Identification and characterization of genes associated with tapping panel dryness from Hevea brasiliensis latex using suppression subtractive hybridization
Identification and characterization of genes associated with tapping panel dryness from Hevea brasiliensis latex using suppression subtractive hybridization
复制标题
使用抑制消减杂交技术对巴西橡胶树胶乳中与割胶板干燥相关的基因进行鉴定和表征
DOI：
10.1186/1471-2229-10-140
复制
发表时间：
2010-07-09
影响因子：
5.3
通讯作者：
Chen, Shoucai
中科院分区：
文献类型：
--
作者：
Li, Dejun;Deng, Zhi;Chen, Shoucai
背景：割面干涸病（TPD）是天然橡胶生产面临的最严重威胁之一。尽管在橡胶树TPD研究方面已付出大量努力，但TPD的分子机制仍知之甚少。鉴定与TPD相关的基因并进行系统分析是阐明TPD所涉及分子机制的前提条件。本研究旨在获取橡胶树中与TPD相关的基因信息。
结果：为了鉴定橡胶树中与TPD相关的基因，利用抑制性消减杂交（SSH）方法构建了健康橡胶树和患TPD橡胶树胶乳的正向和反向cDNA文库。从这两个cDNA文库获得的1106个克隆中，通过反向Northern杂交分析，有822个克隆在两个文库中表现出差异表达。序列分析表明，这822个克隆代表237个独特基因；其中大多数尚未有报道称与橡胶树TPD相关。通过逆转录PCR（RT - PCR）和实时PCR分析进一步研究了20个差异表达基因的表达模式以验证SSH数据。根据基因本体论惯例，237个独特基因被分为10个功能组，例如胁迫/防御反应、蛋白质代谢、转录和转录后、橡胶生物合成等。在已知功能的基因中，在反向文库中优先表达的基因与胁迫/防御反应相关，而在正向文库中则与代谢和能量相关。
结论：本研究通过SSH方法鉴定了与TPD相关的基因。对与TPD相关基因的系统分析表明，活性氧（ROS）的产生和清除、泛素 - 蛋白酶体途径、程序性细胞死亡和橡胶生物合成可能在TPD中起重要作用。因此，我们的研究结果不仅丰富了与TPD相关基因的信息，还为理解橡胶树TPD过程提供了新的见解。
Background: 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.Results: 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.Conclusions: 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.