阅读说明：本技术 克里曼丁花特异性表达启动子CcPIpro及其应用 (Crimania speciosa specific expression promoter CcPipro and application thereof ) 是由 张金智 胡春根 侯小进 于 2019-11-25 设计创作，主要内容包括：本发明公开了克里曼丁花特异性表达启动子CcPIpro及其应用。本发明从克里曼丁基因组中分离出CcPI基因的启动子CcPIpro,其核苷酸序列如SEQ ID NO.1所示,长度为2242bp,能在花中特异性表达,可将该启动子应用于构建花特异表达的转基因植物,为柑橘的遗传改良提供新的基因资源。(The invention discloses a Crimemann flowering plant specific expression promoter CcPipro and application thereof. The promoter CcPipro of the CcPI gene is separated from the Criman butyl gene group, the nucleotide sequence of the promoter CcPI gene is shown as SEQ ID NO.1, the length of the promoter CcPI gene is 2242bp, the promoter can be specifically expressed in flowers, the promoter can be applied to the construction of transgenic plants specifically expressed in the flowers, and new gene resources are provided for the genetic improvement of citrus.)

1. The Crimania speciosa specific expression promoter CcPipro is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.

2. Use of the crilmann flower-specific expression promoter CcPIpro according to claim 1 for genetic improvement in plants.

3. Use according to claim 2, in the genetic modification of citrus.

4. An expression vector comprising the crimann-bud specific expression promoter CcPIpro according to claim 1.

5. The expression vector of claim 4, wherein the expression vector is a recombinant expression vector pBI101+ CcPipro.

6. Use of the expression vector of claim 4 for genetic improvement in plants.

7. The use according to claim 6, in the genetic modification of citrus.

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a Crimemann flowering plant specific expression promoter CcPipro and application thereof.

Background

The plant can regulate the expression of specific gene according to the change of external environment so as to adapt to complicated and changeable growing environment. The TATA and CAAT boxes in eukaryotes, which determine the start site and efficiency of transcription, are responsible for binding to RNA polymerase and initiate the gene transcription process. Among them, the transcriptional level is coordinated by specific cis-acting elements and transcription factors, and is a key link of the expression level. Therefore, the research on the plant specific induced promoter is helpful for understanding the gene transcription regulation expression mode and regulation mechanism, and is applied to the gene engineering to improve or improve the expression of the exogenous target gene. Many promoters specifically expressed and induced in tissues have been verified by means of molecular biology at present, such as MFT in the FT/TF1 gene family, to regulate germination of arabidopsis seeds by forming a negative feedback loop with ABI3 and ABI5 proteins in the ABA signaling pathway (Xi et al 2010). ABA enhances resistance to stress in plants under stress conditions such as drought, low temperature, high salinity conditions by accompanying a large number of physical changes and developmental alterations (Mansfield et al 1987).

Flowering is a highly complex process of physiology, biochemistry and morphogenesis under the control of a certain genetic background. After plants are induced by environmental factors (such as photoperiod and temperature), a series of signal transduction processes (Ca-CaM system) are carried out to start control genes in the flowering determining process, and under the mutual interaction of a plurality of key genes and the mutual restriction of a plurality of metabolic pathways, flower bud differentiation is caused, so that flower organs are generated. These processes are controlled by the start and stop of expression of many specific genes according to a certain time sequence and space specificity program, and each gene has independent function and influences and restricts each other.

Flowers are the most important organs in seed plants, are the most abundant in the evolution process, and the development of flowers is always the key point of research in developmental biology. The classical ABC model reveals the molecular mechanism of floral organ development (Coen and Meyerowitz 1991). Two MADS-box containing class B genes AP3 and PI specifically regulate petal and stamen development (Schwarz-Sommer et al 1992, Yao et al 2008), the expression of which is activated by upstream flower meristem specific genes LFY and AP 1. AP3 and PI exist mainly as heterodimers and play important roles in target gene binding, nuclear localization, and regulation of transcription of downstream genes (McGonigle et al 1996, Yang et al 2003). With the progress of research, the AP3/PI dimer forms a higher protein complex with other MADS-box genes, regulates the specific differentiation of organs, forms a tetramer complex with AP1 and SEP to regulate the formation of petals, and regulates the development of stamens together with AG and SEP proteins. Therefore, the development of a new flower-specific expression promoter will provide new gene resources for the creation of flower-specific expressing plants and genetic improvement of plants.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, a promoter which is specifically expressed in flowers is separated from a Klementine genome, and a transgenic line can be obtained by transforming arabidopsis thaliana by utilizing the specificity of the promoter.

The invention utilizes plant gene cloning technology to clone a promoter specifically expressed in flowers from a genome of Krementine (Citrus clementina), the nucleotide sequence of the promoter is shown as SEQ ID NO.1, and the length of the sequence is 2242 bp. The applicants have named this promoter as CcPipro. Firstly, the specificity of CcPI gene expression is verified by utilizing real-time fluorescent quantitative PCR, meanwhile, a promoter CcPipro is fused with a marker gene GUS, and the fused gene is transferred into an arabidopsis plant by an inflorescence dip-dyeing method, so that the specificity of the promoter CcPipro in flower expression is verified. The invention obtains the recombinant vector pBI101+ CcPipro and the transgenic plant carrying the vector, and provides new gene resources for creating a plant with flower specific expression by using the promoter.

Therefore, the first object of the present invention is to provide the Critudina efflorens specific expression promoter CcPipro, the nucleotide sequence of which is shown in SEQ ID NO. 1.

The invention also provides application of the Crimania speciosa specific expression promoter CcPipro in plant genetic improvement.

Preferably, the application is the application in citrus genetic improvement.

The invention also provides an expression vector containing the Crimanghua specific expression promoter CcPipro.

Preferably, the expression vector is a recombinant expression vector pBI101+ CcPipro, the vector pBI101 is cut by endonuclease Hind III and BamH I, the upstream and downstream Crimum flower specific expression promoter CcPipro containing endonuclease Hind III and BamH I sites is cut by endonuclease Hind III and BamH I, and then a connection reaction is carried out, so that the Crimum flower specific expression promoter CcPipro is inserted between the endonuclease Hind III and BamH I, and the recombinant expression vector pBI101+ CcPipro is obtained.

The invention also provides application of the expression vector in plant genetic improvement.

Preferably, the application is the application in citrus genetic improvement.

The expression mode of PI (CcPI) genes in Citrus varieties of Kriman butyl (Citrus clementina) is verified by separating the PI genes, the upstream promoter CcPIPro sequence is separated, the promoter is fused with GUS genes and then is transferred into arabidopsis thaliana by an inflorescence dip-dyeing method, and the specificity of the promoter expression is verified. The specificity of the gene promoter can be used for constructing transgenic plants with flower specificity expression, and new gene resources are provided for the genetic improvement of citrus.

Drawings

FIG. 1 is a technical scheme of the present invention.

FIG. 2 is the expression pattern of the CcPI gene in different tissues and different parts of the flower; the upper panel shows the results of the quantitative assay, and the lower panel shows the results of the semi-quantitative assay. Description of reference numerals: the expression of the CcPI gene in Crimangding root (R), stem (S), leaf (L), flower (Fl), fruit (Fr), sepal (se), petal (pe), stamen (st), pistil (car) is shown.

FIG. 3 is a schematic representation of the predicted cis-acting elements on the CcPipro promoter.

FIG. 4 is a physical map of the original vector pBI101 and the constructed recombinant vector pBI101+ CcPipro of the present invention; panel A is a physical map of the original vector pBI 101; panel B is a physical map of the constructed recombinant vector pBI101+ CcPipro.

FIG. 5 shows the CcPipro promoter driving the staining of the GUS gene in Arabidopsis; panel A shows the result of transfer of CcPipro, where the CcPipro promoter is specifically expressed in petals (pe) and stamen (st) and no GUS protein is detected in sepals (se) and pistils (car); panel B shows CcPipro that staining of the buds of GUS plants was observed only in the buds (fb) and expression of GUS protein was not detected in the leaves (L).

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.