水杨酸诱导黄瓜PCD的鉴定及相关基因的表达分析

Identification of PCD Induced by Salicylic Acid and Expression Analysis of Genes Related with the PCD in Cucumber

为揭示叶绿体在水杨酸(SA)诱导黄瓜PCD过程中的作用,选择黄瓜幼苗四叶期时,叶片滴加10 mmol/L SA,在滴加SA的位置取材,首先进行ROS原位检测、Trypan blue染色、PI和FDA染色、TUNEL检测,验证SA可诱导黄瓜PCD过程;继而根据高通量测序结果,从1 759个高通量测序得到的差异表达基因中,筛选出15个注释含chloroplast的基因,RT-PCR验证这些基因在SA诱导的黄瓜叶片PCD中有表达,初步认定这些基因参与了SA诱导的黄瓜PCD过程。对筛选得到的基因进行qRT-PCR表达分析,这15个基因在SA诱导的黄瓜PCD中起正调控或负调控的作用,上调表达的5个基因分别是:类囊体加工肽酶1基因、6-磷酸葡萄糖酸脱氢酶基因、果糖1,6-二磷酸醛缩酶3基因、α-葡聚糖-水二激酶1基因和丙二烯氧化物环化酶4基因;下调表达的10个基因分别是:甜菜碱脱氢酶1基因、钙敏感受体基因、脱镁叶绿酸a单加氧酶基因、叶绿素b还原酶1基因、σ因子结合蛋白1基因、NADPH-原叶绿酸酯还原酶基因、2-琥珀苯甲酸-辅酶A连接酶基因、ATP依赖的锌金属蛋白酶基因、烟酰胺腺嘌呤二核苷酸转运子基因和胱硫醚β合成酶(CBS)蛋白基因。为进一步揭示叶绿体在SA诱导黄瓜PCD中的作用奠定了基础。

To reveal the role of chloroplast in the PCD of cucumber induced by salicylic acid(SA),10mmol/L salicylic acid was added to the leaves of cucumber seedlings at the four-leaf period,and the samples were collected at the location where SA was added.ROS in situ detection,Trypan blue staining,PI and FDA staining,and TUNEL detection were performed.These results showed that SA could induce PCD process in cucumber.According to the results of high-throughput sequencing,15genes annotated with chloroplast were screened out among1759differentially expressed genes.These genes were expressed in cucumber leaves treated with SA was confirmed by RT-PCR.It was preliminarily identified that these genes were involved in the PCD process induced by SA in cucumber.According to the expression of qRT-PCR,the15genes played a role of positive regulation or negative regulation in PCD induced SA in cucumber,upon regulating characteristics five kinds of genes were raised:Probable thylakoidal processing peptidase1gene,6-phosphogluconate dehydrogenase gene,Probable fructose-bisphosphate aldolase3gene,Alpha-glucan water dikinase1gene and Allene oxide cyclase4gene,down-regulated expression of10genes were:Betaine aldehyde dehydrogenase1gene,Calcium sensing receptor gene,Pheophorbide a oxygenase gene,Probable chlorophyll(ide)b reductase1gene,Sigma factor binding protein1gene,NADPH-protochlorophyllide oxidoreductase gene,2-succinylbenzoate-CoA ligase gene,ATP-dependent zinc metalloprotease FtsH1gene,Nicotinamide adenine dinucleotide transporter1gene and CBS domain-containing protein1gene.It lays a foundation for further revealing the role of chloroplast in cucumber PCD induced by SA.