Suppression Subtractive Hybridization Cdna Library Construction For Low Temperature-Treated Poncirus Trifoliata And Characterization Of Ptrbam1

The planting area and yield of most commercially important cultivars are severely affected by low temperature and it is difficult to obtain new cold tolerant cultivars through conventional breeding. Genetic improvement could be a robust alternative approach, but the premise is the availability of desired candidate genes. Poncirus trifoliata (L.) Raf (Rutaceae) is a kind of deciduous shrub or small tree, widely used as rootstock in citrus planting. After full cold acclimation, it can tolerate temperature lower than-20℃and it is comparatively more cold tolerant than most commercial citrus cultivars. However, limited understandings about its molecular and physiological mechanisms have been obtained. In this study, suppression subtractive hybridization (SSH) was used to isolate cold responsive genes in4℃and25℃-treated Poncirus leaves. Several ESTs annotated as β-amylase were identified, among which, the full length of the EST showed high similarity to AtBAM3was cloned, designated as PtrBAMl. Subsequently, the ORFs of other β-amylase genes in Poncirus were obtained. The entire gene family was analyzed by bioinformatics and transcriptional expression profiles under different treatments were portrayed. Out of the8, PtrBAM1was introduced into tobacco for functional characterization. The main results are as follows:1. Three-month-old Poncirus leaves were treated under4℃and25℃, respectively. The RNA sampled at5time points (6h,1d,3d,5d,7d) of each temperature was mixed and used for mRNA isolation. The4℃and25℃samples were used as tester and driver, respectively, in the forward library construction and driver and tester in the reverse library. Based on the hybridization efficiency and reverse Northern blotting, the clones showed more than2fold expression changes were sequenced and analyzed. One hundred and eighty eight and180ESTs were successfully sequenced in the forward and reverse libraries, respectively. Assembly results revealed106sequences (75singletons and31contigs) in the forward library, while121sequences (98singletons and23contigs) in the reverse library. The ESTs were analyzed via Blast2go in3aspects:molecular function, biological process and cellular component.Six ESTs were selected from each library for semi-quantitative PCR analysis based on their involvement in membrane integrity protection, excess ROS scavenge and osmotic balance adjustment. The RT-PCR results showed that they were cold responsive and the physiological activities of β-amylase and GST were consistent with their transcriptional expressions.2. The full length of one β-amylase EST from the forward library was obtained via RACE, designated as PtrBAM1and the other7β-amylase genes in Poncirus were cloned subsequently based on homology search, named as PtrBAM2-8. The β-amylase gene family of Poncirus can be categorized into4groups (Ⅰ:PtrBAM5; Ⅱ:PtrBAM1-3and8; Ⅲ:PtrBAM4; Ⅳ:PtrBAM6and7). According to the alignment and the known information, we assumed that PtrBAMl plays the predominant role in starch degradation and PtrBAM6and7are potential transcription factors. The8Poncirus β-amylase genes showed different expression patterns under various treatments (low temperature, dehydration, NaCl, Maltose, ABA), which implied that versatile physiological functions existed among this gene family.3. Starch staining and transmission microscope results revealed that overexpression tobacco lines (F6, F11, F25) exhibited obvious lower starch accumulation than Nud (wild type) and1301(empty vector) lines, while RNAi lines (bam1L1and L2) accumulated more starch than the control. In addition,β-amylase activity, maltose and soluble sugar contents were higher in F6, F11and F25than that in Nud and1301under normal condition. These results demonstrated that PtrBAM1possessed a high activity in starch degradation. The overexpression tobacco lines survived the exposure to-4℃for1h but the controls failed. Moreover, the formers showed lower EL and MDA content as well as less O2-and H2O2accumulation under2℃. Because β-amylase activity, maltose and soluble sugar contents were higher in the overexpression lines after exposure to2℃for6h,1d,3d, implying that PtrBAMl may increase sugars as osmolytes that effectively protected membrane integrity and reduced ROS accumulation, thus improved the cold tolerance of the transgenic plants. In addition, yeast one-hybrid result revealed that PtrBAMl was under the regulation of CBF. Taken together, the molecular function of PtrBAM1was preliminarily characterized in2aspects:gene expression regulation in the upstream and metabolites synthesis and degradation in the downstream.