OsCH2 And OsHSP82 Overexpression Vectors Construction And Gene Transformation Into Rice

High temperature stress can cause tremendous reduction of rice output every year in ourcountry. Cloning and functional characterization of the heat resistant genes will be important inunderstanding the mechanisam of heat stress and breeding high temperature resistant rice.Anthophyll can enhance the plant thermal resistance through destroying the active oxygenproduced by high temperature. Heat shock proteins have also been proved that can increase hightemperature resistance in plant. Overexpression studies ofβ-carotene hydroxylase gene OsCH2and heat shock protein gene OsHSP82 can help illustrating the functionary mechanism andpractical prospect ofβ-carotene hydroxylase gene OsCH2 and heat shock protein geneOsHSP82 in high temperature resistance.BLAST search in GenBank based on sequences of arabipdopsisβ-carotene hydroxylaseand heat shock protein genes, has found 3β-carotene hydroxylase genes and 53 heat shockprotein genes.Two genes with name LOC_Os04g48880 andLOC_Os09g30418 were selected forthis study and were named OsCH2 and OsHSP82.The corresponding cDNAs were obtained from the cDNA database, the accession numberwere AK060559 and AK061896 respectively.OsCH2 protein in rice has 85%,57%,57%,60%, 58% and 61% homologous withzea, gentiana, coffea, lycopersicon, citrus and crocus respectively. OsHSP82 protein has 95%,94%,90%,90% and 89% homologous with wheat,barley, tobacco,tomato and arabidopsisrespectively.The OsCH2 protein has two transmembranes domain, aβ-carotene hydroxylasefunction domain in C-end,and also a conservative sterol desaturase domain. The OsHSP82protein has no transmembrane domain but has a histidine protein kinase function domain in theN terminaland a HSP90 domain.Overexpression vector pOEOsHSP82 and pOEOsCH2 were constructed by putting theamplified complete cDNAs of t riceβ-carotene hydroxylase and heat shock protein genes intothe expression vector pCAMBIA-1300—multi downstream to CaMV35S promoter. PCR andrestriction endonuclease digestion have proved the successful construction two overexpressionvectors.Overexpression vector pOEOsHSP82 and pOEOsCH2 were transformed into agrobacteria strain EHA105. The OsHSP82 and OsCH2 were transformed into rice throughagrobacteria-mediated transformation, and antibiotics resistant transgenic rice plants wereproduced.