【作者】 王广金；

【导师】 李文雄；

【作者基本信息】 东北农业大学 ， 作物栽培学与耕作学， 2002， 博士

【摘要】 通过基因枪法和花粉管通道法，将β-1，3-葡聚糖酶基因导入到品质较好、产量较高，但抗病性差的龙辐3和龙辐10中；将HMW-GS 5+10基因导入到品质较好、产量较高的东农7742、龙辐10和龙辐8及产量较高、适应性广、抗逆性强，但品质差的新克旱九中。目的是选育出高产、抗病、质佳的转基因小麦新品系（品种）。 主要研究结果如下： 对12个基因型的幼胚进行培养效果研究。结果表明，分化率和培养力均存在基因型差异，而各基因型的出愈率差别不大。东农7742、龙辐3和龙6239培养力较高，是较理想的转化试材。三种外植体（幼胚、幼穗和成熟胚）培养效果表明，幼胚、成熟胚的出愈率基本相同，高于幼穗，且差异达极显著。分化率差异较大，高低趋势为幼胚＞幼穗＞成熟胚，培养力大小为幼胚＞成熟胚＞幼穗。可见幼胚是较理想的遗传转化外植体，但选择何种外植体作为试材还应考虑试验条件，取材方便与否等因素。 利用基因枪法将葡聚糖酶基因导入龙辐3和龙辐10，经PCR扩增和RCR-Southern检测，有3株被证实已整合到小麦的基因组中。龙辐3的转化率为0.90％，龙辐10为0.88％。将HMW-GS5+10基因导入东农7742和龙辐10，经PCR检测证明有3株呈阳性，东农7742的转化率为2.63％，龙辐10为1.83％。经PCR检测和PCR-Southern杂交表明转基因龙辐10的T₁代中有3株被证实HMW-GS 5+10基因已整合到小麦基因组中。利用花粉管通道法将葡聚糖酶基因导入到龙辐3和龙辐10中，龙辐3中有3株，龙辐10中有1株经PCR扩增和PCR-Southern检测有与阳性对照相同的杂交带，转化率分别为1.55％和0.53％。另一DNA样本为龙辐10转葡聚糖酶基因形成的品系99K1354，其杂交带与阳性对照相同，证实该品系已将葡聚糖酶基因整合到基因组中。将HMW-GS 5+10基因导入到东农7742、龙辐10和龙辐8中，共获得9株PCR阳性株，经PCR-Southern杂交，这9株均具有与阳性对照相同的杂交带。东农7742、龙辐10、龙辐8的转化率分别为2.27％、2.44％和1.64％，其中21K867是5+10转新克旱九的品系（D₄代），早期世代的选择采用半粒法的PAGE法。 显微压片观察表明：小麦自然授粉3min花粉尚未萌发，5min已开始萌发形成花粉管，20min时在于房顶部可见一些花粉管，30min时在子房内可观察到花粉管，40min花粉管已进入子房深处，有些已接近胚囊，60min时花粉管已开始有胼胝塞。小麦自然授粉不同时间剪掉柱头后的结实率调查结果表明，小麦自然开花20min就开始结实，40min后结实率比较平缓，因此，可以认为导入时间为小麦自然授粉后30-60min为好。 卡那霉素和G418均可以做为标记基因NPTⅡ的筛选剂，但G418的筛选效果要优于卡那霉素，G418的毒性较大，在去掉筛选剂的分化阶段，经G418筛选过的愈伤组织仍有一部分褐化、死亡。G418的适宜筛选浓度为25mg/L，卡那霉素为1000mg/L。对花粉通道法获得的Do代种子进行筛选时卡那霉素优于G418，理由是经卡那霉素处理后，芽鞘及叶片白化，易于区分抗性植株。PPT对愈伤组织的毒性较大，使愈伤组织变褐，甚至其毒性在分化阶段仍起作用。王广金 摘 要 222年n 月 花粉管通道是一种简便易行，成本低廉的转化手段之一，其主要难点是群体量大，筛选困难。本试验进行了花粉管通道法导人目的基因获得DO代种子筛选浓度的研究，并确定 PPT、Kan和 G418的适宜筛选浓度分别为 20mgh、1200mg／L和 50mglL。通过 PPT、Kn和G4筛选可淘汰大部分非转化植株，使检测工作量大大减少，提高花粉管通道法进行小麦转化的效率。 对转葡聚糖酶基因的品系99K 354进行了抗病性鉴定，其白粉病、赤霉病较受体龙辐 10提高 1．2级别，产量提高 3．2％；对转 HMW－GS 5＋10基因的品系 2lK867进行了品质分析，其沉淀值、稳定时间、延伸性和面积较受体新克旱九有较大提高，产量提高5．5％。两个转基因品系除目的基因性状表达外，其它农艺性状也产生了变化，这主要是由于目的基因整合位点的随机性及基因间互作造成的。 对转HMWAIS 5＋10基因的单株及转基因品系2lK867进行SDS。PAGE凝胶电泳检测，高分子麦谷蛋白亚基组成均具有5亚基和10亚基，从蛋白质水平证实了目的基因的表达。转葡聚糖酶基因单株初步抗性鉴定表明，绝大多数转基因植株的白粉病、赤霉病抗性较受体有所提高。更多还原

【Abstract】 B-1, 3-glucanase gene was transferred into cultivars LF3 and LF10 (good quality, high yield but susceptible to disease), and HMW-GS 5+10 genes were delivered into NE7742, LF10 and LF8 (good quality, high yield), and NKH9 (high yield but poor quality) via particle bombardment and pollen tube pathway.The culture effect of embryo in 12 genotypes showed that there was genotype difference in frequency of callus differentiation and culture capacity except the frequency of callus initiation. NE7742, LF3 and L6239 would be the proper materials for gene transform because of the high culture capacity. The different culture effect was found in three types of explants (immature embryo, young spike, and mature embryo) used in the study. However, the frequency of callus initiation in immature embryo and mature embryo was almost the same, but higher than young spike. There was difference in frequency of differentiation among three explants, and the immature embryo ranked the first, young spike next, and mature embryo the last, but immature embryo > mature embryo >young spike when comparing on the basis of cultural capacity. Therefore, immature embryo used as explant in gene transfer would be better than young spike and mature embryo.The result of B-1, 3-glucanase gene transferred into LF3 and LF10 via particle bombardment showed that three transgenic plants with B-1, 3-glucanaes gene integrated have been obtained and confirmed by PCR and PCR-Southem hybridization. The transfer frequencies of LF10 and LF3 were 0.88% and 0.90%, respectively. Three transgenic plants from NE7742 and LF10 that gene HMW-GS 5+10 delivered into were positive and proved by PCR. And the transferred frequencies were 2.63% in NE7742 and 1.83% in LF10. Three of 1 3 T; plants were positive and confirmed by PCR and PCR-Southem hybridization.In gene transformation via pollen tube pathway, B-1, 3-glucanase and HMW-GS 5+10 genes were delivered into LF10, LF3, NE7742, LF8 and XKH9. Five transgenic plants integrated B-1, 3-glucanase gene were obtained and proved by PCR and PCR-Southern hybridization. Four transgenic plants, 3 from LF3 and one from LF10, presented the same hybridization band as positive CK confirmed by PCR and PCR-hybridization. The transferred frequencies were 1.55% in LF3 and 0.53% in LF10. The DNA sample of transgenic line 99K1354 from LF10 was provided with the hybridization band the same as the positive CK. It indicated that gene B-1, 3-glucanase had been integrated into the wheat genome.Nine transgenic plants from NE7742, LF10, LF8 and NKH9 transferred via pollen tube pathway were obtained and confirmed by PCR and PCR-Southern. One of them was line 21K867 (D4) from NKH9 which expressed the integrated HMW-GS 5+10 genes. The line was selected by SDS-PAGE with half kernel in its early generations. The transferred frequencies of NE7742, LF10 and LF8 were 2.27%, 2.44% and 1.64%, respectively.The pollen did not germinate 3 minutes after self-pollination, and started to germinate and formed pollen tube at 5 min. Pollen tubes could be check on the upper part of ovary at 20min and entered into ovary at 30 min, and several approached the embryo sac 40 min after self-pollination. After then, sedimentation of callus was found inside pollen tubes 60 min after pollination. The treatment of stigmas excised after self-pollination showed that wheat started seed setting 20 min after self-pollination. And the seed setting rate tend to stable 40 min after self-pollination. Therefore, it seems that the proper transferring time of foreign gene via pollen tube pathway would be around 30-60 min after self-pollination.Both Kanamycin and Geneticin (G418) can be used as selective regents of mark gene NPTII. However, the selection effect of Geneticin was better when compared with Kanamycin because of the higher drastic toxicity in Geneticin. Even though Geneticin was removed from media, a part of callus treated with Geneticin turned into brown in color and die in continue culture in vitro afterward. The suitable selective concentration of Geneticin an更多还原