杉木木材形成过程特异表达基因的鉴定与分析  

Identification and analysis of differentially expressed genes during wood formation in Chinese fir by SSH

作　　者：王桂凤[1] 高燕[1] 杨立伟[1] 施季森[1] 

机构地区：[1]南京林业大学林木遗传与基因工程江苏省和国家林业局重点实验室,南京210037

出　　处：《遗传》2007年第4期483-489,共7页Hereditas（Beijing)

基　　金：国家重点基础研究发展规划(973计划)(编号:TG199901600004);江苏省自然科学基金项目资助(No.BK2003213)~~

摘　　要：为了获得杉木木质化过程中特异表达的基因,本研究利用抑制差减杂交技术,以杉木突变体独干杉无性系为测试方(Tester),正常的句容0号无性系为驱动方(Driver),构建了正向差减文库,获得了618个克隆。利用通用引物T7和SP6进行PCR及EcoRⅠ酶切鉴定文库重组子,同时,利用点杂交技术,以正向差减、反向差减及未差减的测试方和驱动方四种探针,进行了进一步的筛选阳性克隆。用定量PCR对结果进行了初步验证。260个单一ESTs中60%的与已知蛋白具有显著同源性,可分为4个主要类别:新陈代谢、细胞壁生成和结构重构、信号传导和胁迫。系统分析参与杉木木材形成的基因对了解木质部分化的分子机制具有重要意义,是了解木材形成遗传控制的直接信息来源,也是改良材形和纤维性质的潜在候选基因。Wood is an important raw material for the global industry with rapidly increasing demand. To isolate the differentially expressed genes in xylogenesis of Chinese fir [Cunninghamia lanceolata （Lamb.） Hook], a forward subtractive cDNA library was constructed using suppression subtractive hybridization （SSH） method, which was performed using the eDNA from the mutant Dugansha clone as the tester and the eDNA from the normal Jurong 0 clone as the driver. Six hun- dred and eighteen clones were obtained. Recombinants were identified using PCR with universal T7 and SP6 primers and using EcoR I digestion. To further eliminate false positive, dot hybridization was used with four DIG-labeled probes （FSP, RSP, UTP, and UDP）. Real-time PCR was performed to confirm the results. A total of 260 unique ESTs were obtained, 60% of the ESTs exhibiting homologies with proteins of known function fell into 4 major classes： metabolism, cell wall biogenesis and remodeling, signal transduction and stress. The systematic analysis of genes involved in wood formation in Chinese fir provides valuable insights into the molecular mechanisms involved in xylem differentiation, is important resources for forest research directed toward understanding the genetic control of wood formation and future endeavors to modify wood and fiber properties for industrial use.

关 键 词：杉木 木材形成 抑制差减杂交 特异性表达基因 木质部分化 

分 类 号：S791.27[农业科学—林木遗传育种]