滇杨枝条倒插生根过程中生长素极性运输机制研究

Populus yunnanensis is endemism of southwest China. It is also an unwonted poplar specie in China or even in the world, which distributes in low latitude but high altitude area. It was found in the previous research that the adventitious roots formed at the original morphological apex and shoots sprouted at the original morphological base of inverted cuttings, and the inverted cuttings normally developed into intact plants. Lots of researches suggested that polar auxin transport acts as a trigger for root forming in upright cuttings. However, the mechanism of polar auxin transport in inverted cuttings is not fully understood. In this studies, one-year stems of P. yunnanensis will be used as cuttings, HPLC method will be adopted to detect the gradient differences of endogenous hormones in lateral buds and bark tissues (including cortex and vascular tissues) of inverted cuttings during rooting process; Real-time PCR, ELISA and immunohistochemistry (IHC) methods will be employed to analyze the gene expression of polar auxin transporting carriers, contents change of polar auxin transporting carriers and their polar distributing location on cellular membrane in bark tissues of inverted cuttings during rooting process. Simultaneously, the inverted cuttings development will be observed. Therefore, this project will be a systematic research on the mechanism of polar auxin transport during rooting process in inverted cuttings. The research results should be helpful to elucidate the mechanism of polar auxin transport during rooting process in inverted cuttings, and should lay the theoretical foundation for rooting regulation in cutting propagation.

滇杨是我国西南地区特有的乡土树种之一，也是我国乃至世界少有的分布于低纬度高海拔地区的杨属树种。项目组前期研究发现，滇杨枝条插穗倒插后能够在原形态学上端形成不定根而在原形态学下端长枝，并能够发育为成熟的植株。大量研究结果表明，生长素的极性运输在正插插穗根系发生中发挥着开关式的调控作用。但插穗倒插后的生长素极性运输机制尚不清楚。本研究以滇杨1年生枝条为插穗，分别采用高效液相色谱、实时定量PCR、酶联免疫吸附、免疫组化等技术，系统研究倒插插穗生根过程中侧芽和插穗树皮（含皮层、韧皮部和形成层）内源激素含量梯度差异、插穗树皮内(含皮层、韧皮部和形成层)载体蛋白在生长素极性运输中的功能，结合对倒插苗木生长状况的观测，从激素水平、分子水平和苗木生长3个层次系统开展滇杨倒插生根过程中生长素极性运输机制研究。研究结果有助于阐明倒插插穗生根过程中生长素极性运输机理，也为生产实践中调控插穗生根提供理论依据。

生长素的极性运输在插穗根系发生中发挥着开关式的调控作用。本项目对滇杨倒插生根过程中生长素极性运输机制开展了系统研究。（1）通过对正、倒插苗木的生长指标观测，揭示了插穗极性的改变对苗木生长发育具有明显影响，但插穗侧芽的顶端优势位置不会随之而发生改变，依然是处于插穗形态学上端的侧芽萌发后形成苗木主干。（2）对正、倒插苗木3种氧化酶活性的测定，揭示了插穗极性的改变对扦插苗的生理生化特性具有一定影响，使倒插苗的生长处于逆境条件之中。（3）通过测定正、倒置插穗不同部位侧芽及皮部IAA、GA3、ABA和ZT等内源激素的含量，揭示出生长素在倒置插穗中依然保持了原有的极性运输方向。（4）采用MSAP技术对正倒插苗木年生长周期内DNA甲基化类型及水平进行了分析，表明正、倒插苗木之间的DNA甲基化类型及水平存在明显差异。（5）对正、倒插苗侧枝萌生处上、下插穗皮层及侧枝基部、顶部皮层的RNA-seq分析表明，共8076个DEGs能被KEGG数据库注释，分布于122个通路中，主要富集于植物与病原菌的相互作用、植物激素的信号传导和次生代谢产物的生物合成等通路，且正、倒扦插苗插穗皮层的转录调控差异较大。对DEGs中与植物激素信号传导通路的相关基因分析表明，倒插苗的插穗皮层内源激素信号传导过程具有明显的通路变化，许多相关基因的表达量差异达显著水平。（6）应用qPCR技术对杨树44个与生长素极性运输相关基因在正倒置滇杨插穗水培生根过程中不同部位表达情况进行分析，共检测到41个基因在滇杨插穗皮层中表达，但33个基因的表达在正、倒插穗间不存在差异，PIN1、PIN15，ABCB1、ABCB9、ABCB10、ABCB19、AUX5、AUX6等8个基因于正、倒插穗的不同部位间具有差异表达。（7）对筛选出的PIN1、PIN15、AUX3、ABCB19等4个生长素极性运输载体蛋白基因进行蛋白表达量的ELISA分析，结果显示，正、倒置插穗在水培生根之初，4个基因的蛋白表达量最高，且正、倒置插穗部位间差异较大，可能参与调控了倒插插穗生根过程中生长素的极性运输。