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    光皮桦木质部cDNA-AFLP 技术体系的建立及应用

    Establishment and application of cDNA-AFLP technical system for xylem tissue of Betula luminifera.

    • 摘要: 为了挖掘光皮桦材性相关基因,本研究以光皮桦木质部为材料,通过对RNA 的提取、cDNA 的反转录及纯化 的对比分析,以及选扩体系优化等,建立了相应的cDNA-AFLP 体系。结果表明:采用CTAB 和RNAiso 试剂相结合 的方法得到的RNA 质量较好,cDNA 经纯化后选扩效果明显变好。选扩体系为:4.0 μL 预扩产物(稀释40 倍)、1.6 μL 的引物(10.0 mmol/ L)、0.2 μL 的dNTP(2.5 mmol/ L)、0.4 μL 的Mg2 + (25.0 mmol/ L),以及0.2 μL 的Taq DNA 聚合酶(5 U/ μL)。进一步以来自不同木质化茎段的cDNA 为模板,筛选出31 对扩增效果较好的引物组合,同时分 离出一些差异表达的基因片段,经测序后探讨了其在木质化发育过程中可能的功能。

       

      Abstract: To investigate genes related to wood property in Betula luminifera, a suitable cDNA-AFLP reaction system for xylem tissues of B. luminifera was found in this study based on the comparative analysis of main procedures, including RNA extraction, reverse transcription and purification, and optimizing of selective amplification system. The results showed that CTAB method in combination with the RNAiso reagent was appropriate for RNA isolation of xylem, and cDNA purifying was an essential step after its synthesis. The optimal conditions for selective amplification were obtained as follows: the amount of 4.0 μL of 40 times-diluted preamplification products, 1.6 μL of primer(10.0 μmol/ L), 0.2 μL of dNTP (2.5 mmol/ L), 0.4 μL of Mg 2 + (25.0 mmol/ L), and 0.2 μL of Taq polymerase (5 U/ μL). In addition, 31 out of 64 primer pairs were screened by application of different lignification tissues in the selective amplification. Differentially expressed transcripts during the lignification were identified, and their function was analysed.

       

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