Expressional And Functional Analysis Of A Mitogen-Activated Protein Kinase Gene SlMAPK7 In Solanum Lycopersicum

Mitogen-activated protein kinase (MAPK) play important roles in plant growth and development, as well as multiple signal pathways in response to stress. The function of MAPK signaling pathways during the process of tomato reproductive development is unclear. On the basis of the identification of SIMAPK7 gene, this papar selected S. lycopersicum L. cv. Micro-Tom as the research object. The subcellular localization and organization expression features of SIMAPK7 were characterized using Transient expression analysis, real-time RT-PCR and GUS histochemical staining method. The plant RNAi expression and over expression vector regulated by the constitutive promoter CaMV35S and the plant RNAi expression vector regulated by pollen specific promoter LAT52 of SIMAPK7 was constructed. Through the genetic transformation of’Micro-Tom’, we have got the transgenic tomato plant with lack -expression and over-expression of SIMAPK 7 gene, cytology and molecular biology technology were used to study its biological function in the process of pollen development to analyze the biology function of SIMAPK7 in pollen development process, and provide the foundation of research for deeper study of the mechanism of plant pollen development. The main results were as follows:(1) The expression profiles of SIMAPK7 were analyzed by qRT-PCR. The expression analysis of different tissues using qRT-PCR showed that SlMAPK7 gene was expressed in several tissues, but the relative gene expression level of SIMAPK7 in stamen was the highest than other tissues, and in stem, leaf and fruit was extremely low; SlMAPK7 gene was expressed at every floral development stages, the highest gene expression level of SIMAPK7 was found in the flower bud with the length of about 4.5-6.5 mm. By constructing SlMAPK7 and yellow fluorescent protein (YFP) fusion expression vector, the results of gene gun mediated transient expression showed that YFP-SlMAPK7 fusion protein was localized in the cell nucleus and the membrane.(2) The 1823 bp region flanking (from-29 to-1851) sequences of the 5’-upstream in the tomato SlMAPK7 gene were identified with PCR method. Structure analysis of the promoter with 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’s worth noting that the sequences also contained several pollen development-related cis-elements. Transient expression analysis indicated that cloned sequences were active promoters. The promoter expression vector was transferred into Arabidopsis by Agrobacterium tumefaciens. The expression analysis of SIMAPK7 promoter in Arabidopsis with GUS staining showed that SIMAPK7 was mainly expressed in apical meristem of stem and root in seedling period, while expressed in stigma and receptacle in the adult plant.(3) Expression vector were transferred into ’Micro-Tom’ by Agrobacterium tumefaciens to get the transgenic tomato plant with pLAT52::SlMAPK7-RNAi, p35S::SlMAPK7-RNAi and p35S::SlMAPK7 vector, the reproductive development is abnormal of transgenic tomato plants with nomal growing conditions. The pollen vitality of transgenic tomato with p35S::SlMAPK7-RNAi and pLAT52::SlMAPK7-RNAi vector were only 10% and 6.73% respectively, compared with CK declined obviously, moreover, pollen germination in vitro of the transgenic tomato with p35S::SlMAPK7-RNAi and pLAT52::SlMAPK7-RNAi vector were only 8.33% and 6.22% respectively, also obviously lower than control plants. Pollen germination rate in vitro of over-expression plant with control plants did not differ significantly. Pollen morphology electron microscope scanning was used to study the pollen morphological, the results showed the deficiency of SIMAPK7 caused the pollen deformity. However the upregulation of SIMAPK7 gene have not significant impact on tomato pollen vitality, pollen germination and pollen grain of external form. Combining resin semi-thin section with ultrathin section of flower buds in transmission electron microscope, the observations for pollen development reveal that the pollen content began to degrade at binucleate pollen stage, and the pollen was aborted. Observation on the fruit of the transgenic tomato plants found that different types of transgenic fruits had no significant difference in size, weight of single fruit and colur compared with CK. While there was no or a little seeds in the transgenic tomato fruit with pLAT52::SlMAPK7-RNAi and p35S::SlMAPK7-RNAi vector. Therefore, SIMAPK7 may participate in the regulation of signal transduction of pollen and tomato fruit development, and its mechanism needs further exploration.