Plant and bacterial material
The castor material was an inbred line HY1, developed by the laboratory of molecular breeding for energy crops in Guangdong Ocean University. The plant expression vector pYLRNAi.02 (Fig. 11) was provided by Prof. Liu Yaoguang of South China Agricultural University. Escherichia coli TOP10 and Agrobacterium EHA105 were provided by Prof. Jie Xinming of South China Agricultural University. 2 × Taq Master Mix, DNA Marker, LA Taq, Ex Taq, 10 × Loading buffer, reverse transcription kits were all bought from TaKaRa.
DNA, RNA extration and cDNA synthesis
Genomic DNA of transgenic plants was extracted with modified CTAB method. The RNA of transgenic receptor was extracted by Trizol method with extract RNAiso Plus and adjuvant RANiso-mate for Plant Tissue (TaKaRa) according to the manual of TRIzol kit. After testing for purity and integrity, genomic DNA residue in RNA was eliminated with DNase I to guarantee RNA purity. The first strand of cDNA was synthesized in accordance with the reverse transcription kit instructions.
Primer design
Upstream and downstream primers were designed at the start and stop codon regions of RcPAL according to the ORF sequence released in GenBank (XM_002519475.1) to amplify RcPAL CDs, named RcPAL-F-1 and RcPAL-R-1. The Actin was used as reference gene and its forward and reverse primers were designed, named as RcACTINs and RcACTINa. The forward and reverse primers of target gene were also designed according to primer design principle of real-time PCR, named RcPAL-F-2 and RcPAL-R-2. A pair of specific primers were designed on hygromycin gene sequence to detect transgenic plants, named Hyg-F and Hyg-R. The forward and reverse primers for overexpression vector/antisense expression vector (pYLRNAi-PAL+/ pYLRNAi-PAL−, after the same) were designed at the start and stop codon regions of RcPAL CDs, adding the corresponding restriction site, removing the stop codon (TTA) from the reverse primer for antisense expression vector, named as RcPAL+-F/RcPAL+-R and RcPAL−-F/RcPAL−-R respectively. Primer sequences were as follows.
RcPAL-F-1: 5′-ATGGCAGCAATGGCAGAAAATGGC-3′.
RcPAL-R-1: 5′-TTAGCAAATTGGAAGAGGGGC-3′.
RcACTINs: 5′-CCCAGCACACAGCAGCAA-3′.
RcACTINa: 5′-AGGACTTGAAGAGGAAGAGAGAAACC-3′.
RcPAL-F-2: 5′-ATCTGAGGCATCTGGAGGAA-3′.
RcPAL-R-2: 5′-CAGCATAGGCAAAGACATACTC-3′.
Hyg-F: 5′-GGCGAAGAATCTCGTGCTTTCA-3′.
Hyg-R: 5′-CAGGACATTGTTGGAGCCGAAA-3′.
RcPAL+-F (Adding BglIIsite): 5′-GAAGATCTATGGCAGCAATGGCAGAAAATGGC-3′ RcPAL+-R (Adding MluI site): 5′-CGACGCGTTTAGCAAATTGGAAGAGGGGC-3′.
RcPAL−-F (Adding MluI site): 5′-CGACGCGTATGGCAGCAATGGCAGAAAATGGC-3′.
RcPAL−-R (Adding BglIIsite): 5′-GAAGATCTGCAAATTGGAAGAGGGGC-3′.
Target fragment amplification
Maximum ORF sequence of RcPAL was amplified with primers RcPAL-F-1 and RcPAL-R-1 using HY1 cDNA as template. The PCR reaction procedure was 95 °C 5 min for 1 cycle, 94 °C 35 s, 62 °C 35 s and 72 °C 3 min for 35 cycles; extension for 10 min at 72 °C. PCR products were checked with 1% agarose gel electrophoresis.
Construction of overexpression and antisense expression vector of RcPAL
The overexpression vector pYLRNAi-PAL+ and antisense expression vector pYLRNAi-PAL− were constructed by digesting plant expression vector pYLRNAi.02 (with bacterial screening marker Kanr, and plant screening marker Hygr) and the target CDs sequences of RcPAL with BglII and MluI, recycling vector and target fragments and ligating them with T4 ligase. The CDs sequences of RcPAL was amplified from pMD-RcPAL with the upstream and downstream primers for overexpression and antisense expression respectively. The recombinant vectors were used to transform Escherichia coli TOP10.
Transformation
Imbibing seeds were transformed with vectors pYLRNAi.02 (wild type), pYLRNAi-PAL+ and pYLRNAi-PAL− respectively by acupuncture-vacuum infiltration assisted Agrobacterium tumefaciens mediated method. Firstly, the dry plump castor seeds were sterilized with 70% ethanol for 1 min and then with 10% sodium hypochlorite for 30 min. After being rinsed thoroughly, they were dipped in water of 40 °C for 30 min and transferred onto filter papers previously moistened with distilled water for imbibing at 28 °C for 24 h. Secondly, the seed coat was cracked with an anatomical needle and the seed was pierced once with a disposable syringe of 1 mL(the syringe needle diameter was 0.45 mm)to a depth of ~ 1 mm at the site near the caruncle on the longitudinal midline of seed back, exactly at the the junction of the inclined plane and the plane, beneath which the epicotyl was located. Note that before piercing, the syringe needle had been dipped in the Agrobacterium inoculum. In order to inoculate Agrobacterium into the embryonic meristem effectively and avoid damaging the growing point, the acupuncture point should be behind the plumule which lay beneath the seed coat where a shoot would emerge later (Fig. 12a). Thirdly, the pierced seeds were then soaked into the Agrobacterium inoculum within a air-permeable conical flask (Fig. 12b) and the conical flask was placed into a vacuum compartment, pumped at a pressure of 50 kPa for 20 min, released for 2 min and then vacuum pumped again at same pressure for 5 min (Fig. 12c). Fourthly, the inoculated seeds were transferred without rinsing again onto filter paper moistened with Agrobacterium inoculum and further incubated in the dark at 28 °C for 3 days and until beginning of germination after ~ 9 days (Fig. 12d). Finally, the seedlings were immersed into a 250 mg/L carbenicillin solution for 1 h to remove the remnant Agrobacterium, and after being rinsed thoroughly with sterile water, they were transplanted to a seedling tray with NOVARBO substrate (Finland)and grown in greenhouse (Fig. 12e). Each vector transformed 150 seeds [33].
Identification of transgenic plants
Hygromycin identification
The third leaf from the top of the transgenic plants with 3~4 leaf was cut into rectangular pieces of 1.5 cm × 1.0 cm and soaked in 16 mg/L hygromycin solution containing 0.5 mg/L 6-BA (screening system established in laboratory). At 4 days later, the individuals with dark stripes or necrotic plaques were negative ones, while those remaining green were primarily judged as positive ones.
PCR identification
PCR identification was performed with the primers Hyg-F / Hyg-R using the leaf DNA of transgenic plant as template. The PCR reaction procedure was 95 °C 5 min, one cycle; 94 °C 35 s, 55 °C 35S, 72 °C 1 min, 35 cycles and 72 °C 10 min. PCR products were detected by 1% agarose gel electrophoresis.
Phenotype investigation of transgenic plants
When the transgenic plants were grown in plastic barrels for 80 days, the plant height, stem diameter (base, middle and upper part), stem length, leaf thickness, number of nodes per stem and number of green leaves per plant were investigated.
Determination of PAL activity in transgenic plants
A total of 3 positive transgenic plants with pYLRNAi-PAL+, 3 positive transgenic plants with pYLRNAi-PAL− and 1 transgenic plant with pYLRNAi.02 were selected at 5-leaf stage for PAL activity analysis. Firstly, each blade of these plants was for 6 times with insect needle 5#. At 0, 6, 12, 24 and 48 h after stabbing respectively, 200 mg of fresh leaf tissue was taken from the third leaf from the top on the main stem of each plant, repeated 3 times. The samples were quickly frozen in liquid nitrogen and stored at − 80 °C. Secondly, each sample was ground into homogenate in an ice-cooled mortar with 1 ml of enzyme extraction buffer (0.05 mol/L boric acid, 5.0 mmol/L β-mercaptoethanol, 1.0 mmol/L EDTA-Na2, 5% glycerinum and 5% PVP). The homogenate was transfered into a 2 mL centrifuge tube, setting volume to 2 mL with enzyme extraction buffer, vibrating for 1 min, and centrifuged at 10,000 rpm at 4 °C for 15 min. The supernatant was collected as sample solution for enzyme assay. Thirdly, PAL activity was determined based on the rate of cinnamic acid production as described by Ochoa-Alejo [34]. Briefly, 1 mL 0.02 mol/L L-phenylalanine and 2 mL 0.1 mol/L H3BO3 buffer were added into a 4 mL centrifugal tube, besides, 0.5 mL sample solution was added into the measuring tube and 0.5 mL enzyme extraction buffer was added into the control tube. After water bathing at 30 °C for 60 min, the reaction was terminated by adding 0.2 mL 6 mol/L HCl. With the control tube adjusting zero, the absorbance A290 of the reaction liquid in measuring tube was measured at the wavelength of 290 nm, 1 U of enzyme activity equals to 0.01 of A290 value increased per min.
$$ \mathrm{PAL}\ \mathrm{activity}\left[\mathrm{U}/\left( gFw\cdotp h\right)\right]=\frac{A_{290}\times Vt\times V}{0.01\times Vs\times Fw\times t} $$
(A290: absorbance; Vt: Total volume of the enzyme fluid; Vs: The quantity of the enzyme fluid taken for measurement; V: Total volume of reaction liquid; t: Reaction time; Fw: Fresh weight of sample) [35].
Histochemical staining of lignin
3 positive transgenetic plants with pYLRNAi.02, pYLRNAi-PAL+ and pYLRNAi-PAL− respectively were selected for histochemical staining of lignin with Wiesner staining method. Wiesner staining method was as follows: prepared freehand tissue slice (50~100 μm) and soaked them in 2% (v/v) phloroglucin (dissolved in absolute ethanol) for 5 min, then immersed in 12% (v/v) hydrochloric acid for 5 min, finally, fixed on the slide to be observed and photographed by microscope.
Determination of gene expression
After 24 h of mechanical stab treatment in transgenic plants, the total RNA of leaves with pYLRNAi.02, pYLRNAi-PAL+ and pYLRNAi-PAL− were extracted respectively. The synthesis of cDNA was performed according to the instructions of PrimeScript® RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara) with the castor gene Actin as internal reference. Quantitative PCR program was 95°Cfor 30s (20 °C/s) of 1 cycle; 95°C for 5 s (20 °C/s), 60 °C for 30s (20 °C/s) of 40 cycles; 95 °C for 0 s (20 °C/s), 60 °C for 15 s (20 °C/s), 95 °C for 0 s (0.1 °C/s) of 1 cycle. The melting curve was checked after completion and relative expression was calculated with 2-△△CT method.
Determination of total lignin
The determination of total lignin content was carried out in accordance with acetyl bromide method [35].
