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      Identification and characterization of genes associated with tapping panel dryness from Hevea brasiliensis latex using suppression subtractive hybridization


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          Tapping panel dryness (TPD) is one of the most serious threats to natural rubber production. Although a great deal of effort has been made to study TPD in rubber tree, the molecular mechanisms underlying TPD remain poorly understood. Identification and systematical analyses of the genes associated with TPD are the prerequisites for elucidating the molecular mechanisms involved in TPD. The present study is undertaken to generate information about the genes related to TPD in rubber tree.


          To identify the genes related to TPD in rubber tree, forward and reverse cDNA libraries from the latex of healthy and TPD trees were constructed using suppression subtractive hybridization (SSH) method. Among the 1106 clones obtained from the two cDNA libraries, 822 clones showed differential expression in two libraries by reverse Northern blot analyses. Sequence analyses indicated that the 822 clones represented 237 unique genes; and most of them have not been reported to be associated with TPD in rubber tree. The expression patterns of 20 differentially expressed genes were further investigated to validate the SSH data by reverse transcription PCR (RT-PCR) and real-time PCR analysis. According to the Gene Ontology convention, 237 unique genes were classified into 10 functional groups, such as stress/defense response, protein metabolism, transcription and post-transcription, rubber biosynthesis, etc. Among the genes with known function, the genes preferentially expressed were associated with stress/defense response in the reverse library, whereas metabolism and energy in the forward one.


          The genes associated with TPD were identified by SSH method in this research. Systematic analyses of the genes related to TPD suggest that the production and scavenging of reactive oxygen species (ROS), ubiquitin proteasome pathway, programmed cell death and rubber biosynthesis might play important roles in TPD. Therefore, our results not only enrich information about the genes related to TPD, but also provide new insights into understanding the TPD process in rubber tree.

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          Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries.

          A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a hybridization probe to identify homologous sequences in a human Y chromosome cosmid library. The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. These results suggest that the SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes.
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            Corpse clearance defines the meaning of cell death.

            While philosophers seek the meaning of life, cell biologists are becoming ever more interested in the meaning of death. Apoptosis marks unwanted cells with 'eat me' signals that direct recognition, engulfment and degradation by phagocytes. Far from being the end of the story, these clearance events allow scavenger cells to confer meaning upon cell death. But if the phagocytic 'spin doctors' receive or transmit the wrong messages, trouble ensues.
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              DNA sequence quality trimming and vector removal.

              Most sequence comparison methods assume that the data being compared are trustworthy, but this is not the case with raw DNA sequences obtained from automatic sequencing machines. Nevertheless, sequence comparisons need to be done on them in order to remove vector splice sites and contaminants. This step is necessary before other genomic data processing stages can be carried out, such as fragment assembly or EST clustering. A specialized tool is therefore needed to solve this apparent dilemma. We have designed and implemented a program that specifically addresses the problem. This program, called LUCY, has been in use since 1998 at The Institute for Genomic Research (TIGR). During this period, many rounds of experience-driven modifications were made to LUCY to improve its accuracy and its ability to deal with extremely difficult input cases. We believe we have finally obtained a useful program which strikes a delicate balance among the many issues involved in the raw sequence cleaning problem, and we wish to share it with the research community. LUCY is available directly from TIGR (http://www.tigr.org/softlab). Academic users can download LUCY after accepting a free academic use license. Business users may need to pay a license fee to use LUCY for commercial purposes. Questions regarding the quality assessment module of LUCY should be directed to Michael Holmes (mholmes@tigr.org). Questions regarding other aspects of LUCY should be directed to Hui-Hsien Chou (hhchou@iastate.edu).

                Author and article information

                BMC Plant Biol
                BMC Plant Biology
                BioMed Central
                9 July 2010
                : 10
                : 140
                [1 ]Key Laboratory of Rubber Biology, Ministry of Agriculture, Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou, Hainan 571737, China
                [2 ]Hainan Provincial Key Laboratory of Tropical Crops Cultivation and Physiology, Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou, Hainan, 571737, China
                [3 ]Institute of Biological Science and Technology, College of Agriculture, Hainan University, Haikou, 570228, China
                Copyright ©2010 Li et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Research Article

                Plant science & Botany
                Plant science & Botany


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