Molecular cloning and characterization of NPR1 gene from Arachis hypogaea.

The NPR1 gene was an important regulator for a plant disease resistance. The cDNA of NPR1 gene was cloned from peanut cultivar Ri Hua 1 by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The full length cDNA of Arachis hypogaea NPR1 consisted of 2,078 base pairs with a 1,446 bp open-reading frame encoding 481 amino acids. The predicted NPR1 contained the highly conserved functional domains (BTB/POZ domain from M1 to D116), protein-protein interaction domains (three ankyrin repeats from K158 to L186; N187 to L217 and R221 to D250) and one NPR1-like domain (C262 to S469). The DNA sequence of the NPR1 gene was 2,332 or 2,223 bp. Both two sequences contained three introns and four exons. The NPR1 transcripts were expressed mainly in roots and leaves, while fewer signals were detected in the stems. Amount of the NPR1 transcript was significantly increased 1 h after salicylic acid challenge and was eventually 5.3 times greater than that in the control group. Both the DNA sequence and the coding sequence were obtained from eight cultivars and nine wild species of Arachis. Maximum likelihood analyses of d N/d S ratios for 25 sequences from different species showed that different selection pressures may have acted on different branches.

Isolation and analysis of differentially expressed genes from peanut in response to challenge with Ralstonia solanacearum

Background: Bacterial wilt caused by Ralstonia solanacearum is the most devastating disease in peanut. Planting resistant peanut cultivars is deemed as the sole economically viable means for effective control of the disease. To understand the molecular mechanism underlying resistance and facilitate breeding process, differences in gene expression between seeds of Rihua 1 (a Virginia type peanut variety resistant to bacterial wilt) inoculated with the bacterial pathogen suspension (10(9) cfu ml-1) and seeds of the same cultivar treated with water (control), were studied using the GenefishingTM technology. Results: A total of 25 differentially expressed genes were isolated. Expression of genes encoding cyclophilin and ADP-ribosylation factor, respectively, were further studied by real time RT-PCR, and full length cDNAs of both genes were obtained by rapid amplification of cDNA ends. Conclusions: The study provided candidate genes potentially useful for breeding peanut cultivars with both high yield and bacterial wilt resistance, although confirmation of their functions through transgenic studies is still needed.

Identification of a novel mutation in FAD2B from a peanut EMS mutant with elevated oleate content.

A Virginia type peanut mutant with more than 60% oleate content (E2-4-83-12) was selected from an EMS mutagenized population of LF2 (an export type peanut cultivar with 44.2% oleate) by near infrared reflectance spectroscopy. Cloning and sequencing of FAD2B from LF2 and E2-4-83-12 identified a novel mutation (C313T in the coding region) causing an H105Y substitution in the first histidine box of the FAD2B protein. GC-MS analysis of fatty acids in yeast cells harboring pYES2 with the mutated FAD2B detected no linoleate, confirming that FAD2B from E2-4-83-12 was dysfunctional. Loss-of-function FAD2A and FAD2B together contributed to elevated oleate phenotype of the peanut EMS mutant.