Nodules Of Leguminous Crops And Soil ACC Deaminase Gene And Its Expression

Crop rotation and intercropping involving legume crops are traditional agricultural practices to increase soil fertility achieving higher yield and profit and allowing for sustained production. N contribution from legumes in crop rotation and intercropping has been suggested as the major factor responsible for the beneficial effect. Recent studies showed that H2produced as a byproduct of N2fixation in the legume crops can improve the growth and yield of the subsequent crops by changing soil bacterial community structure, especially the soil hydrogen-oxidizing bacteria which can use H2as energy and CO2as the carbon source to grow chemoautotrophically. Studies on soil hydrogen-oxidizing bacteria associated with legume nodules have suggested several possible plant growth-promoting mechanisms, such as producing ACC deaminase and rhizobitoxine to reduce the concentration of ethylene in plants. ACC deaminase activity has been detected from some hydrogen-oxidizing bacteria isolates. Inoculation of these isolates promoted spring wheat roots growth, arabidopsis leaf number, dry weight of both root and stem. This research aimed at detecting ACC deaminase gene DNA copy number and expression level by using Real-time PCR in8soil samples associated with different hydrogen treatment, respectively. Four DNA primer sets targeting ACC deaminase gene were designed and applied for each soil sample, separately. Results of the present work suggest that rhizospheric ACC deaminase activity stimulated by H2exposure plays an important role in rotation and intercropping with legume plants.The main results and conclusions are as follows:1. The results of ACC deaminase DNA copy number detection showed,(1) a significant increase of DNA copy number after H2treatment (H).(2) DNA copy number of soil sample adjacent to HUP-nodules (JH47) went up more dramatically than that in soil sample ajacent to HUP+nodules (JH). The possible reason is that H2released from HUP-nodules promoted population growth of rhizospheric hydrogen-oxidizing bacteria that contain and express the ACC deaminase gene.(3)In the field trial, more ACC deaminase gene copy was also detected in soil sample adjacent to fababean nodules (RF) than that in bulk soil sample (RFC).(4)Rhizosphere soil of barley growing in H2-treated soil (RIC) showed higher ACC deaminase gene copies than that of soil far away from barley roots (ROC). The increased ACC deaminase gene copy number indicates changes of soil bacterial community around barley roots suggesting interaction between plant roots and hydrogen-oxidizing bacteria.2. The bacterial community structure in5different H2-treated levels all showed that the quantity of Mycobacterium is the highest one among detected bacterial groups. The following ones are Variovorax and Burkholderia. It suggests that Mycobacterium containing ACC deaminase gene is the most sensitive one to increase its bacterial quantity by consuming H2.3. Expression level of target gene is usually represented by quantity of mRNA in samples, which can be measured by real-time quantitative reverse transcriptional PCR techniques. The mean cDNA copy number in soil samples exposed to H2(H, JH47, RF and RIC) are all enhanced. It indicates ACC deaminase gene expression level is stimulated by different hydrogen treatment.4. The ACC deaminase expression level of detected bacterial groups in5different H2-treated levels all showed that the highest expression level is Variovorax. The following ones are Mycobacterium and Burkholderia. It suggests that Variovorax containing ACC deaminase gene is the most sensitive one to be stimulated to express ACC deaminase gene after H2treatment.5. ACC deaminase gene was detected in8hydrogen-oxidizing bacterial isolates belonging to Variovorax and Burkholderia, separately, although the ACC deaminase activity was not detected in Burkholderia isolates on culture. More study of ACC deaminase gene expression of Burkholderia in soil is needed to understand the plant growth-promoting mechanisms of these isolates.