CN107746846A - The IbABF4 genes of coding sweet potato bZIP transcription factors and application - Google Patents

Abstract

The invention discloses a kind of coded sequence of positive regulation flowering of plant sweet potato ABF4 genes and its application.The wherein IbABF4 genes of coding sweet potato bZIP transcription factors, it is the nucleotide sequence shown in SEQ ID NO.1.The protein of IbABF4 gene codes is the amino acid sequence shown in SEQ ID No.2.The present invention isolates the global cDNA of coding bZIP transcription factors from sweet potato, is connected on plant expression vector, converts plant using Agrobacterium infestation method, obtains transfer-gen plant, transfer-gen plant is bloomed in advance.This gene can apply to genetic modification of plants.

Description

IbABF4 gene encoding sweet potato bZIP transcription factor and its application

technical field

The invention belongs to the field of genetic engineering, and specifically relates to an IbABF4 gene encoding a sweet potato bZIP transcription factor, a protein encoded by the gene, a recombinant vector containing the gene, and an anti-stress application of the gene.

Background technique

The occurrence of flowering determines that higher plants have the ability to reproduce and inherit, which is of great significance to the development of individual plants and offspring. The flowering regulation of plants is regulated by multiple pathways. Based on the constantly updated flowering molecular genetic results, the pathways for plants to initiate flowering in response to various endogenous and exogenous signals are summarized as: the classic photoperiod pathway, vernalization pathway, There are 5 autonomous pathways, gibberellin pathways and newer age pathways. In the process of agricultural production, the flowering of crops has an inseparable relationship with yield and adaptation area, such as rice, wheat, corn, potato and so on.

Transcription factors play an important role in the regulation of plant flowering pathways, and bZIP family genes are an important class of transcription factors that regulate plant flowering. Among them, ABF is a type of bZIP transcription factor that can bind to ABRE. The amino acid sequence of the basic region of this type of gene is very conservative, and it also contains special MKIK and QAY/Q motifs. These genes are mainly involved in plant abiotic stress resistance pathways (drought, cold, salt and oxidative stress, etc.) and ABA signal transduction, but there are few reports related to plant flowering.

Sweet potato is not only an important food crop, but also an important economic crop and energy crop. Sweet potatoes are widely planted in more than 100 countries in the world. my country is the largest producer in the world, and the production of sweet potatoes accounts for about 80% of the world's sweet potatoes. Compared with other food crops, sweet potatoes are relatively drought-tolerant, but there are large differences among varieties. A considerable proportion of sweet potatoes in the world are planted in arid environments, and the world's arid and semi-arid areas account for more than one-third of the land area. , the impact of drought on plants ranks first among many natural adversity factors. The whole genome of sweet potato is about 4G, which contains rich gene resources. Isolating and cloning ABF transcription factors in sweet potato and studying their functional characteristics can provide genetic resources for crop genetic improvement.

Contents of the invention

The object of the present invention is to provide an IbABF4 gene encoding sweet potato bZIP transcription factor.

The second object of the present invention is to provide a protein encoded by the gene.

The third object of the present invention is to provide an expression vector containing the gene.

The fourth object of the present invention is to provide a host cell containing the expression vector.

The last object of the present invention is to provide the use of this gene.

Technical scheme of the present invention is summarized as follows:

An IbABF4 gene encoding sweet potato bZIP transcription factor, which is the nucleotide sequence shown in SEQ ID NO.1. The nucleotide sequence is composed of 1263 bases.

The protein encoded by the above gene has the amino acid sequence shown in SEQ ID No.2. The described sequence consists of 420 amino acid residues.

An expression vector pCAMBIA1305-IbABF4 containing the above gene, which contains the nucleotide sequence shown in SEQ ID NO.1.

An Agrobacterium host cell EHA105 containing the above expression vector: pCAMBIA1305-2×35s-IbABF4.

The application of the above-mentioned expression vectors or Agrobacterium host cells in transforming plants to obtain transgenic plants, especially in the preparation of transgenic Arabidopsis thaliana.

Advantages of the present invention:

The present invention isolates the complete cDNA encoding the bZIP transcription factor gene from sweet potato, connects it to the plant expression vector, uses the Agrobacterium infection method to transform the plant, and obtains the transgenic plant. The result shows that the IbABF4 gene can promote the flowering of the plant.

Description of drawings

Figure 1. Phylogenetic tree analysis results of IbABF4 amino acid sequence;

Figure 2. Schematic diagram of pCAMBIA1305-2×35s-IbABF4 vector;

Fig. 3. Comparison of root systems of untransformed A. thaliana lines and transgenic IbABF4 plants.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, unless otherwise specified, the specific experimental methods involved in the following examples are all conventional methods or implemented according to the conditions suggested by the manufacturer's instructions.

Example 1

The sequence of the bZIP transcription factor IbABF4 gene in sweet potato was obtained, as follows:

Total RNA was extracted from 100 mg of fresh sweet potato leaves using the OMEGA RNA kit, and cDNA was synthesized using a reverse transcription kit (Bioteke).

Concrete reaction system is as follows:

The reverse transcription reaction was carried out on the PCR instrument according to the following conditions: 1. 50° C., 45 min; 2. 70° C., 10 min; and then cooled on ice.

The extracted total RNA was sent to Beijing Genomic Institute (BGI), and the sweet potato transcriptome database was obtained through high-throughput sequencing, and the sweet potato Unigene database was obtained through De novo splicing. After comparing CL3260 with the NR database, it was found that the gene might be Participate in the ABA pathway. CL3260 was used to predict the complete ORF of the gene by OFR FINDER (http://www.ncbi.nlm.nih.gov/projects/gorf/) online software, and PrimerPremier 5 was used to design and amplify the complete ORF primer:

IbABF4(F): 5'ATGATGGGGTCATACTTGGA 3'

IbABF4(R): 5'TTACCAAGGCCCAGTAAGCG 3'

Use Primerstar GXL DNA polymerase from TAKARA company to amplify, the specific steps are as follows:

The reaction conditions are as follows: 94°C, 4min; 98°C, 10Sec; 55°C, 15Sec; 68°C, 1.5min; 72°C, 10min; 30 cycles. OMEGA DNA purification kit was used to purify the PCR product, and the purified PCR product was connected to the pEASY-Blunt vector (transGEN’s pEASY-Blunt Vector Cloning Kit was used in this process). The reaction system was as follows: cDNA fragment 4u1 of IbABF4 gene, pEASY -Blunt vehicle 1 μL. Reaction conditions: 20-30°C, 30min. The ligation product was transformed into E-Coli.DH5α competent, spread on LB agar plate medium containing 40u125mg/ml X-Gal, 16ul 50mg/ml IPTG, 100mg/mL Amp and cultured to form a single colony. Pick white colonies, and use the colony PCR method to confirm the length of the insert in the pEASY-Blunt vector, which is consistent with the expectation. Sent to Nanjing GenScript Biotechnology Company for sequencing to obtain the gene sequence SEQ ID NO.1.

Example 2

IbABF4 protein sequence homology analysis, specifically as follows:

The IbABF4 gene sequence obtained by sequencing is 1263bp, encoding 421 amino acids as shown in SEQ ID NO.2, and the translated protein sequence is compared with the NCBI protein data (http://blast.ncbi.nlm.nih.gov/Blast. cgi), the plant species homologous gene similar to the IbABF4 protein sequence was obtained. On the basis of multiple comparison analysis, a phylogenetic tree of the genes of each homologous plant species was established, as shown in Figure 1 for details. Includes morning glory (Ipomoea nil), tobacco (Nicotiana tabacum), potato (Solauum tuberosum), tomato (Solanum lycopersicum), pepper (Capsicum annuum), nightshade (Solanum nigrum), Asian cotton (Gossypium arboretum), rice (Oryzasativa ), walnut (Juglans regia), Arabidopsis thaliana (Arabidopsis thaliana). The phylogenetic tree was constructed using MEGA 5.1 software, and the genetic relationship of IbABF4 was closer to that of Arabidopsis thaliana and grapevine (as shown in Figure 1).

Example 3

The construction of the binary plant expression vector pCAMBIA1305-2×35s-IbABF4 is as follows:

The schematic diagram of the pCAMBIA1305-2×35s-IbABF4 vector is shown in Figure 2. First, the pEASY-Blunt-IbABF4 plasmid was used as a template and primers were used

IbABF4-PstI(F): 5'aa CTGCAG ATGGCTGCCACTATTGAT3'

IbABF4-BamHI(R): 5'cc GGATCC TTATGACCCGCCCGAACCTG 3'

Restriction sites PstI and BamHI were respectively introduced before and after IbABF4, and the reaction system and conditions were as described in Example 1. Then the PCR product and the pCAMBIA1305 empty vector plasmid were double digested with BamHI and PstI respectively, and the digested products of the two were ligated. The ligation system was as follows:

The ligation product was transformed into E-Coli.DH5α, and spread on LB plates containing kanamycin resistance at a concentration of 100 mg/ml. Cultivate at 37°C, pick a single colony after 12 hours for colony PCR verification, shake the bacteria that are positive in the colony PCR verification, extract the plasmid, digest and identify the target band, and finally send it to Huada Gene Sequencing Company for sequencing. The results show that the vector pCAMBIA1305- 2×35s-IbABF4 was constructed correctly.

Example 4

The construction of Agrobacterium strain EHA105 for plant transgenesis: pCAMBIA1305-2×35s-IbABF4 is as follows:

The Agrobacterium strain used in the present invention is EHA105. The constructed expression vector was transformed into Agrobacterium by liquid nitrogen freeze-thaw method. The specific process is as follows: 1) Thaw EHA105 competent cells in an ice bath, add at least 100ng of recovered and purified expression vector plasmids, mix gently, and ice bath for 20-30 minutes; 2) Liquid nitrogen quick freezing for 5 minutes, heat shock at 37°C for 5 minutes, and quickly place Place on ice for 1-2 minutes; 3) Add 800 μL of antibiotic-free LB medium, recover at 28°C, 200 rpm for 3.5 hours; 4) Centrifuge at 4000 rpm for 3 minutes, and suck off the medium; ml kanamycin and 100mg/ml rifampicin on solid LB medium; 6) Inverted culture at 28°C for 30-48h; 7) PCR positive clones were stored at 4°C for later use.

Example 5

IbABF4 transforms Col wild-type Arabidopsis as follows:

Inoculate the positive clone in Example 4 into 50ml YEP (containing 100μg/ml Rif, 100μg/ml Kan) liquid medium, and continue to cultivate at 180rpm at 28°C until OD600=0.8. Centrifuge at 4000rpm for 10min, discard the medium, and collect the bacteria. The Arabidopsis thaliana infiltration buffer was used to dilute the cells with OD600=0.6 to prepare the Arabidopsis thaliana infection solution. When Arabidopsis thaliana bolts 4-5cm, it can prepare for infestation, and remove its terminal inflorescence 3 days before infestation to take advantage of the growth of axillary inflorescences. After the axillary inflorescence grows, the transformation can be carried out when the flowers in the lower part begin to pollinate. Water abundantly before transformation, and remove pollinated flower buds and pods. A total of 50ml of Arabidopsis infection solution (5% sucrose, 0.05% SilwetL-77) was prepared, poured into a petri dish, and the inflorescence of Arabidopsis was immersed in it for 30 seconds, and the rosette leaves should not be contaminated with the bacterial solution. Take out the plants, place them horizontally in a tray, and treat them in the dark for 24 hours. Afterwards, the square plate was placed upright and cultivated under normal light. The T0 generation seeds were harvested when the fruit pods matured.

The T0 generation seeds were vernalized in a refrigerator at 4°C for 3 days before sowing, and after surface disinfection with 0.5% NaClO, they were sown on 1/2 MS solid medium (macroelements and trace elements were halved, containing 20mg/L hygromycin) for cultivation. After 7-14 days, the transformants were selected, and the transformed plants with hygromycin resistance could grow on the medium containing hygromycin, and the roots elongated obviously, while the non-transformants gradually yellowed and died. When the plant grows 4-5 leaves, extract leaf DNA and carry out PCR identification, and the primers and PCR program are the same as the cloning of the IbABF4 gene intermediate fragment in Example 1. The PCR products were detected by 1% agarose gel electrophoresis. The positive transformant of Arabidopsis thaliana that can obtain the target fragment through PCR amplification is continued to be cultivated until the fruit pods mature to harvest T1 generation seeds. The T1 generation seeds harvested from the plants identified as positive by PCR continued to be sown, and were also screened for resistance to hygromycin, and the segregation ratio of the traits was counted. The chi-square test conformed to the 3:1 segregation ratio in Mendelian law of inheritance. Part of the resistant seedlings were transplanted into the soil, and T2 generation homozygous transgenic seeds were harvested.

Phenotype Identification of IbABF4 Transgenic Arabidopsis

Get IbABF4 overexpression Arabidopsis lines (OV-1, OV-2) and grow on 1/2MS medium for the transformed wild-type (WT) plants, observe the phenotype (Figure 3) in Arabidopsis Overexpression of IbABF4 can positively regulate the flowering time of plants.

sequence listing

<110> Jiangsu Academy of Agricultural Sciences

<120> IbABF4 Gene Encoding Sweet Potato bZIP Transcription Factor and Its Application

<130> 2017

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Met Met Gly Ser Tyr Leu Asp Phe Lys Asn Phe Val Glu Thr Pro Gln

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Pro Glu Gly Asn Gly Gly Lys Pro Ala Ser Leu Phe Pro Leu Ala Arg

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Gln Ser Ser Ile Tyr Ser Leu Thr Phe Asp Glu Leu Gln Thr Thr Phe

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Ser Gly Leu Gly Lys Asp Phe Gly Ser Met Asn Met Glu Asp Leu Leu

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Lys Ser Ile Trp Thr Ala Glu Glu Ser Gln Pro Phe Gln Ala Cys Cys

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Val Gly Ala Gly Asp Asn Gly Ser Val Pro Gly Gly Asn Leu Gln Arg

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Gln Gly Ser Leu Thr Leu Pro Arg Thr Leu Ser Gln Lys Thr Val Asp

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Glu Val Trp Lys Asp Phe Gln Lys Asp Thr Gly Ala Thr Lys Asp Cys

115 120 125

Gly Tyr Gly Gly Ser Ser Phe Gly Gln Arg Gln Ser Thr Leu Gly Glu

130 135 140

Met Thr Leu Glu Glu Phe Leu Phe Lys Ala Gly Val Val Val Arg Glu Glu

145 150 155 160

Met Val Pro Asn Ser Val Gly Phe Gly Ser Ser Val Thr Gln Leu Asn

165 170 175

Ser Met Gly Phe Gln Glu Ile Thr Asp Asn Asn Ser Ala Ala Ile Pro

180 185 190

Gly Ala Ser Ser Asn Ser Met Leu Ser Ala Gly Ala Thr Arg Ser Thr

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Gln Gln Gln Pro Leu Gln His His Gln Gln Leu Gln Leu His Gln Pro

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Leu Phe Pro Lys Gln Thr Thr Leu Thr Phe Ala Ser Pro Met Gln Leu

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Gln Asn Asn Ala Gln Gln Ala Arg Ser Gly Ala Arg Arg Pro Ala Leu

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Gly Met Pro Ser Pro Pro His Asn Thr Asn Gln Val Gln Gly Glu Leu

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Ala Gly Gly Gly Gly Gly Gly Ser Pro Gly Ile Pro Leu Ser Ser Ser Asp

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Val Ala Leu Asn Ser Asn Leu Gly Met Ser Ser Leu Ser Pro Ser Pro

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Tyr Ala Phe Asn Glu Gly Gly Arg Gly Arg Arg Ala Cys Asn Ser Ile

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Glu Lys Val Val Glu Arg Arg Arg Arg Arg Arg Met Ile Lys Asn Arg Glu

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Ser Ala Ala Arg Ser Arg Ala Arg Lys Gln Ala Tyr Thr Ile Glu Leu

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Glu Ala Glu Val Glu Lys Leu Lys Glu Ile Asn Gln Glu Leu Met Lys

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Lys Gln Ala Glu Phe Leu Glu Lys Arg Lys Asn Gln Met Arg Glu Lys

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Met Phe Val Pro Trp Glu Ser Lys Pro Arg Cys Leu Lys Arg Thr Leu

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Thr Gly Pro Trp

420

Claims (6)

1. a kind of IbABF4 genes of coding sweet potato bZIP transcription factors, it is characterised in that it is the core shown in SEQ ID NO.1 Nucleotide sequence；The IbABF4 genes have the function of positive regulation flowering of plant.

2. the protein of IbABF4 gene codes described in claim 1, it is characterised in that it is the ammonia shown in SEQ ID No.2 Base acid sequence.

A kind of 3. expression vector pCAMBIA1305-2 × 35s-IbABF4 containing gene described in claim 1.

A kind of 4. Agrobacterium host cell EHA105 containing expression vector described in claim 3：pCAMBIA1305-2×35s- IbABF4。

5. Agrobacterium host cell described in the expression vector or claim 4 described in claim 3 obtains in conversion plant turns base Because of the application in plant.

6. application according to claim 5, it is characterised in that described plant is arabidopsis.