterium resuspension for 3-5 minutes for infection. The Agrobacterium resuspension was then discarded, and excess resuspension was aspirated with a pipette tip. The callus tissue was then transferred to a pre-prepared sterile co-culture medium (MS medium with 30 g / L sucrose, 1 μM 2,4-D, 2 μM 6-BA, and 100 μM AS added). The callus tissue was co-cultured in the dark at 25°C for 3 days. After co-culture, the callus tissue was washed 3-5 times with sterile water and then transferred to a selection medium.

[0040] Example 3: Stable expression of egfp driven by endogenous promoter of duckweed in monocotyledonous duckweed.

[0041] The recombinant plasmid LpSUT2:eGFP constructed in Example 1 was transformed into Agrobacterium GV3101. Then, Agrobacterium GV3101 containing the recombinant plasmid was used to infect duckweed callus tissue. After 3 days of co-culture, the callus tissue was transferred to selection medium containing 100 mg / L G418. After 60 days of selection, the callus tissue was transferred to fresh regeneration medium containing 100 mg / L G418. After 30 days of regeneration, leaves appeared. At this point, single leaves were picked and cultured in fresh medium for further expansion. A suitable amount of samples from the regenerated lines were selected, and genomic DNA was extracted. PCR was used to detect whether the target fragment was integrated into the duckweed genome. After obtaining positive transgenes, the fluorescence intensity of the green fluorescent protein eGFP in the transgenic duckweed was observed using a laser scanning confocal microscope. In this example, the steps for Agrobacterium infection of duckweed callus tissue were the same as in Comparative Example 3.

[0042] The results of stable expression of the promoters in Comparative Example 3 and Example 3 in duckweed are as follows: Figure 3 As shown, by Figure 3 It was found that when the concentration of the screening agent G418 was 100 mg / L, the fluorescence intensity of the green fluorescent protein (eGFP) expressed by the endogenous promoter LpSUT2 in the monocotyledonous plant *Lemna minor* was higher than that of the green fluorescent protein (eGFP) expressed by the 35S promoter. This indicates that under the condition of a screening agent G418 concentration of 100 mg / L, the activity of the endogenous promoter LpSUT2 in *Lemna minor* is higher than that of the 35S promoter.

[0043] Comparative Example 4: mRNA expression levels of the egfp gene driven by the 35S promoter under different concentrations of the screening agent G418.

[0044] The recombinant plasmid 35S:eGFP constructed in Example 1 was transformed into Agrobacterium GV3101. Then, Agrobacterium GV3101 containing the recombinant plasmid was used to infect duckweed callus tissue. After 3 days of co-culture, the callus tissue was transferred to selection media containing 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L G418, respectively. After 60 days of selection, the callus tissue was transferred to fresh regeneration media containing 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L G418. After 30 days of regeneration, leaves appeared. At this time, single leaves were picked and cultured in fresh media for further expansion. A suitable amount of samples from the regenerated lines were selected, and genomic DNA was extracted. PCR was used to detect whether the target fragment was integrated into the duckweed genome. After obtaining positive transgenes, the mRNA expression level of the egfp gene was detected by real-time quantitative PCR. In this comparative example, the steps for Agrobacterium infection of duckweed callus were the same as in comparative example 3.

[0045] Example 4: mRNA expression levels of the egfp gene driven by the endogenous promoter of duckweed under different concentrations of the screening agent G418.

[0046] The recombinant plasmid LpSUT2:eGFP constructed in Example 1 was transformed into Agrobacterium GV3101. Then, Agrobacterium GV3101 containing the recombinant plasmid was used to infect duckweed callus tissue. After 3 days of co-culture, the callus tissue was transferred to selection media containing 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L G418, respectively. After 60 days of selection, the callus tissue was transferred to fresh regeneration media containing 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L G418. After 30 days of regeneration, leaves appeared. At this point, single leaves were picked and cultured in fresh media for further expansion. A suitable amount of samples from the regenerated lines were selected, and genomic DNA was extracted. PCR was used to detect whether the target fragment was integrated into the duckweed genome. After obtaining positive transgenes, the mRNA expression level of the egfp gene was detected by real-time quantitative PCR. In this embodiment, the step of infecting duckweed callus with Agrobacterium is the same as in Comparative Example 3.

[0047] The mRNA expression levels of the egfp gene driven by the promoters in Comparative Example 4 and Example 4 under different concentrations of the screening agent G418 are as follows: Figure 4 As shown. By Figure 4 It was found that when the concentrations of the screening agent G418 were 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L, the expression level of the egfp gene driven by the endogenous promoter LpSUT2 in the monocotyledonous plant *Lemna minor* was higher at the RNA level than that driven by the 35S promoter. Furthermore, with increasing G418 concentration, the expression level of the egfp gene driven by the 35S promoter decreased, while the expression level of the egfp gene driven by the endogenous promoter LpSUT2 did not show a significant difference with increasing G418 concentration, remaining at a high level. This indicates that under different concentrations of the screening agent G418, at the RNA level, the activity of the endogenous promoter LpSUT2 provided by this invention is higher than that of the 35S promoter.

[0048] Comparative Example 5: Fluorescence intensity of eGFP protein expressed by the egfp gene driven by the 35S promoter under different concentrations of the selection agent G418.

[0049] An appropriate amount of 35S:eGFP-positive transgenic duckweed obtained in Comparative Example 4 was taken, and the fluorescence intensity of the eGFP protein was observed using a laser scanning confocal microscope. The laser scanning confocal microscope parameters were: excitation wavelength of 488 nm, emission wavelength of 505-530 nm, and the instrument model was Leica TCS SP8.

[0050] Example 5: Fluorescence intensity of eGFP protein driven by duckweed endogenous promoter under different G418 selection agent concentrations to express egfp gene

[0051] An appropriate amount of LpSUT2:eGFP-positive transgenic duckweed obtained in Example 4 was taken, and the fluorescence intensity of the eGFP protein was observed using a laser scanning confocal microscope. The laser scanning confocal microscope parameters were: excitation wavelength of 488 nm, emission wavelength of 505-530 nm, and the instrument model was Leica TCS SP8.

[0052] The fluorescence intensity results of eGFP protein expressed by the promoters in Comparative Example 4 and Example 4 under different concentrations of the screening agent G418 are as follows: Figure 5 As shown. By Figure 5 It can be seen that the fluorescence intensity of green fluorescent protein (eGFP) expressed in Comparative Example 4 and Example 4 is consistent with the trend of RNA-level expression levels in Comparative Example 3 and Example 3. At the protein level, the fluorescence intensity of eGFP expressed driven by the endogenous promoter LpSUT2 of *Lemna minor* is higher than that driven by the 35S promoter. Furthermore, with the increase of the concentration of the screening agent G418, the fluorescence intensity of eGFP expressed driven by the 35S promoter shows a decreasing trend, while the fluorescence intensity of eGFP expressed driven by the endogenous promoter LpSUT2 of *Lemna minor* does not show a significant difference with the increase of the G418 concentration, maintaining a high expression level at the protein level. This indicates that under different concentrations of the screening agent G418, at the protein level, the activity of the endogenous promoter LpSUT2 of *Lemna minor* provided by this invention is higher than that of the 35S promoter.

[0053] Comparative Example 6: Transcriptome sequencing of 35S:eGFP positive transgenic duckweed

[0054] 35S:eGFP positive transgenic duckweed obtained from Comparative Example 4 at screening agent G418 concentrations of 100 mg / L and 500 mg / L was selected, and samples were stored at -80℃. Transcriptome sequencing was performed on the samples using the Illumina NovaSeq high-throughput sequencing platform.

[0055] Example 6: Transcriptome sequencing of LpSUT2:eGFP positive transgenic duckweed

[0056] The LpSUT2:eGFP positive transgenic duckweed obtained in Example 4 when the concentration of screening agent G418 was 100 mg / L and 500 mg / L was selected, and samples were stored at -80℃. The transcriptomes of the samples were sequenced using the Illumina NovaSeq high-throughput sequencing platform.

[0057] Transcriptome sequencing results of 35S:eGFP positive transgenic duckweed and LpSUT2:eGFP positive transgenic duckweed are as follows: Figure 6 As shown. By Figure 6 It was found that when the concentration of the screening agent G418 increased from 100 mg / L to 500 mg / L, the expression level of the egfp gene driven by the 35S promoter decreased significantly, while the expression level of the nptII gene driven by another 35S promoter in the T-DNA (transfer DNA) fragment increased significantly. When the concentration of the screening agent G418 increased from 100 mg / L to 500 mg / L, there was no significant difference in the expression level of the egfp gene driven by the duckweed endogenous promoter LpSUT2 provided by this invention, and there was also no significant difference in the expression level of the nptII gene driven by another 35S promoter in the T-DNA fragment.

[0058] The above experimental results show that when the concentration of the screening agent G418 is increased from 100 mg / L to 500 mg / L, the two 35S promoters in the T-DNA fragment will affect each other's activity. However, after replacing the 35S promoter driving egfp gene expression with the endogenous promoter LpSUT2 provided by this invention, the two promoters in the T-DNA fragment will not affect each other's activity.

Claims

1. A strong endogenous promoter for duckweed, characterized in that, The promoter is derived from the monocotyledonous plant duckweed, and its nucleotide sequence is shown in SEQ ID NO. 1 of the sequence listing.

2. The application of the endogenous strong promoter of duckweed as described in claim 1 in initiating the expression of target genes in monocotyledonous and dicotyledonous plants, wherein the monocotyledonous plant is duckweed and the dicotyledonous plant is tobacco.

3. A recombinant vector containing the endogenous strong promoter of duckweed as described in claim 1.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector also contains the target gene.

5. The application of the recombinant vector of claim 3 or 4 in initiating the expression of a target gene in monocotyledonous and dicotyledonous plants, wherein the monocotyledonous plant is duckweed and the dicotyledonous plant is tobacco.

Images