| Narcissus tazetta-L.is a member of the family Amaryllidaceae. The development of the narcissus industry was restricted for the critical problems of shortages in available resource and colorful varietie. Using Jinzhanyintaim, Baihua narcissus and Huanghua narcissus as test materials, the full-length cDNA and DNA sequence of phytoene synthase(PSY), phytoene desaturase(PDS), ζ-carotene desaturase(ZDS), lycopene-β-cyclase (LCY-β) and lycopene-ε-cyclase(LCY-e) were cloned by RACE-PCR and RT-PCR in this study. The gene expression patterns in the color formation of chinese narcissus were also analyzed by Real Time PCR. The study will provide a foundation to know more about the color formation of Narcissus tazetta L., as well as facilitate color-enriched and productivity-enhanced development of the narcissus.The major results obtained from the study were summarized as followed:1Cloning and analysis of NtPSYgene from Narcissus tazetta L.The open reading frame (ORF) of PSY were obtained from the three kinds of color type narcissus, the ORF length of NtPSYwas1272bp-1275bp encoding423-424amino acids. The genes were designated as NtPSYJ,NtPSYW(GenBank accession No.JN991013) and NtPSYY(JN991014) respectively. Comparing with other PSY genes in Narcissus genus, there were some replacements and missing amino acids. The genome DNA sequence of PSY genes in three color type narcissus were cloned, the length ranged from2667bp to2840bp, the GenBank accession numbers were No.JN991017(Jinzhanyintai), No.JN991016(Baihua) and No.JN991015(Huanghua). Five introns (A-E) were found in each DNA sequence, the length ranged from81bp to926bp. Sequence nalysis showed that the splicing sites of four introns (A,B,D,E) comformed to the GT-AG rule and introns C were consistented with GC-AG rule.2Cloning and analysis of NtPDS from Narcissus tazetta L,.The open reading frame (ORF) of PDS were obtained from the three kinds of color type narcissus, The ORF length of PDS were1713bp encoding570amino acids. The genes were designated as NtPDSJ, NtPDSW(accession number:JQ797376) and N/PDSY (JQ797377) respectively. Comparing with other PDS genes in Narcissus genus, there were some replacements and missing amino acids as well. The part of genome DNA sequence of PDSgenes in the three color type narcissus were cloned, a length ranged from7167bp to7199bp, and the GenBank accession number are No. JQ797382(Jinzhanyintai), No. JQ797383(Baihua) and No. JQ797384(Huanghua).Ten intons(A-J) were found in each DNA sequence, the length ranged from73bp to1489bp. Sequence analysis showed that the splicing sites of introns comformed to the GT-AG rule.3Cloning and analysis of NtZDS from Narcissus tazetta L.The open reading frame (ORF) of ZDSwas obtained from the three kinds of color type narcissus, the ORF length of Nt ZDS was1725bp encoding574amino acids. The genes were designated as NtZDSJ, NtZDSW(GenBank accession No. JQ797378) and NtZDSY(JQ797379) respectively. Comparing with other ZDS genes in Narcissus genus, there were some replacements and missing amino acids. The part of genome DNA sequence of ZDS genes in the three color type narcissus were cloned, the length ranged from7382bp to9187bp, the GenBank accession number are No. JQ797385(Jinzhanyintai), No. JQ797386(Huanghua). Eleven introns (A-K) were found in each DNA sequence, the length ranged from87bp to2695bp. Sequence analysis showed that the splicing sites of four introns comformed to the GT-AG rule and intron A, C, F size are quite different.4Cloning and analysis of NtLCY-βfrom Narcissus tazetta L.The open reading frame (ORF) of LCY-βwere obtained from the three kinds of color type narcissus, the ORF length of NtLCY-β were1715bp encoding504amino acids. The genes were designated as NtLCY-βJ, NtLCY-βW(GenBank accession No. JQ797380) and MICY-βY (JQ797381) respectively. Comparing with other LCY-β genes in Narcissus genus, there were replacement and missing amino acid. Cloning from genomic DNA sequence of NtLCY-β showed that was no intron and the homologous rate are over98%compared with their cDNA.5Cloning and analysis of NtLCY-ε5'RACE primers were designed according to the known NtLCY-ε3'nucleotide seqence from GenBank, the5'nucleotide sequence was obtained by RACE from Jinzhanyintai narcissus. The full length cDNA of NtLCY-ε was1971bp with an opening reading frame of1659bp (GenBank accession No.JQ282903) encoding a protein with532amino acids(Mw=59771.1, pI=6.15). The5-terminal untranslated region (UTR) was153bp and the3-UTR was lllbp. The ORF sequence of LCY-ε gene in Baihua (No.JQ282902) and Huanghua narcissus (No.JQ282904). According to the ORF sequence of LCY-ε gene in Jinzhanyintai, we obtained the ORF sequences of LCY-ε gene in Baihua (No.JQ282902) and Huanghua narcissus (No.JQ282904). The nucleotide and amino acids from the three kinds of color type narcissus were similar. Replacements were found in the deduced amino acid sequence of LCY-ε gene in Narcissus genus plant with homologous comparison. The deduced amino acid sequence of NtLCY-ε was compared with that of other higher plants and phylogenetic tree was was constructed.4Analysis the expression of PSY,PDS,ZDS and LCY-βin the color formation of Narcissus tazetta L. by qRT-PCRNtACTIN was used as internal control gene, the expression patterns of NtPSY, NtPDS, NtZDS and NtLCY-β in the bud stage, alabastrum stage, early flowering stage and full-bloom stage were carried out. The results indicated that all of the four genes in Jinzhanyintai and Huanghua narcissus expressed in four stages, with higher expression level in the intermediate stages, while much lower in Baihua narcissus, especially the expression of PSY gene is almost undetectable. Additionally, the expression rate of PDS and ZDS in Baihua narcissus appears to be increasing at certain stage, but it still lower than that of other flowers. To sum up, the higher expression level of PSY,PDS,ZDS and LCY-β in Jinzhanyintai and Huanghua narcissus may cause the yellow color of the flower. Therefore, the regulation of these genes may regulate and control the color of Narcissus tazetta L. flower. |
PDF Full Text Request