CN103319603A - Application of rice transcription factor Os01g64730 gene - Google Patents

Abstract

The invention relates to the application of the rice transcription factor Os01g64730 gene, which uses the transcription factor activation motif VP64 to fuse with the rice transcription factor Os01g64730 gene to construct a constitutive transcription factor, and transforms it into crops such as rice, thereby improving the drought resistance of transgenic rice. It has important theoretical value for elucidating the molecular mechanism of rice drought resistance in detail, and can improve rice drought resistance and adaptability to different water environments through transgenic means, so it is also of great significance in production practice.

Description

Application of Rice Transcription Factor Os01g64730 Gene

technical field

The invention relates to the field of genetic engineering, in particular to the application of rice transcription factor Os01g64730 gene.

Background technique

Rice (Oryza sativa L.) is one of the three most important food crops in my country and the world, the staple food of more than half of the world's population, and an important model plant for functional gene research. Related genetics and molecular biology studies have been paid much attention by researchers, and the regulation of transcription level is an important way of gene expression regulation.

Drought is one of the important abiotic stresses that reduce crop yields worldwide. With the continuous increase of the global population, the food problem has become a major problem threatening the survival of all human beings. Therefore, improving the drought resistance of crops and increasing the yield of crops has become the key to solving the food crisis. With the combination of genetic engineering and traditional breeding methods, it has become an important means to cultivate new drought-resistant varieties by using various advanced molecular biology methods to mine drought-resistant genes and study the molecular biological mechanism of plant drought-resistant.

There is a complex system of adversity signal perception and transmission in plants. ABA (Abscisicacid) is an important plant hormone in response to stress signals, and it affects the expression of stress response genes in plants. At present, it is believed that the response of plants to adversity stress is divided into two pathways: ABA-Dependent and ABA-Independent (Zhu2002; Shinozaki, Yamaguchi-Shinozaki et al. 2003; Yamaguchi-Shinozaki and Shinozaki2006; Hirayama and Shinozaki2010). In the ABA-Dependent pathway, AREB/ABF (ABRE binding factor/ABRE-responsive elements bindingprotein) can regulate the expression of ABA-dependent downstream genes by recognizing ABREs (ABA-responsive elements) with ACGT as the core sequence, thereby changing the plant's The state of growth and development finally enables the plant to survive under adversity. Many ABER/ABFs are bZIP-type transcription factors with Leu zipper domain, which have been widely studied in plants.

VP64 is composed of four VP16 functional domain motifs fused together, and is a kind of enhancer. VP16 was first discovered in animal virus genes, and has been widely used in plants, mainly for the study of transcriptional regulation of plant genes. The role of transcription factors in vivo can be roughly divided into two types: one is a transcriptional enhancer, and the other is a transcriptional repressor. When a transcription factor is fused to a VP16 domain motif, it enhances the function of the transcription factor, leading to more pronounced phenotypic changes in transgenic plants.

Contents of the invention

The object of the present invention is to provide the application of rice transcription factor Os01g64730 gene.

In order to achieve the purpose of the present invention, the present invention firstly provides a fusion protein, which is Os01g64730-Linker-(VP16) ₄ ;

Among them, Linker is composed of 11 flexible amino acids in series, and its amino acid sequence is shown in SEQ ID No.9; VP16 is a VP16 protein from herpes simplex virus, and its amino acid sequence is shown in SEQ ID No.10; Os01g64730 is a rice transcription factor Os01g64730.

The amino acid sequence of the rice transcription factor Os01g64730 is shown in SEQ ID No. 1, or an amino acid sequence with equivalent functions formed by replacing, deleting or adding one or several amino acids to the sequence.

Among them, (VP16) ₄ is VP64, which is an enhancer composed of four VP16 functional domain motifs fused together at the GlySer interval, and its amino acid sequence is shown in SEQ ID No.2, or the sequence is replaced, deleted or added An amino acid sequence formed by one or several amino acids with equivalent functions.

The present invention also provides a gene encoding the fusion protein, and a nucleotide sequence that can hybridize with the nucleotide sequence of the gene under stringent conditions; wherein, the stringent conditions are 65°C, 0.1×SSPE or 0.1 ×SSC, 0.1% SDS solution to hybridize and wash the membrane.

The present invention also provides a vector containing the gene encoding the fusion protein. The vector is any vector that can guide the expression of foreign genes in the host. Preferably, the vector is a plant binary expression vector (eg, pCAMBIA1301). When the gene encoding the fusion protein of the present invention is constructed into a plant expression vector, any strong promoter (for example, corn strong promoter Ubiquitin) or an inducible promoter can be added before its transcription start nucleotide . In addition, when the gene encoding the fusion protein of the present invention is constructed into a plant expression vector, enhancers can also be used, and these enhancer regions must be in the same reading frame as the coding sequence to ensure translation of the entire sequence.

The expression vector carrying the gene encoding the fusion protein can be introduced into plant cells by conventional biotechnological methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation (Weissbach, 1998, Method for Plant Molecular Biology VIII , Academy Press, New York, pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology, 2nd Edition).

The invention also provides engineering bacteria containing the gene encoding the fusion protein.

The present invention also provides a method for constructing a transgenic rice plant, specifically, using an Agrobacterium-mediated method to transfer the aforementioned vector into the rice callus, transforming it with an AAM transformation solution containing an inducer and Agrobacterium, and transforming After co-cultivation-screening-differentiation-rooting-transgenic seedling training and transplanting, the final material was screened for transgenic rice plants.

The invention also provides the application of the gene encoding the fusion protein in improving the drought resistance ability of rice.

The present invention also provides a primer pair for amplifying the rice transcription factor Os01g64730 gene, which is a forward primer F: 5'-CAAAAAAGCAGGCTTCATGATGGCGTCGAGGGTG-3' and a reverse primer R: 5'-CAAGAAAGCTGGGTCCCACTCCATCGAGTTTGTTCTTC-3'.

The present invention further provides the application of the rice transcription factor Os01g64730 gene in improving the drought resistance ability of rice.

The aforementioned application is to construct the CDS sequence of the rice transcription factor Os01g64730 gene through the Gateway system to the upstream of the four transcription factor activation motifs VP16, and transform rice, thereby improving the traits of the transgenic rice grains.

For the first time, the present invention uses transcription factor activation motif VP64 (that is, four transcription factor activation motifs VP16) to fuse with rice transcription factor Os01g64730 gene to construct a constitutive transcription factor, and transform it into crops, such as rice, so as to improve the drought resistance ability of rice. It has important theoretical value for elucidating the molecular mechanism of rice drought resistance in detail, and can improve rice drought resistance and adaptability to different water environments through transgenic means, so it is also of great significance in production.

Description of drawings

Fig. 1 is a map of the cVP64-bar-asRED vector in Example 1 of the present invention.

Fig. 2 is the carrier map of ubi:Os01g64730-VP64 in Example 1 of the present invention.

Figure 3 is the result of Western Blot detection of transgenic positive rice lines in Example 3 of the present invention; wherein, WT is wild-type rice 'kitaake', and V17-27 and V17-56 are two lines of Os01g64730-VP64 transgenic rice respectively .

Figure 4 is the phenotypic analysis results of Os01g64730-VP64 transgenic rice line V17-27 and wild-type rice 'kitaake' in Example 4 of the present invention; wherein, WT is wild-type rice 'kitaake', and V17-27 is Os01g64730-VP64 Transgenic rice lines.

Figure 5 is the phenotypic analysis results of the Os01g64730-VP64 transgenic rice line V17-56 and the wild-type rice 'kitaake' in Example 4 of the present invention; wherein, WT is the wild-type rice 'kitaake', and V17-56 is Os01g64730-VP64 Transgenic rice lines.

Detailed ways

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

The isolation of embodiment 1Os01g64730 gene and the construction of plant expression vector

Find the Os01g64730 gene in the plant transcription factor database, design PCR amplification primers according to its sequence, and design PCR amplification primers according to its sequence (F: 5'-CAAAAAAGCAGGCTTCATGATGGCGTCGAGGGTG-3' and reverse primer R: 5'-CAAGAAAGCTGGGTCCCACTCCATCGAGTTTGTTCTTC-3' ).

The leaves of rice 'Nipponbare' were used as materials, total RNA was extracted by TRIzol method, and cDNA was obtained by reverse transcription. The reaction steps were as follows: (1) Add the following substances sequentially on ice to a PCR reaction tube without nuclease: total RNA 6 μl (0.1 ng-5μg), Oligo(dT)18 primer 1μl, nuclease-free ddH2O 5μl, placed in a PCR instrument at 65°C for 5min; (2) Then add the following substances: 5× reaction buffer 4μl, RNase inhibitor 1μl , 10mM dNTP 2μl, M-MμlV reverse transcriptase 1μl, mix gently, and react in a PCR instrument at 45°C for 60min; (3) In a PCR instrument, stop the reaction at 70°C for 5min. The total cDNA was used as a template for PCR amplification. The amplification system was: 25 μl of reaction buffer, 4 μl of dNTP, 17.5 μl of ddH2O, and 0.5 μl of Taq DNA polymerase. , after 30 cycles, extend at 72°C for 10min, and finally finish the reaction at 25°C.

Obtain the CDS sequence of rice transcription factor Os01g64730 gene, its nucleotide sequence is shown in SEQ ID No.3. Perform PCR according to the PrimeSTAR polymerase amplification system and reaction procedures. According to the requirements of Gateway cloning technology, this process includes two rounds of PCR. The primers of the first round of PCR use primers with a part of the adapter attB linker, while the templates of the second round use the PCR products of the first round, and the primers use the complete adapter attB primer. The PCR product was cloned into the pDONR cloning vector, and the sequence identical to the target gene was identified by sequencing. The Os01g64730 gene was constructed on the plant expression vector cVP64-bar-asRED (Figure 1) by LR reaction to obtain the vector ubi:Os01g64730-VP64 (Figure 2, the full sequence of the vector is shown in SEQ ID No.5).

Among them, the construction process of the plant expression vector cVP64-bar-asRED is as follows: the sequence contained in the left and right boundaries of the binary expression vector pCAMBIA1300 is used as the backbone sequence, and the ubi promoter-Gateway-VP64 expression unit and the 35S promoter-asRED expression unit are recombined through in vitro recombination. and 35S promoter-bar expression unit were fused to construct, and the full sequence of the vector cVP64-bar-asRED is shown in SEQ ID No.6.

The acquisition of embodiment 2 transgenic rice plants

Take the mature seeds of rice 'kitaake', shell them manually or mechanically, select the plump, smooth and spot-free seeds and inoculate them on the induction medium for induction culture after being sterilized. Rice callus with good appearance and good growth was selected as the recipient material, and ubi:Os01g64730-VP64 was transferred into the rice callus by the Agrobacterium-mediated method. The AAM transformation liquid of Agrobacterium was transformed, and the callus soaked in the transformation liquid was placed on the co-culture medium for co-cultivation, cultured in the dark at 25°C for 3 days, then placed on the screening medium for about 30 days, and subcultured every 10 days. Then, the screened out resistant calli were transferred to the differentiation medium for differentiation for about 20 days, and subcultured every 10 days. The resistant calli that differentiated into green seedlings were transferred to the rooting medium for rooting, and the seedlings were hardened after about 7 days after the well-developed root system grew, and the number of transgenic seedlings obtained by transformation was counted. After 7 days of hardening, the seedlings were transferred to the field for growth. The transgenic rice was screened with Basta, and Basta (1:1000, v:v) was sprayed after 4 young leaves were grown, sprayed once every other day, and sprayed 3 times in total. A total of 42 transgenic rice plants were obtained.

Among them, the induction medium formula is: N6 large amount + B5 trace amount + NB organic + iron salt + copper cobalt mother liquor + 2.5mg/L2,4-D (auuxin) + 0.6g/L acid hydrolyzed casein + 2.878g/L Proline + 0.5g/L glutamine + 30g/L sucrose, prepared with water, adjust the pH to 5.8-5.9, then add 4g/L plant gel.

The co-culture medium formula is: N6 large amount + B5 trace amount + NB organic + iron salt + 2.5mg/L2,4D + 0.5g/L glutamine + 0.6g/L acid hydrolyzed casein + 10g/L glucose + 30g/L Prepare sucrose with water, adjust the pH to 5.2, and then add plant gel 4g/L. After sterilization, add AS (acetosyringone) 100-200 μg/mL at about 50°C.

The screening medium formula is: N6 large amount + B5 trace amount + NB organic + iron salt + copper cobalt mother liquor + 2.5mg/L2,4D + 0.6g/L acid hydrolyzed casein + 2.878g/L proline + 0.5g/L Glutamine + 30g/L sucrose, prepared with water, adjust the pH to 5.8-5.9, then add 4g/L plant gel. After sterilization, 35 mg/L hygromycin (purchased from Shanghai Niujin Biotechnology Co., Ltd.) or 5 mg/L Bialaphos (purchased from Beijing Baierdi Biotechnology Co., Ltd.) were added.

The formula of the differentiation medium is: MS inorganic + MS-B5 trace + MS organic + iron salt + MS-copper cobalt mother solution + 0.05mg/L NAA + 2.0mg/L Kinetin (kinetin) + 30g/L sorbitol + 2g/ L hydrolyzed casein + 30g/L sucrose, prepared with water, adjust the pH to 5.8-5.9, then add plant gel 4g/L.

in:

N6 large amount of mother liquor (20×, g/L):

NB organic mother liquor (200×, g/L):

MS organic mother liquor (200×, g/L):

B5 trace mother liquor (200×, g/L):

MS-B5 trace mother solution (1000×, g/L):

H ₃ BO ₃ 6.2

MnSO ₄ 4H ₂ O 22.3

ZnSO ₄ 7H ₂ O 8.6

Copper-cobalt mother liquor (2000×, g/L):

MS inorganic mother liquor (20×, g/L):

MS copper-cobalt mother liquor (2000×, g/L):

Iron salt formula (100×, g/L):

Disodium ethylenediaminetetraacetic acid (Na ₂ EDTA) 3.73

Ferrous sulfate (FeSO ₄ 7H ₂ O) 2.78

Example 3

Detection of the expression of fusion protein Os01g64730-VP64 in transgenic rice by Western Blot

In order to detect the overexpression of ubi:Os01g64730-VP64 gene in T2 transgenic rice, the VP64 antibody was used to identify it at the protein level, after SDS-PAGE protein electrophoresis→immunoblotting→immunofluorescence reaction, the Western Blot identification results showed that The target protein was found in the transgenic plants, but no band was found in the wild type (Figure 3).

The specific Western Blot experiment process is as follows:

Put an appropriate amount of sample into liquid nitrogen and freeze it, grind it into powder, add an appropriate amount of loading buffer, mix well, and centrifuge at 12000rpm for 10 minutes; take 5μl of the supernatant and add the sample, electrophoresis at 90V for SDS-PAGE for 30 minutes, and electrophoresis at 120V for 60-90 When the bromophenol blue reaches the bottom of the gel, the electrophoresis can be stopped; after electrophoresis, the membrane is transferred by semi-dry transfer method, and the membrane is stained with ponceau staining solution to observe the transfer effect; after the transfer is completed, transfer the membrane Place in PBST solution containing 5% skimmed milk powder to block at room temperature for 60 minutes or overnight at 4°C; incubate with an antibody (VP64 antibody) at room temperature for 1 hour or overnight at 4°C, then wash 3 times with PBST, 5 minutes each ; Secondary antibody (goat anti-rabbit, purchased from Abmart, Cat. No. M21002S) was incubated at room temperature for 1 hour, and then washed 3 times with PBST, 5 minutes each time; substrate was added to the membrane for exposure.

Among them, the VP64 antibody is a rabbit-derived polyclonal antibody prepared by Abimat Biomedicine (Shanghai) Co., Ltd. based on the amino acid sequence of VP64 to synthesize a polypeptide as a specific antigen.

Example 4 Transgenic rice phenotype analysis

Compared with the wild-type rice 'kitaake', the Os01g64730-VP64 transgenic rice lines (V17-27 and V17-56) had significantly enhanced drought resistance. After 20% PEG treatment for 10 days and rewatering for 7 days, most of the wild-type plants withered, However, the transgenic lines still grew well (Fig. 4, Fig. 5).

The example provided above is to construct the CDS sequence of the rice transcription factor Os01g64730 gene to the upstream of the four transcription factor activation motifs VP16 through the Gateway system, and transform the rice variety 'kitaake', thereby improving the drought resistance of rice. Similarly, the CDS sequence of the rice transcription factor Os01g64730 gene is constructed to the upstream of ≥ 1 transcription factor activation motif VP16 through the Gateway system, or to the upstream of 1 to 3 or more than 4 transcription factor activation motifs VP16 through the Gateway system. Upstream, the transformation of rice varieties can also achieve the effect of improving the drought resistance of rice.

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, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection of the present invention

Claims (10)

1. a fusion rotein is characterized in that, described fusion rotein is Os01g64730-Linker-(VP16) ₄

Wherein, Linker is in series by 11 flexible amino acid; VP16 is the VP16 albumen from hsv; Os01g64730 is rice transcription factor Os01g64730.

2. fusion rotein as claimed in claim 1 is characterized in that, the aminoacid sequence of described rice transcription factor Os01g64730 is shown in SEQ ID No.1.

3. the gene of each described fusion rotein of coding claim 1-2.

4. the carrier that contains the described gene of claim 3.

5. the engineering bacteria that contains the described gene of claim 3.

6. the construction process of a transgenic rice plant, it is characterized in that, adopt agriculture bacillus mediated method, carrier claimed in claim 4 is changed in the Rice Callus, transform with the AAM conversion fluid that contains inductor and Agrobacterium, material after the conversion is through cultivating altogether-screen-break up-take root-exercise and the transplanting of transgenic seedling, the screening transgenic rice plant.

7. the application of gene claimed in claim 3 in improving the Rice Drought Resistence ability.

8. be used for the primer pair of amplifying rice transcription factor Os01g64730 gene, it is forward primer F:5'-CAAAAAAGCAGGCTTCATGATGGCGTCGAGGGTG-3' and reverse primer R:5'-CAAGAAAGCTGGGTCCCACTCCATCGAGTTTG TTCTTC-3'.

9. the application of rice transcription factor Os01g64730 gene in improving the Rice Drought Resistence ability.

10. application as claimed in claim 9, it is characterized in that, it is the upstream of the CDS sequence of rice transcription factor Os01g64730 gene being activated motif VP16 by Gateway system constructing to 4 transcription factor, rice transformation, thus improve the drought-resistant ability of paddy rice.

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