番茄SlMAPK7基因的亚细胞定位与组织表达特性

Subcellular localization and tissue expression pattern of SlMAPK7 gene in tomato.

分离番茄 SlMA PK7基因,利用实时荧光定量反转录聚合酶链反应技术分析在番茄不同组织和花发育不同时期 SlMA PK7的表达特性,发现该基因在雄蕊的表达量明显高于其他组织,在长度4.6～6.5 mm 的花蕾中表达量最高；通过构建黄色荧光蛋白融合表达载体,利用基因枪介导洋葱内表皮细胞的瞬时表达,发现SlMA PK7基因表达蛋白定位在细胞核和细胞膜中；为进一步研究 SlMA PK7的表达特性,分离克隆了SlMA PK7基因的启动子序列,利用 PLACE 和 PlantCARE 软件预测出其含有多种典型的 SlMA PK 启动子顺式作用元件；通过瞬时表达分析该启动子活性,经农杆菌介导转化拟南芥,GUS（β‐glucuronidase ,β‐葡萄糖苷酶）组织化学染色发现,在幼苗期 SlMA PK7主要集中在顶端分生组织和根尖分生组织中表达,在成年植株中则集中在花器官中表达.以上结果表明,SlMA PK7可能在细胞核和细胞膜中参与番茄多种信号的传导,并可能在花器官的发育过程中行使功能.

Summary Mitogen‐activated protein kinase （ MAPK ） cascades are universal signal transmission modules in eukaryotes . Recent increasing evidences have proved that MAPKs play pivotal roles in plant growth and development , as well as in response to biotic and abiotic stresses . Up to date , a number of MAPK genes have been isolated from different plants . However , the most extensively studied MAPKs are MAPK 3 , MAPK4 and MAPK6 in A rabidopsis and rice . The function of other MAPK family members is not clear yet . Tomato , one of the most important vegetables , is considered one of the model plants for productive development . To the best of our knowledge , the research on tomato MAPK family genes is very limited . Therefore , this study aimed to characterize the temporal and spatial expression profiles of tomato SlMA PK7 , analyze the cis‐elements in its promoter sequences , and to confirm the subcellular localization of SlMA PK7 protein . The expression profiles of SlMA PK7 in the roots , stems , leaves , calyxes , petals , stamens , pistils , and fruits from flowering tomato plants were characterized by real‐time fluorescent quantitative reverse transcription polymerase chain reaction （ qRT‐PCR） , as well as the flower buds ranging from 2 mm to 8 .5 mm in length , representing in different floral development periods . The 5′‐upstream cis‐acting sequences of tomato SlMA PK7 gene were identified by PCR method according to the tomato genome sequence data . PLACE and PlantCARE were used to analyze the cis‐element of promoter . A plant expression vector with yellow fluorescent protein （ YFP） was constructed to confirm the subcellular localization of SlMA PK7 protein . Meanwhile , another plant expression vector with green fluorescent protein and β‐glucuronidase （ GUS） report gene was constructed to study the activity of promoter . The promoter expression vector was transferred into A rabidopsis by A grobacterium tume f aciens to analyze the promoter profiles .The expression analysis using qRT‐PCR showed that the relative gene expression level of SlMA PK7 in stamen was far higher than other tissues . At different floral development stages , the highest gene expression level of SlMA PK7 was found in the flower bud with the length of 4 .6 6 .5 mm . Transient expression analysis indicated that YFP‐SlMA PK7 fusion protein was localized in the cell nucleus and the membrane of onion epidermal cells . The 1 823 bp region flanking （ from - 29 bp to - 1 851 bp） sequences of the 5′‐upstream in the tomato SlMA PK7 gene were isolated and sequenced . Structure analysis of the promoter using PLACE and PlantCARE revealed that the promoter sequences contained basic cis‐elements , such as TATA‐box and CAAT‐box and many abiotic stress responsive elements . It was worth noting that the sequences also contained several pollen development‐related cis‐elements . Transient expression analysis indicated that the cloned sequences were active promoters . The expression analysis of SlMA PK7 promoter in A rabidopsis by β‐glucuronidase （ GUS ） staining showed that the SlMA PK7 was mainly expressed in apical meristem of stem and root during the seedling period , while it was expressed in stigma and receptacle in the adult plant .In conclusion , SlMA PK7 takes part probably in the signal transduction pathway in the cell nucleus and membrane , which may play an important role in the flower development in tomato . Our study provides some helpful informations for further elucidating the precise roles of MAPK 7 in tomato growth and development .