cDNA-AFLP analysis of salt-inducible genes expression in Chrysanthemum lavandulifolium under salt treatment

Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino is a halophyte species that belongs to the Asteraceae family, and the genus Chrysanthemum. It is one of the ancestors of C. × morifolium Ramatella. Understanding the tolerance mechanism associated with salt stress in C. lavandulifolium could provide important information for explaining the salt tolerance of higher plants and could also help enhancing breeding programs of cultivated Chrysanthemum. In this study, cDNA amplified fragment length polymorphism (cDNA-AFLP) was used to detect differential gene expression in leaves of C. lavandulifolium in response to NaCl treatment. The determination of membrane permeablility, peroxidase activity (POD), malon-dialdehyde (MDA), as well as proline and leaf chlorophyll contents under different NaCl concentrations showed that a 200 mM NaCl treatment was an optimal condition for the cDNA-AFLP experiment. Using this concentration during different times (0, 3 h, 12 h, 24 h and 48 h), we obtained 1930 cDNA fragments using 64 primers. After sequencing 234 randomly chosen cDNA clones and BLASTx analyzing, we got 129 expressed sequence tags (ESTs) which had no significant homology with other sequences, 85 ESTs were homologous to genes with known functions, whereas the rest of ESTs showed homology to unclassified or putative proteins. 25 ESTs that were similar to known functional genes involved in several abiotic and biotic stresses were confirmed by semi-quantitative RT-PCR and qRT-PCR. The expression patterns of these salt-responsive genes not only responded to salt stress but also to plant hormones, such as abscisic acid (ABA), and to other abiotic stresses such as drought and cold. These results indicate an extensive cross-talk among several stresses. Our results provide interesting information for further understanding the molecular mechanisms of salt tolerance in C. lavandulifolium.     

NaCl胁迫下哈茨木霉ACCC32524的转录组和代谢组分析

【目的】通过分析NaCl胁迫下哈茨木霉(Trichoderma harzianum)ACCC32524转录组和代谢组数据,研究差异表达基因及次级代谢产物的变化情况,初步探索响应NaCl胁迫的分子机制。【方法】利用Illumina HiSeq XTen高通量测序平台完成0、0.4、0.6 mol/L NaCl浓度胁迫培养下哈茨木霉ACCC32524的转录组测序,GC-TOF-MS技术完成对0mol/L和0.6mol/LNaCl胁迫培养下的差异次级代谢产物检测,利用相关软件及数据库对差异表达基因(DEGs)和次级代谢产物的注释、筛选和分类,并进行RT-qPCR验证。【结果】本研究分别得到0.4 mol/L和0.6 mol/L NaCl胁迫下417和733条差异表达基因;GO富集分析显示,分别有318和582条差异表达基因注释到生物学过程、分子功能和细胞组分3个一级分类和40个二级分类;COG分类结果表明分别有232和414条转录本为20个类别,涉及差异表达基因最多的分别为氨基酸的转运和代谢、一般功能预测、碳水化合物的转运和代谢;KEGG代谢途径分析结果表明,分别有75和96条基因归到25个代谢通路中(P≤0.05),其中涉及差异基因最多的是氨基酸的生物合成和2-氧代羧酸代谢通路。从转录组数据中共筛选出与渗透调节、离子转运、活性氧清除等22个耐盐相关基因。0 mol/L和0.6 mol/L NaCl胁迫下的代谢组数据中共筛选出101个差异次级代谢产物,包括8种积累量上调和93种下调物质,其中36个得到定性,分属于糖类、有机酸和氨基酸等9个分类中。RT-qPCR验证挑选的差异表达基因的表达量变化,均与RNA-seq分析结果一致。【结论】NaCl胁迫下引起哈茨木霉ACCC32524基因及次级代谢产物发生明显变化,细胞代谢途径发生明显偏移,这些进程共同作用减少NaCl对细胞的毒害作用,为木霉菌的耐盐机理研究提供重要信息。     

基于转录组测序的扎龙湿地野大麦耐盐性分析

通过转录组测序技术对扎龙湿地野大麦幼苗在200 mmol/L NaCl胁迫2 d后的叶片进行分析。对测序结果进行De novo拼接后,将差异表达基因在GO、KEGG数据库中进行比对注释。结果表明:NaCl胁迫2 d后,对照组与处理组分别获得Unigene序列61038个和46754个,检测到差异表达基因25465个,差异基因GO功能注释到3个大类的55个功能组。差异表达基因被注释到135个Pathway上,直观地显示出NaCl胁迫下野大麦幼苗体内发生调节及改变的代谢过程和信号通路,其中主要涉及光合作用、脯氨酸代谢、叶绿素代谢等途径。通过对野大麦幼苗叶片转录组分析,发掘相关耐盐基因,有助于培育野大麦及其近缘作物新品种,并对揭示植物耐盐性分子机制及相关代谢途径具有重要意义。     

Drought,heat and salt stress induce the expression of a citrus homologue of an atypical late-embryogenesis Lea5 gene

In a search for genes that are induced in citrus cell suspension in response to salt stress, a cDNA clone with high homology to cotton Lea5 gene was isolated. Data base analysis of the protein deduced from the nucleotide sequence indicates that, like in cotton, the protein from citrus contains regions with significant hydropathic character. The gene, designated C-Lea5, is expressed in citrus leaves as well as cell suspension. The steady-state level of C-Lea5 is increased in cell suspension that is grown in the presence of 0.2 M NaCl. This phenomenon is also observed in leaves of citrus plants irrigated with NaCl and in citrus seedlings which are exposed to drought and heat stress. We suggest that the osmotic stress resulted from elevated level of salt is responsible for the increase in the level of C-Lea5.     

盐生植物海滨锦葵幼苗盐胁迫下基因差异表达分析

利用cDNA-AFLP技术对海滨锦葵幼苗盐胁迫下叶片和根部的基因差异表达模式进行分析和比较, 并对部分盐胁迫应答的转录衍生片段进行了回收、测序和功能推测, 以从转录水平分析海滨锦葵的耐盐分子机制。结果显示：(1) 盐胁迫下海滨锦葵幼苗叶片和根部的基因差异表达多以量的变化为主, 包括盐胁迫下基因表达上调、下调或随盐处理浓度高低和胁迫时间长短而波动的差异表达模式; 只有少量基因的差异表达表现出质的变化, 如盐胁迫下基因沉默或诱导表达; (2) 仅在盐胁迫处理2 h的海滨锦葵幼苗根部, 基因的差异表达以质的变化为主的类型比例略高于量的变化类型比例; (3) 盐胁迫应答基因在不同组织中上调、下调、诱导或沉默的比例随胁迫处理时段而动态变化, 在刚胁迫时基因表达的差异加剧, 而后随胁迫处理时段的延长而渐趋稳定。结果预示, 从基因表达水平探讨植物的耐盐分子机理, 尽管有一定的规律可循, 但由于不同组织对盐胁迫的应答是动态变化的过程, 海滨锦葵不同组织在盐胁迫不同阶段的基因时、空、序表达特征并没有固定的程式。对部分盐胁迫下上调或诱导表达的转录衍生片段(Trivially distributed file system, TDFs)进行的序列分析和功能推测表明, 苗期海滨锦葵在盐胁迫下应答基因至少涉及3类：(1) 离子平衡重建或减少胁迫损伤相关基因(特别是运转蛋白类); (2) 恢复盐胁迫下植物生长和发育相关基因：如参与能量合成和激素调节途径相关基因等; (3)信号转导相关基因及功能未确定的新基因。文章并对盐胁迫应答基因的差异表达模式与海滨锦葵的耐盐性关系进行了讨论。     

NaCl胁迫下黑果枸杞转录组测序分析

研究黑果枸杞在不同浓度盐胁迫下基因表达谱变化情况,为进一步研究黑果枸杞抗盐分子机制奠定研究基础。对0(CK)、50、250 mmol/L NaCl溶液胁迫的黑果枸杞组培苗的根和叶在胁迫时间为0、1、12 h时分别取样,采用转录组测序(RNASeq)技术进行测序分析。结果表明,转录组测序共产生222.49 Gb原始数据,拼接出Unigenes 86 037条,注释到7大功能数据库(GO、KEGG、KOG、NR、Pfam、Swiss-Prot和egg NOG)上的Unigenes总数为46 594个,占总Unigenes的54.76%,还有38 929个Unigenes在这些数据库中没有得到注释。通过GO分类和KEGG Pathway富集性分析,分别归于51个GO类别和211条代谢途径。差异表达基因分析显示,黑果枸杞叶片和根的上调基因和下调基因数随着NaCl浓度的增大和处理时间的延长均呈增加趋势,叶片中的上调基因数(7 514)小于下调基因数(9 032),根中的上调基因数(12 347)大于下调基因数(11 559)。在黑果枸杞盐胁迫下转录组中发现28 325个SSR位点,最多的为单核苷酸SSR,占70.47%。综合分析表明,黑果枸杞对盐胁迫的反应是一个多基因参与、多个生物过程协同调控的过程,基因表达量的变化可能是基因调控的主要方式。     

Arabidopsis sos1 mutant in a salt-tolerant accession revealed an importance of salt acclimation ability in plant salt tolerance

An analysis of the salinity tolerance of 354 Arabidopsis thaliana accessions showed that some accessions were more tolerant to salt shock than the reference accession, Col-0, when transferred from 0 to 225 mM NaCl. In addition, several accessions, including Zu-0, showed marked acquired salt tolerance after exposure to moderate salt stress. It is likely therefore that Arabidopsis plants have at least two types of tolerance, salt shock tolerance and acquired salt tolerance. To evaluate a role of well-known salt shock tolerant gene SOS1 in acquired salt tolerance, we isolated a sos1 mutant from ion-beam-mutagenized Zu-0 seedlings. The mutant showed severe growth inhibition under salt shock stress owing to a single base deletion in the SOS1 gene and was even more salt sensitive than Col-0. Nevertheless, it was able to survive after acclimation on 100 mM NaCl for 7 d followed by 750 mM sorbitol for 20 d, whereas Col-0 became chlorotic under the same conditions. We propose that genes for salt acclimation ability are different from genes for salt shock tolerance and play an important role in the acquisition of salt or osmotic tolerance.