Soybean flowering regulatory gene GmCIB6, encoding protein and application thereof

Abstract

The invention discloses a soybean flowering regulatory gene GmCIB6, an encoding protein and application thereof. The soybean flowering regulatory gene GmCIB6 has a nucleotide sequence shown by SEQ ID No.1 and an amino acid sequence shown by SEQ ID No.2. Over expression of the soybean flowering regulatory gene GmCIB6 can obviously promote a plant (such as arabidopsis thaliana) to flower and shorten the flowering time and a growth period. The soybean flowering regulatory gene GmCIB6 can be used for solving the problems of asynchronous flowering period in the process of cross breeding, growth period control of various crops, vegetables, fruits and flowers, the photoperiod sensitivity and the introduction.

Description

technical field

[0001] The invention relates to the field of genetic engineering, in particular to soybean flowering regulator gene GmCIB6, its encoded protein and its application in plant photoperiod and flowering time regulation. Background technique

[0002] Soybean is one of the important crops, and it is an important source of plant protein, edible oil, biodiesel, and secondary metabolites such as isoflavones and lecithin. Because soybean is a short-day plant, flowering is strictly controlled by photoperiod, so excellent varieties in different regions cannot be introduced to each other, and the growth period is also restricted by environmental photoperiod. If the sensitivity of soybean to photoperiod can be reduced, and the limitation of soybean flowering to photoperiod can be broken, the problem of soybean introduction can be solved, so as to realize the mutual exchange of high-quality varieties in various regions, enrich excellent germplasm resources in various region...

Examples

Embodiment 1

[0023] Example 1 Isolation and cloning of GmCIB6 gene

[0024] According to the CDS (code sequence) sequence of the Arabidopsis CIB1 gene, a homology comparison was performed on the http: / / www.phytozome.net website, and a batch of soybean homologous sequences were found, among which the CDS sequence of GmCIB6 was on the soybean genome The position is Glyma16g10620.1, PCR amplification primers were designed, forward primer F1: 5′-ATGGGAATTCAACTAACAGTTATG-3′ and reverse primer R1: 5′-TCAGAGCTCAACTTTCATCTGTG-3′. Using the total soybean cDNA as a template, PCR was performed to obtain the complete sequence of GmCIB6, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0025] The total PCR reaction system is 25 μL, including soybean cDNA (50ng) 1 μL; dNTP (2.5mM) 2.5 μL; primer F1 (10 μM) 1 μL; primer R1 (10 μM) 1 μL; LA Taq enzyme (5U / μL) 0.3 μL; Buffer 2.5 μL; ddH 2 O 16.7 μL, a total of 25 μL. The PCR reaction program was: 94°C pre-denaturation for 5 minutes, 30 cycles...

Embodiment 2

[0026] Example 2 Analysis and identification of GmCIB6 gene

[0027] The full-length CDS sequence of GmCIB6 is 1626bp, encoding a 541AA protein, which has 37.3% homology with Arabidopsis CIB1 protein. Protein structure analysis shows that its C-terminus contains a basic helix-loop-helix structure, which is a conserved domain of bHLH family transcriptional regulators, which regulates gene transcription by binding to the E-box in DNA; protein N The end also contains an NLS, which is associated with nuclear import.

[0028] According to the sequence query results on NCBI (www.ncbi.nlm.nih.gov), so far, there is no sequence information similar to CIB1 in soybean; and there are no published papers related to its function research. Therefore, it is considered that GmCIB6 is a new gene in soybean.

Embodiment 3

[0029] Example 3 The acquisition of transgenic Arabidopsis thaliana

[0030] According to the sequence information of GmCIB6, PCR amplification primers were designed at both ends of its CDS, forward primer F1: 5′-ATGGGAATTCAACTAACAGTTATG-3′ and reverse primer R1: 5′-TCAGAGCTCAACTTTCATCTGTG-3′. The complete sequence of GmCIB6 was obtained by PCR using the total soybean cDNA as a template.

[0031] The total PCR reaction system is 25 μL, including soybean cDNA (50ng) 1 μL; dNTP (2.5mM) 2.5 μL; primer F1 (10 μM) 1 μL; primer R1 (10 μM) 1 μL; LA Taq enzyme (5U / μL) 0.3 μL; Buffer 2.5 μL; ddH 2 O 16.7 μL, a total of 25 μL. The PCR reaction program was: pre-denaturation at 95°C for 5 minutes, 30 cycles at 94°C for 30s, 52.5°C for 30s, 72°C for 2 minutes, and finally 10 minutes at 72°C. The above PCR procedure was repeated three times, and the three PCR products were combined for agarose gel electrophoresis, and then the purified PCR product was recovered by cutting the gel.

[00...

technical field

[0001] The invention relates to the field of genetic engineering, in particular to soybean flowering regulator gene GmCIB6, its encoded protein and its application in plant photoperiod and flowering time regulation. Background technique

[0002] Soybean is one of the important crops, and it is an important source of plant protein, edible oil, biodiesel, and secondary metabolites such as isoflavones and lecithin. Because soybean is a short-day plant, flowering is strictly controlled by photoperiod, so excellent varieties in different regions cannot be introduced to each other, and the growth period is also restricted by environmental photoperiod. If the sensitivity of soybean to photoperiod can be reduced, and the limitation of soybean flowering to photoperiod can be broken, the problem of soybean introduction can be solved, so as to realize the mutual exchange of high-quality varieties in various regions, enrich excellent germplasm resources in various region...

Examples

Embodiment 1

[0023] Example 1 Isolation and cloning of GmCIB6 gene

[0024] According to the CDS (code sequence) sequence of the Arabidopsis CIB1 gene, a homology comparison was performed on the http: / / www.phytozome.net website, and a batch of soybean homologous sequences were found, among which the CDS sequence of GmCIB6 was on the soybean genome The position is Glyma16g10620.1, PCR amplification primers were designed, forward primer F1: 5′-ATGGGAATTCAACTAACAGTTATG-3′ and reverse primer R1: 5′-TCAGAGCTCAACTTTCATCTGTG-3′. Using the total soybean cDNA as a template, PCR was performed to obtain the complete sequence of GmCIB6, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0025] The total PCR reaction system is 25 μL, including soybean cDNA (50ng) 1 μL; dNTP (2.5mM) 2.5 μL; primer F1 (10 μM) 1 μL; primer R1 (10 μM) 1 μL; LA Taq enzyme (5U / μL) 0.3 μL; Buffer 2.5 μL; ddH 2 O 16.7 μL, a total of 25 μL. The PCR reaction program was: 94°C pre-denaturation for 5 minutes, 30 cycles...

Embodiment 2

[0026] Example 2 Analysis and identification of GmCIB6 gene

[0027] The full-length CDS sequence of GmCIB6 is 1626bp, encoding a 541AA protein, which has 37.3% homology with Arabidopsis CIB1 protein. Protein structure analysis shows that its C-terminus contains a basic helix-loop-helix structure, which is a conserved domain of bHLH family transcriptional regulators, which regulates gene transcription by binding to the E-box in DNA; protein N The end also contains an NLS, which is associated with nuclear import.

[0028] According to the sequence query results on NCBI (www.ncbi.nlm.nih.gov), so far, there is no sequence information similar to CIB1 in soybean; and there are no published papers related to its function research. Therefore, it is considered that GmCIB6 is a new gene in soybean.

Embodiment 3

[0029] Example 3 The acquisition of transgenic Arabidopsis thaliana

[0030] According to the sequence information of GmCIB6, PCR amplification primers were designed at both ends of its CDS, forward primer F1: 5′-ATGGGAATTCAACTAACAGTTATG-3′ and reverse primer R1: 5′-TCAGAGCTCAACTTTCATCTGTG-3′. The complete sequence of GmCIB6 was obtained by PCR using the total soybean cDNA as a template.

[0031] The total PCR reaction system is 25 μL, including soybean cDNA (50ng) 1 μL; dNTP (2.5mM) 2.5 μL; primer F1 (10 μM) 1 μL; primer R1 (10 μM) 1 μL; LA Taq enzyme (5U / μL) 0.3 μL; Buffer 2.5 μL; ddH 2 O 16.7 μL, a total of 25 μL. The PCR reaction program was: pre-denaturation at 95°C for 5 minutes, 30 cycles at 94°C for 30s, 52.5°C for 30s, 72°C for 2 minutes, and finally 10 minutes at 72°C. The above PCR procedure was repeated three times, and the three PCR products were combined for agarose gel electrophoresis, and then the purified PCR product was recovered by cutting the gel.

[00...

Images