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

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Abstract

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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Acknowledgments

We thank Mr. Zewei An for technical assistance, and Dr. Renxiao Wang for correcting the manuscript. This work was partly supported by the National Natural Science Foundation of China (NSFC30600386), the Key Project of Chinese Ministry of Education (207094), the 973 Program (2008CB117009), and the Key Lab of Rubber Biology, Ministry of Agriculture (KLOF0601).

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Authors and Affiliations

  1. Key Lab of Rubber Biology, Ministry of Agriculture; State Key Laboratory Breeding Base of Cultivation & Physiology of Tropical Crops; Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou, Hainan, 571737, China

    Xiaohu Xiao, Heping Li & Chaorong Tang

  2. College of Agronomy, Hainan University, Danzhou, Hainan, 571737, China

    Xiaohu Xiao & Heping Li

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  1. Xiaohu Xiao
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  2. Heping Li
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Correspondence to Chaorong Tang.

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Xiao, X., Li, H. & Tang, C. A Silver-Staining cDNA-AFLP Protocol Suitable for Transcript Profiling in the Latex of Hevea brasiliensis (Para Rubber Tree). Mol Biotechnol 42, 91–99 (2009). https://doi.org/10.1007/s12033-008-9139-3

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