Molecular Mechanism Of Heat Shock Protein GbHSP90 Involved In Telomere Regulation In Ginkgo Biloba L.

Ginkgo biloba L.is a rare tree species unique to our country,known as a living fossil among plants,and an important research material in the fields of plant developmental biology,molecular biology and genetics.At the same time,Ginkgo biloba L.is a well-known longevity tree species,and telomere stability of Ginkgo biloba L.is an important factor for the longevity,so it is of great significance to reveal the longevity of Ginkgo biloba L.and the mechanism of telomere protection of Ginkgo biloba L.Telomeres are DNA-protein complex structures present at the ends of eukaryotic chromosomes,consisting of telomeric DNA,telomere proteins and telomerase.In the early stage,the laboratory screened possible telomere-binding proteins by yeast one-hybrid experiments through the telomere DNA sequence of Ginkgo biloba L.and initially identified GbHSP90 as the telomere-binding protein.The full-length GbHSP90 was cloned by RACE technology,and the binding properties of the protein to telomeric DNA were confirmed by GFP yeast one-hybrid and EMSA experiments.In order to further explore the role of Ginkgo biloba L.HSP90 in the regulation of telomeres,the tissue expression characteristics of GbHSP90 were analyzed by real-time quantitative PCR;the subcellular localization of GbHSP90 was studied;fluorescence polarization experiments were used to analyze the characteristics of GbHSP90 binding to telomere DNA was studied in sections;the interacting proteins of GbHSP90 were screened by GST pull down combined with mass spectrometry,and the yeast two-hybrid experiment was used for one-to-one verification;GbHSP90 was overexpressed in 293T cells and HeLa cells,and GbHSP90 was detected effects on cell viability,cell cycle,apoptosis and telomerase activity.The main research conclusions are as follows:1)GbHSP90 exists in various tissues of Ginkgo biloba L.with the highest expression in embryos,followed by lateral roots,and the lowest expression in young leaves.In addition,the subcellular localization results showed that GbHSP90 is localized in the nucleus,which is an important prerequisite for the gene to participate in telomere regulation.2)Prokaryotic expression and purification of GbHSP90 were performed,and the purified product was subjected to EMSA experiments with probes of different types and repeat numbers.These results show that GbHSP90 can specifically bind to plant-type double-stranded DNA sequence（TTTAGGG）₅,but cannot bind to human-type and mutant plant-type telomeric DNA.When the repeat number of the sequence is less than 4,the binding ability will decrease or even disappear,indicating that GbHSP90binds telomeric DNA in a sequence-specific and length-dependent manner.3)GbHSP90 was expressed in sections and purified,and the purified products were subjected to EMSA experiments.The results showed that GbHSP90-N-CR could bind to plant-type double-stranded DNA（TTTAGGG）₅.It can be seen that the GbHSP90-N-CR region is important for binding double-stranded DNA.4)Screen and identify the interacting proteins of GbHSP90 by GST pull down combined with mass spectrometry.The identification results were analyzed and screened to 17 candidate proteins,of which 7 were successfully cloned,and Ginkgo biloba L.telomerase reverse transcriptase TERT was successfully cloned.Yeast two-hybrid experiments confirmed that GbHSP90 can bind to Ginkgo biloba L.chromosome structure maintenance protein GbSMC1 and Ginkgo biloba L.telomerase reverse transcriptase GbTERT.GbTERT is a key subunit of telomerase and an important telomere protein;GbSMC1 can participate in the maintenance of chromosome structure.The above results show that GbHSP90 can form a complex with telomere-related proteins GbSMC1 and GbTERT,and jointly participate in the regulation of telomere in Ginkgo biloba L.5)In order to explore the effect of GbHSP90 on telomere function,GbHSP90 was successfully overexpressed in human renal tubular epithelial cell line 293T and cervical cancer cell HeLa in this study.The ability to divide is accelerated,the process of apoptosis is inhibited,and the activity of telomerase is significantly increased.The above results further confirmed that GbHSP90 plays an important role in the regulation of telomeres in Ginkgo biloba L.6)To further analyze the roles of GbSMC1 and GbTERT interacting with GbHSP90 in telomere regulation,GbSMC1 and GbTERT were overexpressed in 293T cells,and the changes of telomerase activity were detected.The results showed that overexpression of GbSMC1 and GbTERT could significantly increase cellular telomerase activity.In conclusion,Ginkgo biloba L.GbHSP90 specifically binds to double-stranded telomeric DNA sequence,and forms a complex with telomere-related protein GbTERT and chromosome structure maintenance protein GbSMC1 to promote cell division,inhibit cell apoptosis,and enhance telomerase activity.GbHSP90,GbTERT and GbSMC1 are involved in telomere regulation in Ginkgo biloba L.This study systematically elucidated the molecular mechanism of Ginkgo biloba L.HSP90 involved in telomere regulation,provided new insights for analyzing the function of HSP90,and provided new ideas for the study of telomere protection mechanism in woody plants.