ABSTRACT
Calcium signaling plays a critical role in response to various abiotic and biotic stresses in plants. Preliminarily evidence showed that calcium signaling perceived and transduced the harmful signaling generated from continuous cropping stress in R. glutinosa. To investigate the roles of calcium signaling in continuous cropping injury formation, the key genes involved in calcium signaling transduction were identified in R. glutinosa transcriptome through bioinformatic methods. Furthermore, the calcium ion concentration in both normal and continuous cropping R. glutinosa root cells were measured by potassium pyroantimonate precipitation and calcium fluorescence method. As a result, a set of 84 calcium signaling-related genes, including 5 CaMs, 12 CBLs, 21 CDPKs, 21 CIPKs, 16 CMLs, and 9 CRKs were captured in R. glutinosa transcriptome. The analysis of expression profile in continuous cropping compared to normal growth R. glutinosa indicated that continuous cropping stress significantly increased the expression of calcium signaling-related genes in continuous cropping R. glutinosa. At the same time, the abundance levels of 12 calcium signaling-related genes quantified by qPCR further validated the high expression of calcium signaling-related genes presented in continous cropping R. glutinosa. In addition, the continuous cropping condition significantly promoted the accumulation of intracellular calcium ions in R. glutinosa based on two methods of potassium pyroantimonate precipitation and calcium fluorescence. This study verified the possible roles of calcium signaling in the formation of continuous cropping injury on molecular and cellular level, which lays a solid foundation for illuminating formation mechanism of continuous cropping injury on molecular level.
ABSTRACT
Objective: To clone RgMed6 gene, which coded a subunit of mediator complex, from Rehmannia glutinosa, and to analyze the characteristics of protein sequence and gene expression. Methods: The transcriptional EST database of R. glutinosa was used to search analogs of AtMed6 gene by BLAST, the full-length open reading frame (ORF) of RgMed6 was obtained by assembling the ESTs. BLASTp, the online analysis tool of NCBI was used to get the homologous sequences of RgMed6, and MAFFT has been performed to analyze the multiple sequence alignment. Phylogenetic tree has been constructed using MEGA 6.0 software. Quantitative RT-PCR has been applied to detecting the transcription level of RgMed6 in five tissues as well as in tuberous roots or leaves under three stresses. Results: The cDNA sequence of RgMed6 containing 924 bp was obtained. The ORF of RgMed6 was 771 bp encoding 256 amino acids, which had typical structural domains and a potential nuclear localization signal (NLS). RgMed6 showed the highest expression level in leaves, followed by buds, but very weak in stems. The transcription level of RgMed6 mRNA was reduced under continuous cropping conditions in tuberous roots while it increased under salinity stress in leaves. Conclusion: RgMed6, a mediator subunit gene from R. glutinosa has been obtained for the first time, which can lay the foundation for further studies about its molecular function in development and responses to stress.
ABSTRACT
Objective: To determine the optimal concentration of hormone and hygromycin for seedling regeneration of Rehmannia glutinosa. Methods: Using the young leaf of sterile plantlet from R. glutinosa as explants, we conducted the transformation mediated by Agrobacterium tumefaciens, analyzed the efficiency of hygromycin and acetosyringone (AS) on resistant callus induction and plant regeneration. Results: The concentration of hygromycin had greatly affected the production of resistant callus and seedlings. The critical concentration of hygromycin on the resistant callus induction and shoot regeneration were 9 and 6 mg/L, respectively. The optimal concentration of hygromycin for the genetic transformation of Wen 85-5 was 12 mg/L. Adding 100 μmol/L AS could greatly improve the transformation efficiency of R. glutinosa. It was confirmed by PCR detection of hpt gene and GUS staining that the foreign gene was integrated into the genome of R. glutinosa. Conclusion: The stable genetic transformation system of R. glutinosa is established, which lays the foundations for the research on molecular pharmacognosy and genetic improvement.