Cloning And Expression Analysis Of The Enzyme Genes Of Flavonoids Biosynthesis In Narcissus Tazetta L.

Narcissus tazetta L. which is lack of variety and of simplex color, has lowefficiency on color breeding and few accumulations about its related reseach. In thisresearch，the sequence of chalcone synthase (CHS), chalcone isomerase (CHI) andflavonoid3-hydroxylase (F3H) of flavonoid biosynthetic pathway, and Actin asreference gene in Jinzhanyintai、Huanghua Narcissus and Baihua Narcissus werecloned by RT-PCR and RACE. The expresstion level of CHS, CH I and F3H in fourdevelopmental stages and different color styles of Narcissus tazetta L.were analyzdby fluorescence quantitative PCR. So the reseach laid the foundation on its researchabout genetic transformation to cultivate different color of narcissus.1. Cloning and analysis of NtCHSA full length cDNA of NtCHSY (accession number: JQ796709)which is1501bp was cloned from the petals of huanghua Narcissus by RT-PCR and RACE.，and itcontains a open reading frame encompassed1173bp encoding a polypeptide of390amino acids，a5′untranslated region of112bp and a3′untranslated region of216bp.The ORF sequence of CHS from Baihua Narcissus and jinzhanyintai were cloned by the ORFprimer of NtCHSY,which were named as NtCHSW (accession number: JN227883) andNtCHSJ (accession number: JQ796708), there are differences in amino acid position30,50,78,108,261,322and383among NtCHSW, NtCHSJ and NtCHSY.Sequencing analysis showed that the amino acid sequence of Baihua Narcissus was98.97%,98.72%,85.38%,84.69%,85.64%,81.36%,86.51%,81.73%homologouswith CHS genes from Huanghua Narcissus, Jinzhanyintai, Petunia x hybrid,Anthurium andraeanum, Camellia sinensis, Arabidopsis thaliana, Vitis labrusca,Lilium speciosum, respectively. Three genomic sequences coding for this gene wereisolated from three color types of Narcissus tazetta L., containing two exons and oneintron.The splicing sites of the intron were comformed to the GT-AG rule and the genomeaccession number of NtCHSW, NtCHSJ and NtCHSY were JQ796710, JQ796711andJQ796712, respectively.2. Cloning and analysis of NtCHIA full length cDNA of NtCHIW (accession number: JN227884) which is889,was cloned from the petals of Baihua Narcissus by RT-PCR and RACE, and itcontains a open reading frame encompassed735bp encoding a polypeptide of244 amino acids, a5′untranslated region of18bp and a3′untranslated region of136bp.The ORF sequence of CHI from Huanghua Narcissus and Jinzhanyintai were cloned by theORF primer of NtCHIW, which were named as NtCHIJ (accession number: JN968465)and NtCHIY (accession number: JN968464). There are differences in amino acidposition7,11,25,64,80,100,139,179and234among NtCHSW, NtCHSJ andNtCHSY. Sequencing analysis showed that the amino acid sequence of BaihuaNarcissus was93.03%,93.44%,64.75%,55.42%,50.58%,59.43%, and58.20%homologous with CHI genes from Jinzhanyintai, Huanghua Narcissus, Allium cepa,Elaeis oleifera, Vitis labrusca, Oryza sativa Japonica, Arabidopsis thaliana,respectively. Three genomic sequence coding for this gene were isolated from threecolor types of Narcissus tazetta L., containing three exons and two introns. The splicingsites of the intron were comformed to the GT-AG rule and the genome accession number ofNtCHIW, NtCHIJ and NtCHIY were JQ796713, JQ796714�'�JQ796715,respectively.3. Cloning and analysis of NtF3HA full length cDNA of NtF3HW (accession number: JN613146) which is1335was cloned from the petals of Baihua Narcissus by RT-PCR and RACE, and itcontains a open reading frame encompassed1098bp encoding a polypeptide of365amino acids，a5′untranslated region of42bp and a3′untranslated region of195bp.The ORF sequence of F3H from Huanghua Narcissus and jinzhanyintai were cloned by theORF primer of NtF3HW, which were named as NtF3HJ (accession number: JQ388492)and NtF3HY (accession number: JQ388493). There are differences in amino acidposition45,51,54,123,290,310,311and329among NtF3HW, NtF3HJ and NtF3HYSequencing analysis showed that the amino acid sequence of Baihua Narcissus was95.07%,93.03%,80.71%,78.05%,77.11%,73.77%,77.57%and74.80%homologous with F3H genes from Jinzhanyintai, Huanghua Narcissus, Allium cepa,Lilium speciosum, Petunia x hybrid, Arabidopsis thaliana, Nicotiana tabacum,Persea Americana, respectively.4. Cloning and analysis of the full length cDNA of NtActinA gene, named NtActin, was cloned from Narcissus tazetta L. petals by RT-PCRand RACE. The full length cDNA of NtActin (Accession number:JN204912) is1404bp, and it contains a open reading frame encompassed1134bp encoding a polypeptide of377amino acids (Mw=41.797kD, pI=5.31), a5′untranslated region of34bp and a3′untranslated region of236bp. Sequencing analysis showed that theamino acid sequence of NtActin was97.88%,97.61%,97.35%,97.61%,97.88%,97.61%homologous with Actin genes from Setaria italica, Zea may, picea rubens,hordeum vulgare, Arabidopsis thaliana, Stevia rebaudiana, respectively.5. Analysising the expression of NtCHS, NtCHI and NtF3HThe expression analysises of NtCHS, NtCHI, and NtF3H in the budding stage,alabastrum stage, early flowering stage and fullbloom stage were carried out byqRT--PCR and NtActin was used as reference gene. QRT-PCR results indicated thatthe three enzyme genes were active during floral development. The expression levelof NtCHS、NtCHIY and NtF3H reached the maximum in the budding stage and in thedownward trend in later stage, whereas the expression level of NtCHIW and NtCHIJreached the maximum in the alabastrum stage; which shows that the three genes havecertain collaborative relationship in the chage of color.