蝴蝶蘭葉綠體基因體之研究

行政院國家科學委員會專題研究計畫 成果報告

蝴蝶蘭葉綠體基因體之研究

計畫類別： 個別型計畫

計畫編號： NSC92-2317-B-006-004-

執行期間： 92 年 08 月 01 日至 93 年 07 月 31 日 執行單位： 國立成功大學生物科技研究所

計畫主持人： 張清俊

計畫參與人員： 林咸嘉,林惠茹

報告類型： 精簡報告

處理方式： 本計畫涉及專利或其他智慧財產權，1 年後可公開查詢

中 華 民 國 93 年 11 月 2 日

行政院國家科學委員會補助專題研究計畫 ■ 成 果 報 告

□期中進度報告

蝴蝶蘭葉綠体基因体之研究

計畫類別：▓ 個別型計畫 □ 整合型計畫

計畫編號： NSC92-2317-B006-004

執行期間：九十 二年 八 月 一日 至 九十三 年 七 月 三十一 日

計畫主持人： 張 清 俊

共同主持人：

計畫參與人員： 張清俊,林咸嘉,林惠茹

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處理方式：除產學合作研究計畫、提升產業技術及人才培育研究計畫、列

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執行單位： 國立成功大學生物科技研究所

中 華 民 國 九十三 年 十 月 三十 日

計畫名稱：蝴蝶蘭葉綠體基因體之研究 計畫編號: NSC92-2317-B006-004

執行期限: 九十二年八月一日至九十三年七月三十一日

主持人姓名： 張 清 俊 執行機構：成功大學生物科技研究所 一、中文摘要

台灣阿嬤蝴蝶蘭葉綠體基因體已定序、比對、註解完成。基因体總長度 148,962 bp,由一個大小 25,731 bp 的反向重覆區域,將其區隔成 85,957 bp 和 11,543 的兩個不同片段。有 108 個不同功能的基因散布於葉綠體基因体中,包括 74 個蛋白質基因,4 個 rRNA 基因,30 個 tRNA 基因。此外,也解析到 27 個未知的 ORF。我們觀察到在全部 11 個 NADH dehydrogenase 的蛋白質單元基因中,有 6 個在蝴蝶蘭葉綠體中巳喪失掉了。與其它物種來做比對,顯示蝴蝶蘭與其他高等 植物葉綠體基因組之序列有很高的相似性。

関鍵詞: 台灣阿嬤蝴蝶蘭、葉綠體基因體

Abstract

The complete nucleotide sequence of the plastid genome of Phalaenopsis Orchid (Phalaenopsis amabilis var. Formosa) has been determined. The circular double stranded DNA of 148,962 bp contains a pair of inverted repeats of 25,731 bp, which are separated by small and large single copy regions of 11,543 and 85,957 bp, respectively. The plastid genome contains 74 proteins genes, 4 ribosomal RNA, 30 tRNA genes. Unlike other plastid genome of angiosperm which have complete set of genes for 11 subunits of NADH dehydrogenase, we observed complete lack of ndhA, ndhH and ndhF genes, and lost of functional ndhE, ndhG and ndhI genes in the plastid genome of Phalaenopsis orchid. It suggests that most of ndh genes have been transfer to nucleus in the process of evolution.

Key words: Phalaenopsis amabilis var. Formosa, plastid genome

二、緣由與目的(Introduction)

Since the first two complete plastid genome sequences were reported for tobacco (Shinozaki et al., 1986)and liverwort (Ohyama et al., 1986) in 1986, currently over 40 complete sequences representing all of the major lineages of the plant kingdom are available in the NCBI genomic database (http://www.ncbi.nlm.nih.gov). The availability of the plastid genomic sequences have been greatly enhancing our opportunity to explore the chloroplast biogenesis as well as study the phyllogenetic relationship among green plants. The plastid genome of land plants are highly conserved in structure and organization (Sugiura, 1995). Furthermore, complete plastid genomic information have provided a sound basis to genetically engineering the chloroplasts for various applications in plant biotechnology (Daniell et al., 2002).

With complete sequence of plastid genome from major land plants lineages such as a liverwort Marchantia (Ohyama et al., 1986),two ferns Adiantum and Psilotum (Wakasugi et al., 2000; Wolf et al., 2003), black pine of gymnosperm (Wakasugi et al., 1994), and angiosperms such as tobacco (Shinozaki et al., 1986), spinach (Schmitz-Linneweber et al, 2001), oenothera (Hupfer et al., 2000), arabidopsis (Sato et al., 1999), lotus (Kato et al., 2000), medicago (Lin et al., 2001), rice (Hiratsuka et al., 1989), maize (Maier et al., 1995), wheat (Ogihara et al., 2002), the phylogenetic relationship of land plants have been deduced, and the monocot-dicot divergence have been dated back to less than 150 million years ago (Chaw, et. al., 2004). Nevertheless, the origin and early evolution of angiosperms is still poorly understood. Nymphaea and Amborella both are vesselless plants which were regarded as the deepest branching among angiosperms (Parkinson et al., 1999).

However, with recent complete plastomic sequence of Nymphaea alba and Amborella trichopoda revealed that they are not basal angiosperm (Goremykin et. al., 2003;

2004); in contrast, grass family of monocot lineage is the closest phylogenetic relationship to gymnosperm (Goremykin et. al., 2004). Since the availability of the complete plastomic sequence of monocot plants is only limited to three closely related grasses, rice, maize and wheat, the divergent point between gynopsperm and angiosperm are still need further investigated. Apparently, with increasing the number of complete plastomic sequences from diverse family of monocot plant, the phylogenetic origin of angiosperm will begin to emerge.

Orchidaceae is one of the largest (about 30,000 species) and most complex families of monocots. In addition to classical morphological systematic classification within orchidaceae, a number of studies employ molecular markers from nucleus, mitochondria or plastid genes to classify family/subfamilies, tribes, subtribes, genus and even species within orchidaceae (Cameron et al., 1999; Cameron, 2004).

Phalaenopsis, a monopodial epiphytic orchid, belongs to tribe vandeae in orchidaceae

family. Phalaenopsis orchid is a fascinating and important ornamental flower in floral industry. There are 63 species of wild Phalaenopsis orchid in the world distribute between the 23^o latitude of both hemispheres, ranging throughout southern Taiwan, the Philippines, New Guinea, northern Australia, Indonesia, Malaysia, Indo-China, and to the hills of the Himalayas (Christenson, 2001). Two native species of Phalaenopsis have been reported in Taiwan, P. equestris and P. amabilis var. formosa.

They are frequently used as elite parent in commercial breeding. Due to the lack of proper protection, these two species are near extinction in the wild. Fortunately, they have been artificially propagated and produced in large quantities. As a first step to genetically engineering Phalaenopsis orchid chloroplast genome, we complete the plastid genome sequence of P. amabilis var. Formasa. In addition, the complete chloroplast genome data of P. amabilis var. Formasa will contribute to a better understanding of photosynthetic crassulacean acid mechanism (CAM) of orchidaceae plants, and could also provide molecular systematic information for the species of orchidaceae family.

三、研究方法(Materials And Methods)

Leaves of Phalaenopsis amabilis var. Formosa were obtained from four leaves stage of seedlings. Intact chloroplast was fractionated with step percoll (40-80%) gradient as described (Palmer, 1986). Chloroplast DNA was further isolated using CTAB-based protocol as described (Stewart et al., 1993). Chloroplast DNA was randomly fragmented into 2 to 3 kb in size with hydroshear, and clone into pBluescriptSK vector. After transformation of XL1 blue competent cell (Stratagene, La Jolla, USA), shot-gun clones were picked up and propagated on microtiter plates.

DNA were isolated and used as templates for sequencing analysis.

The sequencing reaction was performed by using the BigDye terminator cycle sequencing kit (Applied Biosystems, USA) according to the protocol recommended by manufactures. The DNA sequencer used was ABI 3700 of Applied Biosystems.

The sequence data from both end of each shot-gun clone were accumulated, trimmed, aligned and assembled using Phred-Phrap programs (Phil Green, University of Washington, Seattle, USA). Database searches were done with the BLAST algorithm provided by the National Center for Biotechnology Information (NCBI, USA.). For assignment of tRNA genes, the tRNAscan program was applied for prediction.

四、結果和討論 (Results And Disscussion)

The complete circular chloroplast DNA of Phalaenopsis orchid is 148,962 bp long and can be separated into two regions by a pairs of inverted repeat region (IR) of 25,731 bp. The large single copy region and small single copy region are

85,957 bp and 11,543 bp, respectively. The overall G+C content of the genome is 36.6%, close to that of other plants in angiosperms (38-39%). We also observed that the structure and organization of orchid plastid genome is highly conserved with that of tobacco, in contrast to extensive rearrangement of chloroplast large single copy (LSC) region due to inversion was reported within graminean family (Hiratsuka et. al., 1989).

The genes contained in Phalaenopsis orchid chloroplast DNA are listed in Table 1. We identified 108 genes (not included gene duplicated in the inverted repeat) of known function scattered around plastid genome. Those genes include 4 ribosomal RNA genes; 30 transfer RNA genes; 72 protein-coding genes.

Seventeen genes including 6 tRNA genes and 11 protein-coding genes contain one or two introns. The intron-containing gene in orchid is the same as tobacco.

Three genes such as rpoC1, clpP and rps12 contain two introns, in contrast to lost of introns in rpoC1 and clpP genes in plastid genome of graminean family of monocot plants. Orchid plastid genome contain a complete set of 30 tRNA genes for protein synthesis, the same as that of other higher plants in plastid genome.

Six tRNA genes contain intron. Previous report in the chloroplast genome of marsh orchid, the intron of trnL-UAA contain a minisatellite repeat locus (Cafasso, et. al., 2001); and the minisatellite repeat reveal some degree of variation during evolution among close-related orchid lineage (Cozzolino et. al., 2003). However, we found that the intron of plastid trnL-UAA gene of Phalaenopsis amabilis var.

Formosa did not have minisatellite repeat present. All 11 subunits of NADH dehydrogenase genes are present in the plastid genome of angiosperm, but completely lost in the pinus of gymnosperm (Wakasugi et. al., 1994). Surprsingly, we observed that intact ndhB, ndhC, ndhD, ndhJ and ndhK genes as well as truncated sequence of ndhE, ndhG and ndhI genes present in the chloroplast genome; but lack of ndhA, ndhF and ndhH genes in the plastid genome of orchid.

Apparently, ndhE, ndhG and ndhI are psudogenes, since entire sequence have been deleted 60% for ndhE gene, 45% for ndhG gene and 85% for ndhI gene at the 5’-terminal end as compared with other land plants. It suggest that most of ndh genes in plastid genome of orchid may have transfer to nucleus in the evolutionary process. Independently lost of functional protein-coding genes were observed among vascular plants. For instance, four ribosomal protein genes are either lost or as psudogene from plastid genomes of some plant species. The first, rpl21, is lost in all plant species except in Marcantia, Psilotum and Adiantum. The second, rpl22, is lost in the plastid genome of legume plant such as Lotus and Medicago of Fabaceae family. The third, rpl23, is present in all species except spinach as a psudogene. The forth gene, rps16 is lost in the chloroplast genome of

Marchantia, Psilotum, Pinus and Medicago. accD gene is either lost or as pseudogenes in rice, wheat and maize of poaceae family. infA is either lost or as pseudogene in the chloroplast genome of Arabidopsis, Lotus, Medicago, Atropa and tobacco. psaM gene is only present in the Marchantia, Psilotum and Pinus chloroplast genome. In addition, chlB, chlL, chLN is only present in the chloroplast genome of Marchantia and Pinus.

The orchid chloroplast genome contains potential open reading frames (ORFs) for which no known function has been assigned. Some of them are conserved among plastomes referred to as hypothetical chloroplast reading frames (ycf), suggesting that they encode functional polypeptides, for instance, ycf1 and ycf2 are essential for cell survival in tobacco (Drescher et al., 2000). The orchid plastid genome possesses ycf1 and ycf2, in contrast, them are lost in that of grass family of monocot plants. In addition, we have identified 27 putative ORF with a threshold of 225 bp (Table 1). Using the same threshold of 225 bp, 18 ORF were identified in plastid genome of Chlamydomonase, but no RNA transcripts were detected with RNA filter hybridization (Mude, 2002).

Table 1: List of gene contents in orchid plastid genome

RNA genes

Ribosomal RNA genes rrn23^b, rrn16^b, rrn5^b, rrn4.5^b

Transfer RNA genes trnA(UGC)^ab, trnC(GCA), trnD(GUC), trnE(UUC), trnF(GAA), trnG(GCC), trnG(UCC)^a trnH(GUG)^b, trnI(CAU), trnI(GAU)^ab, trnK(UUU)^a, trnL(CAA)^b, trnL(UAG), trnL(UAA)^a, trnM(CAU)^b, trnfM(CAU), trnN(GUU)^b, trnP(UGG), trnQ(UUG), trnR(UCU), trnR(ACG)^b, trnS(GCU), trnS(UGA), trnS(GGA), trnT(GGU), trnT(UGU), trnV(GAC)^b, trnV(UAC)^a, trnW(CCA), trnY(GUA)

Polypeptide genes

Ribosomal protein genes Small subunit

Large subunit

rps2, rps3, rps4, rps7^b, rps8, rps11, rps12^ab, rps14, rps15, rps16^a, rps18, rps19^b rpl2^abd, rpl14, rpl16^a, rpl20, rpl22, rpl23^b, rpl32, rpl33, rpl36

Transcription/translation apparatus genes RNA polymerase

Translation factor

rpoA, rpoB, rpoC1^a, rpoC2 infA

Photosynthesis Photosystem I Photosystem II

Cytochrome b6/f ATP synthase

NADH dehydrogenase Rubisco^c

psaA, psaB, psaC, psaI, psaJ, ycf3^a, ycf4

psbA, psbB, psbC, psbD, psbE, psbF, psbG, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ (ycf9)

petA, petB^a, petD^a, petG, petL, petN atpA, atpB, atpE, atpF^a, atpH, atpI

ndhB^ab, ndhC, ndhD^d,ψ-ndhE^e, ψ-ndhG^e, ψ-ndhI^e, ndhJ, ndhK rbcL

Miscellaneous proteins accD, clpP^a, matK, cemA, ccsA Conserved proteins ycf1, ycf2^b

Putative ORFs ORF87A(3166g) ORF77A(15391fg)^b ORF80(31670g) ORF87C(93770g) ORF131(5755 fg)^b ORF79(16318fg)^b ORF98(52593g) ORF85(99063f) ORF91(6756fg)^b ORF81B(17935fg)^b ORF87B(53845g) ORF99(100018g) ORF170(10690fg)^b ORF90(22728fg)^b ORF106(63649g) ORF79(99789f) ORF86A(10947 fg)^b ORF119(23949fg)^b ORF96(80598f) ORF81C(103872g) ORF81A(13415fg)^b ORF86B(29187fg)^b ORF103(81773g) ORF77B(142291f) ORF115(13269fg)^b ORF114(30091g) ORF88(91657f)

a. Intron containing gene; b. Two copies due to inverted repeat; c. Ribulose-1, 5-bisphosphate carboxylase/oxygenase.

d. Initiation codon creates by RNA editing; e. Pseudo gene(ψ); f. ORF located in forward strand;

g. ORF located in complementary strand. *The position of start codon of putative ORF is given in parenthesis.

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六、計劃成果自評

已依計劃進度完成蝴蝶蘭葉綠體基因體之定序、基因註解的工 作。並繪製完成整個葉綠體基因體之圖譜,正在撰寫論文,以供發表於學 術期刊。