Funtion Of Ice Recrystallization Inhibition Gene From Triticum Aestivum And Evolution Of Ice Recrystallization Inhibition Gene Family From Triticeae

As one of the staple crops with largest planting area, cold tolerance of wheat limits its growth, annual productivity, geographical distribution and growing season. Thus, for this situation, making full use of the genetic resources, discovering novel genes, and in-depth analysis of mechanism of cold resistance, have great guiding significance for improving cold tolerance of wheat, as well as selection of new resistant wheat varieties. Ice Recrystallization Inhibition proteins can inhibit the growth of ice crystals and ice recrystallization through binding to the surface of ice, which improve plant resistance to cold. Wild relative species of wheat as a critical gene bank of improvement of wheat breeding, can provides a wealth of genetic resources to improve wheat cold hardiness. In this study, six IRI genes are isolated from winter wheat. After cold stress, expression analyses of each member of IRI gene family are done by qPCR. Two representative IRI genes are selected to overexpress in tobacco, and low-temperature stress is used to test the tolerance of transgenic plants by observing the phenotypes, determing survival rates, relative electrolytic leakage, content of proline, as well as MDA.22 IRIs are isolated from wild relative species of wheat, cladogram of all IRIs is conducted. Main results are as follows:1. Isolation, multiple sequence alignment, expression of IRI genes from winter wheatSix IRI genes, named TaIRI3, TaIRI4, TaIRI5, TaIRI6, TaIRI7, TaIRI8, are isolated from winter wheat. Amino acids numbers of these amino acids are 285,285,285,288,287,287. These genes have typical characteristions of IRI genes, including the N-terminal signal peptide, LRR-domain, IRI-domain and a number of conserved cysteine. Depite of the high conservation of IRIs, there exist sequence differences. The TalRIs including isolated in this study and published homologous genes, are grouped into three categories. TaIRI3, TaIRI4、 TaIRI5 and TalRI1 are classified into the same cluster, Group I, TaIRI6, TaIRI7 and TaIRI8 are classified into Group II, TaER.12 diverged by monophyletic belonging to Group Ⅲ. Results show that expression of TaIRI genes had significantly difference among the members of gene family. Generally, expression level of M808 is higher than CS. When after treatment at 4"C for 24h, expression of TaIRI4 (Group I) and TaIRI6 (Group II) are higher than other genes. We speculate that the two genes are major gene of two clusters.2. Detection and function identification of transgenic tobaccosThe transgenic tobaccos of TaIRI4 and TaIRI6 are screened by Southern blot, the results show that the target gene has been successfully integrated into tobaccos. By low temperature stress test, we observe changes of phenotypes, and measure survival rates, relative electric conductivity, content of free proline and MDA, then comprehensive comparison of cold hardiness of each materials, the result is as follows: transgenic tobacco of TaIRI6> transgenic tobacco of TaIRI4> wild-type tobacco.3. Molecular cloning and evolutionary analysis of IRI gene family from Triticeae22 IRI genes are widely isolated from Triticeae and its wild relativesspecies. The IRI genes are demonstrated to be multigene family. Each member of the gene family have typical characteristions of IRI genes, including the N-terminal signal peptide, LRR-domain, IRl-domain and a number of conserved cysteine. Phylogenetic analysis indicates that the isolated IRI genes and published relative genes are clustered into nine categories. There exists multispecies speciticty among them. IRI genes from Aegilops and Triticum share close genetic relationship. These genes and PSR have high homology. We speculate that the proteins may be difunctional proteins.