2 Materials and Methods
4.4 Future research
Coral microbiology is important for the understandings to the coral heath. In this
thesis, we only identified the bacteria communities of T. hoshinota and P. lutea. The
function of the bacterial community is still unknown; especially the role of the
Cyanobacteria associated with T. hoshinota. The further study should pay more efforts on identification of the interactive function between the microbe and corals or
sponge which will be helpful to clarify the microbial role and to provide better
suggestions to coral maintenance and disease control.
On the other hand, the variation of bacterial communities associated with the NSI
and SI P. lutea also offered a positive glimpse of the important information for being a
possible bioindicator of the particular environmental stress. However, these snap-shot
study data are in a limit for further understandings which bacterial population
appearing and disappearing associates with what coral physiology and what
environmental stresses. To build up a bioindicator, a long term investigation is
apparently essential.
Finally, besides the function of bacteria community associated T. hoshinota, the
knowledge of the biology of T. hoshinota was also less known in the past.
Reproduction and development of T. hoshinota is important to realize the possible
reason of outbreak of T. hoshinota. At the same, fishing bite character on T. hoshinota
was discovered in the field. Decease of sponge-eating fish resulted in food chain
function unbalance are looking further research.
Fig. 31 Transmission electron micrographs of cyanobacterial symbionts in Ircinia variabilis from Marseille.
A. Cyanobacteria in Terpios hoshinota. 1.Aphanocapsa raspaigellae. 2. A. feldmannii. Scale bars: 1 mm (1) and 500nm (2).
A
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Appendix 1. Classical (CTAB) DNA Extraction Protocol for Coral
Materials
TE bufferAdd 1ml of 1M Tris-HCl (pH 7.5) in 98.8ml MILIQ-water and then add 0.2ml of 0.5M EDTA.
20mg/ml proteinase K
Add 0.2g of proteinase K in 10ml MILIQ-water.
Lysis buffer 10% SDS
Dissolve 20 g sodium dodecyl sulfate in MILIQ-water in a total volume of 100 ml with stirring. Filter sterilizes using a 0.45-µm filter.
5M NaCl
Dissolve 29.22g NaCl in 100ml MILIQ-water. Autoclave for 15-20mins at 121 .℃
CTAB/NaCl solution
Dissolve 4.1g NaCl in 80ml MILIQ-water and then add 10g of CTAB with heating and stirring. Then adjust volume to 100ml.
Chloroform/isoamyl alcohol(24:1)
Phenol/chloroform/isoamyl alcohol(25:24:1) Isopropanol
70% Ethanol
To 60 ml MILLIQ-water add 140 ml of 100% ethanol.
MILIQ Water (sterile)
Water bath (for incubations at 37 , 65 )℃ ℃ Pipettes, Tips
Microcentrifuge, Microcentrifuge tubes Vortex
Method
1. 在實驗前先將 proteinase K 於冰上退冰。
2. 將樣品(約 2×2cm)加入液態氮磨碎。
3. 將樣品 1mL 離心 12000rpm 5 分鐘後倒掉上清液，加入 TE buffer 1mL，Vortex
離心12000rpm 5 分鐘，重複三次。
4. pellet 用 500μL Lysis buffer 重新懸浮，加入 30μL of 10% SDS, and 3μL of 20mg/mL proteinase K（加入時 tip 要深入液面），Vortex，水浴 1 小時 at 37℃。
5. 加入 100μL of 5M NaCl，快速混合。
6. （使用寬口tip）加入 80μL CTAB/NaCl solution（加入時 tip 要深入液面），
Vortex，水浴 10 分鐘 at 65℃。
7. 加入 Chloroform/isoamyl alcohol 500μL，Vortex，離心 12000rpm 5 分鐘。
8. 取上清液至新的 tube，加入等比例(約 500μL)的 phenol/chloroform/isoamyl alcohol(下層液)，Vortex，離心 5 分鐘.
9. 取上清液至新的 tube，加入 0.6 倍的 2-propanol(約 300μL)，（不可 Vortex，只
可輕輕混勻），離心7 分鐘。
10. 先倒掉上清液，加入 70%的酒精 300μL，離心 5 分鐘.
11. 倒掉上清液，加入滅菌過的超純水保存在 4℃冰箱裡。