甘蓝型油菜丝裂原活化蛋白激酶7基因(BnMAPK7)上游调控因子的鉴定

Identification of upstream regulators for mitogen-activated protein kinase 7 gene(BnMAPK7)in rapeseed(Brassica napus L.)

丝裂原活化蛋白激酶(Mitogen-activated protein kinases,MAPKs)级联途径在植物的生长发育、分裂分化、细胞凋亡以及抗逆等多种生命过程中发挥着极其重要的作用。本研究从甘蓝型油菜中分离克隆了1个C族BnMAPK7基因的启动子,序列长度为1612 bp,命名为ProBnMAPK7。启动子分析工具PlantCARE预测结果表明,ProBnMAPK7含有大量响应光照、激素、防御和损伤等相关顺式作用元件,包括ACE、MRE、ABRE、TGACG-motif和TC-rich repeats等。同时,我们对拟南芥及甘蓝型油菜MAPK7基因的表达模式进行分析发现,MAPK7对生长发育以及生物和非生物胁迫应答过程具有重要调控意义。将ProBnMAPK7逐步分段连接至pCambia1305.1-GUS表达载体筛选启动子的核心区段,GUS组织化学染色分析显示该启动子的核心区段定位于-467~-239 bp(ProBnMAPK7-rPE)。将核心区段3拷贝串联重复后整合至Y1H gold基因组,并进行AbA背景测试,结果显示AbA浓度为500 ng mL^(-1)时,ProBnMAPK7-rPE×3在酵母细胞中的本底表达被完全抑制。利用酵母单杂交技术对BnMAPK7的上游调控因子进行文库筛选,获得3个候选因子BnNAD1B(NADH dehydrogenase 1B)、BnERD6(early response to dehydration 6)和BnPIG3(quinone oxidoreductase PIG3-like)。表明BnNAD1B、BnERD6及BnPIG3蛋白可能通过结合ProBnMAPK7-rPE区段调控BnMAPK7的转录,使得BnMAPK7参与光合作用以及逆境胁迫应答等生物学过程,为进一步阐明甘蓝型油菜BnMAPK7基因的功能奠定了基础,也为MAPKs级联的研究提供了新的思路。

Mitogen-activated protein kinases(MAPKs)cascade plays a key role in plant growth and development,division,dif-ferentiation,apoptosis,and stress resistance.In this study,a 1612 bp promoter of C group BnMAPK7 gene,designated ProBnMAPK7,was cloned from Brassica napus.Promoter structure prediction by PlantCARE revealed that ProBnMAPK7 con-tained a lot of ACE,MRE,ABRE,TGACG-motif,and TC-rich repeats cis-acting elements,which involved in light,hormones,defense,and wounding responsiveness.At the same time,we analyzed the expression patterns of MAPK7 genes in Arabidopsis and B.napus,and found that MAPK7 played an important regulatory role in growth and development process and responding to biotic and abiotic stresses.Different lengths of ProBnMAPK7 were gradually ligated to the pCambia1305.1-GUS expression vector to identify the core fragment.GUS histochemical staining analysis showed that the core fragment of ProBnMAPK7 was lo-cated in the-467 to-239 bp(ProBnMAPK7-rPE)region.Three copies of the promoter core fragment were integrated into the genome of Y1H gold to test the AbA background.The data demonstrated that the expression background of ProBnMAPK7-rPE in yeast cells was completely inhibited by 500 ng mL^(-1)AbA.Using yeast one-hybrid,we screened the library of the upstream regu-latory factors of BnMAPK7,and obtained three candidates,including BnNAD1B(NADH dehydrogenase 1B),BnERD6(early response to dehydration 6),and BnPIG3(quinone oxidoreductase PIG3-like).Taken together,these results suggested that BnNAD1B,BnERD6,and BnPIG3 might bind to ProBnMAPK7-rPE to regulate the transcription of BnMAPK7,to further involve in photosynthesis and responding to stresses.This study lays a foundation for further elucidating the function of BnMAPK7 in rapeseed,and provides a new perspective for research into MAPKs cascade.