Eggplant cryptochrome gene SmCRY2 and application thereof

Abstract

The invention discloses an eggplant cryptochrome gene SmCRY2 and an application thereof. The cDNA sequence of the gene comprises: (a) a base sequence shown by the 1st-1944th sites of SEQ ID NO.1; or (b) a base sequence having at least 70% homology with the base sequence shown by the 1st-1944th sites of SEQ ID NO.1; or (c) a base sequence that can be hybridized with the base sequence shown by the 1st-1944th sites of SEQ ID NO.1. The amino acid sequence coded by the gene is shown by SEQ ID NO.2. The gene is expressed in the roots, stems, leaves, flowers, peel and pulp while the expression quantities in the roots and leaves are remarkably larger than those in other tissues. The invention provides a theoretical basis for improving the poor growth of a plant in dim light through the gene engineering technology in future and is of a great application value.

Description

technical field

[0001] The invention belongs to the field of biotechnology, and relates to a key enzyme in the eggplant light signal pathway and a coding gene thereof, in particular to an eggplant cryptochrome gene SmCRY2 and its application. Background technique

[0002] Blue light and near-ultraviolet light can cause plants to respond to blue light. The main function of cryptochrome in plants is to act as a receptor for blue light and near-ultraviolet light to regulate plant growth and development. There are three cryptochrome genes CRY1, CRY2 and CRY3 in Arabidopsis, among which CRY1 and CRY2 are mainly studied. CRY1 and CRY2 proteins were phosphorylated under blue light irradiation, and this phosphorylation was specifically dependent on blue light and increased with the intensity of blue light, while red light and far-red light did not have this effect. The phosphorylation of CRY1 and CRY2 proteins is also closely related to their function. Most of the screened cry1 and...

Examples

Embodiment 1

[0038] Embodiment 1, the cloning of eggplant SmCRY2 gene

[0039] 1. Acquisition of plant material

[0040] The plant material used in this experiment is the excellent eggplant germplasm resource A. spp. The experimental materials were cultivated in artificial plastic greenhouses in the Aerospace Breeding Base of Pujiang Town, Minhang District, Shanghai. Seedlings are raised, grown and fruited under natural conditions. The leaves of eggplant were collected for RNA extraction.

[0041] 2. Extraction of RNA

[0042]Total RNA was extracted by the TRIzol method (TRIzol was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.). The integrity of RNA was identified by formaldehyde denaturing gel electrophoresis, and then the purity and concentration of RNA were determined on a spectrophotometer (ThermoScientificNANODROP1000Spectrophotometer).

[0043] 3. Full-length cloning of genes

[0044] A pair of degenerate primers were designed based on the conserved sequence of CR...

Embodiment 2

[0048] Example 2. Sequence information and homology analysis of eggplant CRY2 gene

[0049] The full-length CDS open reading frame sequence of the new eggplant CRY2 gene of the present invention is 1944bp, and the detailed sequence is shown in SEQ ID NO.1. According to the CDS open reading frame sequence, the amino acid sequence of eggplant CRY2 is deduced, with a total of 647 amino acid residues, a molecular weight of 73208.5 Daltons, and a theoretical isoelectric point (pI) of 5.91. The detailed sequence is shown in SEQ ID NO.2.

[0050] The CDS open reading frame sequence of eggplant CRY2 and the amino acid sequence of its encoded protein were identified by BLAST program in the Non-redundantGenBank+EMBL+DDBJ+PDB and Non-redundantGenBankCDStranslations+PDB+SwissProt+Superdate+PIR databases. Source retrieval, it was found that it has 92.28% identity with the tomato CRY2 gene (NM_001247316.1) at the nucleotide level; at the amino acid level, the similarity between them is as...

Embodiment 3

[0051] Embodiment 3, expression of eggplant CRY2 gene in different tissues

[0052] 1. Acquisition of plant material

[0053] The eggplant roots, stems, leaves, flowers, pericarp, and pulp were collected at the ripening stage of the fruit, and the samples were wrapped in aluminum platinum paper and immediately put into liquid nitrogen, and then transferred to a -80°C ultra-low temperature refrigerator for storage until use.

[0054] 2. Extraction of RNA

[0055] Total RNA was extracted by the TRIzol method (TRIzol was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.). The integrity was detected by ordinary agarose gel electrophoresis (gel concentration 1.2%; 0.5×TBE electrophoresis buffer; 150v, 15min). The maximum rRNA brightness in the electrophoresis strip should be 1.5-2.0 times the brightness of the second rRNA, otherwise it indicates the degradation of the rRNA sample. RNA with good purity, A 260 / A 280 and A 260 / A 230 About 2.0 or so. The OD value wa...

technical field

[0001] The invention belongs to the field of biotechnology, and relates to a key enzyme in the eggplant light signal pathway and a coding gene thereof, in particular to an eggplant cryptochrome gene SmCRY2 and its application. Background technique

[0002] Blue light and near-ultraviolet light can cause plants to respond to blue light. The main function of cryptochrome in plants is to act as a receptor for blue light and near-ultraviolet light to regulate plant growth and development. There are three cryptochrome genes CRY1, CRY2 and CRY3 in Arabidopsis, among which CRY1 and CRY2 are mainly studied. CRY1 and CRY2 proteins were phosphorylated under blue light irradiation, and this phosphorylation was specifically dependent on blue light and increased with the intensity of blue light, while red light and far-red light did not have this effect. The phosphorylation of CRY1 and CRY2 proteins is also closely related to their function. Most of the screened cry1 and...

Examples

Embodiment 1

[0038] Embodiment 1, the cloning of eggplant SmCRY2 gene

[0039] 1. Acquisition of plant material

[0040] The plant material used in this experiment is the excellent eggplant germplasm resource A. spp. The experimental materials were cultivated in artificial plastic greenhouses in the Aerospace Breeding Base of Pujiang Town, Minhang District, Shanghai. Seedlings are raised, grown and fruited under natural conditions. The leaves of eggplant were collected for RNA extraction.

[0041] 2. Extraction of RNA

[0042]Total RNA was extracted by the TRIzol method (TRIzol was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.). The integrity of RNA was identified by formaldehyde denaturing gel electrophoresis, and then the purity and concentration of RNA were determined on a spectrophotometer (ThermoScientificNANODROP1000Spectrophotometer).

[0043] 3. Full-length cloning of genes

[0044] A pair of degenerate primers were designed based on the conserved sequence of CR...

Embodiment 2

[0048] Example 2. Sequence information and homology analysis of eggplant CRY2 gene

[0049] The full-length CDS open reading frame sequence of the new eggplant CRY2 gene of the present invention is 1944bp, and the detailed sequence is shown in SEQ ID NO.1. According to the CDS open reading frame sequence, the amino acid sequence of eggplant CRY2 is deduced, with a total of 647 amino acid residues, a molecular weight of 73208.5 Daltons, and a theoretical isoelectric point (pI) of 5.91. The detailed sequence is shown in SEQ ID NO.2.

[0050] The CDS open reading frame sequence of eggplant CRY2 and the amino acid sequence of its encoded protein were identified by BLAST program in the Non-redundantGenBank+EMBL+DDBJ+PDB and Non-redundantGenBankCDStranslations+PDB+SwissProt+Superdate+PIR databases. Source retrieval, it was found that it has 92.28% identity with the tomato CRY2 gene (NM_001247316.1) at the nucleotide level; at the amino acid level, the similarity between them is as...

Embodiment 3

[0051] Embodiment 3, expression of eggplant CRY2 gene in different tissues

[0052] 1. Acquisition of plant material

[0053] The eggplant roots, stems, leaves, flowers, pericarp, and pulp were collected at the ripening stage of the fruit, and the samples were wrapped in aluminum platinum paper and immediately put into liquid nitrogen, and then transferred to a -80°C ultra-low temperature refrigerator for storage until use.

[0054] 2. Extraction of RNA

[0055] Total RNA was extracted by the TRIzol method (TRIzol was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.). The integrity was detected by ordinary agarose gel electrophoresis (gel concentration 1.2%; 0.5×TBE electrophoresis buffer; 150v, 15min). The maximum rRNA brightness in the electrophoresis strip should be 1.5-2.0 times the brightness of the second rRNA, otherwise it indicates the degradation of the rRNA sample. RNA with good purity, A 260 / A 280 and A 260 / A 230 About 2.0 or so. The OD value wa...

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