Studies On Cloning And Expression Of Acid Invertase Gene From Different Populations Of Elsholtzia Haichowensis

Most plants cannot grow and reproduce in the seriously contaminated soils. Some plant species called metallophytes, however, have evolved tolerant capability so that they can survive and thrive on metalliferous soils. It is known that in plants the process of resistance for maintenance of the heavy metal tolerance mechanisms requires sufficient carbon and energy, which provided by sucrose. However, sucrose cannot be used directly for metabolism and biosynthesis. It must instead be cleaved into hexoses. Acid invertases play a vital role in hydrolysis of sucrose to glucose and fructose. Both of substrates and hydrolysis products are important nutrients and signal molecules, which involved in plant growth and response to environmental stresses. So, acid invertases might play important regulatory roles in response to heavy metals stress because they can cleave sucrose into hexoses which provide carbon and energy for root growth and maintaining the tolerance mechanisms. The purpose of the present study was to testthe above hypothesis by conducting comparative analyses of Elsholtzia haichowensis from two populations. One population (Cu tolerant population, MP) was from Tonglushan copper mine in Hubei province of China, where mining activities of ancient Chinese people appeared about3000years ago. Mining still continues there nowadays, resulting in considerably elevated Cu concentration in the soils of this area. The other (non-Cu tolerant population, NMP) from a farm in Hong’an county, Hubei province, China. The study of differences of acid invertase activities between the two populations under Cu stress was conducted. Molecular biology methods were used to clone the full-length cDNAs of the acid invertase genes and the promoter sequences. It has also been studied the effect of Cu stress on expression of acid invertase genes. The differences of full-length cDNAs of the acid invertase genes and the promoter sequences between different populations were analyzed. The main results could be obtained in the present study as follows:1. The MP plants showed higher Cu tolerance than NMP plants. There were no significant differeces of acid invertase activities in leaf between the two populations, while there weresignificant differencesin root. The regulation of acid invertases under Cu stress might take place in root. Both of the cell wall invertase and vacuolar invertase activities in MP were much higher than that in NMP, but the neutral/alkaline invertase activities showed no significant differences between the two populations. Acid invertases play important regulatory roles in growth of E. haichowensis under Cu stress.2. Four full-length cDNA sequencesof EhNcwINV (cell wall invertase from NMP), EhCcwINV (cell wall invertase from MP), EhNvINV (vacuolar invertase from NMP) and EhCvINV (vacuolar invertase from MP)(Genebank accession numbers: JX500753, JX500754, JX500755, and JX500756, respectively) were isolated from the root of E. haichowensis. The open reading frames (ORFs) of EhNcwINV and EhCcwINV were1671bp long, which encoded556amino acids, and the EhNcwINV encoded sequence shared99.64%identity with EhCcwINV. The open reading frames (ORFs) of EhNvINV and EhCvINV were1914bp long, which encoded637amino acids, and the EhNvINV encoded sequence shared99.53%identity with EhCvINV. Deduced amino acid sequences of the four cDNA sequences sharedhighly identical withacid invertase genes from other species, and they all contained13well-conserved regions of plant acid invertase, including a (3-fructosidase motif (NDPN) and a catalytic domain (WECP/VD) which are the most conserved characteristic sequences.3D structures of acid invertase proteins of two populations in E. haichowensis, as well as the simulated conformations of acid invertase-sucrose complexes were built.3. The real-time quantitative RT-PCR analysis showed that the difference of activities of acid invertase between NMP and MP under Cu stress was correlation with the transcript levels of acid invertase gene. The transcript levels of cell wall invertase and vacuolar invertase in MP were significantly higher than that in NMP. The transcript level of cell wall invertase in NMP was inhibited significantly under Cu treatment, while the transcript levels of cell wall invertase and vacuolar invertase in MP were induced significantly under Cu treatment.4. Promoter sequences of EhNcwINV, EhCcwINV, EhNvINV and EhCvINV were isolated based on hiTAIL-PCR and DNA walking. The sequences length were1727bp,1732bp,1507bp and1489bp, respectively. The EhNcwINV promoter sequence shared93%identity with EhCcwINV promoter, and EhNvINV promoter sequence shared88%identity with EhCvINV promoter. There were a lot of cis-acting elements involved in regulation of hormones and tolerance response, including a Cu response element CURECORECR.5. The pCAMBIA1301binary vector was digested with restriction enzymes to remove the35S promoter that drives GUS expression and replaced with the fragments of the four promoter sequences to generate the constructs EhNcwINVP::GUS, EhCcwINVP::GUS, EhNvINVP::GUS and EhCvINVP::GUS. Constructs were transformed into Agrobacterium tumefaciens strain GV3101for transient expression assay which was conducted by infiltration of injecting leaves of Arabidopsis thaliana. They were also transfected into Arabidopsis thaliana by floral dip. They were determinated by GUS stain assay intransient expression assay and transgenic Arabidopsis. The results showed that all promoter sequences have the capability of driving GUS expression.