Preliminary Study On The Expression Profile Of Flag Leaf Senescence Associated Genes In Wheat (Triticum Aestivum L.)

Wheat is the world’s important grain crop, and one of three major grain crops in China as well. With population growth and the reduction of land resources, the annual growth rate of wheat yield growth rate has been lower than its consumption. Therefore, it is very important to increase its yield and improve its quality. Leaf senescence (especially the flag leaf senescence) is one of the important factors to limit production. Flag leaf senescence of wheat grain formation and filling their mature simultaneously, premature senescence of flag leaf will seriously affect the wheat yield, while the delayed flag leaf senescence, delaying the aging process can significantly improve wheat yield and improve grain quality."Shiluan 02-1 ", a high quality cultivar containing high gluten, co-cultivated by Hebei Normal University and Original species Station of Luancheng County, has been planted widely recent years. "Shiluan 02-1" shows premature leaf senescence, which affected the yield. In this study, DNA microarray was performed to elucidate the transcriptional differences during wheat flag leaf senescence process, and senescence associated ESTs were isolated and identified. The data provide the insights into regulation mechanisms of leaf senescence.Four different developmental stages (S1, young leaves which area is about half of the mature leaf; S2, mature leaves; S3, senescing leaves with less than 10% of yellowing; S4, sensing leaves with about 50% of yellowing) of the flag leaf were used as materials in our study. Transcription level analysis was performed by using Affymetrix gene chips. Wheat 61115 non-repeat sequence hybridization sites were analyzed by the ANOVA method. Compare with S1, there were 1151 ESTs showed significant differences in S2, S3 and S4 stage. There were 306 ESTs showed significant differences in S2 vs. S1, S3 vs. S2, and S4 vs. S3 point-to-point compare. According to NCBI, CerealsDB and other databases information, these ESTs were further annotated, analyzed and their function were predicted. Most of them are senescence process associated. They are involved in the degradation of chlorophyll, nucleic acid degradation, protein degradation, carbohydrate metabolism, signal transduction, stress defense, nutrition, transport and other processes, including the WAKY, NAC, MYB transcription factors, MRPK, PP2C, which are known to play important roles in leaf senescence. These ESTs could be divided into 3 or 6 classes based on our cluster analysis(hierarchical clustering, complete linkage).Some senescence associated transcription factors obtained by Microarray were further analyzed by Real-time fluorescence quantitative RT-PCR. Our data showed these transcription factors show higher expression levels in senescing flag leaves, and their expression patterns are consistent with microarray results. It indicated that our microarray data are accurate and reliable.MicroRNA exists in plants, insects and mammals, can be paired with the target complementary mRNA molecules resulting in the translation of the mRNA molecule or degradation inhibition, which plays an important regulatory role in the growth and development. We used CBI_PKU, miRBase, PMRD microRNA database and genomic information analysis methods to predict microRNAs regulating on senescence associated transcription factors. Some microRNAs were analyzed by using stem-loop quantitative PCR. Compare to their target transcription factors, these microRNAs showed opposite expression patterns. It further confirmed that our microarray data are reliable and accurate. Furthermore, it suggested these microRNAs play important roles in regulating leaf senescence.In conclusion, transcription microarray was performed using four different developmental stages of wheat flag leaves in this study. Transcriptional level differences were analyzed between senescing and non-senescing leaves, some senescence associated ESTs were obtained accordingly. Microarray data were confirmed by using real time quantitative RT-PCR method and microRNA analysis. Some transaction factors and microRNA may play important roles in regulating leave senescence were identified, while their functions need to be further analyzed in future research.