[Nucleotide polymorphism in the drought induced transcription factor CBF4 region of Arabidopsis thaliana and its molecular evolution analyses].

Intraspecific nucleotide polymorphism in the drought induced transcription factor CBF4 region of Arabidopsis thaliana was analyzed with 17 core accessions growing in different ecoclimate. High density of single nucleotide polymorphism (SNP) and insertion/deletion (Indel) were found, on average 1 SNP per 35.8 bp and 1 Indel per 143 bp. Nucleotide polymorphism in non-coding region was three times higher than that in coding region. In coding region of CBF4, SNP frequency is one SNP per 96.4 bp, one nonsynonymous mutation was detected from 25 av, 203 av and 244 av accessions, which is the 205th site amino acid variation: gly <--> val caused by the 1034th site (corresponding to 19,696 site nucleotide of GenBank No. AB015478 as 1) nucleotide variation: G <--> T. Statistical result of nucleotide diversity showed that linkage disequilibrium (LD) existed in large-scale region of CBF4 and recombination event was also detected in 5' non-coding region. Identical to the results of other genes of Arabidopsis, different regions of the gene were seemingly under different selective pressures. Balancing selection resulted in high nucleotide diversity in 3' non-coding region, and the neutral mutation hypothesis can explain the DNA polymorphism in coding region, whereas, nature positive selection in the population affected nucleotide variation in 5' non-coding region of gene.

[ATHK1 gene regulates signal transduction of osmotic stress in Arabidopsis thaliana].

Differences in physiology and gene expression between ATHK1 knock-out mutant caused by T-DNA insertion and wild type (WT) of WS accession of Arabidopsis thaliana were analysed. Water loss ratio of detached leaf of ATHK1-mutant was obviously higher than that of WT. After being treated with 30% PEG-6000, ion leakage ratio of cell membrane in wild type leaves was 50% higher than that before PEG treatment, while in mutant leaves it increased 80%. The wilted phenotype of ATHK1-mutant after PEG treatment for 48 h was higher than that of WT. All these results showed that ATHK1-mutant was more sensitive to osmotic stress compared to WT and ATHK1 involved in osmotic stress adaptation. Differential-Display Reverse Transcription-PCR (DDRT-PCR) analysis was carried out to investigate the difference of gene expression between ATHK1-mutant and WT. Nine differential cDNA fragments involved in stress adaptation were identified, including the MAPKKK18 and serine/threonine protein kinase genes. These fragments were up-regulated by PEG treatment in WT, but not in ATHK1-mutant. These results indicate that ATHK1 plays an important role up-stream from MAPK in the osmotic stress signal transduction pathway. ATHK1 may be working as a plant osmosensor.

Development of SSR markers for the phylogenetic analysis of almond trees from China and the Mediterranean region.

Expressed sequence tag (EST) derived simple sequence repeats (SSRs, microsatellites) were screened and identified from 3863 almond and 10 185 peach EST sequences, and the spectra of SSRs in the non-redundant EST sequences were investigated after sequence assembly. One hundred seventy-eight (12.07%) almond SSRs and 497 (9.97%) peach SSRs were detected. The EST-SSR occurs every 4.97 kb in almond ESTs and 6.57 kb in peach, and SSRs with di- and trinucleotide repeat motifs are the most abundant in both almond and peach ESTs. Twenty one EST-SSRs were thereafter, developed and used together with 7 genomic SSRs, to study the genetic relationship among 36 almond (P. communis Fritsch.) cultivars from China and the Mediterranean area, as well as 8 accessions of other related species from the genus Prunus. Both EST-derived and genomic SSR markers showed high cross-species transferability in the genus. Out of the 112 polymorphic alleles detected in the 36 cultivated almonds, 28 are specific to Chinese cultivars and 25 to the others. The 44 accessions were clustered into 4 groups in the phylogenetic tree and the 36 almond cultivars formed two distinct subgroups, one containing only Chinese cultivars and one of unknown origin and the other only those originating from the Mediterranean area, indicating that Chinese almond cultivars have a distinct evolutionary history from the Mediterranean almond. Our preliminary results indicated that common almond was more closely related to peach (P. persica (L.) Batsch.) than to the four wild species of almond, (P. mongolica Maxim., P. ledebouriana Schleche, P. tangutica Batal., and P. triloba Lindl.). The implications of these SSR markers for evolutionary analysis and molecular mapping of Prunus species are discussed.

Betaine improves LA-PCR amplification.

PCR is a powerful tool for the amplification of genetic sequences. It has been widely applied in molecular biology. It is generally used to amplify short segments (several hundreds basepairs to several kilobasepairs). It is difficult to amplify a long DNA segment. Based on the sequenced genes, it is known that most intact genes are very long. And intact gene is very important for the gene to express specially and effectively. Long PCR is a very useful tool to amplify intact genes for constructing special expression vectors. We have tried several chemicals to optimize long PCR system and found betaine was the best. Betaine, as an amino acid analogue with small tetraalkylammonium ions, could remarkably improve the amplification of long targets from the plant genome. The suitable concentration of betaine was between 1.0 mol/L and 2.5mol/L. We could effectively amplify a 9 kb DNA segment from maize genome DNA and a 16 kb DNA segment from plasmid. It was shown that different primers and different targets (different GC content) needed different concentrations of betaine. Betaine can reduce or eliminate non-special amplification. In the meantime we tried other additive chemicals, such as DMSO, glycerin, formamide. They were no notable results in long PCR.