利用农杆菌介导法将TT1基因导入水稻的研究
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摘要
水稻是一种世界性的重要粮食作物,我国一半以上的人口以稻米为主食。随着工业化的加快和温室效应的加剧,全球气温变暖使水稻的正常生长和发育受到严重影响。水稻在高温条件下不能正常生长,造成花粉发育不良,活力下降,导致严重的产量损失和品质下降。提高水稻的耐热性已成为一个迫切需要解决的问题。自然界中具有耐热抗性的水稻品种极少且农艺性状不佳,因此通过基因工程技术将已有的耐热基因导入水稻中,选育耐热水稻品种,对适应耕地减少,人口增加等局势具有重要意义。
TT1基因是从甘蓝型油菜中克隆出的一个可提高植物热胁迫抗性的耐热基因,是目前少有的能提高植物耐热性的基因。该基因已经被成功的转入油菜中,结果显示TT1基因的表达能够增加油菜的耐热受性。因此,若能利用基因工程技术使TT1基因在水稻中高效表达,必将为水稻耐热性研究提供一个新的优良品种资源。本研究通过农杆菌介导法将构建在载体T-DNA上的耐热基因TT1导入水稻品种日本晴中,以验证其在水稻中的表达情况,实验取得了以下结果:
1对不同成熟度的日本晴种子进行愈伤诱导,诱导率为:幼嫩种子＞饱绿种子＞饱黄种子。而污染率为:饱黄种子＜饱绿种子＜幼嫩种子。
2在共培养过程中,日本晴幼胚愈伤组织最适共培养时间为2.5天,成熟胚愈伤组织最适共培养时间为3天。二者的最适共培养温度都为22℃。经过浸染和筛选后转入分化培养基中二者都得到了再生苗,幼胚的分化率高于成熟胚。
3以潮霉素为筛选剂分别对日本晴幼胚和成熟胚愈伤组织进行筛选压的确定,结果发现:两者的最佳筛选浓度都为50mg/L。
4对日本晴成熟胚和幼胚愈伤组织再生苗进行PCR验证,证明外源TT1基因已整合到受体植株的DNA中。通过RT-PCR进一步验证,证明TT1基因在植株的RNA水平上进行了表达。
5通过对非转化植株和转化植株进行耐热性鉴定,发现非转化植株在50℃的条件下处理2h后干枯死亡,而转基因植株在同样条件下表现正常。转基因植株在57℃的条件下处理2h后干枯死亡。
Rice(Oryza sativa L.) is an important worldwide cereal crop.More than half of the population in China depends on rice as its principle staple food.With the development of industry and the exacerbation of the greenhouse effect,rice will be not able to grow normally under the rising temperature,the pollens will become abnormal and the pollens' activity will go down.The high temperature finally results in the reduction of the yield and the falling off in quality.It's very important to improve the ability of rice's heat-tolerance.There are few rice varieties which can endure high temperature,or even though they have good ability of heat-tolerance,the agronomic traits are normally bad.So,transferring the heat-tolerant gene into rice varieties to improve the heat tolerance of rice will be of great importance to solve the problem of reducing arable land and increasing population.
TT1 is a single gene which was cloned from Brassica napus,it's the only gene ever discovered in the world that can enhance the thermo-tolerance of rape through the expression of a single gene.It will be an effect way to breed high yield and quantity rice varieties which can endure high temperature if the gene can express normally in rice.In this study,TT1 gene was transferred into rice varieties through Agrobacterium tumefaciens-mediated transformation to testify the function of TT1 in rice.The results were as follows:
1.By comparison of induction efficiency of different maturity seeds of Nipponbare,the young seeds induced more callus than full-green seeds and full-yellow seeds,the induction efficiency of full-yellow seeds were lowest.The pollution frequency of the young seeds were most serious while the full-yellow seeds were the lowest.
2 The optimum time of co-cultivation for the callus derived from immature embryos was 2.5 days while the callus derived from the mature embryos was 3 days.22℃was the optimum co-cultivation temperature both for the callus from immature embryos and mature embryos. The differentiation rate of immature embryos was higher than mature embryos.
3 The selection reagent for immature embryos callus and mature embryos callus indicated that the optimum selection concentration of hygromycin for both of these calli was 50 mg/L.
4 The PCR assay of the regenerated seedlings proved that transgenic plants containing foreign gene TT1 were obtained.The RT-PCR assay further proved that TT1 was expressed on RNA level.
5 By comparison of the ability of the heat-tolerance of transgenic plants and non-genetically modified plants,it showed that non-genetically modified plants died in 2 hours when the temperature was 50℃,while transgenic plants didn't die until the temperature went up to 57℃.
TT1基因是从甘蓝型油菜中克隆出的一个可提高植物热胁迫抗性的耐热基因,是目前少有的能提高植物耐热性的基因。该基因已经被成功的转入油菜中,结果显示TT1基因的表达能够增加油菜的耐热受性。因此,若能利用基因工程技术使TT1基因在水稻中高效表达,必将为水稻耐热性研究提供一个新的优良品种资源。本研究通过农杆菌介导法将构建在载体T-DNA上的耐热基因TT1导入水稻品种日本晴中,以验证其在水稻中的表达情况,实验取得了以下结果:
1对不同成熟度的日本晴种子进行愈伤诱导,诱导率为:幼嫩种子＞饱绿种子＞饱黄种子。而污染率为:饱黄种子＜饱绿种子＜幼嫩种子。
2在共培养过程中,日本晴幼胚愈伤组织最适共培养时间为2.5天,成熟胚愈伤组织最适共培养时间为3天。二者的最适共培养温度都为22℃。经过浸染和筛选后转入分化培养基中二者都得到了再生苗,幼胚的分化率高于成熟胚。
3以潮霉素为筛选剂分别对日本晴幼胚和成熟胚愈伤组织进行筛选压的确定,结果发现:两者的最佳筛选浓度都为50mg/L。
4对日本晴成熟胚和幼胚愈伤组织再生苗进行PCR验证,证明外源TT1基因已整合到受体植株的DNA中。通过RT-PCR进一步验证,证明TT1基因在植株的RNA水平上进行了表达。
5通过对非转化植株和转化植株进行耐热性鉴定,发现非转化植株在50℃的条件下处理2h后干枯死亡,而转基因植株在同样条件下表现正常。转基因植株在57℃的条件下处理2h后干枯死亡。
Rice(Oryza sativa L.) is an important worldwide cereal crop.More than half of the population in China depends on rice as its principle staple food.With the development of industry and the exacerbation of the greenhouse effect,rice will be not able to grow normally under the rising temperature,the pollens will become abnormal and the pollens' activity will go down.The high temperature finally results in the reduction of the yield and the falling off in quality.It's very important to improve the ability of rice's heat-tolerance.There are few rice varieties which can endure high temperature,or even though they have good ability of heat-tolerance,the agronomic traits are normally bad.So,transferring the heat-tolerant gene into rice varieties to improve the heat tolerance of rice will be of great importance to solve the problem of reducing arable land and increasing population.
TT1 is a single gene which was cloned from Brassica napus,it's the only gene ever discovered in the world that can enhance the thermo-tolerance of rape through the expression of a single gene.It will be an effect way to breed high yield and quantity rice varieties which can endure high temperature if the gene can express normally in rice.In this study,TT1 gene was transferred into rice varieties through Agrobacterium tumefaciens-mediated transformation to testify the function of TT1 in rice.The results were as follows:
1.By comparison of induction efficiency of different maturity seeds of Nipponbare,the young seeds induced more callus than full-green seeds and full-yellow seeds,the induction efficiency of full-yellow seeds were lowest.The pollution frequency of the young seeds were most serious while the full-yellow seeds were the lowest.
2 The optimum time of co-cultivation for the callus derived from immature embryos was 2.5 days while the callus derived from the mature embryos was 3 days.22℃was the optimum co-cultivation temperature both for the callus from immature embryos and mature embryos. The differentiation rate of immature embryos was higher than mature embryos.
3 The selection reagent for immature embryos callus and mature embryos callus indicated that the optimum selection concentration of hygromycin for both of these calli was 50 mg/L.
4 The PCR assay of the regenerated seedlings proved that transgenic plants containing foreign gene TT1 were obtained.The RT-PCR assay further proved that TT1 was expressed on RNA level.
5 By comparison of the ability of the heat-tolerance of transgenic plants and non-genetically modified plants,it showed that non-genetically modified plants died in 2 hours when the temperature was 50℃,while transgenic plants didn't die until the temperature went up to 57℃.
引文
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