胆碱激酶基因（CfCK）mRNA砧穗间运输调控嫁接黄瓜低温耐受性的分子生理机制

Cucumber (Cucumis sativusL.) often overcomes low-temperature stress during winter season greenhouse production, leading to a decrease yield. Grafting with pumpkin stocks is often used for enhancing low-temperature tolerance. However, the molecular mechanism of the machinery regulation between scion and stock is still unclear. In previous comparison of Cucumber/ Cucumber with Cucumber/Pumpkin grafts, transcriptome analysis indicated 14 transcript candidates in grafted cucumber alleviating low-temperature stress. Among of them, CmCK performed significantly gene expression difference. CmCK mRNA mobility in Cucumber/Pumpkin graft was primarily identified by RT-PCR with specific primer. To unravel the fundamental regulation mechanism, based on previous works, in this study, we proposed: 1. Confirm CfCK mRNA mobility in hetero-grafted Cucumber/Pumpkin, and identify whether CmCK mRNA mobility is correlating to the physiological and biochemical traits changes of Cucumber/Pumpkin when suffering low temperature stress; 2. To identify whether CmCK1 enhance low-temperature tolerance of cucumber, over expressed CK will be constructed and transformed into cucumber plant. Correspondingly, the functional CmCK in transformats will be analyzed to verify the characteristics of cucumber low temperature tolerance. 3. To identify whether CmCK1 mRNA mobility may recover low temperature tolerance in grafted cucumber plants, systemic CmCK mRNA mobility will be confirmed in Cucumber/ OE CmCK transgenic cucumber grafts, and the physiological and lipidomic experiments will be analyzed as well. Ultimately, we will unravel a regulatory mechanical mechanism of systemic RNA mobility involved in scion and stock communication when suffering low-temperature stress. It also provides a theoretical and technical support for cucumber rootstock improvement.

黄瓜在反季节生产容易遭遇低温胁迫，影响其产量。黄瓜嫁接至南瓜砧木可显著提高低温耐受性，因此推测砧穗间可能存在信息物质交流，但分子调控机制尚不明确。前期本课题组转录组测序比较黄瓜/南瓜、黄瓜/黄瓜，筛选获得14个耐低温差异表达基因，其中CK基因差异表达最大，RT-PCR初步验证南瓜CK基因mRNA在黄瓜/南瓜嫁接体系中运输，为探明此分子生理调控机制，本研究拟首先验证嫁接黄瓜/南瓜耐低温生理生化性状，与南瓜CK基因mRNA在砧穗间的运输性是否相关；然后将南瓜CK基因转入黄瓜，从生理生化、基因表达和代谢水平检测其对耐低温能力的调控；其次，转南瓜CK基因的黄瓜作为砧木，与黄瓜嫁接，验证南瓜CK基因mRNA在黄瓜/转基因黄瓜嫁接体系中长距离运输机制，同时测定黄瓜接穗生理生化、基因表达和代谢物含量，分析耐低温能力。从而丰富嫁接黄瓜的耐低温分子生理机制，为多功能优良砧木改良和选育提供理论依据。

黄瓜在反季节生产容易遭遇低温胁迫，影响其产量。黄瓜嫁接至南瓜砧木可显著提高低温耐受性，因此推测砧穗间可能存在信息物质交流，但分子调控机制尚不明确。前期通过筛选不同耐低温的南瓜砧木与黄瓜嫁接，黄瓜/耐低温南瓜、黄瓜/不耐低温南瓜、与黄瓜/黄瓜、南瓜/南瓜比较，低温处理不同时间测定生理性状（叶绿素和类胡萝卜素含量、电解质渗透率等），结合CBFs-COR低温通路相关基因表达分析，明确了南瓜砧木的耐低温性状。分析嫁接黄瓜砧穗间运输性mRNA和代谢水平差异，结果表明，结果发掘到5个低温诱导的关键南瓜运输性mRNA主要参与了黄瓜氨基酸合成和甘油脂肪酸代谢途径，CfCK1基因是磷脂酰胆碱（PC）和磷脂酰乙醇胺（PE）合成通路上的起始反应中的关键酶，南瓜胆碱激酶CK1 运输通过调控不饱和脂肪酸的合成进而调控黄瓜低温耐受性。外源叶面喷施PC:PE组合溶液降低了黄瓜叶片的相对电解质渗透率和MDA含量，提高黄瓜低温耐受性。低温处理后，耐低温黑籽南瓜和‘强力士’CK1 mRNA响应低温胁迫可以由砧木向上移动至黄瓜接穗，表明南瓜CK1 mRNA能够响应低温而运输性增加，证明了CfCK1运输性及其功能与嫁接黄瓜耐低温性状密切相关。其次，南瓜CfCK1构建过表达载体，发根及稳定转化法将CfCK1基因转入黄瓜，分析低温后生理性状，结合CBF-COR、CsaCK1等基因表达，明确了CfCK1基因提高黄瓜低温耐受性的功能。结果表明CfCK1位于CBFs上游，正调控植物的低温耐受性以及CBF-COR 基因的表达，说明CfCK1通过调控 CBFs 依赖的低温响应途径参与植物耐低温信号途径。然后，将黄瓜与过表达CfCK1黄瓜嫁接，鉴定发现CfCK1基因mRNA从转基因黄瓜砧木至黄瓜接穗，还提高了嫁接WT/CfCK1ox的低温耐受性；最后，初步分析发现CfCK1 mRNA运输翻译后与黄瓜接穗CsaTIFY10a（JAZ家族成员）蛋白互作，提高CBF-COR基因表达，进而提高了嫁接黄瓜的低温耐受性，明确了CfCK1基因mRNA调控黄瓜接穗低温耐受性的分子生理机制。从而丰富嫁接黄瓜的耐低温分子生理机制，为多功能优良砧木改良和选育提供理论依据。