姜汶君 郭夢月 龐曉慧
摘要?葉綠體是細胞內(nèi)具有自主遺傳信息的重要細胞器,近年來葉綠體基因組已發(fā)展成為近緣物種分子標記、揭示植物進化和系統(tǒng)發(fā)育關系的有力工具。藥用植物是人類賴以生存和發(fā)展的自然資源,葉綠體基因組在藥用植物研究中的應用價值受到廣泛關注。在總結(jié)葉綠體基因組結(jié)構(gòu)研究的基礎上,現(xiàn)就葉綠體基因組在藥用植物物種鑒定、系統(tǒng)進化等方面的應用進行綜述,以期為藥用植物種質(zhì)資源的開發(fā)和利用提供參考。
關鍵詞?藥用植物;葉綠體基因組;物種鑒定;DNA條形碼;高變異區(qū);系統(tǒng)進化;分歧時間;基因工程
Application of Chloroplast Genome in Identification and Phylogenetic Analysis of Medicinal Plants
JIANG Wenjun, GUO Mengyue, PANG Xiaohui
(Key Lab of Chinese Medicine Resources Conservation, State Administration of Traditional Chinese Medicine of the People′s Republic of China, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China; Engineering Research Center of Chinese Medicine Resources, Ministry of Education, Beijing 100193, China)
Abstract?Chloroplast is an important organelle with autonomous genetic information in the cell.In recent years, the chloroplast genome has been used as a powerful tool for molecular markers of related species and uncovering plant evolution and phylogeny.Medicinal plants are natural resources that human beings depend on for survival and development, and the application value of chloroplast genomes in the research of medicinal plants has been widely concerned.The paper reviewed the application of the chloroplast genome in species identification and phylogenetic evolution of medicinal plants based on summarizing the research of chloroplast genome structure, providing a reference for the development and utilization of germplasm resources of medicinal plants.
Keywords?Medicinal plants; Chloroplast genome; Highly divergent regions; Species identification; DNA barcode; Phylogenetic analysis; Divergence times; Genetic engineering
中圖分類號:R284文獻標識碼:Adoi:10.3969/j.issn.1673-7202.2020.05.008
葉綠體(Chloroplast)被普遍認為起源于內(nèi)共生的藍藻[1],不僅是細胞內(nèi)具有自主遺傳信息的半自主細胞器,還是植物進行光合作用和生物合成的關鍵場所。近年來隨著新一代測序技術(shù)的迅速發(fā)展和測序成本的下降,已發(fā)表的葉綠體基因組數(shù)量急劇增加。截至2019年12月,GenBank共收錄超過3 300條葉綠體基因組序列,距離煙草和地錢葉綠體基因組(Chloroplast Genome)的首次成功測序僅有短短30余年[2-3]。葉綠體基因組包含豐富的遺傳信息,以結(jié)構(gòu)、大小和基因種類均較保守而著稱[4],被廣泛應用于物種鑒定及系統(tǒng)進化研究,對于篩選藥用植物密切相關物種的分子標記以及破譯親緣關系較近植物類群間的系統(tǒng)發(fā)育關系具有重要貢獻。本文對近年來葉綠體基因組的結(jié)構(gòu)研究以及其在藥用植物鑒定及系統(tǒng)進化研究中的應用進行了綜述,分析了研究中存在的問題,同時對葉綠體基因組研究的發(fā)展方向提出展望。
1?葉綠體基因組結(jié)構(gòu)研究
得益于高通量測序技術(shù)和生物信息技術(shù)的發(fā)展,越來越多物種的葉綠體基因組被成功測序和組裝,研究表明大多數(shù)陸生植物的葉綠體基因組基本上是被2個反向重復序列(Inverted Repeat Sequence,IR)分成大單拷貝區(qū)(Large Single Copy,LSC)和小單拷貝區(qū)(Small Single Copy,SSC)的環(huán)狀四分體結(jié)構(gòu)[4]。伴隨著IR區(qū)的擴展和收縮,葉綠體基因組大小通常為120~160 kb[4-5]。葉綠體基因組共編碼120~130個基因,主要參與光合作用的光反應、轉(zhuǎn)錄和翻譯等相關功能[5-6]。盡管短片段倒位(Small Inversion)、插入/缺失(Insertion/Deletion or Indel)、基因組結(jié)構(gòu)重排、基因轉(zhuǎn)移與丟失等葉綠體結(jié)構(gòu)變異事件已被報道[7],但葉綠體基因組編碼基因種類、排列順序和含量通常被認為是保守的。
蛋白編碼基因和tRNA的數(shù)量非常相似,基因組大小的差異主要來源于IR區(qū)基因數(shù)量的不同[5]。Guo等發(fā)現(xiàn)與其他被子植物相比,五味子ycf15基因丟失,IR區(qū)明顯收縮10 kb,但IR區(qū)收縮機制尚不明確,收縮區(qū)的GC含量明顯低于IR區(qū),推測IR區(qū)收縮可能與GC含量相關[8];與2種其他傘形科藥用植物相比,濱海前胡的IR區(qū)邊界明顯擴增,并且具有一個625 bp的含有trnD-GUC、trnY-GUA、trnE-UUC基因的短片段倒位[9];此外也有部分物種丟失了IR區(qū),例如豆科植物黃耆只有一個反向重復序列[10],屬于蝶形花亞科中的反向重復序列缺失分支(Inverted Repeat-Lacking Clade)。大多數(shù)寄生植物的葉綠體基因組中存在基因丟失現(xiàn)象[11],這與其光合作用能力的部分或全部退化、從寄主上吸收養(yǎng)分和水分的生活方式有關。例如廣寄生和桑寄生,與煙草相比其葉綠體基因組丟失了ndh基因、infA基因、3個rpl基因(rpl32、rps15和rps16)、7個tRNA基因(trnA-UGC、trnG-UCC、trnH-GUG、trnL-GAU、trnK-UUU、trnL-UAA和trnV-UAC)和4個ycf基因(ycf1、ycf5、ycf9和ycf10)[12],研究表明ndh基因在從自養(yǎng)型向異養(yǎng)型轉(zhuǎn)化過程中最先丟失[13]。其他自養(yǎng)型物種在進化過程中也存在基因丟失、短片段倒位等結(jié)構(gòu)變異現(xiàn)象,其中infA基因的丟失在被子植物中最為普遍[14]。黃耆葉綠體基因組中存在3個基因(infA、rps16、rpl22)丟失,其在rps16基因和rbcL基因之間有一個50 kb的倒位,這是豆科植物中廣泛存在的現(xiàn)象[15],此外還發(fā)現(xiàn)其ndhF基因和ycf1基因之間有一個20 kb的倒位[10]。黃花蒿具有一個完全倒置的SSC區(qū)(18 267 bp),這一現(xiàn)象在其他菊科蒿屬植物中也曾被報道,此外rps19基因在LSC區(qū)而非IR區(qū),rps19假基因在IR區(qū)缺失[16]。菊科橐吾屬藥用植物葉綠體基因組的LSC區(qū)具有一個約3.4 kb的小倒位嵌套在23 kb的大倒位中,這也是菊科植物的一個獨特特征[17]。He等報道忍冬葉綠體基因組在trnI-CAU和trnN-GUU之間有一個獨特的重排現(xiàn)象,ycf1假基因丟失,rps19、rpl2和rpl23基因從IR區(qū)移動到LSC區(qū),并首次發(fā)現(xiàn)ycf2和rps18基因中含有內(nèi)含子[18]。
2?藥用植物葉綠體基因組的應用
2.1?物種鑒定
近年來,DNA條形碼(DNA Barcoding)已發(fā)展成為物種鑒定的強有力工具。在植物物種鑒定中,常用的DNA片段包括葉綠體基因組序列rbcL、matK、psbA-trnH等與核基因組的ITS和ITS2[19]。其中,鑒于ITS2序列較強的鑒定能力,已被建議作為藥用植物的通用DNA條形碼[20]。然而,對于一些特殊藥用植物類群,普通DNA條形碼不具備足夠的變異信息以實現(xiàn)物種鑒定,例如淫羊藿屬和貝母屬[21-22]。與普通的DNA條形碼短片段相比,葉綠體全基因組包含更豐富的變異位點,鑒定效率更高。葉綠體全基因組一方面被直接用作物種鑒定的超級條形碼(Super Barcode),另一方面則基于其篩選出高變區(qū)作為鑒定物種的潛在分子標記[23-25]。
2.1.1?超級條形碼?Cui等比對分析了砂仁、縮砂仁、海南砂仁等32種豆蔻屬葉綠體基因組,結(jié)果表明葉綠體全基因組可準確鑒定豆蔻屬物種[26]。以往研究表明ITS、matK、psbA-trnH和rbcL對橐吾屬的物種鑒定效率不理想[27],Chen等報道了6種橐吾屬植物的葉綠體基因組并提出以全基因組作為超級條形碼可有效鑒定橐吾屬植物[17]。鐘志敏發(fā)現(xiàn)基于葉綠體全基因組不僅可以有效鑒定石斛屬41個物種,還可區(qū)分來自3個不同產(chǎn)地的鐵皮石斛[28]。Xia等的研究表明了基于葉綠體全基因組可將野菊和近緣物種準確區(qū)分開,可作為鑒定這些物種的超級條形碼[29]。
2.1.2?高變區(qū)篩選?李雪佩等比較分析了黃草烏和展毛黃草烏的葉綠體全基因組,發(fā)現(xiàn)trnR-atpA和atpF-atpH基因間隔區(qū)上的缺失可作為分子標記鑒定2個物種[30]。葉綠體InDel分子標記也被證明是鑒定白首烏[31]、6種忍冬屬植物[32]和人參屬[33]的有效工具。馬雙姣基于測定的薯蕷和叉蕊薯蕷以及從GenBank下載的4條薯蕷屬植物葉綠體基因組比對分析,篩選出了5個蛋白編碼基因和5個基因間隔區(qū)作為潛在分子標記[34]。Chen等通過對川貝母及其近緣物種的8條葉綠體全基因組進行比對分析,篩選出6個高變區(qū)作為鑒定貝母屬植物的潛在DNA條形碼,并提出葉綠體全基因組作為超級條形碼可能更適合該屬的物種鑒定和系統(tǒng)發(fā)育分析[35]。楊俏俏等通過比對分析6種蔥屬植物的葉綠體基因組,共篩選到7個變異較大的候選DNA條形碼序列用于鑒定蔥屬植物[36]。李曉娟等發(fā)現(xiàn)華重樓葉綠體基因組中cemA基因是假基因,該基因編碼序列中的終止密碼子位置差異可作為區(qū)分華重樓和北重樓的依據(jù)[37]。Do等基于黃瓜假還陽參的葉綠體基因組開發(fā)了新的來自于rbcL和matK基因的SNP分子標記,可將其與相關物種進行有效區(qū)分[38]。Chen等基于3個麻黃屬藥用物種的葉綠體基因組篩選出11個高變區(qū),可作為鑒定麻黃屬植物的潛在DNA條形碼[39]。Zhou等基于掌葉大黃、藥用大黃、唐古特大黃的葉綠體基因組開發(fā)了包含鑒定位點的潛在DNA條形碼并成功驗證,為超級DNA條形碼在大黃中的應用奠定了基礎[40]。
2.2?系統(tǒng)進化
葉綠體基因組在大多數(shù)被子植物中為母系遺傳,重組率低、核苷酸置換率適中[41],具有良好的系統(tǒng)發(fā)育重建潛力。葉綠體基因組的遺傳信息在解決系統(tǒng)發(fā)育位置和不同物種間的親緣關系方面做出了重要貢獻,同時也被廣泛用于揭示物種起源和估計各譜系間的分歧時間。
2.2.1?系統(tǒng)發(fā)育位置?魏似婕等基于藏藥“巴萵色保”的基原植物獐牙菜屬輪葉獐牙菜、川西獐牙菜及6種龍膽屬植物葉綠體全基因組構(gòu)建ML樹,結(jié)果支持經(jīng)典分類學觀點對2個屬物種的分類[42]。沈立群基于實驗獲得的薄荷、白毛夏枯草和活血丹3種唇形科藥用植物的葉綠體基因組及從NCBI下載的唇形科序列構(gòu)建了系統(tǒng)發(fā)育樹,3種植物在進化樹中的位置均被支持且與傳統(tǒng)分類學相符[43]。張慧等基于ML系統(tǒng)進化分析確定了益母草在野芝麻亞科中的進化位置,發(fā)現(xiàn)益母草屬和水蘇屬的親緣關系較近[44]。在Chen等的研究中,基于對川貝母及其密切相關物種的系統(tǒng)發(fā)育分析顯示,8個貝母屬植物聚成3支且均有較強的支持率,川貝母與甘肅貝母、中華貝母密切相關[35]。Chen等報道基于葉綠體基因組構(gòu)建的ML和MP系統(tǒng)發(fā)育樹可以鑒定3種麻黃屬植物,同時發(fā)現(xiàn)麻黃屬植物與買麻藤屬植物、百歲蘭具有更近的系統(tǒng)發(fā)育關系[39]。Cui等基于已發(fā)表姜目物種的葉綠體基因組序列,探討了姜在姜目中的系統(tǒng)發(fā)育位置,結(jié)果表明姜屬為山柰屬的姊妹分支[45]。Gao等分析了益智的葉綠體基因組并構(gòu)建了姜科的系統(tǒng)發(fā)育樹,結(jié)果強烈支持姜科植物喜陰和喜陽的特征分類[46]。Gichira等對非洲一種隸屬于薔薇科的藥用植物Hagenia abyssinica的葉綠體基因組進行了測定并構(gòu)建了系統(tǒng)發(fā)育樹,結(jié)果支持將該屬分在薔薇亞科下[47]。Guo等首次報道了五味子的葉綠體基因組序列,通過構(gòu)建ML和BI系統(tǒng)發(fā)育樹確定了五味子屬與八角屬形成姊妹群[8]。Gu等測定了藥用植物黃薇的葉綠體基因組,基于12種桃金娘目物種共有的68個CDS編碼基因構(gòu)建了系統(tǒng)發(fā)育樹,結(jié)果表明其與紫薇屬物種的親緣關系更近[48]。Li等基于廣寄生、桑寄生等10個物種的54個蛋白編碼基因和matK基因構(gòu)建的NJ和ML系統(tǒng)發(fā)育樹拓撲結(jié)構(gòu)基本一致,結(jié)果有力支持桑寄生科和槲寄生科為獨立的2個單系類群[12]。
2.2.2?分歧時間估計?Lee等以Daucus carata和人參為標定點,基于14個傘形科物種的76個葉綠體基因組共有蛋白編碼基因構(gòu)建貝葉斯進化樹,結(jié)果表明防風和濱海前胡的分歧時間大約在2.3~3.4百萬年前(Million Years Ago,Mya),而珊瑚菜與紫花前胡、大葉山芹的分歧時間大約在1.6~2.8 Mya[9]。He等基于8種唇形科植物及外類群的葉綠體基因組構(gòu)建系統(tǒng)發(fā)育樹,采用分子鐘模型估算廣藿香分歧時間,結(jié)果表明其大約在29.45 Mya從黃芩亞科分支分化[49]。采用最大似自然法對21個毛茛目物種和2個木蘭科物種的葉綠體基因組進行了系統(tǒng)發(fā)育推斷分析,He等發(fā)現(xiàn)黃連與其他毛茛科物種的分歧時間約為81 Mya,而毛茛科和小檗科的分歧時間約為111 Mya,在侏羅紀時期(大約153 Mya)兩者有一個共同的祖先[50]。Kim等基于葉綠體基因組比較分析了五加科的系統(tǒng)發(fā)育關系,樹參屬與五加屬的分歧時間大約在4.48~5.60 Mya,人參屬與楤木屬的分歧時間大約在2.58~3.20 Mya,人參和西洋參的分歧時間大約在0.72~0.87 Mya[51]。Fan等基于葉綠體基因組的蛋白編碼序列計算推斷了14個川續(xù)斷目物種的分歧時間,五?;婆c忍冬科的分歧時間大約為81.14 Mya,莢蒾屬與華?;▽?、五福花屬、四福花屬3個屬的分歧時間大約為47.16 Mya,華福花屬、五福花屬、四?;▽俚姆制鐣r間大約在15~16 Mya[52]。
3?小結(jié)與展望
新一代測序技術(shù)的出現(xiàn)和進步是葉綠體基因組學和遺傳學領域快速發(fā)展的重要因素之一,眾多植物葉綠體全基因組序列的研究和報道,不僅增強了我們對于葉綠體結(jié)構(gòu)功能、系統(tǒng)進化的認知,也為葉綠體轉(zhuǎn)化提供了重要信息。葉綠體基因組編碼植物參與光合作用和其他必要代謝過程的關鍵蛋白,以應對干旱、鹽、熱、光等環(huán)境壓力及防御入侵病原體[53]。葉綠體轉(zhuǎn)化具有蛋白表達水平高、從多順反子mRNA中表達多種蛋白的可行性、避免花粉傳播造成的基因污染等優(yōu)點[54]。葉綠體轉(zhuǎn)基因工程在增強藥用植物的優(yōu)良農(nóng)藝性狀或生產(chǎn)高價值的生物醫(yī)學產(chǎn)品等方面具有巨大潛力。
由于葉綠體自身的復制機制和相對獨立的進化,現(xiàn)已廣泛應用于藥用植物的物種鑒定和系統(tǒng)進化研究中。見表1。物種鑒定是生物多樣性研究的重要前提和基礎,DNA條形碼不依賴于傳統(tǒng)的形態(tài)學鑒定經(jīng)驗,此概念一經(jīng)提出便受到國內(nèi)外學者的廣泛關注和逐步認可,但目前通用的DNA條形碼不足以準確鑒別所有的藥用植物物種。隨著新一代測序技術(shù)和生物信息技術(shù)的進步,葉綠體全基因組作為超級條形碼或基于葉綠體測序分析篩選候選DNA條形碼逐步發(fā)展起來,植物葉綠體基因組的數(shù)量也在逐年增加。就藥用植物而言,目前已發(fā)表的葉綠體基因組序列的數(shù)量還存在不足。葉綠體基因組目前已顯示出了巨大的物種識別潛力,尤其是在親緣關系較近的類群之間,因此有必要對更多的藥用植物進行葉綠體基因組測序,深入研究其鑒定與系統(tǒng)進化問題。
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(2020-02-10收稿?責任編輯:徐穎)