为深入挖掘中国水仙转录组中萜类合成相关基因,了解中国水仙萜类代谢途径和分子调控机制,该研究以'云香'水仙为材料,在其盛花期花瓣与副冠转录组测序的基础上,筛选已注释的萜类合成途径基因并利用NCBI blastn进行再注释,通过对部分候选基因的表达量与生物信息分析进一步筛选出代表基因,采用RT-PCR技术克隆了水仙异戊烯基焦磷酸异构酶基因NaIDI,并对其蛋白序列与特异性表达进行分析.结果表明:(1)Blast比对筛选得到52个与萜类合成上游途径相关的Unigenes,二次筛选获得11个显著差异表达的代表基因.(2)NaIDI基因开放阅读框(ORF)长度为858 bp,编码285个氨基酸,其氨基酸序列与'金盏银台'水仙相似度97.19％;亚细胞定位预测显示该基因定位在叶绿体;系统进化分析表明其与芦笋亲缘关系比较近.(3)实时荧光定量分析结果表明,N aID I基因在水仙开花的不同时期和不同组织器官中差异表达显著,且在盛花期时副冠中的表达量最高,与中国水仙挥发性萜类化合物在开花不同时期和不同组织器官中的表达规律一致,表明NaIDI在萜类代谢中发挥着一定作用.

Aimed to study the characteristics and functions of ACS gene,in the present study,we cloned a 1-aminocyclopropane-1-carboxylate synthetase gene named NtACS 1 (GeneBank KX082936 )based on the RNA-Seq database from the flower of Narcissus tazetta var.'Yunxiang'using RT-PCR method.The length of the open reading frame (ORF)of ACS is 552 bp,encoding 183 amino acids coupled with a molec-ular weight of 20.6 kDa and theoretical isoelectric point of 6.30.qRT-PCR analysis showed that the rela-tive expression level of NtACS 1 both in petals and coronas are decreased gradually along with the aging of flower.Moreover,the expression data of NtACS 1 gene were consistent with those obtained by RNA-Seq, implied that the NtACS1 protein as an ACC synthetase might play a role in the catalytic system 1 of ethyl-ene biosynthesis.Furthermore,sense plant expression vectors of NtACS 1 were successfully constructed with agrobacterium mediated transformation,and 6 positive transgenic tobacco plants were ultimately ob-tained.Our current study will lay an experimental foundation for the future application of the genetic transformation to prolong florescence of 'Yunxiang'.

WRKY transcription factors play an important adjusting role in the process of plant growth and development, hormone signal transduction and abiotic stress response.To clarify the gene Narcissus tazetea var.'Yunxiang', we cloned the NtWRKYY1 gene (GenBank accession No.KX056495) based on 'Yunxiang', analyzed the gene sequence features, evolutionary relationship and expression characteristics, constructed the expression vector before transformed into tobacco.Sequence analysis revealed that the length of NtWRKYY1 gene open reading frame (ORF) is 510 bp, encoding a polypeptide of 169 amino acids.Multiple alignments and phylogenetic analysis showed that NtWRKYY1 protein containing one WRKY consecutive structural domain and C2H2 type zinc finger(Cx4Cx23HxH)belong to Ⅱc sub-group of WRKY transcription factor together with Arabidopsis AtWRKY57.Tissue-specific expression and temporal and spatial expression showed that NtWRKYY1 in 'Yunxiang' had a much higher expression in flowers than that in roots and leaves, and a rising express trend in petal and corona of alabastrum stage, early flowering stage, full bloom stage and faded stage.NtWRKYY1 can be induced by abscisic acid (ABA), high temperature, drought and saline, and restrained by methyl jasmonate (JA) through hormones and abiotic stresses analyzing.We can concluded that NtWRKYY1 gene may play a regulating role during the flower senescence process in 'Yunxiang', involve in the hormone signal transduction of ABA, JA and the abiotic stress regulation of high temperature, drought and saline at the same time.In addition, we constructed overexpressing vector pMDC140-NtWRKYY1 using In-Fusion cloning technique, transformed into tobacco by the method of Agrobacterium though leaf disc transformation.The carrier of PCR and GUS staining results indicated the resistant plantlets were positive.This study will make a good foundation for further exploring the function of the WRKY transcription factor in 'Yunxiang'.

Senescence-associated protein (SSA) gene plays an important role in fruit ripening and senescence.Papaya fruits (C.papaya L.cv.‘Daqing No.8') were used as material,the full-length cDNA of SSA was isolated by rapid amplification ofcDNA ends (RACE) and named as CpSSA (GenBank:KX885408).Bioinformatics analysis indicated that the cDNA of CpSSA is 1 203 bp in length,including a complete open reading frame (ORF) of 420 bp.CpSSA encode a protein of 139 amino acids,with the molecular mass of 15.172 4 kD and an isoelectric point of 9.61.The amino acid sequence of the protein had high homology with SSA from peach,grape,poplar,and spinach.Phylogenetic analysis indicated that CpSSA closely related to SSA from grape.Real-time PCR analysis revealed that the expression of CpSSA was significantly increased in 6 h and 12 h in ethylene-treatment and kept a lower expression level in MCP-treatment.Meanwhile,the expression of CpSSA changed as the papaya fruit ripening and senescence,indicating that CpSSA may be involved in the fruit senescence in papaya.

A sequence of one NA C gene from ‘Yunxiang’ transcriptome library was cloned using PCR technology.Sequence analysis indicates that the eDNA of NAC,named NtNAC3153 (GenBank accession number:KU375569),contained an open reading frame of length 822 bp (encoding 273 amino acids,molecular weight:30.903 9 ku).Physiochemical properties analysis and multiple sequence alignment showed that NtNAC3153 protein was a hydrophilic protein which was free of trans-membrane region and had a conserved N-terminal domain.Phylogenetic analysis indicated that the NtNAC3153 had the closest relationship with Musa acuminata MaNAC,Brachypodium distachyon BdNAC and Setaria italica SiNAC.Real-time PCR analysis showed that the expression of NtNA C3153 gene could be induced by ABA,salt stress and high temperatures.NtNA C3153 may be involved in the regulation of resistance mechanisms of ‘Yunxiang’.

为探究番木瓜NAC转录因子的序列特征及功能,以‘大庆7号’番木瓜果肉为试验材料,采用RT-PCR克隆出2个不同的NAC类基因,命名为CpNAC1(GeneBank KT364871)和CpNAC2(GeneBank KT372241),其开放阅读框(ORF)长度分别为609 bp和805 bp,分别编码202个和268个氨基酸,其N端含有NAM保守结构域.采用实时荧光定量PCR研究其在乙烯利及清水对照处理后果实不同成熟时期中的表达情况,结果发现,CpNAC1和CpNAC2基因随着处理后时间的增加,表达量呈先下降后缓慢上升的趋势,且均与果实成熟呈负相关.但CpNA C1表达趋势与果实成熟过程中乙烯的表达量相反,受乙烯抑制降低表达量,从而参与了番木瓜果实的成熟衰老进程,而CpNAC2基因在乙烯处理后番木瓜果实中表达量与对照处理相比没有显著变化,说明CpNAC2基因不是通过乙烯信号传导途径来调控果实成熟.

HDS is one of the important enzymes in the pathway of plant MEP. In this study, Huanghua Ⅱ and Jinzhanyintai were used as the experimental materials.The specific primers were designed according to the cloned HDS gene fragment and EST sequences. Two genes, named Nt HDSY and Nt HDSJ, were isolated from N. tazetta var by RACE and RT-PCR, and the fragments was about 2 592 and 2 599 bp, respectively. The open reading frame was 2 178 bp, encoding a polypeptide of 745 amino acids. Sequence analysis showed that the amino acid sequence of Huanghua Ⅱ shared 97.77%, 79.29%, 75.29%, 75.51%, 75.02% and 74.40% homologous with Jinzhanyintai, Dendrobium officinale, Catharanthus roseus, Glycine max, Vitis vinifera and Malus domestica,respectively. The real time RT-PCR showed that the transcription expression of HDS gene changed accordingly during flower blooming and flower organs, indicating that the possible role of HDS gene in the synthesis of aroma substances.

In order to understand the changes of pigment content in Narcissus Tazetta flower at flowering phase,the contents of carotenoids and flavonoids in perianth and corona of JINZHANYINTAI and YELLOWFLOWERⅡ at flowering phase were tested by high performance liquid chromatography(HPLC).The results showed that there were a large number of various pigmentations accumulated in perianth and corona of Narcissus Tazetta,and rutin and phytoxanthin were the main pigmentations in YELLOWFLOWERⅡflowers,while rutin,naringin,ferulic acid and phytoxanthin were the main pigmentations in JINZHANYINTAI flowers.The dynamic trends of rutin,naringin and ferulic acid contents in the two varieties above were consistent at flowering phase: the contents in perianth and corona were the highest at flower budding period,were decreased somewhat at early flowering stage and full-bloom stage,and further increased at decline stage.But the phytoxanthin content change was not the same.Analysis of t-test indicated that there was a significant difference in the contents of rutin,naringin,ferulic acid and phytoxanthin between the varieties,respectively,and there was a significant difference in phytoxanthin and ferulic acid contents between perianth and corona in the same variety,respectively,but not significant difference for rutin and naringin contents,respectively.