花粉管通道法转水稻Waxy基因
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摘要
杂交水稻的生产在我国粮食生产中占有十分重要的地位，而杂交稻米品质低劣是目前我国杂交水稻生产中面临的一个普遍问题，稻米的直链淀粉含量被认为是决定稻米品质的重要因素之一，通过调节水稻内源的控制直链淀粉合成的Waxy基因的表达水平，可有效降低稻米直链淀粉含量，进而改良稻米的品质。
在我国，以包括水稻在内的各种作物为对象的遗传转化操作中，花粉管通道法都有过成功的例子。反义调控策略可有效用于调节基因表达水平，并已经被比较成熟的运用于各项转基因研究工作中。三系杂交水稻中，Ⅱ—32是一个应用得比较广泛的不育系，其优点是配合力高、杂种优势较强，缺点在于米质较差，特别是直链淀粉含量比较高。本研究利用反义方向连接的水稻Wary基因片段的构建体，采用花粉管通道法转化Ⅱ—32保持系，在T_0代经过PCR筛选和Gus检测，获得两个阳性单株，并用Southern杂交验证外源片段已整合进水稻基因组中，测量其T_1代种子胚乳的直链淀粉含量，已分别降到13.48％和17.47％。进一步考察T_1代转基因株系的T_2代种子胚乳胚乳直链淀粉含量，各发现有1个和3个单株的直链淀粉含量趋于稳定并降低。此外，本研究还对如何提高花粉管通道法转化效率和花粉管通道法的转化机理进行了探讨。
Poor quality of rice grain is currently the common issues nationwide. Amylose content is regarded as the most important factor in determining rice quality. It is efficient to reduce the amylose content to adjust the waxy locus expression level, which is responsible for the synthesis of amylose in the triploid endosperm of the developing grain and in the haploid pollen.
Pollen tube pathway has been successfully utilized in transgenic research work in kinds of crops, rice included. Antisense down-regulation is a useful tool for adjusting gene expression level. Rice variety, 11-32 is one of widely used restoration lines in China. Though it has shortage which is its amylose content being as high as 25.88%. Using a construction consisted of antisense fragment of rice waxy gene, 2 transformant were obtained by pollen-tube pathway. In T, generation, Southern blot analysis and histochemical checking proved the presence of waxy gene fragment. And the amylose content has been reduced to 13.48% and 17.47% in this two transformant. Furthermore, 1 and 3 plants'amylose content tends to be stable and reduced in T2 generation.
在我国，以包括水稻在内的各种作物为对象的遗传转化操作中，花粉管通道法都有过成功的例子。反义调控策略可有效用于调节基因表达水平，并已经被比较成熟的运用于各项转基因研究工作中。三系杂交水稻中，Ⅱ—32是一个应用得比较广泛的不育系，其优点是配合力高、杂种优势较强，缺点在于米质较差，特别是直链淀粉含量比较高。本研究利用反义方向连接的水稻Wary基因片段的构建体，采用花粉管通道法转化Ⅱ—32保持系，在T_0代经过PCR筛选和Gus检测，获得两个阳性单株，并用Southern杂交验证外源片段已整合进水稻基因组中，测量其T_1代种子胚乳的直链淀粉含量，已分别降到13.48％和17.47％。进一步考察T_1代转基因株系的T_2代种子胚乳胚乳直链淀粉含量，各发现有1个和3个单株的直链淀粉含量趋于稳定并降低。此外，本研究还对如何提高花粉管通道法转化效率和花粉管通道法的转化机理进行了探讨。
Poor quality of rice grain is currently the common issues nationwide. Amylose content is regarded as the most important factor in determining rice quality. It is efficient to reduce the amylose content to adjust the waxy locus expression level, which is responsible for the synthesis of amylose in the triploid endosperm of the developing grain and in the haploid pollen.
Pollen tube pathway has been successfully utilized in transgenic research work in kinds of crops, rice included. Antisense down-regulation is a useful tool for adjusting gene expression level. Rice variety, 11-32 is one of widely used restoration lines in China. Though it has shortage which is its amylose content being as high as 25.88%. Using a construction consisted of antisense fragment of rice waxy gene, 2 transformant were obtained by pollen-tube pathway. In T, generation, Southern blot analysis and histochemical checking proved the presence of waxy gene fragment. And the amylose content has been reduced to 13.48% and 17.47% in this two transformant. Furthermore, 1 and 3 plants'amylose content tends to be stable and reduced in T2 generation.
引文
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2．王关林主编。植物基因工程原理和技术。科学出版社，1998
3．王宗阳，武志亮，邢彦彦，郭小丽，张伟国，洪孟明。水稻蜡质基因中两种类似转座因子的顺序。中国科学B辑，1993，6：595-603
4．王宗阳，郑霏琴，高继平，王小金，吴敏，张景六，洪孟明。水稻蜡质基因分子特性的研究。中国科学B辑，1991，8：825-829
5．王忠，李卫芳，顾蕴洁。水稻胚乳的发育及其养分输入的途径。作物学报。1995，21(5)：520-526
6．王维金主编。作物栽培学。北京，科学技术文出版社。1998，76
7．加藤幸雄，志佐诚。植物生殖生理学。科学出版社，1987，243
8．申岳正。稻米直链淀粉含量的遗传和测定方法的改进。中国水稻科学，1990，23(1)：60-68
9．石春海，陈国林，朱军，臧荣春，吴建国，陈双燕。籼稻稻米直链淀粉含量的胚、胚乳、细胞质和母体遗传效应分析。作物学报，2000，26(6)：833-839
10．何予卿，吕志仁。籼稻米直链淀粉含量的遗传及其基因剂量效应。华中农业大学学报，1993，12(5)：414-420
11．李维科。我国杂交水稻育种技术的三大变革及其作用。作物杂志，1998，62：16
12．周光字，翁坚，龚蓁蓁，曾以申，杨晚霞，黄骏麟，陈善葆，段晓岚。农业分子育种授粉后外源DNA导入植物的技术。中国农业科学，1988，21(3)：1-6
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16．黄祖六，谭学林．Tragoorung S，Vanavichit A．稻米直链淀粉含量基因座位的分子标记定位。作物学报．2000，26(6)：777-782
17．黄骏麟等。外源海岛棉DNA导致陆地棉性状变异。遗传学报，1981，8(1)：
567
18．黄超武，李锐。水稻AC的遗传分析。华南农业大学学报，1990，11(1)：23-29
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