CN113481208B - Application of wild soybean MADS-box family gene GsAGL62 - Google Patents

Abstract

The invention discloses a wild soybean MADS-box family gene GsAGL62 and application thereof. The wild soybean GsAGL62 protein coding gene GsAGL62 has the nucleotide sequence as follows: SEQ ID NO. 1. The constructed plant over-expression vector pMDC83-GsAGL62 is subjected to heterologous expression in a wild type of Arabidopsis, and the transgenic plant is found to have variation in yield traits and obviously increased pod number. The gene can be used as a target gene to be introduced into a plant, and the yield of the transgenic plant is improved through the expression of the GsAGL62 gene. Therefore, the wild soybean GsAGL62 protein coding gene GsAGL62 can be applied to the aspect of improving the yield of pod numbers, thousand seed weights and the like through genetic engineering.

Description

Application of wild soybean MADS-box family gene GsAGL62

Technical Field

The invention belongs to the field of plant genetic engineering, and relates to application of an MLP subfamily gene GsAGL62 of a wild soybean MADS-box family, in particular to application of an MADS-box family gene GsAGL62 which is highly expressed in seeds and related to seed development and is derived from wild soybeans in aspects related to regulation and control of yield traits such as pod number, thousand seed weight and the like of plants.

Background

Pod development is an important link in the growth and development process of higher plants, and the relationship between the pod number and grain weight characters and the yield of crops and the quality of agricultural products is tight, so that economic benefit is influenced. The increase of the pod number can improve the yield of the soybean, and has important significance for soybean breeding. The wild soybean in Heilongjiang province has abundant resources, and provides a valuable gene source for soybean research. The MADS-box family belongs to one of the most important transcription factors of plants and shows a remarkable influence on the growth and development of the plants, and particularly influences the control of flowering time, the development of floral organs and meristems, the formation of fissure zones, fruit maturation, the development of embryos, plant leaves and root organs and the like (Gramzow et al, 2010; Yu et al, 2014). In addition, the family of genes also show significant effects in response to biotic and abiotic stresses (Wei et al, 2009; Wei et al, 2013). A common feature of MADS-box like genes is the MADS domain located at the N-terminus. The structure is highly conserved, and the length is 58-60 amino acids. The gene sequence is widely existed in various plants. In the case of Arabidopsis, more than 100 genes have been identified (De Bodt et al, 2003), and the MADS-box gene roughly includes different types, i.e., type I, type II, etc., based on differences in evolution type and structural characteristics. Among them, the research results for type I genes are relatively few, and the main influence factors of plant floral organ development. Such as the ABCDE model currently studied for relatively mature flower development (zhao yang et al, 2018). To date, the MADS-box family of proteins has been studied relatively extensively in rice and arabidopsis thaliana (Cannon et al, 2004).

Disclosure of Invention

The invention aims to disclose a wild soybean MADS-box family gene GsAGL 62.

Another purpose of the invention is to provide the application of the gene in genetic engineering such as seed development and regulation of pod number and thousand seed weight.

The purpose of the invention can be realized by the following technical scheme:

the wild soybean MADS-box family gene GsAGL62 has a nucleotide sequence of SEQ ID NO. 1.

The amino acid sequence of the protein coded by the wild soybean MADS-box family gene GsAGL62 is SEQ ID NO. 2.

The expression vector contains the wild soybean MADS-box family gene GsAGL 62.

The wild soybean MADS-box family gene GsAGL62 is selected for genetic engineering application in plant seed development and yield traits such as pod number and thousand seed weight regulation.

The number of pods of the GsAGL62 transgenic arabidopsis thaliana is obviously increased.

When the plant expression vector is constructed by using GsAGL62, any enhanced promoter or inducible promoter can be added before the transcription initiation nucleotide. In order to facilitate the identification and selection of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding resistance genes for selectable marker genes (GUS gene, GFP gene, etc.) or antibiotic markers (gentamicin marker, kanamycin marker, hygromycin marker, etc.) which can be expressed in plants. From the safety of transgenic plants, the transformed plants can be directly screened by phenotypic characters without adding any selective marker genes.

The plant expression vector carrying the GsAGL62 of the invention can be used to transform plant cells or tissues by conventional biological methods using Ti plasmid, Ri plasmid, plant viral vector, direct DNA transformation, microinjection, conductance, agrobacterium-mediated transformation, etc., and culture the transformed plant tissues into plants. The transformed plant host can be monocotyledons such as rice, wheat and corn, and can also be dicotyledons such as tobacco, arabidopsis, soybean, rape, cucumber, tomato, poplar, lawn grass and alfalfa.

Has the advantages that:

the GsAGL62 belongs to MADS-box family, and contains MADS _ MEF2 type structural domain. The GsAGL62 is mainly expressed in leaves and seeds through tissue expression analysis, and the subcellular localization shows that the GsAGL62 protein is in cell nucleus. GsAGL62 is over-expressed in arabidopsis thaliana, and compared with a control, the transgenic arabidopsis thaliana has the advantages that the silique number is obviously increased, and the seedling stage leaf blade is larger. The thousand kernel weight statistical analysis of the transgenic arabidopsis shows that the GsAGL62 transgenic line can obviously improve the thousand kernel weight of seeds compared with a control. The invention discloses the utility of the gene in regulating and controlling the plant pod development and the change in yield character. The yield of the crops can be improved by directionally modifying the pod number of the crops.

The GsAGL62 disclosed by the invention is introduced into a plant body by using a plant overexpression vector pMDC83-GsAGL62, so that the podding condition of the plant can be regulated and controlled, and a transgenic plant is obtained.

Drawings

The invention is further explained below with reference to the drawings and the embodiments.

FIG. 1 cloning of the GsAGL62 Gene

Designing a primer according to the sequence information of GsAGL62 predicted by a W05 website, and carrying out PCR amplification by using seed cDNA of a wild soybean material HAAS _187 as a template to obtain a DNA fragment with the length of 522 bp. Through sequencing result analysis, the sequence information of the fragment is consistent with the sequence predicted by the W05 website, namely the 522bp fragment is the GsAGL62 gene. Wherein the Marker is 2k and is 100, 250, 500, 750, 1000 and 2000bp from bottom to top in sequence.

Fig. 2 tissue expression analysis of the GsAGL62 gene.

The expression of the GsAGL62 in different tissues of wild soybean HAAS _187 is researched by adopting a real-time fluorescent quantitative PCR technology, wherein the different tissues of the wild soybean are roots, stems, leaves, flowers, 35d pods and 35d seeds. FIG. 3GsAGL62 subcellular localization (A) d35s: GFP; (B) d35s, GsAGL 62-GFP;

FIG. 4 PCR identification of transgenic Arabidopsis thaliana.

1-13 are different transgenic lines; WT is wild type arabidopsis (negative control); p: the plasmid was pMDC83-GsAGL62 recombinant plasmid (positive control).

FIG. 5 relative expression levels of GsAGL62 in different transgenic Arabidopsis lines

1,2,3,4,5,6,8,9,10,11,12,14,15,16, are different transgenic lines, and WT is wild type Arabidopsis.

FIG. 6 comparison of siliques of transgenic Arabidopsis and wild type Arabidopsis

1,3,4,6 and 8 are different transgenic lines, and WT is wild type Arabidopsis. Indicates a significant difference at a level of 0.01< p < 0.05; indicates a very significant difference in p <0.01 levels;

FIG. 7 statistical analysis of thousand grain weight of transgenic Arabidopsis and wild type Arabidopsis

1,3,4,6 and 8 are different transgenic lines, and WT is wild type Arabidopsis. Indicates a significant difference at a level of 0.01< p < 0.05; indicates a very significant difference in p <0.01 levels;

FIG. 8 comparison of seed morphology in mature period between transgenic Arabidopsis and wild type Arabidopsis

Detailed Description

The present invention is described in further detail below with reference to the data in conjunction with the figures and examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way. In the following examples, various procedures and methods not described in detail are conventional methods well known in the art. The primers used are indicated for the first time and the same primers used thereafter are indicated for the first time.

Example 1 cloning and identification of wild Soybean GsAGL62 and its coding Gene

PCR amplification was performed using the pod cDNA of wild soybean material HAAS _187 as a template and GsAGL62-F/GsAGL62-R as primers.

An upstream primer GsAGL62-F: ATGCCAGACTTGAACGGTGTCG, respectively; (SEQ ID NO.3)

The downstream primer GsAGL62-R: TCAGTTGAGGTTCCCACCTTTT are provided. (SEQ ID NO.4)

The GsAGL62 gene is amplified from the total RNA of soybean seed organs by using an RT-PCR method. The soybean pod tissue was ground in a mortar, added to a 1.5mL EP tube containing the lysate, shaken well and transferred to a glass homogenizer. After homogenization, the mixture was transferred to a 1.5mL EP tube and total RNA extraction was performed using a plant total RNA extraction kit (TIANGEN DP 404). The quality of the total RNA is identified by formaldehyde denatured gel electrophoresis, and then the RNA content is determined on a spectrophotometer. The total RNA obtained was used as a template, and reverse transcription was carried out in accordance with the instructions of the reverse transcription kit supplied by Takara, whereby the first strand cDNA was synthesized. PCR amplification reaction was performed. The PCR reaction system consisted of 2. mu.l of cDNA (0.05. mu.g), 2. mu.l each of the upstream and downstream primers (10. mu.M), 25. mu.l of 2 XPHunta Max Buffer, 1. mu.l of dNTP (10mM) and 1U of Phanta Max Super-Fidelity DNA polymerase (Vazyme), and was made up to 50. mu.l with ultrapure water. The PCR procedure was as follows: the procedure was carried out on a Bio-RAD PTC200 PCR instrument with a pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 15s, annealing at 58 ℃ for 15s, and extension at 72 ℃ for 45s for 30 cycles; the reaction was then terminated by extension at 72 ℃ for 5min and stored at 4 ℃. And recovering the PCR product, cloning the PCR product to a pClone007 vector, and sequencing to obtain a cDNA sequence SEQ ID NO.1 of a soybean gene GsAGL62 with a complete coding region, wherein the cDNA sequence has a total length of 462bp and codes 173 amino acids shown in SEQ ID NO. 2.

Example 2 expression profiles of GsAGL62 in different organs of wild Soybean

Roots, stems, leaves, flowers, 7d pods, 15d pods, 25d pods, 35d pods, 45d pods, 35d seeds of HAAS — 187 material were extracted for RNA and inverted into cDNA for RT-PCR analysis.

Total RNA was extracted as in example 1. The soybean constitutive expression gene Tubulin is used as an internal reference gene, and amplification primers of the soybean constitutive expression gene Tubulin are a forward primer sequence GGAGTTCACAGAGGCAGAG (SEQ ID NO.5) and a reverse primer sequence CACTTACGCATCACATAGCA (SEQ ID NO. 6). And carrying out real-time fluorescent quantitative PCR analysis by taking cDNA from different tissues or organs of the soybean as a template. The amplification primers of the GsAGL62 are GsAGL62-qPCR-F: TCGGGTGACATTCTCGAAGC (SEQ ID NO.7) and GsAGL62-qPCR-R: ACATCCACGTCACAAAGGGT (SEQ ID NO. 8). The results (fig. 2) analysis showed that GsAGL62 expression was relatively high in leaf, seeds, suggesting that GsAGL62 may be associated with soybean seed development.

Example 3 subcellular localization of GsAGL62

The subcellular localization adopts a transient expression method of Nicotiana benthamiana, a carrier used is P2, and primers are GsAGL62-P2-F: ACAAATCTATCTCTCTCGAGATGCCAGACTTGAACGGTGTCG (SEQ ID NO.9) and GsAGL62-P2-R: GCTCACCATGGATCCGTTGAGGTTCCCACCTTTT (SEQ ID NO. 10). PCR amplification, gel tapping recovery after a target band is correct, connecting a gel recovery product to a carrier through a homologous recombination method, constructing a subcellular localization carrier P2-GsAGL62 (the gene is at the N end of GFP), carrying out dark culture for 48h after tobacco transient expression, generating a green fluorescent signal after laser irradiation of a laser confocal microscope (Zeiss, LSM780), and carrying out localization, observation and photographing on protein. The results are shown in FIG. 3, where the transfected unloaded plasmid had a distribution throughout the cells, GsAGL 62: GFP fusion proteins are distributed in the nucleus, suggesting that GsAGL62 may function in the nucleus.

Example 4 genetic engineering of GsAGL62

A vector is constructed by using a double enzyme digestion method, and is divided into two steps of enzyme digestion reaction and recombination reaction, wherein the primer sequence is as follows: CAGGTCGACTCTAGAGGATCCGCCACCATGCCAGACTTGAACGGTGTCG (SEQ ID NO.11)

R:GGGAAATTCGAGCTCGGTACCTCAGTTGAGGTTCCCACCTTTT(SEQ ID NO.12)

The pMDC83 vector is subjected to double enzyme digestion by BamH1 and Kpnl, a target fragment is amplified from T-GsAGL62 by adding a joint primer, then recombination and connection are carried out, a product, namely the expression vector pMDC83-GsAGL62, escherichia coli DH5 alpha is transformed, and a transformation solution is coated on an LB solid culture medium containing 50mg/L Kana to screen positive clones. After sequencing verification, plasmids are extracted to obtain a plant over-expression vector pMDC83-GsAGL62, and pMDC83-GsAGL62 is transferred into Agrobacterium tumefaciens strain EHA105 by a freeze-thaw method. pMDC83-GsAGL62 is used for transforming Arabidopsis thaliana through the mediation of agrobacterium strain EHA105, and is cultured on an MS culture medium containing 50mg/L Kana, and a transgenic plant with Kana resistance is obtained through primary screening.

Extracting the genome DNA of the transgenic arabidopsis with Kana resistance obtained by primary screening, and carrying out PCR identification by using gene specific primers GsAGL62-F: GAGGACCTCGACTCTAGAACTA (SEQ ID NO.13) and GsAGL62-R: GGGAAATTCGAGCTCGGTACCTCAGTTGAGGTTCCCACCTTTT (SEQ ID NO. 14). The positive transgenic Arabidopsis thaliana with a band of about 462bp in size can be amplified (FIG. 4). Selecting plants identified as positive by PCR, and carrying out quantitative analysis on the selected plants by GsAGL62-qPCR-F: TCGGGTGACATTCTCGAAGC (SEQ ID NO.15) and GsAGL62-qPCR-R: ACATCCACGTCACAAAGGGT (SEQ ID NO.16) as a primer, and carrying out real-time fluorescent quantitative PCR detection. The results indicate that GsAGL62 can be expressed in transgenic arabidopsis thaliana (fig. 5). The transgenic plant with positive PCR and real-time fluorescent quantitative PCR detection is named as 35S, namely GsAGL62 transgenic Arabidopsis.

Phenotypic observations were made for 35S:GsAGL 62 transgenic Arabidopsis. In the growth condition of 25 ℃ and long sunshine, 35S shows that the GsAGL62 transgenic Arabidopsis thaliana has a phenotype observed in the seedling stage (3 weeks and 4 weeks after germination) compared with a control, the GsAGL62 transgenic plant has 3 lines of leaf blade growth vigor which is larger than that of WT on the whole, and the number of the leaf blades of the transgenic plant is increased, and the length, the width and the leaf area of the leaf blades are also obviously increased when the number and the length and the width of the leaf blades are measured. Compared with the WT, the number of siliques of 5 lines of the GsAGL62 transgenic line is obviously increased (figure 6), the thousand seed weight of 4 lines is obviously increased (figure 7), the number of the seeds of each line is counted, and the change of the number of the seeds of each transgenic line is not obvious, and the weight of the seeds of each line of 4 lines is obviously increased. In general, the GsAGL62 transgenic plant can increase the yield of a single plant mainly by increasing the pod number, and a new idea is provided for the soybean yield breeding research.

Sequence listing

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Claims (4)

1. The wild soybean GsAGL62 protein coding gene GsAGL62 is characterized in that the nucleotide sequence is shown as SEQ ID number 1.

2. A recombinant expression vector containing the wild soybean GsAGL62 protein coding gene GsAGL62 as claimed in claim 1.

3. The use of the wild soybean GsAGL62 protein coding gene GsAGL62 of claim 1 in increasing the number of arabidopsis thaliana siliques and thousand kernel weight by genetic engineering to enhance the yield of transgenic plants.

4. The use of the recombinant expression vector of claim 2 in increasing the number and thousand kernel weight of arabidopsis thaliana siliques by genetic engineering to enhance the yield of transgenic plants.

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