CN101921752B - Soybean flowering regulatory gene GmCIB6, encoding protein and application thereof - Google Patents

Abstract

The invention discloses a soybean flowering regulating gene GmCIB6 and its coded protein, which has the nucleotide sequence shown in SEQ ID No.1 and the amino acid sequence shown in SEQ ID No.2. Overexpression of the soybean flowering regulation gene GmCIB6 can obviously promote the flowering of the plant (Arabidopsis thaliana), shorten the flowering time, and shorten the growth period. It can be used to solve the problem of flowering failure in hybrid breeding, the growth period control problem of various crops, vegetables, fruits and flowers, the photoperiod sensitivity problem and the introduction problem.

Description

Soybean Flowering Regulatory Gene GmCIB6, Its Encoded Protein and Its Application

technical field

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

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 regions, and regulate the growth period of soybean , improve soybean yield and quality. Although the growth habit of soybean has been changed to a certain extent through traditional cultivation and genetic breeding methods, and some varieties with wider photoperiod adaptability have been obtained, they have not fundamentally changed the flowering habit of soybean (Zhao Cun et al., using photoperiod Screening of light-insensitive soybean varieties (lines) by induction method. Soybean Science. 1996, 15(1): 42-47; ): 225-230; Yang Zhipan and Zhou Xin'an, Research Progress in Soybean Photoperiod Genetic Breeding, Chinese Journal of Oil Crops, 1999, 21(1): 61-73; Luan Xiaoyan, Man Weiqun, Du Weiguang, Chen Yi, Liu Xinlei, Dou Guang Research on insensitive germplasm innovation and photoperiod breeding pathway, Soybean Science, 2004, 23(3): 196-199). The main reason is that there is little understanding of the molecular mechanism of soybean flowering, which makes it difficult to fundamentally solve the problem of soybean flowering habits.

Studies on the molecular mechanism of Arabidopsis flowering have shown that plant flowering is controlled by four pathways, namely the photoperiodic pathway, the autonomous pathway, the vernalization pathway, and the gibberellin pathway (Mouradov et al., 2002), and the signals of these pathways are finally summarized To the two main integrons FT and SOC1, thereby promoting flowering (Suarez-Lopez P, Wheatley K, Robson F, Onouchi H, Valverde F, Coupland G, CONSTANS mediates between the circadian clock and the control of flowering in Arabidopsis.Nature, 2001 , 410: 1116-20.; Hepworth SR, Valverde F, Ravenscroft D, Mouradov A, Coupland G. Antagonistic regulation of flowering-time gene SOC1 by CONSTANS and FLC via separate promoter motifs. EMBO J.2002.21(16): 4327-37 ). Cryptochrome is a blue light receptor that exists in three species from bacteria to plants and animals, and regulates the growth and development of plants and the biological clock of animals and plants (Cashmore AR., Cell 114:537-543(2003). Lin C., Shalitin D., Annu Rev Plant Biol 54:469-496 (2003)). Plants contain at least two cryptochromes: CRY1 and CRY2 (Guo H, Yang H, Mockler TC, Lin C., Science 279: 1360-1363 (1998)). In Arabidopsis CRY1 primarily regulates blue light-controlled deetiolation, whereas CRY2 primarily regulates flowering photoperiod activity (Koornneef M, Heynh., Z Pflanzenphysiol Bd 100:147-160 (1980)). In addition to Arabidopsis, cryptochromes have also been studied in algae, mosses, ferns, tomato, rapeseed, pea, and rice. These studies suggest that in angiosperms, cryptochromes regulate growth and development in the same way as in Arabidopsis (Immeln D, Schlesinger R, J Biol Chem 282:21720-21728. (2007); Imaizumi T, Kanegae T, Wada M. , Plant Cell 12:81-96.(2000).; Imaizumi T, Kadota A, Plant Cell 14:373-386.(2002); Ninu L, et al., Plant J 18:551-556.(1999); Giliberto L , et al., Plant Physiol 137: 199-208. (2005).; Chatterjee M, Sharma P, Plant Physiol 141: 61-74. (2006).; Platten JD, et al., Plant Physiol 139: 1472-1482. (2005); Matsumoto N, Hirano T, Iwasaki T, Plant Physiol 133:1494-1503. (2003).; Zhang YC, et al., Plant J 46:971-983. (2006)). Similar to other photoreceptors, cryptochromes interact with their target proteins to regulate gene expression and physiological responses (H.-Q.Yang et al., Cell 103, 815 (2000); X.Yu et al., Proc Natl Acad Sci US A 104, 7289 (2007); M.F. Ceriani et al., Science 285, 553 (1999); M. Ni, J.M. Tepperman, P.H. Quail, Cell 95, 657 (1998)). In plants, the blue light-dependent interaction between CRYs and CIB1 (cryptochrome-interacting basic-helix-loop-helix) is an early photoreceptor signaling mechanism. The CIB1 protein interacts with CRY2 in a blue-light-specific manner in yeast and Arabidopsis and co-initiates CRY2-dependent flower formation with other CIB1-related proteins. Arabidopsis transgenic plants overexpressing CIB1 had significantly increased FTmRNA, showing early flowering (H Liu et al., Science 269, 968 (2008)). The expression and function of two cryptochromes GmCRY1a and GmCRY2a of soybean were found to be similar to those of Arabidopsis thaliana, both of which affected the elongation of cells inhibited by blue light, but only the degradation of GmCRY1a was dependent on blue light and 26S proteosomes. In contrast to Arabidopsis cryptochrome, GmCRY1a but not GmCRY2a exhibited strong flowering-promoting activity, and the protein expression level of GmCRY1a fluctuated with the rhythm of the circadian clock. GmCRY1a is a master regulator of flower cycle regulation in soybean (Q.Z. Zhang., et al., Proc Natl Acad Sci 2046 (2008)). Therefore, it is speculated that the CIBs genes in soybean, which belong to dicotyledonous plants, may interact with GmCRY1a to regulate soybean flowering.

Contents of the invention

The object of the present invention is to provide a soybean flowering regulation gene GmCIB6, its encoded protein and its application in the regulation of plant photoperiod and flowering time.

In order to achieve the purpose of the present invention, the protein encoded by a soybean flowering regulation gene GmCIB6 of the present invention has an amino acid sequence as shown in SEQ ID No.2 or the sequence has equivalent functions through substitution, deletion or addition of one or several amino acids. amino acid sequence.

The present invention also provides soybean flowering regulatory gene GmCIB6 encoding the above protein.

The aforementioned soybean flowering regulatory gene GmCIB6 has a nucleotide sequence as shown in SEQ ID No.1.

The invention also provides a vector containing soybean flowering regulation gene GmCIB6.

The present invention also provides host cells containing the above-mentioned vectors.

The present invention also provides transformed plant cells containing the soybean flowering regulatory gene GmCIB6.

The present invention further provides the application of the gene described in the soybean flowering regulation gene GmCIB6 in regulating the photoperiod and flowering time of plants, especially soybean and Arabidopsis.

In addition, the present invention provides a primer for amplifying soybean flowering regulatory gene GmCIB6, which includes forward primer F1: 5'-ATGGGAATTCAACTAACAGTTATG-3' and reverse primer R1: 5'-TCAGAGCTCAACTTTCATCTGTG-3'.

GmCIB6 is a bHLH family gene isolated from soybean and obtained functional identification. Gm is the initials of Glycine max (Linn) Merril, and CIB is the abbreviation of cryptochrome-interacting basic-helix-loop-helix.

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, the complete sequence of GmCIB6 was obtained by PCR, its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.2.

The GmCIB6 gene is highly homologous to the Arabidopsis CIB1 gene, and its encoded protein has a 37.3% identity with the Arabidopsis CIB1 protein. (As shown in Figure 1).

Overexpression of the GmCIB6 gene in wild-type Arabidopsis thaliana resulted in reduced photoperiod sensitivity and early flowering in both long-day and short-day conditions. This indicates that the GmCIB6 gene has a similar function to Arabidopsis CIB1 and plays an important role in regulating the timing of flowering.

GmCIB6 gene has important application value. Overexpression of GmCIB6 can reduce the sensitivity of plant flowering to photoperiod, can obviously promote plant flowering, shorten flowering time and growth period. It can be used to solve the problem of flowering failure in hybrid breeding, the growth period control problem of various crops, vegetables, fruits and flowers, the photoperiod sensitivity problem and the introduction problem.

Description of drawings

Fig. 1 is the amino acid sequence comparison of soybean CIB6 protein of the present invention and Arabidopsis CIB1 protein, wherein GmCIB6 represents soybean CIB6 protein, and AtCIB1 represents Arabidopsis CIB1 protein;

Fig. 2 is the flowering contrast of Arabidopsis Col-0 wild type and its GmCIB6 gene T1 plant, wherein, the left side represents the Col-0 wild type plant, and the right side represents the transformed plant;

Fig. 3 is the flowering comparison of the Arabidopsis Col-0 wild type and its GmCIB6 gene T2 plant under long-day (LD) conditions, wherein the left side represents the Col-0 wild type plant, and the right side represents the transformed plant;

Figure 4 is a comparison of the flowering of Arabidopsis Col-0 wild type and its GmCIB6 gene T2 plant under short-day (SD) conditions, wherein the right side represents the Col-0 wild type plant, and the left side represents the transformed plant.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Example 1 Isolation and cloning of GmCIB6 gene

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, and its nucleotide sequence is shown in SEQ ID NO.1.

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, 25 μL in total. The PCR reaction program was: 94°C pre-denaturation for 5 minutes, 30 cycles at 95°C for 30s, 52.5°C for 30s, 72°C for 2 minutes, and finally 72°C for 10 minutes.

Example 2 Analysis and identification of GmCIB6 gene

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 transcription 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.

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.

Example 3 The acquisition of transgenic Arabidopsis thaliana

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.

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, 25 μL in total. 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.

The PCR product was cloned into the pGWC vector containing the Gateway linker (Chen Qijun, Wang Xuechen, etc., using a T vector compatible with Gateway technology to obtain an entry clone, 2004.31 (10), 951-954), and the sequence was identified to be identical to the target GmCIB6. sequence. GmCIB6 was constructed on the overexpression vector pLeela (purchased from Invitrogen) by LR reaction to obtain the vector 35S::GmCIB6. The overexpression vector 35S::GmCIB6 was introduced into the Agrobacterium strain GV3101:90RK (purchased from Invitrogen), and the Agrobacterium-mediated dipping method (Steven J. Clough and Andrew F. Bent. Floral dip: A Simpled Method for Agrobacterium- mediated Transformation of Arabidopsis thaliana.Plant Journal.1998.16(6), 735-743.) Transform Arabidopsis thaliana in full flowering stage, sow the seeds harvested from the transformed plants, and spray 1000 times diluted The herbicide Basta (purchased from Chem Service Lot: 395-85A) was screened, and 5 Arabidopsis transgenic plants overexpressing GmCIB6 resistant to Basta were obtained.

Example 4 GmCIB6 gene function analysis

The T1 plants of the five GmCIB6 Arabidopsis transgenic plants obtained according to the method of Example 3 showed early flowering ( FIG. 2 ). T2 plants flowered early under both long-day and short-day conditions (Fig. 3 and Fig. 4). It indicated that the sensitivity of the transformed plants to photoperiod decreased, indicating that the GmCIB6 gene had a similar function to Arabidopsis CIB1, and played an important role in regulating the flowering time.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (9)

1. A primer for amplifying the soybean flowering regulatory gene GmCIB6, comprising forward primer F1: 5'-ATGGGAATTCAACTAACAGTTATG-3' and reverse primer R1: 5'-TCAGAGCTCAACTTTCATCTGTG-3'. 2. The protein encoded by the soybean flowering regulatory gene GmCIB6, characterized in that its amino acid sequence is as shown in SEQ ID No.2. 3. A gene encoding the protein of claim 2. 4. the gene as claimed in claim 3 is characterized in that, its nucleotide sequence is as shown in SEQID No.1. 5. A vector comprising the gene of claim 3 or 4. 6. A host cell comprising the vector of claim 5. 7. The application of the gene according to claim 3 or 4 in regulating plant photoperiod and flowering time. 8. The use as claimed in claim 7, wherein said plant is soybean. 9. The use according to claim 7, wherein said plant is Arabidopsis.

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