CN103160592B - Molecular biology method for predicating endurance potentials of excellent ice-snow sportsmen - Google Patents
Molecular biology method for predicating endurance potentials of excellent ice-snow sportsmen Download PDFInfo
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- CN103160592B CN103160592B CN201310113515.8A CN201310113515A CN103160592B CN 103160592 B CN103160592 B CN 103160592B CN 201310113515 A CN201310113515 A CN 201310113515A CN 103160592 B CN103160592 B CN 103160592B
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Abstract
The invention discloses a molecular biology method for predicating the endurance potentials of excellent ice-snow sportsmen, which can be used for inquiring the relationship between the I/D (kininase II gene insertion/deletion) polymorphisms of ACE (angiotensin-converting enzyme) and the endurance potentials of the excellent ice-snow sportsmen. Lots of research results indicate that the genetic I/D polymorphisms are associated with the aerobic endurance of the sportsmen, are the key factors for judging the aerobic endurance quality of a human body, and affect the optimal level of the aerobic endurance quality of a human body and the sensitivity of a human body for endurance training; and the genes mainly affect the cardio-pulmonary function of a human body, and thus affecting the aerobic endurance quality of a human body. In consideration that the ACE genes are related to the aerobic endurance of movements, researches for the distribution of the genes in the excellent ice-snow sportsmen by applying the molecular biology method have important theoretical significance for determining the relevance between the genes and endurance-type ice-snow sports, and lay the foundation of future molecular selection by applying the molecular biology method.
Description
Technical field
The present invention relates to a kind of molecular biology method predicting excellent ice-snow sportsmen endurance potential, be exactly specifically the polymorphism by analyzing ACE (Angiotensin-converting enzyme) I/D (kininase II gene insertion/deletion type), thus learn ACE gene pleiomorphism affects the talent of outstanding endurance by which kind of approach.This invention belongs to the interdisciplinary field of basic life science, mankind's inheritance resources, molecular biology and sports science.
Background technology
The motor capacity of human body is by multiple Gene Handling, and these genes determine human motion potential.Along with the developing rapidly of biotechnology comprising molecular genetics, molecular biology, technique of gene detection, find and inquire into the gene relevant to motor capacity, and the experimental study carrying out considerable scale becomes possibility, especially in endurance and strength quality genes involved ACE (Zinc metallopeptidase Zace1).China's ice and snow project is the sports events based on aerobic endurance, but the backwardness relatively of selection means for a long time, majority is still by " experience selection ".Mainly test from aspects such as athletic body shape, function criterion, body constitution, the types of nerve, selection ages in selection process, also relatively low in the research level of genetic arts.In view of ACE gene is relevant with motion aerobic endurance, applied genetics method is carried out this gene and is studied in the distribution of winter sport athlete, for determining that this gene and endurance type winter sports dependency have important theory significance, carry out molecule selection lay the foundation for applying it from now on, and then the development of motherland's winter sports and even whole sports can be promoted.
ACE is 20 peptide strand acid glycoproteins, and be the metallohydrolases containing zinc, its system is called peptidyl one or two peptidohydrolase or kininaseⅡ.ACE is extensively present in whole body respectively to be organized, and is mainly distributed in vascular endothelial cell (especially common with pulmonary circulation), renal epithelial cell etc.Bradykinin is the natural substrate of ACE, suppresses its biologic activity by cracking two C-terminal dipeptides.
Renin-angiotensin system (Renin-anglotensin system, RAS) proangiotensin (Anglotensin origin is comprised, be abbreviated as AO), angiotensin I (Anglotensin I, be abbreviated as Ang I), Angiotensin II (Anglotensin II, be abbreviated as Ang II), feritin, ACE and angiotensin-ii receptor (Anglotensin II receptor, is abbreviated as AT).RAS acts on significantly in cardiovascular systems, as regulated blood pressure, Q volume of blood, antiotasis, and causes the hypertrophy of vessel wall thickening or myocardial cell and the propagation of non-myocardial infarction.Wherein Ang II is the effector peptide in RAS, causes cardiac muscle and vasoconstriction by its acceptor AT, regulates electrolyte balance, causes hyperplasia, propagation etc.Ang II has direct and of short duration effect to heart, comprises and increases heart rate and strengthen cardiac contractility; Meanwhile, Ang II also has the effect of its structure of long-term effect to heart, and it stimulates collagen and sheath to generate mainly through causing the hypertrophy of myocardial cell and inoblast.
ACE is the key enzyme in RAS enzyme/substrate chain reaction system, and the effect of ACE is that catalysis decapeptide Ang I changes octapeptide Ang II into.ACE level decides Ang II growing amount in RAS.Numerous research confirms, ACE horizontal stable, and measuring repeatability is good, environment metabolism and Hormone Factors less on its impact.But research also confirms that ACE level exists obvious mutability between individuals, and difference can reach 5 times more than.Obviously, ACE level is stablized on the one hand with in individuality, and is associated with shortage environmental factors, and on the other hand due to the phenomenon that interindividual variation is larger, people are inferred, and ACE level is largely by genetic determination.
ACE gene is positioned at No. 17 chromosome long arm 2 districts 3 and is with (17q23).ACE gene span is 21kb, has 26 exons and 25 introns.Find 6 kinds of ACE gene polymorphic marks at present, but the Alu Insert Fragment of the 287bp existed in the 16th intron and/or deletion fragment most study.Thus, ACE gene can be divided into DD homozygous deletion type in this polymorphic mark site, the genotype that ID heterozygote inserts or absence type is different with II homozygote insert type three kinds.DNA polymorphism refers to the otherness that karyomit(e) allelotrope nucleotide arranges.There is two or more form in region of DNA territory allelic (or fragment), can be divided into sequence polymorphism and sequence-length polymorphism.Wherein sequence-length polymorphism by tumor-necrosis factor glycoproteins with respective core sequence tandem sequence repeats occur or be distributed on karyomit(e) with being dispersed in, form polymorphism (as Alu repeated sequence) in insertion/deletion mode, this polymorphism is the genetic marker of current most worthy.
The influence factor of maximum aerobic capacity can be summarized in central mechanism and periphery mechanism.Wherein, ventricular volume and stroke output are the core links in central mechanism, and the conformability of good cardiovascular function, particularly artery and heart is that outstanding endurance athletes is successfully crucial; Muscle fiber types, flesh capillary density and oxidasic activity are the dominant roles in the mechanism of periphery.There is with sportsmen's aerobic endurance association in ACE gene I/D polymorphism, is the key factor determining human body aerobic stamina diathesis, affects the optimum level of human body aerobic stamina diathesis and human body to the susceptibility of endurance training simultaneously.The cardio-pulmonary function of this gene major effect human body, thus affect the aerobic stamina diathesis of human body.Therefore can by predicting the endurance potential of excellent ice-snow sportsmen to the analysis of ACE gene polynorphisms, and then for athletic selection with educate material and provide certain theoretical foundation from molecular biology angle.
The method that present stage is used to analyzing gene polymorphism is varied, and wherein comparatively common method has Meta to analyze and PCR.
Meta analysis refers to that carry out system with statistical method to the multiple research datas collected, quantitative comprehensive analysis, it is the quantification summary of document, because its discovery is from difference research, sample is different, program is different, instrument is different, and these results of study to be put together be nonsensical.Therefore the present invention is by obtaining corresponding band after pcr amplification, and carry out nucleotide sequencing to amplified production, the method has simple and easy to do, the reliable and advantage that experimental cost is cheap of real result.
Summary of the invention
The present invention can predict the endurance potential of excellent ice-snow sportsmen.
The object of this invention is to provide a kind of molecular biology method predicting excellent ice-snow sportsmen endurance potential.
Forecasting Methodology provided by the present invention is ACE gene I/D (kininase II gene insertion/deletion type) polymorphism analysis.Be positioned at the insertion of ACE gene intron 16 or deletion polymorphism after PCR method detects, 3 kinds of banding patterns can be presented: what only have 401bp band is homozygote insert type (II), what only have 113bp band is homozygous deletion type (DD), and what have 401bp and 113bp two bands is heterozygote insertion or absence type (I/D).Between DD, ID, II genotype levels of serum ACE three, there were significant differences, and the ACE level of DD type people is the highest, and ID type takes second place, and II type people is minimum.
Embodiment
Carry out ACE gene I/D (kininase II gene insertion/deletion type) polymorphism analysis for excellent ice-snow sportsmen and common artificial experimental subjects below, describe specific embodiment of the invention in detail.
Embodiment: ACE gene I/D (kininase II gene insertion/deletion type) polymorphism analysis
Research object
Research object mainly divides 2 groups, excellent ice-snow sportsmen group and the general population's group, and the general population organizes main selection people from this area suitable with this elite's age, and the principle in strict accordance with stochastic sampling is carried out, all without professional motion training history.
The mensuration of ACE gene I/D polymorphism
(1) extraction of DNA
Get 2ml whole blood from ulnar vein and inject heparin (10U/ml) anti-freezing test tube, then immediately centrifugal (1500rpm, 10min).After dissolving the red corpuscle in whole blood, go in 1.5ml eppendorf pipe, be prepared into leukocyte cell pellet be stored in-80 DEG C for subsequent use.Use classical approach DNA extraction agent box extracting STb gene from leukocyte cell pellet.
(2) design of primers and PCR primer order-checking
Design of primers first finds the ACE gene order published from Genbank database by internet, select suitable sequence context, adopt Primer select5.0 primer-design software, the primer designed is long 21 ~ 24bp generally, annealing temperature is 58 DEG C ~ 60 DEG C, by obtaining corresponding band after pcr amplification.Nucleotide sequencing is carried out to amplified production, confirms that amplified production sequence and bibliographical information are completely the same, illustrate that the gene that above-mentioned primer pair is selected has high degree of specificity.
The sequence of primer is as follows:
Upper primer (Forward): 5 ' TCC CAT TTC TCT AGA CCT GCT G3 '
Lower primer (Reverse): 5 ' GCC CTT AGC TCA CCT CTG CT3 '
(3) PCR reaction system (25l)
Total reaction system is 25l.H
2O12.8l、5×buffer5l、10mM?dNTPs0.75l、250mM?Mg
2+0.4l、10M?primer?for0.4l、10M?primer?Rev0.4l、Ext?aq?Hs0.25l、DNA(10ng/l)5l。
(4) PCR reaction conditions
Initial stage denaturation 95 DEG C, 5min, sex change 95 DEG C of 30s, annealing 58 DEG C of 30s, extension 72 DEG C of 60s, 2-4 × 40cycles, finally extension 72 DEG C of 7min, 4 DEG C of 10min.
(5) electrophoresis
By PCR reaction product 10l+6 × loading buffer2l in 1.6%Agarose gel electrophoresis (glue 45ml+FB2l) imaging, develop under ultraviolet lamp, take the photograph sheet by gel images and image analysis system, and determine genotype.
(6) the genotypic judgement of ACE and qualification result
Be positioned at the insertion of ACE gene intron 16 or deletion polymorphism after above-mentioned PCR method detects, the fragment of two kinds of length can be amplified, namely 401bp inserts the deletion fragment (D) of amplified fragments (I) and 113bp, 3 kinds of banding patterns below can be presented: what only have 401bp band is homozygote insert type (II) under ultraviolet lamp, what only have 113bp band is homozygous deletion type (DD), and what have 401bp and 113bp two bands is heterozygote insertion or absence type (I/D).
Research shows that the insertion/deletion (I/D) of the Alu repeated sequence of the one section of 287bp being positioned at the 16th intron has polymorphism, makes ACE gene have 3 kinds of genotype: II (inserting homozygous), ID (insertion/deletion type) and DD (lacking homozygous).ACE affects by inherited genetic factors at blood level, confirms that ACE gene pleiomorphism has very large association with ACE level in blood plasma and cell.Between DD, ID, II genotype levels of serum ACE three, there were significant differences, and the ACE level of DD type people is the highest, and ID type takes second place, and II type people is minimum.
There is with sportsmen's aerobic endurance association in ACE gene I/D polymorphism, is the key factor determining human body aerobic stamina diathesis, affects the optimum level of human body aerobic stamina diathesis and human body to the susceptibility of endurance training simultaneously.The cardio-pulmonary function of this gene major effect human body, thus affect the aerobic stamina diathesis of human body.
Claims (1)
1. probe into a kininase II gene insertion/deletion type, the molecular biology method of polymorphism and excellent ice-snow sportsmen endurance potential relation:
(1) step detecting the PCR of kininase II gene insertion/deletion type polymorphism is specially:
Kininase II gene insertion/deletion type gene primer is:
Upper primer: 5'TCC CAT TTC TCT AGA CCT GCT G 3'
Lower primer: 5'GCC CTT AGC TCA CCT CTG CT 3'
(2) the PCR reaction conditions of gene is: initial stage denaturation 95 DEG C, 5min, sex change 95 DEG C of 30s, annealing 58 DEG C of 30s, extension 72 DEG C of 60s, 2-4 × 40 cycles, finally extension 72 DEG C of 7min, 4 DEG C of 10min;
(3) by evaluating excellent ice-snow sportsmen kininase II gene insertion/deletion type gene polynorphisms, analyze the dependency of this gene and sportsmen's endurance potential in serum, thus using the relation of this gene in serum as forecasting research vivo gene type and endurance potential, selection of athletes can provide molecular biology method foundation.
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| CN113205883B (en) * | 2021-04-12 | 2022-06-28 | 中国人民解放军陆军军医大学第二附属医院 | Method and system for predicting cardiopulmonary function from genetic information and post-exercise physiological parameters |
| CN113430275A (en) * | 2021-06-25 | 2021-09-24 | 北京体育大学 | Method, primer set and kit for screening athletes by adopting LPL gene rs283 polymorphic site |
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Non-Patent Citations (2)
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| ACE基因I/D多态性与耐力素质的关联性研究:Meta分析;杨贤罡等;<体育科学>;20101231(第7期);摘要，第43-47页结果部分 * |
| 力量素质相关基因多态性的研究进展;杨震等;《西安体育学院学报》;20061231(第4期);第72页2.1部分；第73页第3部分 * |
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