Genetic Study Of Cold Tolerance And Function Analysis Of Cold Stress Related Gene Osdhn2 In Rice (Oryza Sativa L.)

There existed a number of biological constraints in exploiting the heterosis between indiea and japonica hybrid rice.The low-temperature-sensitive sterility(LTSS) of indica-japoniea hybrid has become one of the major problems in indica-japonica hybrid rice breeding after the solution to the problem of poor fertility of the hybrids with the finding of wide-compatibility gene.The previous studies revealed that the LTSS might be caused by low-temperature-sensitive pollen sterility(LTSPS).However,the genetic basis of LTSPS remained unclear.Seventeen rice cultivars and hybrids in different types(indica,japonica,javanica,indica hybrid,japonica hybrid and inter-subspecific hybrid) were evaluated to determine the effect of temperature on pollen fertility.Results showed that the pollen fertility of inter-subspecific hybrids was greatly reduced at miosis stage when the average daily temperature dropped to 22.0-23.0℃,and the extent of pollen fertility reduction varied greatly in different hybrids.However,the pollen fertility reduction of indica and japanica hybrids and conventional cultivars was not obvious under the same temperature conditions. When the average daily temperature dropped to 20℃,pollen development of conventional cultivars and hybrids was also affected.Correlation analysis revealed that there existed positive correlation between pollen fertility and average daily temperature.Temperature at miosis stage was a key factor to pollen development,and the pollen fertility of inter-subspecific hybrids was more sensitive to low temperature than that of conventional variety.To explore the genetic basis of LTSPS in indica-japonica hybrid rice,an F₂ genetic population derived from 3037(indica) and 02428(japonica) was developed.At the booting stage,pollen fertility of F₂ population together with parents were surveyed after the treatment with low temperature daily average 21～23℃.The linkage map was constructed containing 108 SSR markers distributed throughout the whole 12 chromosome with average marker interval 16.26 cM.Using software MapMaker/QTL,two putative QTL, namely qLTSPS2 and qLTSPS5 on chromosomes 2 and 5 were detected by interval mapping,which can explain the phenotypie variation 15.6%and 11.9%respectively.The additive effects were 0.021 and 0.045,dominant effects were -0.246 and -0.215,and the degrees of dominance were 11.7 and 4.8,respectively for the two QTL,therefore the mode of gene action in response to low-temperature stress was overdominanee and LTSPS is mainly the result of interaction between the indica and japonica alleles within each locus.In addition,two-way ANOVA showed that the two QTL acted essentially independent of each other in conditioning LTSPS.Low temperature induced retardation of seedling growth is a common problem in temperate dee growing areas.To gather more information regarding cold-tolerance in rice (Oryza sativa L.),we identified gene loci controlling cold tolerance at early seedling stage. We analyzed the quantitative trait loci(QTL) using 98 backcross-inbred lines(BIL) derived from the backeross between Nipponbare(strongly cold-tolerant rice,as recurrent parent) and Kasalath(cold susceptible dee).A total of 4 QTL were detected,located on four regions on chromosomes 3,7 and 12,which can explain the phenotypie variation 12.11%, 12.66%,6.82%and 15.86%respectively.The additive effects were 11.16,11.14,-8.8 and -14.59,respectively for the four QTL.Dehydrins(DHNs,LEA D11 family) are among the most commonly observed proteins induced by environmental stress associated with dehydration or low temperature.A novel full-length cDNA of a KS-type dehydrin,designated Osdhn2,has been isolated by the combination of bioinformatics and PCR based approaches.A homology tree of KS-type dehydrin from various species including solanum wheat barley arabidopsis alfalfa ricinus citrus revealed that Osdhn2 had a closer evolutionary relationship with solanum. Meanwhile,the putative peptide of Osdhn2 includes two important Putative conserved domains:lysine segment(K segment) and serine segment(S segment).Furthermore,the corresponding genomie clone was isolated and sequenced,and it was composed of 1 exons and 1 introns in the 3'UTR.RT-PCR analysis showed that the Osdhn2 transcripts were detected in all tissues including shoot,matured leaves,old leaves,and developing seeds but low levels in root.The Real-Time PCR revealed that the abundance of Osdhn2 transcripts was detected markedly in matured leaves,pistil.Osdhn2 expression improved after drought,salt and low temperature stress.In order to identify the function of Osdhn2,in vitro functional analyses were performed using an Escherichia coli heterologous expression system.Osdhn2 were cloned and expressed in a pET-32a(+) system.E.coli cells containing the recombinant plasmids or empty vector as controls were treated by salt and low temperature stress.Compared with control cells,the E.coli cells expressing Osdhn2 showed a shorter lag period and improved growth when transferred to LB liquid media containing 800 mmol/L NaCl or 700 mmol/L KCl or after 4℃treatment.The results indicate that the E.coli expression system is a simple,useful method to identify the function of some stress-tolerant genes from plants.The overexpression binary vector of Osdhn2 fused with the constitutively expression promoter CaMV35S has been constructed and transformed to indiea rice Kasalath.The cold tolerance at early seedling stage of transgenic Kasalath has been improved compared with the wild-type and salt tolerance of transgenic Kasalath is also improved when germinated in presence of 200mM NaCl solution.The results showed that the function of Osdhn2 is related to confer clod and salt tolerance.To carry out the complementary test,the RNAi binary vector of Osdhn2 has been constructed and transformed.Also the promoter of Osdhn2 fusion GUS binary construct is also underway.