| The brown planthopper (BPH) Nilaparvata lugens Sta.1 is an important pest of rice. The BPH feeds mainly on the stems, and sucks assimilates from the phloem. BPH feeding interferes with the translocation of photosynthates from source to sink, hence, affects plant and development. Feeding by a large number of BPH may result in drying of the leaves and wilting of the tiller, a condition called hopperburn.The forward- and reverse- suppression subtractive libraries were constructed by using rice seedlings exposed to BPH for 32 h and control plants as materials. Forward subtraction was performed using cDNA synthesized from BPH-infested plants as tester and that from the control plants as driver to enrich genes that are induced by BPH infestation. Reverse subtraction were performed using cDNA from control plants as tester and that from BPH-infested plants as driver aiming to enrich genes that are suppressed by BPH infestation. Sixteen white bacterial colonies were randomly picked up form forwardsubtraction cDNA library, and analyzed by PCR method using T7 and M13 reverse primer. The result showed they contained 100~900 bp inserts. To analyze the efficiency of subtraction, dot blot was conducted by using BpHi008A as probe, which was expressed only in BPH-infested rice seedlings. The result demonstrated that the differential expression genes were enriched in subtracted cDNA pool. The forward- and reverse-suppression subtractive libraries lay solid foundation for screening and cloning genes regulated by BPH feeding, and for analyzing the responses of rice seedlings to BPH attack.27 BPH-responsive genes in rice were isolated from forward- and reverse- SSH libraries by reverse total RNA dot blot analysis and confirmed by Northern blot analysis. Among these genes, 25 were induced and 2 were suppressed by BPH feeding. Sequence similarity searches allowed putative functions to be assigned to 19 transcripts. These genes were placed into several functional categories including cell growth photosynthesis, macromolecule degradation, signaling, and a group responding to stress or invasion of pathogens. 6 clones did not match any protein sequences in the databases. Furthermore, 2 clones were novel rice cDNAs that did not have a match with the rice EST databases. Overall, transcripts involved in senescence and stress in plants were induced, whereas those involved in photosynthesis and cell growth were suppressed. In light of the damage caused by BPH and the differential genes isolated in this study, it can be speculated that the deficiency of sucrose caused by BPH infestation activated senescence program in rice seedlings.A cDNA library was constructed using rice seedlings exposed to BPH for 32 h as materials. The primary library contains 3. 2X106 clones. The rate of recombination is 86%. 1. OX 106 clones were amplified, and80ml amplified library was obtained. The titer of amplified library is 1.2X109 pfu/mL. To estimate the length of inserts, 20 clones were selected randomly and analyzed by PCR method using T7 and M13 reverse primer. The result demonstrated that the size of most inserts is 1-4 Kb, some inserts is larger than 4 Kb, and a few even larger than 6 Kb.The cDNA library was screen using three differentially expressed EST, BpHi008, BpHd002 and BpHi006 as probes, respectively. The full-length cDNAs of these EST were obtained and designated as BpHi008A, BpHd002A and BpHiOO6A, respectively.Bpffi008A is 504 bp in length, and contains one putative open reading frame that encodes 82 amino acids. The protein exhibits about 37% amino acid sequence identity to Wir1 family of proteins that are encoded by pathogen-induced transcripts in wheat. Like Wirl proteins, it consists of a hydrophobic N-terminal half and a hydrophilic Oterminal half relatively rich in glycine and proline. These proteins are predicted to be integrated into the membrane, with the Oterminus being extracytoplastic. Genomic Southern analysis indicated that the BpHi008A gene was present as a single-copy sequence in the rice genome. Temporal and spatial st... |
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