中国野生毛葡萄芪合酶基因表达及对葡萄抗白粉病的影响

doi:10.3864/j.issn.0578-1752.2021.02.007

Abstract

Abstract:

【Objective】Two stilbene synthase (STS) genes from Chinese wild Vitis quinquangularis ‘Danfeng-2’ (VqSTS9 and VqSTS21) were cloned and functionally investigated with the aim to provide a theoretical basis for improving the disease-resistance and quality of V. vinifera.【Method】Homologous cloning was applied to obtain VqSTS9 and VqSTS21 and their overexpression vectors under the control of CaMV35 promoter were constructed, respectively. The calluses were induced from single bud segment of V. vinifera cv. ‘Thompson Seedless’, which were used as the receptor materials. The resistant ‘Thompson Seedless’ plants were obtained via Agrobacterium-mediated genetic transformation. Furthermore, the disease-resistant transgenic plants were determined by different levels of detection. The leaves of wild-type (WT) and transgenic plants were inoculated with Uncinula necator, the pathogenic conditions of leaves infected with U. necator were observed by microscope to compare their resistance. The expressions of STS and other genes in the metabolic pathway of resveratrol synthesis were analyzed by quantitative real-time PCR (qRT-PCR) in WT and transgenic plants under natural conditions and after inoculation with U. necator. The types and contents of stilbenoids in transgenic plants were determined by HPLC.【Result】The cDNA sequences of VqSTS9 (JQ868689) and VqSTS21 (JQ868677) from the Chinese wild V. quinquangular accession ‘Danfeng-2’ were cloned, and both were 1 179 bp in length. Four VqSTS9-overexpressing and three VqSTS21-overexpressing plants were confirmed by PCR and Western blot analysis. These transgenic plants enhanced resistance to powdery mildew and reduced hyphae growth compared with WT plants through observing the growth of U. necator. The qRT-PCR results indicated that VqSTS9 and VqSTS21 transgenic plants increased the expressions of STS, its upstream PAL, the downstream RSGT, and transcription factor genes MYB14, MYB15 under the natural conditions when compared with WT plants. However, the expression of CHS was down-regulated. After inoculation, the expression of STS in transgenic plants was significantly up-regulated compared to WT. HPLC determination demonstrated that the stilbenoids mainly existed as the form of trans-piceid, and its content in transgenic plants was higher than that in WT under natural conditions. After inoculation, the expression of STS was induced, and trans-piceid, trans-resveratrol and viniferin were also produced. Compared with WT plants, the types and contents of stilbenoids in transgenic plants increased.【Conclusion】Overexpression of VqSTS9 and VqSTS21 in ‘Thompson Seedless’ can enhance the expression of STS, increase the types and contents of stilbenoids, and further inhibit the growth of U. necator. Therefore, ‘Danfeng-2’ is an important germplasm resource for disease-resistant breeding and quality improvement of V. vinifera. Stilbene synthase genes VqSTS9 and VqSTS21 can improve resistance to powdery mildew of V. vinifera.

Key words: Chinese wild Vitis quinquangularis, stilbene synthase gene (STS), resveratrol, powdery mildew, genetic transformation, disease-resistance

DING Xi,ZHAO KaiXi,WANG YueJin. Expression of Stilbene Synthase Genes from Chinese Wild Vitis quinquangularis and Its Effect on Resistance of Grape to Powdery Mildew[J].Scientia Agricultura Sinica, 2021, 54(2): 310-323.

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References 46

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基因 Gene	上游引物 Forward primer	下游引物 Reverse primer	目标大小 Target size (bp)
VqSTS9 (vector construction, PCR detection)	F: CGGGATCCATGGCTTCAGTCGAGGAA TTTAGAAACG	R: GCGTCGACATTTGTAACCGTAGGAAT GCTATGCAGC	1179
VqSTS21 (vector construction, PCR detection)	F: CGGGATCCATGGCTTCAGTCGAGGAA ATTAGAAACG	R: GCGTCGACATTTGTAACCATAGGAAT GCTATGCAACAC	1179

株系 Line	1 dpi			2 dpi			3 dpi			7 dpi
株系 Line	萌发数 Germination	初级菌丝数 Primary hyphae	次级菌丝数 Secondary hyphae	萌发数 Germination	初级菌丝数 Primary hyphae	次级菌丝数 Secondary hyphae	萌发数Germination	初级菌丝数 Primary hyphae	次级菌丝数 Secondary hyphae	分生孢子梗数 Conidiophore
WT	35.67±2.05a	16.67±1.25a	2.33±0.47a	25.67±1.70a	10.33±1.25d	36.67±1.70a	14.67±1.25d	8.67±1.25c	45.00±1.63a	203.33±5.31a
OEVqSTS9-L1	21.33±2.36c	5.67±1.25d	0	14.67±1.25cd	12.67±2.05cd	5.33±1.25b	22.33±2.49bc	15.33±1.25a	13.67±1.25c	61.33±3.86e
OEVqSTS9-L2	16.67±1.70d	8.00±2.45cd	1.33±1.25a	11.33±1.25d	14.67±0.94bc	6.67±1.70b	21.67±1.70c	11.67±1.25b	16.33±0.47bc	77.00±3.56d
OEVqSTS21-L2	26.67±2.05b	10.67±1.70bc	1.33±1.89a	19.00±1.63b	17.33±1.25ab	7.33±1.25b	27.33±1.25a	14.33±1.70ab	16.33±2.49bc	87.33±3.68c
OEVqSTS21-L3	22.33±1.7bc	13.00±0.82b	0.67±0.94a	17.33±1.70bc	19.33±1.25a	8.33±1.25b	25.67±0.47ab	16.00±0.82a	18.00±1.63b	124.67±4.11b

Expression of Stilbene Synthase Genes from Chinese Wild Vitis quinquangularis and Its Effect on Resistance of Grape to Powdery Mildew

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