Population genetics of the mangrove Rhizophora stylosa in northern Australia

Starch gel electrophoresis was used to examine genetic variation in five widely separated populations of the mangrove Rhizophora stylosa Griff. Shoots containing leaves and well developed apical buds were collected between March and August 1987 from Darwin, the Daintree River, 2 sites at Missionary Bay (Hinchinbrook Island), Chunda Bay (Cape Ferguson) and Lardelli Creek (Ayr). No two trees at each sampling site were separated by more than 1km, except for the Daintree River site, which was sampled over 2km.A suitable extraction buffer was developed by modifying a buffer developed by Dr Gavin Moran (CSIRO) for eucalypts and acacias.Twenty eight enzymes were investigated for the Rhizophora stylosa samples (ACP, ACON, AK, ADH, ALD, AAT, EST(FL), FH, BetaGAL, G6PD, GPI, GDH, GSR, GAPDH, HK, IDH, LAP, LGGP, LTP, LPP, MDH, ME, MDR, PER, PGM, 6PGD, SKDH, TPI). Fourteen enzymes were resolved adequately for these samples (ACON, AAT, GPI, GDH, GSR, HK, IDH, LAP, LTP, MDH, ME, PER, 6PGD, TPI).Other Rhizophoraceae successfully tested for enzyme activity in leaf and apical bud tissue, using the same methods were Rhizophora apiculata Blume, R. lamarckii Montr., R. mucronata Lamarck, Bruguiera gymnorhiza Lamarck, Ceriops decandra (Griff.) Ding Hou, C. tagal (Perr.) C.B. Rob. var. tagal and C. tagal var. australis C.T. White. Other mangrove species producing clear repeatable results included Lumnitzera racemosa Willdenow, Xylocarpus australasicus Ridley, Sonneratia alba J.E. Smith, Acrostichum speciosum Willdenow, Avicennia marina (Forssk.) Vierh. and A. officinalis Linnaeus.While leaves and buds were chosen initially, due to their year round availability, whole anthers and the plumule of the progagule of Rhizophora stylosa were also successfully tested for enzyme activity. This research was undertaken to:1. develop an effective extraction buffer to counteract the effect of complex secondary metabolites in mangroves, which denature proteins during enzyme extraction.2. develop techniques for the electrophoresis of mangrove tissue and to use these to investigate genetic variability in Rhizophora stylosa and other Rhizophoraceae.

本研究采用淀粉凝胶电泳（starch gel electrophoresis）技术，对5个地理隔离显著的红海榄（Rhizophora stylosa Griff.）种群开展遗传变异检测。1987年3月至8月，研究人员分别从达尔文港、丹特里河、传教士湾（欣钦布鲁克岛）的2个采样点、春达湾（弗格森角）以及拉尔迪利溪（艾尔）采集带有完整叶片与发育成熟顶芽的枝条。除丹特里河采样点覆盖范围达2千米外，各采样点内任意两棵红海榄植株的间距均不超过1千米。

本研究参照加文·莫兰博士（澳大利亚联邦科学与工业研究组织，CSIRO）为桉树与金合欢树开发的缓冲液配方进行改良，成功获得适配本研究的组织提取缓冲液。针对红海榄样本，共筛选28种酶（ACP、ACON、AK、ADH、ALD、AAT、EST(FL)、FH、BetaGAL、G6PD、GPI、GDH、GSR、GAPDH、HK、IDH、LAP、LGGP、LTP、LPP、MDH、ME、MDR、PER、PGM、6PGD、SKDH、TPI），其中14种酶可获得清晰且符合实验要求的分离条带（ACON、AAT、GPI、GDH、GSR、HK、IDH、LAP、LTP、MDH、ME、PER、6PGD、TPI）。

采用相同实验方法，本研究还对其他红树科（Rhizophoraceae）植物的叶片与顶芽组织开展酶活性检测，成功获得可靠结果的物种包括：尖叶红树（Rhizophora apiculata Blume）、拉马克红树（R. lamarckii Montr.）、钝叶红树（R. mucronata Lamarck）、木榄（Bruguiera gymnorhiza Lamarck）、正红树（Ceriops decandra (Griff.) Ding Hou）、印度角果木（C. tagal (Perr.) C.B. Rob. var. tagal）以及澳洲角果木（C. tagal var. australis C.T. White）。此外，以下红树林物种的酶活性检测同样可获得清晰且可重复的实验结果：榄李（Lumnitzera racemosa Willdenow）、澳洲木果楝（Xylocarpus australasicus Ridley）、白海桑（Sonneratia alba J.E. Smith）、美丽卤蕨（Acrostichum speciosum Willdenow）、海榄雌（Avicennia marina (Forssk.) Vierh.）以及药用海榄雌（A. officinalis Linnaeus）。

最初因全年均可稳定获取，研究选择叶片与顶芽作为实验材料，但后续实验证实，红海榄的完整花药以及其胎生繁殖体的胚芽同样可成功用于酶活性检测。

本研究的目标如下：
1. 开发高效的组织提取缓冲液，以抵消红树林中复杂次生代谢物在酶提取过程中导致的蛋白质变性问题；
2. 建立适用于红树林组织的电泳实验技术，并利用该技术探究红海榄及其他红树科植物的遗传变异水平。