番茄SlMAPK12基因的分离及表达特征分析

Isolation of mitogen-activated protein kinase SlMAPK12 gene and characterization of its expression pattern in Solanum lycopersicum

利用PCR克隆技术,在番茄Micro-Tom的cDNA中分离出一个促分裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK),命名为SlMAPK12(GenBank登录号为JF795447)。该基因编码621个氨基酸,含有促分裂原活化蛋白激酶11个区域,磷酸化位点为TDY(苏氨酸-天冬氨酸-酪氨酸,属于D组)。氨基酸序列比对发现,它与拟南芥、杨树、蓖麻等D组MAPK具有高度一致性。利用RT-PCR及qRT-PCR技术分析SlMAPK12的时空表达特性,发现SlMAPK12在Micro-Tom植株的多个器官中均有表达,在雄蕊中的表达量最高。对Micro-Tom幼苗进行(38±1)℃高温处理,发现SlMAPK12在雄蕊中的相对表达量增高,于12h时点达到最高,而后下降。推测SlMAPK12可能参与番茄花粉发育过程中的高温胁迫反应。

Summary In nature, plants are often exposed to ever changing external stimuli during their growth and development, including pathogen infection, drought, salinity and various other biotic and abiotie stresses. These extracellular stimuli are perceived by the plant through a highly elaborate signaling network, which leads to an adaptive stress response at cellular and organismal levels. A specific class of serine/threonine protein kinases, mitogen-activated protein kinases （MAPKs） play an important role in the transduction of various extra and intracellular signals and are conserved throughout eukaryotes. Recently, increasing evidences indicate that MAPK （mitoge^activated protein kinase） has very important effects on plant growth and development, as well as stress resistance. As we know, current research on tomato （Solanurn lycopersicurn L. ） MAPK gene is relatively limited. While the recently completed Oenome Sequencing Project for tomato genome provides an opportunity to deduce the tomato MAPK genes and infer its evolutionary history. So more detailed biochemical and functional characterization is likely to be carried out to understand the function of tomato MAPKs. The cDNA sequence of novel SlMAPK12 （GenBank No. JF795447） was amplified by PCR using primers designed based on the predicted sequences of tomato unigene. Then many bioinformatics tools were used to analyze and predict SIMAPK12, such as ProtParam. To analyze the expression pattern of SIMAPK12 in various tissues and during flower developmental stages, the leaves, stems, petals, stamens, pistils, fruits and 5 stages of floral development from flowering tomato plants were characterized by RT-PCR and furthermore proved by qRT-PCR. For high temperature treatment, the stamen were collected at 0 h, 12 h, 24 h, 48 h and 72 h after （38 ±1） ℃ heat stress treatment. Then the qRT-PCR experiments were employed to characterize gene expression profiles. S1MAPK12 encoding a protein of 621 amino acids contained 11 subdomains of the MAPK. Subdomain contained the threonine and tyrosine residues, named T-D-Y （ThFAsp-Tyr） motif, that was phosphorylated for the activation of S1MAPK12. S1MAPK12 protein sequences were found to contain four types of special domains： active site, ATP binding site, substrate binding site, and activation loop （A-loop） and KIM docking site. Amino acid sequence alignment revealed that S1MAPK12 shared high identity with group-D MAPKs from Arabidopsis, Populus, and Ricinus, which indicated that the group-D MAPKs was conserved in different plant species. The temporal and spatial expression patterns of SlMAPK12 analysed by RT-PCR and qRT-PCR in different tomato tissue indicated that the relative gene expression level of SlMARK12 in stamen was much higher than other organs. Also, the levels of SlMAPK12 expressed in different developmental stages of tomato flower were almost the same. These results indicated that SlMAPK12 might have very important effects on flower development. The expression pattern of SlMAPK12 gene under high-temperature （38 ± 1）℃was analysed by qRT-PCR. We found that the transcripts of SlMAPK12 in stamen accumulated markedly when the tomato seedlings were subjected to heat stress. These results above indicated that SlMAPK12 probably involved in the adaptive responses to heat stress during tomato pollen development. The preferential expression of SIMAPK12 gene provides a new avenue for functional analyses in tomato. The major challenge for future research is to focus on identifying more MAPKs and understanding the specific role of MAPK genes during tomato growth and development.