Using Kuala pear leaves and cortexes as materials, the total RNA was extracted using two methods and these two methods were compared. The most suited methods for Kuala pear were screened; and biotin-labeled cDNA probe...Using Kuala pear leaves and cortexes as materials, the total RNA was extracted using two methods and these two methods were compared. The most suited methods for Kuala pear were screened; and biotin-labeled cDNA probe was synthesized using RT-PCR. The main factors that affected the sensitivity of hybridization were studied. Studies indicated that the highest sensitivity was obtained under the following conditions： probe concentration 400 ng mL^-1, formamide concentration 45%, temperature 42℃, hybridization time 6 hours. The best hybridization results were obtained when the nitrocellulose membrane purchased from Gelman was used. Better blocking of hybridization was obtained using Tween 20 compared with albumin in bovine. The detection of the total RNA using different tissues and different extraction methods was compared. This study indicates that the total RNA of fresh leaf, old leaf, cortexes, and frozen leaf showed signs of hybridization using the two extraction methods.展开更多
Young leaves and shoot with Apple stem pitting virus（ASPV） detected by RT-PCR（reverse transcription-polymerase chain reaction） from Korla pear were used as materials and total RNA was extracted.Two expected fragme...Young leaves and shoot with Apple stem pitting virus（ASPV） detected by RT-PCR（reverse transcription-polymerase chain reaction） from Korla pear were used as materials and total RNA was extracted.Two expected fragments had been obtained by RT-PCR with two specific pairs of primers based on the sequence of NC003462 published in GenBank.The specific fragments had been recovered and cloned,and the white colonies had been screened out.After enzyme digestion of the plasmids isolated,the positive clone was selected to be sequenced.Compared with NC003462,three ORFs（open reading frame）-ORF2,ORF3 and ORF4 of ASPV with 673 bp,365 bp,276 bp representing homology of 77%,88.52%,86.33% respectively had been obtained.展开更多
基金supported by the National Natural Science Foundation of China(30060053).
文摘Using Kuala pear leaves and cortexes as materials, the total RNA was extracted using two methods and these two methods were compared. The most suited methods for Kuala pear were screened; and biotin-labeled cDNA probe was synthesized using RT-PCR. The main factors that affected the sensitivity of hybridization were studied. Studies indicated that the highest sensitivity was obtained under the following conditions： probe concentration 400 ng mL^-1, formamide concentration 45%, temperature 42℃, hybridization time 6 hours. The best hybridization results were obtained when the nitrocellulose membrane purchased from Gelman was used. Better blocking of hybridization was obtained using Tween 20 compared with albumin in bovine. The detection of the total RNA using different tissues and different extraction methods was compared. This study indicates that the total RNA of fresh leaf, old leaf, cortexes, and frozen leaf showed signs of hybridization using the two extraction methods.
文摘Young leaves and shoot with Apple stem pitting virus（ASPV） detected by RT-PCR（reverse transcription-polymerase chain reaction） from Korla pear were used as materials and total RNA was extracted.Two expected fragments had been obtained by RT-PCR with two specific pairs of primers based on the sequence of NC003462 published in GenBank.The specific fragments had been recovered and cloned,and the white colonies had been screened out.After enzyme digestion of the plasmids isolated,the positive clone was selected to be sequenced.Compared with NC003462,three ORFs（open reading frame）-ORF2,ORF3 and ORF4 of ASPV with 673 bp,365 bp,276 bp representing homology of 77%,88.52%,86.33% respectively had been obtained.
