葡萄体细胞胚的诱导及中国野葡萄芪合成酶基因转化研究

葡萄体细胞胚的诱导及中国野葡萄芪合成酶基因VpSTSgDAN2转化研究

STUDY ON VITIS SOMATIC EMBRYO INDUCTION AND CHINESE WILD VITIS STILBENE SYNTHASE GENE VPSTSGDAN2 TRANSFORMATION

作者：肖　宇　　导师：王跃进

西北农林科技大学　　园艺植物种质资源学2011届硕士

摘　要

芪合成酶(stilbene synthase,STS)基因及其特异启动子为本课题组前期从中国野生华东葡萄白河-35-1中克隆得到的抗病相关基因。本研究将芪合成酶基因置于增添不同增强子的该启动子下，构建4个植物表达载体，旨在利用基因工程技术，将其转入感病欧洲葡萄基因组中，为获得抗病性明显提高的转基因植株提供理论与技术依据。

在以“佳利酿”、“无核白”、“火焰无核”、“长穗无核白”、“赤霞珠”的花器官为外植体进行体细胞胚诱导与成苗研究的基础上，通过农杆菌介导法，将携带有中国野生葡萄芪合成酶基因及其特异启动子的植物表达载体转入番茄和葡萄中，取得的主要结果如下：

1．　以5个种欧洲葡萄品种“佳利酿”、“无核白”、“火焰无核”、“长穗无核白”、“赤霞珠”的雄蕊、雌蕊和小花蕾为外植体，在4种不同的培养基上诱导愈伤，其中培养基NN69 + 2.0 mg/L 6-BA + 1.0 mg/L 2,4-D + 60 g/L蔗糖 + 3 g/L phatagel和培养基MS + 0.55 mg/L 2,4-D + 0.5 mg/L NOA+1.24 mg/L 4-CPPU + 30 g/L蔗糖 + 3 g/L phatagel对花器官外植体胚性愈伤的诱导比较有利，诱导率最高可达6.2%。

2．　所研究的5个欧洲葡萄品种，除了“赤霞珠”外，其余4个品种经花器官诱导出的愈伤均可在培养基X6 + 60 g/L蔗糖+ 7 g/L TC-Agar + 0.5 g/L AC中进一步诱导出体细胞胚。而且对于发育时期在心型胚之前的体细胞胚可以在X6培养基中继代保存，产生次生胚。

3．　体细胞胚在液体的MS培养基中110rpm 25℃暗培养1w后转移到成苗培养基WPM+ 0.15 mg/L IBA + 30 g/L蔗糖+7 gL^-1 琼脂 + 0.5 g/L AC中，先弱光培养3 d后置于强光照下培养，1 w左右即可成苗。

4．　利用农杆菌介导法，将含有中国野葡萄芪合成酶基因及其特异启动子的载体转入番茄Micro-Tom中，经初步的PCR和PCR-Southern检测共获得了51株转基因苗。

5．　通过高效液相色谱对番茄转基因植株和野生型植株接种番茄灰霉病24 h后白藜芦醇含量的测定，结果表明含有增强元件Enhancer的转化植株白藜芦醇含量最高，是不添加任何增强元件的载体gD2转化的番茄白藜芦醇含量的4.4倍。野生型植株均未检测到白藜芦醇。

6．　通过接种番茄灰霉病，测定其病情指数，结果表明转基因番茄抗病性都有一定提高，其中含有增强元件Enhancer的增强载体转化的番茄抗病性最强。

关键词　葡萄　体细胞胚发生　农杆菌介导　芪合成酶　启动子

Abstract

Stilbene synthase gene (STS)anditsspecific promoter were previously isolated from Chinese wild Vitis pseudoreticulata accession ‘Baihe-35-1’ ,which were related to disease resistance. The promoter was Added different enhancement components for construction of three enhanced expression vectors. In order to provide theoretical and technical basis for obtaining disease resistance transgenic grapevine,in this study,these four vectors were tried to transform into Vitis vinifera by genetic engineering technology.

Regeneration of “Carignane”,“Flame Seedless”,“Thompson Seedless”,“Long-bunch Thompson Seedless”were established by somatic embryogenesis,respectively,based upon the regeneration systems via floral organ culture. The vectors harboring stilbene synthase gene were introduced into grape and tomato by Agrobacterium-mediated method,respectively. The main results are as follows:

1．　Anthers，ovary and small buds collected from five grape varieties were inoculated in four different medium as explants to induce callus. In which,medium NN69 + 2.0 mg/L 6-BA + 1.0 mg/L 2,4-D + 60 g/L Sucrose + 3 g/L phatagel and medium MS + 0.55 mg/L 2,4-D + 0.5 mg/L NOA+1.24 mg/L 4-CPPU + 30 g/L Sucrose + 3 g/L phatage are effective for callus induction,up to 6.2%.

2．　Somatic embryogenesis were induced from callus of five grape varieties except from “Cabernet Sauvignon” on medium X6 + 60 g/L Sucrose + 7 g/L TC-Agar + 0.5 g/L AC. Moreover,the somatic embryos before heart-shaped period were induced secondary somatic embryos on the same medium.

3．　Somatic embryos could regenerate plantlets using about one week as the following steps: dark cultured in liquid MS with shaking at 110rpm 25℃ for 3 days,then transferred to solid medium WPM+ 0.15 mg/L IBA + 30 g/L Sucrose +7 g/L agar + 0.5 g/L AC under low light treatment for 3 days,change to strong light.

4．　The vector harboring STS anditsspecific promoter was successfully transformed to tomato “Micro-Tom”by Agrobacterium-mediated method. Four lines including 54 transgenic plants were obtained by PCR and PCR-Southern detection.

5．　Resveratrol contents were determined by HPLC from transgenic and wild type tomato plants mock and 12h post inoculation of Botrytis cinerea. Results showed that resveratrol content of transgenic plants that transformed with vector harboring Enhancer were highest in mock and Botrytis cinerea inoculated plants,which is 4.4 times more than tomato transformed with vector harboring no enhanced element,while it was undetectable in wild type.

6．　The disease resistance index of transgenic tomato plants was measured after inoculation of Botrytis cinerea. Results showed that resistance of all transgenic plants was increased to some degree,in which resistance of the plants transformed with vector harboring Enhancer is strongest.

Key words　Grapevine　Somatic embryogenesis Agrobacterium-mediated　Stilbene synthase gene(STS) Promoter