分蘖洋葱PAL和ALL基因调控主效化感物质促黄瓜生长的机理研究

Intercropping with Chinese onion can increase cucumber yields due to the allelochemicals released by Chinese onion, however, its molecular regulation mechanism needs to be further studied. Based on our previous studies of protein expression analysis of Chinese onion with different allelopathic potentials under monocropping or with Chinese onion-cucumber intercorpping systems, the objectives of this proposal, 1）To select significantly variable protein spots of Phenylalanine ammonialyase(PAL) and Alliinase(ALL) as targets to clone the full-length genes of PAL and ALL by RACE technique; 2) To analyze and compare their expressions by qRT-PCR, enzymes activities and the contents of main allelochemicals (cinnamic acid and allicin) secreted in Chinese onion roots by HPLC under monocropping and intercropping systems as well as transforming PAL-RNAi and ALL-RNAi, respectively, into Chinese onion by using RNAi techniques to elucidate the functions and contributions of PAL and ALL genes and their enzymatic production on promotion of cucumber growth; 3) To investigate cucumber growth and its inducible gene groups to demonstrate response signal paths by RNA-seq through adding exogenous cinnamic acid and allicin to ultimately reveal the mechanism of Chinese onion PAL and ALL genes regulating main allelochemicals promoting the growth of cucumber. Overall, the results of this study would clarify the molecular mechanism for increasing cucumber yields under intercropping system and also shed light on the possibility for developing Chinese onion allelochemicals into plant growth regulators.

研究表明套作分蘖洋葱可提高黄瓜产量，这与分蘖洋葱释放的化感物质有关，其分子调控机制尚有待研究。在完成不同化感潜力分蘖洋葱单作、与黄瓜套作蛋白表达分析基础上，本项目拟：1）选择与化感相关且差异较大的蛋白点苯丙氨酸解氨酶（PAL）和蒜氨酸酶（ALL），RACE克隆分蘖洋葱PAL和ALL基因全长；2）单、套作栽培模式下，qRT-PCR分析不同化感潜力分蘖洋葱PAL和ALL基因表达、酶活测定、HPLC检测根系分泌物主效化感物质（肉桂酸和蒜素）含量，结合RNAi技术沉默PAL和ALL基因，阐明PAL和ALL基因及酶促产物在促黄瓜生长中的作用和贡献；3）外源施加肉桂酸和蒜素，调查黄瓜长势，RNA-seq筛选诱导黄瓜表达的基因群，获得应答信号通路，进一步阐明PAL和ALL基因调控主效化感物质促黄瓜生长的机理。该研究结果可为揭示套作提高产量的机制，以及将分蘖洋葱化感物质开发成为植物生长调节剂提供理论参考。

套作分蘖洋葱可提高黄瓜产量，本项目基于分蘖洋葱单作、与黄瓜套作蛋白表达分析，选择与化感作用相关且差异较大的蛋白点苯丙氨酸解氨酶（PAL）和蒜氨酸酶（ALL）为切入点，拟阐明分蘖洋葱PAL和ALL基因调控主效化感物质促黄瓜生长的分子机制。研究结果表明：①分蘖洋葱PAL cDNA全长2127bp（GenBank：MG944239）编码708个氨基酸，分子量77KD，理论等电点6.15，氨基酸序列190-206区域GTITASGDLvPLSyiaA为PAL催化活性位点。ALL基因cDNA全长1440bp（GenBank：MG948559），编码479个氨基酸，分子量54.793KD，理论等电点8.48，氨基酸序列73-84区域CeCntCytGAdC为ALL EGF-like domain。②与单作相比，套作栽培模式下分蘖洋葱PAL、ALL不同程度上调表达；PAL、ALL酶活增强；根际土壤肉桂酸积累，但未检测到大蒜素；已建立分蘖洋葱遗传转化体系；异源转化拟南芥，超表达分蘖洋葱PAL、ALLT3代拟南芥根系浸提液低浓度（50ml•株-1）促进、高浓度（10 ml•株-1）抑制黄瓜幼苗生长；外源施加25μg•ml-1肉桂酸和50μg•ml-1大蒜素显著促进黄瓜胚芽长、胚根鲜重和胚芽鲜重。综上，明确了分蘖洋葱PAL和ALL基因通过调控主效化感物质肉桂酸、大蒜素的适当积累从而促进黄瓜生长。③与单作相比，套作栽培模式下，RNA-seq筛选诱导黄瓜表达的差异基因群， KEGG作Pathway富集分析表明主要差异表达基因集中在苯丙烷、类黄酮、芪类、双苯庚烷、姜辣素等次生代谢产物合成，氨基糖和核苷酸糖代谢，苯丙氨酸、精氨酸、脯氨酸、β-丙氨酸代谢，碱基切除修复等途径。该研究结果丰富了套作分蘖洋葱提高黄瓜产量的分子机制，以及将分蘖洋葱化感物质开发成为植物生长调节剂提供理论参考。