Background

Rape is a traditional oil crop in China and also an important oil crop in Sichuan. Rapeseed oil is rich in nutrition and contains more than ten kinds of fatty acids and multivitamins, especially vitamin E. With the increase of population and the development of social economy, the quantity demand and quality requirement of rapeseed oil are higher and higher. Therefore, breeding high-yield rapeseed varieties has become the main goal of breeding. Flowering is an important process, which changes from vegetative growth to reproductive growth in rapeseed, and early studies mainly described physiological phenomena (Fu and Meng, 1998; Zhao et al., 1999). Research showed that several hypotheses about flowering were put forward, including the flowering hormone hypothesis, the nutrient transfer hypothesis and the multifactor control model hypothesis (Yong et al., 2000). The research results showed that the best development period, the best pollination time, the growth period and the increase of rape yield were related to the flowering. Therefore, it might be necessary to study this character of flowering.

A series of studies on flowering regulation of Arabidopsis thaliana have been carried out by predecessors. The results showed that CO gene played an important role in the photoperiod pathway and played a very important role in the regulation of flowering time as a key transcriptional regulatory factor in Arabidopsis thaliana. Through the study of the expression characteristics, the results showed that the effect part of CO gene was in the leaves (Abe et al., 2005; Wigge et al., 2005). From the expression time, CO existed stably under light conditions, but decomposed under dark conditions. Therefore, CO gene might be an accumulation process during the day and its expression quantity might be the highest in the afternoon (Searle and Coupland, 2004; Kobayashi and Weigel, 2007). In Brassica napus, CO gene was constitutive expression, lacked of development stage and tissue specificity (Ai, 2011). In the study of CO gene function, the result showed that the CO gene promoted flowering under long sunlight (Cohen, 1992). In the study of flowering regulation in rice, the result showed that Hd1, the homologous gene of CO gene, inhibited the flowering of rice under the condition of long sunlight and promoted the flowering under the condition of short sunshine (Yano et al., 2001). Another CO homologous flowering gene, GHD7, which was cloned from rice, promoted the flowering under the condition of long sunshine, but had no effect under the condition of short sunlight (Xue et al., 2008).

On the basis of previous studies (Zheng et al., 2013), this research amplified the target gene primer which included BamHⅠ and ScaⅠ, the amplified fragments were recycled and were double digested with PBI121 expression vector, then connected by using T4-DNA ligase. The ligation product was transformed into Agrobacterium EHA105 receptive cells, and Arabidopsis transgenic positive seedlings were obtained by the method of inflorescence infection, which prepared for the study of CONSTANS gene function in Brassica napus. Thus, it might provide an important reference for the study on the regulation of Brassica napus flowering period.

1 Results and Analysis

1.1 The acquisition of restriction enzyme cutting site called CONSTANS gene

In this study, PCR amplified by using the bacteria solution and the restriction enzyme cutting site containing CONSTANS gene, which obtained from prophase test (Figure 1). The amplified fragment size of PCR was about 1,100 bp, which was consistent with the size of the target fragment.

Figure 1 PCR amplification of primers with restriction enzyme site

1.2 The acquisition of plant overexpression vector PBI121 CONSTANS

This study recycled restriction enzyme cutting site primer and the amplified fragments of positive bacteria solution in CONSTANS gene, which was double enzymes restriction with PBI121 by BamHⅠand ScaⅠ. Then the enzyme-digested products linked by ligase T4-DNA, and PCR amplified by using 35S-2 and CO-R (Figure 2). The amplified fragment size was about 1,100 bp, which was consistent with the size of the target fragment. Therefore, we thought the target gene might be connected with PBI121.

Figure 2 PCR amplification of link product

1.3 The acquisition of positive colony in Agrobacteria

This study extracted plasmid from bacteria solution in PBI121+CONSTANS gene, and then transformed Agrobacteria, picked the white colony to enlarge culture. PCR amplification was carried out with bacterial solution, CO-F and CO-R. The amplified fragment size was about 1,100 bp, which was consistent with the size of the target fragment (Figure 3). Therefore, we thought the overexpression vectors of plant might be transformed into Agrobacteria.

Figure 3 PCR of Agrobacterium positive bacterium containing target gene

1.4 The acquisition of positive transgenic seedlings

This study screened transgenic T0 seedlings by using selection medium (MS+50 mg/L K+8 g/L agar+10 g/L sucrose) (Figure 4). The leaves of transformants were dark green, and the root was longer; the leaves of non transformants became yellow, and the root was short, which couldn’t live long. We transferred the identified transformants into soil and cultured it to be mature. The PCR amplification was conducted with DNA extracted from the leaves of plant using 35S-2 primer and R-primer (Figure 5). The amplified fragment size was about 1,100 bp, which was consistent with the size of the target fragment. Therefore, we thought the plant that we obtained was the transgenic Arabidopsis transgenic seedlings, which contained Brassica napus CONSTANS gene.

Figure 4 Screening of positive seedlings of transgenic Arabidopsis thaliana and obtaining of positive plants Note: A, B, C: Positive seedling screening; D: Positive plant

Figure 5 PCR detection of positive plants Note: 1 ~ 10, 12, 14: Positive transgenic plants; 11, 13: The false positive plants; 15: Wild type control; 16: Positive control

2 Discussion

As for rape seeds, the best development period, the best pollination time, the growth time and the increase of rape yield were related to the flowering (Wang and Wang, 2009). As for the studies on flowering regulation of Arabidopsis thaliana, CO gene played an important role in the regulation of flowering in Arabidopsis thaliana and promoted flowering under long sunlight (Cohen, 1992). In the rice, CO homologous gene inhibited the flowering of rice under long sunlight, but did not affect the flowering of rice under short sunlight (Yano et al., 2001). Another CO homologous gene in rice promoted the flowering of rice under long sunlight, but did not affect under short sunlight (Xue et al., 2008). Other studies have found that the transcriptional patterns similar to Arabidopsis thaliana in petunia, wheat and sugar beets (Liu et al., 2001; Nemoto et al., 2003; Chia et al., 2008). Therefore, these studies might prove that CO gene played an important role in the regulation of flowering in plants.

As for rape seeds, the best development period, the best pollination time, the growth time and the increase of rape yield were related to the flowering (Wang and Wang, 2009). As for the studies on flowering regulation of Arabidopsis thaliana, CO gene played an important role in the regulation of flowering in Arabidopsis thaliana and promoted flowering under long sunlight (Cohen, 1992). In the rice, CO homologous gene inhibited the flowering of rice under long sunlight, but did not affect the flowering of rice under short sunlight (Yano et al., 2001). Another CO homologous gene in rice promoted the flowering of rice under long sunlight, but did not affect under short sunlight (Xue et al., 2008). Other studies have found that the transcriptional patterns similar to Arabidopsis thaliana in petunia, wheat and sugar beets (Liu et al., 2001; Nemoto et al., 2003; Chia et al., 2008). Therefore, these studies might prove that CO gene played an important role in the regulation of flowering in plants.

On the basis of previous studies (Zheng et al., 2013), the results of target gene sequencing were analyzed by restriction enzyme cutting site analysis. And then this study designed restriction enzyme cutting site primer, which double enzymes restriction with plant overexpress vector PBI121. Then this study linked enzyme digestion fragments by ligase T4-DNA, at the same time, transformed competent cell EHA105. We obtained positive plant by Arabidopsis thaliana inflorescence infection, and screened by MS culture medium with kalamycin. PCR amplification showed that PBI121+CONSTANS overexpression vector had been successfully constructed and successfully transfected into Arabidopsis thaliana. And 12 transgenic positive seedlings were obtained, which were ready for the next step of gene function analysis.

3 Materials and Methods

3.1 Materials

Plant material was wild Arabidopsis thaliana in Columbia (stored in the laboratory). Plant expression vector PBI121 (presented by Fu S.X., who works in Chengdu Academy of Agriculture and Forestry Science, Sichuan). Escherichia coli DH5α competence, Agrobacterium tumefaciens EHA105 competence, restricted enzyme BamHⅠand ScaⅠ, T₄DNA ligase, and gel extraction kit were purchased from Tiangen Biotech Co., Ltd. PCR system included Taq enzyme and PCR consumables purchased from Prom ega company. Kanamycin and ampicillin Na salt etc. were purchased from Shanghai Sangon Biotech Co., Ltd.; the primers were synthesized by Shanghai Sangon Biotech Co., Ltd. (Table 1).

Table 1 The sequences of primer

3.2 Methods

Figure 6 The construction strategy of CO gene overexpression vector

3.2.1 The introduction of restriction enzyme cutting site in Bn-CONSTANS gene

This study took the vector Peasy-T1-con-stans bacteria solution plasmid which contained CONSTANS gene as a template. And the PCR amplification with restriction enzyme cutting site primers, which contained BamHⅠand SacⅠ (F-primer and R-primer). PCR reaction system and the control condition (Zheng et al., 2103). The target band was detected by electrophoresis and recycled.

3.2.2 The construction and identification of Bn-CONSTANS gene plant overexpression vector

Recycled PCR product was double digested with PBI121 and ligated with T4-DNA ligase. We transformed E. coli, selected bacterial colony, and detected fragments insertion by primers 35S-2 and CO-R. If about 1,100 bp fragment was obtained, the positive colonies were identified, that was to say the expression vector PBI121 was successfully linked to the target gene Bn-CONSTANS fragment.

3.2.3 PBI121+CONSTANS gene plant overexpression vector transformed to Agrobacterium tumefaciens

This study shook positive cloning bacterial colony in the culture medium of LB+K, and extracted plasmid DNA. And we melt the Agrobacterium competence on the ice, added 2 μL recombinant plasmid into Agrobacterium tumefaciens competence that had just thawed, and then shook gently, froze liquid nitrogen for 2 min, then water bath at 37°C for 5 min, and then added 1 mL LB and cultured it in medium with 28°C for 3 h. Then we collected thallus by centrifugation, and painted on plate LB+Str (25 μg/mL)+Kan (50 μg/mL). The single colony on the recombinant plate was randomly selected to be inoculated in LB+Str (25 μg/mL)+Kan (50 μg/mL) liquid medium and cultured overnight in shaking bed at 28°C. The recombinant plasmid was identified by PCR amplification using the liquid as template.

3.2.4 Agrobacterium tumefaciens infects Arabidopsis thaliana inflorescence

The seeds of wild-type Colombian Arabidopsis thaliana were vernalized at 4°C for 3 days and seeded in a small flowerpot of 7×7×8 (mixed nutritious soil and vermiculite in a ratio of 1: 1). We need to remove the top at the first flowering to promote the growth of lateral branches. In this research, bacteria containing fragments were inoculated in liquid medium at 28°C for overnight. When the OD value was 0.8, the bacteria solution was collected by centrifugation, and the bacteria was suspended with 5% sucrose solution, and the surfactant Silwet-77 was added to the concentration of 0.02%. We need to water the seedlings with enough water in the night before the infection. The inflorescence of Arabidopsis thaliana was immersed in the suspension of Agrobacterium tumefaciens for 1 min, and infected 2~3 times in the whole growing period. After infection, this research treated it by shading with bagging for 16~24 h, and ensured enough water, then harvested seeds when a silique was naturally dehiscent, which was T0 generation seed.

3.2.5 The screening of transgenic positive seedings

This research disinfected the surface of T0 generation seeds, and seeded evenly on screening medium (MS+50 mg/L K+8 g/L agar+10 g/L sucrose). The seeds were placed at 4°C for 2~3 days and then cultured in an artificial climate box. The transformants were transplanted into soil, and the DNA was extracted from the leaves at the later stage. In this research, primer 35S-2 and CO-R were used for PCR examination.

Authors’ contributions

ZBC was the executor of this experiment, responsible for the design, implementation of the experiment and paper writing. LHJ, ZJF, CC CL, JJ and WXM were responsible for thesis revision and related experimental guidance. JLC determined the conception of the research project and guided the writing and revision of the thesis. All the authors read and approved the final manuscript.

Acknowledgements

The study was funded by Cole Industry Technology System (CARS-13), National Key Research and Development Project (2016YFD0101300), Sichuan Crop Breeding Key Project (2016NYZ0031), Sichuan Provincial Financial Innovation Ability Promotion Project (2016ZYPZ-013), Sichuan Rape Innovation Team, and Department of Agriculture upper Yangtze River Oil Crops Scientific Observation Station (Chengdu) (09203-020).

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