CN104561041B - Soybean growth period gene J and its coding albumen - Google Patents

Abstract

Soybean growth period gene J and its coding albumen, it is related to soybean growth period gene J and its coding albumen.The object of the present invention is to provide soybean growth period gene J and its coding albumen.The gene order of soybean growth period gene J of the present invention such as sequence table Seq ID No：Shown in 1.The amino acid sequence such as sequence table Seq ID No of the coding albumen of soybean growth period gene J of the present invention：Shown in 2.The present invention is verified by Genetic Transformation of Soybean, shows that J genes inhibit the physiological function of soybean growth period.The present invention is applied to soybean gene engineering field.

Description

Soybean growth period gene J and its encoded protein

technical field

The present invention relates to soybean growth period gene J and its encoded protein.

Background technique

Soybeans provide human beings with important vegetable proteins and oils. Worldwide, soybeans are cultivated in a wide range of regions ranging from high latitudes in northern Europe, Sweden and North America to Canada, and south to Brazil and Argentina. However, the latitude span suitable for planting a single variety or germplasm resource is generally small. The long puberty trait, that is, the prolongation of the growth period under short-day conditions, plays an important role in extending the adaptation range of soybean varieties to low latitudes, and provides a theoretical basis for the cultivation and management of soybeans in tropical countries.

Kiihl found in an experiment in 1979 that three recessive genes determine the late flowering traits of soybean under short-day conditions. In Brazil, the long pubertal trait has positive implications for the northward extension of soybean production. So Brazil has done extensive research on the trait of prolong puberty. The Brazilian study concluded that five genes control the protracted puberty trait, and at least two genes must be recessive. However, the current research on soybean long puberty trait genes is nothing more than that, so this experiment aimed at this molecular research gap, designed a F2 generation hybrid population, used molecular marker technology to find QTLs related to long puberty, and successfully found related Linked genes. This invention has important significance for the wide adaptability of soybean varieties.

Contents of the invention

The object of the present invention is to provide soybean growth period gene J and its encoded protein.

The gene sequence of the soybean growth period gene J of the present invention is shown in the sequence table Seq ID No: 1.

The amino acid sequence of the encoded protein of the soybean growth period gene J of the present invention is shown in the sequence table Seq ID No: 2.

Beneficial effects of the present invention:

The present invention successfully clones the growth period gene J for the first time by using molecular means on the basis of the study of genetic quantity traits.

In the present invention, 127 F2 generation populations produced by crossing progenies of soybean varieties BR121 and Harosory are used to detect a QTL for soybean growth period near the LJ-SSR marker through the MQM genetic mapping method. Comparing the genes near the LJ-SSR marker with the growth period genes of Arabidopsis thaliana, we successfully found the only gene related to the growth period of Arabidopsis thaliana, that is, the J gene, and cloned this gene. The full-length sequence of the J gene coding region (Seq ID No: 1) obtained by the present invention and Glyma.04G050200.1 published in phytozome (http://www.phytozome.net) (gene interpretation of soybean genome Glyma1.0 version) Correspondingly, but its sequence is not completely identical, the protein translated by the J gene obtained in the present invention has 714 amino acids (Seq ID No: 2). Through transgenic, it was verified that the J gene has the physiological function of strongly inhibiting the growth period of soybean.

At the same time, through homologous comparison, it was found that J gene has high homology with Arabidopsis ELF3 family.

Description of drawings

Figure 1 is a schematic diagram of the structure of the pTF10I-J plasmid overexpression vector;

Fig. 2 is a time map of the maturation period of Williams 82 with J gene and without J gene; W82 is Williams82 without J gene, and 35S:J is Williams 82 with J gene.

Detailed ways

Specific embodiment 1: The soybean growth period gene J gene sequence of this embodiment is shown in the sequence table Seq ID No: 1.

In this embodiment, the F2 population produced by crossing Harosory and BR121 is studied, and the J gene is determined, which is closely related to the growth period of soybean.

In this embodiment, the J gene is expressed in soybeans by means of soybean genetic transformation, and it is verified that the J gene of the present invention can significantly prolong the maturity period of soybeans.

Embodiment 2: The amino acid sequence of the protein encoded by the soybean growth period gene J in this embodiment is shown in Seq ID No: 2 in the sequence table.

Verify effect of the present invention by following test:

(1) Acquisition of J gene in soybean growth period

1. In 2014, 127 F2 individuals produced by crossing BR121 and Harosory were planted in the artificial climate chamber of the Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, and the growth period was detected.

2. Using the already constructed genetic map of chromosome 4, combined with the detected maturity data, to carry out the genetic quantitative traits mapping. Preliminary genetic mapping only found one major QTL, namely LJ_SSR, which was closely related to soybean growth period.

3. By comparing with Arabidopsis thaliana growth period gene cluster analysis, we found the only gene related to Arabidopsis thaliana growth period, that is, J gene.

4. Using the leaves of the soybean variety Harosory as materials, the total RNA of the leaves was extracted with the operation manual of the TRIzol kit purchased from Invitrogen.

5. Treat the total RNA extracted in step 4 with DNase I.

6. Take 1 μg of the total RNA processed in step 5 for cDNA synthesis. The cDNA synthesis operation is carried out according to the manual of the BD SMART ^TM RACE cDNA Amplification Kit purchased from BD Biosciences Clontech Company to obtain cDNA.

7. The obtained cDNA was used as a template to amplify the J gene with primers F1 and R1. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 2 minutes and 30 seconds, a total of 35 cycles, and then Extend at 72°C for 5 min, and sequence the PCR product on an ABI3130 sequencer (ABI Company);

Wherein, the sequence of the primer F1 is 5'CTTCCGTAAC TCGTCACTCA 3'; the sequence of the primer R1 is 5'ACTCTTTCGG GTAAAGCAAT 3'.

The sequencing results show that the soybean growth period gene J has the nucleotide sequence of Seq ID No: 1 in the sequence listing. 2 amino acid sequences of proteins.

(2) Functional verification of soybean growth period gene J

1. Using the leaves of the soybean variety Harosory as a material, the total RNA of the leaves was extracted with the operation manual of the TRIzol kit purchased from Invitrogen;

2. Use DNase I to treat the total RNA extracted in step 1;

3. Take 1 μg of the total RNA processed in step 2 for cDNA synthesis. The cDNA synthesis operation is carried out according to the manual of the BD SMART ^TM RACE cDNA Amplification Kit purchased from BD Biosciences Clontech Company to obtain cDNA;

4. Using J gene F2 and R2 as primers, amplify the cDNA obtained in step 3 by PCR. PCR reaction conditions: 94°C pre-denaturation for 5 minutes, 94°C denaturation for 30 seconds, 60°C annealing for 30 seconds, and 72°C extension for 3 minutes. 35 cycles, and then extended at 72°C for 5 minutes to obtain the soybean growth period gene J containing the XbaI-SacI double enzyme cleavage site;

5. Cloning the obtained soybean growth period gene J containing restriction sites into the pTF101 plasmid to obtain the pTF101-J plasmid, using Willimas 82 as a material, using Agrobacterium-mediated genetic transformation to obtain transgenic soybeans, Transgenic J soybeans and Willimas 82 were planted under 12-hour light and 12-hour dark conditions, and their growth periods were observed;

Wherein, the sequence of the primer F2 is 5'GCTCTAGAGA TGAAGAGAGG GAAGGATGA 3'; the sequence of the primer R2 is 5'CGAGCTCGCT ACACTGAGTC ATTCTGTT 3'.

The schematic diagram of the structure of the pTF101-J plasmid overexpression vector obtained in step 5 is shown in Figure 1;

The maturation time chart of Williams 82 with and without the J gene is shown in Figure 2; where, W82 is the Williams 82 without the J gene, 35S:J is the Williams 82 after the J gene Soybean; as can be seen from Figure 2, the growth period of W82 is 92 days, and the growth period of 35S:J is 105 days, indicating that the maturity period of soybeans transferred to the J gene is 13 days later than that of Willimas 82, thus indicating that J The gene has the function of prolonging the maturity period of soybean.

Claims (1)

1. soybean growth period gene J is in the application for inhibiting Soybean Growth Characters interim, it is characterised in that the gene of soybean growth period gene J Sequence such as sequence table Seq ID No：Shown in 1.