汪燕，梁成剛，陳晴晴，石桃雄，陳其皎，孟子燁，鄧嬌，黃娟，陳慶富

蕎麥屬植物脫落酸不敏感基因序列比較與親緣關(guān)系研究

汪燕，梁成剛*，陳晴晴，石桃雄，陳其皎，孟子燁，鄧嬌，黃娟，陳慶富1

(貴州師范大學(xué)蕎麥產(chǎn)業(yè)技術(shù)研究中心，貴陽550001)

為了研究蕎麥屬不同種間植物脫落酸不敏感基因（ABA-insensitive,ABI）序列變異差異與親緣關(guān)系，試驗(yàn)選用7個(gè)蕎麥野生種，包括野甜蕎、野苦蕎、左貢野蕎、毛野蕎、大野蕎、金蕎麥、硬枝萬年蕎共30份種質(zhì)材料，提取苗期幼嫩葉片的DNA，通過聚合酶鏈?zhǔn)椒磻?yīng)（polymerase chain reaction,PCR）擴(kuò)增獲得蕎麥ABI基因，并對(duì)擴(kuò)增片段進(jìn)行基因測(cè)序與序列分析。結(jié)果發(fā)現(xiàn)，7個(gè)野生種共30份種質(zhì)間ABI基因片段的多態(tài)位點(diǎn)數(shù)占20.28%，其中，14份野苦蕎間ABI基因片段多態(tài)位點(diǎn)數(shù)占1.86%，5份野甜蕎間多態(tài)位點(diǎn)數(shù)占1.81%，說明蕎麥屬植物種內(nèi)ABI基因序列高度保守。對(duì)擴(kuò)增序列進(jìn)行遺傳距離分析發(fā)現(xiàn)，左貢野蕎與毛野蕎種間遺傳距離最大，硬枝萬年蕎與金蕎麥種間遺傳距離最小。聚類分析結(jié)果顯示：野苦蕎與大野蕎、金蕎麥和硬枝萬年蕎親緣關(guān)系較近，與毛野蕎親緣關(guān)系次之；左貢野蕎與野甜蕎親緣關(guān)系近；不同地區(qū)野苦蕎和左貢野蕎分別被單獨(dú)聚為一類，說明生長(zhǎng)環(huán)境對(duì)蕎麥ABI基因序列變異具有影響。

蕎麥；脫落酸不敏感基因；序列分析；親緣關(guān)系

蕎麥?zhǔn)寝た疲≒olygnaceae）蕎麥屬（Fagopyrum Mill.）植物[1-2]，是我國(guó)糧食作物中重要的小宗雜糧，其蛋白質(zhì)含量高，富含黃酮類化合物，特別是蘆丁含量較高，具有顯著的降血脂、降血糖和降血壓等營(yíng)養(yǎng)保健功能[2-3]。陳慶富[1]指出，蕎麥屬至少包括23個(gè)種類，根據(jù)形態(tài)學(xué)鑒定可分為大粒組和小粒組2個(gè)類群，類群間親緣關(guān)系較遠(yuǎn)?，F(xiàn)有研究普遍認(rèn)為蕎麥起源于中國(guó)的西南地區(qū)[2]。YAMANE等[4]的研究指出，四倍體金蕎麥主要分布于西藏地區(qū)，二倍體金蕎麥則主要分布在西藏、云南和四川等地，其中分布于云南和四川地區(qū)的二倍體金蕎麥可分別被聚為一類。近年來，學(xué)者們對(duì)蕎麥的起源與進(jìn)化關(guān)系進(jìn)行了較為深入的研究，但由于蕎麥屬植物種間關(guān)系比較復(fù)雜，至今仍未有一致定論[5-10]。隨著分子生物學(xué)技術(shù)的不斷發(fā)展，在傳統(tǒng)形態(tài)學(xué)鑒定、蛋白亞基和同工酶技術(shù)等基礎(chǔ)上，結(jié)合DNA分子鑒定技術(shù)可為研究蕎麥進(jìn)化關(guān)系提供更為可靠的依據(jù)。

蕎麥主要生長(zhǎng)于高寒地區(qū)，環(huán)境較為惡劣，常遇干旱與凍害等自然災(zāi)害，因此，在長(zhǎng)期的進(jìn)化過程中，蕎麥在抗旱、抗寒，以及耐貧瘠等方面表現(xiàn)出較強(qiáng)的耐逆特性[11]。脫落酸（abscisic acid,ABA）是重要的植物應(yīng)激激素，在調(diào)控作物抗逆性方面具有重要作用[12-13]。生物或非生物逆境會(huì)誘導(dǎo)植物體內(nèi)迅速合成ABA，進(jìn)而增強(qiáng)植物逆境耐受能力[14]。ABA信號(hào)途徑的分子調(diào)控網(wǎng)絡(luò)十分復(fù)雜，其中，脫落酸不敏感（ABA-insensitive,ABI）家族蛋白是參與調(diào)控ABA信號(hào)途徑的重要轉(zhuǎn)錄因子[15-16]。ABI蛋白中ABI1和ABI2在ABA信號(hào)轉(zhuǎn)導(dǎo)途徑中起著負(fù)調(diào)控作用[17]，二者功能缺失都會(huì)表現(xiàn)出對(duì)ABA的高度敏感。ABI3、ABI4和ABI5則通過調(diào)控ABA信號(hào)轉(zhuǎn)導(dǎo)途徑參與調(diào)控種子成熟、休眠、萌發(fā)和幼苗生長(zhǎng)過程[18-22]。郝紅梅[23]研究發(fā)現(xiàn)，植物ABI序列較為保守，可用于植物進(jìn)化與親緣關(guān)系研究。迄今為止，尚未見有關(guān)蕎麥ABI基因研究的相關(guān)報(bào)道。因此，本研究從轉(zhuǎn)錄組測(cè)序構(gòu)建的UniGene數(shù)據(jù)庫中篩選獲得蕎麥ABI UniGene信息，通過設(shè)計(jì)特異性引物對(duì)蕎麥屬不同野生種進(jìn)行目標(biāo)基因的聚合酶鏈?zhǔn)椒磻?yīng)（polymerase chain reaction,PCR）擴(kuò)增，利用序列差異分析與聚類分析，探討蕎麥ABI基因在進(jìn)化過程中的序列變異規(guī)律與不同蕎麥野生種的起源關(guān)系。

1 材料與方法

1.1 試驗(yàn)材料

試驗(yàn)選用7個(gè)蕎麥野生種，包括野甜蕎（F.esculentum）、野苦蕎（F.tataricum）、左貢野蕎（F.zuogongense）、金蕎麥（F.cymosum）、硬枝萬年蕎（F.urophyllum）、毛野蕎（F.pilus）和大野蕎（F.megaspartanium），共30份材料（由貴州師范大學(xué)蕎麥產(chǎn)業(yè)技術(shù)研究中心提供，表1）。

表1 試驗(yàn)材料Table 1 Plant materials used in this study

1.2 目標(biāo)基因PCR擴(kuò)增及測(cè)序

在苗期對(duì)植物材料進(jìn)行取樣，采用CTAB法提取幼嫩葉片的DNA，具體操作步驟參考陳晴晴等[24]的方法。通過轉(zhuǎn)錄組測(cè)序構(gòu)建的數(shù)據(jù)庫（登錄號(hào)：SRS1350375）進(jìn)行蕎麥ABI UniGene篩選。利用Primer 5.0軟件對(duì)篩選獲得的ABI基因進(jìn)行設(shè)計(jì)并合成特異性引物[生工生物工程（上海）股份有限公司]。目標(biāo)基因的PCR擴(kuò)增與瓊脂糖凝膠電泳參考陳晴晴等[24]的方法。將檢測(cè)合格的PCR擴(kuò)增產(chǎn)物送至生工生物工程（上海）股份有限公司進(jìn)行測(cè)序。

1.3 測(cè)序結(jié)果分析與進(jìn)化樹的構(gòu)建

利用 NCBI BLAST（http://blast.ncbi.nlm.nih.gov/Blast.cgi）進(jìn)行序列的在線比對(duì)。將序列對(duì)齊分值大于130、同一性大于70%的基因序列定義為高度同源。使用DNAsp5軟件進(jìn)行序列差異分析，利用MEGA 5.05軟件進(jìn)行ClustalW多序列比對(duì)、遺傳距離和聚類分析。

2 結(jié)果與分析

2.1 蕎麥ABI基因的PCR擴(kuò)增

對(duì)轉(zhuǎn)錄組基因測(cè)序（登錄號(hào)：SRS1350375）獲得的蕎麥UniGene數(shù)據(jù)庫進(jìn)行基因序列比對(duì)，篩選得到蕎麥ABI的UniGene序列。提取30個(gè)蕎麥野生種質(zhì)幼嫩葉片中的DNA，設(shè)計(jì)ABI UniGene的特征引物，進(jìn)行基因序列擴(kuò)增。瓊脂糖凝膠電泳檢測(cè)結(jié)果顯示，在400～500 bp間出現(xiàn)目標(biāo)條帶（圖1）。

圖1 利用特異引物獲得的PCR擴(kuò)增產(chǎn)物Fig.1 PCR amplification products obtained using specific primers

2.2 蕎麥ABI基因序列差異分析

將上述擴(kuò)增得到的所有目標(biāo)條帶送生工生物工程（上海）股份有限公司進(jìn)行測(cè)序，然后用NCBI BLAST對(duì)其中的WT1目標(biāo)片段序列進(jìn)行比對(duì)，發(fā)現(xiàn)其與蓖麻（Ricinus communis）ABI5-like2基因（登錄號(hào)：XM_015729138.1）、葡萄（Vitis vinifera）ABI5-like2基因（登錄號(hào)：XM_002265711.3）和棗（Ziziphus jujube）ABI5-like2基因（登錄號(hào)：XM_016020783.1）等序列對(duì)齊分值大于130，相似性高于70%。說明擴(kuò)增所獲得的目標(biāo)片段為蕎麥ABI基因。對(duì)30份蕎麥野生材料ABI基因片段序列中的429個(gè)排列位點(diǎn)進(jìn)行多重比較分析，發(fā)現(xiàn)不變位點(diǎn)為342個(gè)，多態(tài)位點(diǎn)（S）為87個(gè)，占20.28%，其中包含84個(gè)簡(jiǎn)約信息位點(diǎn)和3個(gè)單型可變位點(diǎn)。說明蕎麥屬植物種間ABI基因序列差別較小。對(duì)14份野苦蕎ABI基因的431個(gè)排列位點(diǎn)進(jìn)行多重比較分析，發(fā)現(xiàn)不變位點(diǎn)423個(gè)，多態(tài)位點(diǎn)8個(gè)，占1.86%，其中包含6個(gè)簡(jiǎn)約信息位點(diǎn)和2個(gè)單型可變位點(diǎn)；對(duì)5份野甜蕎ABI基因的442個(gè)排列位點(diǎn)進(jìn)行多重比較分析，發(fā)現(xiàn)不變位點(diǎn)434個(gè)，多態(tài)位點(diǎn)8個(gè)，占1.81%，其中包含1個(gè)簡(jiǎn)約信息位點(diǎn)和7個(gè)單型可變位點(diǎn)。說明蕎麥屬植物種內(nèi)ABI基因序列高度保守。

2.3 基于ABI基因序列的親緣關(guān)系

對(duì)30份蕎麥屬野生材料的ABI基因片段序列進(jìn)行遺傳距離分析，發(fā)現(xiàn)種間平均遺傳距離為0.015，其中野甜蕎與野苦蕎的種間平均遺傳距離為0.023，與大野蕎為0.025，與毛野蕎為0.024，與左貢野蕎為0.003。從表2可知，左貢野蕎與大野蕎種間遺傳距離最大（0.037），硬枝萬年蕎與金蕎麥種間遺傳距離最?。?.000）。

表2 基于ABI基因片段序列的種間平均遺傳距離（下三角）和種間平均凈遺傳距離（上三角）Table 2 Interspecific genetic distance(below diagonal)and interspecific net genetic distance(above diagonal)based on ABI gene sequence

利用植物ABI基因序列種內(nèi)高度保守的特性，對(duì)7個(gè)野生種共30份野生材料進(jìn)行聚類分析。結(jié)果（圖2）發(fā)現(xiàn)：硬枝萬年蕎、大野蕎、野苦蕎、金蕎麥和毛野蕎被聚為一類，節(jié)點(diǎn)的自舉置信度為98%；野甜蕎與左貢野蕎被聚為一類，節(jié)點(diǎn)的自舉置信度為99%；收集于四川的3個(gè)野苦蕎WT1、WT4和WT5被單獨(dú)聚為一類；野甜蕎與收集于西藏的左貢野蕎Z1和Z2被聚為一類；收集于云南和四川的左貢野蕎Z3、Z4與Z1、Z2及其他野生蕎麥的親緣關(guān)系較遠(yuǎn)，被單獨(dú)聚為一類。

圖2 基于蕎麥ABI基因序列的聚類樹狀圖Fig.2 Phylogenetic dendrogram of buckwheat collections based on specific sites of ABI gene fragment sequence

3 討論

蕎麥生長(zhǎng)適應(yīng)性強(qiáng)，具有較強(qiáng)的抗寒、抗旱、抗輻射和耐貧瘠能力，是一種重要的救災(zāi)糧食作物[2,11]。ABI轉(zhuǎn)錄因子在調(diào)控ABA信號(hào)轉(zhuǎn)導(dǎo)介導(dǎo)的逆境脅迫應(yīng)答方面具有重要作用[25-27]。郝紅梅[23]的研究表明，白菜ABI1序列與擬南芥ABI1序列高度保守。本研究發(fā)現(xiàn)，7個(gè)野生種共30份野生種質(zhì)的ABI基因片段的多態(tài)位點(diǎn)占20.28%，其中，14份野苦蕎間多態(tài)位點(diǎn)占1.86%，5份野甜蕎間多態(tài)位點(diǎn)占1.81%：說明蕎麥屬植物種內(nèi)ABI基因序列高度保守。因此，蕎麥ABI基因序列差異可作為研究蕎麥屬植物親緣關(guān)系的依據(jù)。此外，本研究還發(fā)現(xiàn)：收集于四川和云南的左貢野蕎Z3和Z4單獨(dú)聚為一類，與西藏的左貢野蕎Z1和Z2存在較明顯的差異；同時(shí)，收集于四川的野苦蕎WT1、WT4和WT5也被單獨(dú)聚在一起，與其余地區(qū)的野苦蕎存在較明顯差異。說明在進(jìn)化過程中生長(zhǎng)環(huán)境對(duì)蕎麥ABI基因序列變異具有影響。

蕎麥屬包括至少23個(gè)種，其種間親緣關(guān)系較為復(fù)雜[1]。陳慶富[1,28]通過形態(tài)學(xué)鑒定發(fā)現(xiàn)左貢野蕎與甜蕎較為相似。梁成剛等[29]對(duì)蕎麥屬植物13S球蛋白基因序列進(jìn)行分析發(fā)現(xiàn)，甜蕎及野甜蕎與左貢野蕎親緣關(guān)系較近。根據(jù)ABI基因片段序列差異，在本研究中收集于西藏的左貢野蕎Z1、Z2和野甜蕎聚為一類，說明其親緣關(guān)系較近。前人通過形態(tài)學(xué)觀察和同工酶研究等方式推測(cè)甜蕎與苦蕎可能由金蕎進(jìn)化而來[7-9]。陳慶富[1,28]研究發(fā)現(xiàn)金蕎中存在3種類型且彼此間生殖隔離，因此，將其中2類二倍體種分別命名為大野蕎與毛野蕎，并將兩者與金蕎一起歸為金蕎復(fù)合物一類。張以忠等[6]利用過氧化物酶同工酶鑒定，將甜蕎與大野蕎、苦蕎與毛野蕎分別聚為一類。LI等[5]則通過十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳（SDS-PAGE）鑒定將甜蕎、左貢野蕎與大野蕎聚為一類，將苦蕎、金蕎、毛野蕎和巨蕎聚為一類。任翠娟等[30]通過隨機(jī)擴(kuò)增多態(tài)性DNA標(biāo)記技術(shù)（RAPD）鑒定，發(fā)現(xiàn)大野蕎與甜蕎、毛野蕎與苦蕎親緣關(guān)系較近。由此，陳慶富[1]推測(cè)甜蕎的祖先種可能為大野蕎，而苦蕎的祖先種可能為毛野蕎。然而，甜蕎是典型的異花授粉植物，苦蕎為典型的自花授粉植物，其各自的進(jìn)化歷程十分復(fù)雜[9]。鄭亞迪[31]基于ITS.matK和psbA-trnH單基因與葉綠體matK+psbA-trnH和ITS+matK+psbA-trnH合并數(shù)據(jù)集分析，發(fā)現(xiàn)四川海螺溝野蕎與其他蕎麥類群均存在較遠(yuǎn)的親緣關(guān)系，推測(cè)其可能為最原始的蕎麥種。本研究利用ABI基因序列差異鑒定發(fā)現(xiàn)，大野蕎與野苦蕎、金蕎麥聚為一類，然后再與毛野蕎聚為一類，而大野蕎與野甜蕎遺傳距離較遠(yuǎn)，這與梁成剛等[29]的研究結(jié)果一致。然而，目前尚不能確定蕎麥不同種間的具體進(jìn)化歷程。

硬枝萬年蕎的遺傳多樣性非常豐富，其進(jìn)化關(guān)系較為復(fù)雜。史建強(qiáng)等[32]利用簡(jiǎn)單重復(fù)序列（SSR）分子標(biāo)記鑒定，將硬枝萬年蕎與疏穗小野蕎、細(xì)柄野蕎、小野蕎和齒翅野蕎聚為一類。梁成剛等[29]利用13S球蛋白基因序列差異鑒定，將細(xì)柄野蕎、硬枝萬年蕎、苦蕎和部分野苦蕎聚為一類。鄭亞迪[31]利用葉綠體matK和psbA-trnH序列聯(lián)合分析將硬枝萬年蕎與苦蕎聚為一類，然后再與金蕎和汶川苦蕎聚為一類，并通過綜合分析認(rèn)為硬枝萬年蕎是較汶川野蕎和金蕎更原始的野生蕎麥種。本研究發(fā)現(xiàn)，硬枝萬年蕎的ABI基因片段序列與大野蕎及部分野苦蕎聚為一類，說明其差異較小，親緣關(guān)系較近。

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Analysis of abscisic acid-insensitive gene(ABI)sequence and phylogenetic relationship in genus Fagopyrum.Journal of Zhejiang University(Agric.&Life Sci.),2017,43(5):536-542

WANG Yan,LIANG Chenggang*,CHEN Qingqing,SHI Taoxiong,CHEN Qijiao,MENG Ziye,DENG Jiao,HUANG Juan,CHEN Qingfu
(Research Center of Buckwheat Industry Technology,Guizhou Normal University,Guiyang 550001,China)

Fagopyrum;abscisic acid-insensitive gene(ABI);sequence analysis;phylogenetic relationship

S 517；Q 941.2

A

10.3785/j.issn.1008-9209.2016.09.231

Summary Abscisic acid(ABA)is a vital hormone that regulates stomatal closure and responds to stress conditions in plants.Therefore,ABA signal pathway involves numerous physiological processes in plants,such as seed dormancy,germination,development and ripening.Among the families of ABA receptor genes,ABA-insensitive(ABI)is a major transcription factor that regulates ABA signal pathway.To date,little information is known about the ABI gene of buckwheat.

To characterize ABI gene sequence polymorphism and genetic relationship of genus Fagopyrum,the budlets of 30 germplasm resources from seven wild species including F.esculentum,F.tataricum,F.zuogongense,F.pilus,F.megaspartanium,F.cymosum and F.urophyllum were collected for DNA extraction.The ABI UniGene sequence of buckwheat was isolated from the previously performed transcriptome sequencing data(accession number:SRS1350375).Specific primers of ABI gene were designed based on the high conservative sequence for polymerase chain reaction(PCR)amplification,and the amplified fragmentsfrom 30 germplasm resources were processed for gene sequencing.Then the gene sequence analysis was performed on nucleotide BLAST of NCBI database(http://blast.ncbi.nlm.nih.gov/Blast.cgi),and the genetic variance was analyzed by DNAsp5,and the analysis of ClustalW multiple sequence alignment,genetic distance and hierarchy were clustered by MEGA 5.05.

國(guó)家自然科學(xué)基金（31471562，31660366）；國(guó)家燕麥?zhǔn)w麥現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系專項(xiàng)資金（CARS-08-A4）；貴州省科技廳合作計(jì)劃項(xiàng)目（黔科合LH字〔2015〕7770號(hào)）；貴州師范大學(xué)博士科研資助項(xiàng)目；貴州省“三區(qū)”人才工作專項(xiàng)經(jīng)費(fèi)（0515075）；貴州省蕎麥工程技術(shù)研究中心項(xiàng)目（黔科合農(nóng)G字〔2015〕4003號(hào)）；貴州省高層次創(chuàng)新型人才培養(yǎng)對(duì)象“十百千”計(jì)劃（2014GZ97588）。

梁成剛（http://orcid.org/0000-0001-8916-6872），Tel：+86-851-86780646，E-mail:jesselcg@163.com

（First author）：汪燕（http://orcid.org/0000-0002-0055-6215），E-mail:yanwanguf@163.com

（

）:2016-09-23；接受日期（Accepted）:2017-05-09

The results showed that 20.28%of polymorphic sites were found in ABI gene fragments among 30 germplasm resources of the seven wild species of genus Fagopyrum,whereas only 1.86%of polymorphic sites were found among 14 germplasm resources of F.tataricum and 1.81%of polymorphic sites were found among five germplasm resources of F.esculentum,indicating that ABI gene sequence is highly conserved in the intraspecies of genus Fagopyrum.Genetic distance analysis showed that both the values of interspecific genetic distance and interspecific net genetic distance between F.zuogongense and F.pilus were the largest,whereas those between F.cymosum and F.urophyllum were the least based on the ABI gene sequence analysis.Hierarchical cluster analysis indicated that the phylogenetic relationship of F.tataricum was close to F.megaspartanium,F.cymosum and F.urophyllum,then further grouped with F.pilus,while the F.esculentum was close to F.zuogongense based on the ABI gene sequence analysis.The F.tataricum and F.zuogongense collected from different regions were respectively clustered,suggesting that the mutation of ABI gene might be influenced by the growth environment during plant evolution.

Inconclusion,ABIgenesequenceishighlyconservedintheintraspeciesofgenusFagopyrum;nevertheless,differentgrowth environmentscauseddistinctivemutationsofABIgene.Basedonthegenesequenceanalysis,acloserelationshipwasfoundbetween F.esculentum andF.zuogongense.Inaddition,closerelationshipswerealsofoundamong F.tataricum,F.megaspartanium,F.cymosumandF.urophyllum.TheaboveresultsprovideatheoreticalbasisforABIgeneresearchandevolutionaryrelationshipof genusFagopyrum,buttheexactevolutionamongdifferentspeciesofbuckwheatstillneedsfurtherresearch.