CN106146633A - The application in plant fertility of EPFL1 albumen and encoding gene thereof - Google Patents

Abstract

The invention discloses the application in plant fertility of EPFL1 albumen and encoding gene thereof.The present invention utilizes EPFL1 gene expression in antisensenucleic acids silence arabidopsis body, the transgenic arabidopsis stamen obtained diminishes, angle fruit shortens, ovule number reduces, thus proving that EPFL1 gene can regulate and control the fertility of plant, the aspect such as acceptor material obtain male sterility line in breeding in agricultural production, seeding procedure, creating human controllable's male sterility and GMO bio-safety all has important application prospect.

Description

Application of EPFL1 protein and its coding gene in plant fertility

technical field

The invention belongs to the field of biotechnology, and specifically relates to the application of EPFL1 protein and its coding gene in plant fertility.

Background technique

The pollen formation of the male gametophyte in plants includes two processes: microspore generation and male gamete formation. The process of microspore generation is carried out in young anthers, including the formation of microspore mother cells, the process of meiosis and the dispersal of tetrads. Male gametophytes, or pollen grains, are formed by microspores undergoing two mitotic divisions and contain sperm cells called male gametes. Many related genes are involved in this series of processes of pollen development, and only when they are normally expressed in a certain temporal and spatial order can the formation of fertile pollen be guaranteed.

The research and utilization of male sterility in my country is in a leading position in the world. In recent years, modern cell biology techniques, genetics and molecular biology methods have been used to conduct in-depth research on the male sterility process of model plants rice and Arabidopsis, and some new research results have been obtained, such as: Ems1, CER1, AtGSL2 genes and rice UDT1, MSP1, GAMYB, UDPG, WDA1 genes, etc. Mutations in the above genes directly lead to reduced fertility of plants, and some are completely sterile. Ems1 encodes a receptor kinase gene, which is mainly involved in the development of pollen mother cells and tapetum; UDT1 gene is a key gene for the development of microspores, and this gene mainly acts on meiosis, and it is important for the development of tapetum cells , the meiosis of microspore mother cells and the degradation of the middle layer; the GAMYB gene is necessary for the microspore mother cells to adhere to the tapetum cells to absorb nutrients and then carry out normal meiosis; the WDA1 gene is essential for rice pollen wall wax Layer formation is required. The study of them has deepened the understanding of the mechanism of male sterility in plants.

Contents of the invention

One object of the present invention is to provide the use of substances that inhibit the expression of the gene encoding the EPFL1 protein.

The invention provides the application of the material for inhibiting the expression of the gene encoding the EPFL1 protein in reducing plant fertility.

In the above application, the amino acid sequence of the EPFL1 protein is sequence 5 in the sequence listing.

In the above application, the substance that inhibits the expression of the gene encoding the EPFL1 protein is as follows 1)-3):

1) a DNA fragment as shown in Sequence 2 in the sequence listing;

2) miRNA encoded by the DNA fragment described in 1);

3) A recombinant vector containing the DNA fragment described in 1).

In the above application, the recombinant vector is a vector obtained by co-inserting the DNA fragment and the promoter driving its expression into an expression vector.

In the above application, the expression vector is pCAMBIA2300; the nucleotide sequence of the promoter is sequence 3 in the sequence listing.

In the above application, the recombinant vector is specifically replacing the DNA fragment between the Pst I and Hind III restriction sites of the pCAMBIA2300 vector with the DNA fragment, and replacing the Kpn I and Sal I of the pCAMBIA2300 with the promoter shown in sequence 3 The vector obtained by cutting the DNA fragment between the restriction sites.

In the above application, the reduction of plant fertility is reflected in the reduction of stamens and/or the shortening of siliques and/or the reduction of the number of ovules.

In the above application, the plant is a monocotyledon or a dicotyledon; the dicot is specifically Arabidopsis thaliana.

Another object of the present invention is to provide a method for growing plants with reduced fertility or for growing sterile plants.

The method for cultivating plants with reduced fertility or cultivating sterile plants provided by the present invention includes the following steps: introducing a substance that inhibits the expression of the gene encoding the EPFL1 protein into the target plant to obtain a transgenic plant, the fertility of the transgenic plant is lower than the target plant.

In the above method, the substance that inhibits the expression of the gene encoding the EPFL1 protein is an EPFL1 RNAi fragment;

The nucleotide sequence of the EPFL1 RNAi fragment is sequence 2 in the sequence listing;

The EPFL1 RNAi fragment is introduced into the target plant through a recombinant vector;

The recombinant vector is a vector obtained by inserting the EPFL1 RNAi fragment shown in Sequence 2 in the sequence listing into an expression vector.

In the above method, the target plant is a monocotyledon or a dicot; the dicot is specifically Arabidopsis.

A final object of the present invention is to provide a DNA molecule.

The DNA molecules provided by the present invention are as follows 1) or 2):

1) The sequence is the DNA molecule of sequence 2 in the sequence listing;

2) A DNA molecule that hybridizes to 1) under stringent conditions and encodes the same protein.

The present invention uses antisense nucleic acid to silence the expression of EPFL1 gene in Arabidopsis thaliana, and the obtained transgenic Arabidopsis thaliana stamens become smaller, siliques become shorter, and the number of ovules is reduced, thereby proving that EPFL1 gene can regulate the fertility of plants, and it is useful in agricultural production. It has important application prospects in breeding, obtaining male sterile lines in the process of seed production, creating artificially controllable male sterility, and safe recipient materials for transgenic organisms.

Description of drawings

Figure 1 is a schematic diagram of primer matching for RNAi fragment construction.

Figure 2 is a map of pEPFL1::EPFL1 RNAi recombinant plasmid.

Figure 3 is the identification of expression levels of transgenic lines 2#, 3#, 4#, 8#, 9#.

Fig. 4 shows the stamen fertility of transgenic lines 2#, 3#, 4#, 8#, 9# observed by Alexander staining method.

Fig. 5 is the observation result of siliques of transgenic plants 3# and 4#.

Figure 6 shows the number of ovules of transgenic plants 3# and 4#.

detailed description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The pRS300 plasmid (5ng/ul) was published in the literature "Highly specific gene silencing by artificial microRNAs in Arabidopsis.Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D.Plant Cell.2006 May.18(5):1121-33." Publicly available from the School of Life Sciences, Peking University.

The pQGIE110 plasmid was published in the literature "Expression and functional analysis of the riceplasma-membrane intrinsic protein gene family. Lei Guo, Zi Yi Wang, Hong Lin, Wei ErCui, Jun Chen, Meihua Liu, Zhang Liang Chen, Li Jia Qu and Hongya Gu. Cell Research.2006.March.16(16)277–286.”, publicly available from School of Life Sciences, Peking University.

The pCAMBIA2300 plasmid was obtained from Beijing Dingguo Changsheng Biotechnology Co., Ltd., and the product catalog number is MCV036.

Example 1, Acquisition of pEPFL1::EPFL1 RNAi Transgenic Arabidopsis

1. Acquisition of pEPFL1::EPFL1 RNAi recombinant plasmid

1. EPFL1 RNAi fragment design

Use the online RNAi fragment design website WMD3 (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi) to design the following primers for the construction of the EPFL1 RNAi fragment of EPFL1 (At5g10310). The primer sequences are as follows:

A: 5'-CTGCAAGGCGATTAAGTTGGGTAAC-3';

B: 5'-CTGTTTCCTGTGTGAAATTGTTATCCGC-3';

I(miR-s): 5'-GACGACCACTTTTATTATTCCTTTTCTCTCTTTTGTATTCC-3';

II(miR-a): 5′-GAAAAGGATAATAAAAGTGGTCGTCAAAAGAGAATCAATGA-3′;

III(miR*s): 5′-GAAACGGATAATAAATGTGGTCGTCACAGGTCGTGATATG-3′;

IV (miR*a): 5'-GACGACCACATTTATTTATCCGTTTCTACATATATTCCT-3'.

2. Obtaining EPFL1 RNAi fragments

(1) Using the pRS300 plasmid (5ng/ul) as a template, PCR was carried out according to the primer matching method shown in Figure 1, and three intermediate PCR products (a), (b) and (c) were obtained.

PCR reaction system (50ul): 2xKOD Mix 25ul, pRS300 (1:100) 2ul, upstream primer 2ul, downstream primer 2ul, KODase 1ul, ddH ₂ O to make up 50ul.

PCR reaction conditions: 95°C 5'; 94°C 30", 58°C 30", 68°C 40", 24cycles; 68°C 10'.

(2) Add 5ul (10X) loading buffer to the three intermediate PCR products (a), (b) and (c) obtained in the above step (1), separate, purify and recover by 2% agarose gel electrophoresis , adjust the concentration to 5ng/ul; use (a), (b) and (c) three intermediate PCR products as templates to carry out PCR reaction to obtain PCR products.

PCR reaction system (50ul): 2xKOD Mix 25ul, OligoA-SmaI 2ul, oligoB-SacI 2ul, KODase 1ul, (a) 0.5ul, (b) 0.5ul, (c) 0.5ul, ddH ₂ O to make up 50ul.

PCR reaction conditions: 95°C 5'; 94°C 30", 58°C 30", 68°C 40", 24cycles; 68°C 10'.

(3) Add 5ul (10X) loading buffer to the PCR product obtained in step (2), and use 2% agarose gel electrophoresis to separate, purify, and recover to obtain an EPFL1 RNAi template (d) with a size of 701bp, which is EPFL1 The artificial interference miRNA gene fragment (shown as sequence 1 in the sequence listing).

(4) The EPFL1 RNAi template obtained in the above step (3) was double-digested with restriction enzymes SmaI and Sac I to obtain a DNA fragment of 701bp in size; Perform double enzyme digestion to obtain a large fragment of the backbone vector.

(5) Ligate the above-obtained DNA fragment and the large fragment of the backbone vector with T4 ligase to obtain a recombinant vector. The ligation reaction system is as follows: 10x T4ase Buffer 1ul, T4ase 1ul, DNA fragment 2ul, backbone carrier 1ul, ddH ₂ O 5ul. The ligation reaction was performed at a constant temperature of 16°C for 16 hours.

(7) Transform the recombinant vector obtained in the above step (6) into the DH5α strain by using the E. coli heat shock transformation method, and screen to obtain positive clones.

(8) Use Oligo A and EPFL1-RNAi-a-Hind III primers, and use the positive clone obtained in the above step (7) as a template for PCR amplification to obtain a DNA fragment as shown in sequence 2 in the sequence table, which is EPFL1 RNAi fragment.

Oligo A: 5'-CCCTGCAAGGCGATTAAGTTGGGTAAC-3';

EPFL1-RNAi-a-Hind III: 5'-TCCCCAAGCTTGGCCGCTCTAGAACTAGT-3'.

3. Acquisition of pEPFL1::EPFL1 RNAi recombinant plasmid

(1) Double digest the EPFL1 RNAi fragment and the pCAMBIA2300 plasmid obtained above with restriction enzymes Pst I and Hind III, and connect with T4 ligase (the reaction system is the same as the above step (5)) to obtain an EPFL1 RNAi fragment recombinant plasmids.

(2) Using pEPFL1-Kpn-lp and pEPFL1-Sal-rp primers, clone the DNA fragment of the 2kb region upstream of the EPFL1 coding region (as shown in sequence 3 in the sequence listing) from the Arabidopsis genome, and carry out as the driving EPFL1 RNAi fragment Promoter for in situ gene silencing. The primer sequences are as follows (underlined sequences represent restriction sites):

pEPFL1-Kpn-lp: 5'- CCGG GGTACC TGTGAAAGTCTTCTCTTTCTG-3';

pEPFL1-Sal-rp: 5'- TCGCGTCGACTAAAGGAGGAGCTTCATGTGG -3'.

(3) Use restriction endonucleases Kpn I and Sal I to double-enzyme digest the promoter obtained in the above step (2) and the recombinant plasmid containing the EPFL1 RNAi fragment obtained in step (1), and connect with T4 ligase to obtain The pEPFL1::EPFL1 RNAi recombinant plasmid shown in Figure 2 was sequenced and verified.

The sequencing results show that the pEPFL1::EPFL1 RNAi recombinant plasmid is a DNA fragment between the Pst I and Hind III restriction sites of the pCAMBIA2300 vector replaced by the EPFL1 RNAi fragment shown in sequence 2, and the promoter shown in sequence 3 is replaced by pCAMBIA2300 The vector obtained from the DNA fragment between the Kpn I and Sal I restriction sites.

2. Acquisition of transgenic plants

Transform the pEPFL1::EPFL1 RNAi recombinant plasmid obtained in step 1 into the Agrobacterium GV3101 strain by electric shock to obtain the Agrobacterium containing the pEPFL1::EPFL1 RNAi recombinant plasmid, and then use the tidbit soaking method to extract the pEPFL1::EPFL1 RNAi recombinant plasmid Agrobacterium transfected wild-type Arabidopsis (Columbia). The seeds of the transgenic T ₀ generation were screened using solid MS medium containing 100ug/mL kanamycin sulfate, and 18 transgenic T _{1 generation plants were obtained, and the EPFL1 expression levels of the 18 transgenic T 1 generation} plants were analyzed by RT-PCR detection.

The detection results are shown in Figure 3: Compared with the control, the expression of EPFL1 in the transgenic lines 2#, 3#, 4#, 8#, and 9# decreased significantly, and the transgenic lines 2#, 3#, 4#, 8#, 9# lines were used for further phenotypic analysis.

1. The method of electroporation transformation of Agrobacterium is as follows:

(1) Agrobacterium electroporation transformation competent preparation

A. Resuscitate the Agrobacterium strain, pick the Agrobacterium strain GV3101 from -80°C in 5ml LB medium containing gentamicin sulfate and rifampicin resistance, activate overnight at 28°C and 220rpm;

B. Expand the culture, insert the activated GV3101 into 800ml LB medium containing gentamicin sulfate and rifampicin resistance at a ratio of 1:1000, and cultivate at 28°C and 220rpm until OD550≈1.0;

C. Aseptic operation, divide the bacterial liquid into two bottles, each bottle is 400ml, centrifuge at 4°C, 4000×g for 10min, discard the supernatant, and resuspend the bacterial cells with 200ml pre-cooled 10% glycerol in each bottle;

D. Aseptic operation, centrifuge at 4000×g for 10 minutes at 4°C, discard the supernatant, and resuspend the bacteria with 15ml pre-cooled 10% glycerol in each bottle;

E. Aseptic operation, centrifuge at 4°C, 4000×g for 10min, discard the supernatant; resuspend the bacteria with 0.5ml pre-cooled 10% glycerol, the final volume is about 0.75ml, use immediately or aliquot into 50ul per tube after -80 ℃ frozen.

(2) Electric shock transformation

A. Extract the plasmid to be transformed to a concentration of at least 20ng/μl;

B. Melt 50 μl of competent Agrobacterium frozen at -80°C on ice, and pre-cool the Bio-Rad 0.2cm electric shock cup at the same time;

C. Operate on ice, take 100ng of plasmid DNA in the competent Agrobacterium, flick the tube wall to mix;

D. Set the Bio-Rad GenePulser XcellTM PC Module electric shock meter V=2.4kv, C=25μF, PC=200ohm, electric shock cup parameter 0.2cm;

E. Add the plasmid competent mixture in step 3 to the bottom of the electric shock cup;

F. Put the electric shock cup into the electric shock slot, and electric shock;

G. After the electric shock is completed, quickly add 1ml of normal temperature LB medium and transfer it to a 1.5ml EP tube;

H, 28°C, 220rpm recovery for 3h, take 200ul and spread on the LB solid medium containing gentamicin sulfate, rifampicin, kanamycin sulfate resistance, 28°C inverted plate to screen positive colonies.

2. The transfection method is as follows:

(1) Pick the Agrobacterium strain containing the target gene stored at -80°C, transfer it into 5ml of LB medium containing gentamicin sulfate, rifampicin, and kanamycin sulfate, and activate overnight at 28°C and 220rpm;

(2) Transfer the activated bacterial solution to 500ml of LB medium with corresponding resistance at a ratio of 1:100, and culture at 28°C and 220rpm until OD600=1.2-1.6;

(3) Centrifuge at room temperature at 4000rpm for 10min, discard the supernatant;

(4) Use an equal volume (500ml) of transformation buffer (conversion buffer formula (1L): MS powder (SigmaM5519) 2.2g, sucrose 50g, use KOH to adjust pH=5.7, 1mg/mL 6-benzylaminopurine (6 -BA) 10 μL, Silwet L-77 200 μL) resuspended bacteria;

(5) Take the wild-type (Columbia) Arabidopsis thaliana whose moss reaches 10-15cm, water it to saturation, and cut off the pollinated flowers and siliques at the same time, and only keep the unflowered tidbits;

(6) Put the aerial part of the plant upside down in the transformation buffer. In the resuspended Agrobacterium, ensure that all parts above the basal leaves of the plant are immersed in the bacterial solution, and infect for 8 minutes (the same transformation solution can continuously infect 3 pots of plants);

(7) Take out the plant, place the side of the flower pot in the dark and cultivate it for 24 hours;

(8) Cultivate according to normal cultivation conditions (note that the aboveground part of the plant cannot be watered);

Collect T ₀ generation seeds after the siliques are mature;

(9) After sterilizing the seeds of the T ₀ generation, they were planted on the MS plate containing resistance, and the positive plants of the T ₁ generation were screened.

Embodiment 2, phenotypic identification of transgenic strains

1. Stamens

The stamens of T1 transgenic lines 2#, 3#, 4#, 8#, and 9# were stained and analyzed by Alexander staining method, and wild-type Arabidopsis plants (Col) were used as controls. The Alexander staining method is as follows:

Pipette 200uL of Alexander's staining solution into a 1.5mL EP tube, take florets at the appropriate stage and completely immerse in the staining, and stain at room temperature for more than 8 hours. Take out the florets in the dye solution, dissect the stamens under a stereoscope, place them on a glass slide, drip an appropriate amount of transparent solution, seal the slide with a cover glass, and observe and take pictures under a microscope after being transparent for 8-24 hours.

Alexander staining solution is composed of 95% ethanol 10mL, 1% malachite green (95% ethanol preparation) 1mL, distilled water 50mL, glycerin 25mL, phenol 5g, chloral hydrate 5g, 1% acid fuchsin 5mL, 1% orange G ( OrangeG) 0.5mL and 1mL of glacial acetic acid (4mL for pollen that has not been loosened from the anther) is obtained by mixing.

The transparent liquid is obtained by mixing chloral, glycerin, and water in a volume ratio of 8:1:2.

The results are shown in Figure 4: on the whole, compared with the wild-type Arabidopsis plant (Col), the stamens of the transgenic lines 2#, 3#, 4#, 8#, and 9# became smaller, and some drug cells Abnormal development.

Two, siliques

Transgenic lines 3# and 4# were selected for further analysis on siliques. Wild-type Arabidopsis plants (Col) were used as controls.

The results are shown in Figure 5: the fertility of the transgenic plants decreased significantly, and aborted siliques appeared in all of the transgenic plants, and 3# was more obvious, but the whole plant was not completely aborted, and some shorter siliques could also be produced.

Further observation of these siliques showed that the siliques of the transgenic plants were significantly shorter than those of the wild type. Among them, the siliques of the transgenic line 3# were the shortest, and the siliques of 4# were slightly longer, but still shorter than the wild type. Fertility decreased significantly.

3. Ovule

The siliques of the transgenic lines 3# and 4# were dissected, and the ovules in the siliques were observed. Wild-type Arabidopsis plants (Col) were used as controls.

The results are shown in Figure 6: only part of the ovules in the transgenic plants can develop normally, and a large number of ovules are aborted. The number of ovules in these plants was counted, and it was found that the number of ovules in the transgenic lines 3# and 4# decreased significantly, and the number of ovules in the transgenic lines The number of ovules contained in each silique of line 3# was down-regulated to less than 50% of the wild type.

Claims (10)

1. The application of a substance that inhibits the expression of the gene encoding the EPFL1 protein in reducing plant fertility; the amino acid sequence of the EPFL1 protein is sequence 5 in the sequence listing. 2. application according to claim 1, is characterized in that: the substance of the coding gene expression of described suppression EPFL1 albumen is as follows 1)-3): 1) A DNA fragment as shown in Sequence 2 in the sequence listing; 2) miRNA encoded by the DNA fragment described in 1); 3) A recombinant vector containing the DNA fragment described in 1). 3. The application according to claim 1 or 2, characterized in that: the recombinant vector is a vector obtained by co-inserting the DNA fragment and a promoter driving its expression into an expression vector. 4. The application according to any one of claims 1-3, characterized in that: the expression vector is pCAMBIA2300; the nucleotide sequence of the promoter is sequence 3 in the sequence listing. 5. The application according to any one of claims 1-4, characterized in that: said reduction of plant fertility is manifested in making plant stamens smaller and/or siliques shorter and/or number of ovules reduced. 6. The application according to any one of claims 1-5, characterized in that: the plant is a monocot or a dicot. 7. A method for cultivating reduced fertility or cultivating sterile plants, comprising the steps of: introducing a material that inhibits the expression of the coding gene of the EPFL1 protein into the target plant to obtain a transgenic plant, the fertility of the transgenic plant is lower than the purpose plants. 8. The method according to claim 7, characterized in that: the substance that inhibits the expression of the gene encoding the EPFL1 protein is an EPFL1 RNAi fragment; The nucleotide sequence of the EPFL1 RNAi fragment is sequence 2 in the sequence listing; The EPFL1 RNAi fragment is introduced into the target plant through a recombinant vector; The recombinant vector is a vector obtained by inserting the EPFL1 RNAi fragment shown in Sequence 2 in the sequence listing into an expression vector. 9. The method according to claim 7 or 8, characterized in that: the target plant is a monocot or a dicot. 10. A DNA molecule that is 1) or 2) as follows: 1) The sequence is the DNA molecule of sequence 2 in the sequence listing; 2) A DNA molecule that hybridizes to 1) under stringent conditions and encodes the same protein.