| Soybean(Glycine max(L.)Merr.)is the main source of protein and oil for the world’s population,and also an important source of feed protein.It has important economic value and is closely related to national food security issues.Climate change has led to a gradual lack of freshwater resources worldwide.Drought and water shortages on arable land have become the most important factor in limiting soybean production.In this study,a reverse genetic method was used.The gene sequence of wild soybean HD-Zip I transcription factor Gshdz4 was used for excavating the cultivated soybean genome sequence.The homologous sequence Glyma11g06940 in soybean was obtained.Its nucleic acid sequence and protein sequence were analyzed by bioinformatics,its transcription factor function was verified,and its mechanism of action in response to drought stress was explored.The specific research results are as follows:1.Based on protein structure prediction and multiple sequence alignment with members of soybean HD-Zip Ⅰ subfamily,it was confirmed that the sequence has a homeodomain(HD)and a leucine zipper domain(LZ)and belongs to the HDZip Ⅰ subfamily.And it was named “GmHdz4”.Through gene structure analysis and phylogenetic tree analysis,it was further clarified that it belongs to the δ subgroup and its orthologous relationship with wild soybean Gshdz4;2.The promoter prediction results showed that the promoter region of GmHdz4 had drought response elements and ABA response elements.PEG simulated droughtand ABA-induced expression showed that GmHdz4 has tissue-specific expression characteristics.And the relative expression in the root was negatively correlated with the duration of drought or ABA treatment.3.Subcellular localization experiment on tobacco verified that the protein encoded by GmHdz4 was localized in the nucleus.Yeast two-hybrid experiments showed that GmHdz4 has an activating activity but not a DNA-binding activity as a transcription factor,and is an atypical transcription factor.4.A hairy root system of soybean was constructed using A.rhizogenes K599.Transformed hair roots were induced with recombinant plasmids,resulting in overexpression lines GmHdz4-oe and gene editing lines gmhdz4.Non-transgenic lines NT was used as a control.5.Overexpression of GmHdz4 lead to inhibition of soybean hairy root elongation and lateral root number,which affected the normal development of soybean roots and the roots morphological response under drought stress.It also has a related impact on the soybean shoots.On the contrary,knockout GmHdz4 can promote root growth.6.Overexpression of GmHdz4 led to more sensitive osmotic adjustment of soybean hair roots under drought stress,with more soluble sugar and proline accumulation.And overexpression of GmHdz4 aggravated root damage caused by drought.7.Overexpression of GmHdz4 resulted in inhibition of antioxidant enzyme activity.SOD,POD,and CAT became insensitive to response drought stress.Knockout of the GmHdz4 increased the activities of SOD,CAT and other antioxidases under drought stress,and promoted the elimination of reactive oxygen species.To sum up the results of the study,it can be speculated that the transcription factor GmHdz4 may serve as a negative regulatory factor,which negatively affects the drought tolerance mechanism of soybean. |
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