Lotus germ specific promoter pNnCYP80A and application thereof

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly discloses a lotus germ specific promoter pNnCYP80A and application thereof, and the nucleotide sequence of the promoter is shown as SEQ ID NO.1. The invention also discloses a preparation method of the promoter. The pNnCYP80A promoter provided by the invention is high in activity and can drive a GUS reporter gene to be efficiently and specifically expressed in an immature embryo of transgenic arabidopsis thaliana. The promoter can drive an exogenous gene to be efficiently and specifically expressed in a plant embryo organ, and has a wide application prospect in the fields of researching the development of the plant embryo organ, directionally improving the embryo quality and yield character through transgenosis and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering and molecular biology, and specifically relates to a lotus embryo-specific promoter pNnCYP80A and an application thereof. Background Art

[0002] The development of plant genetic engineering and molecular biology technology has achieved the improvement of traditional agronomic traits such as high yield, high quality and high resistance of plants. At the same time, it has also made plants a bioreactor for pharmaceutical production in the field of biomedicine. Lotus is an important aquatic economic crop that combines ornamental, edible and medicinal values. It has a long history of cultivation, a wide distribution range and rich germplasm resources. The whole lotus plant can be used as medicine. Among them, the lotus embryo is one of the most important organs enriched in lotus secondary metabolites, and it is also the organ with the highest content of lotus dibenzylisoquinoline alkaloids. Lotus embryo is an important resource for studying the synthesis and regulation of lotus secondary metabolites, as well as developing innovative drugs related to lotus dibenzylisoquinoline alkaloids.

[0003] The research purpose of plant genetic engineering is to make the target gene express efficiently and stably in plant cells, so as to achieve high-quality and stable improved plant traits. The gene expression process is mainly regulated by transcription, and the promoter plays a key role in expression regulation. The promoter is a DNA sequence located upstream of the start position of the gene coding sequence, which participates in the activation, recognition, and binding of RNA polymerase, ensuring the accurate and effective start of transcription. At the same time, the promoter also determines the expression site, expression period, and expression intensity of the gene. According to the different expression patterns, promoters can be divided into constitutive promoters, inducible promoters, and tissue-specific promoters. Among them, tissue-specific promoters are usually related to the regulation of plant growth and development, and can drive genes to be transcribed selectively in specific tissues or organs. This tissue-specific expression pattern can effectively avoid the negative effects of constitutive promoters driving the continuous expression of exogenous genes in non-target tissues, accurately regulate the expression site and expression period of exogenous genes in plants, and more safely improve specific traits of plants.

[0004] The embryo is an important component of lotus seeds and can develop into the stems and leaves of plants. After the lotus stigma is pollinated, the embryo matures in about 24 days, and then dehydrates and dries to enter the dormant stage. Some embryo and endosperm specific expression promoters have been published in monocotyledonous plants such as rice, wheat, and corn. However, the mining of embryo-specific expression genes in dicotyledonous plants is still limited, and the reports are mainly concentrated in soybean plants. Correspondingly, there is less knowledge about specific high-expression promoters in the embryo. Therefore, isolating and obtaining seed embryo-specific expression promoters will help to more comprehensively understand the plant embryo development mechanism and cell differentiation mechanism. At the same time, it can also be widely used in plant genetic engineering research, so that the target gene can be accurately expressed in specific tissues, and has broad application prospects in the fields of plant embryo organ development research and embryo quality trait specific improvement. Summary of the invention

[0005] The purpose of the present invention is to provide an efficient embryo-specific promoter pNnCYP80A and its application, the promoter is used to construct a target gene fusion vector specifically expressed in embryos, and the promoter is transferred into the plant genome by a genetic transformation system to achieve directional control of the target gene expression site, and obtain a transgenic plant that specifically expresses the target gene in embryos.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The nucleotide sequence of the lotus embryo-specific promoter pNnCYP80A is shown in SEQ ID NO.1, and the primer sequences used to amplify the promoter pNnCYP80A are shown in SEQ ID NOs.2 and 3.

[0008] The invention relates to an expression cassette containing a lotus embryo-specific promoter pNnCYP80A, wherein the promoter pNnCYP80A is connected to the upstream of a target gene.

[0009] The recombinant expression vector containing the embryo-specific promoter pNnCYP80A is preferably constructed using the plant expression vector pBI101 as a vector.

[0010] Application of lotus embryo specific promoter pNnCYP80A or an expression box or recombinant expression vector containing the promoter in driving the specific expression of target genes in plant embryos.

[0011] Compared with the prior art, the technical scheme of the present invention has the following beneficial effects: the present invention obtains the gene NnCYP80A that is highly expressed in lotus embryos by transcriptome sequencing technology screening, and clones the pNnCYP80A promoter 1489bp upstream of the start codon ATG of the embryo-specific expression gene NnCYP80A. It is constructed into the plant expression vector pBI101 with the GUS reporter gene by enzyme ligation, and Arabidopsis thaliana is transformed to verify the promoter function. The pNnCYP80A promoter of the present invention can specifically drive the downstream GUS gene to be highly expressed in the embryo. Therefore, the expression of the target gene will not affect the nutritional growth and development of the plant. In the future, the expression characteristics of its tissue specificity can be used to carry out plant embryo genetic engineering transformation in a directional manner, such as being applied to plant embryo organ development research, embryo quality trait improvement, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 These are the results of expression analysis of NnCYP80A gene regulated by promoter pNnCYP80A in different lotus tissues.

[0013] Figure 2This is a map of the plant GUS gene expression vector pBI101.

[0014] Figure 3 This is a schematic diagram of the restriction sites of the promoter pNnCYP80A fused to the GUS gene expression vector pBI101.

[0015] Figure 4 These are the results of GUS staining in different parts of Arabidopsis thaliana and GUS gene fluorescence quantitative analysis. DETAILED DESCRIPTION

[0016] The present invention is described in detail below in conjunction with the accompanying drawings and implementation examples. The specific examples are only for illustrating the present invention and do not limit the protection scope of the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0017] Example 1

[0018] Obtaining the full-length sequence of lotus embryo-specific promoter pNnCYP80A:

[0019] According to the analysis of transcriptome data of various lotus tissues, it was found that NnCYP80A gene was highly expressed in lotus core tissues, such as Figure 1 As shown. Based on the whole genome sequence of lotus, the upstream promoter sequence of NnCYP80A (Nn21373) was obtained, and the promoter was named pNnCYP80A, and its nucleotide sequence is shown in SEQ ID NO.1. Primers were designed to amplify the fragment, and a restriction site (underlined) was added to the 5' end of each pair of primers to facilitate the subsequent construction of a plant expression vector. The forward primer was F1: 5'-ACGC GTCGA C CATAGAAGTTGTGCCAATGCAT-3'(SalI), reverse primer is R1:5'-GC TCTAGA TAGAGTTTATAGAGAAGATGTAATACTAG-3'(XbaI).

[0020] The genomic DNA of lotus variety 'Autumn Star' was extracted according to the method described in the genomic DNA extraction kit (TIANGEN), and the promoter pNnCYP80A sequence was amplified using F1 and R1 using the DNA as a template. Reaction system: 10 μM F1 and R1 primers, 2.5 μl each, 2×primerSTAR (TAKARA) 100

[0021] μl, genomic DNA (100ng / μl) 1μl, add double distilled water to 200μl. PCR reaction program: 98℃ pre-denaturation for 5min; 98℃ denaturation for 15s, 58℃ annealing for 15s, 72℃ extension for 30s, 35 cycles; finally 72℃ extension for 5min. Take 5μl of PCR product and perform 1% agarose gel electrophoresis to amplify the target band with a size of about 1500bp. All amplification systems were recovered and purified by PCR product recovery kit (TIANGEN).

[0022] Example 2

[0023] Construction of plant expression vector of promoter pNnCYP80A fused with GUS gene:

[0024] The purified PCR product (pNnCYP80A sequence with restriction sites at both ends) and the plant expression vector pBI101 were double-digested with SalI and XbaI. The double-digestion system was as follows: 3 μg of purified PCR product or pBI101 vector, 10 μl of CutSmartBuffer, 3 μl of endonucleases SalI and XbaI (20000U / ml), and double distilled water was added to 100 μl. The enzyme digestion system was incubated in a 37°C water bath for 3 h, and the enzyme digestion effect was detected by 1% agarose gel electrophoresis. After purifying the pNnCYP80A sequence and the plant expression vector pBI101 after enzyme digestion using a product recovery kit, the pNnCYP80A target fragment was ligated to the vector pBI101 using T4 ligase. The ligation product was transformed into the E. coli competent cell TOP10, and the obtained single colony was verified by PCR and sequencing. The PCR system was as follows: 10 μM F1 and R1 primers, 1 μl each, 2×Phanta Flash Master Mix (Vazyme) 10 μl, bacterial solution 1 μl, double distilled water to 20 μl. PCR reaction procedure: 95°C pre-denaturation for 5 min; 95°C denaturation for 15 s, 58°C annealing for 15 s, 72°C extension for 30 s, 35 cycles; final extension at 72°C for 5 min. The positive recombinant plasmid was named pBI101-pNnCYP80A-GUS. The vector pBI101 map and the promoter recombination site schematic are shown in the figure below. Figure 2 and Figure 3 shown.

[0025] Example 3

[0026] Transformation of Arabidopsis thaliana with recombinant plasmid pBI101-pNnCYP80A-GUS:

[0027] The recombinant plasmid pBI101-pNnCYP80A-GUS was introduced into Agrobacterium tumefaciens GV3101 (deposited in Wuhan Botanical Garden, Chinese Academy of Sciences) by electroporation, incubated at 28°C shaker at 200 rpm for 3 h, and then coated on a resistance screening solid LB plate containing 50 μg / L kanamycin and 50 μg / L rifampicin, and inverted in a 28°C incubator for 2 days. Single colonies were picked for colony PCR verification, and the PCR verification method was the same as that shown in Example 2.

[0028] The correctly verified Agrobacterium GV3101 was used to transform Arabidopsis thaliana by the floral dip method. The harvested T0 generation Arabidopsis seeds were germinated in 1 / 2MS seed screening medium containing 50μg / L kanamycin. The T0 generation plants with normal growth were selected for planting, and the T1 generation plants were screened and harvested in the same way. After the plants grew up, the genomic DNA of wild-type and pBI101-pNnCYP80A-GUS Arabidopsis was extracted for PCR positive identification. The PCR system was as follows: 10μM F1 and R1 primers, 1μl each, 2×Phanta FlashMaster Mix (Vazyme) 10μl, genomic DNA 100ng, double distilled water added to 20μl. PCR reaction program: 95℃ pre-denaturation for 5min; 95℃ denaturation for 15s, 58℃ annealing for 15s, 72℃ extension for 30s, 35 cycles; finally 72℃ extension for 5min. Arabidopsis thaliana that was positive in PCR identification was screened and harvested in the same way. The T2 generation plants were observed for phenotype and identified for gene expression after normal germination in the soil.

[0029] Example 4

[0030] GUS identification of transgenic plants:

[0031] The T2 generation of Arabidopsis thaliana transgenic with pBI101-pNnCYP80A-GUS was subjected to histochemical staining. The specific steps are as follows: cut off some leaves, inflorescences, fruit pods, and seeds of the T2 generation of Arabidopsis thaliana transgenic with pBI101-pNnCYP80A-GUS, soak them in GUS staining reagent (Zhongke Ruitai, catalog number: RTU4032), and use a vacuum pump to perform negative pressure treatment for 10 minutes so that the staining solution can fully penetrate the plant tissue, and react at 37°C overnight. After staining, the tissue was decolorized several times in 75% ethanol and observed and photographed under a stereomicroscope. The results showed that no blue was observed in all tissues of the wild-type Arabidopsis thaliana. No blue was observed in the roots, leaves, stems, and petals of the pBI101-pNnCYP80A-GUS Arabidopsis transgenic strain ( Figure 4 Since the expression site of pNnCYP80A-GUS is located in the embryo inside the seed, which is blocked by the seed coat and endosperm, the GUS staining and observation of the embryo GUS staining under a stereomicroscope are not ideal, so qPCR was used to quantitatively detect the GUS gene in various tissues of Arabidopsis.

[0032] The quantitative detection of GUS gene was carried out on T2 generation plants of Arabidopsis thaliana transgenic with pBI101-pNnCYP80A-GUS, and the quantitative primers were GUS-F: 5'-GCGGACTTACGTGGCAAAGGA-3', GUS-R: 5'-CGACAGCAGCAGTTTCATCAATCA-3'. The specific steps were as follows: about 100 mg of root, stem, leaf, inflorescence and seed tissues of pBI101-pNnCYP80A-GUS transgenic Arabidopsis plants were taken, RNA was extracted according to the instructions of the kit (ZOMANBIO), and RNA was reverse transcribed into cDNA (TransGen) for fluorescence quantitative PCR analysis, and the total volume of the reaction mixture was 15 μl, 2×TB Green Premix Ex TaqII 7.5 μl, ROX dye 0.3 μl, quantitative forward and reverse primers 0.5 μl (10 μM), and cDNA 3 μl (50 ng). The amplification procedure was 95℃ pre-denaturation for 10 min; 95℃ denaturation for 30 s, 60℃ annealing and extension for 30 s, and 40 cycles. Three groups of biological and technical replicates were set. Fluorescence quantitative results showed that the expression of GUS gene in Arabidopsis seeds transfected with pBI101-pNnCYP80A-GUS was significantly higher than that in roots, stems, leaves, and inflorescence tissues ( Figure 4 B).

[0033] In summary, the present invention provides a lotus tissue-specific expression promoter pNnCYP80A, the promoter nucleotide sequence is shown in SEQ ID NO.1, and the promoter drives the target gene to be highly expressed in lotus embryos. The promoter pNnCYP80A is connected to the target gene and transferred into Arabidopsis thaliana, and the promoter is used to drive the high expression of the target gene in Arabidopsis thaliana seeds. The lotus embryo-specific promoter pNnCYP80A helps to more fully understand the plant embryo development mechanism and cell differentiation mechanism. At the same time, it can also be widely used in plant genetic engineering research, and has broad application prospects in the fields of plant embryo organ development research, embryo quality trait specific improvement, etc.

[0034] It should be understood that the above specific implementation method is only used to explain the basic principles of the present invention, and is not intended to limit the present invention in any form. Changes and improvements made based on the present invention still fall within the scope of protection of the present invention.

Claims

1. Lotus embryo-specific promoter pNnCYP80A, characterized in that: The sequence of the promoter is shown in SEQ ID NO.

1.

2. A recombinant expression vector, characterized in that: It comprises the embryo-specific promoter pNnCYP80A as claimed in claim 1.

3. The recombinant expression vector according to claim 2, characterized in that The recombinant expression vector was constructed using the plant expression vector pBI101.

4. A set of primers for amplifying the promoter according to claim 1, characterized in that: The primer sequences are shown in SEQ ID NOs. 2 and 3.

5. Use of the promoter pNnCYP80A according to claim 1 in preparing transgenic plants.

6. Use of the promoter pNnCYP80A according to claim 1 to drive the specific expression of a target gene in plant embryos.

7. Use of the recombinant expression vector according to claim 2 to drive the specific expression of a target gene in plant embryos.

8. The use according to claim 5 or 6, characterized in that: The plants are lotus and Arabidopsis thaliana.

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