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CN-121975926-A - Identification method of species telomere extension mechanism

CN121975926ACN 121975926 ACN121975926 ACN 121975926ACN-121975926-A

Abstract

The invention belongs to the field of biological identification, and particularly relates to an identification method of a species telomere extension mechanism. The method comprises the steps of collecting whole blood RNA samples of adult individuals after target species are sexually mature, detecting expression conditions of telomerase TERT in whole blood RNA of the species, if telomerase TERT expression is performed, determining that the telomere extension species is positive for TERT, if telomerase TERT is not performed, continuing to identify, and analyzing adaptability of alternative telomere extension ALT pathway related genes in a positive selection mode, if the adaptability is positive selection mode, and the adaptability is remarkably enriched in the pathway, then the telomere extension related species is remarkably enriched in a non-positive selection mode, and if the adaptability is not positive selection mode, then the telomere extension species is a TERT negative species. The invention provides a discrimination method for a species telomere extension mechanism by detecting the telomerase TERT expression condition in the somatic cells of adult species individuals and positive selection analysis of alternative telomere extension ALT channel related genes.

Inventors

  • Guo Yuzun
  • WANG ZHIWEN
  • Si Kaixu
  • ZHU XIAMING
  • FANG SHENGGUO
  • HUANG JUN
  • LIN LONGHUI

Assignees

  • 杭州师范大学

Dates

Publication Date
20260505
Application Date
20251219

Claims (7)

  1. 1. A method for identifying a species telomere extension mechanism, comprising the steps of: (S.1) collecting a whole blood RNA sample of an adult individual after sexual maturity of the target species; (S.2) detecting the expression condition of telomerase TERT in the RNA sample, wherein if the telomerase TERT is expressed, the telomere extension species positive to the TERT is obtained, and if the telomerase TERT is not expressed, the identification is continued; (S.3) comparing genome positive selection to analyze the adaptability of alternative telomere extended ALT channel related genes, wherein the adaptability is positive selection, and ALT telomere maintenance related species are obtained if the gene is significantly enriched in the channel, or TERT negative species are obtained if the gene is not positive selection or is not significantly enriched.
  2. 2. The method of claim 1, wherein the method comprises identifying a species telomere extension mechanism, the step S.2 of detecting the expression of telomerase TERT in the RNA sample comprises the following steps: (K.1) extracting whole blood RNA and determining RNA quality by using an Agilent 5400 fragment analyzer system, and quantitatively determining whether the concentration is greater than 100 ng/. Mu.L by using a spectrophotometer, if so, continuing the following steps; (K.2) reversely transcribing RNA by using a TAKARA PRIMESCRIPT RT kit to obtain cDNA, and determining the purity of the cDNA by using a spectrophotometry; (K.3) adopting PREMIER PRIMER 5.0.0 software to design a forward primer and a reverse primer of a target species TERT, and carrying out RT-qPCR amplification; (K.4) use of The method calculates the quantitative expression of telomerase TERT.
  3. 3. The method of claim 2, wherein the reverse transcription of RNA in step K.2 comprises two steps of a degenomic DNA reaction and a reverse transcription reaction.
  4. 4. The method of claim 2 or 3, wherein the step K.2 is performed by spectrophotometry of a 260 /A 280 , A 260 /A 230 to confirm the purity of the cDNA.
  5. 5. The method of identifying a mechanism for elongation of telomeres in a species of claim 1, wherein said positive selection of step s.3 analyzes the positive selection of alternative telomere elongation ALT pathway related genes, comprising the steps of: (Q.1) searching for orthologous genes of the species and the 20 TERT-positive comparative species genes with the closest evolutionary relationship by using OrthoFinder, and confirming homologous sequences; (Q.2) protein coding sequence search and single copy gene family alignment using Prank software, and deleting large insertions/deletions from the alignment by Gblocks to obtain species single copy ortholog sequence parameters, and normalizing to a PAML input file, respectively; (Q.3) using RAxML v8.2.12 software, using maximum likelihood methods to construct phylogenetic trees of the species respectively; (Q.4) using TimeTree to obtain a time correction point for correction; (Q.5) selection pressure analysis was performed using Codeml software in the PAML package.
  6. 6. The method of claim 5, wherein the positive selection assay of step Q.5 sets the target species as the foreground branch and uses the 20 TERT-positive species with the closest evolutionary relationship as the background branch.
  7. 7. The method of claim 5 or 6, wherein the sequence parameter in step Q.2 is orthofinder-fpep/.fasta-msa-t 20-a 20.

Description

Identification method of species telomere extension mechanism Technical Field The invention belongs to the field of biological identification, and particularly relates to an identification method of a species telomere extension mechanism. Background The mechanism of telomere elongation is well documented in human cancer cells, where 80% -85% of cancer cells directly elongate telomeres by telomere reverse transcriptase TERT, while another 10% -15% of cancer cells elongate telomeres by alternative telomere elongation mechanisms (ALTERNATIVE LENGTHENING of Telomere, ALT). In recent studies, we found that these two modes of telomere elongation are prevalent in vertebrate evolution. For example, guo et al in 2023, and Foley et al in 2018 suggest that wild animals such as adult individuals of the species Trionyx sinensis, pigeon, pig, mouse, etc. have detectable expression of telomere reverse transcriptase in somatic cells, which is very similar to the mechanism of direct prolongation of telomeres by telomere reverse transcriptase in human cancer cells (telomere prolongation pattern of 80% -85% of cancer cells). In contrast, we did not detect expression of telomerase in adult somatic cells of extremely long-lived species, such as alligator, myotis bat, and as the two species aged, the somatic telomere lengths of both species did not significantly shorten, as determined by genomic selection pressure analysis, alligator, myotis bat, etc. were highly differentiated in alternative telomere elongation mechanism (ALT) -related genes (telomere elongation pattern of 10% -15% of cancer cells). The average telomere length (> 20 kb) maintained by the ALT telomere extension pattern was statistically longer than the telomere extension pattern positive for TERT (< 10 kb). This lets us think that the pattern of telomere maintenance in wild animals may be related to the length of life of the wild animals. This lays the importance of species telomere prolongation pattern identification. Disclosure of Invention Aiming at the problems existing in the prior art, the invention provides a species telomere extension mechanism identification method by detecting the expression condition of telomerase TERT in somatic cells and the positive selection condition of ALT related genes in a species evolution relationship. The invention is realized by the following technical scheme: In a first aspect, the present invention provides a method of identifying a species telomere extension mechanism comprising the steps of: (S.1) collecting a whole blood RNA sample of an adult individual after sexual maturity of the target species; (S.2) detecting the expression condition of telomerase TERT in the RNA sample, wherein if the telomerase TERT is expressed, the telomere extension species positive to the TERT is obtained, and if the telomerase TERT is not expressed, the identification is continued; (S.3) positive selection analysis of the suitability of alternative telomeres to prolong the ALT pathway associated gene, if positive selection and significant enrichment in the pathway, then maintaining the associated species for ALT telomeres, if non-positive selection or not significant enrichment, then TERT negative species. Further, the detecting the expression of telomerase TERT in the RNA sample in step s.2 includes the following steps: (K.1) taking RNA, measuring the integrity of the RNA by using an Agilent 5400 fragment analyzer system (the 28S/18S value is required to be between 1.8 and 2.0, and the better RNA integrity is ensured), and quantitatively measuring whether the concentration is more than 100 ng/mu L or not by using a spectrophotometer, if so, continuing the following steps; (K.2) reversely transcribing RNA by using a TAKARA PRIMESCRIPT RT kit to obtain cDNA, and determining the purity of the cDNA by using a spectrophotometry; (K.3) using PREMIER PRIMER 5.0.0 software to design forward and reverse primers for RT-qPCR amplification; (K.4) use of The method calculates the quantitative expression of telomerase TERT. Further, the reverse transcription of RNA in step K.2 includes two steps, a genomic DNA removal reaction and a reverse transcription reaction. Further, the step K.2 determines the spectrophotometry of A 260/A280, A260/A230 to confirm the purity of cDNA. Further, the selective pressure analysis of step s.3 maintains positive selection of alternative telomere extended ALT pathway related genes, comprising the steps of: (Q.1) searching for orthologous genes of target species and comparative species genes using OrthoFinder to confirm the homologous sequences; (Q.2) protein coding sequence search and single copy gene family alignment using Prank software and deleting large insertions/deletions from the alignment by Gblocks to obtain species single copy ortholog sequence parameters and mammalian single copy ortholog sequence parameters, and normalizing to a PAML input file, respectively; (Q.3) using RAxML v8.2.12 software, using maximum l