Plant materials preparation
Due to the large amount of seeds were needed in this experiment, seed lotus (Taikong Lotus 36) was used as the material to carry out following experiments. The species was derived from “The Guangchang Bailian Institute”, and cultivated in experimental field of aquatic vegetables in Yangzhou University in Southeast China. For the plant growth, the field maintained 20–25 cm water in depth in spring, and 40–50 cm water in summer. The temperature was 25–35°C in the day, and 18–25°C at night throughout the whole growing season. The seeds were harvested in November, and kept at room temperature.
Cloning of NnWOX1-1, NnWOX4-3andNnWOX5-1
Tull-length of these three genes were found in NCBI database. The primers of NnWOX1-1, forward primer: ATGGAAAGGAAAGATGAT, reverse primer: TCACTTGGATTTGAATGG; NnWOX4-3, forward primer: ATGGAGGGCAAAGAGGAG, reverse primer: TTACACTCTGGCCTTGAA; NnWOX5-1, forward primer: ATGGGGAAGGACGTTGAG, TTAGACGTGAGGGTTGCT. The total RNA was extracted by RNA Extraction Kit (Quagen, Germany), and the first cDNA strand was synthyzed using 1–2 µg of total RNA (Promega, USA) after elimination of DNA contaminations. For component of PCR system, a total of 20 µl mixtures containing 2.5 µl dNTP, 2 µl forward and reverse primers, 2.5 µl MgCl2, 2 µl Taq polymerase (5 U), 2 µl fragments of cDNA, and 7 µl dH2O. The PCR was performed as following program: a total of 35 cycles contained 1 min of 94°C for pre-denaturation, 1 min of 94°C for denaturation, 1 min of 56–60°C for annealing, 1 min of 72°C for extension; 10 min of 72°C for the final extension. A gel purification kit was used to extract target gene fragments (TaKaRa, Dalian, China), which was then ligaed in cloning vector (pMD 18-T vector) (TaKaRa, Dalian, China). These gene fragments were recombined with DH5α, and sequenced by Sangon Biotechnology Co., Ltd. (Shanghai, the China).
Sequence analysis
The comparison among these three genes was performed using the DNAman softwar, and Simple Modular Architecture Research Tool software was applied to analyze of
conserved domain. Phylogenetic tree was constructed by the DNAman software and MEGA X software according to instruction. In addition, the conserved motifs of NnWOX1-1, NnWOX4-3 and NnWOX5-1 were analyzed by DNAman software and MEME server v5.4.1 software. Chromosomal location maps of these three genes were completed by the DNAman software and TBtools software.
Gene expression analysis
qRT-PCR method was applied to monitor these three genes expression in lotus seedlings treated with 20 g/L sucrose and 10 µl IAA. In addition, adventitious root, leaf, stem of six-leaf seedlings and flowers were selected to analyze organ-specific expression characteristic. Plant total RNA was obtained by using RNA extraction mini kit (QIAGEN, Germany) according to protocol of instructions. The genomics DNA was removed by DNase I, and then 3 µg RNA was chosen to synthesize the first strand cDNA with the Kit (Fermentas, USA). SYBR Green Master Mix (Tiangen, China) was used to calculate on the mRNA levels of NnWOX1-1, NnWOX4-3 and NnWOX5-1 with three biological repetition on Mx 3000P machine (STRATAGENE, http://www.stratagene.com). The forward primer of NnWOX1-1 was 5'- GGTGGTGATTGTGAAGGAGT-3', and the reverse primer was 5'- CAATCCAACGCCCTTACTAT-3'; The forward primer of NnWOX4-3 was 5'-TACTGTGGGTATTCAGGGCA-3', and the reverse primer was 5'-TGACTCTTCTGGAAACCCTT-3'; The forward primer of NnWOX5-1 was 5'-CGGCTGTAACCTTTGGACTT-3', and the reverse primer was 5'-TCCCAGGGCAGTTCCTTTTG-3'. β-Actin of lotus was used as an internal standard, and the forward: 5'-AACCTCCTCCTCATCGTACT-3', and reverse: 5'-GACAGCATCAG CCATGTTCA-3'. the mRNA level was calculated with 2−△△Ct method [21]. A total of 25 µl reaction mixture containing 12.5 µl SYBR Premix Ex Taq II (Tli RNaseH Plus) (2×), 1 µl of each primers (forward and reverse), 3 µl cDNAs of NnWOX1-1, NnWOX4-3 and NnWOX5-1, and 8.5 µl dH2O was prepared. The PCR program contained 30 s of 94°C, followed by 5 s of 95°C with 40 cycles, and 60 s of 60°C.
Gene function analysis of NnWOX1-1, NnWOX4-3andNnWOX5-1
NnWOX1-1, NnWOX4-3 and NnWOX5-1 were put into a pGEM-T vector (clone vector), and sequenced by Sangon Biotechnology Co., Ltd after transferred into Escherichia coli for expanding reproduction. The plasmas of these three genes were digested by BamHI and KpnI enzymes, and genes with enzyme digestion site were obtained. NnWOX1-1, NnWOX4-3 and NnWOX5-1 were transferred into a plant transformation vector (pSN1301) contained a CaMV 35S promoter. After completing the process of vector construction, pSN1301:: NnWOX1-1, pSN1301:: NnWOX4-3 and pSN1301:: NnWOX5-1 were put into a Agrobacterium tumefaciens strain GV3101 for infection preparation. These recombinant plasmids were transformed into wild type plants of Arabidopsis by floral dip method. The seeds of T0 generation were screen by 20 µg·g− 1 hygromycin B on the MS medium after sterilization. The screened plants were cultivated in the green house of 22°C under light/12 h and dark/12 h conditions. The positive plants were further identified by the RT-PCR method. The primers, mixture, and PCR reaction process of these three genes were the same with genes cloning mentioned above.
T2 generation seeds of transgenic plants and wild type plants with six leaves were selected for function identification. Firstly, transgenic seeds and wild type seeds were sterilized by 70% alcohol for 10 s, and then sodium hypochlorite (10%) were used for further treatment for 25 min. These seeds were put on medium and base material for further analysis of plant phenotype and root development.
RNA-seq analysis
RNA-seq of NnWOX1-1, NnWOX4-3andNnWOX5-1
Transgenic seeds and wild type seeds were placed on the base material for germination, and then transferred to light house. The temperature was maintained at 22–23°C with 12 h/light and 12 h/darkness. The roots of these plants with six-leaf age were used for gene expression analysis by RNA-seq technique. About 2–3 µg RNA of each material were prepared for library construction. The detail process for each library construction was referred by Cheng et al. (2020a)[21], and sequencing by Nanjing Jisi Huiyuan Biotechnology Co., Ltd with a special construct.
Identification and annotation of differentially expressed genes (DESs)
DEGs were identified with NOISeq method as described by Cheng et al. (2018)[20]. The threshold of DEGs was that the fold change was ≥ 2, and the divergence probability ≥ 0.8. Gene Ontology (GO) including three ontologies (molecular function, cellular component, and biological process) was applied to annotate DEGs in each sample. All the identified differentially expressed genes was grouped into different biological pathway after comparing the genome data derived from National Center for Biotechnology Information (NCBI) database by using KEGG tool. All the DEGs was classified into different metabolic pathways.
Ethylene, IAA and ABA identification
The transgenic NnWOX1-1, NnWOX4-3 and NnWOX5-1 seeds and wild type seeds were cultivated on the base material, and transferred into illuminating incubator with 22°C condition. The six-leaf plants were selected for ethylene, IAA and ABA identification. Firstly, three six-leaf-age seedlings of each sample were washed, and put into a bottle, which was tightly covered with a plastic plug. These bottles were transferred into a dark carton for ten days under 25°C condition. Ethylene producing rate was measured by gas chromatograph (Agilent Company, America). The whole process of ethylene producing rate was completed according to the reported by Fiserova et al. (2008)[42]. This experiment was carried out with three repetitions. Secondly, twenty seedlings of transgenic plants and wild type plants were put into liquid nitrogen, and then grind into powder with a rod. About 2 g powder of each material were transferred into about 600 µl reagent (V/V/V: isopropyl alcohol: water: concentrated hydrochloric acid = 2:1: 0.002) for IAA and ABA extraction. These mixtures were put into a vibrator for reaction for 25–30 min at 4°C condition, 1 ml dichloromethane was added, and then shaking for 30 min at 4°C. These solutions were centrifuged at 10000 rpm for 5 min at 4°C. The supernatant at lower layer was selected, and dried with nitrogen. The dry powder was dissolved by 100 ml filtered methanol, and 50 of above solution was transferred into liquid chromatography (Sigma, Germany) for IAA content determination [21]. The process of ABA content identification was refered to the method reported by Zdunek and Lips (2001) [43].
Lignin identification
Thirty transgenic NnWOX1-1, NnWOX4-3 and NnWOX5-1 and wild type plants root were selected to measure polymer content. The plant growth condition was the same as above mentioned. These plant roots were dried at 60°C for about two days, and 60 mg dry power was transferred into ethanol (1 ml, 70%). the mixture were vortex and centrifuged at 10000 rpm under room temperature condition. 1 ml solution of chloroform / methanol (1:1 V/V) was added into the precipitant after the supernatant was discarded. discarding the supernatant, and the precipitant was washed by 1 ml of acetone. precipitant was put into vacuum condition for drying. The precipitant was treated with 1.5 ml of 0.1 M sodium acetate buffer (pH 5.0) at 80°C for about 30 min, and then transferred into a tube with 10 µl of 0.01% sodium azide amylase and pullulanase for incubation at 37°C overnight. After heating with at 100°C for few minutes, the precipitant was washed with water for a few times. 1 ml of acetone was put into the tuber, and dried with vacuum condition. The precipitate was dissolved by 100 µl solution of 25% acetyl bromide for three hours at 50°C. 400 µl of 2M sodium hydroxide and 70 µl of 0.5M hydroxylamine hydrochloride were added. Acetic acid was used to increase the volume to 2 ml, and 200 µl of supernatant was selected to determinate absorbance value at 280 nm by multifunctional microplate reader (1510–04201, Semefi, USA). The polymer lignin was calculated according to the determinate absorbance value, and the formula was listed as:
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)
(ABS: absorbance value; Coeff: absorption coefficient)
Identification of ethylene role on ARs formation and plant growth in lotus
About 300 lotus seeds were selected to identify rates of ARs formation. These seeds were treated with the methods mentioned as above before germination. About fifty seedlings were firstly treated with 300 mg/L ethylene and 300 mg/L 1-Methylcyclopropene (1-MCP) for two days, and then transferred to water for continue cultivation. The rates of ARs formation and stem length were identified at 0 d, 2 d, 4 d and 6 d. At the same time, twenty seedlings in each repetition were used for fresh and dry weight assessment at 0 d, 5 d, 15 d and 25 d of plant growth in water.
Stresses response for transgenic plants and wide type plants
About fifty seeds of transgenic plants and wide type plants were put into water for 24 h, and then sown on the medium. The detail cultivation condition was the same as mentioned above. The six-leaf seedlings of wild type plants and transgenic plants were treated with drought and salt stress conditions. For drought treatment, the plants were firstly treated with unified water management, and then the water was withdrawn for about ten days. The survival rates were counted after water recover for seven days. For salt treatment, 100 mM NaCl was applied to treat transgenic plant and wide type plants, and then the survival rates was also identified after ten days. Three biological repetitions was carried out for every experiment, and at least fifty seedlings were used for each repetition.
Statistics of physiological indexs in lotus seedling treated with ethylene
About six hundred lotus seeds were immersed in water for germinated at 26°C. The germinated seeds were placed in plastic box for seedlings cultivation under 16 h/light and 8 h/dark condition for about four days. The seedlings with two leaves were treated with 300 mM ethylene and 300 mM 1-Methylcyclopropene (1-MCP) for three days, and then transferred into water for continue growth. Ninety seedlings with uniform in size were classed into three groups, and ARs rates and stem heigth were determined at 0 d, 2 d, 4 d and 6 d. For analysis of fresh weight and dry weight, one hundred and fifty treated seedlings were classed into three repetition, and then fresh weigh and dry weight were identified at 0 d, 5 d, 15 d and 25 d. For dry weight identification, seedlings were firstly used for fresh measure, and put into thermostat at 60°C for 7 d, followed by count with electronic balance.
Statistical analysis
Statistical analyses were carried out by the SPSS software ver. 14.0 (SPSS Inc., Chicago, IL, USA). For analysis in each experiment, the data were recorded as the means ± SE of three repetition, and the differences at p < 0.05 were accepted as the level of significance.