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

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簡歷

基本資料

Project title

Code/計畫編號

MOST107-2611-M019-015

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(I)

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=12678034

Year

2018

Start date/計畫起

01-08-2018

Expected Completion/計畫迄

31-07-2019

Bugetid/研究經費

1868千元

ResearchField/研究領域

海洋科學

大規模定序技術在生物海洋學上的應用日漸頻繁，本申請案擬使用大規模定序技術，藉由分析混合種類轉錄體來瞭解馬祖海域優勢矽藻的基因表現，然後評估矽藻在自然環境中的生理狀態。由於目前學術界對判定轉錄體中基因表現量之高低並沒有一致的做法，這種不確定性在許多種類共存的自然界樣本中更是令人困擾。所以申請人提議在實驗室中培養與馬祖海域種類相同的矽藻，並在不同的培養環境下分別定序每一個培養樣本中的所有核醣核酸 (mRNA) 形成轉錄體。申請人將在各種轉錄體之間篩選mRNA表現量恆定的基因 (稱為管家基因) 約400條，作為校正轉錄體讀序數的基礎。接著將這些管家基因依表現量高低分為四群，用以規範營養鹽吸收基因表現量的上限與下限，然後就能夠在馬祖海域的混合種類轉錄體中，用同樣一組管家基因去瞭解營養鹽吸收基因對環境的反應。本申請案提出的管家基因排比法是一種觀念上的創新，與現行求取營養鹽吸收基因表現量上下限的方法相比，管家基因排比法免除了營養鹽操控與船上培養的部分，具有減少樣本數量、避免培養失敗等優點，將會對海洋學中浮游生物動力的研究造成深遠的影響。 The application of large-scale sequencing is increasingly common in the study of biological oceanography. In this proposal, large-scale sequencing will be used to construct meta-transcriptomes for plankton samples collected in Matsu waters. Reads in meta-transcriptomes will be used to evaluate gene expression in diatoms, and physiological status of dominant diatom species will be studied in their natural habitats. In current algorithms of transcriptome analysis, a standard procedure has not been established to decide if a gene is actively expressed. This uncertainty becomes especially severe in meta-transcriptomes containing reads from many species. Here the applicant proposes that housekeeping genes, which are genes with constant expression levels under all growth conditions, can be used as standards of comparison. The most dominant diatom species in Matsu samples will be selected and grown in laboratory cultures at various nutrient, temperature, and light levels. For each growth condition, transcriptomes will be constructed by sequencing all mRNAs in that diatom population. Expression ratios will be calculated for all gene pairs in a transcriptome, and housekeeping genes will be identified by stable expression ratios across all growth conditions. Next, approximately 400 housekeeping genes will be selected and categorized into 4 groups according to their transcript abundance. The geometric means of transcript abundances associated with these groups will then be used as standards to measure the expression of environment-sensitive genes, such as genes involved in nutrient utilization. In comparison with the method of evaluating gene expression levels with nutrient manipulating experiments, the housekeeping gene restraining method is an incubation-free approach that simplifies sampling procedure and avoids the negative effects due to failed incubation. This new approach is expected to make large-scale sequencing a more reliable technique in the study of plankton dynamics.

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(I)

基本資料

Description