石松類和瓶爾小草葉綠體matK基因之鑑定與matK基因的演化

The Evolution of Chloroplast matK Genes, Including Identification of New Homologues from Ophioglossum petiolatum and Two Lycophytes

高等植物葉綠體trnKUUU基因的內插子屬於第二類內插子，其中包含一段開讀框，因其序列及結構和酵母菌粒線體的maturase相似，故稱之為matK，matK是葉綠體中唯一具有去除內插子功能的基因，只有較高等的輪藻和陸生植物的葉綠體trnK基因才有內插子和trnK5’-matK-trnK3’的結構，其他綠藻葉綠體的trnK並沒有matK開讀框、也沒有第二類內插子。葉綠體trnK5’-matK-trnK3’的結構在高等植物相當一致，但先前研究發現兩個matK脫離trnK而存在的例子，像是葉綠體嚴重退化的寄生植物Epifagus virginiana及因葉綠體基因重組而導致trnK外顯子丟失的鐵線蕨，它們的matK仍然具有完整的開讀框，顯示了matK的確十分重要而不可缺失，蘚苔類和松葉蕨也被證實具有trnK5’-matK-trnK3’的結構，但是在角蘚中卻發現其matK是偽基因。本論文鑑定原始陸生植物其他未被檢視的重要類群中葉綠體matK基因的存在，結果發現瓶爾小草、過山龍、生根卷柏都有trnK5’-matK-trnK3’的結構，以RT-PCR偵測matK表現時，瓶爾小草和過山龍都有偵測到訊號，但生根卷柏卻沒有，顯示matK的表現在原始陸生植物似乎不是那麼一致。利用PCR的偵測並未能在水韭、木賊、觀音座蓮、紫萁、海金沙、芒萁等物種中放大出trnK或matK的片段。但是利用點式雜合反應則顯示這些物種可能有matK序列的存在，顯示這些序列可能因變異太大而無法以PCR方式放大。故有關蕨類葉綠體matK的存在與否仍有待進一步研究。以葉綠體matK核甘酸序列進行譜系分析，結果顯示部分裸子植物（松柏、銀杏、蘇鐵）和被子植物形成姊妹群，兩者再和麻黃類植物形成姊妹群。作者認為這個結果主要是由於matK基因的演化速率太快，而造成譜系分析的不可靠性。

The introns of chloroplast trnKUUU belong to Group II introns and contain an open reading frame denoted as matK. The trnK5’-matK-trnK3’ structure is consistent in almost all examined higher land plants and in Characeae, but not in other green algae examined. The putative gene product MatK is the only maturase in chloroplasts. Functional chloroplast matK genes are retained even in the nonphotosynthetic parasite, Epifagus virginiana and the fern, Adiantum capillus-veneris, in which chloroplast genome rearrangement has left matK free-standing, apart from trnK exons. Among lower land plants, the chloroplasts of Psilotum, mosses and liverworts all have trnK5’-matK-trnK3’ structure, but matK is a pseudogene in hornwort Anthoceros formosae. In this study we found a clear trnK5’-matK-trnK3’ structure in Ophioglossum petiolatum, Lycopodiella cernua and Selaginella doederleinii, but PCR with degenerate primers failed to amplify any trnK or matK fragments from other ferns and fern allies. However, dot blot hybridization showed distinct signals in these plants that failed to amplify matK fragments by PCR, indicating that the matK sequences in those taxa may be too divergent to amplify by an ordinary PCR approach. RT-PCR results showed matK genes are expressed in Ophioglossum petiolatum and Lycopodiella cernua, but no signal was detected in Selaginella doederleinii. Overall, the expression patterns of matK are not consistent in lower land plants. Phylogenetic analysis of matK sequence showed that Pinus, Ginkgo, and Cycas form a monophyletic group, which is sister to angiosperms. Together, they form a clade that is sister to Gnetales. This ad hoc reconstruction is likely due to the high evolutionary rate in matK.