Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (1): 214-223.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0388
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ZHANG Jun1,2,3(), ZHANG Hong1,2,3, ZHANG Rui1,2,3, LU Guo-dong1,2,3, YONG Jing-jiao1,2,3, LANG Si-rui1,2,3, CHEN Ren1,2,3()
Received:
2022-04-01
Online:
2023-01-26
Published:
2023-02-02
Contact:
CHEN Ren
E-mail:zj1340131221@qq.com;chenren@nxu.edu.cn
ZHANG Jun, ZHANG Hong, ZHANG Rui, LU Guo-dong, YONG Jing-jiao, LANG Si-rui, CHEN Ren. Transformation and Functional Identification of the Key Genes Associated with Steviol Glycosides Biosynthesis in Stevia rebaudiana[J]. Biotechnology Bulletin, 2023, 39(1): 214-223.
Fig. 1 Biosynthetic pathways of steviol glycosides CPS, copalylpyrophosphate synthase; KS, kaurene synthase; KO, kaurene oxidized; KAH, kaurenoicacid hydroxylase; 86C2, 91D2, 74G1, 76G1, UDP-glucosyltransferases(UGT86C2, UGT91D2, UGT74G1, UGT76G1)
引物名称Primer name | 引物序列Primer sequence(5'-3') | 引入酶切位点Restriction site |
---|---|---|
Sr76G1-S | TATGGATCCTTGCGTGTAAACGTCAGT | BamH I |
Sr76G1-A | ATGCTCGAGTTATTTACAACGATGAAATGT | Xho I |
Sr85C2-S | TATGGTACCATGGATGCAATGGCTACAAC | Kpn I |
Sr85C2-A | GCCGAGCTCCAAAGTTACATCTTAAATAG | Sac I |
Sr91D2m-S | TTACGGTACCATGGCTACCAGTGACTC | Kpn I |
Sr91D2m-A | GTGGAGCTCTTAACTCTCATGATCGA | Sac I |
CR80 MCS insert 35S-S | CACAATCCCACTATCCTTCGCA | |
CR80 MCS insert NOS-A | TCCACTCTAATCATAAAAACCCATCTC | |
CR80 MCS insert NOS-S | GAGATGGGTTTTTATGATTAGAGTCC | |
CR100 Cla-S | CGCTACTGATTACGGTGCTGCTAT | |
CR100 omega-A | TTGTTTGTTGTTTGTTGTTGTTGGTAA |
Table 1 Primers for PCR amplification
引物名称Primer name | 引物序列Primer sequence(5'-3') | 引入酶切位点Restriction site |
---|---|---|
Sr76G1-S | TATGGATCCTTGCGTGTAAACGTCAGT | BamH I |
Sr76G1-A | ATGCTCGAGTTATTTACAACGATGAAATGT | Xho I |
Sr85C2-S | TATGGTACCATGGATGCAATGGCTACAAC | Kpn I |
Sr85C2-A | GCCGAGCTCCAAAGTTACATCTTAAATAG | Sac I |
Sr91D2m-S | TTACGGTACCATGGCTACCAGTGACTC | Kpn I |
Sr91D2m-A | GTGGAGCTCTTAACTCTCATGATCGA | Sac I |
CR80 MCS insert 35S-S | CACAATCCCACTATCCTTCGCA | |
CR80 MCS insert NOS-A | TCCACTCTAATCATAAAAACCCATCTC | |
CR80 MCS insert NOS-S | GAGATGGGTTTTTATGATTAGAGTCC | |
CR100 Cla-S | CGCTACTGATTACGGTGCTGCTAT | |
CR100 omega-A | TTGTTTGTTGTTTGTTGTTGTTGGTAA |
Fig. 2 Schematic structure of co-expression vectors of single genes or three genes NOS-P, nopaline synthase promoter; NOS-T, nopaline synthase terminator; 35S-P, cauliflower mosaic virus 35S promoter; 35S-W-P, 35S promoter with an additional omega element translational enhancer; NPT II, neomycin phosphotransferase II; sGFP, synthetic green-fluorescent protein with S65T mutation
Fig. 3 PCR amplification of 3 target genes 1, SrUGT76G1(primer pair: Sr76G1-S, A; 1 425 bp); 2, SrUGT85C2(primer pair: Sr85C2-S, A; 1 511 bp); 3, SrUGT91D2m(primer pair: Sr91D2m-S, A; 1 441 bp); M, DNA marker
Fig. 4 Colony PCR identification of the 3 genes individually constructed into pKAFCR80 intermediate vector 1-4, SrUGT76G1(1 675 bp); 5-8, SrUGT85C2(1 764 bp); 9-12, SrUGT91D2m(1 693 bp)(All primer pair: CR80 MCS insert 35S-S, A); M, DNA marker
Fig. 5 Colony PCR identification of the 3 genes individu-ally constructed into pKAFCR100 binary vector 1-5, SrUGT76G1(2 901 bp); 6-9, SrUGT85C2(2 808 bp); 10-13, SrUGT91D2m(2 919 bp)(All primers pair: CR100 Cla-S, CR80 MCS insert NOS-A); M, DNA maker
Fig. 6 Colony PCR identification of 3 genes in combination constructed into pKAFCR100 binary vector A: 1-5, SrUGT76G1(primer pair: Sr76G1-S, A; 1 425 bp); B: 1-5, SrUGT85C2(primer pair: Sr85C2-S, A; 1 511 bp); C: 1-5, SrUGT91D2m(primer pair: Sr91D2m-S, A; 1 441 bp); M, DNA marker
Fig. 7 PCR identification of the transgenic plant transferr-ed 3 genes individually N, wild-type negative control, 1-3, SrUGT76G1 gene transferred individually(primer pair: Sr76G1-S, CR100 omega-A; 2 151 bp); 4-6, SrUGT85C2 gene transferred individually(primer pair: CR100 Cla-S, Sr85C2-A; 2 643 bp); 7-9, SrUGT91D2m gene transferred individually(primer pair: CR80 MCS insert NOS-S, Sr91D2m-A; 2 434 bp); M, DNA marker
Fig. 8 PCR identification of the transgenic plant transferr-ed 3 genes in combination A: N, wild-type negative control, 11-13, three genes were transferred in combination(primer pair: Sr76G1-S, pKAFCR100 omega-A; 2 151 bp). B: N, wild-type negative control, 11-13, three genes were transferred in combination(primer pair: pKAFCR100 Cla-S, Sr85C2-A; 2 643 bp). C: N, wild-type negative control, 11-13, three genes were transferred in combination(primer pair: CR80 MCS insert NOS-S, Sr91D2m-A; 2 434 bp). M, DNA marker
转基因植株Transgenic plant | SGs/% | SMono/% | SBio/% | ST/% | RA/% | RA/SGs/% |
---|---|---|---|---|---|---|
野生型对照Wild-type control | 12.74 ± 1.17c | 1.26 ± 0.13b | 0.66 ± 0.07c | 5.47 ± 0.65b | 5.34 ± 0.52c | 40.56 ± 2.36c |
Sr01102(SrUGT85C2) | 13.22 ± 1.46bc | 1.57 ± 0.17a | 0.74 ± 0.06b | 5.55 ± 0.84b | 5.36 ± 0.64c | 40.79 ± 2.20c |
Sr01103(SrUGT91D2m) | 13.65 ± 1.16b | 0.63 ± 0.07c | 1.10 ± 0.15a | 5.98 ± 0.56a | 5.95 ± 0.64c | 42.79 ± 2.73c |
Sr01101(SrUGT76G1) | 15.22 ± 1.25ab | 1.22 ± 0.12b | 0.74 ± 0.06b | 2.49 ± 0.24d | 10.77 ± 1.02a | 71.70 ± 2.43a |
Sr01105(3个基因组合/3 genes Combined) | 16.12 ± 1.59a | 1.23 ± 0.11b | 0.83 ± 0.09b | 4.60 ± 0.36c | 9.45 ± 1.15b | 58.27 ± 1.96b |
Table 2 Variety of steviol glycosides contents in different transgenic plant
转基因植株Transgenic plant | SGs/% | SMono/% | SBio/% | ST/% | RA/% | RA/SGs/% |
---|---|---|---|---|---|---|
野生型对照Wild-type control | 12.74 ± 1.17c | 1.26 ± 0.13b | 0.66 ± 0.07c | 5.47 ± 0.65b | 5.34 ± 0.52c | 40.56 ± 2.36c |
Sr01102(SrUGT85C2) | 13.22 ± 1.46bc | 1.57 ± 0.17a | 0.74 ± 0.06b | 5.55 ± 0.84b | 5.36 ± 0.64c | 40.79 ± 2.20c |
Sr01103(SrUGT91D2m) | 13.65 ± 1.16b | 0.63 ± 0.07c | 1.10 ± 0.15a | 5.98 ± 0.56a | 5.95 ± 0.64c | 42.79 ± 2.73c |
Sr01101(SrUGT76G1) | 15.22 ± 1.25ab | 1.22 ± 0.12b | 0.74 ± 0.06b | 2.49 ± 0.24d | 10.77 ± 1.02a | 71.70 ± 2.43a |
Sr01105(3个基因组合/3 genes Combined) | 16.12 ± 1.59a | 1.23 ± 0.11b | 0.83 ± 0.09b | 4.60 ± 0.36c | 9.45 ± 1.15b | 58.27 ± 1.96b |
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