黑林1号杨转双价抗虫基因的研究
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摘要
黑林1号杨((P. simonii×P. nigra)×P.15ACL)是以小黑杨为母本、波兰15A为父本的人工杂种。该品种(系)根系发达、生长迅速、抗旱耐寒、适应性强,已成为黑龙江省等东北地区推广的优良杨树品种。但杨树害虫一直是影响其生长的主要生物因素,给林业生产、城市绿化和生态建设造成严重的威胁和巨大的经济损失。基因工程育种手段将外源抗虫基因导入林木,培育抗虫性新品种为林木害虫可持续防治提供了一条新途径。本研究以黑林1号杨为受体材料,通过根癌农杆菌介导的叶盘转化法,将蜘蛛杀虫肽+Bt毒蛋白的嵌合基因导入受体细胞中,获得具有抗虫能力的优良抗性树种。主要研究结果如下：
1.建立了黑林1号杨最佳组培扩繁体系,以WPM+IBA 0.2mg/L+20g/L蔗糖+5.6g/L琼脂(pH5.6)为生根培养基,以MS+6-BA 0.3mg/L+NAA 0.1mg/L+20g/L蔗糖+5.6g/L琼脂(pH 5.6)为叶片分化培养基,对黑林1号杨进行组织培养及扩繁。
2.通过黑林1号杨抗生素敏感性和头孢噻肟钠对农杆菌生长影响的检测,综合分析后确定了叶片转化过程中卡那霉素和头孢噻肟钠的适宜浓度分别为15mg/L和600mg/L；在抗性芽茎段生根筛选阶段,生根培养基中卡那霉素和头孢噻肟钠的适宜浓度分别为25mg/L和700mg/L。
3.建立了农杆菌介导蜘蛛杀虫肽+Bt毒蛋白的嵌合基因转化的黑林1号杨优化遗传转化体系：预培养时间为3d、菌液浓度为OD600=0.1、侵染时间为3min、共培养基中乙酰丁香酮的浓度为200μmol/L,共培养3d,延后选择培养的适宜时间为3d。
4.以生长约20d左右的黑林1号杨健康组培苗叶片为受体材料,利用优化的遗传转化体系对638个叶片进行农杆菌介导,获得8株抗性芽。通过PCR分子检测,有3株抗性芽出现了特异性扩增条带,阳性率为37.5%。通过PCR-Southern印迹杂交的进一步分子检测,3株均出现了特异性的杂交条带,转化率为0.47%,进一步证明了,目的基因已经整合到黑林1号杨基因组内。
(P. simonii X P. nigra) X P.15ACL is Artificial hybrid, of which female parent is populus simonii and male parent is populus nigra. This varietie (line) is characterized by well-developed root system, rapid growth, drought and cold endurable and strong adaptablity. It has become an excellent poplar lines widely planted in the Northeast of China such as Heilongjiang province. However, the poplar pests, the main biological factors affecting poplar growth, have caused a serious threat and the huge economic losses in forestry production, construction of urban greening and ecology. Some insect-resistant genes have been transformed into trees successfully by genetic engineering, which offers a new approach for sustainable forest pest control. In this stury, using an Agrobacterium mediated method, a foreign gene fused by insecticidal peptide of spider and the C-terminal of of Bt ICP gene was transformated into (P. simonii×P. nigra)×P.15ACL.
1. An efficient tissue culture system for (P. simonii×P. nigra)×P.15ACL was established. The rooting culture medium was WPM+0.2mg/L IBA+20g/L saccharose+5.6g/L agar (pH5.6). The leaf differentiation culture medium was MS+0.3mg/L 6-BA+0.1mg/L NAA+ 20g/L saccharose+5.6g/L agar (pH 5.6).
2. The appropriate concentrations of Kanamycin and Cefotaxime in transformation were 15mg/L and 600 mg/L for leaf discs culture and 25mg/L and 700mg/L for rooting culture, respectively, determined by the antibiotics sensitivity test for the poplar and the bacteriostatic test of Cefotaxime sodium on Agrobacterium.
3. A genetic transformation system for (P. simonii×P. nigra)×P.15ACL was established, which included:pre-cultured 2-3d, OD600=0.1-0.2, incubated 3 min, co-cultured 2-3d and the co-cultivation medium plus acetosyringone 200μmol/L was the best combination, carry-forward selection-cultured 3d.
4. A total of 638 healthy leaf discs from 20-day old cultured plantlet were used for Agrobacterium tumfaciens-mediated transformation. As a ruslut,8 shoots with resistance to Kanamycin were obtained. PCR assay showed that 3 of them gave the predicted DNA fragment band, with a transformation rate of 0.47%. PCR-Southern analysis also confirm the three possitive samples. these result showed that the insect-resistant gene has integrated into the genome of (P. simonii×P. nigra)×P.15ACL.
1.建立了黑林1号杨最佳组培扩繁体系,以WPM+IBA 0.2mg/L+20g/L蔗糖+5.6g/L琼脂(pH5.6)为生根培养基,以MS+6-BA 0.3mg/L+NAA 0.1mg/L+20g/L蔗糖+5.6g/L琼脂(pH 5.6)为叶片分化培养基,对黑林1号杨进行组织培养及扩繁。
2.通过黑林1号杨抗生素敏感性和头孢噻肟钠对农杆菌生长影响的检测,综合分析后确定了叶片转化过程中卡那霉素和头孢噻肟钠的适宜浓度分别为15mg/L和600mg/L；在抗性芽茎段生根筛选阶段,生根培养基中卡那霉素和头孢噻肟钠的适宜浓度分别为25mg/L和700mg/L。
3.建立了农杆菌介导蜘蛛杀虫肽+Bt毒蛋白的嵌合基因转化的黑林1号杨优化遗传转化体系：预培养时间为3d、菌液浓度为OD600=0.1、侵染时间为3min、共培养基中乙酰丁香酮的浓度为200μmol/L,共培养3d,延后选择培养的适宜时间为3d。
4.以生长约20d左右的黑林1号杨健康组培苗叶片为受体材料,利用优化的遗传转化体系对638个叶片进行农杆菌介导,获得8株抗性芽。通过PCR分子检测,有3株抗性芽出现了特异性扩增条带,阳性率为37.5%。通过PCR-Southern印迹杂交的进一步分子检测,3株均出现了特异性的杂交条带,转化率为0.47%,进一步证明了,目的基因已经整合到黑林1号杨基因组内。
(P. simonii X P. nigra) X P.15ACL is Artificial hybrid, of which female parent is populus simonii and male parent is populus nigra. This varietie (line) is characterized by well-developed root system, rapid growth, drought and cold endurable and strong adaptablity. It has become an excellent poplar lines widely planted in the Northeast of China such as Heilongjiang province. However, the poplar pests, the main biological factors affecting poplar growth, have caused a serious threat and the huge economic losses in forestry production, construction of urban greening and ecology. Some insect-resistant genes have been transformed into trees successfully by genetic engineering, which offers a new approach for sustainable forest pest control. In this stury, using an Agrobacterium mediated method, a foreign gene fused by insecticidal peptide of spider and the C-terminal of of Bt ICP gene was transformated into (P. simonii×P. nigra)×P.15ACL.
1. An efficient tissue culture system for (P. simonii×P. nigra)×P.15ACL was established. The rooting culture medium was WPM+0.2mg/L IBA+20g/L saccharose+5.6g/L agar (pH5.6). The leaf differentiation culture medium was MS+0.3mg/L 6-BA+0.1mg/L NAA+ 20g/L saccharose+5.6g/L agar (pH 5.6).
2. The appropriate concentrations of Kanamycin and Cefotaxime in transformation were 15mg/L and 600 mg/L for leaf discs culture and 25mg/L and 700mg/L for rooting culture, respectively, determined by the antibiotics sensitivity test for the poplar and the bacteriostatic test of Cefotaxime sodium on Agrobacterium.
3. A genetic transformation system for (P. simonii×P. nigra)×P.15ACL was established, which included:pre-cultured 2-3d, OD600=0.1-0.2, incubated 3 min, co-cultured 2-3d and the co-cultivation medium plus acetosyringone 200μmol/L was the best combination, carry-forward selection-cultured 3d.
4. A total of 638 healthy leaf discs from 20-day old cultured plantlet were used for Agrobacterium tumfaciens-mediated transformation. As a ruslut,8 shoots with resistance to Kanamycin were obtained. PCR assay showed that 3 of them gave the predicted DNA fragment band, with a transformation rate of 0.47%. PCR-Southern analysis also confirm the three possitive samples. these result showed that the insect-resistant gene has integrated into the genome of (P. simonii×P. nigra)×P.15ACL.
引文
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[5]喻子牛主编.苏云金芽孢杆菌[M].北京：科学出版社,1990：229-247
[6]SCHNEPF H F.Cloning and expression of the bacillus thuringiensis crystalprotein gene in E.coli[J].Proc Natl Acad Sci,1981,78:2893-2897
[7]蒋红,朱玉贤,王雅平,等.蜘蛛杀虫肽基因的合成及其在植物表达质粒的构建[J].植物学报,1995,37：321-325
[8]蒋红,朱玉贤,陈章良.导入蜘蛛杀虫肽基因的烟草具有抗虫性[J].植物学报,1996,38：95-99
[9]Mccown BH, Mccabe DE,Russell DR, et al.St able transformation of Populus and incorporation of pest resistance by electric discharge particle acceleration[J].Plant Call Reports,1991,9:590-594
[10]Wang-GL, et al.Poplar plants transformed with a Bacillus thuringiensis toxin gene inseeticidal activity and genomic analysis.Transgenic Research,1996,5(5):281-301
[11]Klopfenstein-NB, et al.TransgenicPopulus hybrid expresses a wound inducible.Potato-proteinase inhibitor Ⅱ CAT gene fusion.Tho rnburg,1991,21:1321-1328
[12]Wang-GL, et al.Poplar plants transformed with a Bacillus thuringiensis toxin gene inseeticidal activity and genomic analysis.Transgenic Research,1996,(5):281-301
[13]陈颖,韩一凡,等.苏云金杆菌杀虫晶体蛋白基因转化美洲黑杨的研究[J].林业科学,1995,21(2)：97-103
[14]姜静,常玉广,董京祥,等.小黑杨转双价抗虫基因的研究[J].植物生理学通讯2004,40(6),669-673
[15]熊忠平,孟繁君,王志英,等.转基因小黑杨对舞毒蛾的抗性测定[J].林业勘查设计,2006,(2)77-79
[16]常团结,朱祯.植物凝集素及其在抗虫植物基因工程中的应用.遗传,2002,24(4)：493-500
[17]饶红宇,黄敏仁.杨树基因工程研究的现状及展望[J].林业科技开发,1999,4：3-6
[18]朱新生,朱玉贤.抗虫植物基因工程研究进展[J].植物学报,1997,39(3)：282-288
[19]陈英,何秋伶,诸葛强,等.林木基因工程研究进展.分子植物育种,2006,4(1)：1-7
[20]Hilder V.A., Gatehouse A.M.R., Sheerman S.E., et al.A novel mechanism of insect resistance engineered into tobacco.Nature,1987,330:160-163
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