摘要
利用自行设计的方法分离提取总RNA,通过一对特异引物,分别扩增出不同梨树品种上苹果锈果类病毒(ASSVd)的特异片段,经由回收、克隆和测序,有10条序列已在GenBank(EU031467-EU031476)登录,利用克隆的ASSVd质粒,通过RT-PCR合成了生物素标记的cDNA探针,建立了斑点杂交检测技术。在此基础上以已知带有苹果锈果类病毒的库尔勒香梨和无类病毒梨树叶片为材料,利用DIG标记,研究了梨树类病毒的叶片石蜡切片IS-RT-PCR检测技术。包括SuperScriptⅡ浓度、4种碱基混合物(dNTPs)、RNA酶抑制剂(RNasin)及互补引物浓度对cDNA合成的影响,退火温度、LA Taq DNA聚合酶、Mg2+、引物浓度及循环次数对原位PCR效果的影响。研究结果表明:RNasin的用量大于0.2U/μL时,信号强度随着RNasin量的加大而增强;只有当dNTPs浓度达0.4mmol/L时才能生成一定量的cDNA;SuperScriptⅡ浓度在0.5￣1.3U/μL均可进行正常的逆转录,而且在该范围内产物的量随SuperScriptⅡ浓度提高而增多;引物浓度达到0.9μmol/L以上时才能进行有效的逆转录,并且生成的cDNA的量随引物浓度增大而增加。原位扩增ASSVd的cDNA适合退火温度为62℃。循环30￣35次可出现较强的蓝色信号,引物浓度在0.8￣1.2μmol/L时显色较好;LA Taq酶浓度为0.004U/μL以上,均显示较深的蓝色;Mg2+浓度为1.5mmol/L就可满足原位PCR所需。
Total RNA was isolated using the method designed by ourselves. Special fragments of Apple Scar Skin Viroid （ASSVd） have been amplified by one specific pair of primer from different pear samples. The ASSVd specific fragment isolated from pear has been recovered, cloned and sequenced, the sequences of ten clones have been submitted to GenBank and sequence accession numbers were EU031467 to EU031476. Then biotin-labelled cDNA probe was synthesized using cloned plasmids by RT-PCR. The probe has been used well for spot hybridization detection of ASSVd, which further proved RT-PCR can be successfully used for the detection of ASSVd in pear. The detection technique of ASSVd pear viroid by IS-RT-PCR with digoxigenin labels for leaf paraffin slice samples and shoot tips frost slice samples was established in this paper. Both Korla pear leaves infected by ASSVd and viroid-free leaves of pear as control tests were used as leaves detection materials. The IS-PCR parameters including concentration of dNTPs, RNasin, LA Taq DNA polymerase, Mg^2＋ and primers, effect of SuperScript Ⅱ reverse transcriptase and complement primer on cDNA synthesis, annealing temperature and cycle number were studied systematically. The results showed the stain signal strength increased accompanied by an increase of RNasin amount when its concentration reached over 0.2 U/μL;Certain amount of cDNA would not be generated until the concentration of dNTPs reached 0.4 mmol/L in reaction solution;Reverse transcription could be carried out when the concentration of SuperScript Ⅱ reverse transcriptase was between 0.5 U/μL and 1.3 U/μL, and the quantity of cDNA production improved with the increase of the concentration of SuperScript Ⅱ;The reverse transcription could not be carried out until the concentration of antisense primer reached 0.9 μmol/L,and the amount of cDNA would increase along with the increase of primers concentration. The suitable annealing temperature for in situ amplification of cDNA was 62℃. The minimum amplification cycle
出处
《分子植物育种》
CAS
CSCD
2008年第4期812-818,共7页
Molecular Plant Breeding
基金
国家自然科学基金资助项目(30360066)
国家科技攻关计划引导项目(2003BA546C)
兵团科委项目(NKB02SDXNK01SW)
