Cloning and characterization of the UBC gene from lotus (Nelumbo nucifera).

Protein ubiquitination is extensively involved in the regulation of a considerable number of physiological processes in plant cells. E2 (ubiquitin-conjugating enzyme, UBC), one of the essential enzymes of eukaryotic ubiquitination, catalyzes protein ubiquitination together with E1 and E3. In this study, we cloned four full-length cDNA NnUBCs of Nelumbo nucifera. With the same coding sequence length of 459 bp and coding 153 amino acids, these four genes are highly homologous with the AtUBC1 and AtUBC2 of Arabidopsis thaliana. Quantitative fluorescence polymerase chain reaction showed that these four genes exhibited different expression patterns in different tissues of N. nucifera. Overall, the expression of NnUBC3 was the highest in all plant tissues. Tests of different stress treatments showed that NnUBC3 plays an important role in response to heat, salt, and drought stresses in N. nucifera. Moreover, transgenic Arabidopsis plants (Atubc1-1Atubc2-1 mutant) expressing NnUBC3 presented a wild-type phenotype, indicating that NnUBC3 performs the same function as AtUBC1 and AtUBC2.

Development and characterization of genic-SSR markers from different Asia lotus (Nelumbo nucifera) types by RNA-seq.

Nelumbo nucifera is an important economic vegetable and traditional medicine, but available genetic resources remain limited. Next generation sequencing has proven to be a rapid and effective means of identifying genic simple sequence repeat (genic-SSR) markers. This study developed genic-SSRs for N. nucifera using Illumina sequencing technology to assess diversity across cultivated and wild lotus. A total of 105,834 uni-contigs were produced with an average read length of 722 bp. Exactly 11,178 genic-SSR loci were identified in 9523 uni-contigs. Di-nucleotide (64.5%) was the most abundant SSR, followed by tri-nucleotide (23%), tetra-nucleotide (8.9%), penta-nucleotide (2.5%), and hexa-nucleotide (1%) repeat types. The most common di- and tri-nucleotide repeat motifs were AG/CT (51%) and AAG/CTT (8%), respectively. Based on these SSRs sequences, 6568 primer pairs were designed, of which 72 primers were randomly selected for synthesis and validation, and 38 in-silico polymorphic primers were obtained using in-house perl scripts. A total of 110 primers were screened in the lotus samples and the results showed that 101 primers yielded amplification products, of which 80 were polymorphs. The number of alleles ranged from 2 to 17 and the PIC (polymorphism information content) ranged from 0.19 to 0.87 with a mean value of 0.55. An Unweighted Pair Group Method with Arithmetic Mean (UPGMA) dendrogram based on Jaccard's similarity coefficients showed that the correlation between geographical source and genotype was low. This study describes the distribution of genic-SSRs in the expressed portion of the lotus genome. These genic-SSRs have an important role to play in molecular mapping, diversity analysis, and marker-assisted selection strategies in Nelumbo.

Cloning and expression of an APETALA1-like gene from Nelumbo nucifera.

The objective of this study was to clone the full-length cDNA of the APETALA1 (AP1) gene from lotus and analyze its sequence and expression pattern. The full-length cDNA sequence of the NnAP1 gene was amplified from the petals of Nelumbo nucifera 'Hongxia' using RT-PCR and rapid amplification of cDNA ends. Bioinformatic methods were used to analyze the sequence characteristics of the gene. Quantitative real-time PCR methods were used to investigate the expression pattern of NnAP1 in various organs and during different developmental stages. The cloned full-length NnAP1 cDNA (GenBank accession No. KF361315) was 902 bp, containing a 795-bp open reading frame encoding 264 amino acids with a relative molecular mass of 30,288.4 and an isoelectric point of 9.13. NnAP1 had a MADS-box domain and a K-box domain, which is typical of the SQUA/AP1 gene family. A protein sequence identity search showed that NnAP1 was 75-96% similar to other plant AP1s. Phylogenetic tree analysis indicated that NnAP1 was very closely related to AP1 of Glycine max, suggesting that they shared the same protein ancestor. Quantitative real-time PCR analysis showed that NnAP1 was expressed in various organs during different developmental stages; it had the highest expression in blooming flowers and had trace expression in the young vegetative and flower senescence stages. Our analysis suggests that NnAP1 plays an important role in controlling floral meristem identity and floral organ formation.

Evaluation of in vitro aldose reductase inhibitory potential of alkaloidal fractions of Piper nigrum, Murraya koenigii, Argemone mexicana, and Nelumbo nucifera.

BACKGROUND: Aldose reductase is primarily involved in development of long-term diabetic complications due to increased polyol pathway activity. The synthetic aldose reductase inhibitors are not very successful clinically. Therefore, the natural sources may be exploited for safer and effective aldose reductase inhibitors. METHODS: In the present study, the aldose reductase inhibitory potential of hydroalcoholic and alkaloidal extracts of Piper nigrum, Murraya koenigii, Argemone mexicana, and Nelumbo nucifera was evaluated. The hydroalcoholic and alkaloidal extracts of the selected plants were prepared. The different concentrations of hydroalcoholic and alkaloidal extracts of these plants were evaluated for their goat lens aldose reductase inhibitory activity using dl-glyceraldehyde as substrate. The aldose reductase inhibitory potential of extracts was assessed in terms of their IC50 value. RESULTS: Amongst the hydroalcoholic extracts, the highest aldose reductase inhibitory activity was shown by P. nigrum (IC50 value 35.64±2.7 µg/mL) followed by M. koenigii (IC50 value 45.67±2.57 µg/mL), A. mexicana (IC50 value 56.66±1.30 µg/mL), and N. nucifera (IC50 value 59.78±1.32 µg/mL). Among the alkaloidal extracts, highest inhibitory activity was shown by A. mexicana (IC50 value 25.67±1.25 µg/mL), followed by N. nucifera (IC50 value 28.82±1.85 µg/mL), P. nigrum (IC50 value 30.21±1.63 µg/mL), and M. koenigii (IC50 value 35.66±1.64 µg/mL). CONCLUSIONS: It may be concluded that the alkaloidal extracts of these plants possess potent aldose reductase inhibitory activity and may be therapeutically exploited in diabetes-related complications associated with increased activity of aldose reductase.

Efficient isolation of high-quality RNA from lotus Nelumbo nucifera ssp nucifera tissues.

Nelumbo nucifera is widely used as food, as an ornamental, in medicine, and as packing material; it is also reported to have anti-HIV effects and antioxidant capacity. We sought an improved method for extracting high-quality total RNA from different tissues of N. nucifera. Four methods for RNA extraction were assessed for their ability to recover high-quality RNA applicable for evaluation of polyphenol oxidase (PPO) gene expression profiles. The recovery and quality of the RNA obtained from five different tissues by the best CTAB-LiCl method were evaluated through UV light absorbance. Both A(260)/A(280) and A(260)/A(230) absorbance ratios were more than 2.0; the yield ranged from 59.87 to 163.75 µg/g fresh weight. The brightness of the 28S band was approximately twice that of 18S; the latter was also considered as high-quality RNA. The PPO gene fragment (606 bp) was successfully amplified by RT-PCR, demonstrating the integrity of the isolated RNA. The relative expression levels of the PPO gene based on RT-PCR in five tissues of lotus were: rhizome buds (2.66), young leaves (2.42), fresh cut rhizome (2.02), petals (1.80), and petiole (1.65), using housekeeping gene ß-actin as an internal control. We concluded that the total RNA isolated by this protocol is of sufficient quality for molecular applications.