四季蜜龙眼花芽和叶芽cDNA-AFLP体系建立及差异基因分析
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摘要
龙眼(Dimocarpus longan Lour.),属于无患子科(Sapindaceae)龙眼属,是我国较重要而有特色的一种亚热带木本果树,也是福建省主要种植的果树之一。
本研究以‘四季蜜’龙眼花芽及叶芽为材料,运用cDNA-AFLP技术,分离龙眼成花相关基因片段,通过克隆测序,BLAST分析,获得了3个可能与龙眼成花相关的基因片段,还有待进一步克隆其基因全长,需要对时空表达情况展开研究,这将有利于揭示龙眼花芽形成过程中相关基因的作用机理。主要试验结果如下:
1建立了适合‘四季蜜’龙眼花芽总RNA提取的技术体系
本研究针对龙眼组织富含多糖、单宁、色素及酚类物质的特点,采用改良的SDS法和CTAB法进行龙眼花芽和叶芽总RNA的提取与纯化研究。结果表明,改良SDS法更适用于总RNA的提取,提取的RNA具有28s、18s两条清晰、完整的条带,且无降解发生;说明改良SDS法提取的总RNA具有较高的纯度,可以满足后续分子生物学研究的要求。研究确定了‘四季蜜’龙眼花芽、叶芽总RNA提取的技术体系:每1.00 g‘四季蜜’龙眼花芽或叶芽加入5.5 mL提取缓冲液,沉淀时间由3 h调整到10 h,可以得到高质量的RNA。
2优化了cDNA-AFLP反应体系,分离‘四季蜜’龙眼成花过程的差异基因
对cDNA-AFLP反应体系的关键因素进行探索,建立了重复性好的cDNA-AFLP分析体系。结果表明,EcoRI和MseI的组合在40μL体系中各取0.9μL可将ds-cDNA酶切完全,与相应接头连接过夜后产物直接用于预扩增。在25μL PCR反应体系中,预扩增产物稀释1200倍,Mg2+浓度1.6 mmol/L,dNTP浓度0.2 mmol/L,选扩的效果最佳。
将测序效果好的32个TDFs序列在GenBank数据库中进行blastn、blastx比对,结果如下:差异片段上调表达28个,下调表达4个, 27个特意基因序列在数据库中搜索到相似性较高,有5个序列没有搜索到结果,推测为未知基因。分析显示27个TDFs中3个TDFs推测与‘四季蜜’龙眼成花相关,分别是引物组合E7M7扩增到的S-腺苷甲硫氨酸合成酶基因,引物组合E7M4扩增到得细胞色素P450基因,引物组合M7E6扩增到的黄烷酮-3-羟化酶基因。
3应用RT-PCR技术分离SAM基因3’端序列
选取3个TDFs中一个可能与‘四季蜜’龙眼花芽形成相关基因片段SAM,设计引物,应用RT-PCR技术扩增其3’端序列,测序和拼接,获得SAM3’末端,共1253bp,与杨树SAM基因同源性达到96%。
Longan(Dimocarpus longan Lour.)is a member of the family Sapindaceae, which is an important and distinctive subtropical orchardfruit in China and additionally the mainly raised fruit tree in Fujian Province.
This research, using flower bud and leaf bud and the technique of cDNA-AFLP, separates gene segment which is related to flowering of Dimocarpus longan Lour. cv Sijimi. Through clone sequencing and BLAST analysis, three gene segments possibly relevant to Longan flowering were obtained. It was still one subject to clone the full length of the gene and extent the research to site- and time- expression which was helpful to disclose the mechanism of relevant gene in the process of Longan flowering. The main experiment results were as follwes:
1 Establishing the isolated technology system adaptable for total RNA of Longan (Dimocarpus longan Lour. cv Sijimi)flower bud and leaf bud
According to the characteristic of the tissue of Longan being rich in polysaccharide, tannins, pigments and phenols, utilizing improved SDS approach and CTAB approach, extraction and purification of the RNA of flower bud and leaf bud of Longan were conducted in this study. The results suggested that the improved SDS approach could be used to extract RNA and the extracted RNA had two lines of 28 and 18s clearly and completely without degradation, which meaned that the RNA extracted by improved SDS approach had higher purity and satisfied the requirements of successive molecular biology research. The isolated technology system adaptable for total RNA in Dimocarpus longan Lour. cv Sijimi floral and leaf bud established in this study was that: 1.00 g flower bud or leaf bud of Dimocarpus longan Lour. cv Sijimi with 5.5mL extraction buffer solution and extending the settling time from 3 h to 10 h could result in high quality RNA
2 Optimizing the reaction system of cDNA-AFLP and separating the differential gene connecting to flowering process.
The key elements of cDNA-AFLP reaction system was probed and analysis system of cDNA-AFLP with good repetitiveness was set up. It showed that it was possible to divideds-cDNA completely by taking 0.9μL from EcoRI and MseI in the 40μL system separately, and the overnight generation from the corresponding joint could be used for pre-amplification. In the reaction system of 25μL PCR, it was better done by diluting the pre-amplification 1200 times with the density of Mg2+ at 1.6 mmol/L and the density of dNTP at 0.2 mmol/L.
Blastn and blastx study by comparing 32 orders of TDF with good predicting capacity to the data in GenBank database was carried out. The results was as below: for 27 out of 32, by search the genes were found, mRNA or cDNA with higher similarity. For the rest 5, there were no results, and they were inferred as unknown genes. Analysis showed that 3 TDFs out of 27 possibly was related to Dimocarpus longan Lour. cv Sijimi flowering as following: the gene of S-adenosyl-L-methionine synthetase amplified from E7M7, the gene of cytochromeP450 amplified from E7M4, and the gene of flavanone 3-hydroxylase amplified from M7E6.
3 Applying RT-PCR technique to separate 3’end of SAM gene sequence
By selecting one of the 3 gene sequences-SAM which was possibly related to Dimocarpus longan Lour. cv Sijimi flowering, designing primers and utilizing RT-PCR technique to expand 3’end sequence of SAM, testing its sequence and putting them together, the 3’end of SAM at the amount of 1252bp was attained. The nucleotide sequence comparison with the SAM gene of Populus trichocarpa showed that identity was all higher than 96%.
本研究以‘四季蜜’龙眼花芽及叶芽为材料,运用cDNA-AFLP技术,分离龙眼成花相关基因片段,通过克隆测序,BLAST分析,获得了3个可能与龙眼成花相关的基因片段,还有待进一步克隆其基因全长,需要对时空表达情况展开研究,这将有利于揭示龙眼花芽形成过程中相关基因的作用机理。主要试验结果如下:
1建立了适合‘四季蜜’龙眼花芽总RNA提取的技术体系
本研究针对龙眼组织富含多糖、单宁、色素及酚类物质的特点,采用改良的SDS法和CTAB法进行龙眼花芽和叶芽总RNA的提取与纯化研究。结果表明,改良SDS法更适用于总RNA的提取,提取的RNA具有28s、18s两条清晰、完整的条带,且无降解发生;说明改良SDS法提取的总RNA具有较高的纯度,可以满足后续分子生物学研究的要求。研究确定了‘四季蜜’龙眼花芽、叶芽总RNA提取的技术体系:每1.00 g‘四季蜜’龙眼花芽或叶芽加入5.5 mL提取缓冲液,沉淀时间由3 h调整到10 h,可以得到高质量的RNA。
2优化了cDNA-AFLP反应体系,分离‘四季蜜’龙眼成花过程的差异基因
对cDNA-AFLP反应体系的关键因素进行探索,建立了重复性好的cDNA-AFLP分析体系。结果表明,EcoRI和MseI的组合在40μL体系中各取0.9μL可将ds-cDNA酶切完全,与相应接头连接过夜后产物直接用于预扩增。在25μL PCR反应体系中,预扩增产物稀释1200倍,Mg2+浓度1.6 mmol/L,dNTP浓度0.2 mmol/L,选扩的效果最佳。
将测序效果好的32个TDFs序列在GenBank数据库中进行blastn、blastx比对,结果如下:差异片段上调表达28个,下调表达4个, 27个特意基因序列在数据库中搜索到相似性较高,有5个序列没有搜索到结果,推测为未知基因。分析显示27个TDFs中3个TDFs推测与‘四季蜜’龙眼成花相关,分别是引物组合E7M7扩增到的S-腺苷甲硫氨酸合成酶基因,引物组合E7M4扩增到得细胞色素P450基因,引物组合M7E6扩增到的黄烷酮-3-羟化酶基因。
3应用RT-PCR技术分离SAM基因3’端序列
选取3个TDFs中一个可能与‘四季蜜’龙眼花芽形成相关基因片段SAM,设计引物,应用RT-PCR技术扩增其3’端序列,测序和拼接,获得SAM3’末端,共1253bp,与杨树SAM基因同源性达到96%。
Longan(Dimocarpus longan Lour.)is a member of the family Sapindaceae, which is an important and distinctive subtropical orchardfruit in China and additionally the mainly raised fruit tree in Fujian Province.
This research, using flower bud and leaf bud and the technique of cDNA-AFLP, separates gene segment which is related to flowering of Dimocarpus longan Lour. cv Sijimi. Through clone sequencing and BLAST analysis, three gene segments possibly relevant to Longan flowering were obtained. It was still one subject to clone the full length of the gene and extent the research to site- and time- expression which was helpful to disclose the mechanism of relevant gene in the process of Longan flowering. The main experiment results were as follwes:
1 Establishing the isolated technology system adaptable for total RNA of Longan (Dimocarpus longan Lour. cv Sijimi)flower bud and leaf bud
According to the characteristic of the tissue of Longan being rich in polysaccharide, tannins, pigments and phenols, utilizing improved SDS approach and CTAB approach, extraction and purification of the RNA of flower bud and leaf bud of Longan were conducted in this study. The results suggested that the improved SDS approach could be used to extract RNA and the extracted RNA had two lines of 28 and 18s clearly and completely without degradation, which meaned that the RNA extracted by improved SDS approach had higher purity and satisfied the requirements of successive molecular biology research. The isolated technology system adaptable for total RNA in Dimocarpus longan Lour. cv Sijimi floral and leaf bud established in this study was that: 1.00 g flower bud or leaf bud of Dimocarpus longan Lour. cv Sijimi with 5.5mL extraction buffer solution and extending the settling time from 3 h to 10 h could result in high quality RNA
2 Optimizing the reaction system of cDNA-AFLP and separating the differential gene connecting to flowering process.
The key elements of cDNA-AFLP reaction system was probed and analysis system of cDNA-AFLP with good repetitiveness was set up. It showed that it was possible to divideds-cDNA completely by taking 0.9μL from EcoRI and MseI in the 40μL system separately, and the overnight generation from the corresponding joint could be used for pre-amplification. In the reaction system of 25μL PCR, it was better done by diluting the pre-amplification 1200 times with the density of Mg2+ at 1.6 mmol/L and the density of dNTP at 0.2 mmol/L.
Blastn and blastx study by comparing 32 orders of TDF with good predicting capacity to the data in GenBank database was carried out. The results was as below: for 27 out of 32, by search the genes were found, mRNA or cDNA with higher similarity. For the rest 5, there were no results, and they were inferred as unknown genes. Analysis showed that 3 TDFs out of 27 possibly was related to Dimocarpus longan Lour. cv Sijimi flowering as following: the gene of S-adenosyl-L-methionine synthetase amplified from E7M7, the gene of cytochromeP450 amplified from E7M4, and the gene of flavanone 3-hydroxylase amplified from M7E6.
3 Applying RT-PCR technique to separate 3’end of SAM gene sequence
By selecting one of the 3 gene sequences-SAM which was possibly related to Dimocarpus longan Lour. cv Sijimi flowering, designing primers and utilizing RT-PCR technique to expand 3’end sequence of SAM, testing its sequence and putting them together, the 3’end of SAM at the amount of 1252bp was attained. The nucleotide sequence comparison with the SAM gene of Populus trichocarpa showed that identity was all higher than 96%.
引文
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