Cloning and expression analysis on full length CDS of zeaxanthin epoxidase gene in Pseudostellaria heterophylla / 中国中药杂志

Zeaxanthin epoxidase plays an important role in indirect pathway of plant abscisic acid biosynthesis. According to the data of Pseudostellaria heterophylla transcriptome, zeaxanthin epoxidase gene was isolated and named as PhZEP. The results of bioinformatics analysis showed that the coding sequence of PhZEP was 1 263 bp long and encoded 420 amino acids. The putative protein molecular weight was 47.34 kDa and its theoretical isoelectric point was 6.64. The characteristic structure domains were predicted, including binding site of lipoprotein and flavoprotein monooxyenase. A signal peptide was discovered at the N-terminal of amino acids. The Real-time PCR revealed that PhZEP had a higher expression level in leaves than other tissues of P.heterophylla. Highly expressed PhZEP was also observed at 10 d and 40 d tuberous root after flowering. PhZEP presented a different expression after treatment with ABA, fluridone and ABA +fluridone compared to the control. The expression of PhZEP in tuberous root after ABA treatment was close to that in control group, while PhZEP showed significant up-regulation in the fluridone treatment group. In this study, the PhZEP gene from P. heterophylla was cloned and this result has important significance for its functional identification. This research provides a basis for the further analysis on functional mechanism of ABA during development of P. heterophylla.

Cloning and bioinformatics analysis of abscisic acid 8'-hydroxylase from Pseudostellariae Radix / 中国中药杂志

Abscisic acid 8'-hydroxylase was one of key enzymes genes in the metabolism of abscisic acid (ABA). Seven menbers of abscisic acid 8'-hydroxylase were identified from Pseudostellaria heterophylla transcriptome sequencing results by using sequence homology. The expression profiles of these genes were analyzed by transcriptome data. The coding sequence of ABA8ox1 was cloned and analyzed by informational technology. The full-length cDNA of ABA8ox1 was 1 401 bp,with 480 encoded amino acids. The predicated isoelectric point (pI) and relative molecular mass (MW) were 8.55 and 53 kDa,respectively. Transmembrane structure analysis showed that there were 21 amino acids in-side and 445 amino acids out-side. High level of transcripts can detect in bark of root and fibrous root. Multi-alignment and phylogenetic analysis both show that ABA8ox1 had a high similarity with the CYP707As from other plants,especially with AtCYP707A1 and AtCYP707A3 in Arabidopsis thaliana. These results lay a foundation for molecular mechanism of tuberous root expanding and response to adversity stress.

Cloning and expression analysis of four carotenoid cleavage dioxygenases genes from Pseudostellaria heterophylla / 中国中药杂志

To investigate the molecular mechanism of quality formation of Pseudostellaria heterophylla, the carotenoid cleavage dioxygenases (CCDs) genes were cloned from the transcriptome database of P. heterophylla, and analyzed them with bioinformatics analysis and expression analysis. The sequence length of four new gene were 1 617, 1 461, 1 746, 1 875 bp, and subsequently, named as PhCCD1,PhNCED2,PhNCED3 and PhCCD4 according to its genetic relationship with Arabidopsis thaliana. The sequence analysis showed that four new gene were all containing REP65 domains and binding sites of ferrous ion, such as histidine, glutamates and aspartates. Analysis phylogeny showed that PhNCED2 and PhNCED3 were the cluster of NCEDs, PhCCD1 and PhCCD4 were the cluster of CCDs. In addition, PhCCD1 and AtCDD1 of Arabidopsis thaliana, PhCCD4 and AtCCD4 of A. thaliana,PhNCED2, PhNCED3 and AtNCED3 of A. thaliana have high similarities. Analysis of real-time fluorescence quantitative showed that PhNCED2 and PhNCED3 were expressed mainly in underground part, the expression quantity of PhNCED2 reached the highest in fibrous root, PhNCED3 keeps higher in phloem and xylem, it may be the key enzymes of ABA biosynthesis genes. Moreover,PhCCD1 and PhCCD4 were expressed mainly in aerial part,the expression quantity of PhCCD1 reached the highest in leaf,PhCCD4 keeps higher in stem and leaf.It may be involved in the biosynthesis of carotenoids for P. heterophylla. The study obtained CDDs gene of P. heterophylla for the first time,this would lay the foundation of developing the response mechanism of P. heterophylla about external stress further,and then exploring the biological approach of quality formation in P. heterophylla.