CAJ | 학술논문

为了挖掘抗病基因，探寻甘蓝抗黑腐病机制，选取抗性材料C7，在幼苗4～5片真叶期，喷施菌液浓度为1.0×108 cfu/mL的细菌悬浮液，喷施蒸馏水作对照，提取接种后1，3，5 d的总RNA进行等量混合，获得2种不同处理的样品池。采用SRAP分子标记技术进行基因差异表达分析，结果显示：60对SRAP引物组合共扩增出大小在100～750 bp的685条带，平均每对引物扩增11条带，共检测到24个多克隆位点。回收测序后，将得到的9条差异表达片段TDFs（ Transcript derived fragments ）进行克隆和序列分析，其中3条差异基因片段未搜索到任何同源蛋白，其余6个基因均按功能分类，可分为涉及能量代谢、植物细胞壁蛋白相关基因、液泡加工酶基因和未知功能基因。表明cDNA-SRAP方法简单、重复性好、试验成本低，完全适用于差异表达分析，从中获得一些与甘蓝抗黑腐病相关的差异基因片段，可用于甘蓝抗黑腐病的分子机理研究。
위료알굴항병기인，탐심감람항흑부병궤제，선취항성재료C7，재유묘4～5편진협기，분시균액농도위1.0×108 cfu/mL적세균현부액，분시증류수작대조，제취접충후1，3，5 d적총RNA진행등량혼합，획득2충불동처리적양품지。채용SRAP분자표기기술진행기인차이표체분석，결과현시：60대SRAP인물조합공확증출대소재100～750 bp적685조대，평균매대인물확증11조대，공검측도24개다극륭위점。회수측서후，장득도적9조차이표체편단TDFs（ Transcript derived fragments ）진행극륭화서렬분석，기중3조차이기인편단미수색도임하동원단백，기여6개기인균안공능분류，가분위섭급능량대사、식물세포벽단백상관기인、액포가공매기인화미지공능기인。표명cDNA-SRAP방법간단、중복성호、시험성본저，완전괄용우차이표체분석，종중획득일사여감람항흑부병상관적차이기인편단，가용우감람항흑부병적분자궤리연구。
In order to explore the resistant disease gene and find out relative mechanisms ,the suspensions with 1.0 ×108 colony forming units/mL was sprayed to the resistant variety C 7 of seedlings at four to five leaves ,while the other group was sprayed distilled water as usual (Control,CK).Leaves were collected in 1,3,5 d time points af-ter XCC and distilled water .A mixture of an equal amount total RNA from the above two groups as two gene pools . Different expression of the genes was analyzed by SRAP technique .The results showed:using 60 pairs of primers ,a-bout 685 cDNA fragments in size between 100~750 bp were amplified ,averagely 11 bands per primer combination , there were 24 polymorphic loci in these two pool .They were sequenced and the Blast program was used to find out homology with reported genes .Sequence alignment indicated that 6 out of these 9 TDFS ( Transcript derived frag-ments) showed homologies to certain genes from 43%to 59%in NCBI,while the other three showed no significant homology with reported gene .Based on their homologies ,these genes were assumed to NADH dehydrogenase ,vegeta-tive cell wall protein ,DELTA-VPE and unknown functional proteins .The results indicated that cDNA-SRAP method had several advantages over other systems:simplicity , reasonable throughput rate , highly reproducibility and inex-pensiveness ,and that the cDNA-SRAP technique was applicable to the analysis of gene different expression .Some fragments of differentially expressed genes associated with cabbage black rot were obtained ,and may be used for in-vestigating the molecular mechanism of cabbage black rot .