Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (2): 96-106.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1171
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MIAO Shu-nan(), GAO Yu, LI Xin-ru, CAI Gui-ping, ZHANG Fei, XUE Jin-ai, JI Chun-li(), LI Run-zhi()
Received:
2022-09-20
Online:
2023-02-26
Published:
2023-03-07
MIAO Shu-nan, GAO Yu, LI Xin-ru, CAI Gui-ping, ZHANG Fei, XUE Jin-ai, JI Chun-li, LI Run-zhi. Functional Analysis of Soybean GmPDAT1 Genes in the Oil Biosynthesis and Response to Abiotic Stresses[J]. Biotechnology Bulletin, 2023, 39(2): 96-106.
引物名称Primer name | 引物序列 Sequence(5'-3') |
---|---|
GmActin-F | AGACCTTCAATGTGCCAGCCA |
GmActin-R | CACGACCAGCAAGATCCAACC |
qGmPDAT1-A-F | TGGCACCTTTGGGGAATTGT |
qGmPDAT1-A-R | TCCTCATACCCAATGCGAGC |
qGmPDAT1-B-F | CCCCCAAGATGATGAAGCGT |
qGmPDAT1-B-R | AGGGATCCCAACCCCATACA |
qGmPDAT1-C-F | GCCGGTTCTAAGTTCAGGCT |
qGmPDAT1-C-R | CGTGAGCACCACTTTGTGTG |
qGmPDAT1-D-F | ACGGAGAAAAGGGTCGGAAC |
qGmPDAT1-D-R | CAAAGTGCAAATGCACCCCA |
qGmPDAT1-E-F | CTGCCATGTTGCTTGGCTTT |
qGmPDAT1-E-R | TGAAAAGGCCCTCTGCACAA |
qGmPDAT1-F-F | TCTTGAGCGAGTTATGCGGG |
qGmPDAT1-F-R | ATCAGTCCACACAGCCTCAC |
GmPDAT1-B-CDs-F | ATGTCGTTTATACGACGCAGAA |
GmPDAT1-B-CDs-R | CTAGAGCTTCAAATTGATCTTTTCA |
GmPDAT1-B-pYES2-F | GGGGTACCATGTCGTTTATACGACGCAGAA |
GmPDAT1-B-pYES2-R | CGGAATTCCTAGAGCTTCAAATTGATCTTTTCA |
Table 1 Primer information
引物名称Primer name | 引物序列 Sequence(5'-3') |
---|---|
GmActin-F | AGACCTTCAATGTGCCAGCCA |
GmActin-R | CACGACCAGCAAGATCCAACC |
qGmPDAT1-A-F | TGGCACCTTTGGGGAATTGT |
qGmPDAT1-A-R | TCCTCATACCCAATGCGAGC |
qGmPDAT1-B-F | CCCCCAAGATGATGAAGCGT |
qGmPDAT1-B-R | AGGGATCCCAACCCCATACA |
qGmPDAT1-C-F | GCCGGTTCTAAGTTCAGGCT |
qGmPDAT1-C-R | CGTGAGCACCACTTTGTGTG |
qGmPDAT1-D-F | ACGGAGAAAAGGGTCGGAAC |
qGmPDAT1-D-R | CAAAGTGCAAATGCACCCCA |
qGmPDAT1-E-F | CTGCCATGTTGCTTGGCTTT |
qGmPDAT1-E-R | TGAAAAGGCCCTCTGCACAA |
qGmPDAT1-F-F | TCTTGAGCGAGTTATGCGGG |
qGmPDAT1-F-R | ATCAGTCCACACAGCCTCAC |
GmPDAT1-B-CDs-F | ATGTCGTTTATACGACGCAGAA |
GmPDAT1-B-CDs-R | CTAGAGCTTCAAATTGATCTTTTCA |
GmPDAT1-B-pYES2-F | GGGGTACCATGTCGTTTATACGACGCAGAA |
GmPDAT1-B-pYES2-R | CGGAATTCCTAGAGCTTCAAATTGATCTTTTCA |
蛋白 Protein | 氨基酸数目 Number of amino acids | 分子质量 Molecular weight/kD | 理论等电点 Theoretical pI | 不稳定系数 Instability index | 亲水系数 GRAVY | 跨膜结构 Transmembrane structure |
---|---|---|---|---|---|---|
GmPDAT1-A | 668 | 74.833 | 8.59 | 36.12 | -0.270 | 1 |
GmPDAT1-B | 668 | 74.776 | 8.59 | 36.87 | -0.256 | 1 |
GmPDAT1-C | 676 | 75.571 | 6.28 | 41.96 | -0.360 | 1 |
GmPDAT1-D | 668 | 74.557 | 6.28 | 42.50 | -0.335 | 1 |
GmPDAT1-E | 625 | 70.376 | 6.45 | 42.51 | -0.173 | 1 |
GmPDAT1-F | 582 | 65.783 | 5.91 | 44.95 | -0.112 | 1 |
AtPDAT1 | 671 | 74.156 | 6.50 | 35.95 | -0.301 | 1 |
Table 2 Physicochemical properties of GmPDAT1 proteins in G. max
蛋白 Protein | 氨基酸数目 Number of amino acids | 分子质量 Molecular weight/kD | 理论等电点 Theoretical pI | 不稳定系数 Instability index | 亲水系数 GRAVY | 跨膜结构 Transmembrane structure |
---|---|---|---|---|---|---|
GmPDAT1-A | 668 | 74.833 | 8.59 | 36.12 | -0.270 | 1 |
GmPDAT1-B | 668 | 74.776 | 8.59 | 36.87 | -0.256 | 1 |
GmPDAT1-C | 676 | 75.571 | 6.28 | 41.96 | -0.360 | 1 |
GmPDAT1-D | 668 | 74.557 | 6.28 | 42.50 | -0.335 | 1 |
GmPDAT1-E | 625 | 70.376 | 6.45 | 42.51 | -0.173 | 1 |
GmPDAT1-F | 582 | 65.783 | 5.91 | 44.95 | -0.112 | 1 |
AtPDAT1 | 671 | 74.156 | 6.50 | 35.95 | -0.301 | 1 |
Fig. 3 Amino acid sequence alignment of GmPDAT1 proteins in G. max Ⅰ: Lid domain. Ⅱ: Salt bridge. Ⅲ, Ⅳ and Ⅴ: Catalytic triad. Conservative regions in the figure are marked with red boxes
Fig. 4 Phylogenetic tree of soybean GmPDAT1s and PDA-T1s from other plant species Gm: Glycine max. At: Arabidopsis thaliana. Rc: Ricinus communis. Ah: Arachis hypogaea. Si: Sesamum indicum. Bn: Brassica napus. Cs: Camelina sativa. Oep: Olea europaea. Jc: Jatropha curcas. The color from red to green indicates the kinship from near to far
Fig. 5 Cis-acting element identified in the promoters of soybean GmPDAT1 genes A: The number of cis-acting elements. B: Distribution of cis-acting elements
Fig. 7 Complementary function assay of GmPDAT1-B gene using yeast mutant H1246 A: Total oil content of transgenic yeast(INVSc1: wild-type yeast. EV: H1246 transformed with pYES2 empty vector. GmPDAT1-B: H1246 transformed with pYES2-GmPDAT1-B). B: Thin layer chromatography of transgenic yeast oil(1: TLC standard. 2: H1246. 3: H1246 transformed with pYES2 empty vector. 4: Wild-type yeast NVSc1. 5: H1246 transformed with pYES2-GmPDAT1-B)
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