Lotus embryo-specific promoter pNnCYP80A and its application

Abstract

This invention belongs to the field of plant genetic engineering technology, specifically disclosing a lotus embryo-specific promoter pNnCYP80A and its applications. The nucleotide sequence of the promoter is shown in SEQ ID NO.1. The pNnCYP80A promoter provided by this invention has strong activity and can drive the efficient and specific expression of the GUS reporter gene in the immature embryos of transgenic Arabidopsis thaliana. This promoter can drive the efficient and specific expression of exogenous genes in plant embryo organs, and has broad application prospects in the study of plant embryo organ development and the targeted improvement of embryo quality and yield traits through transgenics.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant genetic engineering and molecular biology, and particularly relates to a lotus embryo bud-specific promoter pNnCYP80A and application thereof. BACKGROUND

[0002] The development of plant genetic engineering and molecular biology technology has realized the improvement of traditional agronomic traits such as high yield, high quality and high resistance of plants, and also made plants become the bioreactor for pharmaceuticals in the field of biological medicine. Lotus is an important aquatic economic crop with ornamental, edible and medicinal values, and has a long cultivation history, a wide distribution range and rich germplasm resources. The whole lotus plant can be used as medicine, among which, lotus embryo bud is one of the main organs for the enrichment of secondary metabolites of lotus, and also the organ with the highest content of lotus dibenzylisoquinoline alkaloids. Lotus embryo bud is an important resource for the study of synthesis and regulation of lotus secondary metabolites and the development of related innovative drugs of lotus dibenzylisoquinoline alkaloids.

[0003] The research purpose of plant genetic engineering is to make the target gene expressed efficiently and stably in plant cells, so as to realize the improvement of plant traits with high quality and stability. The process of gene expression 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 is involved in the activation, recognition and binding of RNA polymerase, and ensures the accurate and efficient initiation of transcription. At the same time, the promoter also determines the expression site, expression period and expression strength of the gene. According to the different expression modes, the promoter can be divided into constitutive promoter, inducible promoter and tissue-specific promoter. Among them, the tissue-specific promoter is usually related to the regulation of plant growth and development, and can drive the selective transcription of genes in specific tissues or organs. This kind of tissue-specific expression mode can effectively avoid the negative effects caused by the continuous expression of exogenous genes in non-target tissues driven by constitutive promoters, accurately regulate the expression site and expression period of exogenous genes in plants, and more safely improve the specific traits of plants.

[0004] Embryo bud is an important component of lotus seed, which can develop into stem and leaf of the plant. After pollination of lotus stigma, embryo bud matures at about 24 days, and then dehydrates and dries to enter the dormancy stage. Some embryo and endosperm-specific expression promoters have been successively 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, the understanding of high expression promoters specific to embryo bud is less. Therefore, the isolation and acquisition of seed embryo bud-specific expression promoters are helpful to more comprehensively understand the mechanism of plant embryo development and cell differentiation mechanism. At the same time, they can also be widely used in plant genetic engineering research, so that the target gene can be accurately expressed in specific tissues, and have broad application prospects in the fields of plant embryo organ development research and specific improvement of embryo bud quality traits. SUMMARY

[0005] The purpose of the present application is to provide an efficient embryo-specific promoter pNnCYP80A and its application, which is used to construct a fusion vector of a target gene expressed specifically in embryo, and is transformed into the plant genome by a genetic transformation system to realize directional manipulation of the expression site of the target gene and obtain a transgenic plant with the target gene expressed specifically in embryo.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical scheme:

[0007] The nucleotide sequence of the lotus embryo-specific promoter pNnCYP80A is shown as SEQ ID NO. 1, and the primer sequences for amplifying the promoter pNnCYP80A are shown as SEQ ID NO. 2 and 3.

[0008] The expression cassette containing the lotus embryo-specific promoter pNnCYP80A, and the promoter pNnCYP80A is connected to the upstream of the target gene.

[0009] The recombinant expression vector containing the embryo-specific promoter pNnCYP80A, preferably, the plant expression vector pBI101 is used as the carrier to construct the recombinant expression vector.

[0010] The application of the lotus embryo-specific promoter pNnCYP80A or the expression cassette containing the promoter or the recombinant expression vector in driving the specific expression of the target gene in the plant embryo.

[0011] Compared with the prior art, the technical scheme of the present application has the beneficial effects that: the present application screens the gene NnCYP80A which is specifically highly expressed in lotus embryo by transcriptome sequencing technology, and clones the pNnCYP80A promoter of 1489bp upstream of the start codon ATG of the embryo-specific expression gene NnCYP80A. The promoter is constructed on the plant expression vector pBI101 with the GUS reporter gene by enzyme digestion and connection, and is transformed into Arabidopsis for promoter function verification. The pNnCYP80A promoter can specifically drive the high expression of the downstream GUS gene in embryo. Therefore, the expression of the target gene will not affect the vegetative growth and development of the plant. In the future, the tissue-specific expression characteristics can be used to directionally perform plant embryo genetic engineering modification, such as applied to plant embryo organ development research, embryo quality trait improvement, etc. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 The results of the analysis of the promoter pNnCYP80A regulating the expression of NnCYP80A gene in different tissues of lotus.

[0013] Figure 2The map of plant GUS gene expression vector pBI101.

[0014] Figure 3 The schematic diagram of restriction sites of plant GUS gene expression vector pBI101 fused with promoter pNnCYP80A.

[0015] Figure 4 The results of GUS staining and GUS gene fluorescence quantitative analysis of different parts of Arabidopsis. DETAILED DESCRIPTION

[0016] The present application will be described in detail below with reference to the accompanying drawings and embodiments, and the specific embodiments are only for illustrating the present application, but not limiting the protection scope of the present application. The experimental methods in the following examples are all conventional methods, unless otherwise specified.

[0017] Example 1

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

[0019] According to the transcriptome data analysis of each tissue of lotus, it was found that NnCYP80A gene was highly expressed in lotus heart tissue, as shown in Table 1. Figure 1 The upstream promoter sequence of NnCYP80A (Nn21373) was obtained according to the whole genome sequence of lotus, and the promoter was named as pNnCYP80A, and the nucleotide sequence was shown as SEQ ID NO. 1. The primer was designed to amplify the fragment, and an enzyme digestion site (shown by underlined) was added at the 5' end of each pair of primers for subsequent construction of plant expression vector. The forward primer was F1: 5'-ACGC GTCGA C CATAGAAGTTGTGCCAATGCAT-3' (SalI), and the reverse primer was R1: 5'-GC TCTAGA TAGAGTTTATAGAGAAGATGTAATACTAG-3' (XbaI).

[0020] The genomic DNA of lotus variety 'Qiuxing' was extracted according to the method of genomic DNA extraction kit (TIANGEN), and the DNA was used as a template to amplify the promoter pNnCYP80A sequence with F1 and R1. The reaction system was 10 μM F1 and R1 primers, 2.5 μl each, 2×primerSTAR (TAKARA) 100

[0021] μl, genomic DNA (100 ng / μl) 1 μl, and double-distilled water to 200 μl. The PCR reaction program was as follows: 98 °C pre-denaturation for 5 min; 98 °C denaturation for 15 s, 58 °C annealing for 15 s, 72 °C extension for 30 s, 35 cycles; and finally 72 °C extension for 5 min. 5 μl of the PCR product was subjected to 1% agarose gel electrophoresis, and a band of about 1500 bp in size was obtained by amplification. All the amplification systems were purified by a PCR product recovery kit (TIANGEN).

[0022] Example 2

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

[0024] The purified PCR product (pNnCYP80A sequence containing enzyme cutting sites at both ends) and the plant expression vector pBI101 were double-digested with SalI and XbaI, and the double-digestion system was as follows: 3 μg of the purified PCR product or the pBI101 vector, CutSmart Buffer 10 μl, 3 μl of each of endonucleases SalI and XbaI (20000 U / ml), and double-distilled water 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 the pNnCYP80A sequence and the plant expression vector pBI101 were purified by a product recovery kit after enzyme digestion, the T4 ligase was used to connect the pNnCYP80A target fragment to the vector pBI101. The ligation product was transformed into E. coli competent cells TOP10, and the single colonies obtained were subjected to PCR and sequencing verification. The PCR system was as follows: 10 μM F1 and R1 primers 1 μl each, 2 x Phanta Flash Master Mix (Vazyme) 10 μl, bacterial liquid 1 μl, and double-distilled water to 20 μl. The PCR reaction program was as follows: 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; and finally 72 °C extension for 5 min. The positive recombinant plasmid was named pBI101-pNnCYP80A-GUS, and the vector pBI101 map and promoter recombination site schematic diagram are shown in Figure 2 and Figure 3 .

[0025] Example 3

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

[0027] The recombinant plasmid pBI101-pNnCYP80A-GUS was introduced into Agrobacterium tumefaciens GV3101 (preserved in Wuhan Botanical Garden, Chinese Academy of Sciences) by electroporation. After incubation at 28°C for 3 h at 200 rpm, the bacteria were spread on solid LB medium containing 50 μg / L kanamycin and 50 μg / L rifampicin, and incubated in an inverted incubator at 28°C for 2 days. Single colonies were picked and subjected to colony PCR verification, using the same PCR verification method as described in Example 2.

[0028] The verified Agrobacterium GV3101 was used to transform Arabidopsis thaliana by floral dip method. The harvested T0 generation Arabidopsis seeds were germinated on 1 / 2MS seed screening medium containing 50 μg / L kanamycin. Normal growth T0 generation plants were selected and planted, and the T1 generation plants were harvested by the same method. After the plants grew up, the wild type and pBI101-pNnCYP80A-GUS Arabidopsis genomic DNA were extracted for PCR positive identification. The PCR system was as follows: 10 μM F1 and R1 primers, 1 μl each, 2x Phanta Flash Master Mix (Vazyme), 10 μl, genomic DNA, 100 ng, and double distilled water, to 20 μl. The PCR reaction program was as follows: 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; and finally 72°C extension for 5 min. The Arabidopsis plants with positive PCR identification were further screened by the same method to harvest T2 generation seeds. After the T2 generation plants were normally germinated in soil, phenotype observation and gene expression identification were performed.

[0029] Example 4

[0030] GUS identification of transgenic plants:

[0031] The T2 generation plants of pBI101-pNnCYP80A-GUS Arabidopsis were subjected to histochemical staining. The specific steps were as follows: part of the leaves, inflorescences, pods, and seeds of the T2 generation plants of pBI101-pNnCYP80A-GUS Arabidopsis were cut off and immersed in GUS staining reagent (Zhongke Ruite, product number: RTU4032). Vacuum treatment was performed for 10 min by a vacuum pump to allow the staining solution to fully penetrate the plant tissues. The reaction was performed at 37°C overnight. After staining, the tissues were decolorized in 75% ethanol for several times, and observed and photographed under a stereomicroscope. The results showed that no blue color was observed in all tissues of wild type Arabidopsis. No blue color was also observed in the roots, leaves, stems, and petals of pBI101-pNnCYP80A-GUS Arabidopsis transgenic lines. Figure 4 Since the expression site of pNnCYP80A-GUS was located in the embryo inside the seed, which was blocked by the seed coat and endosperm, the GUS staining and stereomicroscopic observation of the embryo GUS staining effect were not ideal. Therefore, qPCR was further used to quantitatively detect the GUS gene in each tissue of Arabidopsis.

[0032] Quantitative detection of GUS gene in T2 generation plants of pBI101-pNnCYP80A-GUS transgenic Arabidopsis thaliana, the quantitative primer is GUS-F: 5'-GCGGACTTACGTGGCAAAGGA-3', GUS-R: 5'-CGACAGCAGCAGTTTCATCAATCA-3'. The specific steps are as follows: about 100 mg of pBI101-pNnCYP80A-GUS transgenic Arabidopsis thaliana root, stem, leaf, inflorescence, seed tissue, according to the kit instructions to extract RNA (ZOMANBIO), reverse transcription of RNA (TransGen) to cDNA for fluorescence quantitative PCR analysis, the total volume of the reaction mixture is 15 μl, 2x TB Green Premix Ex Taq II 7.5 μl, ROX dye 0.3 μl, quantitative forward and reverse primers 0.5 μl (10 μM), cDNA 3 μl (50 ng). The amplification program is 95℃ pre-denaturation for 10 min; 95℃ denaturation for 30 s, 60℃ annealing and extension for 30 s, 40 cycles. Three groups of biological and technical repeats are set. The fluorescence quantitative results show that the GUS gene expression in pBI101-pNnCYP80A-GUS transgenic Arabidopsis thaliana seeds is significantly higher than that in root, stem, leaf and inflorescence tissue Figure 4 B).

[0033] In summary, the present application provides a lotus tissue-specific expression promoter pNnCYP80A, the nucleotide sequence of the promoter is shown as SEQ ID NO. 1, the promoter drives high expression of the target gene in lotus plumule. The promoter pNnCYP80A is linked to the target gene and introduced into Arabidopsis thaliana, and the high expression of the target gene in Arabidopsis thaliana seeds is driven by the promoter. The lotus plumule-specific promoter pNnCYP80A helps to more comprehensively understand the mechanism of plant embryonic development 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 plumule organ development research, specific improvement of plumule quality traits, etc.

[0034] It should be understood that the above specific implementation methods are only used to explain the basic principles of the present application, and do not limit the present application in any form. Changes and improvements made according to the present application still belong to the protection scope of the present application.

Claims

1. Use of the promoter pNnCYP80A for driving the specific expression of a gene of interest in the plant embryo, characterized in that, The sequence of the promoter pNnCYP80A is shown as SEQ ID NO.

1.

2. Use of a recombinant expression vector comprising the promoter pNnCYP80A for driving the specific expression of a gene of interest in the embryo of a plant, characterized in that, The sequence of the promoter pNnCYP80A is shown as SEQ ID NO.

1.

3. Use according to claim 1 or 2, characterized in that, The plants are lotus and Arabidopsis.

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