Overexpression of Soybean GmGRAS69 Gene Enhances Drought Tolerance of Transgenic Arabidopsis

doi:10.7668/hbnxb.20193597

Abstract

Abstract:

To investigate the function and possible molecular mechanisms of soybean GmGRAS69 gene,which belongs to the GRAS transcription factor family,in plant drought stress.Sequence conservation and evolutionary relationship between soybean GmGRAS69 and GRAS members of other species were analyzed by amino acid sequence alignment and phylogenetic tree construction.Quantitative Real-time PCR was used to detect the expression pattern of GmPP2C69 in soybean roots and leaves treated with PEG.Then,GmPP2C69 overexpression vector was constructed,and then wild-type Arabidopsis was transformed by Agrobacterium-mediated floral dip method.The growth phenotypes of wild-type and transgenic Arabidopsis were observed under normal culture and drought treatment.The fresh weight per plant,relative water content of leaf,soluble sugar content of shoot,antioxidant enzymes(SOD,POD and CAT)activities and corresponding antioxidant enzyme gene expression levels were measured.The results showed that GmGRAS69 was significantly up-regulated in both roots and leaves of PEG-treated soybean plants,and the response was more significant in roots.In addition,GmGRAS69-overexpressed Arabidopsis was successfully obtained,and the drought tolerance of the GmGRAS69-overexpressed plants was significantly enhanced compared with WT.Under drought treatment,the fresh weight,leaf relative water content and soluble sugar content of GmGRAS69-overexpressed plants were significantly higher than those of WT.The activities of antioxidant enzymes SOD,POD and CAT and the expression levels of corresponding genes SOD,POD and CAT were also significantly higher than those of WT.The results showed that the expression of GmGRAS69 was up-regulated under drought stress,and the transgenic plants were enhanced drought tolerance by activating SOD,POD and CAT antioxidant enzyme encoding genes,enhancing antioxidant enzyme activities and increasing soluble sugar accumulation.

Key words: Soybean, GRAS family, GmGRAS69 gene, Drought stress, Drought tolerance

ZHANG Bin. Overexpression of Soybean GmGRAS69 Gene Enhances Drought Tolerance of Transgenic Arabidopsis[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(3): 18-25. doi: 10.7668/hbnxb.20193597.

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Figures/Tables 6

Tab.1 Primer sequences for Real-time quantitative PCR experiments

基因 Gene	引物序列(5'-3') Sequence (5'-3')
GmGRAS69	F:TGCCAGGTCTTGTGGAGTTC
	R:TCATCTGGCATGTGGTGCAA
GmUBI3	F:GGGGGTGTAACTGGAAAGCA
	R:TTGCACGTCCTTCCATCCTC
SOD	F:TGGAACTGCCACCTTCACAA
	R:TTCCGAGGTCATCAGGGTCT
POD	F:TAACCAACCAGACGAGACGC
	R:TCGAATCTTGCAGGAGAGGC
CAT	F:AACTCCGCCTGCTGTCTG
	R:ATAGGGCATCAATCCATC
ACT	F:CACTGTGCCAATCTACGAGGGT
	R:CACAAACGAGGGCTGGAACAAG

Fig.1 Alignment and phylogenetic analysis of GmGRAS69 with other GRAS proteins A.Amino acid sequence alignment of soybean GmGRAS69 and other GRAS proteins;B.Phylogenetic tree of the GRAS family.

Fig.2 Expression level of GmGRAS69 gene in leaves and roots of soybean under PEG treatment Different lowercase letters indicate significant difference.The same as Fig.4-5.

Fig.3 Construction of overexpression vector of GmGRAS69 gene and identification of transgenic Arabidopsis A.Cloning of CDS fragment of GmGRAS69;B.Enzyme digestion verification of recombinant vector;C.Schematic diagram of overexpression vector;D.Screening of Arabidopsis plants resistant to glufosinate;E.PCR identification of transgenic Arabidopsis;F.Semi-quantitative PCR detection of heterologous expression of GmGRAS69.

Fig.4 Analysis of drought tolerance of Arabidopsis overexpressing GmGRAS69

Fig.5 Effects of overexpression of GmGRAS69 on antioxidant enzyme activity and expression level of antioxidant enzyme genes

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