甜菊醇糖苷生物合成关键基因的导入和鉴定分析

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

摘要/Abstract

摘要：

甜叶菊（Stevia rebaudiana Bertoni）生产的甜菊醇糖苷因具有高甜度、低热能、不参与人体内代谢兼具保健功能等特点，被誉为最有发展前途的新糖源。从甜叶菊叶片克隆了甜菊醇糖苷生物合成途径中的关键基因SrUGT85C2、SrUGT91D2m和SrUGT76G1，构建植物基因过量表达载体，以单独或组合的形式将这些基因导入到甜叶菊中，获得转基因植株。与野生型对照植株相比，单独导入SrUGT85C2的转基因植株中甜菊醇单糖苷含量提高，总糖苷、莱包迪苷A含量及占比没有明显变化；单独导入SrUGT91D2m的转基因植株中甜菊醇单糖苷含量显著降低，而甜菊醇双糖苷含量显著增加；单独导入SrUGT76G1的转基因植株中，总糖苷含量显著提高，莱包迪苷A含量达到10%以上，比对照提高了2倍，而甜菊糖苷含量减少了一半。3个基因组合同时导入的转基因甜叶菊植株与单独导入SrUGT76G1的转基因甜叶菊植株类似，其总糖苷、莱包迪苷A含量及其占比均显著提高。这些结果为以后通过分子生物学技术来调控甜菊醇糖苷生物合成关键基因的表达，培育莱包迪苷A含量高的高品质甜叶菊新品系提供了理论依据和技术方法。

关键词: 甜叶菊, 甜菊醇糖苷, 莱包迪苷A, UDP-葡萄糖基转移酶, 基因转化

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

张君, 张虹, 张芮, 路国栋, 雍婧姣, 郎思睿, 陈任. 甜菊醇糖苷生物合成关键基因的导入和鉴定分析[J]. 生物技术通报, 2023, 39(1): 214-223.

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.

图/表 12

图1 甜菊醇糖苷的生物合成途径 CPS, 柯巴基二磷酸合酶; KS, 贝壳杉烯合酶; KO, 贝壳杉烯氧化酶; KAH, 内根-贝壳杉烯酸羟化酶; 86C2, 91D2, 74G1, 76G1, UDP-葡萄糖基转移酶（UGT86C2, UGT91D2, UGT74G1, UGT76G1）

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）

表1 PCR扩增引物

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

图2 单基因或三基因共表达载体构建示意图 NOS-P,脂碱合酶启动子; NOS-T,脂碱合酶终止子; 35S-P,花椰菜花叶病毒35S启动子; 35S-W-P, 35S启动子附加一个欧米茄翻译增强子元件; NPT II,新霉素磷酸转移酶II; sGFP, 合成的S65T变异绿色荧光蛋白

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

图3 3个基因PCR扩增 1, SrUGT76G1基因（引物Sr76G1-S, A；1 425 bp）；2, SrUGT85C2基因（引物Sr85C2-S, A；1 511 bp）；3, SrUGT91D2m基因（引物Sr91D2m-S, A；1 441 bp）；M, DNA分子量

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

图4 3个基因以单独形式连接至pKAFCR80过渡载体后菌落PCR鉴定 1-4, SrUGT76G1基因组合（1 675 bp）; 5-8, SrUGT85C2基因组合（1 764 bp）; 9-12, SrUGT91D2m基因组合（1 693 bp）（引物均为CR80 MCS insert 35S-S, A）; M, DNA分子量

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

图5 3个基因以单独形式连接至pKAFCR100二元表达载体后菌落PCR鉴定 1-5, SrUGT76G1基因组合（2 901 bp）; 6-9, SrUGT85C2基因组合（2 808 bp）; 10-13, SrUGT91D2m基因组合（2 919 bp）（引物均为CR100 Cla-S, CR80 MCS insert NOS-A）; M, DNA分子量

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

图6 3个基因以组合形式连接至pKAFCR100二元表达载体菌落PCR鉴定 A: 1-5, SrUGT76G1基因（引物Sr76G1-S, A; 1 425 bp）; B: 1-5, SrUGT85C2基因（引物Sr85C2-S, A; 1 511 bp）; C: 1-5, SrUGT91D2m基因（引物Sr91D2m-S, A; 1 441 bp）; M, DNA 分子量

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

图7 3个基因以单独形式转化植株的PCR鉴定 N, 野生型阴性对照, 1-3, SrUGT76G1基因单独导入（引物Sr76G1-S, CR100 omega-A; 2 151 bp）; 4-6, SrUGT85C2基因单独导入（引物CR100 Cla-S, Sr85C2-A; 2 643 bp）; 7-9, SrUGT91D2m基因单独导入（引物CR80 MCS insert NOS-S, Sr91D2m-A; 2 434 bp）; M, DNA 分子量

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

图8 3个基因以组合形式转化植株的PCR鉴定 A: N, 野生型阴性对照, 11-13, 3个基因以组合形式导入（引物Sr76G1-S, pKAFCR100 omega-A; 2 151 bp）; B: N, 野生型阴性对照, 11-13, 3个基因以组合形式导入（引物pKAFCR100 Cla-S, Sr85C2-A; 2 643 bp）; C: N, 野生型阴性对照, 11-13, 3个基因以组合形式导入（引物CR80 MCS insert NOS-S, Sr91D2m-A; 2 434 bp）; M, DNA 分子量

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

图9 各种转基因植株的基因相对表达量数据为平均值 ± 标准误, n=10

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

图10 各种转基因植株与野生型对照植株

Fig. 10 Transgenic plantlets and wild-type plantlets

表2 不同转基因植株中各种甜菊醇糖苷的含量

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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