cDNA-AFLP分析辣椒抗寒性及CaAQP基因克隆与表达研究
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摘要
以西北农林科技大学园艺学院辣椒遗传育种课题组所筛选的耐寒辣椒品系P70为材料利用cDNA-AFLP技术分析了4℃低温诱导,25℃为对照辣椒低温诱导表达差异的基因,挑选TDF(transcription derivation fragment),利用快速扩增cDNA末端(rapid amplification of cDNA ends, RACE)技术,对特异表达基因全长进行克隆,并在此基础上对获得的全长基因进行半定量及实时定量PCR,对其分析。主要结果如下:
1.利用256对AFLP扩增引物对处理和对照的cDNA进行AFLP分析,分离获得120个差异片段,对其中的80个差异片段进行再次扩增、克隆,收集到30条阳性差异条带(TDFs)。对其中的12条TDFs进行测序,BLASTn比对分析,得到4个与抗逆有关的TDFs,用KH-1、KH-2、KH-3、和KH-4表示,其分别与编码抗坏血酸过氧化物酶基因同源性为98%、细胞色素p450基因同源性为98%、牛血清蛋白(BSA)同源性为94%、水通道蛋白(AQP)基因同源性为90%。
2.挑选AQP基因TDF,以其序列为模板设计特异性引物,利用快速扩增RACE技术法获得辣椒水通道蛋白候选基因的全长cDNA,命名为CaAQP ,GenBank登录序列号为:GU116569。用生物信息学的方法对其进行分析,表明该基因与其他物种编码水通道蛋白的基因有96 %以上的相似性。
3.利用半定量RT-PCR及Real-time PCR技术对经过不同时间的低温诱导处理后CaAQP基因的表达谱分析,结果表明对照与处理不同时段该基因存在表达量的差异,低温处理不同时间段后CaAQP基因表达呈下调的趋势。
Chilling injury is a prominent problem suffered in protected farming in winter or early spring. In order to study the molecular mechanism of chilling resistance, the variety of P70 was used to invetigate the differential gene expression of leaves induced by cold stress at 4℃with different time with cDNA-AFLP method. Then the method of RACE was useed to clone the full length of the interesting fagments. The expression of full length gene was detected and analyzed by real-time quantitative PCR and semi-quantitative RT-PCR. The result showed as follows:
1.256 pairs of AFLP amplification primers were used to analyze the cDNA of the treated and control, there are totally 120 differential expression fragments between treated and control plants, of which 80 were re-amplified and cloned. 30 positive TDFs were collected and 12 fragments were sequenced , only 4 of which were successfully sequenced. Bioinformatic analysis of these sequences indicated that the 4 TDFs (KH-1, KH-2, KH-3 and KH-4) related with cold resistance have high homology to ascorbic acid peroxidase, cytochrome p450, bovine serum albumin (BSA) and water-hole channel protein (AQP) respectively .
2.Using one TDF with reference values as candidates TDF to get full-length and as templates to design gene specific primers, The full length of CaAQP was acquired by the method of RACE (rapid amplification of cDNA ends). Bioinformatic analysis showed that CaAQP had over 96% comparability to cDNA gene that encoded water channel protein.
3.The expression of CaAQP was detected by semi-quantitative RT-PCR and real-time quantitative PCR. The results showed that the gene CaAQP expression show the trend of downregulation with various treatment time.
1.利用256对AFLP扩增引物对处理和对照的cDNA进行AFLP分析,分离获得120个差异片段,对其中的80个差异片段进行再次扩增、克隆,收集到30条阳性差异条带(TDFs)。对其中的12条TDFs进行测序,BLASTn比对分析,得到4个与抗逆有关的TDFs,用KH-1、KH-2、KH-3、和KH-4表示,其分别与编码抗坏血酸过氧化物酶基因同源性为98%、细胞色素p450基因同源性为98%、牛血清蛋白(BSA)同源性为94%、水通道蛋白(AQP)基因同源性为90%。
2.挑选AQP基因TDF,以其序列为模板设计特异性引物,利用快速扩增RACE技术法获得辣椒水通道蛋白候选基因的全长cDNA,命名为CaAQP ,GenBank登录序列号为:GU116569。用生物信息学的方法对其进行分析,表明该基因与其他物种编码水通道蛋白的基因有96 %以上的相似性。
3.利用半定量RT-PCR及Real-time PCR技术对经过不同时间的低温诱导处理后CaAQP基因的表达谱分析,结果表明对照与处理不同时段该基因存在表达量的差异,低温处理不同时间段后CaAQP基因表达呈下调的趋势。
Chilling injury is a prominent problem suffered in protected farming in winter or early spring. In order to study the molecular mechanism of chilling resistance, the variety of P70 was used to invetigate the differential gene expression of leaves induced by cold stress at 4℃with different time with cDNA-AFLP method. Then the method of RACE was useed to clone the full length of the interesting fagments. The expression of full length gene was detected and analyzed by real-time quantitative PCR and semi-quantitative RT-PCR. The result showed as follows:
1.256 pairs of AFLP amplification primers were used to analyze the cDNA of the treated and control, there are totally 120 differential expression fragments between treated and control plants, of which 80 were re-amplified and cloned. 30 positive TDFs were collected and 12 fragments were sequenced , only 4 of which were successfully sequenced. Bioinformatic analysis of these sequences indicated that the 4 TDFs (KH-1, KH-2, KH-3 and KH-4) related with cold resistance have high homology to ascorbic acid peroxidase, cytochrome p450, bovine serum albumin (BSA) and water-hole channel protein (AQP) respectively .
2.Using one TDF with reference values as candidates TDF to get full-length and as templates to design gene specific primers, The full length of CaAQP was acquired by the method of RACE (rapid amplification of cDNA ends). Bioinformatic analysis showed that CaAQP had over 96% comparability to cDNA gene that encoded water channel protein.
3.The expression of CaAQP was detected by semi-quantitative RT-PCR and real-time quantitative PCR. The results showed that the gene CaAQP expression show the trend of downregulation with various treatment time.
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