SSN基因家族调控棉花抗黄萎病反应的功能解析

Cytochrome P450 enzymes are a class of proteins participated in anabolic substances in biological synthesis, detoxification pathway and widely presented in microorganisms, plants and animals. Some of them have been characterizated playing important roles in plant-micro organisms interreaction, like CYP74A in JA synthesis pathway. .A EST sequence was isolated with different expression pattern in 'Hai 7124' (cv of Gossypium babardense L. resistant to Verticillium dahliae) and 'YZ-1' (cv of G. hirsutum L. susceptible to V. dahliae) and could be induced highly by JA,. Totally, three full-length cDNA sequences were obtained by RACE from 'YZ-1' which shared high homologous in ORF and maybe belong to a new subfamily of P450 in plant. RNAi vector with conserved sequences among these genes was constructed and transformed into 'YZ-1' through Agrobacterium-mediated protocol. Lesion necrosis was found in most of the transgenic seedlings（ssn） with decreased expression level of all genes without any pathogen inoculation and deduced to death, While no obvious phenotype was observed in transgenic seedlings for specific gene silencing. So these genes were namely SSN (Silencing-induced Stem Necrosis,SSN). The relevant phytohormone determining results showed that JA was found increased obviously. Genes involved in JA synthesis from linolenic acid like LOX2, AOS, AOC or JA signal transduction, like JAZ1, JAZ10, were identified increased noticeably in ssn plants. Meanwhile, Ghssn lines showed more sensitive to JA at 10 uM and high resistace to V. dahliae than wild type..These preliminary results indicate that in cotton, genes of SSN might share partly redundant activities and play important negative roles in the resistance response to Verticillium wilt through regulating JA synthesis or activity and signal transduction. The function and mechanism of these genes involved in JA synthesis and resistance response to V. dahliae will bring light to disease resistance breeding in cotton.

从棉花中分离获得三条在氨基酸水平具有95%相似的基因，分析显示它们属于一个新的细胞色素P450类亚家族。它们在抗病的海岛棉和感病的陆地棉接种黄萎病菌后具有相反的表达模式，但均受茉莉酸高效诱导。三个基因均被抑制的转基因棉花茎部出现类病斑，因此该基因家族被命名为SSN(Silencing-induced Stem Necrosis, SSN)。但单一基因成员沉默时未有类似症状，表明成员间存在功能冗余。分析发现参与JA合成的基因在转基因棉花ssn内表达明显增强，水杨酸信号路径因子表达降低。ssn中JA含量明显提高并使活性氧积累可能是形成类病斑的基础。GUS染色结果显示SSN在子叶和根中高效表达，亚麻酸能加速ssn子叶上类病斑的形成。依据现有结果推测SSN参与脂类代谢，可能负调控JA合成或其活性形式转换并以此影响棉花对黄萎病的抗性。对其作用机制的阐明将为棉花抗黄萎病遗传育种提供重要理论依据。

黄萎病是影响我国棉花生产的主要病害。我们利用SSH文库筛选和全基因组表达谱分析研究海岛棉品系‘7124’ 抗黄萎病机制，并获得了一批抗病候选基因。为了鉴定抗病关键基因，我们开发了一种利用生物信息学、病毒介导的基因沉默和表达谱分析相结合快速鉴定关键基因的新策略。将蛋白组学与病毒介导的基因沉默相结合证明了脂类代谢参与了棉花免疫调控，SSI2基因通过负调控JA信号路径影响棉花抗黄萎病反应。通过筛选获得了一个在海岛棉和陆地棉接种黄萎病菌后具有差异表达的EST序列。利用RACE技术从陆地棉品系‘YZ1’ 中克隆到三个可能编码细胞色素P450酶的基因。它们在氨基酸水平的一致性为94%，与杨树PtCYP82D2和拟南芥AtCYP82C2的相似性分别为55%和48%，因此它们是CYP82家族的新成员。.构建了该基因家族保守区段和三个基因3’末端非编码区特异RNAi载体以及基因的超量表达载体，通过农杆菌介导法转化‘YZ1’，并获得了转基因再生植株。研究发现当抑制基因家族的表达时转基因幼苗ssn会出现不同程度的类病斑坏死，因此将该基因家族命名为SSN（Silencing-induced Stem Necrosis, SSN）。研究发现特异抑制单一基因表达的转基因植株均未出现类病斑，将其两两进行杂交共抑制两个基因表达时会再现类病斑，因此三个成员之间存在功能冗余。接种实验表明ssn对黄萎病的抗性明显增强，而SSN超量表达转基因系则相反。.全基因组表达分析表明ssn相比野生型显著激活了茉莉酸合成及其信号路径。由于茉莉酸是由亚麻酸为底物的氧脂类代谢物，我们对转基因株系中脂肪酸的含量进行了测定。检测结果发现在SSN超量表达植株中C18:2亚油酸的含量出现显著下降，但C18:2羟基化脂肪酸9/13-HOD，C18:3羟基化脂肪酸9/13-HOT和C18:2衍生的酮基脂肪酸9/13-KOD的含量都极显著升高。因此SSN基因可能通过调控C18:2脂肪酸代谢影响茉莉酸合成，SSN和LOXs之间存在共同底物的竞争关系。ssn转基因植物中LOX代谢通路的激活可能是形成类病斑的直接原因，但SSN的直接底物与生理功能仍有待研究。嫁接实验表明ssn中存在能引起棉花系统性抗病反应和细胞坏死的信号分子，但信号分子及其传递机制还不清楚。