小麦幼苗期水分胁迫所诱导基因表达谱的初步分析

利用抑制差减杂交(Suppression Subtractive Hybridization,SSH)和高密度点阵膜技术研究小麦2叶幼苗期水分胁迫诱导表达基因。通过筛选具有1530个克隆的SSH文库,获得181个阳性克隆。序列同源性比较和功能查询结果发现,83．2％的水分胁迫诱导表达基因分别与不同逆境胁迫条件下表达的基因具有较高的同源性,这些基因在生物体内的功能都是直接或间接对细胞遭受逆境胁迫起保护作用。其中17个EST未找到同源性较高的匹配序列,已经在GenBank注册。用反向Northern、RT-PCR和Northern进一步检验所获得的功能已知EST,初步建立了小麦幼苗期水分胁迫诱导的基因表达谱。     

Identification and functional validation of a unique set of drought induced genes preferentially expressed in response to gradual water stress in peanut

Peanut, found to be relatively drought tolerant crop, has been the choice of study to characterize the genes expressed under gradual water deficit stress. Nearly 700 genes were identified to be enriched in subtractive cDNA library from gradual process of drought stress adaptation. Further, expression of the drought inducible genes related to various signaling components and gene sets involved in protecting cellular function has been described based on dot blot experiments. Fifty genes (25 regulators and 25 functional related genes) selected based on dot blot experiments were tested for their stress responsiveness using northern blot analysis and confirmed their nature of differential regulation under different field capacity of drought stress treatments. ESTs generated from this subtracted cDNA library offered a rich source of stress-related genes including signaling components. Additional 50% uncharacterized sequences are noteworthy. Insights gained from this study would provide the foundation for further studies to understand the question of how peanut plants are able to adapt to naturally occurring harsh drought conditions. At present functional validation cannot be deemed in peanut, hence as a proof of concept seven orthologues of drought induced genes of peanut have been silenced in heterologous N. benthamiana system, using virus induced gene silencing method. These results point out the functional importance for HSP70 gene and key regulators such as Jumonji in drought stress response. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequence reported in this paper has been submitted to (database) under accession numbers: Acc. No. EC268400–EC268685; EC365167–EC365455. An erratum to this article can be found at     

干旱胁迫下刚毛柽柳消减文库的构建及分析

以干旱胁迫下的刚毛柽柳根部组织cDNA为tester,正常生长的刚毛柽柳根部组织cDNA为driver,利用抑制性消减杂交技术(SSH)构建了干旱胁迫下刚毛柽柳根部组织的消减文库。文库克隆的重组率为95%,插入片段大部分集中在250~600 bp之间。通过对文库阳性克隆的随机测序,获得了如Mn-SOD、myb相关蛋白、锌指蛋白等17种与干旱胁迫相关的基因,它们涉及了植物的渗透调节、信号传递、转录调控、活性氧清除等方面。所得EST序列已被GenBank收录。实验为抗逆基因克隆和系统研究干旱胁迫下柽柳根部基因的表达奠定了基础。     

干旱胁迫下黄檗幼苗cDNA消减文库的构建和分析

以干旱胁迫下的黄檗幼苗cDNA 为tester, 正常生长的黄檗幼苗cDNA为driver, 利用抑制性消减杂交技术(suppression subtractive hybridization, SSH)构建了干旱胁迫下黄檗幼苗的消减文库并对其进行了EST序列分析。从消减文库中随机挑取20个阳性克隆, 提取质粒进行酶切和PCR鉴定, 显示文库克隆的重组率大于95%, 插入片段大小大部分集中在300~800bp之间。随机挑取816个克隆进行测序, 得到265个基因。将其进行同源性分析, 划分为16类。获得了热激蛋白70、脱水响应蛋白(RD22)、通用胁迫蛋白、金属硫蛋白(MTII), 晚期胚胎丰富蛋白(LEA14)等44种与干旱胁迫相关的基因,它们涉及了植物的渗透调节、信号传递、转录调控、活性氧清除等方面。本研究为抗逆基因克隆和系统研究干旱胁迫下黄檗基因的表达奠定了重要的理论基础。     

干旱胁迫下黄檗幼苗cDNA消减文库的构建和分析

以干旱胁迫下的黄檗幼苗cDNA 为tester, 正常生长的黄檗幼苗cDNA为driver, 利用抑制性消减杂交技术(suppression subtractive hybridization, SSH)构建了干旱胁迫下黄檗幼苗的消减文库并对其进行了EST序列分析。从消减文库中随机挑取20个阳性克隆, 提取质粒进行酶切和PCR鉴定, 显示文库克隆的重组率大于95%, 插入片段大小大部分集中在300~800bp之间。随机挑取816个克隆进行测序, 得到265个基因。将其进行同源性分析, 划分为16类。获得了热激蛋白70、脱水响应蛋白(RD22)、通用胁迫蛋白、金属硫蛋白(MTII), 晚期胚胎丰富蛋白(LEA14)等44种与干旱胁迫相关的基因,它们涉及了植物的渗透调节、信号传递、转录调控、活性氧清除等方面。本研究为抗逆基因克隆和系统研究干旱胁迫下黄檗基因的表达奠定了重要的理论基础。     

Identification of ESTs differentially expressed in green and albino mutant bamboo (Bambusa edulis) by suppressive subtractive hybridization (SSH) and microarray analysis

To gain a better understanding of gene expression in bamboo (Bambusa edulis Murno), we have used a combination of suppressive subtractive hybridization (SSH), microarray hybridization analysis, sequencing, and bioinformatics to identify bamboo genes differentially expressed in a bamboo albino mutant. Ten expressed sequence tags (ESTs) were found to be differentially expressed; these were isolated and sequenced. RT-PCR analysis of these ESTs supported the results of the microarray analysis. Six ESTs that were nucleus-encoded exhibited differential expression patterns in the green wild-type bamboo relative to the albino mutant. These genes (exception being the Rubisco small subunit) were non-photosynthesis-related genes. The development of a specific SSH cDNA library in which most of the chloroplast-encoded or photosynthesis-related genes had been subtracted proved to be useful for studying the function of non-photosynthesis-related genes in the albino bamboo mutants with aberrant chloroplast genome. The combined use of this SSH library with microarray analysis will provide a powerful analytical tool for future studies of the bamboo genome.     

Differential accumulation of mRNAs in drought-tolerant and susceptible common bean cultivars in response to water deficit

The physiological response to drought was measured in two common bean varieties with contrastive susceptibility to drought stress. A subtractive cDNA library was constructed from the two cultivars, Phaseolus vulgaris'Pinto Villa' (tolerant) and 'Carioca' (susceptible). 18 cDNAs displayed protein-coding genes associated with drought, cold and oxidative stress, signal transduction, plant defense, chloroplast function and unknown function. A cDNA coding for an aquaporin (AQP) was selected for further analyses. The open reading frames (ORFs) of AQPs from 'Pinto Villa' and 'Carioca' were compared and despite their similarity, accumulated differentially in the plant organs, as demonstrated by Northern blot and in situ hybridization. A phylogenetic analysis of the deduced amino acid sequence with other AQPs suggested a tonoplast-located protein. Under drought conditions, the levels of AQP mRNA from the susceptible cultivar decreased to undetectable levels; by contrast, 'Pinto Villa' mRNA was present and restricted the phloem tissue. This would allow 'Pinto Villa' to maintain vascular tissue functions under drought stress.