Purification, modeling and structural insights of calmodulin-binding receptor like cytoplasmic kinase 2 from Oroxylum Indicum.

Calmodulin binding receptor like cytoplasmic kinase 2 (CRCK2) belongs to the family of receptor like kinases (RLKs) which is mainly implicated in pathways associated with the stress responses in plants. The protein from the stem of Oroxylum indicum was isolated and purified using anion-exchange followed by gel filtration chromatography. The purity of protein was checked using SDS-PAGE, which showed a single band of 50 kDa. The purified protein was identified as CRCK2 using MALDI-TOF. Using I-TASSER, a bioinformatics tools, the model of protein was constructed and its secondary structure was predicted using VADAR. The secondary structure content was also determined by far-UV CD, which indicated that the CRCK2 is mainly ß-sheet dominating protein (43% ß-sheet). The secondary structural content predication from computational method is in close agreement with the result obtained by CD spectropolarimeter. This study validates I-TASSER model for determination of structure of a protein. Moreover, stability of CRCK2 was monitored against heat- and guanidinium chloride (GdmCl)-induced denaturation by using circular dichroism (CD) and fluorescence spectroscopy. Denaturation curve analysis gave values of 2.88 ±â€¯0.12 kcal mol-1and 4.11 ±â€¯0.09 M for ∆°GD (Gibbs free energy change at 25 °C) and Cm (midpoint of denaturation), respectively. It has been observed that purified CRCK2 is quite stable protein against both heat-induced as well as GdmCl-induced denaturation. This is very first report of purification and biophysical characterization of CRCK2 protein from medicinal plant O. indicum.

Single-nucleotide polymorphisms(SNPs) in a sucrose synthase gene are associated with wood properties in Catalpa fargesii bur.

BACKGROUND: Association study is a powerful means for identifying molecular markers, such as single-nucleotide polymorphisms (SNPs) associated with important traits in forest trees. Catalpa fargesii Bur is a valuable commercial tree in China and identifying SNPs that associate with wood property would make a foundation of the marker-assisted breeding in the future. However, related work has not been reported yet. RESULTS: We cloned a 2887 bp long sucrose synthase (SUS) gene from the genome of C. fargesii, which is a key enzyme in sucrose metabolism and also associated to wood formation in trees, coding 806 amino acids that expressed mainly in young branches, xylem, and leaves according to real-time quantitative PCR. Then we identified allelic variations of CfSUS associated with nine wood quality associated traits in Catalpa fargesii Bur. Totally, 135 SNPs were identified through cloning and sequencing the CfSUS locus from a mapping population (including 93 unrelated individuals) and 47 of which were genotyped as common SNPs (minor allele frequency > 5%) in the association population that comprised of 125 unrelated individuals collected from main distribution area. Nucleotide diversity and linkage disequilibrium (LD) analysis showed CfSUS has a relative low SNP diversity (πT = 0.0034) and low LD (r2 dropped below 0.1 within 1600 bp). Using the association analysis, we found 11 common SNPs and 14 haplotypes were significantly associated with the traits (false discovery rate, Q<0.1), explaining 3.21-12.41% of the phenotypic variance. These results provide molecular markers above associated with wood basic density, pore rate, and six other traits of wood, which have potential applications in breeding of Catalpa fargesii Bur. CONCLUSION: We first cloned a SUS gene in C. fargesii, then identified several SNPs and haplotypes that associated with wood properties within this gene, suggesting CfSUS participates in the wood formation of C. fargesii. Moreover, molecular markers we identified in this study may be applied into marker-assisted breeding of C. fargesii in the future.

Identification, cloning and characterization of a GDSL lipase secreted into the nectar of Jacaranda mimosifolia.

The presence and function of several proteins secreted into floral nectars has been described in recent years. Here we report the presence of at least eight distinct proteins secreted into the floral nectar of the tropical tree Jacaranda mimosifolia (Bignoniaceae). Steps were initiated to identify and characterize these proteins in order to determine potential functions. The N-terminal sequence of the major Jacaranda nectar protein, JNP1, at 43 kDa contained similarity with members of the plant GDSL lipase/esterase gene family. Based upon this sequence, a full-length cDNA was isolated and predicted to encode a mature protein of 339 amino acids with a molecular mass of 37 kDa. Both raw nectar and heterologously expressed JNP1 displayed lipase/esterase activities. Interestingly, J. mimosifolia flowers produce an opaque, white colored nectar containing spherical, lipophilic particles approximately 5 microm in diameter and smaller. GS-MS analysis also identified the accumulation of free fatty acids within the nectar. It is proposed that JNP1 hydrolyzes Jacaranda nectar lipids with the concomitant release of free fatty acids. Potential functions of JNP1 in relation to pollinator attraction and prevention of microbial growth within nectar are briefly discussed.