Application of over-expressed BnA05CARK3 gene in regulation and control of rape fertility

Abstract

The invention relates to the technical field of gene engineering, in particular to application of an over-expressed BnA05CARK3 gene in regulation and control of rape fertility. The cloned BnA05CARK3 gene is utilized to successfully construct a recombinant vector for over-expressing the BnA05CARK3 gene, and the BnA05CARK3 gene is subjected to genetic transformation and over-expression in the rape through an agrobacterium infection method, so that the development process of the rape in a drought stress environment is promoted, the pollen activity and thousand seed weight of the rape are improved, and the yield of the rape is increased. The quality and the yield can reach the normal development degree of wild type rape.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and specifically relates to the application of overexpressing BnA05CARK3 gene in regulating rapeseed fertility. Background Art

[0002] As an important oil crop in my country, rapeseed is the largest source of vegetable oil and the second largest source of feed protein in China. It plays an important role in food, feed, ornamental, and industrial fields. Rapeseed requires a large amount of water during its growth period, especially during the germination period. The soil must have sufficient moisture to allow the rapeseed seeds to absorb 60% of their own weight of water and germinate. However, global warming has increased the frequency and intensity of droughts in most regions, which undoubtedly brings certain climate risks to rapeseed production. According to statistics, about 35% of my country's arable land is located in arid or semi-arid areas. Under drought stress, the soil moisture is low. If rapeseed is planted, it will cause the rapeseed seeds to lack water during the germination period, uneven or no seedlings, and affect the pollination and fertilization of rapeseed flowers, resulting in a decrease in rapeseed oil content, yield and quality. Therefore, how to regulate the fertility of rapeseed under drought stress is of great significance for expanding rapeseed. Summary of the invention

[0003] Based on the above problems, one of the purposes of the present invention is to provide an application of overexpressing the BnA05CARK3 gene in regulating rapeseed fertility, and the second purpose is to provide a method for regulating rapeseed fertility.

[0004] To achieve the above object, the first technical solution adopted by the present invention is: Application of overexpression of BnA05CARK3 gene in regulating rapeseed fertility, the nucleotide sequence of the BnA05CARK3 gene is shown in SEQ ID NO.1.

[0005] When regulating the fertility of rapeseed, the cloned BnA05CARK3 gene is first used to construct a recombinant vector for overexpressing the BnA05CARK3 gene, and then the recombinant vector is transformed into rapeseed to overexpress the BnA05CARK3 gene.

[0006] The method for cloning the BnA05CARK3 gene is as follows: specific primers are designed according to the BnA05CARK3 gene sequence, and PCR amplification is performed using rapeseed cDNA as a template.

[0007] The vector used for overexpressing the BnA05CARK3 gene may be a vector known to those skilled in the art that can be used for plant genetic transformation.

[0008] Preferably, the vector used to overexpress the BnA05CARK3 gene is pCAMBIA1305.1.

[0009] The method for transforming the recombinant vector into rapeseed is: transforming the recombinant vector into Agrobacterium, and then infecting the rapeseed with the bacterial solution of the transformed Agrobacterium.

[0010] The strain used to transform the recombinant vector into Agrobacterium can be Agrobacterium tumefaciens or Agrobacterium rhizogenes, and their derivative strains.

[0011] Preferably, the strain used to transform the recombinant vector into Agrobacterium is Agrobacterium tumefaciens AGL1.

[0012] The second technical solution adopted by the present invention is: The method for regulating rapeseed fertility comprises: The cloned BnA05CARK3 gene was used to construct a recombinant vector for overexpressing the BnA05CARK3 gene, and then the recombinant vector was transformed into rapeseed.

[0013] Preferably, the vector used to overexpress the BnA05CARK3 gene is pCAMBIA1305.1.

[0014] Preferably, the method for transforming the recombinant vector into rapeseed is: transforming the recombinant vector into Agrobacterium, and then infecting the rapeseed with the bacterial solution of the transformed Agrobacterium.

[0015] Preferably, the strain used to transform the recombinant vector into Agrobacterium is Agrobacterium tumefaciens AGL1.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention utilizes the cloned BnA05CARK3 gene to successfully construct a recombinant vector for overexpressing the BnA05CARK3 gene, and genetically transforms and overexpresses the BnA05CARK3 gene in rapeseed through Agrobacterium infection, thereby promoting the development process of rapeseed seeds under a drought stress environment, improving the pollen vigor and 1000-grain weight of rapeseed, and facilitating research on high-yield rapeseed under a drought environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of pollen viability test of plants in the experimental group and the control group 1 are shown in FIG. Figure 2 These are pictures of siliques of plants in the experimental group and the control group 1. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical scheme and advantages of the present invention clearer, the scheme of the present invention is further described in detail below in conjunction with embodiment.It will be appreciated by those skilled in the art that the following examples are exemplary only, and are not intended to limit the scope of the present invention.In addition, in the following description, those not indicating specific conditions in the examples are carried out according to the conditions of normal conditions or manufacturer's recommendations.Reagents used or instruments not indicating manufacturers are conventional products that can be obtained by commercial purchase.

[0019] The rapeseed used in the present embodiment is all Brassica napus (Zhongshuang 11 variety). The components of the culture medium used in the embodiment are as follows: LB liquid medium: 5 g yeast powder, 10 g tryptone, 10 g NaCl, 950 mL deionized water; YEB liquid medium: 5 g beef extract, 1 g yeast powder, 5 g peptone, 5 g sucrose, 4 g magnesium sulfate heptahydrate, 1000 mL deionized water; YEB solid medium: beef extract 5 g, yeast powder 1 g, peptone 5 g, sucrose 5 g, magnesium sulfate heptahydrate 4 g, deionized water 1000 mL, agar powder 15 g; It should be noted that the above culture media need to be sterilized by high pressure at 120℃ for 20 min.

[0020] Example 1 Construction of a recombinant vector for overexpressing the BnA05CARK3 gene 1. Extraction of rapeseed cDNA Total RNA from mixed tissues and organs of Brassica napus was extracted and reverse transcribed to obtain cDNA. The cDNA sequence was submitted to the NCBI (National Center for Biotechnology Information) website for BLAST comparison. The comparison results showed that the sequence had a high homology with the CARK3 genes of other registered plants. After further multiple comparisons, it was speculated that there may be only two CARK3 genes in Brassica napus, and the corresponding independent genes were named BnA05CARK3 and BnC04CARK3. The BnA05CARK3 gene was selected for subsequent processing.

[0021] 2. Construction of a recombinant vector for overexpression of the BnA05CARK3 gene PCR amplification was performed using the above cDNA as a template.

[0022] The amplification reaction system is: 1 μL of cDNA template, 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of ultra-fidelity DNA polymerase, 10 μL of buffer, 4 μL of dNTP Mixture, and deionized water is added to make up to 50 μL.

[0023] The upstream primer and the downstream primer were designed according to the BnA05CARK3 gene sequence, and the primer sequences were: Upstream primer: 5′-AGATCTCTCTCTCCCATACGATT-3′; Downstream primer: 5′-GTTGATCATAGATTACCCAGAGGCC-3′.

[0024] The reaction conditions of the amplification were as follows: pre-denaturation at 98°C for 5 min, denaturation at 98°C for 15 s, annealing at 55°C for 30 s, extension at 72°C for 2 min, 30 cycles of reaction, and extension at 72°C for 10 min.

[0025] The PCR product was recovered and connected to the Gateway cloning system entry vector pDONR 221 to obtain the pDONR 221-BnA05CARK3 plasmid. The pDONR 221-BnA05CARK3 plasmid was transformed into Escherichia coli DH5α, and the PCR-positive monoclonal bacterial solution was selected for sequencing and analysis. The nucleotide sequence of the BnA05CARK3 gene is shown in SEQ ID NO.1.

[0026] The above-mentioned correctly sequenced pDONR 221-BnA05CARK3 plasmid was subjected to LR exchange reaction with the overexpression vector pCAMBIA1305.1. The specific reaction method was as follows: 100 ng of pDONR 221-BnA05CARK3 plasmid, 150 ng of vector pCAMBIA1305.1, and 2 μL of 5×LR Clonase Reaction Buffer were mixed, double deionized water was added to a total volume of 8 μL, and then 2 μL of LR Clonaseenzyme mix was added, vortexed twice to mix, centrifuged slightly, and placed in a 25°C water bath for 8 h, and then 1 μL of proteinase K solution was added to terminate the reaction, vortexed briefly, and then incubated at 37°C for 10 minutes to obtain the recombinant vector pCAMBIA1305.1-BnA05CARK3. The recombinant vector was transformed into Escherichia coli DH5α, and positive clones were picked for bacterial PCR test and enzyme digestion verification.

[0027] Example 2 Transformation of the recombinant vector overexpressing the BnA05CARK3 gene into rapeseed The recombinant vector pCAMBIA1305.1-BnA05CARK3 was transformed into Agrobacterium, and then the bacterial solution of the transformed Agrobacterium was used to infect Brassica napus. The specific method is as follows: Take out the Agrobacterium tumefaciens AGL1 competent cells from the -70℃ freezer, thaw on ice until the cell suspension is in an ice-water mixing state, take 100 μL of the cell suspension, add 2 μL of the recombinant vector pCAMBIA1305.1-BnA05CARK3, mix well and let stand on ice for 5 min, then put it in liquid nitrogen for quick freezing for 5 min, quickly transfer it to a 37℃ water bath after quick freezing and let stand for 5 min, then put it back in the ice water bath and put it in the ice bath for 5 min to restore cell activity; 800 μL of LB liquid medium without antibiotics was added to the cell suspension with cell activity restored above, and the culture was shaken at 28°C and 250 r / min for 3 hours to revive the bacteria, and then centrifuged at 5500 rpm for 1 min, and the supernatant was spread on YEB solid medium (containing 50 mg / L rifampicin and 100 mg / L spectinomycin hydrochloride), and inverted culture was carried out at 28°C for 72 hours to obtain a single colony of Agrobacterium; Pick the above Agrobacterium single colony, place it in 5 mL YEB liquid culture medium (containing 50 mg / L rifampicin and 100 mg / L spectinomycin hydrochloride), and culture it in a shaking incubator at 28°C and 200 rpm for 48 hours. After the culture is completed, add 50 mL YEB liquid culture medium (containing 50 mg / L rifampicin and 100 mg / L spectinomycin hydrochloride) and culture it in a shaking incubator at 28°C and 200 rpm for 48 hours to obtain a bacterial liquid of transformed Agrobacterium; Take 1 cm hypocotyl of Brassica napus seedlings, immerse it in the above-mentioned transformed Agrobacterium bacterial solution for 15 minutes, then take it out, put it on its side in a tray and place the tray in a growth room, culture it in the dark at 25℃ for 24 hours, then take it out and culture it under natural light. Water it 1-2 times a week. After the seeds mature, harvest the T0 generation seeds.

[0028] Example 3 Regulatory effect of overexpression of BnA05CARK3 gene on rapeseed fertility under drought stress The above T0 generation seeds were sterilized and placed in a 90 mm×15 mm culture dish, with a sterilized filter paper layer and a layer of absorbent paper as the bud bed, and 6 mL of 6% PEG solution was added to simulate drought conditions. The culture dish was then placed in a 25°C constant temperature and light incubator and cultured under 16 h / 8 h light / dark conditions (experimental group). Control group 1 was replaced with wild cabbage-type rapeseed seeds instead of T0 generation seeds, and control group 2 was replaced with wild cabbage-type seeds instead of T0 generation seeds, and no PEG solution was added to the culture dish.

[0029] The development process of rapeseed in the experimental group and the control group was observed. The overall development process of rapeseed in the experimental group and the control group 2 was similar, and the harvested siliques were not much different, while the development process of rapeseed in the control group 1 was significantly slowed down. Therefore, the rapeseed in the experimental group and the control group 1 were mainly compared in the follow-up.

[0030] The activity of rapeseed pollen in the experimental group and the control group 1 was tested by using the fuchsin staining method. The test results are as follows: Figure 1 As shown. Figure 1 It can be seen that the activity of rapeseed pollen in the experimental group is normal, while the activity of pollen in the control group 1 is significantly reduced. Figure 2 These are pictures of rapeseed siliques in the experimental group and control group 1. From the picture, it can be seen that the length of the siliques in the control group 1 is shorter than that in the experimental group, indicating that the valence of the rapeseed siliques in the control group 1 is abnormally developed.

[0031] The thousand-grain weight of rapeseed in the experimental group and control group 1 was measured, and the results were 1.74 g and 0.68 g, respectively. The thousand-grain weight of rapeseed in the control group 1 was less than half of that in the experimental group.

[0032] In summary, overexpressing the BnA05CARK3 gene in Brassica napus can promote the seed development process of Brassica napus under drought stress, improve the pollen vigor and 1000-grain weight of rapeseed, and achieve the normal development level of wild Brassica napus seeds in terms of quality and yield.

[0033] Since Brassica rapa, Brassica rapa and Brassica juncea all belong to the family Cruciferae, it can be inferred that the BnA05CARK3 gene of the present invention can also be well applied in Brassica rapa and Brassica juncea.

[0034] Those skilled in the art may make various changes, modifications, substitutions and variations in form and details to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. Application of overexpression of BnA05CARK3 gene in regulating rapeseed fertility, characterized in that: The nucleotide sequence of the BnA05CARK3 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that When regulating the fertility of rapeseed, the cloned BnA05CARK3 gene is first used to construct a recombinant vector for overexpressing the BnA05CARK3 gene, and then the recombinant vector is transformed into rapeseed to overexpress the BnA05CARK3 gene.

3. The use according to claim 2, characterized in that The vector used to overexpress BnA05CARK3 gene was pCAMBIA1305.

1.

4. The use according to claim 2, characterized in that The method for transforming the recombinant vector into the recipient plant rapeseed is: transforming the recombinant vector into Agrobacterium, and then infecting the rapeseed with the bacterial solution of the transformed Agrobacterium.

5. The use according to claim 4, characterized in that The strain used to transform the recombinant vector into Agrobacterium is Agrobacterium tumefaciens AGL1.

6. A method for regulating rapeseed fertility, characterized in that: include: The cloned BnA05CARK3 gene was used to construct a recombinant vector for overexpressing the BnA05CARK3 gene, and then the recombinant vector was transformed into rapeseed.

7. The method according to claim 6, characterized in that The vector used to overexpress BnA05CARK3 gene was pCAMBIA1305.

1.

8. The method according to claim 6, characterized in that The method for transforming the recombinant vector into rapeseed is: transforming the recombinant vector into Agrobacterium, and then infecting the rapeseed with the bacterial solution of the transformed Agrobacterium.

9. The method according to claim 6, characterized in that The strain used to transform the recombinant vector into Agrobacterium is Agrobacterium tumefaciens AGL1.

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