Cloning And Expression Analysis Of Key Genes Of Anthocyanin Compounds In Analysis Of Hippophae

Iris halophila is a promising horticulture plants in saline soils because its salt and drought tolerance.I. halophila and its variety I. halophila var. sogdiana have distinct flower colors, i.e. the former bears yellow flowers while the latter bears various blue or violet flowers. In the view of molecular genetics,I. halophila seems like a natural knock-out mutant of I. halophila var. sogdiana in flower color, so these species are ideal plant material to dissect the anthocyanin biosythesis and flower color pigmentation. In order to elucidate the molecular mechanism of flower pigmentation and clone genes conferring blue flower color for future gene engineering, we carried out a comparative analysis of inner perianth specific transcriptiome, and found some differently expressed genes related to anthocyanin biosythesis. Based on this research, we cloned some cDNA segments of important genes in anthocyanin biosythesis, namely chalcone synthase (CHS), chalcone isomerase (CHI), and flavonoid 3’,5’-hydroxylase like (F3’5’H-like). The expression level of these genes by real-time PCR revealed that F3’5’H-like and CHS genes are up-regulated in I. halophila var. sogdiana compared with that in I. halophila and this is likely the main reason for different flower color in these 2 species.In this study, total RNA was extracted from inner perianth of young flower of both I. halohpila and I. halophila var. sogdiana, respectively. RNAs were then sequenced by Illumina high-throughput method, and assembled contigs were annotated by BLASTX search using NCBI NR protein database and Swiss-prot database. Differentially expressed genes were screened by Reads Per Kilobase per Million mapped reads (RPKM).171,173 transcripts were recovered from the transcriptome, and the mean length of transcripts was 479bp. These transcripts mapped to 35,543 Unigenes with a mean length of 750bp. Comparative analysis of expression level of house-keeping genes shuch as actin, tubulin, ubiquitin, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase and eukaryotes translation initiation factors showed that 2 plant materials have similar expression pattern. Among these Unigenes,74 were related to anthocyanin biosythesis, which belongs to 25 genes. Based on RPKM analysis,6 genes relating to anthocyanin biosythesis were expressed more than 10 fold in I. halophila var. sogdiana than that in I. halophila, with F3’5’H-like a RPKM ratio up to 155.17.Genes were cloned by retro-transcriptional PCR using gene specific primers designed according to the Unigene sequences assembled in the transcriptome analysis, and CDS segments of differentially expressed genes were cloned. The cloned sequence were analysed by a translated BLAST methods to find their homologs and correct reading frames. Then the partial polypeptide segments were aligned to their corresponding homologs by CLUSTAL and/or MUSCLE methods, and phylogenetic trees were built with MEGA software. Real-time PCR assays of each cloned genes were carried out to verify the expression level both in I. halophila and I. halophila var. sogdiana, using tubulin as reference sequence.The results showed that 326bp of CHS segment 1,420bp of CHS segment 2, 461bp of CHI segment, and 496bp of F3’5’H-like segment were cloned from I. halophila, and 311bp of CHS,457bp of CHI, and 498bp of F3’5’H-like gene segments were cloned from I. halophila var. sogdiana. The expression level analysis by real-time PCR indicated that CHS are up-regulated by 5.27 times, and F3’5’H-like 3.71 times, in I. halophila var. sogdiana than that in I. halophila, respectively.Sequence analysis revealed that F3’5’H-like gene in Iris plants are different from classical F3’5’H gene in other plants, which belongs to CYP75A subfamily of cytochrome P450 super family. It exhibited 57% amino acid sequence identity with F3’5’H-like isolated from Vanda coerulea (Orchidaceae), which belongs to CYP76AB subfamily, and it is a new member of this subfamily. Transcriptome analysis revealed its transcript is much more abundant in I. halophila var. sogdiana than that in I. halophila, and this result was confirmed by real-time PCR. Accordingly, it may be a novel type of blue flower relating gene.