离子束介导基因群转移小麦研究
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摘要
为了结合实验室大规模开展的小麦品质育种工作的需要，建立适用的小麦后代蛋白筛选体系在育种过程中是至关重要的。根据中国农科院谷物品质监督检验中心提供的经过准确测定品质的样品进行定标，先搜集标准样品光谱，然后经过数学处理建立定标模型，采用验证样品库对定标方程进行验证，获得较准确的定标效果，建立了利用新一代NIRS 5000光栅型近红外光谱仪进行准确、快速、无损测定小麦籽粒品质的系统，经多次的比对实验证明，该系统与化学分析测定值吻合较好。两年来该系统在实验室开展的品质育种中发挥了重要作用，已测定了8万个小麦材料，为大量介导基因群转移的后代材料的品质测定提供了可靠保障。
从我国优良的推广品种、品系出发，对离子束介导基因群转移中介导时间的选择、不同受体小麦品种、不同供体大豆品种，以及大豆、黑豆、绿豆等不同供体的转化效率分别进行了研究。结果表明，离子束介导基因群转移的转化时间以48小时的转化效果最好。不同受体小麦品种相同供体大豆品种的转化效果不同，不同供体大豆品种相同受体小麦品种的转化效果也有差异。通过对豆科类如大豆、黑豆、绿豆外源DNA的转化效果来看，以大豆的转化效果最稳定，转化效率较高。两年来，在T1代中筛选出了蛋白含量高于18％的有133株，高于19％的有30株。在T1代中蛋白质含量与对照呈显著差异的高蛋白的单株数目所占的百分比一般在5％～20％之间。通过对高蛋白的单株继续种植，发现这些单株在T2代有一定遗传性，这主要表现在T2代有的株系仍能保持高蛋白的特性。
总体上讲，从六个单位利用离子束辅助外源基因群转移方法开展的十余个品种的大量试验数据中用统计的方法得到的实验结果证实：离子束辅助外源基因群转移方法的确有效，它不仅对小麦的蛋白质含量的提高有明显的效果，而且对小麦的田间农艺性状，如株高，熟期，株型，穗型等有定向诱变的作用。目前，离子束介导基因群转移的机理虽然不是很清楚，但它有可能成为一种无性的远缘分子育种手段。由于其简单实用，不需克隆基因，也不存在环境危险性，是一种创造新种质资源、丰富作物遗传多样性的有效手段，因而离子束辅助外源基因群转移方法在品质育种中将具有很好的应用前景。
To meet the need of the quality breeding aim of our laboratory, to establish an effective selecting system on the basis of protein is the most important thing. According to the tested wheat sample supported by the china corn quality supervision &test center, the sample spectrums were selected, through mathematic management; a calibration model and an equation were founded accordingly. The new calibration was tested by another group of sample and the result was very good, then the Infrared Reflectance Spectroscopy instrument set up a new accurate, fleet and intact system for wheat protein test. After some contrast experiment, the result by NIRS is inline with the chemical one. In about two years, about 80,000 wheat samples from genetic transformation had been tested, and the system played an important role in the wheat quality breeding.
In the light of the good wheat breeds and series of our country, the transformation time and the efficiency of different wheat breeds and different soy and different legume were studied respectively. Result shows that the finest transformation time is 48 hour. The transformation efficiency is different among different wheat breeds when the exogenous DNA is certain. And the efficiency is different also among different exogenous DNA when the wheat breed is determined. The efficiency of soy DNA transformation is the most stable and better one among different legume. In the last two years, we have got 133 plants, whose protein content is higher than 18%, and 30 plants higher than 19% in Tl. The number of samples in Tl whose protein content distinguished dramatically from CK ranges from 5 to 20 per. The high protein samples were raised the next year; we can see that the trait of high protein can be inherited in T2.
Generally speaking, form the results of different area and of different wheat breeds, it shows that the method of exogenous DNA transformation of wheat via ion beam is indeed very potent; it cannot only increase the protein content of wheat, but also contribute to the improvement of the agricultural properties. For the moment, the mechanism of the method is not very clear, but it may be a nonsexual and distant related breeding way. Because of its simplicity, safety, and no need of cloning gene, it may be an effective way of broadening the wheat breeds and its genetic diversities, and thus it will have a bright future in quality breeding.
从我国优良的推广品种、品系出发，对离子束介导基因群转移中介导时间的选择、不同受体小麦品种、不同供体大豆品种，以及大豆、黑豆、绿豆等不同供体的转化效率分别进行了研究。结果表明，离子束介导基因群转移的转化时间以48小时的转化效果最好。不同受体小麦品种相同供体大豆品种的转化效果不同，不同供体大豆品种相同受体小麦品种的转化效果也有差异。通过对豆科类如大豆、黑豆、绿豆外源DNA的转化效果来看，以大豆的转化效果最稳定，转化效率较高。两年来，在T1代中筛选出了蛋白含量高于18％的有133株，高于19％的有30株。在T1代中蛋白质含量与对照呈显著差异的高蛋白的单株数目所占的百分比一般在5％～20％之间。通过对高蛋白的单株继续种植，发现这些单株在T2代有一定遗传性，这主要表现在T2代有的株系仍能保持高蛋白的特性。
总体上讲，从六个单位利用离子束辅助外源基因群转移方法开展的十余个品种的大量试验数据中用统计的方法得到的实验结果证实：离子束辅助外源基因群转移方法的确有效，它不仅对小麦的蛋白质含量的提高有明显的效果，而且对小麦的田间农艺性状，如株高，熟期，株型，穗型等有定向诱变的作用。目前，离子束介导基因群转移的机理虽然不是很清楚，但它有可能成为一种无性的远缘分子育种手段。由于其简单实用，不需克隆基因，也不存在环境危险性，是一种创造新种质资源、丰富作物遗传多样性的有效手段，因而离子束辅助外源基因群转移方法在品质育种中将具有很好的应用前景。
To meet the need of the quality breeding aim of our laboratory, to establish an effective selecting system on the basis of protein is the most important thing. According to the tested wheat sample supported by the china corn quality supervision &test center, the sample spectrums were selected, through mathematic management; a calibration model and an equation were founded accordingly. The new calibration was tested by another group of sample and the result was very good, then the Infrared Reflectance Spectroscopy instrument set up a new accurate, fleet and intact system for wheat protein test. After some contrast experiment, the result by NIRS is inline with the chemical one. In about two years, about 80,000 wheat samples from genetic transformation had been tested, and the system played an important role in the wheat quality breeding.
In the light of the good wheat breeds and series of our country, the transformation time and the efficiency of different wheat breeds and different soy and different legume were studied respectively. Result shows that the finest transformation time is 48 hour. The transformation efficiency is different among different wheat breeds when the exogenous DNA is certain. And the efficiency is different also among different exogenous DNA when the wheat breed is determined. The efficiency of soy DNA transformation is the most stable and better one among different legume. In the last two years, we have got 133 plants, whose protein content is higher than 18%, and 30 plants higher than 19% in Tl. The number of samples in Tl whose protein content distinguished dramatically from CK ranges from 5 to 20 per. The high protein samples were raised the next year; we can see that the trait of high protein can be inherited in T2.
Generally speaking, form the results of different area and of different wheat breeds, it shows that the method of exogenous DNA transformation of wheat via ion beam is indeed very potent; it cannot only increase the protein content of wheat, but also contribute to the improvement of the agricultural properties. For the moment, the mechanism of the method is not very clear, but it may be a nonsexual and distant related breeding way. Because of its simplicity, safety, and no need of cloning gene, it may be an effective way of broadening the wheat breeds and its genetic diversities, and thus it will have a bright future in quality breeding.
引文
第一章参考文献：
1．王关林，方宏钧，植物基因工程原理与技术，1998，科学出版社。
2. Abdnl-Baki A A, etal. Plant Report, 1990,70:181～190.
3. Matthews B F, etal. Sex Plant Report, 1990,3:147～151.
4. Dekeyser R, A, etal. Plant Cell, 1990, 2;591-602.
5. Zaghmout O M F, etal. Nucleic Acid Res, 1993, 21; 1048.
6. Draper J, etal Plant Cell, Tissue and Organ Culture, 1995,40:1～15.
7. Tao W etal. Proc Natl. Acad. Sci, 1987, USA, 84; 1840-1845.
8. Tsukakoshi M etal. Appl, Phys. B, 1984, 35; 135-140.
9．王兰岚，宋桂英，张健等。激光生物学报，1993，2(2)：281。
10．王兰岚，宋桂英，周奕华等。激光生物学报，1993，2(2)；、279-280。
11．王兰岚，傅荣着，宋桂英等，利用激光微束穿刺法将外源基因导入小麦的研究。遗传学报，1995，22(5)；394-399。
12. Vasil V etal. Bio/Technology, 1992,10;667-674.
13. Vasil V etal. Bio/Technology, 1993,11;1553-1558.
14．傅荣着，曹光诚，马汉生等，用基因枪法将人工雄性不育基因导入小麦的初步研究，遗传学报，1997，24(4)；358-361。
15．王亚馥，崔凯荣，陈克明等，玉米DNA导入小麦后引起白化突变体的质粒超螺旋结构的研究。农业分子育种研究进展，1993，中国农业科技出版社。
16．倪建福，周文麟，仲乃琴等。偃麦草DNA导入小麦的研究。农业分子育种研究进展，1993，中国农业科技出版社，76-80。
17．吴兆苏，小麦育种学，北京：中国农业出版社，1990．
18．余增亮，离子束生物技术引论。安徽科技出版社。
19．余增亮等，离子刻蚀生物样品的初步研究。安徽农业大学学报，1994，3；260-264。
20．韩建伟，余增亮，低能离子对番茄果皮刻蚀与穿透作用的研究，生物物理学报，1998，Vol，14，No；757-761
21．杨剑波，吴跃进，吴李君等，低能离子束介导外源基因进入水稻细胞研究，安徽农业大学学报，1994，21(3)330～335．
22．郑卫红，硕士论文，离子束介导烟草转基因的研究。1998。中科院合肥等离子体物理所。
23．李红，博士论文，1999。低能离子束介导水稻转基因体系的研究。中科院合肥等离子体物理所。
24．吴跃进，博士学位论文，离子注入水稻生物学效应和基因转导若干研究，1993．
25．吴敬德等，玉米稻的光和效率分析。安徽农业科学，1997，25(2)；112-113。
26．程备久，低能离子束注入棉花早期效应及其与遗传变异关系的研究(博士论文)1995
第二章参考文献：
1．何进，梁运祥，浅谈凯氏定氮法，山西粮油学报，1996，21(3)．
2. Williams P, Norris K. Near infrared technology in agriculture and food industries. United States of America: American Association of Cercal Chemists, 1990:185～315.
3 钟海庆，红外光谱入门，北京：化学工业出版社，1984：1～40．
4．杨曙明，张瑜，近红外光谱分析技术及其在饲料质量分析中的应用，分析测试仪器通讯，No．4，215．
5. Marten H, T Naes. Multivariate Calibration by Data Compression. In Near infrared Technology in the Agriculture and Food Industry, Milliams, P. and Norris, K. H (Edts.), St. Paul. 1987.234.
6. Norris K H. Predicting Forage Quality by Infrared Reflectance Spectroscopy. J Anim Sci. 1976, 43:4.
7．许俐，陈斌，近红外分析技术在食品和农产品领域中的应用，农业机械学报，1997．8．Vol．28．
8. Mnzana N.A. 1990. Studies on tropical vegetables. 2. Amino and fatty acid composition in seed of cleome (Gynandropsis gynandra L. Briq) selections from Zambia. Food Chemistry. 35:287-293.
9. Pazdernik. D.L., A.S. Killam., and J.H. Orf. 1997. Analysis of Amino and Fatty Acid Composition in Soybean Seed, Using Near Infrared Reflectance Spectroscopy. Agron. J. 89:679-685.
10 王志敏，小麦籽粒蛋白质贮积的生理学研究进展，国外农学-麦类作物，1996年第四期．
11 王宪泽，田纪春，张忠义，不同品种小麦籽粒蛋白质及其组分积累规律的研究，山东农业大学学报，Vol．25，No 4．
12 蒋纪芸，张保军，硬粒小麦4286籽粒蛋白质及其组分的发育规律及调控技术，国外农学-麦类作物，1995，No．3．
13 彭玉魁，李菊英等，近红外光谱分析技术在小麦营养成分鉴定上的应用，1997．Vol．17，No．2．
第三章参考文献：
1. Ohtal Y, High-efficiency genetic transformation of maize by a mixture of pollen and exogenous DNA, Proc Natl Acad Sci, 1986,83;715-719.
2．黄骏麒，钱思颖等，外源海岛棉DNA导致陆地棉性状变异，遗传学报，1981，8(1)；56-62。
3．黄骏麒等，外源抗枯萎病DNA导入感病棉的抗性转移，遗传学报，1986，(3)；22-26。
4. Hess D., etal. Transformation experiments by pipeting agrobacterium into the spikelets of wheat (Triticum aestivum 1) Plant Science. 1990,72;233-244.
5. Snape J T etal. The use of irradiated pollen for differential gene transfer in wheat (Triticum aestivum L.) Theor. Appl, Genet, 1983,33; 195-201.
6. Horst Lorz, Barara Baker, Jeff Schell, Gene transfer to cereal cells mediated by protoplast transformation, Mol. Gen. Genet. 1985,199; 178-182.
7．吴跃进，博士学位论文，离子注入水稻生物学效应和基因转导若干研究，1993．
8．吴敬德等，玉米稻的光和效率分析。安徽农业科学，1997，25(2)；112-113。
9．吴丽芳，低能离子束介导转基因小麦研究，博士论文，1999。中科院合肥等离子体物理所。
10. Jeff J, Doyle, Jane I, Doyle. Isolation of plant DNA from fresh tissue. Focus, 1998, 12(1): 13～15.
11. Murray M G, Thompson W F. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 1980, 8(19): 4321～4325.
1．王关林，方宏钧，植物基因工程原理与技术，1998，科学出版社。
2. Abdnl-Baki A A, etal. Plant Report, 1990,70:181～190.
3. Matthews B F, etal. Sex Plant Report, 1990,3:147～151.
4. Dekeyser R, A, etal. Plant Cell, 1990, 2;591-602.
5. Zaghmout O M F, etal. Nucleic Acid Res, 1993, 21; 1048.
6. Draper J, etal Plant Cell, Tissue and Organ Culture, 1995,40:1～15.
7. Tao W etal. Proc Natl. Acad. Sci, 1987, USA, 84; 1840-1845.
8. Tsukakoshi M etal. Appl, Phys. B, 1984, 35; 135-140.
9．王兰岚，宋桂英，张健等。激光生物学报，1993，2(2)：281。
10．王兰岚，宋桂英，周奕华等。激光生物学报，1993，2(2)；、279-280。
11．王兰岚，傅荣着，宋桂英等，利用激光微束穿刺法将外源基因导入小麦的研究。遗传学报，1995，22(5)；394-399。
12. Vasil V etal. Bio/Technology, 1992,10;667-674.
13. Vasil V etal. Bio/Technology, 1993,11;1553-1558.
14．傅荣着，曹光诚，马汉生等，用基因枪法将人工雄性不育基因导入小麦的初步研究，遗传学报，1997，24(4)；358-361。
15．王亚馥，崔凯荣，陈克明等，玉米DNA导入小麦后引起白化突变体的质粒超螺旋结构的研究。农业分子育种研究进展，1993，中国农业科技出版社。
16．倪建福，周文麟，仲乃琴等。偃麦草DNA导入小麦的研究。农业分子育种研究进展，1993，中国农业科技出版社，76-80。
17．吴兆苏，小麦育种学，北京：中国农业出版社，1990．
18．余增亮，离子束生物技术引论。安徽科技出版社。
19．余增亮等，离子刻蚀生物样品的初步研究。安徽农业大学学报，1994，3；260-264。
20．韩建伟，余增亮，低能离子对番茄果皮刻蚀与穿透作用的研究，生物物理学报，1998，Vol，14，No；757-761
21．杨剑波，吴跃进，吴李君等，低能离子束介导外源基因进入水稻细胞研究，安徽农业大学学报，1994，21(3)330～335．
22．郑卫红，硕士论文，离子束介导烟草转基因的研究。1998。中科院合肥等离子体物理所。
23．李红，博士论文，1999。低能离子束介导水稻转基因体系的研究。中科院合肥等离子体物理所。
24．吴跃进，博士学位论文，离子注入水稻生物学效应和基因转导若干研究，1993．
25．吴敬德等，玉米稻的光和效率分析。安徽农业科学，1997，25(2)；112-113。
26．程备久，低能离子束注入棉花早期效应及其与遗传变异关系的研究(博士论文)1995
第二章参考文献：
1．何进，梁运祥，浅谈凯氏定氮法，山西粮油学报，1996，21(3)．
2. Williams P, Norris K. Near infrared technology in agriculture and food industries. United States of America: American Association of Cercal Chemists, 1990:185～315.
3 钟海庆，红外光谱入门，北京：化学工业出版社，1984：1～40．
4．杨曙明，张瑜，近红外光谱分析技术及其在饲料质量分析中的应用，分析测试仪器通讯，No．4，215．
5. Marten H, T Naes. Multivariate Calibration by Data Compression. In Near infrared Technology in the Agriculture and Food Industry, Milliams, P. and Norris, K. H (Edts.), St. Paul. 1987.234.
6. Norris K H. Predicting Forage Quality by Infrared Reflectance Spectroscopy. J Anim Sci. 1976, 43:4.
7．许俐，陈斌，近红外分析技术在食品和农产品领域中的应用，农业机械学报，1997．8．Vol．28．
8. Mnzana N.A. 1990. Studies on tropical vegetables. 2. Amino and fatty acid composition in seed of cleome (Gynandropsis gynandra L. Briq) selections from Zambia. Food Chemistry. 35:287-293.
9. Pazdernik. D.L., A.S. Killam., and J.H. Orf. 1997. Analysis of Amino and Fatty Acid Composition in Soybean Seed, Using Near Infrared Reflectance Spectroscopy. Agron. J. 89:679-685.
10 王志敏，小麦籽粒蛋白质贮积的生理学研究进展，国外农学-麦类作物，1996年第四期．
11 王宪泽，田纪春，张忠义，不同品种小麦籽粒蛋白质及其组分积累规律的研究，山东农业大学学报，Vol．25，No 4．
12 蒋纪芸，张保军，硬粒小麦4286籽粒蛋白质及其组分的发育规律及调控技术，国外农学-麦类作物，1995，No．3．
13 彭玉魁，李菊英等，近红外光谱分析技术在小麦营养成分鉴定上的应用，1997．Vol．17，No．2．
第三章参考文献：
1. Ohtal Y, High-efficiency genetic transformation of maize by a mixture of pollen and exogenous DNA, Proc Natl Acad Sci, 1986,83;715-719.
2．黄骏麒，钱思颖等，外源海岛棉DNA导致陆地棉性状变异，遗传学报，1981，8(1)；56-62。
3．黄骏麒等，外源抗枯萎病DNA导入感病棉的抗性转移，遗传学报，1986，(3)；22-26。
4. Hess D., etal. Transformation experiments by pipeting agrobacterium into the spikelets of wheat (Triticum aestivum 1) Plant Science. 1990,72;233-244.
5. Snape J T etal. The use of irradiated pollen for differential gene transfer in wheat (Triticum aestivum L.) Theor. Appl, Genet, 1983,33; 195-201.
6. Horst Lorz, Barara Baker, Jeff Schell, Gene transfer to cereal cells mediated by protoplast transformation, Mol. Gen. Genet. 1985,199; 178-182.
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