厚皮甜瓜果实RNA的提取及采后硅酸钠处理对其防卫基因的诱导表达
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摘要
随着甜瓜采后诱导抗病性研究的深入,对防卫基因的诱导表达分析已成为亟待研究的内容。本文以厚皮甜瓜“银帝”(Cucumis melo L. cv. Yindi)为材料,对果实RNA的提取方法主要条件进行了筛选,并对果实中7个防卫基因在100mmol硅酸钠处理和Trichothecium roseum挑战接种下的表达模式进行了分析。结果表明:
1.1ml Trizol提取200mg果实量时可得到最多的RNA量,氯仿抽提2次可使所提取RNA的A260/A280在1.9-2.0之间,所提取的RNA无蛋白质和DNA污染。室温沉淀和低温沉淀对RNA的产量无明显差异。提取的RNA可顺利反转录,反转录产物稀释5倍后扩增β-actin基因片段与设计大小一致,验证了该方法提取的RNA可用于RT-PCR。
2.cDNA模板量在经过几次调整后,基本达到均一化,用标准Marker比较法验证了MCHT、PAL、SOD、POD、APX和LOX基因,用测序比对的法验证了CHS基因,并得到了甜瓜CHS基因片段序列。由此表明,用标准Marker比较法和测序比对的法可验证扩增的基因,设计的引物可做表达分析。
3.硅酸钠处理和处理后的挑战接种提前了MCHT和SOD基因表达高峰2d,而且也提高了表达量;处理后的挑战接种提高了PAL和CHS基因的表达量;处理后POD基因表达高峰延长了2d,提高了挑战后POD基因表达量;APX和LOX基因表达明显可被硅酸钠处理抑制。由此表明,硅酸钠处理后诱导了果实的抗病性。
As progressing on postharvest induced resistance in muskmelon, it is necessary to analysis the expression of defense genes in treated fruit. In this paper, RNA was extracted from muskmelon“Yindi”(Cucumis melo L. cv. Yindi)after screening on major factors of Trizol method. The expression of seven defense genes was analyzed by RT-PCR in fruit treated with sodium silicate at 100mmol and challenged with Trichothecium roseum. The results indicated as below:
1. The most amount of extracted RNA was in 200mg sample with 1ml Trizol. A260/A280 of RNA was beween1.9 and 2.0 in the condition of chloroform extractived twice. The extracted RNA was not polluted by protein and DNA and was transcribed reversely. Both room and low temperature sediment had no singinfigant effect on RNA yield. Amplified geneβ-actin was same as designed after the production of reverse transcription diluted five times. It was proved that extracted RNA could be used for RT-PCR .
2. The cDNA template quantity was homogeneity after adjusted several times. The gene of MCHT, PAL, SOD, POD, APX and LOX was verificated by standard Marker method. CHS was verificated with test sequence analysis method and the sequence in muskmelon was gotten. The results showed that the target genes could be vertificated by standard Marker method and the test sequence analysis method and the designed primers could be used for expression analysis.
3. The melon treated with sodium silicate and the challenge with T. roseum advanced MCHT and SOD genes expression maximum for 2 days, and the expression of treatment was more than the control. The treatment and challenge improved PAL and CHS genes expression, prolonged expression maximum of POD genes for 2 days, and increased POD gene expression after treatment and challenge conditions. However, APX and LOX genes expression were inhibited by the treatment. The results suggest that fruits resistance is induced by treatment of sodium silicate.
1.1ml Trizol提取200mg果实量时可得到最多的RNA量,氯仿抽提2次可使所提取RNA的A260/A280在1.9-2.0之间,所提取的RNA无蛋白质和DNA污染。室温沉淀和低温沉淀对RNA的产量无明显差异。提取的RNA可顺利反转录,反转录产物稀释5倍后扩增β-actin基因片段与设计大小一致,验证了该方法提取的RNA可用于RT-PCR。
2.cDNA模板量在经过几次调整后,基本达到均一化,用标准Marker比较法验证了MCHT、PAL、SOD、POD、APX和LOX基因,用测序比对的法验证了CHS基因,并得到了甜瓜CHS基因片段序列。由此表明,用标准Marker比较法和测序比对的法可验证扩增的基因,设计的引物可做表达分析。
3.硅酸钠处理和处理后的挑战接种提前了MCHT和SOD基因表达高峰2d,而且也提高了表达量;处理后的挑战接种提高了PAL和CHS基因的表达量;处理后POD基因表达高峰延长了2d,提高了挑战后POD基因表达量;APX和LOX基因表达明显可被硅酸钠处理抑制。由此表明,硅酸钠处理后诱导了果实的抗病性。
As progressing on postharvest induced resistance in muskmelon, it is necessary to analysis the expression of defense genes in treated fruit. In this paper, RNA was extracted from muskmelon“Yindi”(Cucumis melo L. cv. Yindi)after screening on major factors of Trizol method. The expression of seven defense genes was analyzed by RT-PCR in fruit treated with sodium silicate at 100mmol and challenged with Trichothecium roseum. The results indicated as below:
1. The most amount of extracted RNA was in 200mg sample with 1ml Trizol. A260/A280 of RNA was beween1.9 and 2.0 in the condition of chloroform extractived twice. The extracted RNA was not polluted by protein and DNA and was transcribed reversely. Both room and low temperature sediment had no singinfigant effect on RNA yield. Amplified geneβ-actin was same as designed after the production of reverse transcription diluted five times. It was proved that extracted RNA could be used for RT-PCR .
2. The cDNA template quantity was homogeneity after adjusted several times. The gene of MCHT, PAL, SOD, POD, APX and LOX was verificated by standard Marker method. CHS was verificated with test sequence analysis method and the sequence in muskmelon was gotten. The results showed that the target genes could be vertificated by standard Marker method and the test sequence analysis method and the designed primers could be used for expression analysis.
3. The melon treated with sodium silicate and the challenge with T. roseum advanced MCHT and SOD genes expression maximum for 2 days, and the expression of treatment was more than the control. The treatment and challenge improved PAL and CHS genes expression, prolonged expression maximum of POD genes for 2 days, and increased POD gene expression after treatment and challenge conditions. However, APX and LOX genes expression were inhibited by the treatment. The results suggest that fruits resistance is induced by treatment of sodium silicate.
引文
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