中国野生毛葡萄芪合成酶基因表达与抗白粉病分析

doi:10.3864/j.issn.0578-1752.2019.14.005

Abstract

Abstract:

【Objective】 Grapevine is an important fruit crop in the world. European grape is one of the most important cultivated grape varieties for its high quality and high yield. However, it is susceptible to fungal diseases, especially the powdery mildew. Resveratrol is a metabolite of stilbene synthase (STS) in grape. It is known as a phytoalexin and plays an important role in disease resistance. Besides, resveratrol in fruits has a health-care effect on humans. Chinese wild Vitis quinquangularis ‘Danfeng-2’ displays resistance to powdery mildew and high resveratrol content. The objective of this study is to analyze the function of STS from Chinese wild V. quinquangularis ‘Danfeng-2’, and to apply it to disease-resistance breeding to improve European grapes’ disease-resistance and stilbenoids content. 【Method】 VqSTS26 and VqSTS32 were obtained by homologous cloning from Chinese wild V. quinquangularis ‘Danfeng-2’, and the pCAMBIA35S:: VqSTSs:: GFP overexpression vector was constructed. The meristem callus of V. vinifera Thompson Seedless was induced from the organogenesis, which was used as the receptor material. The transgenic grapes were obtained by agrobacterium-mediated genetic transformation. The expression of STS at transcriptional level and the content of stilbenoids at metabolite level were compared between transgenic plants and wild-type Thompson Seedless under natural growth conditions and Uncinula necator induction. The growth and development of U. necator on transgenic and wild-type grapes’ leaves were observed by microscope. The spore germination, hyphal growth and conidiophore formation were counted, and the powdery mildew resistance of transgenic plants was analyzed. 【Result】 Eight transgenic plants with VqSTS26 and five transgenic plants with VqSTS32 were successfully obtained by PCR detection and Western blot identification. Real-time quantitative PCR analysis (qRT-PCR) showed that the expression of STS was significantly increased in transgenic grapes under natural condition, and the expression of its upstream gene PAL and downstream gene RSGT was up-regulated, while the expression of CHS was down-regulated due to the substrate competition with stilbene synthase. Liquid chromatographic analysis showed that the stilbenoids content in transgenic grapes was higher than that in wild-type Thompson Seedless, mainly existed in the form of trans-piceid. The expression of STS and its product synthesis were induced by U. necator. The expression of STS in transgenic grapes was significantly increased at 1-2 dpi (days post induction) under U. necator induction and was highest at 7 dpi, while trans-resveratrol and viniferin were added from original trans-piceid. The STS expression and its products’ content in transgenic plants displayed excellently higher levels than those in wild-type Thompson Seedless under U. necator induction. U. necator developmental process on grape leaves was observed by microscope. It was found that the growth of U. necator on transgenic grapes was inhibited compared with that of wild-type Thompson Seedless, and the development of hyphae was later than that on wild-type Thompson Seedless. The number of conidiophore on transgenic grape leaves at 7 dpi was lower than that on wild-type Thompson Seedless. 【Conclusion】 Overexpression of VqSTS26 and VqSTS32 from ‘Danfeng-2’ in Thompson Seedless can improve the expression of STS, promote the formation and accumulation of stilbenoids, and inhibit the growth of U. necator on transgenic Thompson Seedless leaves. Therefore, Chinese wild V. quinquangularis ‘Danfeng-2’ and its carrying STS, as well as its products are important germplasm resources and genetic resources for the improvement of resistance to powdery mildew and content of stilbenoids in European grape varieties.

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

LIU MengQi,WU FengYing,WANG YueJin. Expression of Stilbene Synthase Gene and Resistance to Powdery Mildew Analysis of Chinese Wild Vitis quinquangularis[J].Scientia Agricultura Sinica, 2019, 52(14): 2436-2449.

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References 43

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基因 Gene	引物序列 Primer sequence	目标大小 Target size (bp)
VqSTS26 (Vector construction, PCR detection)	F: AGAACACGGGGGACGAGCTCATGGCTTCAGTTGAGGAATTTAGAAACG R: ACCATGGTGTCGACTCTAGAATTTGTAACTGTAGGAATGCTATGCAGC	1179
VqSTS32 (Vector construction, PCR detection)	F: AGAACACGGGGGACGAGCTCATGGCTTCAATTGAGGAAATTAGAAACGC R: ACCATGGTGTCGACTCTAGAATTTGTAACCATAGGAACGCTATGCAG	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	分生孢子梗数 Conidiophore
野生型 Wide type	42.67± 3.51a	21.67± 2.08a	4.33± 1.53a	22.67± 2.08a	8.67± 1.15c	50.33± 3.51a	6.33± 1.53c	12.67± 1.53c	69.00± 4.00a	15.33±1.53a	195.33± 12.66a
OEVqSTS26-L6	32.33± 2.52bc	7.33± 1.53b	0.33± 0.58b	16.33± 0.58b	11.67± 1.53c	14.33± 2.08c	30.67± 1.53a	13.33± 1.53c	16.33± 2.52c	0	92.67± 7.37b
OEVqSTS26-L8	26.00± 1.00c	12.00± 2.00b	0.33± 0.58b	17.33± 1.53b	17.67± 2.52b	10.33± 1.53c	31.67± 3.51a	11.00± 1.73c	23.00± 2.65c	0	42.00± 3.00c
OEVqSTS32-L3	35.00± 1.53b	17.67± 2.52a	3.33± 1.53ab	17.00± 1.00b	25.00± 1.00a	22.67± 1.53b	13.33± 2.52b	18.00± 1.00b	34.33± 3.06b	0	110.33± 14.29b
OEVqSTS32-L4	35.33± 1.53b	19.67± 1.53a	3.67± 1.53a	13.67± 1.53b	21.00± 2.00ab	26.33± 1.53b	18.33± 1.53b	23.00± 2.00a	32.33± 2.08b	0	58.33± 2.08c

Expression of Stilbene Synthase Gene and Resistance to Powdery Mildew Analysis of Chinese Wild Vitis quinquangularis

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