CAJ | 학술논문

目的探索学习记忆异常基因hab-1(cn308)的功能.方法 1. 在hab-1领域有Y63D3A.6、Y63D3A.7和Y63D3A.8等三个YAC克隆.在基因库里检索Y63D3A.6、Y63D3A.7和Y63D3A.8的cDNA序列,用DNASIS-Mac v3.6软件处理并设计引物,进行PCR.把PCR产物插入pMD~(TM)18-TVector载体,并培养提取Y63D3A.6、Y63D3A.7和Y63D3A.8的目的 DNA,纯化DNA.2.进行rescue实验:把三种YAC克隆(clone)Y63D3A.6、Y63D3A.7和Y63D3A.8的DNA分别与GFP蛋白连接配制融合蛋白.应用OLYMPUSAxiovert S100共聚焦倒置显微镜操作系统,在L4大小的C.elegans生殖腺(gonad)内分别把三种GFP融合蛋白溶液显微注射,发育传代至F1.在F1代选择带有GFP荧光的C.elegans,观察基冈的表达部位,并进行Tap测试,通过观察在哪个YAC克隆把hab-1(cn308)的表现型恢复为野生型来确定hab-1的等位基因.3. DNA测序和同源性检索:hab-1的等位基冈确定以后对cn308进行DNA测序.根据cn308的测序结果与已知的等位基因DNA序列进行Homology search,分析cn308的基因结构,并进行BLSTP homologic search,和其他物种比较目的基因的功能来阐明hab-1的基因功能.结果对Y63D3A.6、Y63D3A.7和Y63D3A.8等三种基因表达率进行χ~2检验结果表示差异有显著性(χ~2=26.84,P<0.05).表明Y63D3A.6、Y63D3A.7、Y63D3A.8等三种基因虽然迮锁于第一常染色体上邻近的位置.但在C. elegans的头部表达率无关联性,并有显著性差异.同时可见Y63D3A.7强表达于C. elegans的头部,Tap测试证明Y63D3A.7把hab-1的表现型恢复野生型.DNA测序结果表明hab-1(cn308)是GAG错义突变为GGG,即Glu→Cly的突变体.BLSTP homologic search结果表明Y63D3A.7是调节线粒体complex Ⅰ的活性,就是说明hab-1基因与线粒体complex Ⅰ的功能有关.结论 Y63D3A.7是hab-1的等位基冈,主要表达于中枢神经系统.C.elegans的学习记忆机能低下与hab-1的突变影响线粒体complex Ⅰ的功能有关.
목적 탐색학습기억이상기인hab-1(cn308)적공능.방법 1. 재hab-1영역유Y63D3A.6、Y63D3A.7화Y63D3A.8등삼개YAC극륭.재기인고리검색Y63D3A.6、Y63D3A.7화Y63D3A.8적cDNA서렬,용DNASIS-Mac v3.6연건처리병설계인물,진행PCR.파PCR산물삽입pMD~(TM)18-TVector재체,병배양제취Y63D3A.6、Y63D3A.7화Y63D3A.8적목적 DNA,순화DNA.2.진행rescue실험:파삼충YAC극륭(clone)Y63D3A.6、Y63D3A.7화Y63D3A.8적DNA분별여GFP단백련접배제융합단백.응용OLYMPUSAxiovert S100공취초도치현미경조작계통,재L4대소적C.elegans생식선(gonad)내분별파삼충GFP융합단백용액현미주사,발육전대지F1.재F1대선택대유GFP형광적C.elegans,관찰기강적표체부위,병진행Tap측시,통과관찰재나개YAC극륭파hab-1(cn308)적표현형회복위야생형래학정hab-1적등위기인.3. DNA측서화동원성검색:hab-1적등위기강학정이후대cn308진행DNA측서.근거cn308적측서결과여이지적등위기인DNA서렬진행Homology search,분석cn308적기인결구,병진행BLSTP homologic search,화기타물충비교목적 기인적공능래천명hab-1적기인공능.결과 대Y63D3A.6、Y63D3A.7화Y63D3A.8등삼충기인표체솔진행χ~2검험결과표시차이유현저성(χ~2=26.84,P<0.05).표명Y63D3A.6、Y63D3A.7、Y63D3A.8등삼충기인수연책쇄우제일상염색체상린근적위치.단재C. elegans적두부표체솔무관련성,병유현저성차이.동시가견Y63D3A.7강표체우C. elegans적두부,Tap측시증명Y63D3A.7파hab-1적표현형회복야생형.DNA측서결과표명hab-1(cn308)시GAG착의돌변위GGG,즉Glu→Cly적돌변체.BLSTP homologic search결과표명Y63D3A.7시조절선립체complex Ⅰ적활성,취시설명hab-1기인여선립체complex Ⅰ적공능유관.결론 Y63D3A.7시hab-1적등위기강,주요표체우중추신경계통.C.elegans적학습기억궤능저하여hab-1적돌변영향선립체complex Ⅰ적공능유관.
Objective To research into the function of abnormal gene of learning and memory hab-1 (cn308). Methods 1. There were three YAC clones in the hab-1 area, Y63D3A.6, Y63D3A.7 and Y63D3A.8. The cDNA sequence of Y63D3A.6, Y63D3A.7 and Y63D3A.8 were retrieved in the gene bank, processed by DNASIS-Mac v3.6 software and designed primers, PCR was processed. The PCR product was inserted into pMD ~(18)TVector, culturing and recovering target DNA of Y63D3A.6, Y63D3A.7 and Y63D3A.8, purifying DNA .2. Carrying out rescue experiments; DNA of three YAC clones Y63D3A.6, Y63D3A.7 and Y63D3A.8 were linked with the GFP Protein respectively for preparing DNA:GFP fusion protein. The three DNA:GFP fusion protein solution was microinjected into L4 size C. elegans gonad respectively by OLYMPUS Axiovert S100 confocal inverted microscope operating system, growing and subcultring to F1 generation, observing gene expression site at the selected C. elegans with GFP fluorescence in F1 generation, carrying out Tap test, observing which YAC clone rescued the hab-1 (cn308) phenotype to wild-type phenotype to identify the hab-1 allele. 3. DNA sequencing and Homolog search; DNA sequencing for cn308 after hab-1 allele was ascertained. According to the results of cn308 sequencing and known allele DNA sequences to Homolog search, the genetic structure of cn308 was analyzed and BLST homologic search was carried out, comparing target gene function with other species to clarified gene function of hab-1. Results The result of χ~2 test for the rate of gene expression of Y63D3A.6, Y63D3A.7 and Y63D3A.8 χ~2 showed χ~2 >0.05, P<0.05, there was a significant difference. It showed that although Y63D3A.6, Y63D3A.7 and Y63D3A.8 have linkage with the adjacent location of the first autosomal, expressing rate on the head of C. elegans had no relatedness and had significant differences. It revealed that Y63D3A.7 was expressed strongly on the head of C. elegans, and Tap test proved that Y63D3A.7 rescued hab-1 to wild-type phenotype. The result of DNA sequencing was that hab-1 (cn308) was a missense mutation (GAG→GGG, namely, Glu→Gly ). The result of BLST homologic search showed that Y63D3A.7 was the activity which adjusts mitochondrion complex Ⅰ, it illustrated that hab-1 gene had relations with the function of mitochondrion complex Ⅰ. Conclusion Y63D3A.7 is the allele of hab-1, mainly expressed in the central nervous system. Hypofunction of learning and memory in C. elegans has relations with the function of mitochondrion complex Ⅰ which influences hab-1 mutations.