牡丹试管苗生根休眠相关机理研究

Difficulty in rooting in vitro and low transplanting survival rate is the main restricted factor of tissue culture of peony. Recently, the research of root induction in vitro peony had made some progress. But, low survival rate of plantlets has not been perfectly solved. Research shown that rooting dormancy and growth retardation of aerial part after rooting were the main cause of low living ratio of plantlets. Plant endogenous hormone and phenolics were the key substance of plant dormancy regulation. In our lab, the formal study shown that the phenols contents were very high in tissue culture seedling of peony. Metabolism of phenolics certainly will have greater effect on rooting dormancy of peony tissue culture seedling. Based on our prophase work, we will carry out the following work: Samples from the rooted plantlets and the unrooted plantlets will be prepared for transcriptome sequencing. Variation of endogenous hormone, phenolics and enzyme activity will be analyzed in the rooted plantlets and the unrooted plantlets. By bioinformatics analysis, we try to find out main factors and key genes effected on rooting dormancy of peony tissue culture seedling. The research will lay a foundation of further study on molecular mechanism of rooting dormancy of peony tissue culture seedling. Our study will also provide theoretical and technical support for establishing highly efficient system of the rapid propagation in vitro of peony.

牡丹试管苗生根困难，移栽成活率低一直是制约牡丹组培快繁的主要因子，中外学者开展了大量的研究工作。近年来，在试管苗生根诱导方面取得了一定的突破，但移栽成活率低至今尚未得到有效解决。研究发现，试管苗移栽成活率低的主要原因在于试管苗存在生根休眠，生根后地上部生长停滞，导致试管苗弱小，显著影响移栽成活率。植物内源激素和酚类物质是影响植物休眠的关键物质，项目组前期研究发现，牡丹试管苗中的酚类物质含量较高，酚类物质的代谢势必对试管苗生根休眠产生较大影响。本项目拟在前期工作基础上，对试管苗生根前后的样品进行转录组测序，分析生根前后内源激素、酚类物质、酶活性等生理生化指标的变化规律，通过生物信息学分析，寻找影响牡丹试管苗生根休眠的主要因子，挖掘试管苗生根休眠的关联基因，为试管苗生根休眠的分子机理研究奠定基础，为建立牡丹高效快繁体系提供理论和技术支撑。

牡丹试管苗移栽不成活一直是制约牡丹组培快繁的主要因子之一，而试管苗移栽不成活的主要原因在于试管苗生根后地上部发生休眠，导致试管苗不生长，并逐渐死亡。本项目通过对试管苗生根前后的地上部组织进行了转录组测序，并结合内源激素、酚酸类物质、碳水化合物含量及相关酶活性进行了分析，结果如下：（1）牡丹试管苗生根后，体内生长素（IAA）和脱落酸（ABA）含量显著上升，并随着生根时间的延长逐渐上升，而赤霉素(GA3)则相反，其含量随着试管苗生根时间的延长呈显著下降，导致了ABA/ GA3、ABA/ IAA、ABA/ ZA的比值显著升高；芍药苷、没食子酸、没食子酸甲酯、苯甲酸及对羟基苯甲酸含量在试管苗生根后均显著上升，引起了试管苗体内酚酸类物质的大量积累；可溶性糖、淀粉含量、PPO及PAL活性在试管苗生根后也都发生了显著的升高。（2）试管苗生根前后的转录组测序及生物信息学分析发现，试管苗生根前后的差异表达基因在植物激素信号转导、次生物质代谢及碳水化合物代谢等通路中富集显著，并挖掘出大量与试管苗休眠相关的差异表达基因，如CYP707A4、PYL/PYR、VH1、SLR1、GA3、ATHK1、ARF2、PPO、CCoAOMT、DHS、4CL、SDH、 CYP73A5、BGLU、PE、SUS、GBE、UXS1、BBX、AGP等。从中挑选出SLR1、ETR2等10个基因在试管苗生根前后进行了qRT-PCR表达验证，其结果与转录组测序表达模式相似，说明转录组数据准确可靠。并将BBX基因家族在模式植物矮牵牛中进行了鉴定和功能验证。综合以上结果得出，牡丹试管苗生根后，相关基因表达发生了变化，内源激素失衡和酚酸类物质的积累是导致试管苗地上部分生长停滞，进入休眠的主要影响因子。本项目研究结果揭示了牡丹试管苗生根后发生休眠的主要影响因子，并挖掘出大量与试管苗生根休眠相关的基因，初步揭示了试管生根休眠的相关机理，为深入研究试管苗休眠的分子机理和试管苗休眠解除奠定了理论基础，为完善牡丹组培快繁体系开辟了新途径。研究结果发表论文3篇，在审SCI论文1篇，圆满完成了项目制定的各项目标任务。