Identification and characterization of differentially expressed genes from tobacco roots after decapitation

Abstract

Nicotine is the major alkaloid in tobacco and its biosynthesis is regulated by a variety of factors. Topping, as an important agronomic factor, can induce the nicotine biosynthesis increase. Some key nicotine biosynthesisrelated genes and the framework of nicotine biosynthesis pathway have been well studied, but the details of nicotine biosynthesis pathway are not well understood now. To investigate the genes expressed after tobacco topping, we constructed a suppression subtractive hybridization library using cDNA from control tobacco plants as driver and those from topped tobacco plants as tester. The insert size of positive clones was 200-1,000 bp confirmed by PCR. After differential screening, 560 significantly differently expressed clones among 1,950 positive clones were acquired, sequenced and 273 high quality expressed sequence tags (ESTs) were acquired. The results of nucleotide blast homological analysis indicated that these ESTs mainly involved in alkaloid biosynthesis (4%), plant hormone metabolism (3%), signaling/transcription (18%), stress/ defense (32%), protein metabolism (9%), carbon metabolism (6%), other metabolism (15%) and function unknown (13%). The expression of selected genes was analyzed by reverse transcription polymerase chain reaction and RNA gel blot hybridization, and the result indicated that their transcription amount increased after tobacco topping. NtNAC-R1was in silico cloned, and the expression level of NtNAC-R1 increased at 12 and 24 h in tobacco roots after topping, which indicated that NtNAC-R1 may play an important role in the signal transduction after tobacco topping. In addition to many previously reported nicotine biosynthesis-related genes, some new genes, such as transcription factors related to nicotine biosynthesis/regulation and the members of plant hormone pathway, were discovered in our library. The results contribute new data to the list of possible candidate genes involved in nicotine biosynthesis and regulation.