Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (14): 2436-2449.doi: 10.3864/j.issn.0578-1752.2019.14.005

• PLANT PROTECTION • Previous Articles     Next Articles

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

LIU MengQi,WU FengYing,WANG YueJin()   

  1. College of Horticulture, Northwest A & F University/State Key Laboratory of Crop Stress Biology in Arid Areas/Key Laboratory of Horticultural Plant Germplasm Resource Utilization in Northwest China, Ministry of Agriculture, Yangling 712100, Shaanxi
  • Received:2019-03-22 Accepted:2019-04-24 Online:2019-07-16 Published:2019-07-26
  • Contact: YueJin WANG E-mail:wangyj@nwsuaf.edu.cn

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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

Table 1

Primers used in the study"

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

Fig. 1

Localization, sequence alignment and cluster analysis of VqSTS26 and VqSTS32 from ‘Danfeng-2’"

Fig. 2

Transformation and identification of transgenic VqSTS26 and VqSTS32 Thompson Seedless mutant plants"

Fig. 3

Analysis of the resistance to powdery mildew in transgenic VqSTS26 and VqSTS32 plants"

Table 2

Amounts of germination, primary hyphae, secondary hyphae and conidiophore of per 100 spores of grape leaves"

株系
Line		 1 dpi 2 dpi 3 dpi 7 dpi
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

Fig. 4

Analysis of STS expression and stilbenoids content between transgenic plants and wild-type Thompson Seedless after U. necator induction"

Fig. 5

qRT-PCR analysis of STS metabolic pathway related gene expression in transgenic lines"

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