大豆子房原位转化法的建立与应用
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摘要
花粉管通道法是一种无需组织培养的原位转化技术,可用于解决传统农杆菌介导法与微弹注射法转化困难的作物的遗传转化问题。然而,对于这种方法的推广来说,转化操作的重复性差、转化效率低仍旧是最大的障碍。因此,对于该方法,确认影响转化的关键因素,并对相关因素加以优化,是实现转化操作标准化的重要前提条件。通常认为,大豆产量偏低主要归因于花、幼荚等生殖器官的大量脱落,据报道不同品种的大豆花荚脱落率可达43%-81%。大豆花、荚脱落率除受品种的基因型影响外,干旱、光照不足、营养缺乏、高温等胁迫条件均可导致脱落率升高。在这些胁迫反应过程中,乙烯作为逆境激素起到了关键性的“信号分子”作用。1-氨基环丙烷-1羧酸合酶(ACC合酶,EC4.4.114)是乙烯生物合成途径的限速酶,在植物的乙烯释放过程中起到了关键性的调控作用。因此,克隆、构建ACC合酶反义基因并转化大豆,考察乙烯释放减少对于大豆产量性状的影响,对于大豆的品种改良而言,将具有重要的理论与生产实践意义。
为此,本论文进行了如下实验：
1、首先采用pBI121质粒作为转化载体,比较切割1/3花柱和贴近子房切割两种不同长度的转化路径的转化效应。结果发现平均转化率分别为1.6%和3.1%,转化率差异显著,说明了转化路径是影响转化的重要因素。
2、本研究进一步设计了仅由35S启动子、gus报告基因、nos终止子以及25bp大小的T-DNA左、右边界作为侧翼序列组成的gus报告基因最小化线性转化元件。烟草叶片瞬时表达阳性结果确证了最小化线性转化元件设计的可行性。
3、采用此线性转化元件,在辽豆14中比较了4种不同长度的转化路径的转化效应差异,即Ⅰ,只切割柱头；Ⅱ,切割1/2-2/3花柱；Ⅲ,完全去除花柱,而不伤害子房；与Ⅳ,部分切除子房。平均转化率分别为0%、1.0%、11.0%和2.0%,转化率差异显著。最佳的转化路径为完全去除花柱,而不伤害子房。采用这种最佳转化路径,进一步对铁丰29、辽豆13和辽豆14三个大豆栽培品种进行了扩大转化实验,转化率分别为7.1%、7.3%和11.3%,转化率差异不显著,揭示了大豆品种基因型差异对转化率无影响,说明采用此最佳转化路径具有无基因型依赖的特征,这对于实现转化操作的标准化具有重要意义。我们将这种采用最小化线性转化元件设计与最佳转化路径的转化方法定义为大豆子房原位转化法。
4、从大豆叶片cDNA文库中克隆了一个伤害诱导的1-氨基环丙烷-1-羧酸(ACC)合酶基因(SACS2), RT-PCR法分析该基因的正义链表达情况,发现了ACC合酶基因的天然反义转录物(ASACS2);进一步分析了6种栽培大豆,确认了ASACS2在大豆中存在的普遍性,序列分析表明ASACS2属于顺式天然反义转录物；Real Time RT-PCR测定铁丰29营养生长期SACS2和ASACS2的表达量,发现二者的比值为一常数；进一步分析发现在不同的大豆品种这一比值不同,具有品种特异性的特征。
5、将SACS2反向克隆到pBI121质粒表达载体上,以构建的质粒为模板,PCR法构建了仅由35S启动子、反向SACS2、nos终止子和T-DNA左、右边界组成的最小化ACC合酶反义基因线性转化元件,采用大豆子房原位转化法进行大豆转化。分子生物学检测(PCR、Southen杂交)确证了外源ACC合酶反义基因已经整合到转化获得大豆植株基因组中,并稳定遗传到转化后代植株中；在5日龄的转基因黄化大豆苗中,ACC合酶的酶活性升高约17.6%-23.4%,而乙烯释放在检测前6小时升高,之后下降,乙烯释放总量下降,表明外源ACC合酶反义基因在转基因大豆中的表达抑制了乙烯的释放,而且这种表达受到了复杂的转录后调控,而导致酶活性升高；与对照株相比,在所有的转基因世代(T0～T2)中营养生长期延长了7天左右；不同的T2转基因后代株系田间小试结果发现,部分株系中平均单株结荚数显著增多,平均百粒重显著增加,具有明显的增产特征；在形态上,单株株高降低,节数增加,而节间距缩短明显,表现了明显的乙烯释放减少特征。
总之,我们在大豆中建立的具有生物安全性的子房原位转化法是一种在产量提高、品质改良上具有广泛应用前景的转化技术。
Pollen-tube pathway transformation method is one kind of in planta techniques that is tissue culture-independent. It is also a promising method that can replace the conventional Agrobacterium-mediated transformation and particle bombardment methods in the transformation of recalcitrant crops. However, the low repeatability and transformation frequency still prevents its application in soybean. Thus, it is of great significance to identify the factors on transformation frequency and optimize the key factors of this transformation method and to standardize the manipulation of the protocol in soybean. Low grain yield in legumes has been found to be attributed to abortion of flowers and pods, and abscission rates of 43 to 81% have been reported for different genotype soybean varieties. At the same time, the environmental cues, such as water deficiency, light insufficiency, inadequate nutrition and high temperature-would-enhance the abscission of reproductive organs. The stress hormone, ethylene, serves as a key signaling molecule and plays a critical role in response to the various abiotic stresses.1-aminocyclopropane-l-carboxylate synthase (ACC synthase, EC 4.4.114) is the rate-limiting enzyme in ethylene biosynthesis pathway. Its expression regulates the release of ethylene in plants; hence it might be a good attempt to transform soybean with ACC synthase antisense gene to improve the yield in soybean through the reverse genetics pathway and investigate the relationship between the ethylene release and yield-related traits.
The aims and results of this study were as follow:
1. To determine whether the length of the passageway through which foreign DNA enters the embryo sac is a key factor to the success of pollen-tube pathway transformation, we compared the transformation efficiencies of two different lengths of the passageway including cutting off 1/3 stigma or cutting near the ovary, using the plasmid pBI121 as the foreign DNA. Different lengths of the passageway were found to produce significant different transformation efficiencies,1.6% and 3.1%.
2. By reference to the mechanism of T-DNA borders in Agrobacterium-mediated transformation, we designed the minimal cassette, which only contains the 35S promoter, gus reporter gene, and nos terminator, and both ends of the cassette were flanked with T-DNA borders and obtained them by Polymerase Chain Reaction (PCR). Transformation of this minimal cassette into transcient expression system of tobacco leaves resulted in three positive expression of the reporter gene among all thirty leaves, thus pointing to the feasibility of its design.
3. To optimize the transformation passageway, we compared the effects of four types of of passageway lengths on the transformation, and the optimized length of the passageway was further tested in more soybean cultivars to investigate the effects of genotypes. A maximum transformation frequency of 11.0% was obtained in soybean cultivar Liaodou 14 with their styles extensively removed, whereas removal of the stigma, partial style cutting and partial ovary cutting contributed to a transformation frequency of 0%, 1.0% and 2.0%, respectively. An average transformation frequency of 8.2% was obtained when 619 flowers from three soybean cultivars (Liaodou14, Liaodou13, and Tiefeng29) were transformed by this optimized method. No significant difference in the transformation frequencies was observed among the different cultivars, indicating no genotype specificity. This also supported the feasibility for standardizing the manipulation through optimizing the transformation pathway, and hence, the modified protocol along with using minimal cassettes was designated as ovarian in planta transformation.
4. To investigate whether the natural antisense transcript (NATs) of ACC synthase gene exists in soybean, the expression of the non-template strand was analyzed by RT-PCR. The NATs of ACC synthase gene (designated as ASACS2) was detected in six soybean cultivars by RT-PCR and sequence analysis showed it and its sense counterpart, SACS2 belonged to cis-NATs, the ratios of SACS2 to SACS2, as measured by real-time RT-PCR were constant among the various soybean cultivars, indicating the presence of cultivars specificity. NATs might be a new regulation pathway governing the release of ethylene in soybean.
5. To improve the soybean yield, the ovarian in planta transformation of minimal ACC synthase antisense gene cassettes were carried out. One cDNA of wound-induced ACC synthase, SACS2, was cloned from soybean leave and inversely inserted into the plasmid pBI121 to give the construct pBI121-SACS2. A linear minimal cassette composed of 35S promoter, ACC synthase antisense gene, and nos terminator and the flanked T-DNA border sequences was amplified from pBI121-SACS2 by PCR. The integration of the foreign ACC synthase antisense DNA into the soybean genome and its subsequent inheritance by the progenies were confirmed by PCR and Southern blotting analysis. The activity of ACC synthase was not reduced according to the theory expectation of antisense gene, but increased for 17.6%-23.4%, indicating there must be a complex regulation system to ethylene release, and the total quantity of ethylene released has first increased and then decrased in the young etiolated transgenic plants compared to the wild type. In field trials, all transgenic plants (To-T2) had one prolonged vegetative growth stage, the lasting seven days longer than that of wild type and result into a delayed reproductive stage. Average pod-bearing number and average one-hundred-grain weight of the partial transgenic lines had increased significantly compared to wildtype ones, that showed the traits of increasing yield, and on the plant external morphology, the plant height had decreased, the node number had increased, and the average distance of stem-stem was significantly shortened in all detected transgenic lines, showed the traits of the decreased ethylene release.
In conclusion, the ovarian in planta transformation in soybean with the linear minimal cassette and the optimal transformation passageway is a new prospective transformation method in yield and quality improvement.
为此,本论文进行了如下实验：
1、首先采用pBI121质粒作为转化载体,比较切割1/3花柱和贴近子房切割两种不同长度的转化路径的转化效应。结果发现平均转化率分别为1.6%和3.1%,转化率差异显著,说明了转化路径是影响转化的重要因素。
2、本研究进一步设计了仅由35S启动子、gus报告基因、nos终止子以及25bp大小的T-DNA左、右边界作为侧翼序列组成的gus报告基因最小化线性转化元件。烟草叶片瞬时表达阳性结果确证了最小化线性转化元件设计的可行性。
3、采用此线性转化元件,在辽豆14中比较了4种不同长度的转化路径的转化效应差异,即Ⅰ,只切割柱头；Ⅱ,切割1/2-2/3花柱；Ⅲ,完全去除花柱,而不伤害子房；与Ⅳ,部分切除子房。平均转化率分别为0%、1.0%、11.0%和2.0%,转化率差异显著。最佳的转化路径为完全去除花柱,而不伤害子房。采用这种最佳转化路径,进一步对铁丰29、辽豆13和辽豆14三个大豆栽培品种进行了扩大转化实验,转化率分别为7.1%、7.3%和11.3%,转化率差异不显著,揭示了大豆品种基因型差异对转化率无影响,说明采用此最佳转化路径具有无基因型依赖的特征,这对于实现转化操作的标准化具有重要意义。我们将这种采用最小化线性转化元件设计与最佳转化路径的转化方法定义为大豆子房原位转化法。
4、从大豆叶片cDNA文库中克隆了一个伤害诱导的1-氨基环丙烷-1-羧酸(ACC)合酶基因(SACS2), RT-PCR法分析该基因的正义链表达情况,发现了ACC合酶基因的天然反义转录物(ASACS2);进一步分析了6种栽培大豆,确认了ASACS2在大豆中存在的普遍性,序列分析表明ASACS2属于顺式天然反义转录物；Real Time RT-PCR测定铁丰29营养生长期SACS2和ASACS2的表达量,发现二者的比值为一常数；进一步分析发现在不同的大豆品种这一比值不同,具有品种特异性的特征。
5、将SACS2反向克隆到pBI121质粒表达载体上,以构建的质粒为模板,PCR法构建了仅由35S启动子、反向SACS2、nos终止子和T-DNA左、右边界组成的最小化ACC合酶反义基因线性转化元件,采用大豆子房原位转化法进行大豆转化。分子生物学检测(PCR、Southen杂交)确证了外源ACC合酶反义基因已经整合到转化获得大豆植株基因组中,并稳定遗传到转化后代植株中；在5日龄的转基因黄化大豆苗中,ACC合酶的酶活性升高约17.6%-23.4%,而乙烯释放在检测前6小时升高,之后下降,乙烯释放总量下降,表明外源ACC合酶反义基因在转基因大豆中的表达抑制了乙烯的释放,而且这种表达受到了复杂的转录后调控,而导致酶活性升高；与对照株相比,在所有的转基因世代(T0～T2)中营养生长期延长了7天左右；不同的T2转基因后代株系田间小试结果发现,部分株系中平均单株结荚数显著增多,平均百粒重显著增加,具有明显的增产特征；在形态上,单株株高降低,节数增加,而节间距缩短明显,表现了明显的乙烯释放减少特征。
总之,我们在大豆中建立的具有生物安全性的子房原位转化法是一种在产量提高、品质改良上具有广泛应用前景的转化技术。
Pollen-tube pathway transformation method is one kind of in planta techniques that is tissue culture-independent. It is also a promising method that can replace the conventional Agrobacterium-mediated transformation and particle bombardment methods in the transformation of recalcitrant crops. However, the low repeatability and transformation frequency still prevents its application in soybean. Thus, it is of great significance to identify the factors on transformation frequency and optimize the key factors of this transformation method and to standardize the manipulation of the protocol in soybean. Low grain yield in legumes has been found to be attributed to abortion of flowers and pods, and abscission rates of 43 to 81% have been reported for different genotype soybean varieties. At the same time, the environmental cues, such as water deficiency, light insufficiency, inadequate nutrition and high temperature-would-enhance the abscission of reproductive organs. The stress hormone, ethylene, serves as a key signaling molecule and plays a critical role in response to the various abiotic stresses.1-aminocyclopropane-l-carboxylate synthase (ACC synthase, EC 4.4.114) is the rate-limiting enzyme in ethylene biosynthesis pathway. Its expression regulates the release of ethylene in plants; hence it might be a good attempt to transform soybean with ACC synthase antisense gene to improve the yield in soybean through the reverse genetics pathway and investigate the relationship between the ethylene release and yield-related traits.
The aims and results of this study were as follow:
1. To determine whether the length of the passageway through which foreign DNA enters the embryo sac is a key factor to the success of pollen-tube pathway transformation, we compared the transformation efficiencies of two different lengths of the passageway including cutting off 1/3 stigma or cutting near the ovary, using the plasmid pBI121 as the foreign DNA. Different lengths of the passageway were found to produce significant different transformation efficiencies,1.6% and 3.1%.
2. By reference to the mechanism of T-DNA borders in Agrobacterium-mediated transformation, we designed the minimal cassette, which only contains the 35S promoter, gus reporter gene, and nos terminator, and both ends of the cassette were flanked with T-DNA borders and obtained them by Polymerase Chain Reaction (PCR). Transformation of this minimal cassette into transcient expression system of tobacco leaves resulted in three positive expression of the reporter gene among all thirty leaves, thus pointing to the feasibility of its design.
3. To optimize the transformation passageway, we compared the effects of four types of of passageway lengths on the transformation, and the optimized length of the passageway was further tested in more soybean cultivars to investigate the effects of genotypes. A maximum transformation frequency of 11.0% was obtained in soybean cultivar Liaodou 14 with their styles extensively removed, whereas removal of the stigma, partial style cutting and partial ovary cutting contributed to a transformation frequency of 0%, 1.0% and 2.0%, respectively. An average transformation frequency of 8.2% was obtained when 619 flowers from three soybean cultivars (Liaodou14, Liaodou13, and Tiefeng29) were transformed by this optimized method. No significant difference in the transformation frequencies was observed among the different cultivars, indicating no genotype specificity. This also supported the feasibility for standardizing the manipulation through optimizing the transformation pathway, and hence, the modified protocol along with using minimal cassettes was designated as ovarian in planta transformation.
4. To investigate whether the natural antisense transcript (NATs) of ACC synthase gene exists in soybean, the expression of the non-template strand was analyzed by RT-PCR. The NATs of ACC synthase gene (designated as ASACS2) was detected in six soybean cultivars by RT-PCR and sequence analysis showed it and its sense counterpart, SACS2 belonged to cis-NATs, the ratios of SACS2 to SACS2, as measured by real-time RT-PCR were constant among the various soybean cultivars, indicating the presence of cultivars specificity. NATs might be a new regulation pathway governing the release of ethylene in soybean.
5. To improve the soybean yield, the ovarian in planta transformation of minimal ACC synthase antisense gene cassettes were carried out. One cDNA of wound-induced ACC synthase, SACS2, was cloned from soybean leave and inversely inserted into the plasmid pBI121 to give the construct pBI121-SACS2. A linear minimal cassette composed of 35S promoter, ACC synthase antisense gene, and nos terminator and the flanked T-DNA border sequences was amplified from pBI121-SACS2 by PCR. The integration of the foreign ACC synthase antisense DNA into the soybean genome and its subsequent inheritance by the progenies were confirmed by PCR and Southern blotting analysis. The activity of ACC synthase was not reduced according to the theory expectation of antisense gene, but increased for 17.6%-23.4%, indicating there must be a complex regulation system to ethylene release, and the total quantity of ethylene released has first increased and then decrased in the young etiolated transgenic plants compared to the wild type. In field trials, all transgenic plants (To-T2) had one prolonged vegetative growth stage, the lasting seven days longer than that of wild type and result into a delayed reproductive stage. Average pod-bearing number and average one-hundred-grain weight of the partial transgenic lines had increased significantly compared to wildtype ones, that showed the traits of increasing yield, and on the plant external morphology, the plant height had decreased, the node number had increased, and the average distance of stem-stem was significantly shortened in all detected transgenic lines, showed the traits of the decreased ethylene release.
In conclusion, the ovarian in planta transformation in soybean with the linear minimal cassette and the optimal transformation passageway is a new prospective transformation method in yield and quality improvement.
引文
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