Housekeeping Genes as Normalization Standards in Marine Phytoplankton Gene Expression Databases Generated by Large-Scale Sequencing (II)

View Statistics Email Alert RSS Feed

Information

Details

Project title

Code/計畫編號

MOST108-2611-M019-005

Translated Name/計畫中文名

海洋浮游植物大規模定序資料庫中使用管家基因做為表現量校正點之研究 (Ⅱ)

Project Coordinator/計畫主持人

Jeng Chang

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Institute of Marine Biology

Website

https://www.grb.gov.tw/search/planDetail?id=13122956

Year

2019

Start date/計畫起

01-08-2019

Expected Completion/計畫迄

01-07-2020

Bugetid/研究經費

2005千元

ResearchField/研究領域

海洋科學

本計畫的研究目標是在改善目前針對矽藻所設計的基因表現測量法，以便日後在海洋生態系中得到更準確的量測值，藉此可以了解矽藻對環境做出的生理反應。本計畫延續去年的研究工作，第一部份的成果是在2016年六月和七月的馬祖樣本中，針對單種矽藻：熱帶骨藻 (Skeletonema tropicum) 使用反轉錄定量聚合脢鎖鏈反應 (RT-qPCR) 測量下列基因表現量：硝酸鹽傳輸基因 (Nrt2)、轉錄延長因子基因 (EFL)、TATA 區結合蛋白基因 (TBP)、和甘油醛-3-磷酸脫氫酶基因 (GAPDH)。同時在實驗室中使用同一種類設立培養組，將實驗室表現量和航次表現量互相比對，結果發現參考基因EFL的表現量具有飄移現象，使得Nrt2測值受到低估。第二部份的成果是針對2017年六月和七月的馬祖樣本中的第二個優勢種：多氏骨藻 (S. dohrnii) 篩選管家基因。首先根據全轉錄體和相關文獻篩選出 5種有潛力的管家基因，包括EFL、mtnD、NDUFS7、Actin16、和 YWHAE，然後以RT-qPCR觀察它們的表現量是否在營養鹽操控實驗中維持穩定。結果顯示以參考基因的效能來說，5種基因的平均表現量並沒有比單一的EFL產生更好的校正效果，但是會有助於辨識產生飄移現象的參考基因。第三部份的成果是在過去一年中完成關於磷酸鹽傳輸基因的論文稿一篇，目前正在審查中。The goal of the present research is to improve the measurement of gene expression in diatoms. This improvement will lead to more accurate estimates of gene expression in marine ecosystems, and will provide new knowledge about how diatoms respond physiologically to environmental conditions. Research works described here is a continuation from the works conducted in the previous year. In the first part, samples were obtained from Matsu waters in Jun. and Jul. 2016. Specific primers were designed for the diatom, Skeletonema tropicum, and expression levels of several genes were measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR). These genes included the nitrate transporter gene Nrt2, the translation elongation factor-like gene EFL, the TATA-box binding protein gene TBP, and the glyceraldehyde 3-phosphate dehydrogenase gene GAPDH. A pure strain of the same species was used to set up experimental cultures, and gene expressions measured in the laboratory were compared with those measured in research cruises. The results indicated that EFL expression drifted at one station, which caused an underestimation in the expression of other genes. In the second part, samples were obtained from Matsu waters in Jun. and Jul. 2017. A dominant diatom, S. dohrnii, in these samples was selected as the target species. Based on transcriptome analysis and other information sources, 5 genes including EFL, mtnD, NDUFS7, Actin16, and YWHAE, were selected as candidate housekeeping genes, and their stabilities in gene expression were evaluated by RT-qPCR under various nutrient treatments. When judged by their suitability as a combined reference gene, the averaged expression of these 5 genes did not perform better than did a single EFL gene. However, the monitoring of multiple reference genes is helpful to identify the gene with a drifted expression. The third part of our results was the completion of a manuscript concerning a phosphate transporter gene in S. tropicum. This manuscript is currently in review.

Keyword(s)

馬祖海域
浮游植物
矽藻
營養鹽
純種培養
大規模定序
高通量定序
管家基因
轉錄體
基因表現
Diatoms
gene expression
high-throughput sequencing
housekeeping genes
large-scale sequencing
Matsu waters
nutrients
phytoplankton
pure culture
transcriptome

DSpace CRIS

Housekeeping Genes as Normalization Standards in Marine Phytoplankton Gene Expression Databases Generated by Large-Scale Sequencing (II)

Details

Description