Generation and analysis of drought stressed subtracted expressed sequence tags from safflower (Carthamus tinctorius L.)

Drought is the most crucial environmental factor that limits productivity of many crop plants. Exploring novel genes and gene combinations is of primary importance in plant drought tolerance research. Stress tolerant genotypes/species are known to express novel stress responsive genes with unique functional significance. Hence, identification and characterization of stress responsive genes from these tolerant species might be a reliable option to engineer the drought tolerance. Safflower has been found to be a relatively drought tolerant crop and thus, it has been the choice of study to characterize the genes expressed under drought stress. In the present study, we have evaluated differential drought tolerance of two cultivars of safflower namely, A1 and Nira using selective physiological marker traits and we have identified cultivar A1 as relatively drought tolerant. To identify the drought responsive genes, we have constructed a stress subtracted cDNA library from cultivar A1 following subtractive hybridization. Analysis of?~1,300 cDNA clones resulted in the identification of 667 unique drought responsive ESTs. Protein homology search revealed that 521 (78?%) out of 667 ESTs showed significant similarity to known sequences in the database and majority of them previously identified as drought stress-related genes and were found to be involved in a variety of cellular functions ranging from stress perception to cellular protection. Remaining 146 (22?%) ESTs were not homologous to known sequences in the database and therefore, they were considered to be unique and novel drought responsive genes of safflower. Since safflower is a stress-adapted oil-seed crop this observation has great relevance. In addition, to validate the differential expression of the identified genes, expression profiles of selected clones were analyzed using dot blot (reverse northern), and northern blot analysis. We showed that these clones were differentially expressed under different abiotic stress conditions. The implications of the analyzed genes in abiotic stress tolerance are discussed in our study.     

Identification and characterization of phospholipase D and its association with drought susceptibilities in peanut (Arachis hypogaea)

Preharvest aflatoxin contamination has been identified by the peanut industry as a serious issue in food safety and human health because of the carcinogenic toxicity. Drought stress is the most important environmental factor exacerbating Aspergillus infection and aflatoxin contamination in peanut. The development of drought-tolerant peanut cultivars could reduce aflatoxin contamination and would represent a major advance in the peanut industry. In this study, we identified a novel PLD gene in peanut (Arachis hypogaea), encoding a putative phospholipase D (PLD, EC 3.1.4.4). The completed cDNA sequence was obtained by using the consensus-degenerated hybrid oligonucleotide primer strategy. The deduced amino acid sequence shows high identity with known PLDs, and has similar conserved domains. The PLD gene expression under drought stress has been studied using four peanut lines: Tifton 8 and A13 (both drought tolerant) and Georgia Green (moderate) and PI 196754 (drought sensitive). Northern analysis showed that PLD gene expression was induced faster by drought stress in the drought-sensitive lines than the drought tolerance lines. Southern analysis showed that cultivated peanut has multiple copies (3 to 5 copies) of the PLD gene. These results suggest that peanut PLD may be involved in drought sensitivity and tolerance responses. Peanut PLD gene expression may be useful as a tool in germplasm screening for drought tolerance. The nucleotide sequence, reported in this paper, have been submitted to GenBank under accession number AY274834.     

受褐飞虱取食的水稻幼苗的抑制消减cDNA文库的构建及筛选

采用抑制消减杂交方法,以褐飞虱取食32h的水稻幼苗及未受褐飞虱取食的水稻幼苗为作为对比材料构建了消减cDNA文库,以分离水稻幼苗中褐飞虱应答基因。随机从消减cDNA文库中挑选16个白色菌落提取质粒,进行PCR扩增,发现插入片段的长度位于100～900bp之间。以在受褐飞虱取食的水稻幼苗中特异表达的基因(BpHi008A)为探针,通过斑点印迹分析发现在抑制消减后的cDNA池中,目的基因得到有效富集。利用反向总RNA斑点印迹分析和Northern杂交验证,从消减cDNA文库中筛选到了25个基因受褐飞虱取食的诱导。其中有17个克隆与编码已知功能蛋白的基因有显著的同源性,它们分别参与蛋白质的折叠与降解、蛋白质与蛋白质的相互作用及信号传递、脂类代谢、胁迫反应、物质运输和细胞生长等。总体上,参与胁迫反应和衰老的基因在褐飞虱取食后表达增强。     

Identification and annotation of abiotic stress responsive candidate genes in peanut ESTs

Peanut (Arachis hypogaea L.) ranks fifth among the world oil crops and is widely grown in India and neighbouring countries. Due to its large and unknown genome size, studies on genomics and genetic modification of peanut are still scanty as compared to other model crops like Arabidopsis, rice, cotton and soybean. Because of its favourable cultivation in semi-arid regions, study on abiotic stress responsive genes and its regulation in peanut is very much important. Therefore, we aim to identify and annotate the abiotic stress responsive candidate genes in peanut ESTs. Expression data of drought stress responsive corresponding genes and EST sequences were screened from dot blot experiments shown as heat maps and supplementary tables, respectively as reported by Govind et al. (2009). Some of the screened genes having no information about their ESTs in above mentioned supplementary tables were retrieved from NCBI. A phylogenetic analysis was performed to find a group of utmost similar ESTs for each selected gene. Individual EST of the said group were further searched in peanut ESTs (1,78,490 whole EST sequences) using stand alone BLAST. For the prediction as well as annotation of abiotic stress responsive selected genes, various tools (like Vec-Screen, Repeat Masker, EST-Trimmer, DNA Baser, WISE2 and I-TASSER) were used. Here we report the predicted result of Contigs, domain as well as 3D structure for HSP 17.3KDa protein, DnaJ protein and Type 2 Metallothionein protein.     

Characterization of two cDNAs for novel drought-inducible genes in the highly drought-tolerant cowpea

Ten cDNAs for drought-inducible genes were isolated using differential screening of a cDNA library prepared from 10-hr dehydrated cowpea plants,Vigna unguiculata (S. Iuchi, K. Yamaguchi-Shinozaki, T. Urao, T. Terao, K. Shinozaki; Plant Cell Physiology, 1996 in press). Two of the cDNA clones, designated CPRD12 and CPRD46, were sequenced and characterized. The CPRD12 and CPRD46 cDNAs encode putative proteins related to nonmetallo-short-chain alcohol dehydrogenase (CPRD12) and chloroplastic lipoxygenase (CPRD46). Northern blot analysis revealed that these genes are induced by high-salinity stress and exogenous abscisic acid, but not by cold stress. The CPRD46 gene is also responsive to heat stress and methyl jasmonate and salicylic acid. Genomic Southern blot analysis suggested that CPRD12 constitutes a small gene family, but that CPRD46 is a single copy gene. We discuss the possible functions of these two CPRD gene products under drought stress.     

TILLING by sequencing to identify induced mutations in stress resistance genes of peanut (Arachis hypogaea)

Background

Targeting Induced Local Lesions in Genomes (TILLING) is a powerful reverse genetics approach for functional genomics studies. We used high-throughput sequencing, combined with a two-dimensional pooling strategy, with either minimum read percentage with non-reference nucleotide or minimum variance multiplier as mutation prediction parameters, to detect genes related to abiotic and biotic stress resistances. In peanut, lipoxygenase genes were reported to be highly induced in mature seeds infected with Aspergillus spp., indicating their importance in plant-fungus interactions. Recent studies showed that phospholipase D (PLD) expression was elevated more quickly in drought sensitive lines than in drought tolerant lines of peanut. A newly discovered lipoxygenase (LOX) gene in peanut, along with two peanut PLD genes from previous publications were selected for TILLING. Additionally, two major allergen genes Ara h 1 and Ara h 2, and fatty acid desaturase AhFAD2, a gene which controls the ratio of oleic to linoleic acid in the seed, were also used in our study. The objectives of this research were to develop a suitable TILLING by sequencing method for this allotetraploid, and use this method to identify mutations induced in stress related genes.

Results

We screened a peanut root cDNA library and identified three candidate LOX genes. The gene AhLOX7 was selected for TILLING due to its high expression in seeds and roots. By screening 768 M2 lines from the TILLING population, four missense mutations were identified for AhLOX7, three missense mutations were identified for AhPLD, one missense and two silent mutations were identified for Ara h 1.01, three silent and five missense mutations were identified for Ara h 1.02, one missense mutation was identified for AhFAD2B, and one silent mutation was identified for Ara h 2.02. The overall mutation frequency was 1 SNP/1,066 kb. The SNP detection frequency for single copy genes was 1 SNP/344 kb and 1 SNP/3,028 kb for multiple copy genes.

Conclusions

Our TILLING by sequencing approach is efficient to identify mutations in single and multi-copy genes. The mutations identified in our study can be used to further study gene function and have potential usefulness in breeding programs.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1348-0) contains supplementary material, which is available to authorized users.     

毛尖紫萼藓干旱胁迫cDNA文库的构建

干旱胁迫是影响植物生长发育的主要环境因素,严重影响农作物的产量。解决这个问题的有效途径是培育和利用优良的抗旱品种。应用比较功能基因组学方法筛选抗旱相关基因,并通过基因工程培育抗旱品种已成为植物遗传资源与品种改良研究的重要内容。毛尖紫萼藓（Grimmia pilifera）是典型旱生藓类,生长在向阳的裸岩上,具有很强的抗旱能力,是很好的抗旱基因资源。本研究采用SMART技术构建毛尖紫萼藓干旱cDNA文库,文库滴度为2.8×10⁵ pfu·mL^-1,重组率为91.7%,插入片段大小为500~2 000 bp,平均为800 bp。通过测序我们获得了1 045条ESTs,其中高质量的996条,通过拼接获得875个Unigenes,为进一步筛选抗旱相关基因奠定了基础。     

cDNA微阵列技术研究干旱胁迫下星星草基因的表达

运用cDNA微阵列技术研究干旱胁迫下星星草基因的表达。制备了载有660条星星草单一基因的cDNA微阵列。分别对干旱胁迫和对照星星草的mRNA进行荧光标记,并与载有星星草基因的cDNA微阵列进行杂交,通过芯片的杂交信号强度分析,共获得22个下调表达和17个上调表达的基因。BLASTX分析表明这些基因按功能可以分为脱水保护、信号转导与调控、活性氧清除、代谢、核糖体蛋白等几大类。发现了一些与干旱胁迫相关的功能未知基因和新基因。     

Isolation and expression analysis of LEA genes in peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Late embryogenesis abundant (LEA) protein family is a large protein family that includes proteins accumulated at late stages of seed development or in vegetative tissues in response to drought, salinity, cold stress and exogenous application of abscisic acid. In order to isolate peanut genes, an expressed sequence tag (EST) sequencing project was carried out using a peanut seed cDNA library. From 6258 ESTs, 19 LEA-encoding genes were identified and could be classified into eight distinct groups. Expression of these genes in seeds at different developmental stages and in various peanut tissues was analysed by semi-quantitative RT-PCR. The results showed that expression levels of LEA genes were generally high in seeds. Some LEA protein genes were expressed at a high level in non-seed tissues such as root, stem, leaf, flower and gynophore. These results provided valuable information for the functional and regulatory studies on peanut LEA genes.