Cloning, Chromosomal Location, Prokaryotic Expression And Exprission Of Chintinase Genes From Common Wheat

Chitinase is kinds of enzyme which decompose chitin and it commonly exist in the cellof animals, plants and microorganisms. Currently,people understand that chitinase can inhibitthe growth of microorganism by breaking down chitin in cell wall. With that understanding,scientists cultivate novel wheat breeds which resist blumeria graminis and puccinia recondita.However, the common wheat contains more complex genome comparing other plants, whichmade the research about chitinase in wheat was even harder to conduct.In order to analysis the composition and expressing of chintiase during pustulationperiod of wheat, this project isolated genes of chintiase from wheat genome though a pair ofprimes designed from chintiase sequences in wheat and analysis sequences of chintiasesubunits and its predicting protein. The cloned genes were located in different chromosomesand set up for prokaryotic expression. Finally expressions of different chintiase subunits areresearched by transcript profiling in the lab.1. Thirty ClassⅠ-type chintinase genes were isolated from common wheat and have highhomology with other chintiase genes in GenBank. They are divided into6subunits based onsequence characters and named Chi-1，Chi-2，Chi-3，Chi-4，Chi-5，Chi-6, and admissionnumbers were labeled as KJ507385，KJ507386，KJ507387， KJ507388，KJ507389，KJ507390，respectively..2. Analysis results for protein sequence of cloned chitinase genes found that all chintinasegenes have integral ORF without intron，molecular Weight are ranging from33451Da to33581Da. Chi-2contains16Cysteine, while other subunits have17Cysteine. The Isoelectricpoint of Chi-1，Chi-2，Chi-3，Chi-5，Chi-6is alkaline, and Chi-4is acidic.3. The subunits of Chi-1，Chi-2，Chi-3，Chi-4，Chi-6were anchored in3A、2B、5B、1A、3D of wheat chromosomes. But Chi-5was not located.4. Prokaryotic expression system and purification system of Chi-1，Chi-2，Chi-3，Chi-4，Chi-6were established. 5. It showed Chi-6was the main expression in seed during pustulation period of wheat, byanalysis transcript profiling of wheat. The amount of Chi-6expression increase continuouslyfrom day4to12.6. ClassⅠchitinase from wheat is similar with chitinase of rice and barley, while it is farwith that of Arabidopsis thought analysising homologous tree from cloned chintinase geneswith other creatures.This study cloned6different kinds of chitinase genes from common wheat (china spring),successfully finish location, prokaryotic expression and analysis the expression level of6chintiase subunits during pustulation period in seed. Though deep research about wheatchitinase, it helps to improve resistance of wheat by genetic engineering.