宏轉(zhuǎn)錄組學(xué)在環(huán)境微生物生態(tài)學(xué)中的應(yīng)用

李 瑩,吳興杰,賀治斌,貝水寬,馬 可,彭靜靜*

宏轉(zhuǎn)錄組學(xué)在環(huán)境微生物生態(tài)學(xué)中的應(yīng)用

李 瑩1,吳興杰2,賀治斌2,貝水寬2,馬 可1,彭靜靜2*

(1.中國農(nóng)業(yè)大學(xué)資源與環(huán)境學(xué)院,農(nóng)田土壤污染防控與修復(fù)北京市重點實驗室,北京 100193；2.中國農(nóng)業(yè)大學(xué)資源與環(huán)境學(xué)院,國家農(nóng)業(yè)綠色發(fā)展研究院,植物-土壤相互作用教育部重點實驗室,北京 100193)

系統(tǒng)總結(jié)了宏轉(zhuǎn)錄組學(xué)實驗操作及數(shù)據(jù)分析流程,概述了宏轉(zhuǎn)錄組學(xué)在環(huán)境微生物生態(tài)學(xué)的研究策略和最新進展,并指出其應(yīng)用前景.宏轉(zhuǎn)錄組學(xué)在解析環(huán)境微生物群落功能上具有廣闊的前景,為了解微生物群落的動態(tài)演化及其與環(huán)境因素和生態(tài)系統(tǒng)功能的關(guān)系提供了強有力的工具.

宏轉(zhuǎn)錄組；微生物組；群落結(jié)構(gòu)；功能基因；mRNA富集；RNA

近年來,微生物組學(xué)發(fā)展迅速.宏基因組、宏轉(zhuǎn)錄組、宏蛋白組和宏代謝組等組學(xué)方法被廣泛用于揭示微生物群落組成及功能,探索生態(tài)系統(tǒng)中復(fù)雜微生物群落的基因圖譜傳遞的信息.其中宏轉(zhuǎn)錄組側(cè)重于研究活躍微生物組基因的表達及其對環(huán)境的響應(yīng),為研究活躍微生物組群落動態(tài)變化和功能響應(yīng)提供了全新見解.

微生物組是指包括微生物(細菌、古菌、真核生物和病毒)的基因組(基因)及其環(huán)境在內(nèi)的所有生物和非生物因素的總和[1].環(huán)境微生物組存在著極大的物種和功能多樣性,是生態(tài)系統(tǒng)發(fā)揮功能和服務(wù)人類的基礎(chǔ)[2],在生物地球化學(xué)循環(huán)[3]、農(nóng)業(yè)生產(chǎn)[4-5]、土壤修復(fù)[6]以及溫室氣體調(diào)控[7]等方面均發(fā)揮著重要作用,直接或間接地影響著動植物和人類健康以及氣候變化.土壤微生物的生物量與陸地動物和植物的生物量相當(dāng),是保持土壤肥力、有機碳固定[8]、促進植物生產(chǎn)以及維持生態(tài)系統(tǒng)功能的重要組成部分[[9-10].海洋微生物則在生物地球化學(xué)循環(huán)過程中扮演著重要角色,對保持海洋生態(tài)系統(tǒng)的健康和穩(wěn)定做出了巨大貢獻[11].大氣微生物近年來也備受關(guān)注,了解大氣微生物群落的全球分布和季節(jié)性變化[12]對于理解其調(diào)控氣候變化的機制[13]和對糧食安全及環(huán)境保護[14]的影響至關(guān)重要.

目前絕大多數(shù)微生物尚不能被分離培養(yǎng),其功能及代謝特征尚未可知,因此限制了對環(huán)境微生物的挖掘和利用.近年來,隨著微生物組學(xué)研究方法的快速發(fā)展和突破,極大地推動了環(huán)境微生物的研究進程.微生物組學(xué)技術(shù)以不依賴于分離培養(yǎng)的優(yōu)勢可針對環(huán)境樣品中的全部微生物進行研究,能夠系統(tǒng)性地揭示整體微生物群落的組成、活性、功能及動態(tài)變化等[1,15].其中,宏轉(zhuǎn)錄組學(xué)通過分離提取微生物群落中的RNA或者富集mRNA,合成cDNA[7]進行高通量測序分析.這種方法可針對微生物群落研究其在某一特定環(huán)境、特定時期和特定狀態(tài)下進行轉(zhuǎn)錄的所有RNA的類型及數(shù)量,來確定活躍微生物的代謝功能.相對于宏基因組研究微生物群落的組成和功能(包括死亡和休眠微生物),宏轉(zhuǎn)錄組的優(yōu)勢是可以揭示微生物群落中活躍物種的組成及其基因的表達.以宏轉(zhuǎn)錄組為代表的多組學(xué)研究方法為解析不同生境微生物群落動態(tài)變化、相互作用和功能響應(yīng)提供了前所未有的機遇.自2007年以來,宏轉(zhuǎn)錄組學(xué)在各領(lǐng)域得到廣泛應(yīng)用,與宏轉(zhuǎn)錄組學(xué)相關(guān)文章數(shù)量持續(xù)上升,其中2018年發(fā)文量達到200余篇(注:截止至2020年12月5日,以metatranscriptomics和metatranscriptome為主題檢索Web of Science數(shù)據(jù)庫).本文系統(tǒng)地介紹了宏轉(zhuǎn)錄組學(xué)的原理和數(shù)據(jù)分析流程,歸納了環(huán)境中微生物群落及其代謝能力的最新進展,并強調(diào)了如何繼續(xù)利用宏轉(zhuǎn)錄學(xué)來研究環(huán)境中微生物類群的生態(tài)適應(yīng)及其功能關(guān)聯(lián).

1 宏轉(zhuǎn)錄組學(xué)的研究策略

1.1 宏轉(zhuǎn)錄組學(xué)的基本原理

宏轉(zhuǎn)錄組學(xué)主要關(guān)注微生物群落的總RNA或者mRNA,研究步驟通常包括樣品采集、總RNA提取、mRNA的富集、cDNA合成、上機測序和數(shù)據(jù)分析5個步驟(圖1).由于mRNA的穩(wěn)定性較差,易降解,且占總RNA的比例僅有1%～5%,因此如何除去核糖體RNA來富集mRNA以及防止其降解是宏轉(zhuǎn)錄組學(xué)技術(shù)的關(guān)鍵[16-17].此外,也有研究未進行mRNA富集(表1),直接合成cDNA后進行測序.

1.1.1 樣品的采集和保存 mRNA分子的平均半衰期在幾秒到幾分鐘范圍內(nèi),且具有相同生物學(xué)功能的基因顯示出相似的mRNA降解率,這是宏轉(zhuǎn)錄組學(xué)樣品提取中存在的主要難點[18].此外, mRNA穩(wěn)定性會受到微生物生長速度的影響,在微生物種內(nèi)和種間也存在較大差異[19].因此,為了最大限度地減少前期準備導(dǎo)致的RNA轉(zhuǎn)錄譜及其完整性的變化,需要盡可能縮短在實驗樣本采集、貯存、運輸和制備過程中的時間.例如,樣本采集后應(yīng)立即投入液氮中快速冷凍或者將樣品轉(zhuǎn)移到RNAlater等核酸保存液中,然后轉(zhuǎn)移至-80℃冰箱中保存,盡量避免反復(fù)凍融.理想情況下,采樣過程引起的延遲應(yīng)在分秒范圍內(nèi)[20].

1.1.2 總RNA的提取 總RNA的提取一般是借助物理方法(微珠)結(jié)合細胞裂解液將細胞破碎,利用試劑使蛋白質(zhì)變性,從而將RNA釋放到溶液中.Mettel等人[21]基于腐殖酸含量不同的4種土壤(草地,稻田,森林和農(nóng)田),從RNA的純度、完整性及產(chǎn)率等方面評估和優(yōu)化了RNA提取方法.一般來說,從低pH(4.5～5.0)土壤要比從高pH (7.0～8.0)土壤中提取RNA的穩(wěn)定性和完整性高,并且腐殖酸含量較低.近年來,各種高效商業(yè)試劑盒也逐漸用于土壤RNA的提取.目前,PowerSoilTM總RNA提取試劑盒(MoBio)最為常用.此試劑盒基于苯酚(pH = 4.5～5.0)進行提取,然后采用試劑盒特異性的RNA純化方法.在RNA的提取過程中,要注意提取條件(如是否需要低溫)和污染控制. RNA酶分布廣泛且非常穩(wěn)定,容易降解mRNA,因此在提取RNA過程中要操作規(guī)范,以減輕RNA酶污染.

圖1 宏轉(zhuǎn)錄組學(xué)實驗流程

1.1.3 mRNA的富集 環(huán)境微生物群落中的總RNA主要由mRNA、tRNA和rRNA組成,其中mRNA約占1%～5%[22].模板mRNA的質(zhì)量與cDNA的合成效率密切相關(guān).因此,富集mRNA是微生物群落基因表達功能分析中的關(guān)鍵一步,也是宏轉(zhuǎn)錄組實驗中的重要一步.從環(huán)境樣品中富集mRNA的方法包括以下幾種:

(1) rRNA消減雜交處理[23]. rRNA的消減雜交保留了mRNA轉(zhuǎn)錄本的全部多樣性,因此可以用于針對mRNA的研究中.消減雜交的關(guān)鍵是利用一組捕獲探針與rRNA內(nèi)高度保守的序列區(qū)域互補. MICROB細菌mRNA富集試劑盒可用于通過消減雜交特異性去除細菌rRNA ,但將其應(yīng)用于土壤RNA提取時仍存在一些缺陷.例如,土壤微生物組的多樣性及復(fù)雜性使得靶向rRNA捕獲探針的物種覆蓋范圍成為mRNA富集的限制因子. rRNA探針的設(shè)計要盡可能涵蓋更多的物種類別.此外,隨著用于mRNA富集的RNA片段增加,消減雜交的rRNA去除效率也會下降[22].

(2)優(yōu)先降解rRNA的核酸外切酶處理.其原理是利用5'-單磷酸酯依賴核酸外切酶酶促反應(yīng)來降解rRNA .成熟的rRNA易被5'-單磷酸酯酸化,而真核生物的mRNA受帽子結(jié)構(gòu)保護,細菌mRNA帶有三磷酸基團, mRNA由此被保留下來.因此認為rRNA被5'-單磷酸酯酸化是通過5'端至3'端進行核酸外切酶特異性降解,從而達到mRNA的富集.然而,當(dāng)用其提取土壤RNA時,由于細菌的mRNA衰變過程中,位于5'端的三磷酸基團轉(zhuǎn)化為單磷酸形式,所以5'-單磷酸酯依賴核酸外切酶不僅降解rRNA,還會大量的降解mRNA[21-22,24].此外,腐殖質(zhì)是強大的酶抑制劑,會影響5'-單磷酸依賴核酸外切酶的活性,因此提取土壤RNA時必須先要去除腐殖質(zhì)[21].

(3)凝膠電泳片段分離[16].通過精確切除主要rRNA條帶之間的瓊脂糖可回收非rRNA.此方法可以通過切除23S, 16S和5S核糖體條帶之間的瓊脂糖來有效去除rRNA,但mRNA中可能仍含有微量的rRNA.由于腐殖酸在電場中的遷移速度比RNA分子快,該方法還可以同時去除腐殖酸[16].

(4) 雙鏈特異性核酸酶(DSN)處理[25].雙鏈特異性核酸酶是一種在高溫下優(yōu)先降解雙鏈DNA的酶.該方法通常在富含mRNA的cDNA文庫中降解rRNA反轉(zhuǎn)錄的cDNA.一般在RNA狀態(tài)下使用mRNA特異性Poly(A)尾部選擇,或者使用寡核苷酸引物方法從總RNA中進行逆轉(zhuǎn)錄.

1.1.4 cDNA的合成 一般來說,當(dāng)前的高通量測序平臺需要以cDNA為模板.因此,富集的mRNA需要經(jīng)過反轉(zhuǎn)錄為cDNA后再進行測序. cDNA合成基本步驟如下:首先以RNA單鏈為模板,在DNA反轉(zhuǎn)錄酶的作用下催化合成cDNA第一鏈,隨后以其為模板,利用聚合酶生成cDNA第二鏈.通過將cDNA雙鏈和載體連接,以此為模板進行PCR擴增,即可構(gòu)建cDNA文庫,用于微生物基因表達及調(diào)控分析.

1.2 宏轉(zhuǎn)錄組學(xué)測序

早期宏轉(zhuǎn)錄組學(xué)借助DNA芯片技術(shù)或微陣列的方法,但考慮到靈敏度、效率和成本的因素,該方法逐漸被新興的測序技術(shù)所取代.隨著測序技術(shù)的發(fā)展,宏轉(zhuǎn)錄組學(xué)技術(shù)得以大范圍應(yīng)用.第一代測序技術(shù)(Sanger雙脫氧法測序)[26]首次進行核酸序列測序.但因其測序通量低且無法批量測序的缺陷難以進一步應(yīng)用.二代測序技術(shù)(NGS)極大地增加了測序通量,并可實現(xiàn)自動化流程,推動了微生物組學(xué)研究的進步.目前常用的第二代測序技術(shù),以Illumina平臺為例,具有通量高、錯誤率低以及測序讀長涵蓋范圍廣等優(yōu)勢[28-29],廣泛用于宏轉(zhuǎn)錄組學(xué)研究中.近年來,以單分子和納米孔測序技術(shù)為代表的三代測序技術(shù)開始興起[30].單分子測序速度較快,讀長較長并且不需要PCR擴增,可用于全長轉(zhuǎn)錄本測序[29];納米孔技術(shù)亦可直接測定RNA序列,但其主要缺點是錯誤率和成本較高.

1.3 宏轉(zhuǎn)錄組學(xué)的數(shù)據(jù)分析方法

近年來,針對宏轉(zhuǎn)錄組數(shù)據(jù)分析的一些軟件和在線工具被廣泛開發(fā)和使用,自動化、高效和高通量分析成為人們對宏轉(zhuǎn)錄組數(shù)據(jù)分析流程的基本要求.本文對宏轉(zhuǎn)錄組數(shù)據(jù)處理流程及常用的軟件和數(shù)據(jù)庫進行了系統(tǒng)的總結(jié)(圖2),有助于相關(guān)研究者根據(jù)需求選擇合適流程.

原始下機數(shù)據(jù)一般為fastq格式,其包含測序過程中添加的引物、接頭、測序錯誤序列以及宿主污染等,因此需要對數(shù)據(jù)進行質(zhì)控.在質(zhì)量控制階段, Cutadapt[31]和Trimmomatic[32]常用來去除接頭(adapter)和低質(zhì)量堿基.數(shù)據(jù)質(zhì)控后,可使用本地軟件或者在線工具通過對比數(shù)據(jù)庫進行mRNA和rRNA分離提取.CAMERA[33]和MG-RAST[34]是用于宏轉(zhuǎn)錄組數(shù)據(jù)處理的在線分析網(wǎng)站,可通過對其已有數(shù)據(jù)庫進行序列的對比分析.通過SortMeRNA[35]軟件對比相應(yīng)數(shù)據(jù)庫,可從宏轉(zhuǎn)錄組數(shù)據(jù)中篩選出rRNA和mRNA序列.在對RNA序列進行預(yù)測和分類后,基于Trinity[36]、PANDAseq[37]或FLASH[38]等軟件分別對獲得的RNA的轉(zhuǎn)錄本序列碎片進行重疊配對, 分離后的mRNA序列可用于構(gòu)建宏轉(zhuǎn)錄組長片段并進行基因表達鑒定,對于rRNA的分析則可獲得相應(yīng)的微生物物種信息.拼接產(chǎn)生的mRNA contigs ,映射階段使用的軟件為Bowtie2[39],可將上一步生成的mRNA長序列映射到參考基因組.基于NCBI_nr, KEGG(https://www.kegg. jp/), COG (http://www.ncbi.nlm.nih.gov/COG)等數(shù)據(jù)庫對比可用于對獲取的序列進行功能注釋.而基于SILVA (http://www.arb-silva.de/)和NCBI_nt (https: //www.ncbi.nlm.nih.gov/) 數(shù)據(jù)庫可以得到堿基序列所攜帶的物種和結(jié)構(gòu)組成信息. 序列對比至數(shù)據(jù)庫可使用USEARCH[40]、BLAST[41]或DIAMOND[42]等軟件.MEGAN[43]軟件可以將數(shù)據(jù)庫對比結(jié)果進行物種分類和功能注釋.

最后,通過DESeq2[44]或edgeR[45]軟件對基因進行差異表達分析,可借助繪圖軟件進行數(shù)據(jù)可視化處理.針對提取的rRNA序列,使用BLAST進行聚類分析, SOAP2[46]用于提取rRNA,再用QIIME2[47-48]、MOTHUR[49]對比基因數(shù)據(jù)庫參考從而對rRNA序列進行物種注釋,獲得精確度較高的物種組成圖譜.

2 宏轉(zhuǎn)錄組學(xué)在環(huán)境微生物生態(tài)學(xué)中的應(yīng)用

采用宏轉(zhuǎn)錄組學(xué)的方法,可以發(fā)現(xiàn)特定生境下微生物群落基因的動態(tài)表達與調(diào)控機制,解密復(fù)雜的群落多樣性及環(huán)境因素對其代謝過程的影響,從而為探索微生物群落結(jié)構(gòu)及生態(tài)學(xué)功能奠定基礎(chǔ).表1對宏轉(zhuǎn)錄組學(xué)在不同生境微生物生態(tài)學(xué)中的研究進行了總結(jié).

圖2 宏轉(zhuǎn)錄組學(xué)的數(shù)據(jù)分析流程

2.1 宏轉(zhuǎn)錄組學(xué)在土壤生態(tài)環(huán)境研究中的應(yīng)用

土壤微生物是陸地生態(tài)系統(tǒng)可持續(xù)性的基礎(chǔ),驅(qū)動著陸地生態(tài)系統(tǒng)中的殘體降解、養(yǎng)分循環(huán)和植物生產(chǎn),對生態(tài)系統(tǒng)多功能性的維持和提升中有著重要作用[68].每克土壤中約有109～1010個的微生物個體,這些微生物群體參與了土壤有機碳的周轉(zhuǎn)和氮素的循環(huán)以及溫室氣體排放等相關(guān)過程,并對氣候變化具有反饋調(diào)節(jié)作用[69].近年來,宏轉(zhuǎn)錄組學(xué)方法的應(yīng)用極大地促進了土壤生態(tài)系統(tǒng)微生物組的功能研究,包括微生物介導(dǎo)的甲烷產(chǎn)生與氧化、有機碳的固定與分解以及氮、磷、硫等元素的生物地球化學(xué)循環(huán)等[50,53,70-71].以產(chǎn)甲烷過程為例,宏轉(zhuǎn)錄組學(xué)的方法揭示了稻田土壤中不同功能微生物組參與的多聚體水解、脂肪酸互營養(yǎng)化和產(chǎn)甲烷等有機質(zhì)厭氧降解的復(fù)雜代謝網(wǎng)絡(luò)[7].基于宏轉(zhuǎn)錄組學(xué)技術(shù)對于產(chǎn)甲烷古菌的研究進一步擴展了對碳代謝途徑的認識,并發(fā)現(xiàn)了眾多之前未被報道的新型產(chǎn)甲烷古菌[72],為研究甲烷代謝相關(guān)微生物的代謝途徑和分離培養(yǎng)奠定了基礎(chǔ).對于甲烷氧化過程,有學(xué)者認為傳統(tǒng)的甲烷氧化菌在大氣甲烷氧化中發(fā)揮主要的作用[73].對甲烷循環(huán)過程的深入挖掘?qū)⒉粩嗉由顚μ佳h(huán)途徑的了解.宏轉(zhuǎn)錄組學(xué)方法還可以關(guān)注氮素循環(huán)過程中以往被忽視的方面.例如,利用宏轉(zhuǎn)錄組學(xué)首次發(fā)現(xiàn)是驅(qū)動水稻土氮素還原過程(硝酸鹽異化還原為銨和固氮作用)中的關(guān)鍵微生物類群,而該微生物類群在以往的研究中一直被忽略[71].宏轉(zhuǎn)錄組除了可以揭示功能基因表達水平,還可以對活躍的微生物代謝功能進行研究.酸桿菌的硫代謝基因能夠在不同缺氧環(huán)境下表達上調(diào),而硫桿菌的代謝參與了多糖水解、糖利用、有氧呼吸、發(fā)酵和氫氧化等,擴展了對已知酸桿菌的生理和遺傳特性的認識[74].

2.2 宏轉(zhuǎn)錄組學(xué)在極端環(huán)境微生物研究中的應(yīng)用

極端環(huán)境中可能存在一些適應(yīng)性較強的未知物種,宏轉(zhuǎn)錄組與宏基因組、宏蛋白組學(xué)等聯(lián)合分析有助于揭示該環(huán)境中微生物群落和功能多樣性及相互關(guān)系,幫助人們揭示其生物學(xué)本質(zhì).已有學(xué)者研究了受氣候變化影響的北極多年凍土,發(fā)現(xiàn)固氮和反硝化微生物是其土壤微生物群落中的活躍成員[75],并且甲烷氧化菌群落組成及活性會隨著凍土融化進程而發(fā)生顯著變化[76].在對南極Vostok湖積冰的微生物研究中發(fā)現(xiàn),該極端環(huán)境具有高度的物種多樣性,不僅存在嗜冷微生物,還包括嗜熱、耐堿、耐鹽以及海洋微生物[77-78].南、北極沿海可溶性有機碳施入會引起常見微生物群落(如,和)的異養(yǎng)代謝過程的明顯轉(zhuǎn)錄反應(yīng)并可檢測到細胞應(yīng)對環(huán)境脅迫的適應(yīng)機制[79].宏基因組和宏轉(zhuǎn)錄組學(xué)方法的共同應(yīng)用發(fā)現(xiàn)了酸性礦山廢水中具有極高的轉(zhuǎn)錄活性的微生物類群[80]及其環(huán)境適應(yīng)機制[81].由此可見,宏轉(zhuǎn)錄組學(xué)為研究極端環(huán)境微生物群落以及挖掘未知基因提供了全新見解.

表1 宏轉(zhuǎn)錄組學(xué)在環(huán)境生態(tài)學(xué)中的應(yīng)用研究

2.3 宏轉(zhuǎn)錄組學(xué)在污染環(huán)境中的應(yīng)用

由人類活動或自然因素所引起的環(huán)境污染可能會破壞微生物生境而引起微生物群落結(jié)構(gòu)變化,進而影響微生物群落基因的表達.宏轉(zhuǎn)錄組學(xué)可以從轉(zhuǎn)錄水平上揭示污染物降解過程,以及污染物對微生物群落代謝的影響.Falk等[60]研究了受人為污染的淡水沉積物中的微生物群落,指出β氧化、糖異生和聚酯合成相關(guān)基因在有機污染物豐富的地方出現(xiàn)高表達,且降解譜的終點是硝酸鹽還原和產(chǎn)甲烷過程.Lu等[82]基于擴增子研究并未發(fā)現(xiàn)草甘膦對淡水微生物群落結(jié)構(gòu)有顯著影響,但宏轉(zhuǎn)錄組學(xué)分析表明草甘膦顯著影響了一些藍藻的轉(zhuǎn)錄.宏轉(zhuǎn)錄組學(xué)還可以用來研究污染物對微生物群落基因表達產(chǎn)生的影響以及污染物代謝過程的影響因素.Doyle等[62]研究石油污染海水發(fā)現(xiàn),沿不同海岸線的距離會導(dǎo)致烷烴和多環(huán)芳烴分解代謝途徑的差異表達;含氧相可以通過微生物介導(dǎo)的替代電子受體(如硫化物)的再氧化以及通過生物固氮提供氮,促進缺氧相中石油的生物降解[83].此外,宏轉(zhuǎn)錄組還可以挖掘代謝污染物的主要微生物類群.Zhou等[84]利用宏轉(zhuǎn)錄組研究汞污染的稻田,發(fā)現(xiàn)了該地微生物群落中相對豐度較低但能夠降解汞的主要微生物是,,和; Sharma等[85]指出古菌在重金屬和農(nóng)藥污染的土壤中發(fā)揮著重要的作用.因此宏轉(zhuǎn)錄組為生物修復(fù)的相關(guān)研究開拓了新思路.

3 結(jié)語

隨著微生物組學(xué)研究的深入,宏轉(zhuǎn)錄組學(xué)的重要性及其傳遞的生物學(xué)信息逐漸被重視.相對于擴增子、宏基因組等在微生物群落組成和基因潛力研究方面的優(yōu)勢,宏轉(zhuǎn)錄組更注重研究微生物群落中活躍物種的組成及其基因表達.然而單一組學(xué)分析手段無法滿足微生物群落多樣性、功能及動態(tài)變化的系統(tǒng)性研究.將宏轉(zhuǎn)錄組學(xué)與其它組學(xué)進行聯(lián)合分析,從物種組成、基因潛力、基因表達等多水平發(fā)揮各自優(yōu)勢,是全面了解微生物群落信息的有效途徑,也是未來微生物組學(xué)領(lǐng)域研究的熱點.

作為一種較新的研究手段,宏轉(zhuǎn)錄組學(xué)在未來環(huán)境微生物研究中具有廣泛的應(yīng)用潛力.但在實驗步驟優(yōu)化、數(shù)據(jù)融合及解決現(xiàn)實問題等方面仍存在諸多亟待解決的問題.這些問題的解決也將是宏轉(zhuǎn)錄組學(xué)未來發(fā)展的主要方向.優(yōu)化RNA提取方法,實現(xiàn)對更多類型環(huán)境樣本和研究要求的RNA的有效提取,同時開發(fā)出快捷有效的宏轉(zhuǎn)錄組數(shù)據(jù)分析方法和軟件是該領(lǐng)域難點;如何將宏轉(zhuǎn)錄組與生物學(xué)數(shù)據(jù)進行匹配,以達到多組學(xué)數(shù)據(jù)互相補充解釋的目的,將是未來生信數(shù)據(jù)分析中的重點研究方向;針對特殊環(huán)境開發(fā)原位提取技術(shù),降低運輸過程中造成的樣品降解程度也是宏轉(zhuǎn)錄組學(xué)研究亟待突破的瓶頸之一;研究環(huán)境污染對微生物群落的影響以及微生物群落應(yīng)對環(huán)境脅迫的適應(yīng)機制,開發(fā)具有污染修復(fù)能力的菌株并應(yīng)用到污染環(huán)境中,或?qū)⑹俏磥砗贽D(zhuǎn)錄組研究的重要領(lǐng)域.

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Application of metatranscriptomics in environmental microbial ecology.

LI Ying1, WU Xing-Jie2, HE Zhi-Bin2, BEI Shui-Kuan2, MA Ke1, PENG Jing-Jing2*

(1.Beijing Key Laboratory of Farmland Soil Pollution Prevention and Remediation, College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193, China；2.Key Laboratory of Plant-Soil Interactions, Ministry of Education College of Resources and Environmental Sciences, National Academy of Agriculture Green Development, China Agricultural University, Beijing 100193, China)., 2021,41(9)：4341～4348

In this review, the pipeline for metatranscriptomics workflow and data analysis were systematically summarized. Then, the strategy of research in environmental microbial ecology was discussed. Based on the above, the prospects of metatranscriptomics application were proposed. Metatranscriptomics has been useful in analyzing the function of environmental microbiomes. It provides a powerful tool for us to better understand the dynamic evolution of the functional microbial community and its relationship with environmental factors and ecosystem function.

metatranscriptomics；microbiome；community structure；functional gene expression；mRNA enrichment；RNA

X703.5

A

1000-6923(2021)09-4341-08

李 瑩(1998-),女,河北廊坊人,中國農(nóng)業(yè)大學(xué)碩士研究生,主要從事環(huán)境微生物學(xué)研究.

2021-01-29

國家自然科學(xué)基金資助項目(41977038)

* 責(zé)任作者, 副教授, jingjing.peng@cau.edu.cn