油菜超量表达柠檬酸合成酶和植酸酶基因提高抗土壤磷铝胁迫的研究
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摘要
铝毒害和有效磷缺乏是酸性土壤中限制作物生长的两个主要因子。虽然土壤中有效磷很低,但总磷量很高,其中有机磷占总磷的20%-80%,是植物可吸收利用的潜在磷源。甘蓝型油菜占我国油菜种植面积的80%以上,但其主产区土壤偏酸,或酸性,活性铝偏高,有效磷缺乏。因此,减轻或消除酸性土壤的铝毒害、提高土壤磷的有效性对我国油菜产业可持续发展具有重要意义。本研究通过农杆菌介导的基因遗传转化技术,在获得甘蓝型油菜超量表达Pseudomonas aeruginosa柠檬酸合成酶(CS)基因、及整合有胡萝卜外展蛋白的植酸酶基因phyA和appA的转基因株系的基础上,利用水培、砂培、土培等方法研究了转基因植株对铝毒害和低磷胁迫的抗性、及其生理和分子机理。获得的主要结果如下：
1.通过筛选鉴定获得5个T3代甘蓝型油菜转基因株系。利用Southern blotting分析T3转基因植株外源基因的拷贝数,结果显示：超量表达CS的转基因株系CS3含有两个P. aeruginosa CS基因拷贝,而CS6株系含有一个拷贝；超量表达phyA的转基因株系P3含有一个Aspergillus niger phyA基因拷贝,P11株系含有3个phyA基因拷贝,而超量表达植酸酶基因appA的转基因株系a18含有两个Escherichia coli appA拷贝。利用Northern blotting分析外源基因表达量,结果显示：在mRNA水平上,CS3和CS6株系有较高的P. aeruginosa CS基因表达量；P3和P11株系有较高的A. niger phyA基因表达量；a18株系有较高的E. coli appA基因表达量。卡那抗性筛选结果显示,5个转基因株系均无Km抗性分离。
2.超量表达CS基因的转基因株系CS3和CS6的铝毒抗性显著增强。铝胁迫下,转基因株系CS3和CS6不仅柠檬酸的分泌量显著增加,而且苹果酸的分泌量也显著高于野生型植株(WT)。转基因株系较高的三羧酸循环途径(TAC)相关的柠檬酸合成酶(CS)、苹果酸脱氢酶(MDH)、磷酸烯醇式丙酮酸羧化酶(PEPC)的活性以及较高的柠檬酸和苹果酸转运子的表达量,导致了其有机酸分泌量的增加。25μM AICl3处理48h后,CS3和CS6的相对主根伸长量极显著大于WT,并且长期50μM AICl3胁迫下CS3和CS6的生物量显著高于WT,说明在甘蓝型油菜中超量表达CS基因增强了植株的铝毒抗性。
3.超量表达CS基因的转基因株系CS3和CS6的抗低磷胁迫能力增强。低磷胁迫下,CS3和CS6根系分泌的柠檬酸和苹果酸含量显著高于WT,并且CS、MDH和PEPC的活性得到显著提高。盆栽土培试验表明,以FePO4作为土壤中的磷源时,油菜超量表达CS基因显著提高植株从土壤中获取磷的能力,转基因植株苗期地上部和籽粒中的磷累积量显著高于WT植株。
4.超量表达植酸酶基因的转基因株系P3、P11和a18的抗低磷能力增强。在油菜中超量表达整合有胡萝卜外展蛋白信号肽序列的植酸酶基因ex::phyA和ex::appA,获得转基因植株在水培试验中的结果表明,根组织和分泌的植酸酶活性显著高于WT。砂培试验结果显示,当以植酸钠作为唯一磷源时,P3、P11和a18株系地上部磷累积量分别比WT植株增加了70.8%、36.8%和19.6%。并且,三个转基因株系的地上部生物量均显著高于WT植株。盆栽土培试验结果显示,当以植酸钠作为唯一磷源时,P11和a18株系的籽粒产量分别比WT植株提高了20.9%和59.9%,种子中磷的累积量分别比WT植株增加了20.6%和46.9%。三个转基因株系种子的植酸酶活力约为1000U/kg种子,而WT的种子中没有检测到植酸酶活性。并且P11和a18株系种子的植酸含量显著低于WT。
综上所述,在甘蓝型油菜中超量表达CS基因不仅提高了转基因株系柠檬酸的分泌量,而且影响了苹果酸的合成代谢,这两种有机酸分泌量的增加提高了转基因株系CS3和CS6的铝毒抗性和低磷忍耐力。甘蓝型油菜超量表达融合有胡萝卜外展蛋白信号肽序列的植酸酶基因可以显著增强转基因植株根系分泌的植酸酶活性,提高转基因株系对植酸磷的吸收利用能力。并且,转基因植株种子具有很高的植酸酶活性。
Aluminum (Al) toxicity and low phosphorus (P) availability are two major constraints on crops growth in acid soil. Although P availability is very low, total P is quite abundant in soils with organic P constituting up to20%-80%, which is the potential P source for plant growth. Brassica napus is one of the main oil crops and is cultivated up to more than80%of cultivated region of oil crops in China. However, these cultivated region of Brassica napus are acid or relatively acid soil, with high Al toxic levels and low P availability. So, reducing or eliminating Al toxicity of acid soils and improving soil P availability are essential to enhance Brassica napus production.
In this research, based on the Agrobacterium-mediated gene transformation, we introduced a Pseudomonas aeruginosa citrate synthase (CS) gene and two phytase genes phyA and appA which include an extracellular targeting sequence from the carrot extension (ex) gen into Brassica napus cv Westar10. The tolerances of the transgenic lines to Al toxicity and P deficiency, and their physiological and molecular mechanism were studied using hydroponics, sand and soil culturing in the study. The main results were listed as follows:
1. Five T3generation transgenic Brassica napus lines were obtained by molecular identification and Km-resistance screening. The results of Southern blotting showed that CS3and CS6transgenic lines overexpressing CS gene harboured two and single P. aeruginosa CS loci, respectively. P3and P11transgenic lines harboured single and three A. niger phyA loci, respectively, and a18transgenic line harboured two E. coli appA loci. Northern blotting analysis showed that transgenic lines CS3and CS6showed an accumulation of P. aeruginosa CS transcripts. The expression of A. niger phyA at the mRNA level was very high in P3and P11transgenic lines. a18transgenic lines showed an accumulation of E. coli appA transcripts. All of the five transgenic lines are lack of segregation with kanamycin-resistant screening.
2. Transgenic lines CS3and CS6overexpressing CS gene improved the tolerance to Al toxicity. Both root concentration and exudations of citrate and malate in transgenic lines CS3and CS6significantly increased compared with wild type (WT) following exposure to Al. These may be attributed to higher activities of the CS, malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC) enzymes in the TCA cycle and the expression of BnALMT and BnMATE in the transgenic plants following Al exposure.
Transgenic lines CS3and CS6have enhanced Al tolerance. When exposed to25μM AICI3for48h, the relative root lengths of transgenic plants are significantly longer than WT. Moreover, prolonged Al treatment (10days) experiments revealed that transgenic lines accumulated much more biomass than WT.
3. Transgenic lines CS3and CS6overexpressing CS gene improved the tolerance to P deficiency. Transgenic lines CS3and CS6showed enhanced citrate and malate exudation when grown in P-deficient conditions. Moreover, the enzyme activities of the transgenic lines were significantly higher compared with WT in response to P-deficient stress. The soil culture experiment showed that transgenic lines CS3and CS6possessed improved P uptake from the soil and accumulated more P in their shoots and seeds when FePO4was used as the sole P source.
4. Transgenic lines P3, P11and a18overexpressing phytase gene enhanced the tolerance to P deficiency. The extracellular phytase activities of transgenic Brassica napus overexpressing ex::phylappA are significantly higher than WT in hydroponic culture. Quartz sand culture experiment showed that the shoots P accumulation in the three transgenic lines P3, P11and a18increased by70.8%,36.8%and19.6%compared with WT, respectively, when phytate was used as the sole P source. Moreover, the shoots biomass of the three transgenic lines was all significantly higher than WT. The soil culture experiment showed that seed yields of transgenic lines P11and a18increased by20.9%and59.9%, and seeds P accumulation increased by20.6%and46.9%compared with WT, respectively, when phytate was used as the sole P source. Phytase activities in P3, P11and a18seeds reached approximately1000units per kg seed, whereas no phytase activities were detected in WT seeds. Moreover, phytic acid contents of P11and a18seeds were significantly lower than WT.
In conclusion, the overexpression of the CS gene in B. napus can not only lead to increased citrate synthesis and exudation, but also altered malate metabolism. The increased rate of the two organic acids exudation in transgenic canola overexpressing CS lead to significantly improved capacities for Al tolerance and soil P acquisition. The increased extracellular phytase activities in transgenic canola overexpressing ex::phylappA lead to significantly improved the ability of plants to utilize phytate. Moreover, transgenic seeds of P3, P11and a18have highly phytase activities.
1.通过筛选鉴定获得5个T3代甘蓝型油菜转基因株系。利用Southern blotting分析T3转基因植株外源基因的拷贝数,结果显示：超量表达CS的转基因株系CS3含有两个P. aeruginosa CS基因拷贝,而CS6株系含有一个拷贝；超量表达phyA的转基因株系P3含有一个Aspergillus niger phyA基因拷贝,P11株系含有3个phyA基因拷贝,而超量表达植酸酶基因appA的转基因株系a18含有两个Escherichia coli appA拷贝。利用Northern blotting分析外源基因表达量,结果显示：在mRNA水平上,CS3和CS6株系有较高的P. aeruginosa CS基因表达量；P3和P11株系有较高的A. niger phyA基因表达量；a18株系有较高的E. coli appA基因表达量。卡那抗性筛选结果显示,5个转基因株系均无Km抗性分离。
2.超量表达CS基因的转基因株系CS3和CS6的铝毒抗性显著增强。铝胁迫下,转基因株系CS3和CS6不仅柠檬酸的分泌量显著增加,而且苹果酸的分泌量也显著高于野生型植株(WT)。转基因株系较高的三羧酸循环途径(TAC)相关的柠檬酸合成酶(CS)、苹果酸脱氢酶(MDH)、磷酸烯醇式丙酮酸羧化酶(PEPC)的活性以及较高的柠檬酸和苹果酸转运子的表达量,导致了其有机酸分泌量的增加。25μM AICl3处理48h后,CS3和CS6的相对主根伸长量极显著大于WT,并且长期50μM AICl3胁迫下CS3和CS6的生物量显著高于WT,说明在甘蓝型油菜中超量表达CS基因增强了植株的铝毒抗性。
3.超量表达CS基因的转基因株系CS3和CS6的抗低磷胁迫能力增强。低磷胁迫下,CS3和CS6根系分泌的柠檬酸和苹果酸含量显著高于WT,并且CS、MDH和PEPC的活性得到显著提高。盆栽土培试验表明,以FePO4作为土壤中的磷源时,油菜超量表达CS基因显著提高植株从土壤中获取磷的能力,转基因植株苗期地上部和籽粒中的磷累积量显著高于WT植株。
4.超量表达植酸酶基因的转基因株系P3、P11和a18的抗低磷能力增强。在油菜中超量表达整合有胡萝卜外展蛋白信号肽序列的植酸酶基因ex::phyA和ex::appA,获得转基因植株在水培试验中的结果表明,根组织和分泌的植酸酶活性显著高于WT。砂培试验结果显示,当以植酸钠作为唯一磷源时,P3、P11和a18株系地上部磷累积量分别比WT植株增加了70.8%、36.8%和19.6%。并且,三个转基因株系的地上部生物量均显著高于WT植株。盆栽土培试验结果显示,当以植酸钠作为唯一磷源时,P11和a18株系的籽粒产量分别比WT植株提高了20.9%和59.9%,种子中磷的累积量分别比WT植株增加了20.6%和46.9%。三个转基因株系种子的植酸酶活力约为1000U/kg种子,而WT的种子中没有检测到植酸酶活性。并且P11和a18株系种子的植酸含量显著低于WT。
综上所述,在甘蓝型油菜中超量表达CS基因不仅提高了转基因株系柠檬酸的分泌量,而且影响了苹果酸的合成代谢,这两种有机酸分泌量的增加提高了转基因株系CS3和CS6的铝毒抗性和低磷忍耐力。甘蓝型油菜超量表达融合有胡萝卜外展蛋白信号肽序列的植酸酶基因可以显著增强转基因植株根系分泌的植酸酶活性,提高转基因株系对植酸磷的吸收利用能力。并且,转基因植株种子具有很高的植酸酶活性。
Aluminum (Al) toxicity and low phosphorus (P) availability are two major constraints on crops growth in acid soil. Although P availability is very low, total P is quite abundant in soils with organic P constituting up to20%-80%, which is the potential P source for plant growth. Brassica napus is one of the main oil crops and is cultivated up to more than80%of cultivated region of oil crops in China. However, these cultivated region of Brassica napus are acid or relatively acid soil, with high Al toxic levels and low P availability. So, reducing or eliminating Al toxicity of acid soils and improving soil P availability are essential to enhance Brassica napus production.
In this research, based on the Agrobacterium-mediated gene transformation, we introduced a Pseudomonas aeruginosa citrate synthase (CS) gene and two phytase genes phyA and appA which include an extracellular targeting sequence from the carrot extension (ex) gen into Brassica napus cv Westar10. The tolerances of the transgenic lines to Al toxicity and P deficiency, and their physiological and molecular mechanism were studied using hydroponics, sand and soil culturing in the study. The main results were listed as follows:
1. Five T3generation transgenic Brassica napus lines were obtained by molecular identification and Km-resistance screening. The results of Southern blotting showed that CS3and CS6transgenic lines overexpressing CS gene harboured two and single P. aeruginosa CS loci, respectively. P3and P11transgenic lines harboured single and three A. niger phyA loci, respectively, and a18transgenic line harboured two E. coli appA loci. Northern blotting analysis showed that transgenic lines CS3and CS6showed an accumulation of P. aeruginosa CS transcripts. The expression of A. niger phyA at the mRNA level was very high in P3and P11transgenic lines. a18transgenic lines showed an accumulation of E. coli appA transcripts. All of the five transgenic lines are lack of segregation with kanamycin-resistant screening.
2. Transgenic lines CS3and CS6overexpressing CS gene improved the tolerance to Al toxicity. Both root concentration and exudations of citrate and malate in transgenic lines CS3and CS6significantly increased compared with wild type (WT) following exposure to Al. These may be attributed to higher activities of the CS, malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC) enzymes in the TCA cycle and the expression of BnALMT and BnMATE in the transgenic plants following Al exposure.
Transgenic lines CS3and CS6have enhanced Al tolerance. When exposed to25μM AICI3for48h, the relative root lengths of transgenic plants are significantly longer than WT. Moreover, prolonged Al treatment (10days) experiments revealed that transgenic lines accumulated much more biomass than WT.
3. Transgenic lines CS3and CS6overexpressing CS gene improved the tolerance to P deficiency. Transgenic lines CS3and CS6showed enhanced citrate and malate exudation when grown in P-deficient conditions. Moreover, the enzyme activities of the transgenic lines were significantly higher compared with WT in response to P-deficient stress. The soil culture experiment showed that transgenic lines CS3and CS6possessed improved P uptake from the soil and accumulated more P in their shoots and seeds when FePO4was used as the sole P source.
4. Transgenic lines P3, P11and a18overexpressing phytase gene enhanced the tolerance to P deficiency. The extracellular phytase activities of transgenic Brassica napus overexpressing ex::phylappA are significantly higher than WT in hydroponic culture. Quartz sand culture experiment showed that the shoots P accumulation in the three transgenic lines P3, P11and a18increased by70.8%,36.8%and19.6%compared with WT, respectively, when phytate was used as the sole P source. Moreover, the shoots biomass of the three transgenic lines was all significantly higher than WT. The soil culture experiment showed that seed yields of transgenic lines P11and a18increased by20.9%and59.9%, and seeds P accumulation increased by20.6%and46.9%compared with WT, respectively, when phytate was used as the sole P source. Phytase activities in P3, P11and a18seeds reached approximately1000units per kg seed, whereas no phytase activities were detected in WT seeds. Moreover, phytic acid contents of P11and a18seeds were significantly lower than WT.
In conclusion, the overexpression of the CS gene in B. napus can not only lead to increased citrate synthesis and exudation, but also altered malate metabolism. The increased rate of the two organic acids exudation in transgenic canola overexpressing CS lead to significantly improved capacities for Al tolerance and soil P acquisition. The increased extracellular phytase activities in transgenic canola overexpressing ex::phylappA lead to significantly improved the ability of plants to utilize phytate. Moreover, transgenic seeds of P3, P11and a18have highly phytase activities.
引文
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