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

摘要/Abstract

摘要：

【目的】克隆中国野生毛葡萄（Vitis quinquangularis）‘丹凤-2’芪合酶（stilbene synthase）基因（STS）并研究其功能,为提高欧洲葡萄（V. vinifera）的白粉病抗性及品质提供依据。【方法】利用同源克隆法获得中国野生毛葡萄‘丹凤-2’芪合酶基因VqSTS9和VqSTS21,构建植物过表达载体;用无核白单芽茎段诱导出分生愈伤组织,作为农杆菌介导法遗传转化的受体材料,获得抗性植株,经过不同水平检测,确定转基因植株;对野生型和转基因植株叶片人工接种葡萄白粉病菌（Uncinula necator）,通过显微技术观察叶片受白粉病菌侵染后的情况,比较两者对白粉病的抗性;利用实时荧光定量PCR（qRT-PCR）分析野生型和转基因植株在自然条件和接种白粉病菌后STS及其相关基因的表达,用高效液相色谱法（HPLC）检测转基因植株中芪类物质的种类与含量。【结果】同源序列克隆得到VqSTS9（JQ868689）与VqSTS21（JQ868677）的cDNA序列,长度为1 179 bp。经PCR和Western blot检测,鉴定出过表达VqSTS9无核白植株4株和过表达VqSTS21无核白植株3株。显微观察发现,与野生型植株相比,转VqSTS9 和VqSTS21植株叶片上的菌丝生长较慢,表现出对白粉病的抗性。qRT-PCR结果表明,自然生长条件下,与野生型植株相比,转VqSTS9和VqSTS21植株STS的表达量提高,STS上游苯丙氨酸裂解酶基因（PAL）、下游白藜芦醇糖基转移酶基因（RSGT）、转录因子基因（MYB14、MYB15）的表达量均不同程度上升,而查尔酮合成酶基因（CHS）表达量降低;人工接种白粉病菌后,与野生型植株相比,转基因植株STS表达量显著上调。高效液相色谱分析表明,自然条件下,芪类物质主要以反式云杉新苷形式存在,转基因植株芪类物质的含量高于野生型植株;在接种白粉病菌诱导表达后,除了反式云杉新苷,还产生了反式白藜芦醇和葡萄素,即转基因植株体内芪类物质的种类和含量均有所增加。【结论】将VqSTS9、VqSTS21转入无核白后,转基因植株STS的表达量增高,芪类物质的含量与种类增加,并抑制白粉病菌的生长。因此,中国野生毛葡萄‘丹凤-2’携带的VqSTS9和VqSTS21能够增强欧洲葡萄对白粉病的抗性,‘丹凤-2’可用作葡萄抗病性育种的种质资源。

关键词: 中国野生毛葡萄, 芪合酶基因, 白藜芦醇, 白粉病, 遗传转化, 抗病性

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

丁茜,赵凯茜,王跃进. 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[J]. 中国农业科学, 2021, 54(2): 310-323.

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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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