Transformation and Functional Identification of the Key Genes Associated with Steviol Glycosides Biosynthesis in Stevia rebaudiana

doi:10.13560/j.cnki.biotech.bull.1985.2022-0388

Abstract

Abstract:

Steviol glycosides produced by Stevia rebaudiana Bertoni are regarded as the most promising substitute for sucrose with a wide range of potential applications and high economic value due to their high sweetness, low caloric energy, non-participation in human metabolism and health care functions. Three key genes from Stevia leaves, SrUGT85C2, SrUGT91D2m and SrUGT76G1 in the biosynthetic pathway of steviol glycosides, were cloned, and overexpression vector of plant genes were constructed. In individual or in combined way, these genes were transferred into the S. rebaudiana, and the transgenic plants were obtained. Compared with the control of wild-type plant, the content of steviolmonoside increased in the transgenic plant transferred SrUGT85C2 alone, despite little changes in the total content of steviol glycosides, rebaudioside A, and the content ratio of rebaudioside A to the total steviol glycosides. In the transgenic plant transferred SrUGT91D2m alone, the content of steviolmonoside decreased, while the content of steviolbioside increased significantly. In the other hand, in the transgenic plant transferred SrUGT76G1 alone, the total amount of steviol glycosides significantly increased, especially the content of rebaudioside A reached to 10%, which was nearly two times than that in the control, whereas the content of stevioside reduced by half. The transgenic plant transferred the three genes in combination was similar to the transgenic plant transferred SrUGT76G1 alone, and the total content of steviol glycosides, rebaudioside A, and the content ratio of rebaudioside A to the total steviol glycosides increased significantly. These results provides the theoretical basis and technical methods for regulating the expressions of key genes in the biosynthesis of steviol glycosides and cultivating new high-quality S. rebaudiana strains with high content of rebaudioside A through molecular biology techniques.

Key words: Stevia rebaudiana, steviol glycosides, rebaudioside A, UDP-glucosyltransferases, gene transformation

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.

Figures/Tables 12

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）

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

Fig. 9 Gene relative expressions in transgenic plantlets Data represent means ± standard error, n=10

Fig. 10 Transgenic plantlets and wild-type plantlets

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 ± 1.17^c	1.26 ± 0.13^b	0.66 ± 0.07^c	5.47 ± 0.65^b	5.34 ± 0.52^c	40.56 ± 2.36^c
Sr01102（SrUGT85C2）	13.22 ± 1.46^bc	1.57 ± 0.17^a	0.74 ± 0.06^b	5.55 ± 0.84^b	5.36 ± 0.64^c	40.79 ± 2.20^c
Sr01103（SrUGT91D2m）	13.65 ± 1.16^b	0.63 ± 0.07^c	1.10 ± 0.15^a	5.98 ± 0.56^a	5.95 ± 0.64^c	42.79 ± 2.73^c
Sr01101（SrUGT76G1）	15.22 ± 1.25^ab	1.22 ± 0.12^b	0.74 ± 0.06^b	2.49 ± 0.24^d	10.77 ± 1.02^a	71.70 ± 2.43^a
Sr01105（3个基因组合/3 genes Combined）	16.12 ± 1.59^a	1.23 ± 0.11^b	0.83 ± 0.09^b	4.60 ± 0.36^c	9.45 ± 1.15^b	58.27 ± 1.96^b

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