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研究生：	黃銀慶 Huynh, Ngan Khanh
論文名稱：	姬蝴蝶蘭磷酸鹽轉運蛋白之全基因組鑑定及其表現模式 Genome wide identification of Phosphate transporter I and their expression patterns in the Phalaenopsis equestris
指導教授：	蔡文杰 Tsai, Wen-Chieh
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 熱帶植物與微生物科學研究所 Institute of Tropical Plant Sciences
論文出版年：	2022
畢業學年度：	110
語文別：	英文
論文頁數：	52
外文關鍵詞：	Phosphate transporter I, Phalaenopsis equestris, Genome wide identification
相關次數：	點閱：138 下載：0
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Orchidaceae is famous for its beautifully shaped flowers, unique ecological and reproductive adaptability and is one of the most distinct plant families. Here, the Phosphate Transporter I genes were identified from the genome sequence of tropical epiphytic orchid Phalaenopsis equestris, a common parental species for orchid breeding. An epiphyte is a kind of plant dependent on trees for structural support but not for nutritional support. By contrast, a terrestrial orchid just grows on land and does not need a host plant for surviving. Almost of orchids appear in tropical or subtropical regions of the world. In Arabidopsis thaliana, the high-affinity phosphate transporters are encoded by the PHT1 family. Plant growth and development require phosphorus (P) as an essential nutrient, accounting for about 0.2% of the dry weight. A great deal of biological macromolecules like nucleic acids, membrane lipids, and ATP are the significant components coming from phosphorus (P). The plants mainly take up phosphorus from the soil through the roots in the form of inorganic phosphate (Pi), including HPO4- and HPO42-.
Especially, the PHT1 is a membrane protein of a crucial facilitator family and the primary means of entry of Pi from the soil to the plants (Nussaume et al., 2011). The PHT1 transporters are also involved in Pi translocation as well as Pi remobilization in the aerial parts of the plants and play a vital role as transporters for phosphate import in plants. I performed spatial and temporal expression analysis by qRT-PCR, phylogenetic tree analysis, and predicted subcellular localization of PHT1 genes from P.equistris genome to understand the function of PHT1 in the orchid. To detect and characterize the PePHT1 genes (Peq019301, Peq003369, Peq021219, and Peq003368) expression patterns in orchid, qRT-PCR was performed in P.aphrodite subsp. formosana and seedlings growing in the low-phosphate medium and sufficient-phosphate medium. The result showed that the gene Peq021219 has the highest expression in the sepal compared to other parts of P. aphrodite subsp. formosana. Interestingly, the gene Peq021219 expression is higher than other genes in both sufficient and deficient medium. Peq021219 might have a major role responding to the phosphate and is the remarkable gene due to its highest expression in Phalaenopsis seedling.

Contents
Abstract	I
Acknowledgement	III
List of Tables	VII
List of Figures	VIII
List of Supplementary of Tables 	IX
List of Supplementary of Figures 	X
Abbreviations	XII
1. Introduction	1
1.1 Introduction of Phalaenopsis  orchids  and orchid genomes	1
1.2 Macronutrients requirement for Phalaenopsis orchids	3
1.3 Phosphorus (P) is crucial nutrient in plants.	4
1.4   The mechanism of plants acquires P	5
1.5 Phosphate transporter is special transporter to acquire P in plant	6
1.6 The PHT1 family of phosphate transporters	7
1.7 Regulation of plant phosphate transporters	9
2. Aim of the study	10
3. Material and Methods	11
3.1 Plant materials	11
3.2 RNA preparation	11
3.3 Phylogenetic analysis	12
3.4 Quantitative real-time RT-PCR (qRT-PCR)	12
3.5   Expression pattern of PePHT genes in seedling growing with sufficient or deficient medium	13
3.6 Biochemical properties, subcellular localization of PePHT1	13
4. Results	14
4.1 Identification and sequence analysis of PePHT1 genes from Orchidaceace	14
4.2 Phylogenetic analysis of four genes of PePHT1 among other orchid species and other plants	14
4.3 Expression pattern of PePHT1 genes in P. aphrodite subsp. formosana	15
4.4 Expression pattern of PePHT1 genes in Phalaenopsis seedling treated with sufficient phosphate and phosphate starvation	16
4.5 Characteristic features of PePHT1 genes in orchids.	16
5. Discussion	18
5.1 Identification of PePHT1 genes	18
5.2   Phylogenetic analysis of four genes of PePHT1 among other orchid species and other plants and roles  of  plant Pi transporters	18
5.3 Expression pattern of PePHT1 genes in P. aphrodite subsp. formosana	20
5.4 Expression pattern of PePHT1 genes in Phalaenopsis seedling treated with sufficient phosphate and phosphate starvation.	21
5.5 Characteristic features of PePHT1 genes in orchids.	22
6. Conclusion	23
7. Reference	24

List of tables
Table 1: The number of PHT1 genes in different Orchid species	32
Table 2. Characteristic features of PePHT1 proteins	33
Table 3. The primers were used in this study	34

List of Figures
Fig.1 Research materials of P.aphrodite subsp.formosana	35
Fig.2 Multiple sequence alignment of PHT1 proteins among Phalaenopsis equestris, Arabidopsis,maize	36
Fig. 3 Neigbor-joining phylogenetic tree of PePHT1 proteins from the angiosperms
	37
Fig 4.Quantitative RT-PCR analysis of PePHT1 expression in P.aphrodite subsp.formosana at various organs	38
Fig 5. Quantitative RT- PCR analysis of expression PePHT1 genes  in P.amabilis	39
Fig 6. Heat map of PePHT1 genes	40
Fig 7. Heat map of AshPHT1 genes of D. catenatum	41
Fig 8. Heat map of DcaPHT1 genes of A.shenzhenia	42

List of Supplementary of Tables
Supplementary Table.1.The accession number and description of the maize proteins from the gene bank used in the phylogenetic tree	43
Supplementary Table.2.The accession number and description of the Arabidopsis thaliana proteins from the gene bank used in the phylogenetic tree	44
Supplementary Table.3.The accession number and description of the rice proteins from the gene bank used in the phylogenetic tree 	45
Supplementary Table.4.The accession number and description of the Elaeis guineensis, Phoenix dactylifera and Cymbidium hybridum and Musa acuminata  proteins from the gene bank used in the phylogenetic tree.	46
 
 List of Supplementary of Figures
Supplementary Figure.1.Orchid seedlings are growing in with sufficient and deficient medium in  2 weeks 	47
Supplementary Figure.2.Orchid seedlings are growing in with sufficient and deficient medium in  4 weeks 	48
Supplementary Figure.3.Orchid seedlings are growing in with sufficient and deficient medium in  6 weeks 	49
Supplementary Figure.4.Orchid seedlings are growing in with sufficient and deficient medium in  8 weeks	50
Supplementary Figure.5.Orchid seedlings are growing in with sufficient and deficient medium in  10 weeks	51
Supplementary Figure.6.Orchid seedlings are growing in with sufficient and deficient medium in  12 weeks	52
                                    

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