杨树抗旱多基因转化及整合机制研究
摘要
随着全球气候变化和环境污染的加剧，水分、温度、光照等非生物因子正逐渐成为制约林木健康生长和生产力发挥的重要因素。培育抗非生物逆境的林木新品种已经成为提高我国森林覆盖率，发挥人工林生产力的重要途径。传统育种手段存在育种周期长，效率低等缺点，已不能满足我国林业发展对优良品种的迫切需求，基因工程技术的迅速发展使得人们可以通过分子水平的操作实现对林木相关性状的快速、高效改良。本研究以欧美杨渤丰1号（Populus×euramericana cl.‘Bofeng1’）为试验材料，在对其组培再生体系进行优化的基础之上，采用基因枪法对其进行五个抗旱相关基因——转录因子基因JERF36基因、ZxZF基因、AREB基因和功能基因SacB基因、GST基因的共转化，以期培育出具有强抗旱性的转基因杨树新品种，同时，对外源基因在受体材料基因组上的整合特征进行深入探讨，完善转基因技术的理论基础。主要研究结果如下：
1．优化了渤丰1号杨再生体系，突破了黑杨派树种组培再生率不高的难题。其叶片和叶柄最适不定芽诱导培养基均为MS+6-BA0.4mg·L~(-1)+NAA0.04mg·L~(-1)；不定芽最适生根培养基为1/2MS+IBA0.05m g·L~(-1)+NAA0.02mg·L~(-1)；组培的最适光照强度为2300lx；40mg. L~(-1)卡那霉素可以抑制渤丰1号杨叶片的诱导与分化，20mg. L~(-1)卡那霉素可以抑制渤丰1号杨不定芽的生根。首次发现增加Cu~(2+)浓度能显著促进杨树不定芽的诱导和分化；使用该再生体系时，外植体的分化率和生根率均达100%，叶片的平均分化芽数多达20个以上，组培苗移栽成活率可达98%。
2．进行了渤丰1号杨抗旱多基因的共转化。对获得的3000多株再生苗进行了PCR检测，共获得62个单基因转化植株（JERF3618株、SacB10株、ZxZF28株、GST5株、AREB1株），22个双基因转化植株（JERF36和SacB2株、JERF36和ZxZF13株、SacB和ZxZF7株），8个三基因转化植株（JERF36、SacB和ZxZF）和1个四基因转化植株（JERF36、ZxZF、GST和AREB）。然后，对部分转化植株进行了Southern杂交检测和QRT-PCR分析，进一步证实了外源基因在渤丰1号杨中的整合和表达。
3．对转基因株系1B14进行了基因组重测序研究，率先采用高通量测序的手段对外源基因的整合情况进行了深入探究。发现外源基因在受体植株的染色体上存在整合，总的拷贝数为3，并且外源基因倾向于整合在染色体的远端，这可能与该区域的复制活跃有关。外源基因JERF36、SacB、ZxZF均整合在第2号染色体上，基因的左右边界均带有载体的部分序列，并有部分的缺失。同时，还发现有载体的部分骨架序列整合在受体染色体上，这与农杆菌介导的转化明显不同，可能与基因枪以物理的方式将外源基因导入受体细胞有关。根据对整合特征的分析我们总结出基因枪介导的外源基因转化可分为两种方式（农杆菌式整合和机械式整合）的假设。此外，重测序还检测到转基因植株的SNPs总计为2988581，其中1077575是纯合，而1911006属于杂合。短的插入缺失位点(1~5bp)数是212089，其中115674是杂合，96415属于纯合，110523是插入，101566为缺失。检测到结构变异SVs总计为24584，其中缺失18863，插入4362，以及1359的其他结构变异。
总之，能否成为林木基因工程研究的模式材料关键在于所选材料是否拥有高效的组培再生体系。本研究所建立的再生体系是目前黑杨派树种中的最佳体系之一，这为研究者以生产实践中占主体地位的黑杨派树种为模式材料进行基因克隆、转化以及基因在林木中的功能解析和应用等提供了前提。同时，获得的大量转基因株系为培育抗性优良的杨树品种提供了坚实的物质基础。在全基因组水平上对转基因整合机制的分析，开辟了转基因研究的新方向，深入揭示了外源基因在受体基因组中的整合特征，为转化机制的阐明奠定了理论基础。
With the aggravation of global climate change and environmental pollution, abioticfactors such as water, temperature, light, etc. are gradually becoming the limit factors in treesgrowth and productivity. Breeding new varieties to resist abiotic stress has become animportant way for increasing forest coverage rate and plantation productivity in China.Because the long cycle, low efficiency and other defects, traditional breeding methods can nolonger meet the needs of the forestry development. With the rapid development of geneticengineering, people can improve forest faster and more efficient by the molecular leveloperation. In this research, Populus×euramericana cl.‘Bofeng1’ was the experimentalmaterial. On the basis of regeneration system optimization, five drought-related gene JERF36,ZxZF, AREB, SacB and GST were co-transformation into it by the particle bombardmenttransformation so as to breed the new varieties of transgenic poplar with drought tolerance. Inaddition,the integration mechanism of foreign genes were also studied. The main results aredescribed as follows:
1. A stable and high efficient regeneration system of ‘Bofeng1’ was established. theoptimum medium for adventitious buds regeneration was MS+6-BA0.4mg L~(-1)+NAA0.04mg L~(-1); The optimum medium for rooting of shoots was1/2MS+IBA0.05m g L~(-1)+NAA0.02mg L~(-1); The optimal light intensity was2300lx; the induction and differentiation ofleaves can be inhibited by40mg L~(-1)Kan；the rooting of shoots can be inhibited by20mg L~(-1)Kan. It was the fist time to find that the differentiation rate of adventitious buds of ‘Bofeng1’ can be significantly promoted by copper. Using this system, both shoot regeneration rate androoting rate were up to100%; and the average number of differentiated shoots in each leafexplant was up to20; and the survival rate of seedlings reached98%.
2. More than3000regenerated seedlings were obtained by multiple drought-related geneco-transformation. Subsequently, PCR analysis of regenerated plants was done. At last,62single-gene transgenic plants were obtained (JERF3618lines, SacB10lines, ZxZF28lines, GST5lines, AREB1lines);22double gene transgenic plants were obtained (JERF36&SacB2lines, JERF36&ZxZF13lines, SacB&ZxZF7lines);8three gene transgenic plants wereobtained (JERF36, SacB, ZxZF);1four gene transgenic plants was obtained (JERF36, GST,AREB, ZxZF). Then, the result of Southern blot and QRT-PCR analysis in some samples furtherconfirmed the integration and expression of foreign genes in ‘Bofeng1’.
3. The re-sequencing study of1B14showed that JERF36、SacB and ZxZF were integratedin the chromosomes of ‘Bofeng1’. The total copy number was3, and we found that exogenousgenes tend to be integrated in the distal part of recipient chromoses. This should be related totwo factors: the distal part of plant chromoses was rich in genes; and its transcriptional activitywas higher than other regions. Exogenous genes were integrated in chromosome2. There werepartial fragments of vector DNA at left and right borders of JERF36、SacB and ZxZF, and bothbeing lost partial sequence. In addition, we found there were a number of large fragments ofvector backbone sequences integrated into the recipient chromosome, which was significantlydifferent to the integration mechanism of Agrobacterium mediated transformation. It may bedue to gene transferred into recipient cell by mechanical forces in the particle bombardmenttransformation. According to the analysis of the exogenous gene's integration features, wesuppose that there are two ways for foreign gene integrating into the recipient genome in theparticle bombardment transformation (Agrobacterium-style integration and mechanicalintegration). Moreover, on the basis of re-sequencing, in receptor material we found2988581single nucleotide polymorphism (SNPs). and1077575is homozygous;1911006isheterozygous; There are212089short insertion&deletion(Indel). and115674isheterozygous;96415is homozygous;110523is inserted;101566is in deletion. There are24584structural variation (SVs).and18863is in deletion,4362is in insertion,1359belong toother structural variation.
In a conclusion, an efficient regeneration system is a key factor to determine whether itcan become the model material of forest genetic engineering research. The regeneration systemestablished in this study is one of the best systems in the species of Aigeiros Section. Itprovides the premise for researchers to use black poplar which have the dominant status in production practice as a model material for gene cloning, transformation, and functionalanalysis and applications in trees. Meanwhile, a large number of transgenic plants wereobtained, which provide a solid material foundation for the cultivation of excellent resistantvarieties of poplar. The new direction of transgenic research was opened up by the analysis ofintegration mechanisms in the whole genome. The integration characteristics of the exogenousgene in the receptor genome was in-depth revealed, and it provides a theoretical basis for theelucidation of the transformation mechanism.
1．优化了渤丰1号杨再生体系，突破了黑杨派树种组培再生率不高的难题。其叶片和叶柄最适不定芽诱导培养基均为MS+6-BA0.4mg·L~(-1)+NAA0.04mg·L~(-1)；不定芽最适生根培养基为1/2MS+IBA0.05m g·L~(-1)+NAA0.02mg·L~(-1)；组培的最适光照强度为2300lx；40mg. L~(-1)卡那霉素可以抑制渤丰1号杨叶片的诱导与分化，20mg. L~(-1)卡那霉素可以抑制渤丰1号杨不定芽的生根。首次发现增加Cu~(2+)浓度能显著促进杨树不定芽的诱导和分化；使用该再生体系时，外植体的分化率和生根率均达100%，叶片的平均分化芽数多达20个以上，组培苗移栽成活率可达98%。
2．进行了渤丰1号杨抗旱多基因的共转化。对获得的3000多株再生苗进行了PCR检测，共获得62个单基因转化植株（JERF3618株、SacB10株、ZxZF28株、GST5株、AREB1株），22个双基因转化植株（JERF36和SacB2株、JERF36和ZxZF13株、SacB和ZxZF7株），8个三基因转化植株（JERF36、SacB和ZxZF）和1个四基因转化植株（JERF36、ZxZF、GST和AREB）。然后，对部分转化植株进行了Southern杂交检测和QRT-PCR分析，进一步证实了外源基因在渤丰1号杨中的整合和表达。
3．对转基因株系1B14进行了基因组重测序研究，率先采用高通量测序的手段对外源基因的整合情况进行了深入探究。发现外源基因在受体植株的染色体上存在整合，总的拷贝数为3，并且外源基因倾向于整合在染色体的远端，这可能与该区域的复制活跃有关。外源基因JERF36、SacB、ZxZF均整合在第2号染色体上，基因的左右边界均带有载体的部分序列，并有部分的缺失。同时，还发现有载体的部分骨架序列整合在受体染色体上，这与农杆菌介导的转化明显不同，可能与基因枪以物理的方式将外源基因导入受体细胞有关。根据对整合特征的分析我们总结出基因枪介导的外源基因转化可分为两种方式（农杆菌式整合和机械式整合）的假设。此外，重测序还检测到转基因植株的SNPs总计为2988581，其中1077575是纯合，而1911006属于杂合。短的插入缺失位点(1~5bp)数是212089，其中115674是杂合，96415属于纯合，110523是插入，101566为缺失。检测到结构变异SVs总计为24584，其中缺失18863，插入4362，以及1359的其他结构变异。
总之，能否成为林木基因工程研究的模式材料关键在于所选材料是否拥有高效的组培再生体系。本研究所建立的再生体系是目前黑杨派树种中的最佳体系之一，这为研究者以生产实践中占主体地位的黑杨派树种为模式材料进行基因克隆、转化以及基因在林木中的功能解析和应用等提供了前提。同时，获得的大量转基因株系为培育抗性优良的杨树品种提供了坚实的物质基础。在全基因组水平上对转基因整合机制的分析，开辟了转基因研究的新方向，深入揭示了外源基因在受体基因组中的整合特征，为转化机制的阐明奠定了理论基础。
With the aggravation of global climate change and environmental pollution, abioticfactors such as water, temperature, light, etc. are gradually becoming the limit factors in treesgrowth and productivity. Breeding new varieties to resist abiotic stress has become animportant way for increasing forest coverage rate and plantation productivity in China.Because the long cycle, low efficiency and other defects, traditional breeding methods can nolonger meet the needs of the forestry development. With the rapid development of geneticengineering, people can improve forest faster and more efficient by the molecular leveloperation. In this research, Populus×euramericana cl.‘Bofeng1’ was the experimentalmaterial. On the basis of regeneration system optimization, five drought-related gene JERF36,ZxZF, AREB, SacB and GST were co-transformation into it by the particle bombardmenttransformation so as to breed the new varieties of transgenic poplar with drought tolerance. Inaddition,the integration mechanism of foreign genes were also studied. The main results aredescribed as follows:
1. A stable and high efficient regeneration system of ‘Bofeng1’ was established. theoptimum medium for adventitious buds regeneration was MS+6-BA0.4mg L~(-1)+NAA0.04mg L~(-1); The optimum medium for rooting of shoots was1/2MS+IBA0.05m g L~(-1)+NAA0.02mg L~(-1); The optimal light intensity was2300lx; the induction and differentiation ofleaves can be inhibited by40mg L~(-1)Kan；the rooting of shoots can be inhibited by20mg L~(-1)Kan. It was the fist time to find that the differentiation rate of adventitious buds of ‘Bofeng1’ can be significantly promoted by copper. Using this system, both shoot regeneration rate androoting rate were up to100%; and the average number of differentiated shoots in each leafexplant was up to20; and the survival rate of seedlings reached98%.
2. More than3000regenerated seedlings were obtained by multiple drought-related geneco-transformation. Subsequently, PCR analysis of regenerated plants was done. At last,62single-gene transgenic plants were obtained (JERF3618lines, SacB10lines, ZxZF28lines, GST5lines, AREB1lines);22double gene transgenic plants were obtained (JERF36&SacB2lines, JERF36&ZxZF13lines, SacB&ZxZF7lines);8three gene transgenic plants wereobtained (JERF36, SacB, ZxZF);1four gene transgenic plants was obtained (JERF36, GST,AREB, ZxZF). Then, the result of Southern blot and QRT-PCR analysis in some samples furtherconfirmed the integration and expression of foreign genes in ‘Bofeng1’.
3. The re-sequencing study of1B14showed that JERF36、SacB and ZxZF were integratedin the chromosomes of ‘Bofeng1’. The total copy number was3, and we found that exogenousgenes tend to be integrated in the distal part of recipient chromoses. This should be related totwo factors: the distal part of plant chromoses was rich in genes; and its transcriptional activitywas higher than other regions. Exogenous genes were integrated in chromosome2. There werepartial fragments of vector DNA at left and right borders of JERF36、SacB and ZxZF, and bothbeing lost partial sequence. In addition, we found there were a number of large fragments ofvector backbone sequences integrated into the recipient chromosome, which was significantlydifferent to the integration mechanism of Agrobacterium mediated transformation. It may bedue to gene transferred into recipient cell by mechanical forces in the particle bombardmenttransformation. According to the analysis of the exogenous gene's integration features, wesuppose that there are two ways for foreign gene integrating into the recipient genome in theparticle bombardment transformation (Agrobacterium-style integration and mechanicalintegration). Moreover, on the basis of re-sequencing, in receptor material we found2988581single nucleotide polymorphism (SNPs). and1077575is homozygous;1911006isheterozygous; There are212089short insertion&deletion(Indel). and115674isheterozygous;96415is homozygous;110523is inserted;101566is in deletion. There are24584structural variation (SVs).and18863is in deletion,4362is in insertion,1359belong toother structural variation.
In a conclusion, an efficient regeneration system is a key factor to determine whether itcan become the model material of forest genetic engineering research. The regeneration systemestablished in this study is one of the best systems in the species of Aigeiros Section. Itprovides the premise for researchers to use black poplar which have the dominant status in production practice as a model material for gene cloning, transformation, and functionalanalysis and applications in trees. Meanwhile, a large number of transgenic plants wereobtained, which provide a solid material foundation for the cultivation of excellent resistantvarieties of poplar. The new direction of transgenic research was opened up by the analysis ofintegration mechanisms in the whole genome. The integration characteristics of the exogenousgene in the receptor genome was in-depth revealed, and it provides a theoretical basis for theelucidation of the transformation mechanism.
引文
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