Genetic diversity, functional properties and expression analysis of NnSBE genes involved in starch synthesis of lotus (Nelumbo nucifera Gaertn.)

Plant material and treatments

Nelumbo nucifera cv. Taikong lotus 36, the highest strain of selective lotus breed after space mutagenesis, was selected in this study (Wu et al., 2007). Seeds were sprouted by soaking in water for germination. Five days after, plants were provided with 50 cm depth pots in a greenhouse for the entire growing season. Functional leaves, petiole, rhizomes and roots were separately collected in the 8th week, 10th week, 12th week. Seeds from plants at 12, 16, 20, 24 and 28 DAF (days after fertilization) were also collected from the Taikong lotus 36 in the genetic experimental base of Wuhan University. Various materials from lotus were quick-frozen in liquid nitrogen, then stored at −80 °C for next manipulation. The fresh leaves of 45 lotus individuals (Supplement 1) were collected from the genetic experimental base of Wuhan University and stored in silica gel (Sinopharm, China).

RNA isolation and cDNA synthesis

Total RNA and genomic DNA were isolated from plant tissue samples using a plant RNA extraction kit and Plant Genomic DNA Kit (TIANGEN, Beijing, China), according to their respective operation manuals. The quantities were subjected to 1% agarose gel electrophoresis and the results were analyzed using a UV Transilluminator (Eppendorf, Hamburg, Germany). The first-strand cDNA of all materials was constructed using the FastKing RT Kit (with gDNase) (TIANGEN). The products were stored at −20 °C for later use.

Characterization of a genetic polymorphism

Genetic polymorphism of NnSBE cDNA from 45 individuals of lotus were also analyzed using the gene-specific primers. PCR reactions were conducted in 50 µl volumes containing two µl of total cDNA, five µl of 10 ×PCR buffer, five µl of two mM dNTPs mixture, three µl of 25 mM MgSO₄, 1.6 µl of 10 pM of each primer, one µl of one U/µl KOD DNA polymerase (TOYOBO, Osaka, Japan) and 30.8 µl ddH₂O, for a total volume of 50 µl. Amplification conditions followed the two-step amplification procedure: 94 °C for 2 min, 36 cycles of 98 °C for 10 s, (Tm)  °C for 30 s, and 68 °C for 1 min. The sizes of the PCR products were assessed using 1.0% agarose gel electrophoresis. PCR products were sequenced by Sanger sequencing (Augct, Beijing, China). Sequences were aligned by DNAman to find molecular markers.

Bioinformatics analysis

All sequence data were obtained from the National Center for Biotechnology Information (NCBI) GenBank database (http://www.ncbi.nlm.nih.gov/). All primers were designed by Primer Premier 5.0v. Genomic structures were performed with the program Gene Structure Display Server 2.0 (http://gsds.cbi.pku.edu.cn/) (Hu et al., 2015). The GO (Gene Ontology, http://www.geneontology.org) was used for annotation of gene products. The conserved domains were predicted by Pfam server (http://pfam.xfam.org/search/sequence;) (Finn et al., 2016). All sequences were aligned using Cluxal-X(Thompson et al., 1997) and DNAman. Subsequently the phylogenetic neighbor-joining tree was generated by MEGA6.0v (Tamura et al., 2013). The bootstrap consensus tree inferred from 1,000 replicates was taken to depict the evolutionary history of the analyzed taxa. The H_O, H_E, Shannon Index was calculated by popGen32 (Nei, 1978; Nei & Li, 1973; Nei & Li, 1979). A dendrogram of the cluster analysis was based on Nei’s genetic distance using the UPGMA method.

Real-time PCR analysis

The cDNA of reverse transcription was diluted for 1:10 and used for RT-PCR and qRT-PCR assays. A house-keeping gene, CYP (cyclophilin, GenBank accession no. EU131153), was selected as the reference gene in this experiment. The primers of CYP were based on the sequence of CDS and designed by Primer Primer 5.0 (Table 1). The total PCR reaction mixture contained 2 µl cDNA (1:10 diluted), 0.4 µl forward primer (10 M), 0.4 µl reverse primer (10 M), 10 µl 2 ×SYBR qPCR Master Mix (with ROX Premixed) (Vazyme, China) and 7.2 µl ddH₂O, for a total volume of 20 µl. For the qRT-PCR experiment, the two-step amplification procedure was used: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The relative gene expression data was calculated using the 2^−ΔΔCt method with the guidance of the StepOne software v2.1 (ABI, US). The experimental design followed MIQE (minimum information for publication of quantitative real-time PCR experiments) (Bustin et al., 2010; Bustin et al., 2009). All measurements were processed three times for biological and technical paralleled repetition.

Construction of plasmids

The full length CDS of NnSBEI and NnSBEIII, which were cloned from lotus, were subcloned into the plasmids pET-28a and pET-32a respectively. The pET-28a-NnSBEI was generated using the following primers: 5′-CCCAAGCTTATGTACAGTTTTTCTGGGT-3′(HindIII site underlined), and 5′-CATGCCATGG TCAGTCATCCAATCCCA-3′(Nco I site underlined). The pET-32a-NnSBEIII was obtained by primers: 5′-CGGGATCCATGG CTACTACAGTTGCGCT-3′(BamHI site underlined) and 5′-GCGATATCTCATATTCG CAAAATCCGAG-3′(EcoR V site underlined). The full length and sequence of the nucleotide acid of the inserted gene in the extracted recombinant plasmid were completely identical to the genes. Then, the different recombinant plasmids were transformed into Escherichia coli BL21 (DE3) (TransGen Biotech, Beijing, China) for expressing and into DH5α for storing.

Enzyme activity assay

Cheng et al.’s protocol of measuring SBE activity was used in this study (Cheng et al., 2001). First, total enzyme isolation: about 0.4 g powder of plant material was added into a 4 ml HEPES–NaOH (PH7.5) buffer. After proper shaking, the tube was centrifuged at 10,000 g for 15 min, and supernatant was extracted to check the enzyme activity of SBE. We took 100 µl dilution, added 1,280 µl of HEPES–NaOH (PH7.5) buffer, and 120 µl 0.75% of soluble starch. Then we incubated the total reaction mixture at 37 °C, and gave it a water bath for 20 min. Next, we gave it a boiling water bath for 1 min to stop the reaction, diluted the mixture with two ml water (containing 0.2% HCl), added 150 µl of an iodine solution (0.1%I₂-1%KI), blending it at room temperature for 10 min. The boiled crude enzyme was used as the control. Finally, we measured absorbance at 660 nm. The enzyme activity of SBE was expressed as a percentage of decrease in absorbance at the wavelength of 660 nm to be compared with the control. All measurements were processed three times for biological and technical paralleled repetition.

The affinity of two isozymes that bind amylopectin and amylose assay

Recombinant proteins were expressed in the Escherichia coli BL21(DE3) cells. The transformed cells were grown in LB at 37 °C until the OD600 reached 0.6–1.0, using IPTG to induce expression with a final concentration of 0.5 mM, and were further incubated at 16 °C for 12 h. Cells were harvested by centrifugation at 12,000 rpm for 5 min at 4 °C. The products were suspended in a buffer containing 10 mM PBS in the radio of 200:1 (ml/g), and cells were disrupted (Diagenode, Liege, Belgium) by sonication for 25 min and centrifuged for 5 min at 4 °C. We took 30 µl supernatant, added 0, 12.5, 16, 20, 25, 35 µll 0.75% amylopectin (Sigma-Aldrich, St. Louis, MO, USA) or amylose (Sigma), and added HEPES–NaOH (PH7.5) buffer into 90 ml. Then the total reaction mixtures were incubated at 37 °C for 30 min. We diluted the mixture with 100 µll water (containing 0.2% HCl), and added 10 µll iodine solution (0.1%I₂-1%KI), blending at room temperature for 10 min. Finally, we measured absorbance of amylopectin at 560 nm and amylose at 620 nm. The blank pET-28a and pET-32a which were transformed into BL21 and cultured in the same condition were used as the control. The affinity of the two isozymes that bind amylopectin and amylose were measured by enzyme activity. All measurements were processed three times for technical repetition and 2∼3 paralleled repetition.

Cloning of NnSBE genes

Based on the reference sequence of whole-genome sequencing, the CDS of two SBE genes were obtained using RT-PCR technique from the embryo of the lotus seed. The CDS of NnSBEI (FJ592190.1) was 2,577 bp, and the CDS of NnSBEIII (XM_010254179) was 2691bp (Fig. 1). Complete genomic structures of NnSBEI (comprising of 14 exons and 13 introns) and NnSBEIII (comprising of 21 exons and 20 introns) were separately distributed over 14.3 and 32.1 kb. The gene structures are shown in Fig. 2 and the gene information is shown in Table 2. The cDNA of NnSBEI showed the highest (85%) identity with Castanea mollissima, and 80–84% identify with SBEI genes of other plant species, while NnSBEIII showed the highest (85%) identity with Juglans regia and 80–84% identity with the others.

The deduced protein of NnSBEI comprised 858 amino acids with a predicted molecular mass of 97.144 kDa, and NnSBEIII comprised 896 amino acids with a predicted molecular mass of 103.135 kDa. GO analysis indicated NnSBE participated in biological processes (GO:0005978) and one molecular function (GO:0043169; GO:0003844; GO:0004553). Pfam analysis showed that NnSBEs have three domains of secondary structure: the central catalytic A domain, an N-terminal domain and the C-terminal domain. The central catalytic A-domain of SBEs is α-amylase which covered four conserved amino acid regions and six active sites spread in those conserved regions respectively. The characterization of catalytic A-domain showed significant homology (47.83%) between NnSBEI and NnSBEIII, but highly dissimilar sequences in N-terminus (21.84%) and C-terminus (26.47%).

Phylogenetic analysis

SBE protein sequences from 20 other species were used for phylogenetic analysis, shown in Fig. 3. The NJ phylogenetic tree indicates that SBEs in these plants could be classified into two families: SBE A and SBE B. SBE A was further divided into four classes: algae SBEII, dicot SBEII, monocot SBEII and SBEIII, and SBE B was subdivided into algae, dicot SBEI, monocot SBEI. NnSBEI belonged to the dicot SBEI of family B; NnSBEIII belonged to the SBEIII class of family A. Evolutionary divergence reveal that SBEIII came before SBEI, and differentiation of NnSBE occurred before that of the other dicots, but later than the monocots.

Characterization of genetic diversity

Genetic diversity was identified based on the ORF of two NnSBE genes from 45 lotus individuals belonging to four clusters (Supplement 1). This revealed six polymorphic sites from the coding region: five SNPs in NnSBEIII and one in NnSBEI. The SNPs of NnSBEIII was analyzed and the detailed parameters of genetic diversity are listed in Table 3. Three SNPs resulted in missense mutations which changed the amino acid sequence, while the other two SNPs were synonymously mutated. The observed heterozygosity ranged from 0.2 to 0.4444, and the expected heterozygosity ranged from 0.4012 to 0.4583. The Shannon-Wiener Index ranged from 0.5908 to 0.6508. In further association analysis of starch content (amylopectin, amylose, total starch) in lotus seeds, no SNP showed highly significant associations. Total of 12 genotypes were detected in those 45 individuals, each cluster had different several kinds of genotypes (Supplemental Information 1). A homozygous genotype (AA GG GG AA GG) of NnSBEIII was observed in most individuals in seed lotusand a haplotype (A G G A G) was identified from seed lotus. Cluster analysis according to the UPGMA method is shown in Fig. 4. The 45 individuals of lotus were copolymerized into three classes. The first class can be divided into two subclasses with a similarity coefficient of 9.8, and most seed lotus showed together in a same subgroup. These results paved a way to apply the useful allelic variations or gene haplotypes in cultivar lotus and quality breed programs.

Expression pattern

The temporal and spatial expression of both genes were analyzed to investigate their expression patterns. The results of qRT-PCR demonstrated that the transcriptional expression level of NnSBEI and NnSBEIII was detectable in all tissues. Tissue-specific expression analysis showed that the highest transcript levels of NnSBEI and NnSBEIII were observed in leaves. The transcript level of NnSBEI was higher in rhizomes while NnSBEIII expressed strongly in petioles. The expression profile of NnSBEI in the rhizome showed strong temporal differences, and the relative expression level increased gradually from initial development to late stage. NnSBEIII showed temporal differences in the petiole, which enhanced in the middle and decreased in the late stage (Fig. 5). During the seed embryo developing stage, NnSBEI and NnSBEIII expressed significant differences. Transcripts of NnSBEIII reached the peak at 20 DAF in the middle of development stage, and then decreased gradually, while NnSBEI increased in 16 DAF to 28 DAF and expressed strongly in the mid-late developmental stage (Fig. 6). In the peak stage, NnSBEI expressed ten times higher than NnSBEIII.

Dynamic changes of enzyme activities

The dynamic changes of enzyme activity of the SBEs were analyzed in different temperatures, tissues and developing stages. Incubation in different temperatures revealed that the highest enzyme activity was generated under 37 °C. The enzyme activity of inter-organizational measurement and assessment showed the highest catalytic activity in leaves, followed by rhizomes and petioles which increased steadily, and activity in the roots was the weakest. During seed development, enzyme activity increased rapidly, and the activity peak appeared at 20 DAF, then decreased slightly (Fig. 7).

The affinity of two isozymes that bind amylopectin and amylose

To explore the affinity activity, recombinant DNA techniques were used to generate the pET-28a-NnSBEI and pET-32a-NnSBEIII plasmids. Testing of the inducible expression vector showed that both proteins were soluble, and the apparent molecular weights of pET-28a-NnSBEI and pET-32a-NnSBEIII were about 105 kDa and 120 kDa (with His tag) respectively. The affinity activity of NnSBEI and NnSBEIII to amylopectin and amylose were assayed at various starch concentrations. As we can see in Fig. 8, the activity of both isozymes correlated with the types and concentration of substrate. Rate of reaction changed with the increase of the substrate concentration until the enzyme was saturated. NnSBEI showed higher affinity activity when the substrates were amylose and amylopectin. However, another isozyme, NnSBEIII, only worked on amylopectin, and the branching efficiency was only half of NnSBEI.