基于16S rDNA高通量測(cè)序技術(shù)研究轉(zhuǎn)基因作物對(duì)根際細(xì)菌群落結(jié)構(gòu)的影響

梁晉剛， 劉鵬程， 張秀杰

(農(nóng)業(yè)部科技發(fā)展中心，北京 100176)

幾十年來，轉(zhuǎn)基因作物釋放對(duì)生態(tài)環(huán)境的影響一直是社會(huì)公眾關(guān)注的一個(gè)重要問題，包括對(duì)土壤生態(tài)環(huán)境的影響[1-4]。土壤細(xì)菌是土壤中數(shù)量最豐富、分布最廣泛的微生物類群，廣泛參與有機(jī)物質(zhì)分解、養(yǎng)分釋放和能量轉(zhuǎn)移等過程，其多樣性和活性是保持土壤生態(tài)系統(tǒng)穩(wěn)定的基礎(chǔ)之一。根際細(xì)菌對(duì)外界干擾比較敏感，不僅受到植物基因型影響，也受土壤類型、農(nóng)業(yè)耕作管理、季節(jié)變化等影響[5-9]。準(zhǔn)確檢測(cè)轉(zhuǎn)基因作物對(duì)根際細(xì)菌群落組成和結(jié)構(gòu)的影響，是評(píng)估轉(zhuǎn)基因作物釋放后土壤生態(tài)風(fēng)險(xiǎn)的重要基礎(chǔ)[10-11]。

近年來，分子生物學(xué)的不斷發(fā)展為根際細(xì)菌群落結(jié)構(gòu)的研究提供了新思路，其中，以編碼rRNA的rDNA基因?yàn)榛A(chǔ)的高通量測(cè)序技術(shù)尤受青睞，該技術(shù)準(zhǔn)確、靈敏，具有較高的通量，已經(jīng)被廣泛應(yīng)用于土壤微生物遺傳多樣性的研究領(lǐng)域中[12-15]。目前，市面上常用的用于研究環(huán)境微生物的高通量測(cè)序平臺(tái)有Illumina MiSeq、Roche 454。針對(duì)細(xì)菌的16S rDNA基因序列分析，MiSeq憑借其測(cè)序讀段長、測(cè)序周期短、通量大等特點(diǎn)，成為使用最為普遍的測(cè)序平臺(tái)[16]。

16S核糖體RNA(16S ribosomal RNA，簡稱16S rRNA)是原核生物的核糖體中30S亞基的組成部分。由于不同種的真細(xì)菌與古細(xì)菌之間的16S rRNA編碼基因16S rDNA是高度保守的，且16S rDNA序列長度適中(1 540 bp)，包括9個(gè)高可變區(qū)(hypervariable region)和8個(gè)保守區(qū)(constant region)，因此，常被用于對(duì)各種生物進(jìn)行系統(tǒng)發(fā)生學(xué)方面的研究[17]。利用16S rDNA的通用引物進(jìn)行PCR擴(kuò)增，獲得絕大部分微生物16S rDNA高可變區(qū)的擴(kuò)增產(chǎn)物，構(gòu)建擴(kuò)增產(chǎn)物的文庫，運(yùn)用Illumina MiSeq平臺(tái)或Roche 454平臺(tái)進(jìn)行高通量測(cè)序，然后比較分析測(cè)序數(shù)據(jù)，對(duì)土壤微生物群落的多樣性進(jìn)行研究[18-20]。

目前，已有研究者利用16S rDNA測(cè)序技術(shù)來研究各種環(huán)境樣品中微生物的多樣性[21-30]。迄今為止，大部分的研究結(jié)果均表明，轉(zhuǎn)基因作物的釋放對(duì)土壤微生物沒有影響或有很小的影響，且影響是短暫的[31-42]。

1　轉(zhuǎn)基因大豆對(duì)根際細(xì)菌群落結(jié)構(gòu)的影響

Liang等通過收集高產(chǎn)高蛋氨酸轉(zhuǎn)基因大豆ZD91和其對(duì)應(yīng)的非轉(zhuǎn)基因大豆ZD在鼓粒期時(shí)的根際土壤樣品，采用Roche 454焦磷酸測(cè)序技術(shù)對(duì)大豆根際土壤細(xì)菌16S rDNA V4區(qū)序列擴(kuò)增子進(jìn)行高通量測(cè)序及分析，發(fā)現(xiàn)在這種轉(zhuǎn)基因大豆和其親本的根際土壤中，均存在酸桿菌門(Acidobacteria)、變形菌門(Proteobacteria)、擬桿菌門(Bacteroidetes)、放線菌門(Actinobacteria)、厚壁菌門(Firmicutes)等細(xì)菌類群，但兩者的細(xì)菌群落結(jié)構(gòu)無顯著性差異[43]。Lu等采用Illumina MiSeq平臺(tái)對(duì)16S rDNA V4區(qū)序列擴(kuò)增子進(jìn)行高通量測(cè)序，發(fā)現(xiàn)抗草甘膦除草劑轉(zhuǎn)基因大豆ZUTS31在鼓粒期對(duì)根際細(xì)菌群落的α、β多樣性無顯著性影響[44]。Lu等利用同樣的方法，發(fā)現(xiàn)抗除草劑轉(zhuǎn)基因大豆NZL06-698對(duì)土壤微生物群落，特別是對(duì)固氮菌群產(chǎn)生的影響，表現(xiàn)為微生物物種豐度和多樣性降低，固氮菌群豐度降低[45]。

2　轉(zhuǎn)基因玉米對(duì)根際細(xì)菌群落結(jié)構(gòu)的影響

Barriuso等連續(xù)4年收集根際土壤樣品，并采用Roche 454焦磷酸測(cè)序技術(shù)對(duì)根際土壤細(xì)菌16S rDNA V6區(qū)序列擴(kuò)增子進(jìn)行高通量測(cè)序及分析，發(fā)現(xiàn)抗蟲玉米MON810與對(duì)照玉米品種相比，抗蟲玉米MON810對(duì)根際細(xì)菌群落結(jié)構(gòu)無顯著性影響[46]。Barriuso等采用相同的方法，連續(xù)3年監(jiān)測(cè)噴施草甘膦對(duì)耐除草劑玉米NK603根際細(xì)菌群落結(jié)構(gòu)的影響。結(jié)果表明，草甘膦對(duì)玉米NK603根際細(xì)菌群落結(jié)構(gòu)無顯著性影響[47]。Dohrmann等利用Roche 454焦磷酸測(cè)序技術(shù)測(cè)定了細(xì)菌16S rDNA V7+V8區(qū)序列，發(fā)現(xiàn)抗蟲玉米MON 89034×MON 88017對(duì)根際細(xì)菌群落結(jié)構(gòu)無顯著性影響[48]。

3　其他轉(zhuǎn)基因植物對(duì)根際細(xì)菌群落結(jié)構(gòu)的影響

Sohn等應(yīng)用454平臺(tái)測(cè)序測(cè)定16S rRNA基因V3-V4變異區(qū)序列，發(fā)現(xiàn)白藜蘆醇強(qiáng)化轉(zhuǎn)基因水稻RS526對(duì)根際土壤細(xì)菌群落結(jié)構(gòu)無顯著性影響[49]。Zhu等利用Illumina MiSeq平臺(tái)測(cè)定16S rRNA基因V4區(qū)序列，分析了種植8年轉(zhuǎn)基因白楊樹D520、D521后的根際土壤細(xì)菌結(jié)構(gòu)，發(fā)現(xiàn)轉(zhuǎn)基因白楊樹對(duì)細(xì)菌多樣性和群落結(jié)構(gòu)無顯著性影響[50]。Debruyn等利用Illumina MiSeq平臺(tái)測(cè)定16S rRNA基因V4區(qū)，發(fā)現(xiàn)木質(zhì)素含量降低的轉(zhuǎn)基因柳枝稷對(duì)根際細(xì)菌多樣性、豐富度和群落組成均無顯著性影響[51]。

4　總結(jié)與展望

16S rDNA相對(duì)分子量大小適中，突變率小，基于16S rDNA的高通量測(cè)序技術(shù)已被廣泛應(yīng)用于微生物系統(tǒng)進(jìn)化、分類及多樣性分析研究中。在16S rDNA高可變區(qū)的選擇方面還存在一定爭(zhēng)議，由于目前二代高通量測(cè)序的讀長限制，僅能針對(duì)16S rDNA的某一段可變區(qū)進(jìn)行測(cè)序，有的選擇測(cè)單可變區(qū)(V3、V4、V5、V6、V7)，有的選擇對(duì)連續(xù)可變區(qū)(V1-V2、V1-V3、V3-V4、V3-V5、V4-V5、V4-V6、V5-V6、V6-V7、V5-V8、V6-V8、V7-V8、V1-V8、V5-V9、V6-V9、nearly full-length)進(jìn)行16S rDNA測(cè)序[52-65]。Sun等研究比較了16S不同區(qū)域的測(cè)序結(jié)果，發(fā)現(xiàn)V4-V5區(qū)(515F/907R 或515F/926R)是最佳的測(cè)序區(qū)域，造成的基因組內(nèi)異質(zhì)性最小，該區(qū)域是Roche 454測(cè)序時(shí)代最常用的區(qū)域[66]。在Illumina時(shí)代，由于平臺(tái)測(cè)序長度的限制，V4單區(qū)測(cè)序(515F/806R)被更為廣泛地使用，同時(shí)這對(duì)引物也是地球微生物組計(jì)劃(Earth Microbiome Project，簡稱EMP)中推薦使用的引物序列，它們將是16S測(cè)序的主力[67]。

16S rDNA高通量測(cè)序技術(shù)對(duì)土壤微生物物種多樣性、結(jié)構(gòu)多樣性、功能多樣性和遺傳多樣性研究的迅猛發(fā)展起到了重要作用[68]。然而，這種方法對(duì)于轉(zhuǎn)基因的研究來說，存在著不能檢測(cè)受體、轉(zhuǎn)化體、噴灑除草劑轉(zhuǎn)化體對(duì)各類土壤微生物功能(包括氮循環(huán)、磷循環(huán)、碳循環(huán)等)的各類基因豐度等影響的缺點(diǎn)[16]。同樣地，16S rDNA擴(kuò)增子高通量測(cè)序本身也常受到一些條件的影響，如PCR擴(kuò)增體系對(duì)高通量測(cè)序的影響，PCR擴(kuò)增偏好性對(duì)測(cè)序的影響，PCR擴(kuò)增循環(huán)數(shù)對(duì)原始物種相對(duì)豐度的影響等[69]。在未來的研究中，16S rDNA高通量測(cè)序技術(shù)在轉(zhuǎn)基因作物對(duì)土壤細(xì)菌影響的研究中的運(yùn)用會(huì)越來越普及，隨著測(cè)序成本明顯下降，土壤宏基因組測(cè)序?yàn)檠芯课⑸锶郝浣Y(jié)構(gòu)與功能等提供了一種新的選擇，相比16S rDNA高通量測(cè)序，該測(cè)序方法能夠更全面地分析微生物的物種及基因等方面的多樣性。由于土壤微生物群落組成復(fù)雜，在實(shí)際應(yīng)用中，應(yīng)該綜合考慮各研究方法的優(yōu)缺點(diǎn)，取長補(bǔ)短，以便在土壤微生物多樣性研究中獲得更為全面且準(zhǔn)確的信息，盡快建立對(duì)土壤生態(tài)系統(tǒng)安全的評(píng)價(jià)技術(shù)體系，并對(duì)轉(zhuǎn)基因作物的環(huán)境安全性作出全面、科學(xué)、客觀的評(píng)價(jià)[70-72]。

致謝：感謝南京大學(xué)生命科學(xué)學(xué)院陸桂華副研究員、南京農(nóng)業(yè)大學(xué)植物保護(hù)學(xué)院顧沁博士在論文寫作過程中給予的指導(dǎo)和幫助。

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