| Salinity soil is a worldwide problem threatening crop production and leads to reduction in the yield of a wide variety of crops all over the world. Wheat (Triticum aestivum L.) is one of the most important crop. Salinity soil limits wheat production as other crops. The study on wheat salt-tolerant genes is helpful to understand the mechanism of wheat salt tolerance and the breeding of salt-tolerant wheat. Na~+ and Cl~- in salinity soil is the major factor of inhibiting plant growth. But for most plants, Na~+ is the primary cause of ion-specific damage.Thereafter, cloning Na~+-uptake related genes and analysing their natural diversity in wheat are valuable to understand their function in wheat salt-tolerant mechanism and improve wheat salt tolerance by traditional breeding and genetic engineering.Based on wheat TaHKT1(high-affinity potassium uptake transporter) cDNA sequence, specific primers were designated. TaHKT1 structure was proved by comparing TaHKT1 genomic sequence and cDNA sequence cloned from wheat {Triticum aestivum L. cv. Chinese Spring). TaHKT1 gene is organized with three exons and two introns. Five TaHKT1 kinds found in 38 wheat accessions were named TaHKT1 -1, TaHKT1-2, TaHKT1-3, TaHKT1-4 and TaHKT1-5. TaHKT1-1, TaHKT1-2, TaHKT1-3, TaHKT1-4 cDNA were cloned from different wheat accessions. TaHKT1-5 might be a pseudogene because its corresponding cDNA cannot be found and a premature stop code exists in HKT1-5 coding sequence according to structural prediction. 19 TaHKT1 kinds were cloned from wheat wild relatives. TaHKT1 sequence diversity of wheat wild relatives is greater than that of common wheat. Sequence diversity analysis of wheat relatives, landraces and varieties shows TaHKT1 belongs to domestication gene.Single BAC clones containing TaHKT1, TaHKT2 and TaHKT3 respectively were isolated by screening Aegilops tauschii AL8/78 BAC library (DD )and Aibai Chinese Spring wheat BAC library(AABBDD) based on the primers designated according to TaHKT1 cDNA sequence. Nucleotide sequence of TaHKT2, TaHKT3 and flanking sequence of TaHKT1, TaHKT2 and TaHKT3 were cloned by BAC plasmid extending sequencing. Two TaHKT2 kinds TaHKT2-l, TaHKT2-2 and three TaHKT3 kinds, TaHKT3-l, TaHKT3-2 and TaHKT3-3 were cloned in 13 wheat accessions. Two-base deletion in TaHKT2 5'-UTR may lead to TaHKT2 silence. TaHKT4 and its flanking sequence were cloned from wheat Laizhou953. Five kinds were cloned from 13 wheat accessions.They were named TaHKT4-l, TaHKT4-2, TaHKT4-3, TaHKT4-4 and TaHKT4-5. TaHKT2, TaHKT3 and TaHKT4 were located on wheat chromosome 7D, 7B and 7A respectively.Based on nce(Oryza sativa L.)SKC1(shoot K~+ content)cDNA, two wheat ESTs were found in GenBank database. Based on specific primer designated according to wheat EST sequence, AeSKCl was isolated by screening Aegilops tauschii AL8/78 BAC library. TaSKC1 was cloned on the basis of Aegilops tauschii AL8/78 AeSKCl. Two TaSKC1 kinds cloned in 27 accessions were named TaSKC1-1 and TaSKC1-2. Only one-base mutation was found in first intron between TaSKC1-1 and TaSKC1-2.Five TaSKCl kinds were found in 8 synthetic double-diploids, three of five different from TaSKCl. Study on TaSKCl natural diversity of synthetic double-diploids, landraces and varieties shows that bottleneck effect is obvious in the process of TaSKCl domestication, so TaSKCl belongs to domestication gene. Single BAC clone containing TaSKC2 was isolated by screening Aibai Chinese Spring wheat BAC library based on the primers designated according to TaSKCl. TaSKC2 and its flanking sequence were cloned by BAC plasmid extending sequencing. Natural diversity of wheat TaSKC2 was studied. Two TaSKC2, TaSKC2-l and TaSKC2-2, were found in 12 wheat accessions. But more TaSKC2 sequences were found in three landraces including Chadianhong, Xiaobaimai and Guchengying. TaSKC2 may be silent gene because its corresponding cDNA cannot be found.The TaHKTl (TaHKTl-1, TaHKTl-2 and TaHKTl-3), TaHKT3 and TaHKT4 yeast (Saccharomyces cerevisiae) expression vectors were constructed. Transgenic yeasts (Na+ sensitivity strain G19) harbouring TaHKTl (TaHKTl-1, TaHKTl-2 and TaHKTl-3), TaHKT3, TaHKT4 and pYES2 were generated by LiAc/SS Carrier DNA/PEG method. Na+ uptake ability of wheat HKT family members is ranked as TaHKT4-2 |
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