Cloning And Functional Analysis Of Transcription Factor SlPRE6-like In Tomato (Solanum Lycopersicum )

The bHLH transcription factor family is the second largest transcription factor family following the MYB family in plant.The genes of bHLH transcription factor family can participate in the growth and development of plants,the regulation of various signal pathways,the response of adversity,the regulation of biosynthetic pathways of various active ingredients.The subfamily PREs gene of bHLH transcription factor is involved in the growth and development of cell elongation,hormone signal regulation and light morphogenesis in Arabidopsis thaliana.However,we have little knowledge of the function of PREs in tomatoes.In this paper,the SlPRE6-like gene of the PREs subfamily was studied by using Lycopersicon esculentum Mill.var.Ailsa Craig(AC++)as the research material.The main contents and results are as follows:(1)The SlPRE6-like protein contains 95 amino acid sequences.By comparison with the homologous PREs amino acid sequence,the protein has an atypical b HLH domain.Systemic phylogenetic tree analysis shows that SlPRE6-like and AtPRE6 which is a light morphological protein in arabidopsis has a high homology.Suggesting that it may be involved in the regulation of optical signal transduction.(2)The expression pattern of SlPRE6-like gene in tomato AC++ showed that the expression level of the gene was higher in young leaves,mature leaves and flowers,especially in young leaves.In the senescence of the leaves,sepals and green fruit in a small amount of expression in other tissues and organs almost no expression.Expression pattern analysis showed that SlPRE6-like gene may be involved in the regulation of growth and development of tomato leaves.(3)Plant leaf development has an important relationshipand with biological stress and abiotic stress.To this end,we treated four kinds of abiotic stress of tomato AC++ seedlings with salt,high temperature,low temperature and drought.The expression level of SlPRE6-like gene in the above-mentioned abiotic stress was detected by real-time quantitative PCR.It was found that the expression trend of SlPRE6-like gene was down-regulated rapidly in salt(leaf)and high temperature treatment.The expression trend of SlPRE6-like was not obvious when salt(root),low temperature and dehydration stress were applied.The results showed that SlPRE6-like gene was closely related to salt(leaf)and high temperature stress response,but not with salt(root),low temperature and dehydration stress.(4)In order to study the relationship between the development of plant leaves with gibberellin and paclobutrazol,the wild type tomato was treated with different concentrations of gibberellin and paclobutil,and the results were analyzed by real-time quantitative PCR with 10 days seedling.The results showed that at different concentrations in the treatment of gibberellin,the expression level of SlPRE6-like gene was significantly down-regulated compared with the control group,and the expression level of Sl PRE6-like gene increased first and then decreased with the increase of paclobutrazol treatment concentration.The results showed that SlPRE6-like gene may be related to the concentration of gibberellin and paclobutrazol in the regulation of tomato leaf growth and development.(5)Through the early bioinformatics analysis,expression pattern analysis,stress test and exogenous gibberellin,paclobutrazol treatment test,it is found that SlPRE6-like gene is very likely to be involved in the growth and development of tomato leaves,to this end,we constructed the overexpression vector pBI121-SlPRE6-like of SlPRE6-like gene to study the specific biological function of SlPRE6-like gene in the leave of tomato.The transgenic plants of SlPRE6-like were successfully screened by agrobacterium tumefaciens transformation with wild type tomato cotyledon.In appearance,the leaves of transgenic tomatoes showed a significant upward curl,and the plants were prone to death.Under the dark and light conditions,the length of hypocotyl of the transgenic T1 generation longer than that of the wild type by 10 days,the expression level of DCL and SlGLK1 in chlorophyll synthesis was significantly downregulated under dark and light conditions.by comparing the two-month transgenic T1 generation tomato seedling leaves and wild-type tomato leaves,it was found that the transgenic tomato leaves showed significant yellowing and senescence.The expression level of Chloroplast synthesis related gene SlGLK1 were significantly decreased,DCL expression also decreased.The expression of SlCab7 and SlRbcS and Sl AUREA were decreased,especially,the decrease of SlRbcS was obvious.Indicating that SlPRE6-like gene may be involved in the regulation of chlorophyll synthesis and photosynthesis-related gene expression,so as to control the yellowing and senescence of leaves.In summary,the study of tomato SlPRE6-like gene was carried out by bioinformatics analysis,stress treatment and exogenous hormone response.It was found that the tomato SlPRE6-like gene may be involved in the regulation of leaf development,and then through the genetically modified functional test series of related experiments show that SlPRE6-like gene is more sensitive to light,may be involved in regulating the expression of the major genes associated with photosynthesis and by promoting the transmission of gibberellin signaling,and then control the tomato leaf curl,senescence.In this study,we studied the leaf curl and senescence of the PREs family gene SlPRE6-like in tomato,which laid a foundation for the transgenic transformation of plant leaves in engineering practice,and had certain market value.