The Effect Of The Heat Shock Protein Genes Of Invasive Alien Weed Ageratina Adenophora (compositae) Under Low Temperture Stress

An invasive aline weed, Ageratina adenophora,has become widespread in Southwest of China due to its high environmental adaptation. The invasion of A. adenophora in China, is due to the gradual acclimation of active factor induced by temperature conditions. The alien plants evolutionarily adapt to environmental changes related to different latitudes and altitudesin introduced ranges. However, little is known about the low temperature response of A. adenophora and molecular and genetic aspects that influence resistance to environmental variability. Heat shock proteins genes (hsps) have an essential role in stress resistance and adaptation to the environment. The gene provides an accessible and useful tool for generating insights about molecular mechanisms by how the weeds become successfully invaders.In this study, with respect to adaptation to abiotic environments, we collected and compared response of A. adenophora populations to low temperature from different high altitude in Sichuan Yunnan of China where the infestation of A. adenophora is quite severe. Meanwhile, we have studied hsps genes that are important in relation to stress resistance and adaptation to the environment to reveal their contribution to invasion of A. adenophora in China.The physiological responses to low temperature were determined to elucidate mechanisms chilling tolerance. The study made use of artificial climate chamber to compare the A. adenophora chilling tolerance from different high altitude populations from Sichuan and Yunnan province of China. With the decrease of temperature, the physiological changes including increases in malondialdehyde (MDA) and total soluble protein contents, reductions in total soluble sugar, fluctuation of superoxide dismutase (SOD) activity, and catalase activity (CAT) were observed among all the eight populations. However, different extents of low temperature sensitivities were found among the high altitude populations. The results revealed that after the trestment with gradually decreasing temperature, the A. adenophora from the population of Mopanshan and Lushan of Sichuan showed the lowest chilling damage and high tolerance. The populations from Daqingshan of Sichuan and Longling of Yunnan both presented a higher chilling damage and lower tolerance. These indicated that A. adenophora populations from high altitudes were insensitive to chilling tolerance. These data indicate that the invasive plant from high altitude populations with different chilling tolerance reflected a higher capacity to its infested environments. These results may be a manifest A. adenophora expanded to Northern China much faster.In order to know molecular and genetic aspects of adaption, we cloned full length of coding sequences of invasive alien weed A. adenophora heat shock proteins by the combination of homology cloning in the present study, renamed as Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90. Full-length of Aahsp17, Aahsp60, Aahsp70 and Aahsp90 were 691, 1,581, 2,074 and 2,094 bases of nucleotide sequence, which have been deposited in Genebank database (Accession no. EU209067, EU209068, EU209069, EU209070). The four genes were found highly to be conserved when compared at the amino acid level with the corresponding HSPs from other plants. Sequence analysis, identification, alignments and phylogenetic analyses of the hsps showed that these HSPs shared high levels of identity with corresponding proteins from other species. The phylogenetic analysis suggested that the four hsps were present closed to some other HSPs from Arabidopsis thaliana, Nicotiana tabacum, Lycopersicon esculentum et al.We investigated the characterization and expression of Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90 of A. adenophora. After heat treatment, Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90 transcripts were not detected in roots, but high levels were found in the stems and leaves. Tissue specificity showed a similar pattern of response to heat shock. The kinetics of regulation of Aahsps was analysed by Northern blot hybridisation, which revealed the significantly changes at mRNA level for Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90. Transcripts of these genes were considerably induced underheat and cold stress. Although the gene expression responses induced by high and low temperture stress shared several common features, there we still observed some differences among Aahsps. The four hsps expression response to heat exposure was robust, and differed from the response to low temperture stress. These results indicate that all of the genes are stress-inducible forms or cognate forms that are constitutively expressed. The sourthern results show that Aahsp17.6 and Aahsp60 appear to be present as a single copy and Aahsp70 and Aahsp90 are encoded by a multi-gene family.We made investigations on the possible function of Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90 in vivo under heat and cold sress, and SDS-PAGE analysis of cell lysates revealed their protective effect is associated with an increase in the thermo stability of soluble proteins. The PCR products of ORF sequences encoding Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90 were inserted into the same digested pET-30a vector to generate pET-30a-Aahsps. These plasmids were transformed into Escherichia coli BL21 (DE3) plyss competent cells. Aahsps were expressed in E. coli cells. The recombinant Aahsps proteins produced in bacterial expression were separated by 12% SDS-PAGE.The recombinant Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90 over-expression improved viability in comparison with control cultures in E. coli under heat stress conditions. SDS-PAGE analysis of cell lysates suggested that the protective effect in vivo was due to increased thermol stability of soluble cytosolic proteins. The recombinant Aahsp17.6, Aahsp60, Aahsp70 and Aahsp90 over-expression improved viability in comparison with control cultures in E. coli under cold stress conditions.Cloning and sequence analysis of promoters in hsp90 and hsp17.6 genes from A. adenophora by two skills of chromosome walking from a known sequence to an unknown region based on PCR methods. The promoter region of hsp90 with 864bp (GenBank No. FJ434253), from invasive weed A.adenophora was amplified based on PCR methods. Another promoter region of hsp17.6 with 1,485 bp (GenBank No. FJ434252) was cloned by TAIL-PCR. Both of the 5′-flanking regions of hsp90 and hsp17.6 contained specific heat shock element (HSE) (nGAAn) and several cis-acting elements, such as TATA-box, CAAT-box. Especially, the promoter region of hsp17.6 has ten of HSE, which could be bound to heat shock transcription factors (HSFs). Then hsp17.6 of A. adenophora wae robustly induced and accumilated in response to low temperture stress. The recombinant HSP17.6 and HSP17.7 from A.adenophora were applied to determine their chaperone function. The recombinant HSP17.6 and HSP17.7 were applied to determine their chaperone function. 33.6 mg of HSP17.6 and 20.2 mg of HSP17.7 was purified from 1l of each respective cell culture using a Ni-NTA column. In vitro, HSP17.6 and HSP17.7 actively participated in the refolding of the model substrate citrate synthase (CS) and effectively prevented the thermal aggregation of CS at 45°C HSP17.6 and HSP17.7 prevented this process at different ratios. The maximal suppression by the HSP17.6/HSP17.7-to-CS monomer was also observed at a ratio of 5:1. The effect of the recombinant HSP17.6 and HSP17.7 on the thermal inactivation of CS was investigated. When the CS was incubated at 38°C alone or in presence of 50μg/ml lysozyme, less than 12% of CS activity remained after 60min. After shifting the temperature of the samples to 25°C, the CS activity remained low after 60min in the presence or absence of 50μg/ml lysozyme. They both prevented the irreversible inactivation of CS at 38°C at stoichiometric levels. Therefore, this report confirms the chaperone activity of HSP17.6 and HSP17.7 and their potential as a protectant for active proteins.To confirm the role of Aahsp17.6 in low temperture condition, an integratedvector pBI121of the Aahsp17.6 and NPTⅡgene(a kanamycin resistant gene)with CaMV35S promoter was constructed and introduced into tobacco mediated by Agrobacterium tumefaciens LBA4404. At low-temperature, the soluble sugar content of transgenic seedlings was higher than that of wild-type plants, whereas the MDA content and the SOD activity were lower than that of wild-type plants. Based on these results, the Aahsp17.6 transgenic tomato plants were more tolerable against low temperture stress than the wild-type plants. Thus, we suggest that Aahsp17.6 may play a pivotal role in chilling tolerance and has potential consequences in this invasive plant for expansion to non-freezing low temperatures zones.In conculsion, these results may be important for analysis that A. adenophora from high altitude in southwest regions of China has evolved into different ecotypes through physiological adaptation. And the inducible hsps provide a unique system to measure the molecular mechanism by which the invasive alien plants respond to changing environmental and developmental cues. Moreover we could evaluate the molecular capacity of A. adenophora to acclimatise to high and low temperatures in the future according to the results.