铁皮石斛细胞分裂素合成关键酶基因IPT、CYP735A对促生菌NS的分子响应模式

Dendrobium officinale is a rare and endangered medicinal plant, the growth is extremely slow. The preliminary study of this group showed that Bacillus sp. strain NS can promote the growth of D. officinale. In order to understand its molecular mechanism, the transcriptome of D. officinale inoculated with NS was sequenced. Based on the transcriptome data, this project will be focused on the differentially expressed genes including IPT and CYP735A which encode cytokinin synthetic key enzymes. The full-length cDNA can be obtained by RACE technique and the physical and chemical properties of the protein, the conserved domain and other characteristics will be analyzed. Then, an experiment containing one control group and three groups treated with NS-inoculated, cytokinin inhibitor Lovastatin spray, NS-inoculated with Lovastatin spray will be carried out. The growth index and cytokinin content of D. officinale will be determined at different time. Real-time RT-PCR and RNA in situ hybridization technique will be used to analyze the temporal and spatial expression characteristics of IPT and CYP735A, to analyze the dynamic changes and correlation between gene expression, cytokinin content and growth index. At last, the effect of exogenous application of cytokinin on the growth of D. officinale will be tested. The results would confirm our speculation: one of the ways which Bacillus sp. NS promotes the growth of D. officinale is to promote the endogenous cytokinin synthesis. The results of this study will provide the molecular basis for the promoting mechanism of promoting bacteria and provide scientific basis for accelerating the growth of D. officinale.

铁皮石斛为珍稀濒危药用植物，生长极为缓慢。本课题组前期研究显示，芽孢杆菌NS能显著促进铁皮石斛生长。为深入了解其促生机制，对接种NS后的铁皮石斛进行了转录组测序。在此基础上，本项目拟进一步筛选确定差异表达显著的细胞分裂素合成关键酶IPT、CYP735A基因家族，通过RACE技术获得其全长cDNA并进行信息学分析；然后设计一个对照组和三个处理组，即NS接种组、细胞分裂素合成抑制剂Lovastatin组、NS接种+Lovastatin组，测定不同时间段铁皮石斛生长指标、细胞分裂素含量，同时利用Real-time RT-PCR和RNA原位杂交技术分析IPT、CYP735A的时空表达特征，分析基因表达量、细胞分裂素含量以及生长指标间的动态变化及相关性；再明确外源施用细胞分裂素对铁皮石斛的促生作用，最终验证我们的推测：芽孢杆菌NS实现对铁皮石斛促生作用的途径之一是促进其内源细胞分裂素的合成。

铁皮石斛为名贵中草药，因生境独特而生长缓慢。本课题组前期发现芽孢杆菌NS能显著促进铁皮石斛地上部生长。本项目以细胞分裂素合成关键酶基因IPT、CYP735A家族为主要研究对象，通过分析对照组、NS接种组、细胞分裂素合成抑制剂Lovastatin处理组、NS接种+Lovastatin处理组铁皮石斛基因表达量、细胞分裂素含量以及生长指标间的动态变化，阐释芽孢杆菌NS的促生机理。本研究基于RACE技术和三代转录组测序结果，从铁皮石斛中克隆获得5个基因，命名为DenCYP735A和DenIPT1-4。其中，DenCYP735A、DenIPT1-2在根中表达量最高， DenIPT 3-4在圆球茎中表达量最高。芽孢杆菌NS处理组中，DenIPT4在24 h时表达量最高，显著高于对照和外源细胞分裂素处理组；抑制剂Lovastatin对DenCYP735A和DenIPT1-3基因的表达没有影响，但可以显著抑制DenIPT4的表达；接种NS可以部分减轻抑制剂对DenIPT4表达的不良影响。所有处理组中细胞分裂素ip和tZR浓度在处理后7 d达到峰值，随后下降；芽孢杆菌NS处理组ip和tZR浓度最高，达到1.19 ng/g和1.53 ng/g，抑制剂处理组浓度最低，为0.91 ng/g和1.10 ng/g，NS与抑制剂处理组浓度介于对照和抑制剂处理组之间。处理30 d后，NS处理组植株株高与干重均显著高于对照组和其他处理组，达到6.1 cm和0.70 g/株；细抑制剂处理组株高和干重最低，为4.9 cm和0.55 g/株；抑制剂与NS共同处理组植株株高和干重为5.1 cm和0.59 g/株。综合分析，我们推测芽孢杆菌NS实现对铁皮石斛促生作用的途径之一就是促进铁皮石斛内源细胞分裂素的合成。