야생벼의 일종인 Oryza grandiglumis (CCDD, 2n=48)는 도열병, 잎집무늬마름병, 흰빛잎마름병, 그리고 벼멸구와 같은 병충해에 저항성을 가지는 것으로 알려져 있다. 이와 같은 곰팡이와 해충에 반응하여 차별 발현하는 유전자를 클로닝 하기 위하여 상처처리와 yeast extract를 Oryza grandiglumis에 0시간과 24시간 각각 처리하였다. 유전자의 클로닝을 위하여 희귀 발현유전자의 클로닝에 효율적인 것으로 알려진 Suppression Subtractive Hybridization (SSH) 방법이 처리 후 24시간 된 식물을 재료로 사용되었다. 그 결과, 776개의 cDNA clones이 확보되었으며, 유전자 발현의 진위여부를 빠르게 스크린하기 위하여 colony array가 수행되었다. 115개의 colony가 positive로 판명되었고, 이들의 평균 insert size는 400 bp에서 700 bp에 이르렀고, 이들에 대한 염기서열 분석이 수행되었다. 염기서열 분석 결과, 68개 clone들이 알려진 기능의 유전자와 homology를 나타냈으며, 이중에서 16개 clone이 일차대사에 관련된 것과 유사성을, 5개가 plant retrotransposon과 유사성을, 5개가 식물 방어기작 관련 metallothionein-like gene과 염기서열 유사성을 보였다. 이외에 다양한 유전자들이 아미노산 합성관련, membrane transport, signal transduction등에 관여하는 유전자들과 상동성을 나타내었다. 이들 유전자중에서 4 개의 클론(ogwfi-161, ogwfi-646, ogwfi-663, ogwfi-695)들이 선발되었고 이들에 대한 Northern 분석이 수행되었다. Northern 분석 결과 ogwfi-161, ogwfi-646, ogwfi-663, ogwfi-695는 wounding과 yeast extract처리 에 의한 차별 발현이 확인되었다. 이상의 결과를 종합하여 볼 때, SSH방법은 병충해등과 같은 조건에 의해 차별 발현되는 유전자들을 빠른 시간 내에 다량으로 발굴할 수 있는 매우 효율적인 방법이라고 생각된다.
야생벼의 일종인 Oryza grandiglumis (CCDD, 2n=48)는 도열병, 잎집무늬마름병, 흰빛잎마름병, 그리고 벼멸구와 같은 병충해에 저항성을 가지는 것으로 알려져 있다. 이와 같은 곰팡이와 해충에 반응하여 차별 발현하는 유전자를 클로닝 하기 위하여 상처처리와 yeast extract를 Oryza grandiglumis에 0시간과 24시간 각각 처리하였다. 유전자의 클로닝을 위하여 희귀 발현유전자의 클로닝에 효율적인 것으로 알려진 Suppression Subtractive Hybridization (SSH) 방법이 처리 후 24시간 된 식물을 재료로 사용되었다. 그 결과, 776개의 cDNA clones이 확보되었으며, 유전자 발현의 진위여부를 빠르게 스크린하기 위하여 colony array가 수행되었다. 115개의 colony가 positive로 판명되었고, 이들의 평균 insert size는 400 bp에서 700 bp에 이르렀고, 이들에 대한 염기서열 분석이 수행되었다. 염기서열 분석 결과, 68개 clone들이 알려진 기능의 유전자와 homology를 나타냈으며, 이중에서 16개 clone이 일차대사에 관련된 것과 유사성을, 5개가 plant retrotransposon과 유사성을, 5개가 식물 방어기작 관련 metallothionein-like gene과 염기서열 유사성을 보였다. 이외에 다양한 유전자들이 아미노산 합성관련, membrane transport, signal transduction등에 관여하는 유전자들과 상동성을 나타내었다. 이들 유전자중에서 4 개의 클론(ogwfi-161, ogwfi-646, ogwfi-663, ogwfi-695)들이 선발되었고 이들에 대한 Northern 분석이 수행되었다. Northern 분석 결과 ogwfi-161, ogwfi-646, ogwfi-663, ogwfi-695는 wounding과 yeast extract처리 에 의한 차별 발현이 확인되었다. 이상의 결과를 종합하여 볼 때, SSH방법은 병충해등과 같은 조건에 의해 차별 발현되는 유전자들을 빠른 시간 내에 다량으로 발굴할 수 있는 매우 효율적인 방법이라고 생각된다.
Oryza grandiglumis (CCDD, 2n=48), one of the wild rice species, has been known to possess fungal-,bacterial-, and insect-resistance against sheath blight, rice blast, bacterial leaf blight and brown plant hopper (Nilaparvata lugens). To rapidly isolate differentially expressed genes responding to fu...
Oryza grandiglumis (CCDD, 2n=48), one of the wild rice species, has been known to possess fungal-,bacterial-, and insect-resistance against sheath blight, rice blast, bacterial leaf blight and brown plant hopper (Nilaparvata lugens). To rapidly isolate differentially expressed genes responding to fungal and wounding stress, wounding and yeast extract were treated to O. grandiglumis for 24 hrs. Suppression subtractive hybridization (SSH) method was used to obtain differentially expressed genes from yeast extract and wounding treated plants. Seven hundreds and seventy six clones were obtained by subcloning PCR product, and colony array and screening were carried out using radio-isotope labeled cDNA probes prepared from the wounding and yeast extract treated plants. One hundred and fifteen colonies were confirmed as true positive ones. Average insert size of the clones were ranged from 400 bp to 700 bp and all the inserts were sequenced. To decide the identity of those clones, sequences were analyzed by sequence homology via GenBank database. The homology search result showed that 68 clones were matched to the genes with known function; 16 were related to primary metabolism, 5 to plant retrotransposons, 5 to defense related metallothionein-like genes. In addition to that, others were matched to various genes with known function in amino acid synthesis and processing, membrane transport, and signal transduction, so on. In northern blot analysis, induced expressions of ogwfi-161, ogwfi-646, ogwfi-663, and ogwfi-695 by wounding and yeast extract treatments were confirmed. The result indicates that SSH method is very efficient for rapid screening of differentially expressed genes.
Oryza grandiglumis (CCDD, 2n=48), one of the wild rice species, has been known to possess fungal-,bacterial-, and insect-resistance against sheath blight, rice blast, bacterial leaf blight and brown plant hopper (Nilaparvata lugens). To rapidly isolate differentially expressed genes responding to fungal and wounding stress, wounding and yeast extract were treated to O. grandiglumis for 24 hrs. Suppression subtractive hybridization (SSH) method was used to obtain differentially expressed genes from yeast extract and wounding treated plants. Seven hundreds and seventy six clones were obtained by subcloning PCR product, and colony array and screening were carried out using radio-isotope labeled cDNA probes prepared from the wounding and yeast extract treated plants. One hundred and fifteen colonies were confirmed as true positive ones. Average insert size of the clones were ranged from 400 bp to 700 bp and all the inserts were sequenced. To decide the identity of those clones, sequences were analyzed by sequence homology via GenBank database. The homology search result showed that 68 clones were matched to the genes with known function; 16 were related to primary metabolism, 5 to plant retrotransposons, 5 to defense related metallothionein-like genes. In addition to that, others were matched to various genes with known function in amino acid synthesis and processing, membrane transport, and signal transduction, so on. In northern blot analysis, induced expressions of ogwfi-161, ogwfi-646, ogwfi-663, and ogwfi-695 by wounding and yeast extract treatments were confirmed. The result indicates that SSH method is very efficient for rapid screening of differentially expressed genes.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
제안 방법
The suppression PCR reaction was subcloned into TA cloning vector and 776 colonies were obtained. With the subtracted clones, colony array with 32P-labeled forward- subtracted cDNA probes was performed. As a result, 115 out of 776 clones were identified as positive true (data not shown).
이론/모형
The nucleotide sequences of the cloned cDNAs were determined by the dideoxy-chain termination method using the AutoCycle TM Sequencing Kit (Amersham, UK). Electro phoresis was performed on ALFexpress H DNA Analysis System (Amersham, UK).
성능/효과
The observed true colony ratio is relatively high compared to other screening method such as DDRT (Dif ferential Display Reverse Transcription), even though it still needs to be proven further with more delicate exper imental tools [2, 8]. The sequences of 115 clones were determined and 68 clones were matched to genes with known function (Table 1).
As expected, many of the identified cDNAs were house keeping genes related to primary metabolism. Twenty three percentage of clones were matched to Oryza sativa, Arabidopsis thaliana, and Homo sapiens genomic DNA. Four clones (ogwfi-6, ogwfi-23, ogwfi-55, ogwfi-611) had ho mology to plant retrotransposons which are tightly linked to molecular pathways activated by stress, and activation of the retrotransposons are under the control of czs-regulatory sequences strikingly similar to those of plant defense genes [3].
참고문헌 (14)
Chomczynski, P. 1992. One-hour downward alkaline capillary transfer for blotting DNA and RNA. Anal. Biochem. 201, 134-150
Diatchenko, L., Y. C. Lau., A. P. Campbell., A. Chenchik., F. Mooadam., B. huang., S. A. Lukyanov., K. Lukyanov., N. Gurskaya, E. D. Sverdlov and P. D. Siebert. 1996. Suppression subtractive hybridization: A method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. 93, 6025-6030
Grandbastien, M. S., H. Lucas., J. B. Morel., C. Mhiri., S. Vernhettes and J. M. Casacuberta. 1997. The expession of the tobacco Tnt1 retrotransposon is linked to plant defense responses. Genetica. 100(1-3), 241-252
Hsieh, H. M., W. K. Liu and P. C. Huang. 1995. A novel stress-inducible metallothionein-like gene from rice. Plant Mol. Biol. 28(3), 381-389
Hwang, H. and G. Kochert. 1994. Comparative RFLP mapping of an allotetraploid wild rice species (Oryza latifolia) and cultivated rice (O. sativa). Plant Mol. Biol. 25, 633-648
Jena, K. K. and G. S. Kochert. 1991. Restriction fragment length polymorphism analysis of CCDD genome species of the genus Oryza L. Plant Mol. Biol. 16, 831-839
Kim, Y. O., H. J. Koh., S. H. Kim., S. H. Jo., J. W. Huh., K. S. Jeong., I. J. Lee., B. J. Song and T. L. Huh. 1999. Identification and functional characterization of a novel, tissue-specific NAD(+)-dependent isocitrate dehydrogenase beta subunit isoform. J. Biol. Chem. 24:274(52), 36866-36875
Liang, P., D. Bauer., L. Averboukh., P. Warthoe., M. Rohrwild., H. Muller., M. Strauss and A. B. Pardee. 1995. Analysis of altered gene expression by differential display. Methods Enzymol. 254, 304-321
Liang, P. and A. B. Pardee. 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science. 14:257(5072), 967-971
Reddy, A. S., M. C. Kiefer-Myer and M. Delseny. 1993. Characterization of new variants of a satellite DNA from Oryza officinalis, specific for the CC genome of wild rice. Genome. 36(4), 750-761
Sasabe, M., K. Toyoda., T. Shiraishi., Y. Inagaki and Y. Ichinose. 2002. cDNA cloning and characterization of tobacco ABC transporter: NtPDR1 is a novel elicitor- responsive gene. FEBS lett. 518, 164-168
Siebert, P. D., A. Chenchik, D. E. Kellogg., K. A. Lukyanov and S. A. Lukyanov. 1995. An improved PCR method for walking in uncloned genomic DNA. Nucl. Acids Res. 23, 1087-1088
Xiao, J., J. Li., S. Grandillo., S. N. Ahn., L. Yuan., S. D. Tanksley and S.R. McCouch. 1998. Identification of trait-improving quantitative trait loci alleles from a wild rice relative, Oryza rufipogon. Genetics. 150, 899-909
※ AI-Helper는 부적절한 답변을 할 수 있습니다.