| Under stresses, plants establish osmotic adujustment substances to maintain physiological and biochemical steady states to sustain growth. The raffinose family oligosaccharides (RFOs), including raffinose, stachyose, verbascose, are galactosidases, which usually function in transportation and storage of carbohydrates to protect plants from abiotic stresses, such as drought stress, low temperature and salinity. During the process of plant growth and development under natural conditions, few of RFOs is accumulated in vivo plants; however, its accumulation is rapidly increased under the environmental stresses. GolS has been generally considered as a key enzyme in the RFOs biosynthetic pathway, therefore, it has also been considered as a key gene involved in abiotic stresses. Cassava, strongly tolerant to drought and infertility, but sensitive to low temperature and salinity, can be regarded as an ideal material to study the drought-tolerance mechanism of euhalophytes. In this research, seven galactinol synthase genes have been isolated from cassava, and named as MeGolS1-7, respectively. Their functions, gene expressional characterizations, molecular regulation of the MeGolSs have been studied. The aims of this research are to analyze the mechanism of the MeGolSs to stress tolerance. The main results of this thesis are as follows:1、In cassava, seven galactinol synthase family genes have been isolated. The cDNA and the deduced amino acid sequences of the MeGolSs described in this study have been deposited in GenBank with the following accession numbers:MeGolS1(KF793045), MeGolS2(KF793046), MeGolS3(KJ722606), MeGolS4(KJ722605), MeGolS5(KJ722604), MeGolS6(KJ722603), MeGolS7(KJ816317).2、Multiple alignment analysis of the deduced amino acid sequences of MeGolSs by DNAMAN6.0shows that three conserved domains of KLRIWEFVEY、DxD (GGD in MeGolS4)、YFNAGMFVFE、FAEODFLNMF、YNL、HxxxxGxKPW remain in all members of the MeGolS family. The structures of the MeGolS genes show that all the MeGolS genes have four exons and three introns. The3D structures of MeGolSs amino acids show a DxD (GGD in MeGolS4) in the middle, tightly embraces the Mn2+ion and inlaid around UDP-galactinol.3、The phylogenetic analysis of galactinol synthase genes shows that they can be divided into three kinds, and MeGolS5shows a comparatively higher identity with AtGolS2in Arabidopsis,, which has been found involved in drought tolerance.4、In this research, the expression profiles of MeGolSs in response to20%PEG6000drought stress have been analyzed using real time RT-PCR. Among the MeGolSs, MeGolS1 and MeGolS5are more sensitive to drought stress with a comparatively significant up-regulation, and MeGolS5can rapidly respond to drought stress at3h. However, MeGolS3was down-regulated by salt and low temperature stress. Therefore, MeGolS5has been selected to further study on drought tolerance.5, The expression of the MeGolS5gene fused with GFP in tobacco showed that the MeGolS5locates on the plasma membrane.6、The prokaryotic expression of MeGolS5based on pGEX-6p-1vector and BL21(DE3) is high-efficiency. The optimal and efficiency conditions in E. coli are:the gene expression at cell concentration OD600=0.4, IPTG0.2mmol/L, the culture temperature is37℃, and the culture time is4h.7、The MeGolS5has been transformed into Arabidopsis thaliana mediated by Agrobacterium. Six lines have been obtained by PCR identification, and three of them have been identified using RT-PCR and enzymatic determination. The germination rate of MeGolS5over-expressing Arabidopsis is higher than the wild type on the MS+Mannitol. After21d under the drought stresses conditions, MeGolS5over-expression Arabidopsis could grow normally, however, the wild type Arabidopsis poorly grew with curly and perished leaves even died away. The expression analysis of the genes such as SOD1, POD, APX1, CAT1, CMO and DHAR), which have been regarded involved in drought stress, showed a higher expression in MeGolS5over-expressing Arabidopsis than in the wild type Arabidopsis.8, The promoter region upstream of MeGolS5has some cis-acting elements that are related to stresses such as ABA-responsive elements (ABRE) and drought inducible elements (MYB, MYC, and W-Box). qRT-PCR analysis of MeGolS5under hormone treatments showed that MeGolS5was significantly up-regulated by H2O2and ABA. The results demonstrated that the galactinol synthase gene MeGolS5is involved in drought stress in cassava, and might function by means of increasing the activity of galactinol synthase, and increasing the content of soluble sugar in order to improve osmotic adjustment and scavenging of ROS. |
