Affiliation:
1. National Key Laboratory for Germplasm Innovation and Utilization of Horticultural Crops, College of Horticulture and Forestry Sciences Huazhong Agricultural University Wuhan 430070 China
2. Hubei Hongshan Laboratory Wuhan Hubei 430070 China
Abstract
SUMMARYSmall RNAs play important roles in regulation of plant development and response to various stresses. Northern blot is an important technique in small RNA research. Isotope‐ and biotin‐ (or digoxigenin) labeled probes are frequently used in small RNA northern blot. However, isotope‐based probe is limited by strict environmental regulation and availability in many places in the world while biotin‐based probe is usually suffered from low sensitivity. In this study, we developed a T4 DNA polymerase‐based method for incorporation of a cluster of 33 biotin‐labeled C in small RNA probe (T4BC33 probe). T4BC33 probe reaches similar sensitivity as 32P‐labeled probe in dot blot and small RNA northern blot experiments. Addition of locked nucleic acids in T4BC33 probe further enhanced its sensitivity in detecting low‐abundance miRNAs. With newly developed northern blot method, expression of miR6027 and miR6149 family members was validated. Northern blot analysis also confirmed the successful application of virus‐based miRNA silencing in pepper, knocking down accumulation of Can‐miR6027a and Can‐miR6149L. Importantly, further analysis showed that knocking‐down Can‐miR6027a led to upregulation of a nucleotide binding‐leucine rich repeat domain protein coding gene (CaRLb1) and increased immunity against Phytophthora capsici in pepper leaves. Our study provided a highly sensitive and convenient method for sRNA research and identified new targets for genetic improvement of pepper immunity against P. capsici.
Funder
National Natural Science Foundation of China
National Key Research and Development Program of China
Subject
Cell Biology,Plant Science,Genetics
Cited by
3 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献