| Mediator complex is a multiprotein complex widely found in animals,plants and microorganisms,playing a key role in the transcriptional regulation of eukaryotes.Its spatial conformation consists of head,central,tail and kinase modules.Studies on the mediator complex have focused on Arabidopsis,including control of embryo formation,development of shoot meristems,flowering time,and root hair formation.In addition,it participates in the response to abiotic stresses.As a widely cultivated vegetable crop,tomato not only has important economic value,but also is a model plant for scientific research.However,adverse environments such as drought and high salt cause great damage to the growth and yield of tomato.Currently,it is little to know that tomato mediators involve in the response to abiotic stress.Thus,it is of great significance to study the resistance genes of tomato.In this study,the SlMed23 gene is screened from tomato using bioinformatics methods.After that,constructing the silencing vector.The vector is transformed into tomato using agrobacterium-mediated genetic transformation.The expression of this gene is silenced using RNA interference technology.In order to explore the role of SlMed23 gene in tomato growth and development and abiotic stress,we investigated the expression level of SlMed23 in tomato at different growth and development stages by qRT-PCR and detected its physiological and biochemical indicators of related developmental stages.The main research contents are as follows:(1)According to biological evolution tree,we find the SlMed23 gene of tomato has high homology with AtMed23 and the similarity is found to be 68.14% by comparing the amino acid sequences.Analysis of its upstream 3000 bp promoter sequence revealed that tomato SlMed23 contains multiple cis-acting elements related to high temperature stress,drought stress,injury stress and jasmonic acid response.(2)SlMed23 is expressed in all tissues of wild-type tomato,and the expression level is higher in young leaves and flowers,and its expression level gradually increases with the development of the fruit.In addition,the expression of SlMed23 in tomato leaves is different when drought stress and salt stress.The transcription level of SlMed23 is induced by ABA,GA3 and MeJA,by analyzing the expression pattern of SlMed23 in response to exogenous hormones.(3)Constructing SlMed23 silencing vector and transforming tomato plants by agrobacterium transformation.Observing the growth status of seedlings with higher silencing efficiency,we find that leaf axes of leaves under the first inflorescence of silent plants become shorter,the leaf structure is relatively simple,and the leaf margin becomes shallower compared to the wild-type,but the plant height and the number of leaves are almost unchanged.Detection of the expression of related genes by qRT-PCR technology,compared to the wild-type,the expression levels of GA20ox2 and GA3ox1,GA synthesis genes,are significantly decreased in silenced plants.The transcription level of the GA2ox2,GA degradation gene,is significantly upregulated.The expression level of GAST1,the GA response gene,is inhibited,the transcription level of GAI,the signal suppressor,was down-regulated.The expression of the auxin synthesis gene ToFZY2 is significantly reduced.The expression levels of the response genes ARF7 and ARF8 are significantly down-regulated.The transcription levels of the auxin polar transport-related genes PIN7 and PIN8 are significantly reduced.In addition,the expression levels of leaf polarity development genes KNOX1,KNOX2 and LA are significantly downregulated compared to wild-type plants.(4)Compared with wild-type tomato,the fruits of the silent lines are darker in the IMG and MG periods,and the chlorophyll content increased significantly.The transcription levels of chlorophyll synthesis gene DCL and SGR degradation gene are detected by qRT-PCR.We find the expression of DCL is significantly up-regulated,and SGR gene is significantly down-regulated,in RNAi plants.(5)Compared with wild-type tomato,the pollen of transfered SlMed23 gene tomato plants germinated less in vitro.Therefore,silencing SlMed23 may inhibit pollen development.(6)Silencing the SlMed23 gene enhances the salt tolerance of the plant.Detecting the water loss rate,relative water content,total chlorophyll content and malondialdehyde content of the seedlings,we find that,in the beginning,these indicators of the silencing of SlMed23 plants is similar to that of wild type,while at 7 and 15 d,silent lines had lower water loss,higher relative water content,higher total chlorophyll content,and lower malondialdehyde content.In addition,we detect the expression of stress-related genes in roots by qRT-PCR,and find that the expression levels of Cat1,Cat2,APX1 and PR1 are significantly up-regulated in the silenced lines.By exploring the salt sensitivity of transfered SlMed23 gene tomato seedlings,we find that,after treatment with 0 mM and 100 mM NaCl,the hypocotyls of the silenced lines are not significantly different from the wild type.However,compared to the wild type,treatment with 100 mM NaCl,the main roots of silent tomato seedlings became significantly longer.The results of seed germination experiments show that,when treatment with 0mM NaCl,most seeds of WT and RNAi can germinate;when treatment with 40 mM NaCl,the number of seed germination in silent lines is significantly more;when treatment with 60 mM NaCl,both have a low germination rate.(7)The results of ABA treatment show that there is no significant difference in the growth status of WT and RNAi when ABA is not contained in the medium.When treatment with 3?M and 5?M ABA,the hypocotyl length,main root length and weight of the silenced SlMed23 seedlings are significantly down-regulated.The results of MeJA treatment show that there is no significant difference in hypocotyl and root length between WT and RNAi when MeJA is not contained in the medium.When treatment with 10?M,20?M and 50?MMeJA,the main root and hypocotyl are longer and the seedlings are heavier of the RNAi lines compared with wild type.In summary,the preliminary study on the function of tomato mediator gene SlMed23 in plant growth and development and high salt stress are carried out in this paper,which provided an important reference for comprehensively studying the function of plant mediators and cultivating new resistant plants. |
PDF Full Text Request