利用含有anti-waxy基因表达载体改良两系不育系水稻261S食味品质的研究
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摘要
近年来,世界各地在转基因作物研究和推广种植方面都取得了飞速发展,越来越多的转基因作物走向商业化种植,不仅给全球农业带来了勃勃生机,也为推广和应用转基因作物的种植者带来了良好的经济效益,尤其对帮助发展中国家农民摆脱贫穷具有重要的发展前景。但同时,转基因作物中常用标记基因的存在也引起越来越多人的关注和担忧,如何获得不含标记基因的转基因作物显得日益重要。本文首先对近年来在世界各地转基因作物研究和推广种植方面以及安全性转基因技术研究进行了综述。
杂交水稻食味品质改良研究是目前杂交水稻育种研究领域中的重要内容之一。据文献报道,稻米直链淀粉含量是决定水稻食味品质最主要指标。杂交水稻在产量等性状所体现的优势与父本和母本遗传组成配合力相关。尽管目前利用常规育种技术也能够实现降低稻米直链淀粉含量,但改良后的水稻其他遗传组成也会随之发生变化。而利用转基因技术将能够降低稻米直链淀粉含量的反义蜡质基因(anti-waxy)导入杂交水稻亲本,即可实现在有效降低稻米直链淀粉含量的同时,最大程度地保持其与另一亲本杂交组合优势的原有遗传组成。
本研究采用可获得无选择标记基因的安全性转基因策略,利用根癌农杆菌介导的共转化法,首先将含有抗性标记基因hpt和报告基因gus的pCAMBIA1301表达载体与含有单份拷贝anti-waxy基因片断的p13AWY-1表达载体以1:9的比例共同导入“闵优香粳”杂交稻两系不育系水稻品种261S,经过PCR检测筛选出共转化植株,通过对共转化植株的Southern blot分析证明anti-waxy基因已以不同拷贝数整合入水稻基因组中,通过对T1代种子的GUS检测已达到将含抗性标记基因的转基因后代去除的目的,从而为继续筛选出只含anti-waxy基因的安全性转基因后代奠定了基础。此外,还对目前已收获的部分T1代种子进行糙米直链淀粉含量检测,发现共转化后代的直链淀粉含量(AC)已有不同程度的降低,说明该转化试验是成功有效的。
同时为了探索稻米直链淀粉含量降低程度与转入anti-waxy基因拷贝数之间的关系,本研究又对实验室早期获得转入单拷贝anti-waxy基因水稻进行杂交获得外源基因呈双拷贝的转基因后代,通过对其稻米直链淀粉含量分析证明,含有双份拷贝anti-waxy基因可以更大程度地降低稻米直链淀粉含量,并且可以获得与我国云南特种软米水稻品种直链淀粉含量(小于10%)范围相同的转基因软米新种质资源。
基于前期两部分试验研究的结果,为了更便利地在一次转化中实现anti-waxy基因在水稻基因组中呈双拷贝整合,达到更进一步改良261S水稻食味品质的目的和培育无抗性选择标记基因的转基因软米,本研究又将在p13AWY-1表达载体基础上重新构建的含有双份拷贝串联重复anti-waxy基因的p13AWY-2表达载体转入261S水稻,目前也已成功获得了共转化植株。通过该研究不仅可为进一步改良由261S为母本配组而成的优质杂交稻“闵优香粳”的食味品质奠定基础,而且也可为研究水稻基因组中整合入不同拷贝数anti-waxy基因后的目的基因表达情况提供参考。
通过文献查阅,尽管在国内外采用转基因技术将anti-waxy基因导入水稻改良稻米直链淀粉含量已有较多报道,但是在相关的报道中使用的载体大多都是将目的基因和抗性标记基因构建在一个T-DNA上,有个别报道将目的基因和抗性标记基因分别构建在不同载体上,但是在含有目的基因的T-DNA上依然存在来自于花椰菜花叶病毒的35S启动子。因此,从载体的安全性角度分析,本研究获得的转基因水稻后代将是最有可能通过我国农业部的安全性评价,在今后水稻的生产中被应用。
In recent years, with large amount of transgenic plants being commercialized, people feel uneasy about the potential risks of transgenic plants. Marker-free and acceptor promoter may raise food and environment biosafety of Genetically Modified Crops,so it becomes more and more important to abtain safety transgenic plants.
In the first part of the study,the two kinds of Agrobactium tumefaciens, which respectively carried the vector pCAMBIA1301 (contains hygromycin gene and gus gene) and vector p13AWY-1, which carried aim gene (anti-waxy gene) only but without resistance marker gene (hygromycin) and 35S promoter,were mixed at the density ratio of 1:9. The T-DNAs of the two vectors pCAMBIA1301 and p13AWY-1 were introduced into 261S by the co-transformation method of which is safety transgenic technique. Using PCR analysis showed that 6 lines among 46 transgenic lines were co-transformation plants, The vector p13AWY-1 T-DNA in the rice genome was confirmed through Southern blot analysis. Then using gus gene to wash out T1 seeds that have the report gene and the marker gene. It can lay the groundwork for filtrating rice materials which containing target gene, without the marker gene and report gene.The amylase analysis shows that the amylose content of some of the transgenic rice have been lower than the wild tipe.
In the second part of the study,we contrast the amylose content of four lines of trandgenic rice,of which contain one copy or two copies of anti-waxy gene. It shows that amylose content is reduced obviously in the two lines of transgenic rice.,which carried two copies of anti-waxy gene by crossing the transgenic species which were introduced one copy of anti-waxy gene.It also showed that the average amylose contents of the two hybrid rice of F1 were 8.96% and 8.23%, and they all within reach of the extension of amylose contents of soft rice ,it meaned we could cultivate new kinds of soft rice by introducing anti-waxy gene into some rice species ,and the results also told us we could obtain the highly expressed transgenic plants by crossing the transgenic species which obtained through the same transformation.
In the third part of the study,the plasmid p13AWY-2 which carried two copies of aim gene(anti-waxy) was introduced into 261S by the co-transformation method too,and we have got co- transformed plants now.It will lay the foundation for rice eating quality improvement by transgenic technology. Moreover, it can make the genetically modified plants safer for consumers and the environment.
In this first and third parts of the study,we used not only the safety methord but also the marker free vectors for transforming the anti-waxy gene into the male sterile line to improve the tasting quality of rice, so the transgenic rice with aim gene only will be safety and we can hope that the transgenic products will be more easily and early planted for use.
杂交水稻食味品质改良研究是目前杂交水稻育种研究领域中的重要内容之一。据文献报道,稻米直链淀粉含量是决定水稻食味品质最主要指标。杂交水稻在产量等性状所体现的优势与父本和母本遗传组成配合力相关。尽管目前利用常规育种技术也能够实现降低稻米直链淀粉含量,但改良后的水稻其他遗传组成也会随之发生变化。而利用转基因技术将能够降低稻米直链淀粉含量的反义蜡质基因(anti-waxy)导入杂交水稻亲本,即可实现在有效降低稻米直链淀粉含量的同时,最大程度地保持其与另一亲本杂交组合优势的原有遗传组成。
本研究采用可获得无选择标记基因的安全性转基因策略,利用根癌农杆菌介导的共转化法,首先将含有抗性标记基因hpt和报告基因gus的pCAMBIA1301表达载体与含有单份拷贝anti-waxy基因片断的p13AWY-1表达载体以1:9的比例共同导入“闵优香粳”杂交稻两系不育系水稻品种261S,经过PCR检测筛选出共转化植株,通过对共转化植株的Southern blot分析证明anti-waxy基因已以不同拷贝数整合入水稻基因组中,通过对T1代种子的GUS检测已达到将含抗性标记基因的转基因后代去除的目的,从而为继续筛选出只含anti-waxy基因的安全性转基因后代奠定了基础。此外,还对目前已收获的部分T1代种子进行糙米直链淀粉含量检测,发现共转化后代的直链淀粉含量(AC)已有不同程度的降低,说明该转化试验是成功有效的。
同时为了探索稻米直链淀粉含量降低程度与转入anti-waxy基因拷贝数之间的关系,本研究又对实验室早期获得转入单拷贝anti-waxy基因水稻进行杂交获得外源基因呈双拷贝的转基因后代,通过对其稻米直链淀粉含量分析证明,含有双份拷贝anti-waxy基因可以更大程度地降低稻米直链淀粉含量,并且可以获得与我国云南特种软米水稻品种直链淀粉含量(小于10%)范围相同的转基因软米新种质资源。
基于前期两部分试验研究的结果,为了更便利地在一次转化中实现anti-waxy基因在水稻基因组中呈双拷贝整合,达到更进一步改良261S水稻食味品质的目的和培育无抗性选择标记基因的转基因软米,本研究又将在p13AWY-1表达载体基础上重新构建的含有双份拷贝串联重复anti-waxy基因的p13AWY-2表达载体转入261S水稻,目前也已成功获得了共转化植株。通过该研究不仅可为进一步改良由261S为母本配组而成的优质杂交稻“闵优香粳”的食味品质奠定基础,而且也可为研究水稻基因组中整合入不同拷贝数anti-waxy基因后的目的基因表达情况提供参考。
通过文献查阅,尽管在国内外采用转基因技术将anti-waxy基因导入水稻改良稻米直链淀粉含量已有较多报道,但是在相关的报道中使用的载体大多都是将目的基因和抗性标记基因构建在一个T-DNA上,有个别报道将目的基因和抗性标记基因分别构建在不同载体上,但是在含有目的基因的T-DNA上依然存在来自于花椰菜花叶病毒的35S启动子。因此,从载体的安全性角度分析,本研究获得的转基因水稻后代将是最有可能通过我国农业部的安全性评价,在今后水稻的生产中被应用。
In recent years, with large amount of transgenic plants being commercialized, people feel uneasy about the potential risks of transgenic plants. Marker-free and acceptor promoter may raise food and environment biosafety of Genetically Modified Crops,so it becomes more and more important to abtain safety transgenic plants.
In the first part of the study,the two kinds of Agrobactium tumefaciens, which respectively carried the vector pCAMBIA1301 (contains hygromycin gene and gus gene) and vector p13AWY-1, which carried aim gene (anti-waxy gene) only but without resistance marker gene (hygromycin) and 35S promoter,were mixed at the density ratio of 1:9. The T-DNAs of the two vectors pCAMBIA1301 and p13AWY-1 were introduced into 261S by the co-transformation method of which is safety transgenic technique. Using PCR analysis showed that 6 lines among 46 transgenic lines were co-transformation plants, The vector p13AWY-1 T-DNA in the rice genome was confirmed through Southern blot analysis. Then using gus gene to wash out T1 seeds that have the report gene and the marker gene. It can lay the groundwork for filtrating rice materials which containing target gene, without the marker gene and report gene.The amylase analysis shows that the amylose content of some of the transgenic rice have been lower than the wild tipe.
In the second part of the study,we contrast the amylose content of four lines of trandgenic rice,of which contain one copy or two copies of anti-waxy gene. It shows that amylose content is reduced obviously in the two lines of transgenic rice.,which carried two copies of anti-waxy gene by crossing the transgenic species which were introduced one copy of anti-waxy gene.It also showed that the average amylose contents of the two hybrid rice of F1 were 8.96% and 8.23%, and they all within reach of the extension of amylose contents of soft rice ,it meaned we could cultivate new kinds of soft rice by introducing anti-waxy gene into some rice species ,and the results also told us we could obtain the highly expressed transgenic plants by crossing the transgenic species which obtained through the same transformation.
In the third part of the study,the plasmid p13AWY-2 which carried two copies of aim gene(anti-waxy) was introduced into 261S by the co-transformation method too,and we have got co- transformed plants now.It will lay the foundation for rice eating quality improvement by transgenic technology. Moreover, it can make the genetically modified plants safer for consumers and the environment.
In this first and third parts of the study,we used not only the safety methord but also the marker free vectors for transforming the anti-waxy gene into the male sterile line to improve the tasting quality of rice, so the transgenic rice with aim gene only will be safety and we can hope that the transgenic products will be more easily and early planted for use.
引文
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