林业科学
林業科學
임업과학
SCIENTIA SILVAE SINICAE
2015年
5期
135-144
,共10页
成龙平%胡海涛%郭卫东%杨莉%王长春%杨玲
成龍平%鬍海濤%郭衛東%楊莉%王長春%楊玲
성룡평%호해도%곽위동%양리%왕장춘%양령
牛奶子%基因表达%实时定量 PCR%标准化%内参基因
牛奶子%基因錶達%實時定量 PCR%標準化%內參基因
우내자%기인표체%실시정량 PCR%표준화%내삼기인
Elaeagnus umbellata%gene expression%qRT-PCR%normalization%reference gene
【目的】实时定量 PCR结果的精确性在很大程度上取决于选择的内参基因的稳定性。通过评估候选管家基因的表达稳定性，筛选出用于牛奶子研究的最佳内参基因。【方法】设计简并引物从牛奶子中克隆12个潜在的内参基因片段，包括14-3-3、18S核糖体 RNA(18SrRNA)、β-actin (Actin)、延长因子1-α(EF1-α)、真核起始因子4A (eIF4A)、3－磷酸甘油醛脱氢酶(GAPDH)、RNA聚合酶－Ⅱ(RPⅡ)、60S核糖体蛋白(RPL7)、翻译延长因子2(TEF2)、泛素连接酶 E2(UBCE)、泛素(UBQ)和泛素延伸蛋白5(UBQ5)。采集牛奶子5个不同器官(根、茎、叶、花和红果)、4个成熟期的果实(绿果、黄果、深粉果和完全成熟的红果)、2种激素(脱落酸、赤霉素)处理4个时间点的绿果、热处理4个时间点的离体叶片、幼苗盐胁迫2个时间点的根茎叶，应用实时荧光定量 PCR 技术检测12个内参基因在各样品中的表达情况，采用基于 CT值的 geNorm、Normfinder和 BestKeeper及 CT比较4种算法评价这些内参基因的稳定性，最终的排名则由 RefFinder算法产生。【结果】器官组稳定性好的前2位内参基因为 UBCE和 RPL7，果实成熟期组排名前2的为eIF4A和UBCE，激素处理组排名前2的为eIF4A和UBCE，非生物胁迫组排名前2的则为 Actin和 EF1-α，综合分析所有样品排名前3位的是 eIF4A、RPL7和 UBCE。分别用筛选出的稳定的eIF4A、RPL7、UBCE和不稳定的 RPⅡ作为内参基因评价目的基因八氢番茄红素合成酶基因 EutPsy 在果实成熟过程中的表达，结果显示分别以3个稳定的内参基因为单内参基因与以 eIF4A 同 UBCE 组合做内参基因所得到的结论一致，而 RPⅡ给出的则不同。【结论】在应用荧光实时定量 PCR技术研究牛奶子基因转录表达时，通常情况下eIF4A、RPL7和 UBCE相对于其他9个候选内参基因更为可靠。研究结果为牛奶子及胡颓子属其他物种的基因表达分析奠定基础。
【目的】實時定量 PCR結果的精確性在很大程度上取決于選擇的內參基因的穩定性。通過評估候選管傢基因的錶達穩定性，篩選齣用于牛奶子研究的最佳內參基因。【方法】設計簡併引物從牛奶子中剋隆12箇潛在的內參基因片段，包括14-3-3、18S覈糖體 RNA(18SrRNA)、β-actin (Actin)、延長因子1-α(EF1-α)、真覈起始因子4A (eIF4A)、3－燐痠甘油醛脫氫酶(GAPDH)、RNA聚閤酶－Ⅱ(RPⅡ)、60S覈糖體蛋白(RPL7)、翻譯延長因子2(TEF2)、汎素連接酶 E2(UBCE)、汎素(UBQ)和汎素延伸蛋白5(UBQ5)。採集牛奶子5箇不同器官(根、莖、葉、花和紅果)、4箇成熟期的果實(綠果、黃果、深粉果和完全成熟的紅果)、2種激素(脫落痠、赤黴素)處理4箇時間點的綠果、熱處理4箇時間點的離體葉片、幼苗鹽脅迫2箇時間點的根莖葉，應用實時熒光定量 PCR 技術檢測12箇內參基因在各樣品中的錶達情況，採用基于 CT值的 geNorm、Normfinder和 BestKeeper及 CT比較4種算法評價這些內參基因的穩定性，最終的排名則由 RefFinder算法產生。【結果】器官組穩定性好的前2位內參基因為 UBCE和 RPL7，果實成熟期組排名前2的為eIF4A和UBCE，激素處理組排名前2的為eIF4A和UBCE，非生物脅迫組排名前2的則為 Actin和 EF1-α，綜閤分析所有樣品排名前3位的是 eIF4A、RPL7和 UBCE。分彆用篩選齣的穩定的eIF4A、RPL7、UBCE和不穩定的 RPⅡ作為內參基因評價目的基因八氫番茄紅素閤成酶基因 EutPsy 在果實成熟過程中的錶達，結果顯示分彆以3箇穩定的內參基因為單內參基因與以 eIF4A 同 UBCE 組閤做內參基因所得到的結論一緻，而 RPⅡ給齣的則不同。【結論】在應用熒光實時定量 PCR技術研究牛奶子基因轉錄錶達時，通常情況下eIF4A、RPL7和 UBCE相對于其他9箇候選內參基因更為可靠。研究結果為牛奶子及鬍頹子屬其他物種的基因錶達分析奠定基礎。
【목적】실시정량 PCR결과적정학성재흔대정도상취결우선택적내삼기인적은정성。통과평고후선관가기인적표체은정성，사선출용우우내자연구적최가내삼기인。【방법】설계간병인물종우내자중극륭12개잠재적내삼기인편단，포괄14-3-3、18S핵당체 RNA(18SrRNA)、β-actin (Actin)、연장인자1-α(EF1-α)、진핵기시인자4A (eIF4A)、3－린산감유철탈경매(GAPDH)、RNA취합매－Ⅱ(RPⅡ)、60S핵당체단백(RPL7)、번역연장인자2(TEF2)、범소련접매 E2(UBCE)、범소(UBQ)화범소연신단백5(UBQ5)。채집우내자5개불동기관(근、경、협、화화홍과)、4개성숙기적과실(록과、황과、심분과화완전성숙적홍과)、2충격소(탈락산、적매소)처리4개시간점적록과、열처리4개시간점적리체협편、유묘염협박2개시간점적근경협，응용실시형광정량 PCR 기술검측12개내삼기인재각양품중적표체정황，채용기우 CT치적 geNorm、Normfinder화 BestKeeper급 CT비교4충산법평개저사내삼기인적은정성，최종적배명칙유 RefFinder산법산생。【결과】기관조은정성호적전2위내삼기인위 UBCE화 RPL7，과실성숙기조배명전2적위eIF4A화UBCE，격소처리조배명전2적위eIF4A화UBCE，비생물협박조배명전2적칙위 Actin화 EF1-α，종합분석소유양품배명전3위적시 eIF4A、RPL7화 UBCE。분별용사선출적은정적eIF4A、RPL7、UBCE화불은정적 RPⅡ작위내삼기인평개목적기인팔경번가홍소합성매기인 EutPsy 재과실성숙과정중적표체，결과현시분별이3개은정적내삼기인위단내삼기인여이 eIF4A 동 UBCE 조합주내삼기인소득도적결론일치，이 RPⅡ급출적칙불동。【결론】재응용형광실시정량 PCR기술연구우내자기인전록표체시，통상정황하eIF4A、RPL7화 UBCE상대우기타9개후선내삼기인경위가고。연구결과위우내자급호퇴자속기타물충적기인표체분석전정기출。
Objective]The accuracy of quantitative real-time polymerase chain reaction ( qRT-PCR) analysis strongly depends on transcript normalization using stably expressed reference genes. The present study was conducted to evaluate the stability of candidate housekeeping genes and identify the most reliable gene or a set of genes to be used as reference genes in qPCR analysis of Elaeagnus umbellata.[Method]Twelve potential reference genes were selected among the most common reference genes reported in literature and their fragments were cloned by degenerate primers from E. umbellata, including 14-3-3 ,18 S ribosomal RNA gene ( 18 SrRNA ) ,β-actin ( Actin ) ,elongation factor 1-α ( EF1-α) ,eukaryotic initiation factor 4A (eIF4A),glyceraldehyde-3-phosphate dehydrogenase (GAPDH),RNA polymerase-Ⅱ (RPⅡ),60S ribosomal protein L7 ( RPL7 ) ,translation elongation factor 2 ( TEF2 ) ,ubiquitin-conjugating enzyme E2 ( UBCE ) , ubiquitin (UBQ) and ubiquitin extension protein 5 (UBQ5). Samples were collected from five types of organs (root, stem,leaf,flower and red fruit),fruits at four different ripening stages (green,yellow,dark yellow and fully matured red fruit) ,green fruits at four time points after hormone ABA or GA3 treatments,detached leaves at four time points under 40 ℃,and three types of organs ( root,stem and leaf) of the seedlings treated with salt stress at three time points. The expression stability of these 12 genes was evaluated based on the CT values using four statistical algorithms including geNorm,Normfinder,BestKeeper,and the comparative ΔCT. Overall ranking of four sets aforementioned was generated using RefFinder software.[Result]UBCE and RPL7 were ranked as the two best reference genes for organ set,eIF4A and UBCE for fruit-ripening samples,eIF4A and UBCE for hormone treatment set,and Actin and EF1-α for abiotic stress set. When including the data obtained from all the 29 samples into the analysis,eIF4A,RPL7,and UBCE were identified as the top three reference candidates. The expression levels of phytoene synthase ( EutPsy) were further assessed during fruit ripening by using the top three reference genes in comparison to the worst one RPⅡ. When the three stable reference genes eIF4A,UBCE and RPL7 or the combination of top two eIF4A and UBCE were used for normalization,the trend of the relative transcript abundance of EutPsy were consistent. However,when the worst reference gene RPⅡwas employed for normalization,the expression profile was different.[Conclusion]eIF4A followed by RPL7 and UBCE were found to be more reliable than other nine genes for normalization purposes in measuring gene expression of E. umbellata. This was the first systematic analysis for the selection of superior reference genes for qRT-PCR in E. umbellata under different conditions,which benefits future studies on gene expression in E. umbellata and other species of the genus Elaeagnus.
