甜瓜性别相关基因遗传转化研究
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摘要
本研究从建立高频再生体系入手，以甜瓜子叶节为外植体，对其不定芽和植株再生进行了系统的研究，并对甜瓜不定芽再生过程中的生理生化和内源激素动态变化进行了研究，初步分析和探讨了甜瓜不定芽发育机理。建立了农杆菌介导组培法、农杆菌介导茎尖不定芽法和花粉管通道法三种遗传转化方法，将性别相关基因导入到具有重要推广价值的甜瓜品系中。主要研究结果如下：
1本试验以甜瓜子叶节为外植体，6-BA为主要诱导激素，辅之以IAA，建立了稳定的甜瓜子叶节再生体系。甜瓜子叶节在不定芽诱导培养基上直接分化出了丛生芽，对于本研究所用的4份试材，最终获得4号品系最适激素水平为6-BA的有效浓度1mg/L，IAA的有效浓度0.01mg/L,再生频率为96.7%。
2甜瓜子叶节外植体再生过程SOD、可溶性糖含量、POD、CAT、可溶性蛋白、叶绿素呈有规律的动态变化。这些生理生化指标的变化与不定芽的产生密切相关。可溶性糖、可溶性蛋白含量在甜瓜不定芽产生过程中呈现上升趋势，可见，这些物质在不定芽发生过程中起着重要的作用。在3种抗氧化酶中，SOD活性变化在愈伤组织的早期发生过程中起关键作用；CAT活性变化对不定芽的诱导发生、后期发育阶段起主导作用；而POD活性变化在不定芽发生过程中作用不明显，并且SOD与CAT活性变化有一定相关性。两种抗氧化酶在愈伤组织诱导及不定芽分化、发育和成熟过程中分工合作、相互配合。4个甜瓜品系的叶绿素(叶绿素a、叶绿素b、类胡萝卜素、叶绿素总量)含量均呈现大幅下降趋势，即在无菌苗时期，叶绿素的含量为最高值。表明无菌苗时期光合作用能力最强，而在愈伤组织生长过程中，光合能力减弱。较高的ZR含量有利于不定芽的诱导，较低的IAA/ZR比值和较高的ZR/ABA比值有利于甜瓜子叶节的再生。
3在建立了高频再生体系的基础上，对农杆菌介导下子叶节遗传转化过程中，Km使用浓度等主要影响因素进行了系统研究，优化了转化流程，建立了农杆菌介导甜瓜子叶节遗传转化体系，对获得的抗性植株进行了PCR和Southern杂交检测，阳性转化率为2.25%。
4通过对主要影响因素进行系统研究，确立了甜瓜授粉授精时间，优化了花粉管通道法遗传转化体系。建立了T1代抗性种子的筛选体系，对获得的抗性植株进行了PCR检测，阳性率为0.05%。
5对茎尖产生不定芽的激素水平等主要影响因素进行了研究，建立了茎尖遗传转化体系，优化了转化流程中的各项参数。对获得的抗性植株进行了PCR和Southern杂交检测，阳性率为8%。
6.通过转录组测序，选择不同性别甜瓜植株差异表达的EST，获得同源片段后，从甜瓜雌雄异花同株中克隆获得一条具有完整阅读框的CmACS-3基因序列，与公布的哈密瓜CmACS-3序列二者核苷酸序列相同率达98%。CmACS-3全长1865bp，该序列编码区长度为1446bp，编码492个氨基酸。该基因在甜瓜幼苗根、茎、叶中均表达。通过构建植物表达载体，茎尖法转化甜瓜品系M-23，转基因植株营养生长正常，雌蕊正常，花药上花粉量少，通过花粉离体萌发实验发现花粉萌发率下降，这可能与转基因植株花药表型与基因作用之间的互作有关。
In this study, through the establishment of high frequency regeneration system, meloncotyledon node was regarded as explant, meanwhile adventitious buds and plant regeneration wereanalyzed systematically; Dynamic changes of physiology, biochemistry and endogenous hormonesin regeneration process of melon adventitious bud were analyzed, developmental mechanism ofmelon adventitious bud was preliminary analyzed. The Agrobacterium-mediated tissue culturemethod, Agrobacterium-mediated adventitious bud of the shoot tip method, and the pollen tubepathway method were established; Gender-related genes were transformed into an importantpractical valuable melon strains. The main results were as follows:
1In this experiment, the cotyledon node of melon was regarded as explant.6-BA was mainlyinduced hormone, complemented by the IAA. The regeneration system of cotyledon nodes fromdifferent melon genotypes was established through the organs direction occurrence ways.Clustered buds were differentiated from melon cotyledon node in adventitious bud inductionmedium. For4materials used in this study,4th line was suitable for optimal hormone levelultimately. Effective concentration of6-BA was1mg/L, IAA was0.01mg/L, and regenerationfrequency was96.7%.
2Soluble sugar content, SOD, CAT, POD, soluble proteins, and chlorophyll demonstratedregular dynamic change in melon adventitious bud regeneration process. These changes wereclosely related with cell division and regeneration start of adventitious bud. Soluble sugar andprotein content, as the material and energy bases of development of adventitious bud showed anupward trend in the process of adventitious bud start. For three kinds of antioxidant enzymes,activity changes of SOD played a key role in the early development of callus; Activity changes ofCAT played a leading role in adventitious bud occurrence, late development and maturation; whileactivity changes of POD were not obvious in adventitious bud, and related with activity changes ofSOD and CAT. Two antioxidant enzymes cooperated mutually in callus induction and adventitiousbud differentiation, development and maturation. Chlorophyll content (chlorophyll a, chlorophyll b,carotenoids, and total chlorophyll) of4melon strains displayed a sharp downward trend. In asterile seedling, the content of chlorophyll reached the highest value. That indicated ability of photosynthesis was the strongest in sterile seedling, but weekend in the callus growth process.Higher content of ZR was conducive to the formation of adventitious bud, the lower hormone ratioof IAA/ZR and higher ratio of ZR/ABA were conducive to melon cotyledon node regeneration.
3On the basis of establishment of high frequency regeneration system, some main impactfactors, such as Km concentration, were analyzed systematically in the process ofAgrobacterium-mediated the cotyledon node genetic transformation. Conversion process wasoptimized, and the Agrobacterium-mediated melon cotyledon node genetic transformation systemwas established. Through PCR detection and southern hybridization detection for resistant plants,the positive rate was2.25%.
4Through analyzing main impact factors systematically, pollination time was ascertained,and pollen tube pathway genetic transformation system was optimized. Screening system ofresistant seeds from T1generation was established. Through PCR detection for resistant plants, thepositive rate was0.05%.
5Some main impact factors, such as hormone levels, for the generation of adventitious budwere analyzed, genetic transformation system of the shoot tip was established, and the parametersof the conversion process were also optimized. Through PCR detection and southern hybridizationdetection for resistant plants, the positive rate was8%.
6Through transcriptome sequencing, different gender difference expression ESTs wereselected and ESTs homologous fragments were obtained. CmACS-3gene sequence including acomplete open reading frame was gained from monecious melon, and then was identical to have98%homologous nucleotide sequence with the cantaloupe CmACS-3. CmACS-3(1865bp) had1446bp in coding region, which encoded492amino acids. CmACS-3was expressed in root, stem,and leaves. Through constructing plant expression vector, shoot tip method was applied totransformed melon M-23. Transgenic plants grew normally, pistils were normal, and pollen ofanther was less. Germination rate of pollen was low by germination in vitro experiment.However,decreased in the amount of pollen in the anther. This might be in accordance with the relationshipbetween anther phenotype in transgenic plants and gene action.
1本试验以甜瓜子叶节为外植体，6-BA为主要诱导激素，辅之以IAA，建立了稳定的甜瓜子叶节再生体系。甜瓜子叶节在不定芽诱导培养基上直接分化出了丛生芽，对于本研究所用的4份试材，最终获得4号品系最适激素水平为6-BA的有效浓度1mg/L，IAA的有效浓度0.01mg/L,再生频率为96.7%。
2甜瓜子叶节外植体再生过程SOD、可溶性糖含量、POD、CAT、可溶性蛋白、叶绿素呈有规律的动态变化。这些生理生化指标的变化与不定芽的产生密切相关。可溶性糖、可溶性蛋白含量在甜瓜不定芽产生过程中呈现上升趋势，可见，这些物质在不定芽发生过程中起着重要的作用。在3种抗氧化酶中，SOD活性变化在愈伤组织的早期发生过程中起关键作用；CAT活性变化对不定芽的诱导发生、后期发育阶段起主导作用；而POD活性变化在不定芽发生过程中作用不明显，并且SOD与CAT活性变化有一定相关性。两种抗氧化酶在愈伤组织诱导及不定芽分化、发育和成熟过程中分工合作、相互配合。4个甜瓜品系的叶绿素(叶绿素a、叶绿素b、类胡萝卜素、叶绿素总量)含量均呈现大幅下降趋势，即在无菌苗时期，叶绿素的含量为最高值。表明无菌苗时期光合作用能力最强，而在愈伤组织生长过程中，光合能力减弱。较高的ZR含量有利于不定芽的诱导，较低的IAA/ZR比值和较高的ZR/ABA比值有利于甜瓜子叶节的再生。
3在建立了高频再生体系的基础上，对农杆菌介导下子叶节遗传转化过程中，Km使用浓度等主要影响因素进行了系统研究，优化了转化流程，建立了农杆菌介导甜瓜子叶节遗传转化体系，对获得的抗性植株进行了PCR和Southern杂交检测，阳性转化率为2.25%。
4通过对主要影响因素进行系统研究，确立了甜瓜授粉授精时间，优化了花粉管通道法遗传转化体系。建立了T1代抗性种子的筛选体系，对获得的抗性植株进行了PCR检测，阳性率为0.05%。
5对茎尖产生不定芽的激素水平等主要影响因素进行了研究，建立了茎尖遗传转化体系，优化了转化流程中的各项参数。对获得的抗性植株进行了PCR和Southern杂交检测，阳性率为8%。
6.通过转录组测序，选择不同性别甜瓜植株差异表达的EST，获得同源片段后，从甜瓜雌雄异花同株中克隆获得一条具有完整阅读框的CmACS-3基因序列，与公布的哈密瓜CmACS-3序列二者核苷酸序列相同率达98%。CmACS-3全长1865bp，该序列编码区长度为1446bp，编码492个氨基酸。该基因在甜瓜幼苗根、茎、叶中均表达。通过构建植物表达载体，茎尖法转化甜瓜品系M-23，转基因植株营养生长正常，雌蕊正常，花药上花粉量少，通过花粉离体萌发实验发现花粉萌发率下降，这可能与转基因植株花药表型与基因作用之间的互作有关。
In this study, through the establishment of high frequency regeneration system, meloncotyledon node was regarded as explant, meanwhile adventitious buds and plant regeneration wereanalyzed systematically; Dynamic changes of physiology, biochemistry and endogenous hormonesin regeneration process of melon adventitious bud were analyzed, developmental mechanism ofmelon adventitious bud was preliminary analyzed. The Agrobacterium-mediated tissue culturemethod, Agrobacterium-mediated adventitious bud of the shoot tip method, and the pollen tubepathway method were established; Gender-related genes were transformed into an importantpractical valuable melon strains. The main results were as follows:
1In this experiment, the cotyledon node of melon was regarded as explant.6-BA was mainlyinduced hormone, complemented by the IAA. The regeneration system of cotyledon nodes fromdifferent melon genotypes was established through the organs direction occurrence ways.Clustered buds were differentiated from melon cotyledon node in adventitious bud inductionmedium. For4materials used in this study,4th line was suitable for optimal hormone levelultimately. Effective concentration of6-BA was1mg/L, IAA was0.01mg/L, and regenerationfrequency was96.7%.
2Soluble sugar content, SOD, CAT, POD, soluble proteins, and chlorophyll demonstratedregular dynamic change in melon adventitious bud regeneration process. These changes wereclosely related with cell division and regeneration start of adventitious bud. Soluble sugar andprotein content, as the material and energy bases of development of adventitious bud showed anupward trend in the process of adventitious bud start. For three kinds of antioxidant enzymes,activity changes of SOD played a key role in the early development of callus; Activity changes ofCAT played a leading role in adventitious bud occurrence, late development and maturation; whileactivity changes of POD were not obvious in adventitious bud, and related with activity changes ofSOD and CAT. Two antioxidant enzymes cooperated mutually in callus induction and adventitiousbud differentiation, development and maturation. Chlorophyll content (chlorophyll a, chlorophyll b,carotenoids, and total chlorophyll) of4melon strains displayed a sharp downward trend. In asterile seedling, the content of chlorophyll reached the highest value. That indicated ability of photosynthesis was the strongest in sterile seedling, but weekend in the callus growth process.Higher content of ZR was conducive to the formation of adventitious bud, the lower hormone ratioof IAA/ZR and higher ratio of ZR/ABA were conducive to melon cotyledon node regeneration.
3On the basis of establishment of high frequency regeneration system, some main impactfactors, such as Km concentration, were analyzed systematically in the process ofAgrobacterium-mediated the cotyledon node genetic transformation. Conversion process wasoptimized, and the Agrobacterium-mediated melon cotyledon node genetic transformation systemwas established. Through PCR detection and southern hybridization detection for resistant plants,the positive rate was2.25%.
4Through analyzing main impact factors systematically, pollination time was ascertained,and pollen tube pathway genetic transformation system was optimized. Screening system ofresistant seeds from T1generation was established. Through PCR detection for resistant plants, thepositive rate was0.05%.
5Some main impact factors, such as hormone levels, for the generation of adventitious budwere analyzed, genetic transformation system of the shoot tip was established, and the parametersof the conversion process were also optimized. Through PCR detection and southern hybridizationdetection for resistant plants, the positive rate was8%.
6Through transcriptome sequencing, different gender difference expression ESTs wereselected and ESTs homologous fragments were obtained. CmACS-3gene sequence including acomplete open reading frame was gained from monecious melon, and then was identical to have98%homologous nucleotide sequence with the cantaloupe CmACS-3. CmACS-3(1865bp) had1446bp in coding region, which encoded492amino acids. CmACS-3was expressed in root, stem,and leaves. Through constructing plant expression vector, shoot tip method was applied totransformed melon M-23. Transgenic plants grew normally, pistils were normal, and pollen ofanther was less. Germination rate of pollen was low by germination in vitro experiment.However,decreased in the amount of pollen in the anther. This might be in accordance with the relationshipbetween anther phenotype in transgenic plants and gene action.
引文
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