| Plants biosynthesize and accumulate a vast array of the small organic molecules which are known as secondary metabolites for the defense against certain biotic and abiotic stresses. Amongst these, flavonoids, a class of plant phenolics have been extensively studied due to their tremendous chemical diversity and ubiquitous occurrence. However, these studies mainly concentrated on model plants, ornamental plants, and agricultural plants. Few studies have been conducted on wild stress-resistant plants. Reaumuria trigyna is a highly xeric small shrub. It is endemic to the Eastern Alxa-Western Ordos area. Thus, R.trigyna has good adaptability to the high saline and hyper-drought conditions, it is a suitable material for researcher to study the tolerance mechanisms of secondary metabolism.In this study, we screened out nine DEGs (Differentially Expressed Gene,|log2Ratio|≥1) in the flavonoid biosynthetic pathway based on the transcriptome data under NaCl-treatment in R.trigyna. Analysis the transcriptional levels of the nine genes and the composition of flavonoid under the treatments with different levels of NaCl and UV-B stresses. We cloned the full-length cDNA sequence of RtLDOX and RtPAL1 in flavonoid biosynthetic pathway from R.trigyna, and conducted sequence, phylogenetic analyses, expression level and protein activity of the two genes. We introduced RtLDOX into the Arabidopsis LDOX mutant line. Analysis the catalyze ability and the tolerance of RtLDOX responded to abiotic stresses. The results of the this study are as follow:1. We identified 118 unigenes annotated as genes encoding enzymes related to flavonoid biosynthetic biosynthesis, such us the flavonol biosynthetic pathway, isofavonoids biosynthetic pathway and anthocyanin biosynthetic pathway. Of the annotated unigenes,47 were annotated as members of families related to the flavonol biosynthetic pathway. Of those 47 unigenes, about 70% were upregulated under NaCl treatment. We screened 665 unigenes that were annotated to MYB, WD40, bHLH and WRKY transcription factor families, and predicted 13 unigenes were related to regulate the flavonoid biosynthetic pathway.2. Laboratory experiments were conducted to monitor changes in gene expression and accumulation of total polyphenols, total flavonols, and antioxidants under NaCl and ultraviolet-B (UV-B) radiation treatments. The expressions of genes related to the flavonol biosynthesis pathway (RtC4H, RtCHS, RtF3H3, RtFLS1, RtFLS2, RtF3’5’H, RtF3’H, RtOMT, and RtMYBF1) increased under NaCl and UV-B treatments. The antioxidant activity of flavonol extracts in R.trigyna were higher than it in Arabidopsis. Treatments with NaC1 and UV-B also increased the total flavonols and antioxidant contents. Several flavonols, including rutin, hyperoside, isorhamnetin-3-O-neohes peidoside and myricetin, were induced in response to NaCl and UV-B stresses. Overall, our results showed that the expression of genes related to flavonol biosynthesis and flavonol contents increased in R.trigyna under NaCl and UV-B stresses. These findings provide new information about the relationships between environmental factors and flavonoid accumulation in R. trigyna.3. The full-length cDNA of R. trigyna LDOX (RtLDOX) was cloned by RT-PCR and RACE (Rapid amplification of cDNA ends), the full-length sequence contained a 1,089-bp ORF encoding a protein of 362 amino acid. To identify putative cis-regulatory elements in the RtLDOX promoter, we uncovered the RtLDOX promoter harbors many c/s-acting elements that might be recognized and bound by transcription factors related to stress response, such as MYB, LTRE and W-Box. Using qPCR, we found the expression level of RtLDOX were induced by NaCl, PEG,4℃ and UV-B stress, and RtLDOX was more highly expressed by NaCl than other stresses with about 25-fold change. We then constructed the prokaryotic expression vector pET32a-RtLDOX to transform E. coli. After overexpression and purification of the RtLDOX recombinant protein, we used ultra-performance liquid chromatography (UPLC) to identify RtLDOX recombinant protein was able to replace flavanone-3-hydroxylase (F3H), another dioxygenase in the flavonoid pathway, to convert naringenin to dihydrokaempferol in vitro.4. We introduced RtLDOX into the Arabidopsis LDOX mutant line ttll-11. R. trigyna LDOX can complement the Arabidopsis LDOX mutant transparent testall (ttll-11), which has reduced proanthocyanin (PA) and anthocyanin levels in seeds, to accumulate these two compounds. Under NaCl stress, the biomass and antioxidant activities of RtLDOX transgenic lines were superior to those of tt11-11 and wild-type Arabidopsis. According to a DPPH assay, the free radical-scavenging activity of transgenic lines, expressed as the percentage of antioxidant activity, increased dramatically under salt stress. Thus, the tolerance of RtLDOX transgenic lines under salt stress was related to the increased antioxidant activity of flavonoids.5. Under UV-B stress, we found the enzyme activity of RtPAL was induced dramatically, the highest level was about 2.5-fold relative to control. The expression level of RtPAL1, RtPAL2 and RtPAL3 under UV-B stress were identified by qPCR. The results showed that the expression level of RtPAL2 or RtPAL3 not changed significantly between control and stress samples. However, the expression level of RtPAL1 was induced dramatically under UV-B stress. We presumed that RtPAL1 is a key gene to regulated the enzymatic activity of RtPAL, it may be play an important role for R.trigyna resistance to UV-B stress. We cloned the full-length cDNA of RtPALJ, the full-length sequence contained a 2,055-bp ORF encoding a protein of 684 amino acid. The eukaryon expression vector pPZP221-RtPAL1 was constructed and we transform it to Agrobacterium, then we will use Agrobacterium-mediated method to introduce RtPAL1 into plant to identity the function of the gene in response to abiotic stress. |
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