Cloning And Functional Analysis Of The Auxin Early Response Gene SAUR Of Cunninghamia Lanceolata

Cunninghamia lanceolata is one of the most important fast-growing tree species in southern China,and it was widely planted across the southern China due to its fast-growing and high yield and quality of timbers,hence occupied a crucial position in forestry production of our country.The practice of long-term forestry production shows that aluminum toxicity is one of the main reasons that restrict the growth of Chinese fir in acid soils in southern China,resulting in a decline in the yield of Chinese fir plantation.Therefore,how to improve the growth of Chinese fir in acidic soil and increase the output of Chinese fir plantation has become an urgent problem to be solved.Like other plants,the growth and development of Chinese fir in acidic soil is a dynamic process.It continuously regulates its own growth and development by integrating its own developmental and environmental signals.As one of the key hormones regulating plant growth and development,auxin plays a key role in mediating plant response to environmental and developmental signals.Although the role of auxin in regulating plant aluminum stress tolerance has long been confirmed.However,this process involves a series of genes and regulatory elements of the auxin signal transduction pathway,while the Auxin early response gene SAUR acts as an upstream gene for the auxin signal transduction pathway,mediating auxin on plant growth and development and abiotic Stress plays a key role in the regulation process.However,the molecular mechanisms that mediate auxin-mediated plant aluminum stress tolerance have not been fully understood.Therefore,the ClSAUR gene family and promoter cloning of Cunninghamia lanceolata,the expression pattern analysis under different physiological conditions and the study of gene function under aluminum stress were carried out to clarify the role and status of CISAUR gene in Chinese fir growth and aluminum stress,and to deepen the CISAUR gene in Chinese fir.The understanding of the regulation mechanism of Chinese fir growth and aluminum stress,and provide theoretical basis for the ultimate use of genetic engineering methods to improve the resistance of Cunninghamia lanceolata,in order to ultimately increase the yield of Chinese fir plantation under aluminum stress.Below are key research findings:1.Cloning and bioinformatics analysis of ClSAUR gene family of C.lanceolataEight members of cDNA and gDNA seguences of CISAUR gene family were cloned from tissue culture seeding of C.lanceolata by RT-PCR combined with RACE.According to bioinformatics analysis,all of the CISAUR genes contained no intron,with the number of amino acids encoded was between 95-196.Further more,all of the proteins encoded by CISAUR genes were non-secretory protein without transmembrane domain,located in cytoplasmic.At the same time,in addition to ClSAUR25 was an acid protein,CISAUR36 was a stable protein ane ClSAUR29 was a hydrophobic protein,the other CISAUR proteins were alkaline,hydrophile and unstable.Functional domain analysis results showed that CISAUR protein families were all belong to the members of auxin-induced super-family which contain PLN structure.The numbers and positions of phosphorylation differs greatly among different members of CISAUR protein in C.lanceolata,and the main phosphorylation sites in most of the CISAUR members of C.lanceolata were serine.Protein structure analysis showed that ClSAUR2,ClSAUR24,CISAUR25,ClSAUR29,ClSAUR36,and ClSAUR39 mainly offered random coil structure in its protein,while ClSAUR50 and ClSAUR91 mainly offered alpha helix in its protein structure.2.Promoter cloning and cis-element analysis of ClSAUR25 geneThe 5'-flanking sequence of CISAUR25 gene which length was 1471 bp was obtained from genome DNA of C.lanceolata using Tail-PCR.Three core promoter regions of the 5'-flanking sequence were located at 226?276 bp,1034?1084 bp and 1234?1284 bp,with the same possible transcriptional starting site which was C.There also had five CpG islands located at 1148?1347 bp,1149-1348 bp,1150-1349 bp,1151?1350 bp and 1152-1351 bp.The analysis of the cis-elements showed that ClSAUR25 5'-flanking region containing a abundant quantity of CAAT-box and TATA-box,in addition,there were having cis-elements that respond to light,ethylene,metal,anaerobic,meristematic tissue,endosperm,circadian and numbers of unknown elements.Further more,CISAUR25 might be subject to the regulation of bZIP transcription factor.3.The expression pattern of CISAUR genes of C.lanceolata under different conditionsThe expression patterns of CISAUR genes of C.lanceolata during different development periods of callus,in various tissues,and treated with auxin for different time were analyzed by using real-time fluorescent quantitative PCR technology.The results showed that there exist significantly differences in the expression patterns of different members in ClSAUR gene family during different periods of callus formation,the CISAUR2 and CISAUR24 expression were significantly induced during the periods of callus early induction and differentiation,the expression of CISAUR25 were highly induced during entire periods of callus development,while the expression CISAUR50,CISAUR91,and ClSAUR39 were mainly induced during the period of callus early induction,these results suggested that different members of CISAUR gene family may play a vital role during various periods of callus development.On the other hand,the tissue-specific expression patterns of different members of ClSAUR gene family results showed that the expression patterns varied significantly among different members of CISAUR gene family,the CISAUR2 and CISAUR39 were mainly expressed in leaves,and the CISAUR25,ClSAUR50 and CISAUR91 were mainly expressed in the roots,while the CISAUR24 was highly expressed in the shoots,suggesting different members of CISAUR gene family may have different function in different tissues.In addition,different members of ClSAUR gene family also displayed different responses to auxin treatments,the ClSAUR24,ClSAUR25,ClSAUR39,and ClSAUR91 were rapidly response to auxin,which can be induced by auxin within 0.5 h,these genes showed a first increased and then decreased expression pattern.The CISAUR2 also response fast to auxin,which was rapid suppressed by auxin(within 0.5 h),then the expression of CISAUR2 fluctuated during the rest of time,the expression of CISAUR50 also fluctuated during the whole treatment time.4.The functional analysis for ClSAUR25The full-length ORF of CISAUR25 gene was constructed into pCambia1301-1 expression vector,and the over-expression of lines and tissue culture seedlings of Arabidopsis thaliana and tobacco were obtained respectively by using agrobacterium-mediated transformation method.By analyzing the contents of endogenous hormones,we found that the IAA and ABA contents in transgenic tobaccos were significantly higher than that in the wild type ones,while the opposite was true for GA and ZR.Moreover,exogenous treatment of tobaccos with IAA showed that the expression tendency of CISAUR25 in tobaccos both displayed first increased then decreased,and the expression levels reached the maximum at 1 h after IAA treatment.Additionally,the relative expression level of CISAUR25 was generally significantly higher in transgenic tobaccos as compared with the wild type.Aluminum(Al)tolerance analysis results showed that the hydrogen peroxide content in wild type and transgenic tobaccos were both significantly increased under A1 stress,and the increased rate of hydrogen peroxide in transgenic tobaccos was lower than those in wild type,similarly,these was also true for MDA content in transgenic and wild type tobaccos under A1 stress,indicating a lesser Al-induced oxidative damage was found in transgenic tobaccos,and suggested the transgenic tobaccos had a higher A1 tolerance as compared with wild type ones.The soluble sugar and proline results of tobaccos under A1 stress showed that A1 treatment both increased the contents of proline in transgenic and wild type tobaccos,and the content of soluble sugar was increased in wild type,while decreased in transgenic tobaccos,respectively,under A1 stress.These results suggested that the altered osmotic substances in tobaccos under A1 stress may not be the reasons for transgenic tobaccos tolerant to high A1 treatment.On the other hand,we found A1 significantly increased the SOD activity in wild type tobacco,while this was not observed in transgenic plants,but the activity of SOD was relatively high in transgenic plants.Moreover,the activities of POD and CAT were both induced under A1 stress in tobacco seedlings,and the increased rate of POD in wild type was higher than those in transgenic tobaccos,while the opposite was true for CAT,suggesting the lesser Al-induced oxidative damage in transgenic tobaccos may obtained by increasing the CAT activity,which enhanced its capacity for reactive oxygen species.Furthermore,we also analyzed the expression of citrate transport gene NtMATE and gene respondsible for A1 transport from cytoplasm into vacuole NtALS3,the results showed that the expression of NtMATE was significantly up-regulated under A1 stress both in wild type and transgenic plants,the expression levels of NtMATE in wild type and transgenic plants were 2.75 and 2.96 folds higher under A1 stress as compared with their control,and the expression levels of NtMATE in transgenic plants under normal and A1 stress conditions were both significantly higher than those in wild type plants.On the other hand,although the expression of NtALS3 did not varied significantly between wild type and transgenic plants under control,but A1 treatment significantly increased the expression of NtALS3 in transgenic plants,the expression levels of NtALS3 in transgenic plants was 3.21 folds higher than that in wild type,suggesting transgenic plants can enhance its A1 tolerance by increasing the expression of NtMATE and NtALS3 in transgenic tobaccos under A1 stress and consequently promoted the secretion of organic acid,and also the transportation of A1 from cytoplasm to vacuole,resulting in inhibition the uptake of A1 in transgenic tobaccos.As a result,Al-induced oxidative damage was alleviated,which ultimately enhanced its tolerance to Al.