甘蔗与斑茅后代荧光原位杂交体系优化  

Optimization of Fluorescent in Situ Hybridization System in Hybrid Progeny of Saccharum officinarum and Erianthus arundinaceus

作　　者：吴嘉云[1,2] 黄永吉[1] 邓祖湖[1] 林彦铨[1] 陈如凯[1] 

机构地区：[1]福建农林大学农业部福建甘蔗生物学与遗传育种重点实验室,福州350002 [2]广州甘蔗糖业研究所,广州510316

出　　处：《农业生物技术学报》2013年第11期1279-1286,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.30671329);国家甘蔗产业体系专项资金资助(No.CARS-20-1-5)

摘　　要：高效的杂交体系是利用荧光原位杂交技术进行相关研究的基础。本研究对影响甘蔗染色体荧光原位杂交效果的关键因素如染色体预处理、变性条件和变性方式等进行筛选，优化适合于甘蔗的荧光原位杂交体系。本研究以甘蔗（Saccharum officinarum）与斑茅（Erianthus arundinaceus）F1崖城96-66的根尖为材料，采用对二氯苯离体、α-溴代萘离体和4℃低温水中非离体3种预处理方法，筛选染色体形态，采用分开变性和共变性两种变性方式并结合筛选合适的变性温度（设置70、80、90和98℃共变性与60、70、80和90℃分开变性），比较其杂交信号效果。结果表明，采用对二氯苯离体预处理，染色体形态呈粗短型，适用于以甘蔗基因组原位杂交计数来探讨染色体遗传模式的研究；采用α-溴代萘离体预处理和4℃低温水中非离体预处理，染色体形态较长，缢痕较明显，适用于染色体基因定位和核型分析等研究；80℃共变性和70~80℃的温度范围分开变性的杂交效果较佳；据筛选结果采用探针和染色体分开变性，染色体变性温度为80℃优化基因组原位杂交的体系来检验效果，α-溴代萘饱和水溶液预处理后所得的染色体较为分散，形态相对较长，45S rDNA定位信号强且分辨率高；对二氯苯饱和水溶液预处理后所得的染色体分散，形态较短小，荧光信号均一，说明优化的体系效果好。本研究为进一步开展相关的染色体荧光原位杂交提供技术平台，为甘蔗近缘属野生种质利用提供技术支撑，为远缘杂交后代材料的分子细胞遗传学深入研究提供基础资料。Optimizing an efficient system is the basis of related research by using fluorescent in situ hybridization. In this study, the key factors which effect on fluorescence in situ hybridization results, such as chromosomal pretreatment, denaturing conditions and denaturing ways, were screened in order to optimize a suitable system for fluorescent in situ hybridization in sugarcane（Saccharum）. Sugarcane root tips of YC96-66 which is F1 between Erianthus arundinaceus and Saccharum officinarum were pretreated with three following ways, p-dichlorobenzene under the condition of in vitro, α-bromidenaphthalene under the condition of in vitro and 4℃ low temperature water under the condition of non in vitro to screen chromosome morphology. The chromosome was denatured in two different ways with the optimal temperature on simultaneous denaturation at 70, 80, 90 and 98℃ and separate denaturation at 60, 70, 80 and 90℃ to compare the effects of hybridization signal. Results showed that chromosome morphology was stubby when using p-dichlorobenzene under the condition of in vitro which was suitable for sugarcane genomic in situ hybridization study on the count of chromosomal inheritance patterns research. Using the methods of α-bromonaphthalene under the condition of in vitro and 4℃ low temperature water under the condition of non in vitro, chromosome morphology was long and its centromere constriction was more obvious, so they both were suitable for chromosome mapping, karyotype analysis or other research. The effects of hybridization signal were better in the treatment of simultaneous denaturation at 80℃ and separate denaturation at temperature ranged from 70 to 80℃. Base on the screening results, separate denaturation at 80℃ was adopted to optimize an fluorescent in situ hybridization system. The effect of the system was detected using different pretreatment according the different aims. Pretreated with α-bromidenaphthalene, got the effect of longer, scattering chromosome, strong signal of 45S rDNA and high-

关 键 词：甘蔗 斑茅 荧光原位杂交 预处理 变性 

分 类 号：S511.6[农业科学—作物学]