Optimization Of Chromosome-specific BAC Library Construction And Biological Function Determination Of Stress Resistance-related Genes In Common Wheat (Triticum Aestivum L.)

Bacterial artificial chromosomes (BACs) are widely used for the construction of physical maps and sequencing for the whole genome, positional-cloning genes and the study on gene structure and regulation strategies. The study takes in-depth investigation into the chromosome sorting and construction of BIBAC library with the high-molecular-weight DNA from sorted chromosomes composing composite peaks to present a useful technique for subsequent construction of chromosome 5DL-specific library in wheat (Triticum aestivum L.). The results were summarized as the following:1,The result of synchronized root tips of common wheat ditelosomic line 5DL: Over 85% cells can be synchronized after subjected to 1.25 mM HU treatment for 18 h and 6-h recovery treatment. In the following treatment with 1μM trifluralin for 2.5 h, the mitotic indice can reach more than 60%.2,Acquisition of high-quality chromosome suspension: The chromosome suspension released from homogenization suited for flow karyotypic analysis and sorting after fixed in 2% paraformaldehyde for 20 minutes and purified through 10% and 30% sucrose concentration gradient. The result showed the intact chromosomes, little debris and few nuclei in suspension.3,Flow karyotype of common wheat ditelosomic line 5DL: The result showed an additional peak on the flow histogram of common wheat ditelosomic line 5DL besides 4 peaks similar to the flow karytotype of common wheat.4,Chromosome 5DL sorting and determination: The chromosomes from additional peak were confirmed to be chromosome 5DL by PCR with specific primer pair Xgwm174 in combination with microscope detection. The 233-bp band can be attained, which confirmed the sorted chromosomes were just the chromosome 5DL.5,Achievement of HMW DNA from sorted chromosomes: Partial digestion of plugs made from chromosomes of composite peaks with 2 U BamHI for 3~10 minutes can get 50~300 kb HMW DNA in a large number.6,High capacity of BIBAC vector affirmation: Full digestion of recombinators from random 50 clones with NotI displayed that the insert fragment can reach 100 kb or so. The five optimized techniques can be applied to the construction of chromosome-specific BAC libraries.Dehydration, high salinity and low temperature are three major abiotic stresses affecting crop growth and field. Na+ vacuolar compartment plays an important role in adaption to Na+ toxity in plants, especially in pehalophyte. The study concentrated on the molecular biological functions of Na+/H+ antiporter and some kinases like CDPKs and MAPKs responsive to adverse conditions. The findings were covered as the following:1,The expression mode of Na+/H+ antiporter: RT-PCR method was applied with cDNAs through reverse transcription using RNA extracted from dehydration-, high salinity-, low temperature- and ABA-treated common wheat cv. Chadianhong. The result showed the expression of Na+/H+ antiporter was in relation to the above mentioned three abiotic treatments but not to ABA stress.2,Bioinformatic analysis of Na+/H+ antiporter: Na+/H+ antiporter ORF encompassed 11 transmembrane domains using DNAMAN software.3,Subcelluar localization of Na+/H+ antiporter: The gene encoding Na+/H+ exchanger protein was fused to N-terminus of green fluorescence protein (GFP) and expressed in epidermal cells of onions by bombardment particle, then was detected under confocal microscope. The result manifested Na+/H+ antiporter was mainly localized in the membrane of vacuole.4,Expression mode of CDPK1 and CDPK2: CDPK1 and CDPK2 was related to all the four stresses by RT-PCR.5,Prokaryotypic expression of CDPK1 and MAPKs (MAPK1 and MAPK2): With the elongation of IPTG induction time, the expression of CDPK1 and MAPKs in E. coli.strain BL21 can be enhanced by fusing to the C-terminus of Gluthathione-S-Transferase (GST) in the vector pGEX-4T-1. The study can make preparations for the following activating experiment of CDPK-like and MAPK-like kinases.To some extent, the research may reinforce the understanding on the molecular mechanism of dehydration-, high salinity- and low temperature-stress resistance and provide reference for stress resistance-related breeding in wheat.