Study On Structure And Expression Characteristics Of Senescence-associated Genes In Dahlia Petals

Petal senescence is the final developmental stage of plant life cycle, which is along with the petals wilting and falling off, color changing, flower diameter diminishing or other features. And it is the result of the comprehensive regulation of gene expression, protein synthesis as well as internal and external factors, which involves a series of physiological processes. A variety of different expressed proteins associate with senescence have been isolated and identified from dahlia petals in previous study, which mainly involve the functions of ethylene synthesis, anthocyanin synthesis and cell wall modification, and Dp XTH1 and Dp XTH2 have been cloned. But there is lack of research and understanding of the expression characteristics at the transcripitional level yet.In this research, the ‘unipetalous yellow’(Dahlia pinnata Cav) was used as the experimental material, and senescence-associated genes were cloned from dahlia petals through the gene cloning technology. Also, the genetic structure and the evolutionary relationships had been investigated by the bioinformatiics method. Besides, the expression characteristics of senescence-associated genes were analyzed by real-time fluorescence quantitative PCR technology with the different development stages and different treatments of exogenous hormones in dahlia. The main results were shown as follows:1. The c DNA sequences of CHS, ANS and CHI genes were cloned from dahlia petals by molecular biology and bioinformatics technologies, which were respectively named as Dp CHS(1170 bp, 389 amino acid residues and one stop codon), Dp ANS(1068 bp, 355 amino acid residues and one stop codon) and Dp CHI(675 bp, 224 amino acid residues and one stop codon). Blast result analysis showed that the homology of Dp CHS, Dp ANS and Dp CHI associated with Michal J, one of the dahlia cultivars, was up to 99%. Cluster result analysis showed that the relationship of Dp CHS, Dp ANS and Dp CHI was the closest to several compositaes that were reported. They were respectively clustered into the same group as they were based on the similar evolution of the amino acid sequence of the evolutionary tree in the system and the relationships of plants, also the same family plants were in the same branch on evolution.2. Using SAMS sequence of sunflower(Gen Bank JN561248.1) and ETR sequence of daisy(Gen Bank: AF547624.1) as the original nucleic acid sequence and applying molecular biology and bioinformatics technologies respectively cloned the c DNA sequences of SAMS and ETR by electronic cloning technology. Degenerate primers were designed in the sequence of conservative area respectively, and partial c DNA sequences of SAMS and ETR were cloned from dahlia petals, named as Dp SAMS and Dp ETR respectively. Dp SAMS gene fragment size was 163 bp, could encode 54 amino acid residues; Dp ETR gene fagment size was 118 bp, could encode 39 amino acid residues. Blast and Cluster analysis revealed that the homology of Dp SAMS when it was compered with 100 c DNA fragments was between 83%~96%. Thereinto, the highest homology of it was sunflower which was up to 96%. While the homology of Dp ETR was between 71%-91%, and it had the highest homology of daisy that was up to 91%.3. RNA extracted from the dahlia that grew in the natural state during the process of squaring period, early florescence, full-bloom stage and flower senescence stage was reverse transcribed into c DNA. Designing the Real-time quantitative PCR specific primers of Dp CHS, Dp ANS, Dp CHI, Dp SAMS, Dp ETR, using the β-actin as a reference gene for real-time fluorescence quantitative PCR(q RT-PCR) analysis, the results showed that the expression quantities of Dp CHS、Dp ANS and Dp ETR genes on transcription level were all first increased and then showed a downward trend from the flowering beginning to accumulate. It reached peak in the flowering stage, later the transcript level decreased in the decline petals. However, the expression quantity of Dp CHI showed a downward trend, in the other words, it reached the highest expression when it was in the squaring period. While it was opposite for Dp SAMS, it reached the highest expression when it was in the flower senescence stage. So, Dp CHS, Dp ANS, Dp ETR and Dp SAMS were preliminary judged as petal senescence-associated genes of dahlia, while Dp CHI was a down senescence-regulated gene in petals of dahlia.4. After using ethylene(ETH), 1-methylcyopropene(MCP) as exogenous hormones processed the petals in vivo for 0.5 h, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h respectively in the process of early florescence, the extracted RNA from each experiment was taken for q RT-PCR analysis as the no treatment one was regarded as the contrast. During the treatment period, the expression level of Dp CHI, Dp SAMS, Dp ETR, Dp XTH1 and Dp XTH2 showed that a trend of increased firstly and then decreased; also, the expression levels of Dp CHS and Dp ANS showed a trend of rise firstly and then decreased after the treatment of CK or ETH, but it increased gradually after the treatment of MCP. Compared with the contrast, the expression level of senescence-associated genes was the highest and the peak was earlier detected. However, it decreased after the treatment of MCP, and the peak was delayed to detect. The results illustrated that the senescence process of dahlia petals could be regulated after the treatment of ETH or MCP which was associated with the expression changes of above genes.5. After using ethylene(ETH), 1-methylcyopropene(MCP) as exogenous hormones processed the petals in vivo for 12 hours in the process of squaring period, early florescence and full-bloom stage respectively, the extracted RNA from each experiment was taken for q RT-PCR analysis as the no treatment one was regarded as the contrast. After the petals were treated in ETH for 12 hours in the process of early florescence, the results showed that the relative expression peaks of Dp CHS, Dp ANS, Dp ETR, Dp XTH1 and Dp XTH2 were detected, while the peak of Dp CHI appeared in the process of squaring period, the peak of Dp SAMS appeared in the process of full-bloom stage. In terms of different genes, the relative expression peak of Dp CHI appeared first, followed by Dp CHS, Dp ANS, Dp ETR, Dp XTH1 and Dp XTH2, the final was Dp SAMS. Compared with the contrast, the transcript levels of Dp SAMS and Dp ETR were significantly increased with the treatment of ETH, while it was inhibited by the treatment of MCP. The above experimental results further illustrated that the treatment of exogenous hormone could affect the related gene transcription level, and the regulation of gene transcription level was associated with the periods of petals treatments. It was obviously effected by the MCP treatment in the early flowering, which could delay the senescence process of petals and then extend the ornamental period of dahlia.