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| RNAi技术对番茄甾体生物碱合成途径的研究及应用
[摘要] 番茄（Solanum lycopersicum）是一种在全世界广为栽培的食用农作物，然而其果实处于尚未成熟的绿果期时含有大量的反营养物质——甾体生物碱（SAs），鉴于当前市场普遍存在催熟现象，导致了甾体生物碱影响人体健康的潜在风险随之加大。本文以甾体生物碱合成关键基因GAME4为对象，通过RNAi技术构建了GAME4基因的RNAi表达载体GAME4-RNAi-pHELLSGATE12，并通过农杆菌介导番茄遗传转化技术获得转基因番茄植株，以探究甾体生物碱合成关键基因GAME4的功能及将其沉默对番茄果实甾体生物碱含量的影响，为低甾体生物碱含量番茄品种的培育奠定基础、同时为其他茄科植物相关研究提供参考。主要研究结果如下：
番茄GAME4基因干扰片段的获得：提取新鲜番茄组织RNA、反转获得cDNA，设计特异性引物，扩增带有attB接头的GAME4基因干扰片段，该扩增片段长度为167bp。
番茄GAME4基因RNAi表达载体的构建：根据RNAi表达载体构建原则以及Gateway重组技术原理，首先通过BP反应使带有attB接头的GAME4-RNAi片段连入pDONR207，构建入门载体GAME4-RNAi-pDONR207，再通过LR反应使GAME4-RNAi-pDONR207上的干扰片段通过重组进入pHELLSGATE12载体，构建带有“正向序列-内含子-反向序列” ihpRNA的GAME4-RNAi-pHELLSGATE12目的载体。
转基因番茄植株的获得：利用农杆菌介导法将GAME4-RNAi-pHELLSGATE12转化野生型Micro-Tom番茄植株，经过抗性筛选及分子检测最终获得阳性的稳定转化植株。
[关键词] 番茄；GAME4基因；RNAi；遗传转化
[Abstract] Tomato is a kind of food crops widely planted worldwide.However, when its fruit is in the green fruit stage,it contains abundant antinutrient——steroidal alkaloids (SAs). Due to forced ripening, which is normally seen in market,the risk that SAs do harm to our health is getting larger. In this paper, we picked GAME4,the key gene of steroidal alkaloid synthesis pathway, and constructed the RNAi expression vector GAME4-RNAi-pHELLSGATE12 of GAME4 gene through RNA interference technology. After that, by agrobacterium mediated tomato genetictransformation technique obtained transgenic plants. To explore the function of GAME4 and study effects on the content of steroidal alkaloids in tomato fruit when it is silenced,which lay the foundation for the cultivation oftomato varieties with low content of steroidal alkaloids and provide reference for other Solanaceae related research as well.
Main results achieved were as follows:
The obtained of tomato GAME4 gene RNA interference fragment: After fresh RNA was extracted and cDNA was gained through reverse transcription,we designed the specific primer and amplified GAME4 gene interference fragment with attB site, whose size was: 167bp, respectively.
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Constructed GAME4 gene RNAi expression vector: Based on the RNAi expression vector construction principle and Gateway technique, firstly,we cloned the GAME4 RNAi fragment with attB site into pDONR207 and constructed the entry vector GAME4-RNAi-pDONR207. Then we inserted the RNAi fragment into pHELLSGATE12 vector through the recombination of LR reaction, after which the destination vector GAME4-RNAi-pHELLSGATE12 with the ihpRNA structure of “sense fragment-intron-antisense fragment”.
Transgenic plants were obtained: By agrobacterium mediated method transferred GAME4-RNAi-pHELLSGATE12 into Micro-Tom tomato, respectively. Finally, stable positive inversion plants were obtained by molecular detection.
[Key words] GAME4 gene; RNAi; genetic transformation.
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