全寄生植物菱形奴草質體基因組演化之研究

Abstract

非光合作用植物只存在著退化的質體，且其質體基因組已高度的縮減。菱形奴草（Mitrastemon kanehirai）為台灣特有的全寄生植物，屬於嚴重瀕臨絕滅的珍貴保育物種。目前菱形奴草只有一條質體序列被發表，而此序列 pt16S rDNA 顯示了演化速度加快的現象。 本論文首先比較了六種DNA萃取方法在兩種非光合作用植物日本蛇菰（Balanophora japonica）和菱形奴草上的成效。而利用Barnwell等人(1998)針對富含黏液的多肉植物發展出的方法所抽出的蛇菰及奴草DNA，其純度皆能夠進行限制酶切割反應。此外並成功的在菱形奴草運用了Milligan在1989年所敘述利用高鹽溶液去除細胞核DNA的方法，有效的提高其質體DNA的含量。 研究顯示除了質體外，異營性植物的細胞核以及粒腺體小次單元rDNA也有演化速度加快的現象。為了釐清異營性植物細胞內三種SSU rDNA間演化速度的相對關係，本研究檢視了九種異營性植物（包括一種半寄生、五種全寄生以及三種真菌異營性的植物）的粒線體19S、質體16S以及核18S rDNA；藉由相對速率分析和譜系分析兩種方法來估算這些rDNA序列的演化速度。分析顯示與其他被子植物相比較，非光合作用植物的pt16S及nr18S rDNA序列，其取代率明顯的增高，而mt19S rDNA則無此現象。九種被檢視的植物中，日本蛇菰和菱形奴草的pt16S和nr18S rDNA的變異最大，並且伴隨著序列中GC比例的降低。 此外本論文利用了次世代定序技術，成功的獲得了非光合作用植物菱形奴草質體基因組的完整序列。與一般陸生植物的葉綠體基因組相比，菱形奴草的質體基因組喪失了所有與光合作用相關的基因，並欠缺反向重覆區域，而保留下來的基因大多與轉譯作用相關。這個保有4個rRNA、4個tRNA及18個蛋白質編碼(protein-coding)基因、大小只有25,740 bp的質體基因組為目前已知的最小質體基因組。

Non-photosynthetic plants only retain remnant plastids and their plastome is highly reduced. Mitrastemon kanehirai, a root holoparasite, is endemic to Taiwan and considered an endangered species. Mitrastemon kanehirai has only one plastid sequence has been reported, and the sequence, pt16S rDNA, shows increased substitution rate. In this dissertation, the performance of six DNA extraction procedures for two non-photosynthetic plants, Balanophora japonica and M. kanehirai, were compared. All six procedures yielded DNA of sufficient quality for PCR, and the method described by Barnwell et al. (1998) performed well in isolating DNA from both species for restriction enzyme digestion. Meanwhile, enrichment of M. kanehirai plastid DNA content was achieved by using the ‘high salt’ methods based on protocol presented by Milligan (1989). High rate of nucleotide substitution in three subcellular SSU rDNAs have been reported in heterotrophic plants, and the rate heterogeneity among these sequences are presented in this dissertation. Mt19S, pt16S and nr18S rDNA sequences from nine heterotrophic plants, including one hemiparasitic, five holoparasitic and three mycoheterotrophic plants, were examined. Rate heterogeneity among various rDNA sequences was evaluated by relative rate tests and phylogenetic analysis. Both pt16S and nr18S rDNA sequences of non-photosynthetic species show significant increases of substitution rate, but the phenomenon was not found in mt19S rDNA. The extreme divergent pt16S and nr18S rDNA sequences were found in B. japonica and M. kanehirai, and accompanied by a decrease in GC content of the sequences. Mitrastemon kanehieai plastome was sequenced by using next generation sequencing technology. The genome is smallest plastome that have been described with size of 25,740 bp. Only 26 genes were retained in the plastome, which include 4 rRNAs, 4 tRNAs and 18 protein-coding genes. These retained genes are mostly involved in translation machinery. All photosynthesis-related genes were lost, and the inverted repeat region is absent. Despite the enormous reduction, the M. kanehirai plastome is a functional gene expression system. DNA transfer from plastid to nucleus and horizontal transfer from the host to the parasite were also observed in M. kanehirai.