江西林业科技
江西林業科技
강서임업과기
JIANGXI FORESTRY SCIENCE AND TECHNOLOGY
2013年
5期
1-4
,共4页
伍艳芳%徐海宁%宋晓琛%江香梅
伍豔芳%徐海寧%宋曉琛%江香梅
오염방%서해저%송효침%강향매
樟树%不同组织%总RNA%提取方法%RT-PCR检测
樟樹%不同組織%總RNA%提取方法%RT-PCR檢測
장수%불동조직%총RNA%제취방법%RT-PCR검측
Cinnamomum camphora%different tissues%total RNA%extraction method%RT-PCR
采用Invitrogen Total RNA Purification Kit直接提取法，Trizol试剂盒法和本实验室改良的CTAB法，分别提取樟树不同组织（根、茎、叶和花）的总RNA，并对提取效果进行了检测和比较分析。结果表明：（1）采用本实验室改良的CTAB法提取的RNA完整性好，无杂质污染且得率高，稳定性好，可满足半定量RT-PCR等试验需要；而Invitrogen Total RNA Purification Kit直接提取法和Trizol试剂盒法提取的樟树不同组织总RNA，结果均不理想，存在RNA 28S rRNA谱带模糊不清、有DNA污染、降解严重和得率低等问题，这两种方法均不适用于樟树不同组织RNA的提取。（2）樟树不同组织总RNA提取的得率不同，其中叶片的总RNA得率最高，达到782.34 mg/kg；茎的得率最低，为514.33 mg/kg。（3）以Actin作为内参基因，对樟树根、茎、叶和花中的Fad2基因片段进行半定量RT-PCR检测，结果表明，Fad2基因片段在樟树叶片中的表达量相对较高，而在花中的表达量较低。
採用Invitrogen Total RNA Purification Kit直接提取法，Trizol試劑盒法和本實驗室改良的CTAB法，分彆提取樟樹不同組織（根、莖、葉和花）的總RNA，併對提取效果進行瞭檢測和比較分析。結果錶明：（1）採用本實驗室改良的CTAB法提取的RNA完整性好，無雜質汙染且得率高，穩定性好，可滿足半定量RT-PCR等試驗需要；而Invitrogen Total RNA Purification Kit直接提取法和Trizol試劑盒法提取的樟樹不同組織總RNA，結果均不理想，存在RNA 28S rRNA譜帶模糊不清、有DNA汙染、降解嚴重和得率低等問題，這兩種方法均不適用于樟樹不同組織RNA的提取。（2）樟樹不同組織總RNA提取的得率不同，其中葉片的總RNA得率最高，達到782.34 mg/kg；莖的得率最低，為514.33 mg/kg。（3）以Actin作為內參基因，對樟樹根、莖、葉和花中的Fad2基因片段進行半定量RT-PCR檢測，結果錶明，Fad2基因片段在樟樹葉片中的錶達量相對較高，而在花中的錶達量較低。
채용Invitrogen Total RNA Purification Kit직접제취법，Trizol시제합법화본실험실개량적CTAB법，분별제취장수불동조직（근、경、협화화）적총RNA，병대제취효과진행료검측화비교분석。결과표명：（1）채용본실험실개량적CTAB법제취적RNA완정성호，무잡질오염차득솔고，은정성호，가만족반정량RT-PCR등시험수요；이Invitrogen Total RNA Purification Kit직접제취법화Trizol시제합법제취적장수불동조직총RNA，결과균불이상，존재RNA 28S rRNA보대모호불청、유DNA오염、강해엄중화득솔저등문제，저량충방법균불괄용우장수불동조직RNA적제취。（2）장수불동조직총RNA제취적득솔불동，기중협편적총RNA득솔최고，체도782.34 mg/kg；경적득솔최저，위514.33 mg/kg。（3）이Actin작위내삼기인，대장수근、경、협화화중적Fad2기인편단진행반정량RT-PCR검측，결과표명，Fad2기인편단재장수협편중적표체량상대교고，이재화중적표체량교저。
The total RNA isolated from different tissues (root, stem, leaf and flower) in Cinnamomum camphora were extracted by Invitrogen Total RNA Purification Kit, common Trizol Kit and the improvement CTAB method, and the isolation effects of the three methods were tested and compared. The results showed as follow: (1) The improved CTAB could extract high-quality and high-integrity RNA, which could be used for semi-quantitative RT-PCR and so on. It was in an indistinct way of the total RNA from the different tissues in C. camphora by Invitrogen Total RNA Purification Kit extraction. RNA extracted was immingled by some DNA and was not enough for molecular biology experiment. Total RNA could not be extracted by Trizol method, the degradation of total RNA was seriously and the yield was very low. Trizol method and Invitrogen Total RNA Purification Kit were not suitable for RNA extraction from C. camphora. (2) The yield of RNA from leaves was 782.34 mg/kg, which higher than the yield from stems (514.33 mg/kg). (3) RT-PCR was used to determine Fad2 gene expression with Actin gene as comparison. The results showed that the expression of Fad2 gene showed high level in leaf and low level in flower.
