授粉后外源DNA导入技术在春小麦抗条锈病育种中的应用
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摘要
为了从小麦亲缘种属中向小麦引进抗条锈病基因,解决普通小麦内抗病基因资源贫乏,现有抗源抗性不够理想的难题,本研究利用花粉管通道途径在授粉后3-6小时内将燕麦总DNA导入到宁夏主栽品种宁春4号小麦中,创造变异材料,将燕麦的抗条锈病基因导入到普通小麦中,为小麦育种提供抗条锈病的新种质。
1、选用4种DNA提取方法,并进行比较,发现4种提取方法所得的DNA纯度基本均符合导入的质量标准;DNA的浓度也达到分子育种所要求的不能低于200μg/ml的要求;不同的植物采取不同的DNA提取方法,其DNA的提取率不同;植物种类与提取方法互作效应的方差分析表现为极显著。
2、对供体、受体、导入后代各变异品系在宁夏当地进行连续多年(1998-2004)的成株期混合菌种的抗病性鉴定和在陕西杨凌两年(1998-1999)的分生理小种鉴定。结果显示:供体对小麦条锈菌免疫,受体对小麦条锈菌各生理小种严重感染,共筛选获得对条锈病抗性较好品系5个。这些结果证明供体燕麦上可能携带抗小麦条锈菌的新基因,并已通过花粉管通道进入小麦,整合到小麦的染色体组中,稳定表达。
3、燕麦总DNA导入宁春4号小麦后,导入后代从外部形态、生理生化、品质等多方面产生变异。导入后代多数株高略高于受体;单株叶面积普遍比受体的叶面积增大5～8cm~2;有的变异品系表现出供体材料的蓝绿色特征,叶片变厚变硬。结实小穗数增加5～6个,主穗粒数增加10～20粒;籽粒由卵圆形变为椭圆形,腹沟加深,有的籽粒颜色由红变白;有些变异品系千粒重增加3～4g。方差分析表明,各变异品系的主要农艺性状除生物产量外都存在着差异。
4、对供体、受体及其导入后代的变异品系进行酯酶同工酶和过氧化物酶同工酶分析表明:变异后代与亲本的谱带有明显差异,外观性状有变异的品系,一般都伴随有同工酶谱的变异,无论是酯酶同工酶还是过氧化物酶同工酶,均出现了受体没有而供体特有的谱带:在过氧化物酶同工酶中既出现了供、受体均不具有的新的杂交酶带,有的还显现出偏父或偏母以及完全互补型的酶带;酶的活性也发生了很大的变化。
5、在对宁春4号小麦进行燕麦总DNA导入的同时,也用紫麦的总DNA,对宁春4号小麦进行导入,于D~2代出现一株紫杆紫粒的变异株,其植株高于宁春4号小麦,但又低于紫麦,粒色略淡于紫麦,第二年将这变异株种成株行,并按单株收获,发现这两个变异性状均能遗传,表明紫麦DNA已导入到小麦中,由此证明DNA导入技术确实可行。
To solving the problems of lacking the resources of resistant gene and not enough ideal resistance materials in wheat, the resistance genes of yellow rust (Puccinia striiformis) from the relative of wheat were introduced into wheat. By taking oat (Avena saliva L.) and common wheat (Ningchun No.4) as donor and recipient, total DNA prepared from oat was introduced into Ningchun No.4 through pollen tubing. After 3-6 hours self-pollination, the yellow rust resistant gene from oat was inoculated into common wheat, therefore creating the new variant strains for common wheat breeding.1. Four different plant DNA extracting methods were compared using common wheat, violet wheat and corn. The results showed that all 4 methods were comparable to the quality standard of DNA with infused gene; the density of DNA also fulfilled the molecular breeding requirements of no less than 200 ug/ml. The amount of extracted DNA was different with different plants by using different extraction methods. The variance analysis indicates that the mutual effects between plant species and extraction methods were highly significant.2. The resistance of donor, recipient and all progenies to yellow rust were identified by inoculating mixed raced with different virulence at adult stage during 1996 -1999. The results showed that the donor was immune, whereas the recipient was seriously susceptible. Six of the progenies were highly resistant to five physiological races of yellow rust. The result also indicated that the new genes resistant to yellow rust may be carried on oat DNA, which have been integrated into the genome of wheat and expressed steadily.3. After introducing oat total DNA into Ningchun No.4, the progenies have many variations such as morphological character, chemophysiology, quality trait etc. Comparing to the recipient, the height of most progenies was higher; The leaf area of plant increased 5-8cm~2; The blade color of some variant strains were green-blue; The blades were thick and hard; The spike length, spikelet number, grain number of main spike and grain weight of main spike were generally increased; 1000-grain weight increased up to 3-4g; The shape of seeds changed from egg circular to oval and their furrow became deeper; The seeds color of some progenies turned into white; The variance analysis indicated that all the agronomic traits of the variant strains except biological yield had changed.
4. The isoenzyme patterns between donor, recipient and their progenies showed obviously difference. The strains having variance in morphological characters all have difference in isoenzyme patterns. There are special bands of donor in the enzyme patterns of progenies of both esterase isoenzyme and isoenzyme peroxidase. Some new hybrid enzyme bands, as well as the tendency towards donor or recipient or complete complementary enzyme bands appeared in the isoenzyme peroxidase and the activities of enzyme also took place great change.5. While introducing oat total DNA into Ningchun No.4, the violet wheat total DNA was also introduced into Ningchun No.4, one variant plant with purple of grain and stem appeared in D2 generation. The height of the plant was far higher than Ningchun No.4's, but a little lower than violet wheat's. The color of grain was pale purple. Second year, the seeds of variant plant were planted one row and harvested with single-plant. Both variant traits can be inheritance, which showed that the DNA of violet wheat had been introduced into wheat, and the technology of exogenous DNA introduction was indeed carried out.6 The Random Amplified Polymorphism DNA (RAPD) analysis showed that three of 64 tested random primers (S1402, S1466, S1468) amplified the polymorphic DNA in variant strains (99-05, 99-07, 99-08), which the same as oat. The Simple Sequence Repeat (SSR) analysis indicated that the random primer xgwml40 of wheat amplified the polymorphic DNA in donor oat, receptor Ningchun No.4, variant strains (99-02, 99-03, 99-07) and their disease resistant and susceptible generations with Ningchun No.4, which showed they were the transformed progenies of oat total DNA into the genome of Ningchun No.4.
1、选用4种DNA提取方法,并进行比较,发现4种提取方法所得的DNA纯度基本均符合导入的质量标准;DNA的浓度也达到分子育种所要求的不能低于200μg/ml的要求;不同的植物采取不同的DNA提取方法,其DNA的提取率不同;植物种类与提取方法互作效应的方差分析表现为极显著。
2、对供体、受体、导入后代各变异品系在宁夏当地进行连续多年(1998-2004)的成株期混合菌种的抗病性鉴定和在陕西杨凌两年(1998-1999)的分生理小种鉴定。结果显示:供体对小麦条锈菌免疫,受体对小麦条锈菌各生理小种严重感染,共筛选获得对条锈病抗性较好品系5个。这些结果证明供体燕麦上可能携带抗小麦条锈菌的新基因,并已通过花粉管通道进入小麦,整合到小麦的染色体组中,稳定表达。
3、燕麦总DNA导入宁春4号小麦后,导入后代从外部形态、生理生化、品质等多方面产生变异。导入后代多数株高略高于受体;单株叶面积普遍比受体的叶面积增大5～8cm~2;有的变异品系表现出供体材料的蓝绿色特征,叶片变厚变硬。结实小穗数增加5～6个,主穗粒数增加10～20粒;籽粒由卵圆形变为椭圆形,腹沟加深,有的籽粒颜色由红变白;有些变异品系千粒重增加3～4g。方差分析表明,各变异品系的主要农艺性状除生物产量外都存在着差异。
4、对供体、受体及其导入后代的变异品系进行酯酶同工酶和过氧化物酶同工酶分析表明:变异后代与亲本的谱带有明显差异,外观性状有变异的品系,一般都伴随有同工酶谱的变异,无论是酯酶同工酶还是过氧化物酶同工酶,均出现了受体没有而供体特有的谱带:在过氧化物酶同工酶中既出现了供、受体均不具有的新的杂交酶带,有的还显现出偏父或偏母以及完全互补型的酶带;酶的活性也发生了很大的变化。
5、在对宁春4号小麦进行燕麦总DNA导入的同时,也用紫麦的总DNA,对宁春4号小麦进行导入,于D~2代出现一株紫杆紫粒的变异株,其植株高于宁春4号小麦,但又低于紫麦,粒色略淡于紫麦,第二年将这变异株种成株行,并按单株收获,发现这两个变异性状均能遗传,表明紫麦DNA已导入到小麦中,由此证明DNA导入技术确实可行。
To solving the problems of lacking the resources of resistant gene and not enough ideal resistance materials in wheat, the resistance genes of yellow rust (Puccinia striiformis) from the relative of wheat were introduced into wheat. By taking oat (Avena saliva L.) and common wheat (Ningchun No.4) as donor and recipient, total DNA prepared from oat was introduced into Ningchun No.4 through pollen tubing. After 3-6 hours self-pollination, the yellow rust resistant gene from oat was inoculated into common wheat, therefore creating the new variant strains for common wheat breeding.1. Four different plant DNA extracting methods were compared using common wheat, violet wheat and corn. The results showed that all 4 methods were comparable to the quality standard of DNA with infused gene; the density of DNA also fulfilled the molecular breeding requirements of no less than 200 ug/ml. The amount of extracted DNA was different with different plants by using different extraction methods. The variance analysis indicates that the mutual effects between plant species and extraction methods were highly significant.2. The resistance of donor, recipient and all progenies to yellow rust were identified by inoculating mixed raced with different virulence at adult stage during 1996 -1999. The results showed that the donor was immune, whereas the recipient was seriously susceptible. Six of the progenies were highly resistant to five physiological races of yellow rust. The result also indicated that the new genes resistant to yellow rust may be carried on oat DNA, which have been integrated into the genome of wheat and expressed steadily.3. After introducing oat total DNA into Ningchun No.4, the progenies have many variations such as morphological character, chemophysiology, quality trait etc. Comparing to the recipient, the height of most progenies was higher; The leaf area of plant increased 5-8cm~2; The blade color of some variant strains were green-blue; The blades were thick and hard; The spike length, spikelet number, grain number of main spike and grain weight of main spike were generally increased; 1000-grain weight increased up to 3-4g; The shape of seeds changed from egg circular to oval and their furrow became deeper; The seeds color of some progenies turned into white; The variance analysis indicated that all the agronomic traits of the variant strains except biological yield had changed.
4. The isoenzyme patterns between donor, recipient and their progenies showed obviously difference. The strains having variance in morphological characters all have difference in isoenzyme patterns. There are special bands of donor in the enzyme patterns of progenies of both esterase isoenzyme and isoenzyme peroxidase. Some new hybrid enzyme bands, as well as the tendency towards donor or recipient or complete complementary enzyme bands appeared in the isoenzyme peroxidase and the activities of enzyme also took place great change.5. While introducing oat total DNA into Ningchun No.4, the violet wheat total DNA was also introduced into Ningchun No.4, one variant plant with purple of grain and stem appeared in D2 generation. The height of the plant was far higher than Ningchun No.4's, but a little lower than violet wheat's. The color of grain was pale purple. Second year, the seeds of variant plant were planted one row and harvested with single-plant. Both variant traits can be inheritance, which showed that the DNA of violet wheat had been introduced into wheat, and the technology of exogenous DNA introduction was indeed carried out.6 The Random Amplified Polymorphism DNA (RAPD) analysis showed that three of 64 tested random primers (S1402, S1466, S1468) amplified the polymorphic DNA in variant strains (99-05, 99-07, 99-08), which the same as oat. The Simple Sequence Repeat (SSR) analysis indicated that the random primer xgwml40 of wheat amplified the polymorphic DNA in donor oat, receptor Ningchun No.4, variant strains (99-02, 99-03, 99-07) and their disease resistant and susceptible generations with Ningchun No.4, which showed they were the transformed progenies of oat total DNA into the genome of Ningchun No.4.
引文
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