摘要
镉是一种生物毒性极强的金属元素。环境中,尤其是土壤中的镉极易被植物吸收,进而通过食物链逐级富集,严重危及人体健康。超积累生态型(Hyperaccumulating Ecotype, HE)东南景天是一种原生于我国的镉超积累植物,它能够在地上部累积超过5,000：mg/kg的镉而不表现出明显的毒害症状,是一种较为理想的生物修复材料。对东南景天镉超积累机制,尤其是分子机制的深入研究有助于揭示植物体金属稳态调节网络,挖掘镉超积累关键基因,为创新植物修复材料提供基因素材。过往在东南景天上开展的研究多集中在生理层面,由于缺乏基因组序列信息,对这些生理过程背后所蕴含生化机制的研究进展相对滞后,成为东南景天超积累机制研究中的短板。鉴于此,本研究综合利用二代测序技术Roche454平台和Illumina/Solexa平台测定并构建了HE东南景天地上部转录组序列信息；并以此为基础,研究HE东南景天地上部转录组对镉胁迫的响应；从中选取代表性的基因,利用实时荧光定量PCR (q-RT-PCR)技术,分别对比研究超积累与非超积累生态型(Non-Hyperaccumulating Ecotype, NHE)东南景天中所选基因在正常生长及镉处理条件下的响应差异,以期揭示东南景天地上部镉超积累的分子机制。
此外,本研究还从转录组序列数据库中选取ZIP家族基因为例,综合利用cDNA末端快速扩增技术(Rapid Amplification of cDNA Ends, RACE)、q-RT-PCR、酵母功能互补实验及生物信息学分析方法克隆得到3个ZIP基因,并初步研究了其基因特性与功能。具体研究结果如下：
1.利用Roche454和Illumina/Solexa平台测定并构建了HE东南景天地上部转录组序列信息。在Roche454平台上共获得135,894条平均长度为286bp的高质量测序读长：在Illumina/Solexa测序平台上获得4,750余万条,总计9.4Gb的序列信息。以Roche454测序结果作为拼装骨架,结合Illumina/Solexa平台的海量数据构建HE东南景天地上部转录组序列信息,共拼装得到57,162条长于200bp的Contigs。利用蛋白数据库,对其中的编码基因进行功能注释。根据GO分析,这些基因主要涉及代谢、生物大分子代谢、生物合成等生理过程,参与结合、催化、蛋白结合等分子功能。代谢通路分析表明,所拼装得到的Contigs主要涉及生物体内37条代谢通路,其中与信号转导相关的高达6,695条,占总数的11.7%。另有671条Contigs参与编码转录调控因子,包括锌指蛋白(16.3%)、MYB(16.2%)、 bHLH(12.5%)和bZIP(8.3%)家族等；
2.对所构建的HE东南景天地上部转录组序列进行生物信息学分析,结果表明,HE东南景天与已完成全基因组测序的物种序列相似度较低,与模式植物拟南芥的相似度不足40%,亲缘关系较远。利用生物信息学软件,在HE东南景天的转录组中找到6,176条较理想的简单重复序列(Simple Sequence Repeat, SSR),为构建东南景天分子标记提供了丰富素材。对景天酸代谢关键基因磷酸烯醇丙酮酸羧化酶(Phosphoenolpyruvate Carboxylase, PEPC)的分析表明,HE东南景天兼有C3和景天酸代谢(Crassulacean Acid Metabolism, CAM)型PEPC特征；
3.对比加镉处理与对照条件下HE东南景天地上部转录组的表达变化差异,发现有110条Contigs表达显著上调,另有123条Contigs表达被抑制。利用q-RT-PCR对代表性基因的表达水平进行验证,与Solexa测序结果一致,表明Solexa法测得的基因表达数据具有较高的可靠性。在基因表达水平发生显著变化的Contigs中,大多为功能未知基因,尤其是表达受抑的Contigs中,近一半功能未知。将其余基因进行富集分析,按照GO分类,主要涉及细胞外空间,氨基酸及其衍生物代谢,催化活性与大分子代谢；按照代谢通路分类,则主要与次生代谢物合成,能量代谢,代谢病及位于膜上的转运系统等相关。以富集分析结果为依据,并结合基因功能注释,可知镉诱导下表达显著上调的Contigs主要参与了植物体细胞壁合成、沉积与修饰,金属螯合物合成、金属离子及其配合物转运,抗氧化及基因的转录调节四大类生理过程；
4.以HE与NHE东南景天为试验材料,利用q-RT-PCR法对比研究镉超积累／耐性相关基因在两种生态型东南景天地上部的表达差异。参照镉诱导条件下HE东南景天地上部转录组响应情况及相关文献,选取了与细胞壁合成、修饰,抗氧化,金属转运,金属螯合物合成等相关的18个基因,以HE东南景天地上部序列信息为依据设计引物,用PCR法在非超积累植物上进行扩增,结果表明,所设计引物均能在两种生态型东南景天上特异扩增。以植物体内过氧化物酶基因的表达水平为依据,确定5μM镉处理下,NHE东南景天地上部中镉的胁迫水平与HE东南景天在100μM镉处理下的胁迫水平相当。对比研究两种生态型东南景天在相似镉胁迫条件下的基因表达差异,结果显示,漆酶,肉桂酸羟化酶及Nramp2基因在HE中的表达水平要高于NHE,而Nramp3, HMA4和YSL3则是在NHE中显著诱导。纤维素合成酶与S-腺苷甲硫氨酸合成酶基因在HE中表达上调,而在NHE中表达被抑制。进一步对比研究正常生长状况下两种生态型东南景天地上部的基因表达,结果显示大多数与金属转运、细胞壁合成及修饰相关的基因在正常生长条件下,其在HE内的表达水平要组成性地高于NHE东南景天；
5.以HE东南景天转录组序列为依据,根据功能注释,从中选择表达丰度较高的ZIP家族基因片断Sa_contig615、contig3033和Sa_contig22157,利用RACE方法克隆全长,发现Sa_contig3033的全长序列与AtZIP1序列相似度较高,故命名为SaZIP1;相似地,将Sa_contig615和Sa_contig22157全长序列分别命名为SaZIP4与SaZIP11。拓扑结构预测表明,这三个基因均具有ZIP基因典型的8个跨膜域。酵母功能互补实验表明,SaZIP4和SaZIP11在酵母中具有极强的吸收锌的能力和部分吸收铁的能力,而SaZIP1则不具有上述能力。此外,利用q-RT-PCR,研究上述基因对锌镉处理的响应,结果显示,锌处理抑制了SaZIP4和SaZIP1在地上部中的表达,以及SaZIP1在根中的表达；而镉处理则会诱导SaZIP1和SaZIP11在地上部和根中的表达。
The non-essential heavy metal cadmium (Cd) is very toxic. It readily enters the plant tissue, and thus enters human body through food chain. The major concern of Cd pollution is its potential threats to human health with the risks of causing many cancers including the lung, bladder, renal, prostate, and breast cancer. Sedum alfredii Hance is a Cd hyperaccumulator native in China. Investigations into its physiological and biochemical mechanisms of Cd hyperaccumulation would offer valuable insight into heavy metal homeostasis. Besides, genes identified responsible for Cd hyperaccumulation would aid the creation of genetic modified plant materials for phytoremediation. In the past, main efforts were devoted to the physiological mechanisms of hyperaccumulation of S. alfredii. Lacking of genome sequences has greatly hindered the progress toward molecular mechanisms of hyperaccumulation of S. alfredii. In the present research, we employed the RNA-seq (Roche454and Illumina/Solexa) platforms to explore the transcriptome of S. alfredii Hance and to investigate its transcriptional changes in response to high Cd accumulation in shoots. The gene expression in shoots of two contrasting ecotypes of S. alfredii was also examined by quantitative-Real-Time-Polymerase Chain Reaction (q-RT-PCR).
Zrt-Irt like Protein (ZIP) family genes were reported mediating the transmembrane uptake of divalent heavy metals. Based on the RNA-seq results, three ZIP family genes in S. alfredii were cloned. The expression pattern of these ZIP genes was analysed by q-RT-PCR.Yeast complementary test was carried out to examine the potential function of above genes in Fe/Zn uptake. The results were as follows:
1. The transcriptome of S. alfredii was investigated with Roche454and Illumina/Solexa platforms. Pooling of sequencing runs on Roche454resulted in135,894qualified reads with an average length of286bases; More than47.5million qualified reads were generated on the Illumina/Solexa platforms, which was equal to9.4gigabases (Gb). The454reads were firstly used to generate the transcriptome assembly, and then Illumina reads were mapped to it and assembled. The final assembly with all the contigs obtained generated the transcriptome of S. alfredii shoots with57,162contigs longer than200bp. Based on GO term, these contigs involved metabolic process, macromolecule metabolic process, and binding, catalytic activities. Pathway enrichment of the transcriptome showerd that these contigs involved37different pathways. Among them,6,695contigs were related to signal transduction mechanisms, accounting for11.7%of the total. Besides,671contigs encoding putative transcription factors wer identied, including zinc finger proteins (16.3%), MYB (16.2%), bHLH (12.5%) and bZIP (8.3%);
2. Compared to the protein databases from six fully sequenced species, including Arabidopsis thaliana, Vitis vinifera, Medicago truncatula, Zea mays, Oryza sativa, Chlamydomonas reinhardtii, S. alfredii Hance showed a similarity less than40%. Among the57,162contigs in S. alfredii transcriptome,6,176perfect SSRs were found, which would be useful co-dominant, locus specific markers fro DNA fingerprinting, genome mapplying, phylogenetic analysis, etc. Sedum alfredii belongs to Crassulaceae family. Twenty contigs encoding PEPC homologs were found in the transcriptome of Sedum alfredii. Analysis revealed that S. alfredii had both C3and CAM types of PEPCs.
3. Long exposure of S. alfredii to Cd was employed in the present work to investigate the molecular mechanisms of Cd hyper-tolerance. Compared with control, totally110contigs were up-regulated and123contigs were down-regulated by Cd treatment. To evaluate the validity of Solexa data,15inducible genes were selected, and there expression level was examined by q-RT-PCR. The expression pattern was generally agreed with Solexa sequencing. Among all the significantly regulated contigs, nearly one half of them encoded unknown proteins. Based on GO term, Cd induced contigs were mostly related to extracullar, amino acid and derivative metabolism, catalytic activity and macromolecule metabolism. Based on KEGG orthologies, these genes were related to biosynthesis of other secondary metabolites, energy metabolism, metabolic diseases and membrane transport. In general, the up-regulated contigs could be grouped into five categories, namely, cell wall deposition and modification, metal transport, metal ligand synthesis and metal-ligand transport, reactive oxygen species detoxification, and transcriptional gene regulation.
4. By q-RT-PCR, gene expression in the shoots of two contrasting ecotypes of S. alfredii was compared. Several genes were selected based on the current RNA-seq results and also previously reported metal tolerance determinants, including cell wall modification, deposition, antioxidants, metal transport and ligand biosynthesis. All primers were designed based on the transcriptome of S. alfredii, PCR results showed that these primers were applicable for both ecotypes of S. alfredii. Based on the induction fold of peroxiredoxin gene,5μM Cd treatment on non-hyperaccumulating ecotype (NHE) of S.. alfredii produced compatible Cd stress as100μM Cd on hyperaccumulating ecotype (HE). Among all the examined contigs, lacccase, cinnamate5-hydroxylase and Nramp2were more highly induced in HE than that in NHE; cellulose synthase, SAM synthase were up-regulated in HE while down-regulated in NHE. In contrast, Nramp3, HMA4and YSL3were more highly induced in NHE than that in HE. Under normal growth condition, the expression level, of genes related to heavy metal transport, cell wall synthase and modification were mostly constitutively higher in HE than those in NHE.
5. Based on the transcriptome sequences of hyperaccumulating ecotype of S. alfredii, three contigs encoding ZIP family genes were selected with relatively high expression level. The full length of these contigs were cloned. Blast analysis showed that, Sa_Contig615was most similar to AtZIP4and thus named as SaZIP4. Sa_Contig3033and Sa_Contig22157were accordingly named as SaZIP1and SaZIPll, respectively. Phobius prediction of transmembrane topology revealed8transcmembrane domains in these genes. Among these genes, SaZIP4and SaZIPll fully restored Zinc (Zn) uptake ability and partial iron (Fe) uptake ability in according Zn or Fe uptake deficient strains. The expression of SaZIP4in shoot and SaZIPl in both shoot and root was depreesed with application of Zn. While, application of Cd induced the expression of SaZIPl and SaZIPll in both shoots and roots. These results indicated positive roles of these genes in the transportation of Zn/Cd within S. alfredii.
引文
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