乙烯利调控甘蔗基因的差异表达及甘蔗乙烯受体基因Sc-ERS的克隆研究
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摘要
乙烯是一种结构简单的气体植物激素，它对植物的代谢调节可贯穿其整个生活周期。前人的大量研究结果已证实，在甘蔗生长发育的前期，叶面喷施低浓度的乙烯利能有效促进甘蔗早期的萌芽、分蘖和生长，增强光合作用，提高抗旱性等。为进一步了解乙烯利调控甘蔗生长的分子作用机理，以便更好地利用乙烯利进行甘蔗化学调控，本文对用乙烯利处理的甘蔗前期基因差异表达进行了分析，并对甘蔗乙烯受体Sc-ERS基因进行了克隆、序列分析等方面研究。主要研究结果如下：
1．建立和优化了一套研究乙烯利调控甘蔗基因差异表达的cDNA-AFLP分析体系和银染体系。结果表明：模板质量的高低是cDNA-AFLP分析能否成功的关键。在甘蔗RNA提取中及时去除甘蔗组织可以降低多糖物质的残留；在RT-PCR反应及cDNA第二链合成的操作中，适当延长合成的时间有利于获得更长、更完整的ds cDNA；在cDNA-AFLP分析中，确定初级模板预扩增反应循环数20循环、反应产物20倍稀释液作为二级模板时选择性扩增效果较好；在银染检测过程中，使用高纯度的Na_2CO_3，并控制好凝胶漂洗和显色时间可以获得理想的检测效果。利用cDNA-AFLP技术对乙烯利处理的甘蔗进行基因差异表达研究，每个样品的扩增条带数均在35-80之间，其中大多数在50-70范围内，处理和对照之间条带多态性明显。
2．对部分差异转录片段进行了回收、克隆、反向Northern杂交验证、序列分析和功能分析等方面研究。研究结果表明：在反向Northern杂交实验中，24个测试样品中有11个表现没有差异，假阳性率高达46％；在乙烯作用下，甘蔗的CHI、GST、ARP、LHC、核结合蛋白基因以及一批未知基因差异表达；其中CHI、GST、LHC和核结合蛋白是与促进植物的抗病抗逆、提高光合作用等密切相关的因子。
3．根据已知乙烯受体基因的序列信息，设计了一对简并引物和一对特异引物，对甘蔗叶片基因组DNA进行PCR扩增，获得了一个甘蔗乙烯受体基因(Sc-ERS)的片段。序列分析表明，该片段大小为4310bp，包含一个由5个外显子和4个内含子组成的4219bp大小的完整编码区。Sc-ERS编码区与玉米乙烯受体ERS1基因(AY359578)、水稻乙烯受体ERS1基因(AY043031)的编码区同源率分别为91.4％和61.6％，推导Sc-ERS编码的蛋白包含636个氨基酸残基，与玉米乙烯受体基因、水稻乙烯受体基因所编码蛋白的氨基酸同源率分别为94.1％和89.4％。对推导蛋白的结构进行分析的结果显示Sc-ERS编码的蛋白和玉米、水稻ERS1所编码的蛋白一样，在N段保守区含有3个跨膜结构，并由3个跨膜结构、GAF结构、HisKA和HATPase_c组成功能域。通过核苷酸序列分析和蛋白质结构分析表明Sc-ERS基因属于ERS1基因。
The gaseous plant hormone ethylene induces diverse effects in plants throughouttheir life cycle. Many previous studies have showed that sugarcane growth could beregulated by foliar spraying of ethphon. The ethephon treatments induced variousphysiological changes at the early growth stage of sugarcane, such as earlier and fastersprouting, improved tillering, faster elongation, promoted photosynthesis and enhancedability resisce to diseases and adversity, and so on. To explore the molecular mechanismsof ethephon in regulating sugarcane growth, the differences in gene expressions betweenethephon treatment and control at early growth stage of sugarcane were investigated byusing cDNA-AFLP technique, and the ethylene receptor gene Sc-ERS in sugarcane wascloned and sequenced in the present study. The main results were summarized asfollows.
1. The optimized cDNA-AFLP protocol and an efficient silver stain system weredeveloped, and the gene differential expressions in sugarcane regulated with ethephonwere analysed. The results indicated that the quality of template DNA was important incDNA-AFLP analysis. It is effective to reduce polysaccharides in the total RNA byremovinging the tissue residue of sugarcane from the extractive mixture timely. In thecDNA-AFLP protocol, 20 cycles for pre-amplification of primary template wasdetermined, followed by a 20-fold dilution of the secondary template before selectiveamplification. In the silver staining, better results could be realised by using ultra-pureNa_2CO_3 and limiting the time of rinsing and developing under controlled condition. ThecDNA-AFLP analysis results showed that the polymorphisms between the treatment andthe control were abundant, and 35 to 80 bands were obtained in each amplification.
2. Some transcript derived fragments (TDFs) were isolated, and then cloned,detected with reverse Northern blot and sequenced. The results showed that there were11 samples of 24 TDFs which displayed no difference in expression, and the ratio ofhypocrite positive TDFs was about 46%. The CHI, GST, ARP, LHC, nuclear bindingprotein gene and a set of unknown genes were differentially expressed by the treatmentwith ethephon. CHI and GST were important factors for resistance to diseases, andLHC and nuclear binding protein were related to light binding and CO_2 primary fixingin photosynthesis, respectively.
3. A pair of degenerate primers and a pair of specific primers were designed basedon the conserved sequences of plant ethylene receptor genes, and a 4310 bp fragment ofSc-ERS was cloned and amplified with these primer pairs. The sequencing resultsshowed that Sc-ERS, consisted of five extrons and four introns, contained an entirecoding region of a gene. The coding region of Sc-ERS showed 91.4% and 61.6%homology with those of maize ERS1 and rice ERS1, respectively. The deduced protein ofSc-ERS encoded 636 amino acids, and shared 94.1% and 89.4 % similarity with those ofmaize ERS1 and rice ERS1, respectively. The architecture of protein analysis indicatedthat the Sc-ERS contained three transmembrane structures in the conserved N end asthe maize ERS1 and rice ERS1 did. The functional region of the deduced protein ofSc-ERS, maize ERS1 and rice ERS1 consisted of three transmembrane structures, i.e.GAF, HisKA domain and HATPase_c domain. These results indicated that the Sc-ERSin sugarcane was similar to the ERS1 in other plants.
1．建立和优化了一套研究乙烯利调控甘蔗基因差异表达的cDNA-AFLP分析体系和银染体系。结果表明：模板质量的高低是cDNA-AFLP分析能否成功的关键。在甘蔗RNA提取中及时去除甘蔗组织可以降低多糖物质的残留；在RT-PCR反应及cDNA第二链合成的操作中，适当延长合成的时间有利于获得更长、更完整的ds cDNA；在cDNA-AFLP分析中，确定初级模板预扩增反应循环数20循环、反应产物20倍稀释液作为二级模板时选择性扩增效果较好；在银染检测过程中，使用高纯度的Na_2CO_3，并控制好凝胶漂洗和显色时间可以获得理想的检测效果。利用cDNA-AFLP技术对乙烯利处理的甘蔗进行基因差异表达研究，每个样品的扩增条带数均在35-80之间，其中大多数在50-70范围内，处理和对照之间条带多态性明显。
2．对部分差异转录片段进行了回收、克隆、反向Northern杂交验证、序列分析和功能分析等方面研究。研究结果表明：在反向Northern杂交实验中，24个测试样品中有11个表现没有差异，假阳性率高达46％；在乙烯作用下，甘蔗的CHI、GST、ARP、LHC、核结合蛋白基因以及一批未知基因差异表达；其中CHI、GST、LHC和核结合蛋白是与促进植物的抗病抗逆、提高光合作用等密切相关的因子。
3．根据已知乙烯受体基因的序列信息，设计了一对简并引物和一对特异引物，对甘蔗叶片基因组DNA进行PCR扩增，获得了一个甘蔗乙烯受体基因(Sc-ERS)的片段。序列分析表明，该片段大小为4310bp，包含一个由5个外显子和4个内含子组成的4219bp大小的完整编码区。Sc-ERS编码区与玉米乙烯受体ERS1基因(AY359578)、水稻乙烯受体ERS1基因(AY043031)的编码区同源率分别为91.4％和61.6％，推导Sc-ERS编码的蛋白包含636个氨基酸残基，与玉米乙烯受体基因、水稻乙烯受体基因所编码蛋白的氨基酸同源率分别为94.1％和89.4％。对推导蛋白的结构进行分析的结果显示Sc-ERS编码的蛋白和玉米、水稻ERS1所编码的蛋白一样，在N段保守区含有3个跨膜结构，并由3个跨膜结构、GAF结构、HisKA和HATPase_c组成功能域。通过核苷酸序列分析和蛋白质结构分析表明Sc-ERS基因属于ERS1基因。
The gaseous plant hormone ethylene induces diverse effects in plants throughouttheir life cycle. Many previous studies have showed that sugarcane growth could beregulated by foliar spraying of ethphon. The ethephon treatments induced variousphysiological changes at the early growth stage of sugarcane, such as earlier and fastersprouting, improved tillering, faster elongation, promoted photosynthesis and enhancedability resisce to diseases and adversity, and so on. To explore the molecular mechanismsof ethephon in regulating sugarcane growth, the differences in gene expressions betweenethephon treatment and control at early growth stage of sugarcane were investigated byusing cDNA-AFLP technique, and the ethylene receptor gene Sc-ERS in sugarcane wascloned and sequenced in the present study. The main results were summarized asfollows.
1. The optimized cDNA-AFLP protocol and an efficient silver stain system weredeveloped, and the gene differential expressions in sugarcane regulated with ethephonwere analysed. The results indicated that the quality of template DNA was important incDNA-AFLP analysis. It is effective to reduce polysaccharides in the total RNA byremovinging the tissue residue of sugarcane from the extractive mixture timely. In thecDNA-AFLP protocol, 20 cycles for pre-amplification of primary template wasdetermined, followed by a 20-fold dilution of the secondary template before selectiveamplification. In the silver staining, better results could be realised by using ultra-pureNa_2CO_3 and limiting the time of rinsing and developing under controlled condition. ThecDNA-AFLP analysis results showed that the polymorphisms between the treatment andthe control were abundant, and 35 to 80 bands were obtained in each amplification.
2. Some transcript derived fragments (TDFs) were isolated, and then cloned,detected with reverse Northern blot and sequenced. The results showed that there were11 samples of 24 TDFs which displayed no difference in expression, and the ratio ofhypocrite positive TDFs was about 46%. The CHI, GST, ARP, LHC, nuclear bindingprotein gene and a set of unknown genes were differentially expressed by the treatmentwith ethephon. CHI and GST were important factors for resistance to diseases, andLHC and nuclear binding protein were related to light binding and CO_2 primary fixingin photosynthesis, respectively.
3. A pair of degenerate primers and a pair of specific primers were designed basedon the conserved sequences of plant ethylene receptor genes, and a 4310 bp fragment ofSc-ERS was cloned and amplified with these primer pairs. The sequencing resultsshowed that Sc-ERS, consisted of five extrons and four introns, contained an entirecoding region of a gene. The coding region of Sc-ERS showed 91.4% and 61.6%homology with those of maize ERS1 and rice ERS1, respectively. The deduced protein ofSc-ERS encoded 636 amino acids, and shared 94.1% and 89.4 % similarity with those ofmaize ERS1 and rice ERS1, respectively. The architecture of protein analysis indicatedthat the Sc-ERS contained three transmembrane structures in the conserved N end asthe maize ERS1 and rice ERS1 did. The functional region of the deduced protein ofSc-ERS, maize ERS1 and rice ERS1 consisted of three transmembrane structures, i.e.GAF, HisKA domain and HATPase_c domain. These results indicated that the Sc-ERSin sugarcane was similar to the ERS1 in other plants.
引文
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