Application of overexpression of BnA05CARK3 gene in regulating fertility of Brassica napus

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to application of overexpression of BnA05CARK3 gene in regulation of fertility of Brassica napus. The present application successfully constructs a recombinant vector for overexpression of BnA05CARK3 gene by using the cloned BnA05CARK3 gene, and makes the BnA05CARK3 gene in Brassica napus to be genetically transformed and overexpressed by means of agrobacterium infection, and then promotes the development process of Brassica napus under drought stress environment, so that the pollen vitality and the thousand-grain weight of Brassica napus can be improved, and the quality and yield can reach the normal development degree of wild type Brassica napus.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to application of overexpression of BnA05CARK3 gene in regulation of fertility of Brassica napus. BACKGROUND

[0002] As an important oil crop in China, Brassica napus is the first largest plant oil source and the second largest feed protein source in China, and plays an important role in the fields of food, feed, ornamental, industry and the like. Brassica napus needs a large amount of water during the growth period, especially during the germination period, and sufficient soil humidity is required to enable the seed of Brassica napus to absorb water accounting for 60% of its own weight and germinate. However, global warming makes the frequency and intensity of drought in most regions increase, which undoubtedly brings certain climate risks to the production of Brassica napus. Under drought stress, the soil humidity is low, and if Brassica napus is planted, the seed of Brassica napus will be short of water during the germination period, and the seedling will not be uniform or cannot germinate, and the process of flower pollination and fertilization of Brassica napus will be affected, thereby causing the oil content of Brassica napus to decrease, and the yield and quality to decrease. Therefore, how to regulate the fertility of Brassica napus under drought stress is of great significance to the expansion of Brassica napus. SUMMARY

[0003] Based on the above problems, one of the purposes of the application is to provide application of overexpression of BnA05CARK3 gene in regulation of fertility of Brassica napus, and the other purpose is to provide a method for regulating the fertility of Brassica napus.

[0004] To achieve the above purposes, a first technical scheme adopted by the application is as follows:

[0005] The application of overexpression of BnA05CARK3 gene in regulation of fertility of Brassica napus, and the nucleotide sequence of the BnA05CARK3 gene is shown in SEQ ID NO. 1.

[0006] In the regulation of the fertility of Brassica napus, the cloned BnA05CARK3 gene is used to construct a recombinant vector for overexpression of the BnA05CARK3 gene, and then the recombinant vector is transformed into Brassica napus to overexpress the BnA05CARK3 gene.

[0007] The method for cloning the BnA05CARK3 gene is as follows: specific primers are designed according to the sequence of the BnA05CARK3 gene, and the cDNA of Brassica napus is used as a template for PCR amplification.

[0008] The vector for overexpression of the BnA05CARK3 gene can be a vector known to those skilled in the art and used for genetic transformation of plants.

[0009] Preferably, the vector for overexpression of the BnA05CARK3 gene is pCAMBIA1305.1.

[0010] The method for transforming the recombinant vector into the oilseed rape is: transforming the recombinant vector into Agrobacterium, and then infecting the oilseed rape with the bacterial liquid of the transformed Agrobacterium.

[0011] The strain for transforming the recombinant vector into Agrobacterium can be a strain of Agrobacterium tumefaciens or a strain of Agrobacterium rhizogenes, and a derivative strain thereof.

[0012] Preferably, the strain for transforming the recombinant vector into Agrobacterium is a strain of Agrobacterium tumefaciens AGL1.

[0013] The second technical solution adopted by the present application is:

[0014] The method for regulating the fertility of the oilseed rape comprises:

[0015] The recombinant vector for over-expressing the BnA05CARK3 gene is constructed by using the cloned BnA05CARK3 gene, and then the recombinant vector is transformed into the oilseed rape.

[0016] Preferably, the vector for over-expressing the BnA05CARK3 gene is pCAMBIA1305.1.

[0017] Preferably, the method for transforming the recombinant vector into the oilseed rape is: transforming the recombinant vector into Agrobacterium, and then infecting the oilseed rape with the bacterial liquid of the transformed Agrobacterium.

[0018] Preferably, the strain for transforming the recombinant vector into Agrobacterium is a strain of Agrobacterium tumefaciens AGL1.

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] The present application successfully constructs the recombinant vector for over-expressing the BnA05CARK3 gene by using the cloned BnA05CARK3 gene, and makes the BnA05CARK3 gene in the oilseed rape to be genetically transformed and over-expressed by the Agrobacterium infection method, thereby promoting the seed development process of the oilseed rape under the drought stress environment, and improving the pollen viability and the thousand-grain weight of the oilseed rape, so as to help the high-yield research of the oilseed rape under the drought environment. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The figure is the pollen viability detection result of the plants in the experimental group and the control group 1;

[0022] Figure 2 The figure is the silique of the plants in the experimental group and the control group 1. DETAILED DESCRIPTION

[0023] To make the objectives, technical solutions and advantages of the present application clearer, the solutions of the present application are further described in detail below with examples. Those skilled in the art will understand that the following examples are only exemplary and are not intended to limit the scope of the present application. In addition, in the following description, the specific conditions not indicated in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not indicated by the manufacturer are all conventional products that can be obtained by commercial purchase.

[0024] The oilseed rape used in the examples of the present application is all Brassica napus (Zhongshuang 11 variety). The components of the culture medium used in the examples are as follows:

[0025] LB liquid culture medium: yeast powder 5 g, tryptone 10 g, NaCl 10 g, deionized water 950 mL;

[0026] YEB liquid culture medium: beef extract 5 g, yeast powder 1 g, tryptone 5 g, sucrose 5 g, magnesium sulfate heptahydrate 4 g, deionized water 1000 mL;

[0027] YEB solid culture medium: beef extract 5 g, yeast powder 1 g, tryptone 5 g, sucrose 5 g, magnesium sulfate heptahydrate 4 g, deionized water 1000 mL, agar powder 15 g;

[0028] It should be noted that the above culture media all need to be autoclaved at 120°C for 20 min.

[0029] Example 1 Construction of recombinant vector for overexpression of BnA05CARK3 gene

[0030] 1. Extraction of oilseed rape cDNA

[0031] Total RNA of Brassica napus mixed tissue organs 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 result showed that the sequence had high homology with other registered plant CARK3 genes, and after further multiple alignment, it was speculated that there might be only two CARK3 genes in Brassica napus, and the corresponding independent genes were named BnA05CARK3 and BnC04CARK3. BnA05CARK3 gene was selected for subsequent processing.

[0032] 2. Construction of recombinant vector for overexpression of BnA05CARK3 gene

[0033] The above cDNA was used as a template for PCR amplification.

[0034] The amplified reaction system is: cDNA template 1 μL, upstream primer 1 μL, downstream primer 1 μL, super-fidelity DNA polymerase 1 μL, buffer 10 μL, dNTP Mixture 4 μL, and deionized water to 50 μL.

[0035] The upstream primer and the downstream primer are designed according to the sequence of BnA05CARK3 gene, and the primer sequences are respectively:

[0036] The upstream primer is 5'-AGATCTCTCTCTCCCATACGATT-3';

[0037] The downstream primer is 5'-GTTGATCATAGATTACCCAGAGGCC-3'.

[0038] The amplification reaction conditions are: 98 ℃ pre-denaturation for 5 min, 98 ℃ denaturation for 15 s, 55 ℃ rehydration for 30 s, 72 ℃ extension for 2 min, 30 cycles of reaction, and 72 ℃ extension for 10 min.

[0039] After the PCR product is recovered, it is connected to the Gateway cloning system entry vector pDONR221 to obtain the pDONR221-BnA05CARK3 plasmid. The pDONR221-BnA05CARK3 plasmid is transformed into E. coli DH5α, and the single colony liquid of PCR positive is picked for sequencing and analysis, and the nucleotide sequence of the BnA05CARK3 gene is shown as SEQ ID NO. 1.

[0040] The above-mentioned pDONR221-BnA05CARK3 plasmid with correct sequencing is subjected to LR exchange reaction with the overexpression vector pCAMBIA1305.1. The specific reaction method is as follows: 100 ng of pDONR221-BnA05CARK3 plasmid, 150 ng of vector pCAMBIA1305.1, 2 μL of 5x LR Clonase Reaction Buffer are mixed, deionized water is added to a total volume of 8 μL, 2 μL of LR Clonase enzyme mix is added, and the mixture is mixed for a short time twice, centrifuged, and then incubated at 25℃ in a water bath for 8 h. Then 1 μL of protease K solution is added to terminate the reaction, and after a short vortex, it is incubated at 37℃ for 10 minutes. The recombinant vector pCAMBIA1305.1-BnA05CARK3 is obtained. The recombinant vector is transformed into E. coli DH5α, and the positive clones are picked for bacterial PCR test and enzyme digestion verification.

[0041] Example 2: Transforming the recombinant vector overexpressing BnA05CARK3 gene into rape

[0042] The recombinant vector pCAMBIA1305.1-BnA05CARK3 was transformed into Agrobacterium, and then the Brassica napus was infected with the transformed Agrobacterium, and the specific method was as follows:

[0043] The A. tumefaciens AGL1 competent cells were taken out from the-70°C refrigerator, thawed on ice until the cell suspension was in an ice-water mixed state, 100 μL of the cell suspension was taken, 2 μL of the recombinant vector pCAMBIA1305.1-BnA05CARK3 was added, and after mixing, it was placed on ice for 5 min, then it was quickly frozen in liquid nitrogen for 5 min, and then it was quickly transferred to a 37°C water bath for 5 min, and then it was placed back in the ice-water bath for 5 min to restore the cell activity;

[0044] 800 μL of antibiotic-free LB liquid medium was added to the above cell suspension with restored cell activity, and the bacterial cells were recovered by incubation at 28°C and 250 r / min for 3 hours, and then centrifuged at 5500 rpm for 1 min, and the supernatant was spread on YEB solid medium containing 50 mg / L of rifampicin and 100 mg / L of spectinomycin, and incubated at 28°C for 72 hours to obtain a single colony of Agrobacterium;

[0045] The above Agrobacterium single colony was picked and placed in 5 mL of YEB liquid medium containing 50 mg / L of rifampicin and 100 mg / L of spectinomycin, and incubated at 28°C and 200 rpm for 48 hours, and then 50 mL of YEB liquid medium containing 50 mg / L of rifampicin and 100 mg / L of spectinomycin was added, and incubated at 28°C and 200 rpm for 48 hours to obtain the bacterial liquid of the transformed Agrobacterium;

[0046] The 1 cm Brassica napus hypocotyls were immersed in the above bacterial liquid of the transformed Agrobacterium for 15 min, and then taken out and placed on a tray in a growth chamber, and incubated at 25°C in the dark for 24 hours, and then placed in natural light for incubation, and watered 1-2 times per week, and after the seeds matured, the T0 generation seeds were harvested.

[0047] Example 3 Regulation of Brassica napus fertility under drought stress by overexpression of BnA05CARK3 gene

[0048] The T0 generation seed is placed in a 90 mm*15 mm culture dish after sterilization, a layer of filter paper and a layer of water absorption paper are added as a bud bed after sterilization, 6 mL of 6% PEG solution is added to simulate drought conditions, and then the culture dish is placed in a constant temperature light incubator at 25 DEG C, and cultured under 16 h / 8 h light / dark conditions (experimental group). The control group 1 is wild Brassica napus seed instead of T0 generation seed, and the control group 2 is wild Brassica napus seed instead of T0 generation seed, and no PEG solution is added to the culture dish.

[0049] The development process of the rape in the experimental group and the control group is observed. The overall development process of the rape in the experimental group and the control group 2 is close, and the harvested silique is also not much different, and the development process of the rape in the control group 1 is obviously slowed down. Therefore, the rape in the experimental group and the control group 1 is mainly compared subsequently.

[0050] The pollen viability of the rape in the experimental group and the control group 1 is detected by using the boron pink staining method, and the detection result is as shown in Figure 1 It can be seen from Figure 1 that the pollen activity of the rape in the experimental group is normal, and the pollen activity of the control group 1 is significantly reduced,

[0051] Figure 2 The silique of the rape in the experimental group and the control group 1 is shown in the figure, and it can be seen from the figure that the length of the silique in the control group 1 is shorter than that in the experimental group, indicating that the silique petal development of the rape in the control group 1 is not normal.

[0052] The thousand seed weight of the rape in the experimental group and the control group 1 is determined, and the determination results of the experimental group and the control group 1 are 1.74 g and 0.68 g respectively, and the thousand seed weight of the rape in the control group 1 is less than half of that in the experimental group.

[0053] In summary, by overexpressing the BnA05CARK3 gene in Brassica napus, the seed development process of Brassica napus under drought stress environment can be promoted, the pollen viability and the thousand seed weight of the rape can be improved, and the quality and yield can reach the normal development degree of wild Brassica napus seed.

[0054] Based on the fact that Brassica napus, Brassica rapa and Brassica juncea all belong to Cruciferae plants, it can be speculated that the BnA05CARK3 gene of the application can also be well applied in Brassica rapa and Brassica juncea.

[0055] Those skilled in the art can make various forms and details of changes, modifications, replacements and modifications to these embodiments without departing from the spirit and principles of the application, and the scope of the application is defined by the claims and their equivalents.

Claims

1. The application of overexpressing BnA05CARK3 gene in improving the fertility of Brassica napus, characterized in that, The nucleotide sequence of the BnA05CARK3 gene is shown as SEQ ID NO.

1.

2. Use according to claim 1, wherein In order to improve the fertility of Brassica napus, a recombinant vector for overexpression of the BnA05CARK3 gene is constructed by using the cloned BnA05CARK3 gene, and then the recombinant vector is transformed into Brassica napus to overexpress the BnA05CARK3 gene.

3. Use according to claim 2, wherein the compound is ###0002### The vector for overexpression of the BnA05CARK3 gene is pCAMBIA1305.

1.

4. The use according to claim 2, wherein the compound is ###0002### The method for transforming the recombinant vector into the recipient plant Brassica napus is to transform the recombinant vector into Agrobacterium, and then to infect Brassica napus with the bacterial liquid of the transformed Agrobacterium.

5. The use according to claim 4, wherein the compound is ###0002### The strain for transforming the recombinant vector into Agrobacterium is Agrobacterium tumefaciens AGL1.

6. A method for improving the fertility of oilseed rape, characterized in that, The method for transforming the recombinant vector into the recipient plant Brassica napus is to transform the recombinant vector into Agrobacterium, and then to infect Brassica napus with the bacterial liquid of the transformed Agrobacterium. The strain for transforming the recombinant vector into Agrobacterium is Agrobacterium tumefaciens AGL1.

7. The method of claim 6, wherein, The method for transforming the recombinant vector into the recipient plant Brassica napus is to transform the recombinant vector into Agrobacterium, and then to infect Brassica napus with the bacterial liquid of the transformed Agrobacterium.

8. The method of claim 6, wherein, The strain for transforming the recombinant vector into Agrobacterium is Agrobacterium tumefaciens AGL1.

9. The method of claim 6, wherein, ​

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