Comparative Analyses Reveal Potential Uses Of Brachypodium Distachyon As A Model For Cold Stress Responses In Pooideae

Brachypodium distachyon is a phylogenetic sister group to the temperate cereals (including Bromus hordeaceus, Hordeum vulgure, Triticum aestivum and Secale cereale) and forage grasses (Avena sativa, Lolium perenne, Festuca rubra, Deschampsia cespitosa and Poa parentis included) tribes. And the whole genome sequence of B. distachyon has been fully finished which is published on http://www.Brachypodium.org in last year. B. distachyon has all the features of a good model plant, like Arabidopsis thaliana. Especially B. distachyon has a more related relationship to cool cereals than rice (Oryza sativa). In one word, these features make B. distachyon a better model plant for studying agronomic traits in Pooideae than rice. But little is known about how does B. distachyon deal with low temperature and the differences between B. distachyon and core pooideae plants on moleculeur reactions to cold. In this study, we choose4genotype of B. distachyon(BD21, BD3-1, BD29-1, BD1-1), Lolium perenne and Phleum pratense as our main experiment materials, in which BD21and BD3-1belong to spring type and the other two genotypes are winter type. There are three important cold related gene families in Pooideae:the ice recrystallization inhibition proteins genes (IRIps), fructosyltransferase genes (FSTs), and many C-repeat binding factor genes (CBFs). Here we use comparative analyses to study conservation and evolution of these gene families in B. distachyon to better understand B. distachyon’s potential as a model species for agriculturally important temperate grasses.We have found out the differnces about how the main four cold related gene families express before and after cold accilimation by qPCR, cDNA microarray and bacteriophage library construction experiments. And we have measured the total carborhadrates and frutan contants in Lolium perenne and B. distachyon treated with cold and none treated materials. Besides we have constracted several microRNA vectors in order to knock down the main BDIRIp gene and overexpression vectors. As to find out the founction of BDIRIp gene, we tried to transformed the differents vectors to wild BD21. The main results are as followes:1. IRIps are thought to minimize cell damage during ice formation by restricting ice crystal growth in the apoplastic space, a process known as ice crystal recrystallization. The ancestral IRIps gene in Pooideae is thought to have evolved from a LRR-protein kinase by gaining an ice binding domain through an expansion of a repeat motif (NxVxG/NxVxxG). In core Pooideae species extensive gene duplications have given rise to large IRIps gene families. In B. distachyon, there are7BDIRIps genes on the chromosome5, which Bradi5g22870and Bradi22880are on the reverse DNA and the other five gene are all forward and closely. But the amino sequences alignment shows that Bradi5g22870and Bradi22880are excluded the type sequence of IRIps gene family.2. From the IRIP genes phylogeny map in poodieae,we can see that BDIRIps have the same ancestor with the IRIps in wheat, barley, Lolium perenne and Deschampsia antarctica. Especially the BDIRIps have more closer relationship with IRIps in Deschampsia antarctica. The evolution of BDIRIps have experienced lots recombinant mutants in the long timeperiods. We infer that the Bradi5g22870.1gene is evoluted from Bradi5g27350.1by losing the NxVxG ice binding motif. In the same, the Bradi5g22880.1gene has evoluted from Bradi5g27350.1by losing the NxVxG ice binding motif.3. We sampled the four deffernt genotypes of B. distachyon after4h,1d and10d cold treated at1℃seperately. The we isolated RNA from all the leaf samples and have done the qPCR experiment compared to the none treated samples. It shows that all the four BDIRIps have grow up aftrer cold treated, and as time goes by, the BDIRIps gene grow to the top at1d treated, then it decrease slowly. Only gene Bradi5g27300.1has a very little growth. The spring genotype BD3-1is very different from the other three, because all the four cold related IRlps genes have increased very slowly, almost stay still.4. CBFs are transcription factors that regulate suites of genes during drought and low temperature stress. Two CBF subfamilies, CBF3and CBF4, have undergone lineage specific duplications in the Pooideae and the members of these CBF3/4-subfamilies are thought to play roles in Pooideae freezing stress. Our DNA microarray experiment shows that there are11CBF genes in BD21genotype. And after cold treaeted, only two of them increase expression and the others are goes down.5. We have made the CBF phylogeny map in poodieae, and shows that all the11BDCBF genes are belong to the CBF3subfamily. Furthermore, ten of the BDCBFs are CBF3d subfamly except the Bradi4g35630.1gene is classified to CBF3c. To find out why BD3-1has very weak cold resistance, we have made bacteriophage library using BD3-1and we get two CBF3cloens.6. FSTs convert sucrose molecules into fructan sugars and low temperature stress induces FSTs expression and fructan accumulation in core Pooideae species. Both induction of genes encoding fructan synthesis into plants which lack endogenous FST genes and over-expression of endogenous FSTs in Pooideae increases plant freezing tolerance. By DNA microarray experiment, we get two similar BDFST genes. But from the FSTs and invertase gene family phlogeny map, the two simimlar BDFST genes are belong to INVs for lacking the FSTs typical amino sequences WMNDPNG.7.After treated the B. distachyon and L. perenne at2℃for4days, we measured the total carborhdrate and fructan unsing colorimetric quantification measurement. Compared the none treated control, Both the carbohdrate and fuctan in cold treated B. distachyon and L. perenne meterials are increased. But the fructan in cold treated B. distachyon only has a little growth, the total carbohdrate increase as much as three times than the none treated B. distachyon meterials. This is contified that FSTs is lack in B. distachyon. So fructan does not play a main role in cold resistance in B. distachyon,and in cold treated L. perenne fructan does play a important role. By high-performance anion-exchange chromatography experiment (HPAEC), we see kestose and nystose are the main short oligosaccharides of fructan which accumulate in cold treated L. perenne.8. B. distachyon contains cold responsive IRIP genes which have evolved through B. distachyon specific gene family expansions. A large cold responsive CBF3subfamily was present in B. distachyon, while CBF4homologs are absent from the genome. No FST gene homologs in B. distachyon encoded typical core Pooideae FST-motifs and low temperature induced fructan accumulation was dramatically different in B. distachyon compared to core Pooideae species. As last, we conclude that B. distachyon can serve as an interesting model for specific molecular mechanisms involved in low temperature responses in core Pooideae species.9. About the B. distachyon transfomation experiment using microRNA vectors and overexpression vectors, we only get transformed callus with GFP report gene. But the transformed shoots can not regenerate from the transformed callus, even the none transformed callus only have a poor regeneration. We infer that the genotype of B. distachyon is very important in the B. distachyon transformation.