摘要
柽柳(Tamarix spp.),灌木(或小乔木),广泛分布于中国西部和中亚地区,能够在干旱和盐渍化土地上生长,对干旱、盐渍、高温等非生物胁迫具有很强的耐受能力,是改造环境的理想树种,也是研究植物抗逆机理和克隆抗逆基因的理想材料。
本研究以0.3M NaHCO3处理的柽柳根部组织为材料,利用Solexa技术建立胁迫0(对照),12,24和48h的柽柳转录组,共获得81344个Unigenes.通过转录组分析,与对照相比,胁迫12、24和48h显著差异表达的基因分别有3976、6057和3069个。为了鉴定差异表达基因与胁迫之间的关系,对基因表达模式的动态变化进行了聚类分析,差异基因可以分为10类显著共表达的基因表达模式。对共表达的差异表达基因进行GO功能富集分析,结果显示细胞防护、蛋白结构保护和修复、胁迫应答信号通路、细胞内离子平衡保持和重建等生物过程在柽柳抵抗逆境胁迫中具有重要作用。鉴定了15个转录因子家族的1605个基因,这些转录因子家族包括MYB,MYC,bZIP, ERF,WRKY,NAC,ABRE,DREB,bHLH和MADS Box等,这些家族基因的表达大部分都响应NaHCO3胁迫。此外,NaHCO3胁迫下,脯氨酸和海藻糖合成相关基因的表达量增加,提示胁迫下柽柳的脯氨酸和海藻糖含量提高。同时。15个编码热激蛋白的转录本的表达显著变化(占总数45%)。另外,分别有32.7%-50.5%的差异表达基因为未知功能的新基因,说明这些基因在柽柳的胁迫应答反应中也具有重要作用。总之,对这些胁迫应答的关键基因进行系统的分析对深入了解柽柳耐盐机理具有重要意义。
从柽柳中克隆获得14个脂质转运蛋白基因(ThLTP1-14),利用实时荧光定量RT-PCR技术对这些基因在不同的非生物胁迫和外源激素ABA处理下柽柳根、茎、叶中的表达模式进行研究。结果表明,正常生长条件下,14个ThLTPs在根、茎、叶中均表达。但是,在相同组织中它们的基因表达量存在明显差异,在根、茎、叶中分别相差3700倍、540倍和9000倍,说明它们在柽柳组织器官中活性和生理功能可能不同。14个脂质转运蛋白基因和在NaCl,PEG,NaHCO3和CdCl2胁迫下至少在一个组织中表达发生明显改变(>2倍),并且所有基因的表达均受ABA高度诱导,说明这些基因的表达依赖于ABA信号转导途径,参与柽柳非生物胁迫应答。
水通道基因在响应干旱和盐碱胁迫中其重要作用。本研究克隆了18个水通道蛋白基因(ThAQP1-18)。实时荧光定量RT-PCR结果表明,这些基因至少在一个组织中表达发生明显改变,且相对于茎和叶,在根组织中受诱导程度更高,表明这些基因可能主要在柽柳根部胁迫应答反应中发挥作用。其中ThAQP2.ThAQP11和TgAQP14在胁迫前后表达差异最明显,表明这些基因与柽柳非生物胁迫的耐受性密切相关,可作为候选的抗逆基因资源对其功能和调控机理进行深入研究。
为了深入了解柽柳水通道蛋白的表达调控途径,我们选取了一个受非生物胁迫高度诱导表达的水通道蛋白基因(ThAQP11),利用TAIL-PCR技术克隆得到ThAQP11启动子序列,长度1605bp。序列分析发现,该启动子区域包含多种与逆境响应相关的顺式作用元件,如MYB.MYC.WRKY.ABRE等。将其定向替换植物表达载体pCAMBIAl301中的35S启动子,构建了ThAQP11启动子驱动GUS的二元表达载体,浸花法转化拟南芥再通过GUS染色研究ThAQP11启动子活性和基因的特异性表达。结果显示ThAQP11基因启动子可以驱动GUS,而且其表达有一定的组织特异性,主要在子叶、主根和叶脉表达。利用酵母单杂交技术筛选与其启动子ABA应答顺式作用元件ABRE互作的上游调控因子,得到一个bZIP类转录因子,表明ThAQP11响应逆境胁迫可能是由于逆境胁迫会促使细胞内ABA含量增加,激活bZIP转录因子,调控bZIP转录因子与启动子上ABRE元件的相互作用,从而促进了ThAQP11的表达。
Tamarix plants, which include shrubs or small trees, are widely distributed in drought-stricken areas and in salinic soil in Central Asia and Western of China. Tamarix plants are highly tolerant to different abiotic stresses including drought, salinity and high temperatures, thus making them ideal plants to aid reversing environmental degradation. These characteristics make them also valuable for the study of mechanisms of stress resistance.
To investigate stress responses in plant roots, four transcriptomes from roots of Tamarix hispida treated with NaHC03for0,12,24and48h were built using Solexa technology. In total,81,344unigenes were generated. There were3,976,6,057and3,069genes significantly differentially expressed after stress for12,24and48h, respectively. The dynamic changes in the differentially expressed genes (DEGs) and their relationship to stress were identified. Ten clusters of genes were found to be significantly co-expressed. GO-enriched analysis showed that the processes of cellular protection, protection and repair of damaged structural proteins, signal pathway responses to stress, and maintaining and re-establishing cellular ion homeostasis may play major roles in the stress tolerance of T. hispida. We examined15transcriptional factor (TF) families from the transcriptomes, including Myb, Myc, bZIP, ERF, WRKY, NAC, ABRE, DREB, bHLH, and MADS Box. These TF families contained1605unique TF genes. DGE analysis showed that the largest numbers of TFs were differentially expressed at24h, followed by12and48h. Under NaHCO3stress, genes involved in proline and trehalose synthesis were highly induced, suggesting that proline and trehalose levels were highly increased in T. hispida. Fifty heat shock proteins (45%in total) were DEGs, suggesting important roles in stress tolerance. Novel genes accounted for a high percentage (32.7%-50.5%) of total DEGs, indicating their importance for an in-depth characterization of stress tolerance mechanisms. This systematic analysis has identified several stress response genes pivotal for an in-depth characterization of stress tolerance mechanisms in plants.
In this study, we cloned14unique LTPs genes (ThLTP1-14) from T. hispida to investigate their roles under various abiotic stress conditions. The expression profiles of the14ThLTPs in response to various abiotic stresses and abscisic acid (ABA) exposure in root, stem and leaf tissues were investigated using real time RT-PCR. The results showed that all14ThLTPs were expressed in root, stem and leaf tissue under normal growth conditions. However, under normal growth conditions, ThLTP abundance varied from each organ, with expression differences of9000-fold in leaves,540-fold in stems, and3700-fold in roots. These results indicated that activity and/or physiological importance of these ThLTPs are quite different. Differential expression of the14ThLTPs and18ThAQPs were observed (>2-fold) for NaCl, PEG, NaHCO3and CdCl2in at least one tissue indicating they were all involved in abiotic stress responses. All ThAQPs were highly induced (>2-fold) under ABA treatment in roots, stems and/or leaves, and suggesting they are regulated by the ABA signal transduction pathway. In addition, ThAQPs were more highly induced in roots than in stem and leaf tissues, indicating that these genes may play roles in stress responses mainly in the roots rather than the stem and leaves. We hypothesize that ThLTPs and ThAQPs expression constitutes an adaptive response to abiotic stresses in T. hispida and plays an important role in abiotic stress tolerance.
To investigate the expression pattern of ThAQP11, the promoter of ThAQPll was isolated using TAIL-PCR approach. The obtained promoter sequence is1605bp in length and contains some stress-response cis-acting elements such as MYB, MYC, WRKY, ABRE and so on. The promoter of ThAQP11was inserted into a plant expression vector pCAMBIA1301to replace CaMV35S promoter (PAQ11:GUS). The Arabidopsis seedlings tansformed with PAQ11: GUS were obtained. The histochemical GUS staining analysis showed that the ThAQP11mainly expressed in cotyledon, leaf veins and taproot.
To screen the upstream regulatory genes of ThAQP11promoter using yeast one-hybrid, a bZIP transcription factor binding to cis-element ABRE was obtained. The results indicated that the bZIP can regulate the expression of ThAQP11through interaction with the cis-acting elements ABRE under abiotic stresses.
引文
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