| As an important economic crop,soybean[Glycine max(L.)Merr.]has been widely developed and applied in both edible and non-edible aspects.Therefore,soybean has played an extraordinary impact on agricultural and economic development.During the growth and development of soybean,it will suffer from various biological and abiotic stresses,resulting in the impact of soybean yield and quality,causing serious economic losses.As a major abiotic stresse,drought stress has attracted more and more researchers’ attention.Many resistant plants increase tolerance through an osmotic adjustment under abiotic stress.The aquaporins and ion channel proteins will increase the osmotic regulation of plants by establishing plant cell water and ion balance.Therefore,it is of great significance to study the mechanism of aquaporins and ion channel proteins in soybean under drought conditions.In this study,a soybean variety ’Tianlong 1’ was used as the material to successfully isolate the aquaporin gene GmTIP1-1 and the voltage-dependent anion channel gene Gm VDAC to study the related function of these two genes under drought stress.The main results of this study are as follows:1.Cloning of soybean GmTIP1-1 and Gm VDACGmTIP1-1 and GmVDAC genes were cloned by designing sequence-specific primers using soybean variety Tianlong l’as material.Bioinformatics analysis found that the CDS sequence of GmTIP1-1 gene is 753 bp in length,encoding 250 amino acids,and its isoelectric point is 6.51;the CDS sequence of GmVDAC gene is 831 bp,encoding 276 amino acids,and its isoelectric point is 9.62.2.Bioinformatics analysis of GmTIP1-1 and Gm VDACPhylogenetic tree analysis suggested that TIP1-1 protein is highly conserved among species.It is very similar to TIP1-1 protein in plants such as alfalfa(Medicago truncatula)and cucumber(Cucumis sativus).The similarity to cucumber TIP1-1 is 92%,and to alfalfa TIP1-1 is as high as 99%.The VDAC protein is weakly conserved in various species.Phylogenetic tree analysis revealed that the VDAC protein of grapes(Vitis vinifera L.)is closely related,but the similarity is only 38%.Analysis of the VDAC domain suggested that the GmTIP1-1 protein contains 1 MIP domain and 6 transmembrane domains.3.Expression patterns and tissue-specific expressions of GmTIP1-1 and GmVDAC under different treatmentsThe qRT-PCR results suggested that the expression level of GmTIP1-1 gene in soybean tissues was significantly different:the expression level in soybean roots was the highest,the expression level in stems was the second,and the expression level in leaves was the lowest.Drought and ABA treatment can induce the expression of GmTIP1-1 and Gm VDAC.4.Subcellular localization analysis of GmTIP1-1 and GmVDACThe subcellular localization results suggested that GmTIP1-1 protein was localized on the cell membrane.The six transmembrane regions of GmTIP1-1 protein are located on the cell membrane.This suggests that the localization effect of each domain of GmTIP1-1 protein is the same.The GmVDAC protein is localized on the peroxisome.Like previous research results,there may be GmVDAC protein in most of the body by regulating its own on-off state to control the transport and energy conversion of metabolites,anions and cations between the peroxisome and the cytoplasmic matrix.5.Screening of GmTIP1-1 and GmVDAC interacting proteins and analysis of their mechanismThe yeast two-hybrid experiment suggested that the protein GmTIP1-1 interacted with GmSNARE and GmF-box in yeast.The qRT-PCR results suggested that soybean GmSNARE and GmF-box genes were induced to express under drought conditions.However,the expression of GmSNARE gene in leaves was not significantly different from that of wild type.Yeast two-hybrid experiments suggested that GmVDAC and GmPRX interacted in yeast.The GST-pull down experiment verified the interaction between GmVDAC and GmPRX.GmVDAC can interact with the GmPrx protein to change the Prx expression of the plant itself to balance the ROS level in the plant,and enhance the plant’s tolerance to abiotic stress. |
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