Cloning And Functional Analysis Of MiHSP Genes Encoding Small Heat Shock Proteins In Mangifera Indica L.

Mango, one of species in Anacardiarceae family, is evergreen plant that is one of five important tropical fruits in the world. However, due to the effect of global warming, the growth of mango tree in many countries is influenced by high temperature and severe drought. As a result, there is a decrease in both quality and quantity of mango fruit.To date, there have been many studies on the effects of high temperatures on the growth of mango tree. However, these studies only focused on the influence of high temperature on mango fruit in the post harvest. To the best of our knowledge, there has been no studies on the impact of high temperatures in the physiological and biochemical property of mango tree. In order to evaluate and propose the heat tolerant varieties in growing mango, Siji and Tainong No 01 are two species of one year grafted Mango from the root stock of Tumang were selected to examine the effect of high temperature on their development. The experimental temperature was set at 44℃, and samples were analyzed at different periods as function of time. This study analyzed and compared physiological and biochemical criteria of two species after the experiment. Finally, the heat tolerant variety was proposed in the production.Three genes with the characteristics of heat tolerance were documented in the previous study on the influence of environmental condition on Siji mango by Luocong et al. (2010). Based on these findings, this study conducted the cloning of gene, comparing and analyzing biological information, identifying the antibodies of prokaryotic organisms, designing Vecto, conducting gene modification on Arabidopsis, and testing gene function of generation T1, T2 and T3. The results are showed as follows:1. The change of physiological and biochemical property of the two selected mango varieties under high temperature condition.The data analysis has shown the different fluctuation of physiological and biochemical between the two varieties. There was an increase in the amount of Malondialdehyde (MDA) and Electrical conductivity in the cytoplasm of both varieties. The SOD level of the siji was lower than that of the control sample, while the SOD of Tainong No 01 increased in the early stage and then decreased, both CAT activity decreased, The content of proline increased first and then decreased, Soluble protein and Soluble sugar content increased, Relative electrical conductivity and Relative water content decreased. There was difference between two varieties in terms of the amplitude of physiological and biochemical criteria. Based on the analysis and comparison, this study proposed that Tainong No 01 is a thermal resistance variety which is better than Siji.2. Gene cloning and analyzing characteristics of three genes of MiHSP 17.6, MiHSP17.7 and MiHSP23.2.Based on the results of 3 genes from previous study about heat shock protein (HSP), RNA was extracted from the parts of leaves, stem, flower and fruit of Siji. The RT-PCR and 5 ’RACE technology were successfully applied to reverse transcription cloning cDNA (complementary DNA), resulted in the three genes with the length of 680,720 and 615. These genes have reading frame of 462bp, 465bp and 615bp, corresponding to the encryption of 144,145 and 205 amino acids. The preliminary results showed that the molecular weight of the three genes are 17.6,17.7 and 23.2 KDa with the distinguishing isoelectric point theory of 5.8,5.7 and 5.2. By using Blast comparison and homologous protein analysis, the segments of three selected genes have features of small heat shock protein, named MiHSP17.6, MiHSP17.7 and MiHSP23.2 that have been registered as KJ459857, KJ459858 and KJ459859 respectively in the bank of gene. Based on biochemical analysis, MiHSP17.6 and MiHSPl7.7 are categorized as the family members of cytoplasm, and MiHSP23.2 is a member of the mitochondrial family. By analyzing the genetic homology, three proteins are homologized with the similarity ranged from 80-82% in Cocoa (Theobroma cacao), Cotton (Gossypium herbaceum) and Orange (Citrus sinensis). The realtime fluores-cence quantitative PCR was applied to examine both young and mature parts of leaves, stem, flower and fruit. The results showed that the flowering period has highest genetic homology. At the reproductive stage MiHSP23.2 appeared a rise in the beginning then following by a slowly drop, while both MiHSP17.7 and MiHSP17.6 consisted by a decrease. During the ripening stage,three gene segments have clearly shown the highest level. In different adverse conditions including low and high temperature, NaCl, drought (PEG) and in the stimulation of physical signals (ABA, H2O2, SA), all three genes were expressed.3. Prokaryote expression of MiHSP17.6 & MiHSP17.7 and the their functionsTwo genes were designed Vecto pET-28a, carried forward into bacteria BL21 and promoted recombinant proteins. The results of electrophoresis Western blot approach indicated the anti-resistant of three genes. Specific binding of monoclonal antibodies against 6-His, PET-MiHSP17.6 and pET-HSP17.7 E.coli were induced by protein, No expression was expressed at 52, The low at 4℃and-20 ℃ was significantly higher than that in control. The salt tolerance of pET-MiHSP17.7 protein was significantly higher than that of the control pET-28a but the salt tolerance of pET-MiHSP17.6 was lower than that of the control. At the early stage of stress (2h), the drought tolerance of two was higher than that of the control, but in the middle and later period, the drought tolerance of two protein was significantly lower than that of the control.4. Eukaryotic expression of MiHSPl7.6, MiHSP17.7 and MiHSP23.2.Base on the sequence of gene as well as the vecto designation of pB121 gene, gene was transferred on Arabidopsis. The selection of gene modified seedlings was conducted using Kan anibiotic, GUS chemical organization and PCR techniques. The results was obtained in the generations of T1; MiHSP17.6, MiHSP17.7 and MiHSP23.2 recruited 05,35 and 05 seedlings. Transgenic plants on the 3rd generation (T3) at different growth stages (germination, embryos, seedlings and mature plants) was subjected in the experiment of high and low temperature, the different concentration of salt (NaC1) and drought (PEG). The expressions of three genes were observed in order to evaluate the plant tolerance in severe environment (hot, cold, salinity and drought).