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A Silver-Staining cDNA-AFLP Protocol Suitable for Transcript Profiling in the Latex of Hevea brasiliensis (Para Rubber Tree)

文献类型： 外文期刊

作者： Xiao, Xiaohu 1 ; Li, Heping 1 ; Tang, Chaorong 1 ;

作者机构： 1.Chinese Acad Trop Agr Sci, Key Lab Rubber Biol, Minist Agr,Rubber Res Inst, State Key Lab Breeding Base Cultivat & Physiol Tr, Danzhou 571737, Hainan, Peoples R China

2.Hainan Univ, Coll Agron, Danzhou 571737, Hainan, Peoples R China

关键词： cDNA-AFLP;Silver staining;Transcript profiling;Hevea brasiliensis;Latex

期刊名称：MOLECULAR BIOTECHNOLOGY （ 影响因子：2.695； 五年影响因子：2.303 ）

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收录情况： SCI

摘要： cDNA amplified fragment length polymorphism (cDNA-AFLP) is a powerful transcript-profiling tool widely used in diverse plant species. When applied to a new biological system, however, existing protocols usually require substantial modifications. Furthermore, the usage of radioactive isotope in typical protocols excludes their application in many labs. Latex, as the cytoplasm of rubber-producing cells sees a critical role in elucidating rubber biosynthesis and its regulation in rubber tree (Hevea brasiliensis). This paper describes a detailed step-by-step silver-staining cDNA-AFLP procedure, which is suitable to latex transcript profiling analysis. Theoretical analysis revealed that with the combination of two restriction enzyme pairs (ApoI/MseI and TaqI/MseI), approximately 94% of latex whole transcriptome could be visualized. After varying multiple parameters, including the amounts of primary and secondary template usage, pre-amplification cycle number and gel development, we obtained a high-quality silver-staining fingerprint. In the ApoI/MseI system, an average of 88.6 discernable bands (100-1,000 bp) was produced for each selective primer pair, and 97.2 bands for another system TaqI/MseI). TaqI/MseI was the first pair of 4-bp cutters used in cDNA-AFLP analysis and proved to be efficient and reliable. The sensitivity and reliability of our method were further verified by an application example in detecting differential gene expression in the latex of Hevea tree.

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