Tissue-specific promoter JcTM6 gene promoter expressed in plant flowers and application thereof
阅读说明：本技术 一种在植物花中表达的组织特异性启动子JcTM6基因启动子及其应用 (Tissue-specific promoter JcTM6 gene promoter expressed in plant flowers and application thereof ) 是由 陶彦彬 徐增富 王静娴 明新 于 2019-10-16 设计创作，主要内容包括：本发明提供了一种在植物花中驱使基因表达的组织特异性启动子JcTM6基因启动子及其应用,属于转基因植物和植物基因编辑技术领域。在植物花中表达的组织特异性启动子JcTM6基因启动子的核苷酸序列如SEQ ID No.1所示。本发明还提供了一种含所述JcTM6基因启动子的重组载体。基于JcTM6基因启动子的活性强且具有在花中特异性表达的特点,本发明提供了组织特异性启动子JcTM6基因启动子、重组载体或所述引物在构建转基因植物或植物花、果实和种子性状改良中的应用。同时JcTM6基因启动子可作为花特异性启动子驱使功能基因表达,用于改良小桐子繁殖器官性状,实现种子产量的提高。(The invention provides a tissue-specific promoter JcTM6 gene promoter for driving gene expression in plant flowers and application thereof, belonging to the technical field of transgenic plants and plant gene editing. The nucleotide sequence of the tissue-specific promoter JcTM6 gene promoter expressed in the plant flower is shown in SEQ ID No. 1. The invention also provides a recombinant vector containing the JcTM6 gene promoter. Based on the characteristics of strong activity of a JcTM6 gene promoter and specific expression in flowers, the invention provides an application of a tissue-specific promoter JcTM6 gene promoter, a recombinant vector or a primer in constructing transgenic plants or improving the traits of flowers, fruits and seeds of the plants. Meanwhile, the JcTM6 gene promoter can be used as a flower-specific promoter to drive functional gene expression, is used for improving the characteristics of the propagation organs of jatropha curcas and realizes the improvement of seed yield.)
1. A tissue-specific promoter JcTM6 gene promoter expressed in plant flowers is disclosed, wherein the nucleotide sequence of the JcTM6 gene promoter is shown as SEQ ID No. 1.
2. A recombinant vector comprising the tissue-specific promoter JcTM6 gene promoter of claim 1.
3. The recombinant vector according to claim 2, wherein the tissue specific promoter JcTM6 gene promoter is inserted at the XbaI/BamHI multiple cloning site of the basic vector.
4. The recombinant vector according to claim 2, wherein the recombinant vector further comprises a functional gene of interest; the target functional gene is positioned at the 3' end of the tissue-specific promoter JcTM6 gene promoter.
5. A group of primers for amplifying the tissue-specific promoter JcTM6 gene promoter of claim 1, comprising a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID No. 12; the nucleotide sequence of the reverse primer is shown as SEQ ID No. 13.
6. Use of the tissue specific promoter JcTM6 gene promoter of claim 1, the recombinant vector of any one of claims 2 to 4 or the primer of claim 5 for constructing transgenic plants and/or plant gene editing.
7. The use of claim 6, wherein the transgenic plant comprises transgenic jatropha curcas and transgenic arabidopsis thaliana.
8. Use of the tissue specific promoter JcTM6 gene promoter of claim 1, the recombinant vector of any one of claims 2 to 4 or the primer of claim 5 for improving the traits of flowers, fruits and/or seeds of plants.
9. Use of the tissue specific promoter JcTM6 gene promoter of claim 1, the recombinant vector of any one of claims 2 to 4, or the primer of claim 5 for increasing seed yield of jatropha curcas.
Technical Field
The invention belongs to the technical field of transgenic plants and plant gene editing, and particularly relates to a tissue-specific promoter JcTM6 gene promoter expressed in plant flowers and application thereof.
Background
Jatropha curcas L is a perennial woody plant of Jatropha of Euphorbiaceae, has strong drought resistance, can grow in a barren wasteland, and can be propagated by direct seeding or branch cutting. As oil plants, the oil content of the jatropha curcas seeds reaches about 30 to 40 percent. Jatropha curcas oil has been used for a long time in the past as a lamp oil for lighting needs; after improvement and processing, the jatropha curcas is prepared into biodiesel and aviation fuel for application, so that the jatropha curcas is used as an energy plant for large-scale planting in a plurality of countries. Although the jatropha curcas has various excellent characteristics, the jatropha curcas is vigorous in vegetative growth and few in female flowers, so that the seed yield is low, and the development prospect of the jatropha curcas as an energy plant is limited, so that the flower characters of the jatropha curcas need to be optimized and improved through various ways, and the yield of the jatropha curcas is improved to meet the production requirement.
The genetic diversity of the jatropha curcas is low, and the yield is difficult to be greatly improved by the traditional breeding approach. At present, the plant transgenic technology is widely applied to the improvement of agronomic traits of crops. The prerequisite for this technology to be used is the establishment of a Stable plant genetic transformation system, whereas the genetic transformation system of Jatropha curcas has become Stable (Joshi M, et al, effective genetic transformation of Jatropha current L.by microbial transformation of embedded plants, induced crack Prod,2011,33(1) 67-77; Kumar N, et al 2015, Stable genetic transformation of Jatropha current viral expression-induced genetic transformation, induced crack Prod,2010,32(1) 41-47; Pan J, et al, agricultural transformation-genetic transformation of Jatropha current microbial transformation of Jatropha current expression, 11-47; Jan J, 405, ecological transformation of Jatropha current genetic transformation of Jatropha current, biological transformation of Jatropha current, 7. biological transformation of Jatropha J, 405. biological transformation of Jatropha current, 7. biological transformation of Jatropha current, 2. 7. biological transformation of Jatropha current, biological transformation of Biovector of (647. transformation of biological transformation of Biovector of 3. transformation of Biovector of transformation of Biovector of 3. transformation of Biovector of (7. transformation of Biovector of Biotransformation of Biovector of Biotransformation of Biovector of, therefore, the method is an effective way for improving the characteristics of jatropha curcas through transgenic breeding. Besides a stable genetic transformation system, whether a functional gene can be accurately expressed in the jatropha curcas body is also important for success or failure of breeding.
The promoter is a DNA sequence which is located at the upstream of a gene and directly regulates the expression of a functional gene, and comprises a constitutive promoter, a tissue-specific promoter and an inducible promoter. The tissue-specific promoter can make the functional gene be expressed in specific tissue position or development stage, and can make directional improvement on the plant property, so that the research on tissue-specific promoter is more and more, and the practical requirements of plant gene engineering can be met. At present, only the 7S promoter and JcSDP1 promoter (Qu J et al, Development of marker-free transgenic Jatropha plants with the innovative levels of growth and tolerance of the oleic acid Biotechnol Biofuels 2012,5: 10; Kim MJ et al, Gene synergy of sub-dependent 1(JcSDP1), encoding a sheet-domain triacylglycerol Lipase, enhanced oil access in Jatropha cure. technol Biofuels 2014,7:36) have been reported for the genetic transformation of Jatropha curcas, which is far from sufficient for transgenic breeding of Jatropha curcas.
Disclosure of Invention
In view of the above, the present invention aims to provide a novel tissue-specific promoter from jatropha curcas, i.e., jc (tm) 6 gene promoter, which has high expression activity in flowers, is used to drive functional genes to improve jatropha curcas flower traits, and is used in transgenic plants or to increase the yield of jatropha curcas seeds.
The invention provides a tissue-specific promoter JcTM6 gene promoter expressed in plant flowers, wherein the nucleotide sequence of the JcTM6 gene promoter is shown as SEQ ID No. 1.
The invention provides a recombinant vector containing the tissue-specific promoter JcTM6 gene promoter.
Preferably, the tissue-specific promoter JcTM6 gene promoter is inserted at the XbaI/BamHI multiple cloning site of the basic vector.
Preferably, the recombinant vector further comprises a target functional gene; the target functional gene is positioned at the 3' end of the tissue-specific promoter JcTM6 gene promoter.
The invention provides a group of primers for amplifying a tissue-specific promoter JcTM6 gene promoter, which comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID No. 12; the nucleotide sequence of the reverse primer is shown as SEQ ID No. 13.
The invention provides application of the tissue-specific promoter JcTM6 gene promoter, the recombinant vector or the primer in constructing transgenic plants and/or plant gene editing.
Preferably, the transgenic plant comprises transgenic jatropha curcas and transgenic arabidopsis thaliana.
The invention provides application of the tissue-specific promoter JcTM6 gene promoter, the recombinant vector or the primer in improvement of plant flower, fruit and seed traits.
The invention provides an application of the tissue-specific promoter JcTM6 gene promoter, the recombinant vector or the primer in improving the yield of jatropha curcas seeds.
The invention provides a tissue-specific promoter JcTM6 gene promoter expressed in plant flowers, the nucleotide sequence of the JcTM6 gene promoter is shown as SEQ ID No.1, the tissue-specific promoter JcTM6 gene promoter is derived from plant jatropha curcas, the expression condition of JcTM6 gene in jatropha curcas is measured by real-time fluorescence quantitative PCR, the result shows that JcTM6 is mainly expressed in female flowers and male flowers of jatropha curcas, the expression quantity in the male flowers is 3 times of that in the female flowers, other tissue parts are hardly expressed except for flower buds and fruit peels (42), thus the JcTM6 gene is obtained as a gene with flower-specific expression, in order to verify the activity of JcTM 7, roots, stems, young leaves, mature leaves, stem tips, flower buds, female flowers, male flowers after pollination, fruits after 12 days, fruits after 25 days and seeds after pollination are fully pollinated, the expression of the male flowers in 5 transgenic jatropha period shows that the root, stems, DAP leaves, mature leaves, stem tips, buds, flowers, female flowers, fruits, male flowers, fruits, flowers, fruits, flowers, plants, and fruits, the like, the fruits, the later-3- β and.
The invention provides application of the tissue-specific promoter JcTM6 gene promoter, the recombinant vector or the primer in constructing transgenic plants or improving plant flower traits. The JcTM6 gene promoter is used as a flower-specific promoter to drive a functional gene to improve the flower character of jatropha curcas so as to improve the seed yield. The isolated cloning of the promoter lays a certain foundation for the genetic engineering breeding of the jatropha curcas.
Drawings
FIG. 1 is the result of expression profile analysis of the JcTM6 gene promoter in Jatropha curcas, wherein the root (R), stem (S), Young Leaf (YL), Mature Leaf (ML), inflorescence bud (If), Female Flower (FF), Male Flower (MF), pericarp (
FIG. 2 shows the JcTM6 promoter sequence and its expression vector construction, in which FIG. 2-A shows the nucleotide sequence of JcTM6 promoter and the position of cis-regulatory element, and A of initiation codon ATG (bold frame) is + 1; cis-regulatory elements on the sequence are identified in bold and underlined; FIG. 2-B is a T-DNA schematic of JcTM6 GUS expression vector;
FIG. 3 is a JcTM6 showing GUS staining results of GUS transgenic Arabidopsis, FIG. 3-A showing GUS staining results of inflorescence buds, and FIG. 3-B showing GUS staining results of flowers whose organs include sepals (Se), petals (Pe) and stamens (St);
FIG. 4 is JcTM6 GUS staining of GUS transgenic jatropha curcas, organs including root (R), stem (St), Young Leaf (YL), Mature Leaf (ML), stem tip (SA), inflorescence bud (If), Female Flower (FF), Male Flower (MF), fruit (Ft) 12 days after pollination and seed (Sd) 25 days after pollination;
FIG. 5 shows JcTM6 quantitative determination results of GUS fluorescence activity of GUS transgenic jatropha curcas tissue parts; roots (R), stems (St), Young Leaves (YL), Mature Leaves (ML), stem tips (SA), inflorescence buds (IF), Female Flowers (FF), Male Flowers (MF), fruits (Ft) 12 days after pollination, fruit peels (Pp) and seeds (Sd) 25 days after pollination;
FIG. 6 is a representation of JcTM6 GUS staining of female GUS transgenic jatropha curcas, left: female flowers in early development, middle: mid-developmental female flowers, right: mature female flowers, the floral organs including sepals (Se), petals (Pe), stigma (Sa), ovaries (Oy), ovules (Oe) and honeyglands (Ne);
FIG. 7 is a phenotypic drawing of driving expression of cytokinin synthase gene Arabidopsis ATP/ADPisopentenyltransferase 4(AtIPT4) gene in Arabidopsis thaliana using the JcTM6 gene promoter, FIG. 7-A is a wild-type (WT) and JcTM6pro: AtIPT4 transgenic plant, and FIG. 7-B is a flower of wild-type (WT) and JcTM6pro: AtIPT4 transgenic plant.
Detailed Description
The invention provides a tissue-specific promoter JcTM6 gene promoter expressed in plant flowers, wherein the nucleotide sequence of the JcTM6 gene promoter is shown as SEQ ID No. 1. The JcTM6 gene promoter is 1820bp in length. The invention clones a tissue-specific promoter TOMATO MADS BOX GENE6(TM6) GENE promoter from Jatropha curcas. The TM6 gene belongs to MADS box family, is B-type gene in the ABC model of floral organ development, and is mainly involved in regulating the development of the second round and the third round of floral organs. The TM6 gene evolved from the paleoAP3 gene together with the euAP3 gene, but unlike euAP3 gene, which is expressed only in petals and stamens, the TM6 gene is also expressed in pistils. The tissue specific promoter JcTM6 gene promoter is mainly expressed in flowers.
Cis-element analysis was performed on the JcTM6 gene promoter. The analysis result shows that the promoter sequence contains two DNA binding sites CArGbox of MADS-box protein, which is an important regulatory element of MADS-box gene. In addition, the promoter also contains a plurality of POLLEN-specific expression elements, including 8 POLLEN1 LEALAT 52 motif (AGAAA), 5 GTGANTG10motif (GTGA), and 1Q-element (see FIG. 2-A). The POLLEN1LELAT52 motif and GTGANTG10motif are important for the specific expression of tomato LAT52 gene and tobacco g10 gene in POLLEN respectively. The Q-element can enhance the specific expression of the ZM13 gene in corn pollen.
The JcTM6 gene promoter has high promoter activity, and GUS gene fluorescence activity quantitative detection results show that the GUS activity in female flowers is very high, and the JcTM6 gene promoter has a small amount of expression in inflorescence buds, male flowers, fruits 12 days after pollination and seeds 25 days after pollination in other tissue parts. Meanwhile, the results of detecting the GUS activity in female flowers at different development stages show that GUS staining is very obvious in the development process of the female flowers, and the function of promoting the functional gene expression of the JcTM6 gene promoter is not limited by the development stage of the female flowers, so that the JcTM6 gene promoter is an ideal tissue-specific promoter in the transgenic field. The source of the JcTM6 gene promoter is not particularly limited in the present invention, and it can be obtained by a common method well known in the art, for example, artificially synthesized based on the nucleotide sequence of the JcTM6 gene promoter or by a conventional PCR amplification method.
The invention provides a group of primers for amplifying a tissue-specific promoter JcTM6 gene promoter, which comprises a forward primer and a reverse primer, wherein the nucleotide sequence of the forward primer is shown as SEQ ID No.12 (5'-tgctctagaaatagctataaaatcaatt-3'); the nucleotide sequence of the reverse primer is shown as SEQ ID No.13 (5'-cgcggatccttttcctttcttcttgata-3'). The primer can specifically amplify the JcTM6 gene promoter. The PCR amplification method comprises the following steps: mu.l 10 XHiFi Taq enzyme buffer, 1. mu.l 10mM dNTP, 10mM upstream and downstream guides0.5. mu.l each of 10ng of genomic DNA, 1U of HiFi Taq enzyme, ddH2Supplementing O to 20 μ l; the amplification procedure is preferably as follows: pre-denaturation at 94 ℃ for 3min, 30 cycles (denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 90s), and extension at 72 ℃ for 8 min.
The invention provides a recombinant vector containing the tissue-specific promoter JcTM6 gene promoter. The type of the basic vector used in the recombinant vector is not particularly limited in the present invention, and plant expression vectors well known in the art may be used. The tissue-specific promoter JcTM6 gene promoter is preferably inserted at the XbaI/BamHI multiple cloning site of the base vector. The base vector preferably comprises the pBI101 vector. The recombinant vector preferably further comprises a functional gene of interest; the target functional gene is positioned at the 3' end of the tissue-specific promoter JcTM6 gene promoter. In the present invention, the construction method of the recombinant vector preferably comprises digesting the vector containing the JcTM6 gene promoter and the pBI101 vector with endonucleases XbaI and BamHI, linking the excised JcTM6 gene promoter to the digested pBI101 vector with T4 DNA ligase (Promega), and inserting the JcTM6 gene promoter in front of the GUS gene on the pBI101 vector to construct the JcTM6pro GUS plant expression vector.
Based on that in transgenic jatropha curcas and arabidopsis thaliana, the expression activity of the JcTM6 gene promoter in flowers is very high, the invention provides the tissue-specific promoter JcTM6 gene promoter, the recombinant vector or the application of the primer in constructing transgenic plants and/or plant gene editing. In the present invention, the method for constructing a transgenic plant is preferably to transform a plant with agrobacterium-mediated transformation to construct a recombinant vector, thereby obtaining a transgenic plant containing a JcTM6 gene promoter. The method of transformation is not particularly limited in the present invention, and any transformation method known in the art may be used, for example, as described in the prior art (Clough and Bent (1998), Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana, Plant Journal16: 735-743). The type of the transgenic plant is not particularly limited, and plants well known in the art can be used as transformation targets, and in order to illustrate the biological characteristics of the JcTM6 gene promoter, the invention is illustrated by constructing transgenic jatropha curcas and transgenic Arabidopsis thaliana, but the invention is not to be construed as limiting the scope of the invention.
Based on the fact that in transgenic jatropha curcas and arabidopsis thaliana, the expression activity of the JcTM6 gene promoter in flowers is very high. The invention provides application of the tissue-specific promoter JcTM6 gene promoter, the recombinant vector or the primer in improvement of plant flower, fruit and/or seed traits. In the application, the JcTM6 gene promoter is transferred to the 5' end of the functional gene for improving the plant flower character, so as to promote the efficient transcription of the functional gene. The present invention is not particularly limited in kind of the functional gene for improving the floral trait of the plant, and may be any kind of functional gene for improving the floral trait of the plant, which is well known in the art, and examples thereof include a cytokinin synthase gene isopentenyltransferase (ipt) gene and a loneyguy (log) gene. The specific method for improving the flower character of the plant is not particularly limited, and the operation scheme well known in the field can be adopted.
Based on the fact that in the transgenic jatropha curcas, the expression activity of the JcTM6 gene promoter is very high in female flowers, and only a small amount of expression is generated in inflorescence buds, male flowers, fruits and seeds. Therefore, the JcTM6 gene promoter can be used as a female flower specific promoter to drive a functional gene to improve the jatropha curcas flower character so as to improve the seed yield. Therefore, the invention provides the application of the tissue-specific promoter JcTM6 gene promoter, the recombinant vector or the primer in improving the yield of the jatropha curcas seeds. In the application, the JcTM6 gene promoter is transferred to the 5' end of the functional gene for improving the seed yield, so as to promote the efficient transcription of the functional gene. The present invention is not particularly limited in the kind of the functional gene for increasing seed yield, and the kind of the functional gene for increasing seed yield known in the art may be used, for example, including cytokinin synthase gene IPT gene and LOG gene. The specific method for improving the flower character of the plant is not particularly limited, and the operation scheme well known in the field can be adopted.
The tissue-specific promoter JcTM6 gene promoter expressed in plant flowers and its application provided by the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.
The drugs and reagents used in the examples of the present invention are commercially available.
The plant materials used in the examples of the present invention, Arabidopsis thaliana (Col-0 ecotype) and Jatropha curcas, were all derived from the West double Banna tropical plantations, academy of Chinese sciences.