枳低温诱导转录因子PtrbHLH的功能鉴定及其抗寒机制解析

Our previoius work showed that PtrbHLH isolated from Poncirus trifoliata was a homologue of ICE1-like gene and could be induced by low temperatrue. PtrbHLH mRNA abundance was higher in the cold-tolerant genotype than in the sensitive one. Based on these data, in the present project we will first identify the fundamental characteristics of PtrbHLH as a transcription factor　and its posttranslational modification under low temperature. An overexpression vector will be constructed to transform Arabidopsis thaliana and lemon, while a RNAi vector will be constructed to transform P. trifoliata. Cold tolerance of the transgenic plants will be evaluated in order to identify the function of PtrbHLH in cold tolerance. Lemon transgenic and non-transformed plants treatd with or without low temperature will be used for microarry hybridization. The chip data will be processed to screen differentially expressed genes, which are then subjected to functional annotation and classification. Transcript levels of the genes will be analyzed using QRT-PCR to establish an expression profiling. In addition, physiological measurement with regard to some metabolic pathways involving the differentially expressed genes will be carried out. These work is aimed to dissect the molecular and physiological mechanisms underlying the function of PtrbHLH. Promoters of several differentially expressed genes will be isolated and bioinformatically assayed to explore the stress-responsive cis-acting elements. Furthermore, interaction between PtrbHLH and the promoters and specific binding of the cis-acting elements will be investigated so as to unravel the target genes of PtrbHLH. The current project is an extension of our earlier work. It will, on the one hand, provide genes of vaulabe significance for citrus genetic engineering related to stress, and, on the other hand, will lay theoretical groundwork for deciphering the mechanisms of action on PtrbHLH, tapping its target genes, and setting up a cold-responsive network using PtrbHLH as a node. Therefore, this project holds both theoretical and practical potentials.

在前期研究中，我们克隆得到枳ICE1-like基因PtrbHLH，发现它受低温诱导且在抗寒材料中表达水平高于不抗寒材料。在此基础上，本项目拟首先分析PtrbHLH作为转录因子的基本特征和低温下的翻译后修饰。构建超表达载体转化拟南芥和柠檬，构建RNAi载体转化枳，鉴定PtrbHLH抗寒功能。对低温处理前后柠檬转基因和非转基因植株进行芯片杂交，分析杂交数据，筛选差异表达基因，建立其表达谱；对差异表达基因涉及的代谢途径进行生理测定。分离部分差异表达基因的启动子，利用生物信息学分析顺式作用元件，研究PtrbHLH与启动子的互作及它与顺式作用元件的特异结合，以确定PtrbHLH的候选靶基因。本项目是已有研究的深入和延续，将为柑橘抗逆工程提供重要的基因资源，还有利于揭示PtrbHLH基因的作用机制、挖掘其靶基因，为解析以它为节点的低温胁迫应答网络提供理论依据，因而具有重要的理论意义和潜在应用价值。

本项目克隆了柑橘抗寒资源枳（Poncirus trifoliata）PtrbHLH和PtrICE1两个基因，它们均受低温、脱水及盐诱导，定位于细胞核，并具有转录激活活性。通过超表达和RNAi分析阐明了PtrbHLH和PtrICE1的抗寒功能。芯片杂交分析表明，与野生型相比，正常条件和低温处理后PtrbHLH转基因柠檬植株分别有108个和716个基因上调，其中参与ROS清除的POD和CAT基因富集。超表达转基因植株的POD和CAT活性高于野生型，而ROS含量则低于野生型。采用酵母单杂交和双荧光素酶系统分析表明，PtrbHLH可以与POD和CAT启动子上的E-box元件结合。揭示了PtrbHLH的抗寒机制是调控POD和CAT介导的ROS清除。利用酵母双杂交分离了与PtrICE1互作的17个蛋白，其中一个是多胺合成酶ADC。利用BiFC验证了PtrICE1与ADC的互作，发现超表达植株多胺含量增加。研究提示了PtrICE1抗寒的一个新机制，是与ADC互作促进多胺合成。利用公布的甜橙基因组序列，全基因组发掘了甜橙56个bHLH基因。分析了其中24个基因在不同胁迫（干旱、低温、盐）和 ABA处理后的表达情况，并对受多个胁迫诱导的bHLH18进行了抗寒功能分析。项目的开展为揭示bHLH类转录因子的功能奠定了基础，为利用基因工程开展抗寒分子育种提供了重要的基因资源。共发表SCI论文6篇，获批发明专利3项，获得个人奖项和荣誉称号4项，培养研究生7人，其中博士生4人，硕士生3人。