The Regulation Mechanism Of Banana Transcription Factor Gene-MaASR1for Improving The Drought Resistance Of Arabidopsis

ASR(ABA, stress, ripening induced protein) is a class of small molecular weight hydrophilic plant-specific proteins, existing in tomato, tobacco, papaya, potato, corn, honey pomelo, loblolly pine, lilies, rice, grapes and strawberries. ASR genes located in the nucleus in different cell types can be used as a transcription factor binding to specific DNA sequences in order to regulate the expression of genes.The ASR gene has a very close relationship with biological and non-biological stress, and plays an important role in sugar and ABA signal transduction.A new ASR has been obtained from banana fruit (Musa acuminata L. AAA group cv. Brazilian) cDNA library, named MaASR1(accession number AY628102). The MaASR1gene was overexpressing in wild-type Arabidopsis thaliana, and obtained Arabidopsis transgenic lines L14. Its preliminary study found that the overexpression of MaASR1gene in Arabidopsis could significantly improve the drought resistance in Arabidopsis.In order to further study the molecular mechanism of drought resistance for over expressing MaASR1gene in arabidopsis thaliana. DNA microarray was used to broad-spectrum screening the differentially expressed genes under natural and drought treatment in wild-type Arabidopsis thaliana and transgenic lines L14. ChIP (Chromatin Immunoprecipitation) was used to further study the direct target genes of MaASR1which plays a regulatory role in gene expressing. And then bioinformatics analysis and RT-PCR validation of the results obtained by microarray and ChIP-SEQ were done. After that the prokaryotic expression vector was constructed to express MaASR1protein. EMS A (Electrophoretic Mobility Shift Assay) technology was used in vitro validation for direct interaction between the purified MaASR1protein and its specific gene element to obtain the specific DNA sequences combined with MaASR1protein. The main findings are as follows:1) In the Expression profile microarray (14vs WT), the wild-type Arabidopsis thaliana and transgenic lines L14were all without any treatment. There was a total of747differentially expressed genes, including559up-regulated genes and188 down-regulated genes. In the Expression profile microarray (Y vs W), the wild-type Arabidopsis thaliana and transgenic lines L14were all drought-treated. There was a total of653differentially expressed genes, including256up-regulated genes and397down-regulated genes.2) The differential genes in Expression profile microarray were classified by biological processes, location and biological function. In accordance with the classification of the biological processes, the differentially expressed genes involved in photosynthesis and protein modification changes the most significant; Classified according to location, the differentially expressed genes located in plastid changes the most significant; Classified according to its biological function, the differentially expressed genes which have transport activity, catalytic activity and protein binding activity changes the most significant.3) Hormone (ABA, ethylene and auxin) related genes obtained in Expression profile microarray were validated through RT-PCR. The results were as follows:①Without any treatment and drought stress conditions, MaASR1transgenic Arabidopsis compared with the wild type the ABA biosynthesis was decreased and the amount of degradation was increased. Under drought treatment the transfer of MaASR1can improve the expression of ABI5and ABF1. AB15could regulate the expression of part of the LEA protein and enhance the expression of ABA-induced gene which conferred Arabidopsis transgenic plants drought-tolerant and drought resistance. ABF1can play a role with other ABI genes in synergy and could participate in the ABA signaling pathway which conferred Arabidopsis transgenic plants drought resistance. The drought resistance obtained of MaASR1transgenic lines was not through the CBL/CIPK complex which could involve ABA-dependent or ABA-independent pathway to complete the stress response.②Under drought treatment, MaASRl could improve ethylene synthesis level in vivo by raising the expression level of ACS6and ACO1in Arabidopsis. The Transfer of MaASR1could positive regulate the ethylene response and increase the gene expression of ERF which conferred Arabidopsis transgenic plants drought resistance.③MaASRl gave the drought resistance of the transgenic plants not through improving the expression levels of GH3.3and GH3.5. Plant drought resistance could be enhanced by increasing the expression levels of the HAT2which could cause the reduced of IAA content. MaASRl changed the regulation mode of some auxin-responsive genes under drought stress.4) MaASR1can increase the expression of DREB2A which involved in the ABA-independent pathway. It was speculated that DREB2A could regulate transduction pathways which was independent on ABA to control the plant drought response, and thus give the plant drought tolerance.5) The Expression profile microarray and ChIP-SEQ results showed that, under drought treatment, MaASR1affected photosynthesis through the influence of related gene expression of photosystem I and photosystem II, chloroplast and RUBISCO activating enzyme to reduce transpiration to retain moisture, improving the drought resistance of plants to a certain extent.6) The Expression profile microarray (14vs WT) results showed that C3HC4-type zinc finger protein gene is one of the highest network connectivity genes. MaASRl could increase the expression levels of C3HC4-type zinc finger protein gene. MaASR1could give a strong plant drought tolerance by regulation the C3HC4-type zinc finger protein gene expression levels.7) The Expression profile microarray (Y vs W) results showed that the sugar transporter family gene is one of the highest network connectivity genes, indicating the important position of the sugar transporter family protein in the entire metabolic network and the close relationship between ASR and the sugar transporter family protein under drought stress conditions.8) Arabidopsis seedlings were used for the exploration of sonication conditions during chromatin immunoprecipitation, and obtain appropriate DNA fragments for the subsequent experiment. That could provide the conditions to get more information about interaction between DNA and protein, and also laid the foundation for the application of chromatin immunoprecipitation.9) The prokaryotic expression vector pET30a-MaASRl was constructed to express MaASRl protein. MaASR1protein was purified through Ni-Agarose affinity chromatography and then was verified by SDS-PAGE and Western. That laid the foundation for EMSA in vitro to validate the combination of MaASRl protein with specific DNA elements.10) The Expression profile microarray and ChIP-SEQ results all showed that, under drought stress conditions, the sugar transporter family proteins play an important role in Arabidopsis transgenic lines L14. The EMSA results proved^that the MaASR1and hexose transporter have direct interaction. MaASR1could combine with specific sequence in promoter of the hexose transporter protein to regulate the expression of the hexose transporter protein, and then regulating the metabolism of hexoses. Hexose could protect the structure of cell membranes and proteins by regulating the changes of cell osmotic potential. That could prevent cells from the intracellular nutrient loss and cell damage due to the water loss in order to make the transgenic Arabidopsis with strong drought resistance.11) Ch1P-SEQ, RT-PCR and EMSA results showed that, MaASRl could combined with the sequence of the promoter region of the small G protein subfamily members RhoGAP. MaASRl could negatively regulate the expression of Arabidopsis "molecular switch" RopGTPase by regulating the expression of the small G protein RhoGAP. That could make RopGTPase in the non-activated state, thereby indirectly involved in ABA signaling pathway in order to response to drought stress.