Cloning And Functional Analysis Of A New Cotton Gene GhDr1 Related In Resistense To Abiotic Stresses

Abiotic stresses such as drought, soil salinization, chilling damage and heat damage caused by climate anomalies are seriously affecting crop yields and quality. Suffering abiotic stresses, plants can reverse the damage through their own stress tolerance ability. This process is achieved after the point abiotic stress signals stimulating plant cells, and is by the means of signal transduction to the final aim that the expression of functional genes can be regulated. Expression changes of the protein related in signal transduction pathways will affect stress tolerance to abiotic stresses. All currently found signal pathways such as MAPK, CDPK and CIPK signal pathways involves a phosphorylation cascade amplifying the abiotic stress signals in order to regulate the expression of downstream genes. Regulating the expression of genes related in signal transduction pathways by the means of genetic engineering, in order to improve stress tolerance has a bright future.In this study, I research into cloning and preliminary functional analysises of a cotton gene related in stress tolerance on the basis of a gene fragment previously cloned in our laboratory. The main results obtained are as follows:1. With a previously cloned stress-related 346bp gene fragment of GhDr1 , I focus the gene to a single BAC clone by screening the genomic BAC library. I cloned the 5 'and 3' flanking sequences of GhDr1 by the cloning technology of TAIL-PCR, and the final 7037bp genomic sequence involving GhDr1 is got. And then I cloned the 1913bp cDNA sequence of GhDr1 by the gene amplification methods RT-PCR and TAIL-PCR.2. Analysis results shows that GhDr1 contains 4 exons and 3 introns, and the transcription start site is located at 492bp upstream of ATG the initiation codon. Alternative splicing exists in GhDr1's second intron . And four splicing isoforms are generated by alternative splicing. The peptide differences of the four amino acid sequences from encoded protein corresponding to the four splicing isoforms have many serines and threonines that can be regulated by phosphorylation and many prolines that affects secondary structure of the protein sequences. And bioinformatic prediction results shows that these differences indeed led to changes of the phosphorylation sites and secondary structures. The promoter region of GhDr1 is located in the 5'-UTR region and no more than 1.5kb upstream of the transcription start site. Cis-acting element analysis shows that there are many regulatory elements involved in abiotic stress resistance and signal transduction pathways locate in the existence of the promoter region of GhDr1.3. Bioinformatic analysis results of GhDr1 are as follows: the full length of CDS is 1485bp, encoding 494 amino acid residues,and GhDr1 is a negatively charged acidic stable protein; there are 40.69%αhelix and 44.94% random coil in the secondary structure of GhDr1 protein; there are many hydrophilic amino acids and gathered hydrophilic area in the amino acid sequence of GhDr1 with no obvious transmembrane helix; sub-cellular localization prediction shows that GhDr1 is most likely located in the cytoplasm; the results of homology modeling shows that GhDr1 are most homologous to corresponding proteins in Ricinus communis,Vitis vinifera,Populus trichocarpa and Arabidopsis thaliana; Motif domain prediction find that there are tyrosine kinase II phosphorylation sites,N-myristoylation sites,protein kinase C phosphorylation sites,zinc-finger like domain,MAPK phosphorylation sites and MAPK interaction docking motif in the sequence of GhDr1. Many functional proteins involved in stress response and signal transduction pathways are found in the Vitis vinifera and Arabidopsis thaliana chromosomal regions where the homologous genes of GhDr1 locates. So we predict GhDr1 may be involved in the signal transduction pathways and be regulated in the MAPK phosphorylation cascade .4. Expression pattern analysis shows that GhDr1 expresses in all the cotton organs of different growth stages. The highest relative expression value appears in mature leaves in cotton. The relative expression values are stable in roots such as radical, young roots and mature roots and maintain overall the highest values in roots, stems, leaves and flowers in the mature age of cotton5. After construction of transient expression vector 35S::35S::p16318-GhDr1-hGFP, the plasmids are transferred into onion epidermal cells for transient expression. The fluorescence signal expressed by fusion protein distributes evenly in the cytoplasm of onion epidermal cells . Thus we confirm GhDr1 as a cytoplasmic protein.6. The plant binary expression vector pBIGhDr1GUS was constructed and was transferred into Agrobacterium through electroporation. Transgenic plants were generated by dipping flowers of arabidopsis thaliana. During the transformation we improved the flower dip transformation method and the transformation efficiency was elevated to about 50%. The T1 progeny was identified by antibiotics resistance screening,PCR identification and GUS staining. The T2 progeny and T3 progeny were also identified by antibiotics resistance screening. Finally we got the homozygous arabidopsis lines that transferred cotton gene GhDr1. By the means of Real-Time PCR,we analyzed the expression levels of the homozygous lines of arabidopsis which transferred GhDr1. Finally, we got two super high expression lines, two high expression lines and ten middle to low expression lines.7. We selected the representative low expression lines 38 and 62, high expression line 24 and super high expression line 61 as experimental materials. These lines combined with WT and mutant arabidopsis lines which was ordered and identified homozygous were compared to investigate to analyse physiological functions of GhDr1 related to resistanse to abiotic stresses. The results of physiological experiments showed that: the phenotype of transgenic lines was better than the wild type and mutant lines in normal culture conditions; the middle to low expression transgenic lines were not sensitive to exogenous ABA while the super high expression transgenic lines were sensitive to exogenous ABA; the middle to low expression transgenic lines were sensitive to salt stress while the high expression transgenic lines were not sensitive to salt stress; the germination rate of transgenic lines were higher than WT and mutant lines with exogenous ABA added to the medium; the germination rate of transgenic lines were lower than WT and mutant lines with NaCl added to the medium. These results show that GhDr1 indeed participate in the physiological processes related to stress resistance. And different expression levels of GhDr1 can reverse the sensitivity of transgenic plants to abiotic stresses. In summary, this study cloned the genomic and promoter sequences of a new cotton gene GhDr1 which is related in abiotic stresses resistance; four types of splicing isoforms were found in the cDNA sequences of GhDr1;the expression patterns and subcellular localization profiles of GhDr1 were analyzed; GhDr1 gene was overexpressed in model plant arabidopsis; the homozygous transgenic arabidopsis lines were got and their physiological phenotypes related to abiotic stresses resistance were investigated.Ultimately we determine the GhDr1 gene is indeed involved in physiological processes related to abiotic stresses resistance. This study makes reference value to the aim that the resistance to abiotic stresses of cotton can be achieved by the means of genetic engineering.