广藿香转录组文库的构建及响应连作障碍的miRNAs鉴定与分子机制

Pogostemon cablin has been one of the well-known Chinese medicinal materials. The essential oil is effective to “transform turbid dampness, stop vomiting and stimulate appetite, release exterior dampness and summer-heat attack”. It was reported that the problem of continuous cropping obstacles of P. cablin was very severe, which affected the yield, quality and efficacy of this herb medicine. In the experiment, consecutive monoculture (CM, the second year planting) P. cablin will be taken as treatment samples, normal growth ones (the first year planting, CK) as control samples. Its roots and leaves will be collected at the four stages of slow growth, rapid growth, maturity and complete maturity, respectively. Based on the early study of "interaction between root exudates and rhizosphere microorganisms", the project will construct transcriptome libraries of P. cablin roots and leaves by using Solexa/Illumina sequencing technique. With isolation and sequencing of these sRNA from different-stage roots and leaves, miRNAs differential expression profiles of P. cablin responding to continuous cropping obstacles will be constructed, resulting in identifying key miRNA and its targets. At the same time, the consecutive monoculture P. cablin DGE expression profiles will be established by using Digital Gene Expression Profiling (DGE) analysis, collecting candidates for consecutive monoculture problem-related genes. The spatiotemporal expression profiles of candidate genes will be confirmed and these key genes will also be verified by exploring qRT-PCR and Northern blot technologies. The project will identify these miRNAs and continuous cropping obstacles-related genes by constructing transcriptome libraries of P. cablin roots and leaves. This study will provide the theoretical basis for the effective regulations on continuous cropping obstacles of P. cablin and conquering the difficulties of continuous cropping obstacles of Chinese medicine materials production.

广藿香具有芳香化浊、开胃止呕、发表解暑功效，连作后的广藿香药材产量和品质明显下降。本项目在前期“根系分泌物与根际微生物间互作效应”研究基础上，设正茬和连作2处理，分别于缓慢生长、快速生长、成熟和完全成熟4阶段取其根与叶样品，利用Solexa测序，分别构建根和叶的不同生育期转录组文库；提取并分离小RNA，测序后构建小RNA库，匹配转录组，建立响应广藿香连作的miRNAs差异表达谱，以确定响应广藿香连作的miRNAs及其靶基因；采用编码基因序列标签测序法，构建连作广藿香编码基因数字表达谱，筛选响应连作障碍的“候选基因”；运用qRT-PCR、Northern blot等，验证候选基因的时空表达，锁定响应连作障碍的关键基因，以揭示广藿香连作障碍的分子机制。本项目通过构建广藿香转录组文库，鉴定响应连作的关键基因及miRNAs，为实现广藿香连作障碍的有效调控，解决中药材生产中连作障碍难题提供理论依据。

连作障碍是广藿香生产栽培中存在的显著问题，会导致广藿香生长受抑，致使药材产量和品质下降，极大抑制广藿香产业的发展。目前连作障碍的形成机制和作用机理尚不明确，为缓解广藿香连作障碍，必须从分子层面解析连作对广藿香植株伤害的分子机制。miRNA在植物响应逆境胁迫中扮演着重要角色，势必成为研究广藿香连作障碍分子调控机制的开端和重点。为此，本课题构建了正茬和连作广藿香根和叶的转录组文库，利用GO、Pathway等生物信息分析，对序列进行De novo组装、Unigene功能注释与分析，得到了CDS序列，进行归类和基因功能注释，共检测到了6892个差异基因，下调3660个，上调3232个。有4059个和1560个差异基因被分别注释到GO和KEGG数据库，应激诱导早熟性老化等GO功能以及帖类生物合成等途径被显著富集。本课题组还构建了广藿香特异miRNAs差异表达谱，确定了广藿香响应连作障碍的关键miRNAs（包括保守的、新型的）及其靶基因的候选名单，其中基因组比对率分布在63.54-84.84%，已知miRNA比对率分布在0.47-0.83%，所有样本比对上已知miRNA的总数为927，所有样本新预测miRNA的总数为130，共设有10个差异比较组，检测到的差异miRNA数量为：37，56，29，48，50，22，17，24，21，20。此外，本课题组采用NlaIII酶切，识别并切断cDNA上的CATG位点，纯化带有cDNA3’端的片段，经过Mme1酶切后，获得两端连有不同接头序列的21bp标签库，PCR扩增后，纯化并Solexa测序得到21碱基标签频率库，构建正茬与连作广藿香数字化基因差异表达谱，并通过qRT-PCR验证了所用RNA-Seq结果具有极高的可靠性，也表明这些基因确实与连作胁迫响应相关，从而锁定导致连作“毒害”的基因。此外，我们还分析了广藿香内生真菌及其次生代谢产物的生物活性，并鉴定了广藿香化感物质对羟基苯甲酸对广藿香幼苗根系的伤害机制。本课题揭示了连作广藿香植株响应连作胁迫分子机制，为实现广藿香连作障碍的有效调控，缓解广藿香药材生产中连作障碍难题提供理论依据。