rolB-pttGA20ox双价基因转化毛白杨及遗传稳定性研究
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摘要
毛白杨(Populus tomentosa Carr.)是我国特有的优良白杨派乡土树种,具有材质优良、寿命长、抗性和适应性强等特点,是我国北方地区常用的建筑、家具用材和造纸工业原料树种,对美化环境和保持水土也有重要作用。但毛白杨的插条生根较难、扦插成活率低,在很大程度上阻碍了这一优良树种的推广和产业化进程。本研究通过农杆菌介导的叶盘转化方法,将双价基因rolB-pttGA20ox转入毛白杨优良无性系PT-16和PT-19基因组中,开展利用基因工程育种技术改良生根和高生长性状的研究;并以转rolB-pttGA20ox双价基因烟草为试材,探讨了双价外源基因在转化植株中的表达稳定性和遗传特性。主要研究结果如下:
1、优化了农杆菌介导的毛白杨遗传转化体系。首先确定毛白杨PT-16和PT-19叶片外植体不定芽诱导卡那霉素(Kan)临界耐受浓度为25 mg/L,试管苗不定根再生Kan临界敏感浓度为20 mg/L。高效的毛白杨叶盘转化方法为:叶盘预培养2d→含有200μmol/L AS的分化培养基(pH为5.0)接触培养4 h→OD_(600)=0.3～0.4的农杆菌菌液侵染25 min→含200μmol/L AS的共培养培养基(pH为5.0,不含CoCl_2·6H_2O)5 d→延迟选择培养3 d。在此优化的转化体系下,Kan~r芽再生率可达16%。胭脂碱型C58菌株介导基因转化毛白杨的能力明显高于章鱼碱型LBA4404菌株。
2、转rolB-pttGA20ox双价基因毛白杨的获得及分子检测。采用优化的农杆菌介导叶盘转化方法再生的Kan~r芽,经含有30 mg/L Kan的生根培养基和叶片分化培养基筛选,获得了29株初步判定已导入外源基因的转化植株。进行PCR检测阳性率为65.52%;Southern杂交证明外源基因已插入毛白杨基因组中,且均为单拷贝形式整合。进一步通过RT-PCR初步证明了外源基因在转化植株各器官中的表达,rolB基因具有特异性,表达强弱顺序为根＞茎＞叶;pttGA20ox基因在各器官中的表达丰度一致。
3、鉴定了目的基因在转基因毛白杨中的表达特性。转化植株的组织细胞表现出对生长素的高敏感性,在低浓度生长素条件下表现出明显高于对照的生根能力。外源生长素的短时间诱导即可调控rolB基因的表达丰度;而pttGA20ox未受到外源生长素的诱导。转化植株各部位的GA_3、IAA和ZR三种内源激素含量与对照差异明显。
4、转基因毛白杨的形态表现出rolB和pttGA20ox两个基因所具有的功能特征。移栽温室4个月时,转化植株的生根状况明显优于对照,表现为侧根发达、毛状根众多,茎基部始生根位置上升约4cm,整个根系的根数多到难于统计,主根平均长度为43.3 cm,比对照长12.7 cm;转化植株的平均株高达到对照的2.18倍。
5、以转rolB-pttGA20ox双价基因烟草为材料,验证了目的基因的稳定表达。内源激素和生长量测定表明rolB-pttGA20ox双价基因在转化植株体内能够稳定存在和表达,没有受到移栽或其他环境改变的影响而诱发基因沉默等现象。
6、证实了rolB-pttGA20ox双价基因在转化植株中的遗传稳定性。随机选取90株播种生长的T_1代转基因烟草,PCR显示其中62株能够扩增出rolB和pttGA20ox两个基因的特异条带,说明外源基因能够在转化植株中稳定遗传;进一步进行x~2检测证明外源基因的分离符合3:1的孟德尔式遗传分离比率,推测其是以单位点插入转化烟草基因组中的。生长指标和内源激素含量的动态测定结果均表明双价基因在T_1代转化植株中得到了表达。
Chinese white poplar(Populus tomentosa Carr.)is a native tree species of section Leuce which is widely employed for forest production,forestation and ecological environment construction in the north of China.However,the difficulty of rooting in hardwood cuttings has seriously hindered the practical and industrial process of P.tomentosa.The major objective of this study was to breed new P.tomentosa varieties with improved rooting ability and apical dominance through Agrobacterium tumefaciens-mediate transformation of rolB-pttGA20ox double genes.Meanwhile,the inheritance and expression pattern about rolB-pttGA20ox double genes were also studied by transgenic tobacco.The major results of this study were introduced as follows:
1)Optimization of A.tumefaciens-mediated genetic transformation system.
Kanamycin(Kan)-resistance test shows that the critical kanamycin sensitive concentrations for inducing shoots and roots of P.tomentosa(PT-16 and PT-19)were 25 and 20 mg/L respectively.The effective genetic transformation process was as follows:pre-culture of leaf discs for 2 d→culture of leaf discs on the regeneration medium containing 200μmol/L AS(pH 5.0)for 4 h→immersed in the agrobacterium solution(OD_(600)=0.3-0.4)for 25 min→culturing leaf discs on the co-culture medium containing 200μmol/L AS(pH 5.0,without COCl_2·6H_2O)for 5 d→initial application of Kan selection culture for 3 d.The Kan-resistance shoot regeneration rate reached to 16%with the optimized transformation system.The infection ability of nopaline strain C58 with P.tomentosa was better than that of octopine strain LBA4404.
2)Molecular detection oftransgenic poplar.
Twenty-nine transgenic plantlets with Kan resistance were obtained through identification of rooting and inducing shoots ability on rooting medium and regeneration medium containing 30 mg/L Kan.The 65.52%positive rate was indicated by PCR analysis.Southern blotting shows that the target genes were integrated into the poplar genome and the integration mode was single-copy.Tissue-specific expression indicates that expression levels of rolB gene were root＞stem＞leaf,and pttGA20ox gene was expressed similarly in different parts of transgenic plantlets.
3)Demonstration of the transgene expression in transgenic poplar.
The transgenic plantlets exhibited a high sensitivity to auxin and possessed remarkably higher rooting ability compared to control at low auxin levels,rolB gene could be regulated by a short-period induction of exogenous auxin,whereas pttGA20ox gene was not regulated by exogenous auxin.The content of endogenous hormones(GA_3,IAA and ZR)in the transgenic plantlets were significantly different from control.
4)Characterization of the function of rolB-pttGA20ox double genes in transgenic poplar.
Phenotypic measurement shows that the amount and the length of roots were remarkably higher than those of the control.The average taproot length of transgenic plantlets was 43.3 cm,which was 12.7 cm longer than control.The average height of transgenic plantlets was 2.18 times as large as that of the control.
5)Validation of the stable expression of rolB-pttGA20ox double genes in transgenic tobacco.
The physiological detection shows that the target genes could steadily express in transgenic tobacco.The environment variations,such as transplantation,did not induce the silencing of transformed genes.
6)Demonstration of the genetic stability of rolB-pttGA20ox double genes in transgenic tobacco.
PCR screening of T_1 plantlets shows the stable inheritance of genes in the transgenic plantlets.The segregation ratio between transgenic versus non-transgenic plants was observed to be 3:1,which conformed to the Mendelian Law of single gene segregation.This also suggests that there was one insertion locus of transgene in the genome of transgenic tobacco.Physiological detection indicates that rolB-pttGA20ox double genes were expressed in T_1 transgenic tobacco.
1、优化了农杆菌介导的毛白杨遗传转化体系。首先确定毛白杨PT-16和PT-19叶片外植体不定芽诱导卡那霉素(Kan)临界耐受浓度为25 mg/L,试管苗不定根再生Kan临界敏感浓度为20 mg/L。高效的毛白杨叶盘转化方法为:叶盘预培养2d→含有200μmol/L AS的分化培养基(pH为5.0)接触培养4 h→OD_(600)=0.3～0.4的农杆菌菌液侵染25 min→含200μmol/L AS的共培养培养基(pH为5.0,不含CoCl_2·6H_2O)5 d→延迟选择培养3 d。在此优化的转化体系下,Kan~r芽再生率可达16%。胭脂碱型C58菌株介导基因转化毛白杨的能力明显高于章鱼碱型LBA4404菌株。
2、转rolB-pttGA20ox双价基因毛白杨的获得及分子检测。采用优化的农杆菌介导叶盘转化方法再生的Kan~r芽,经含有30 mg/L Kan的生根培养基和叶片分化培养基筛选,获得了29株初步判定已导入外源基因的转化植株。进行PCR检测阳性率为65.52%;Southern杂交证明外源基因已插入毛白杨基因组中,且均为单拷贝形式整合。进一步通过RT-PCR初步证明了外源基因在转化植株各器官中的表达,rolB基因具有特异性,表达强弱顺序为根＞茎＞叶;pttGA20ox基因在各器官中的表达丰度一致。
3、鉴定了目的基因在转基因毛白杨中的表达特性。转化植株的组织细胞表现出对生长素的高敏感性,在低浓度生长素条件下表现出明显高于对照的生根能力。外源生长素的短时间诱导即可调控rolB基因的表达丰度;而pttGA20ox未受到外源生长素的诱导。转化植株各部位的GA_3、IAA和ZR三种内源激素含量与对照差异明显。
4、转基因毛白杨的形态表现出rolB和pttGA20ox两个基因所具有的功能特征。移栽温室4个月时,转化植株的生根状况明显优于对照,表现为侧根发达、毛状根众多,茎基部始生根位置上升约4cm,整个根系的根数多到难于统计,主根平均长度为43.3 cm,比对照长12.7 cm;转化植株的平均株高达到对照的2.18倍。
5、以转rolB-pttGA20ox双价基因烟草为材料,验证了目的基因的稳定表达。内源激素和生长量测定表明rolB-pttGA20ox双价基因在转化植株体内能够稳定存在和表达,没有受到移栽或其他环境改变的影响而诱发基因沉默等现象。
6、证实了rolB-pttGA20ox双价基因在转化植株中的遗传稳定性。随机选取90株播种生长的T_1代转基因烟草,PCR显示其中62株能够扩增出rolB和pttGA20ox两个基因的特异条带,说明外源基因能够在转化植株中稳定遗传;进一步进行x~2检测证明外源基因的分离符合3:1的孟德尔式遗传分离比率,推测其是以单位点插入转化烟草基因组中的。生长指标和内源激素含量的动态测定结果均表明双价基因在T_1代转化植株中得到了表达。
Chinese white poplar(Populus tomentosa Carr.)is a native tree species of section Leuce which is widely employed for forest production,forestation and ecological environment construction in the north of China.However,the difficulty of rooting in hardwood cuttings has seriously hindered the practical and industrial process of P.tomentosa.The major objective of this study was to breed new P.tomentosa varieties with improved rooting ability and apical dominance through Agrobacterium tumefaciens-mediate transformation of rolB-pttGA20ox double genes.Meanwhile,the inheritance and expression pattern about rolB-pttGA20ox double genes were also studied by transgenic tobacco.The major results of this study were introduced as follows:
1)Optimization of A.tumefaciens-mediated genetic transformation system.
Kanamycin(Kan)-resistance test shows that the critical kanamycin sensitive concentrations for inducing shoots and roots of P.tomentosa(PT-16 and PT-19)were 25 and 20 mg/L respectively.The effective genetic transformation process was as follows:pre-culture of leaf discs for 2 d→culture of leaf discs on the regeneration medium containing 200μmol/L AS(pH 5.0)for 4 h→immersed in the agrobacterium solution(OD_(600)=0.3-0.4)for 25 min→culturing leaf discs on the co-culture medium containing 200μmol/L AS(pH 5.0,without COCl_2·6H_2O)for 5 d→initial application of Kan selection culture for 3 d.The Kan-resistance shoot regeneration rate reached to 16%with the optimized transformation system.The infection ability of nopaline strain C58 with P.tomentosa was better than that of octopine strain LBA4404.
2)Molecular detection oftransgenic poplar.
Twenty-nine transgenic plantlets with Kan resistance were obtained through identification of rooting and inducing shoots ability on rooting medium and regeneration medium containing 30 mg/L Kan.The 65.52%positive rate was indicated by PCR analysis.Southern blotting shows that the target genes were integrated into the poplar genome and the integration mode was single-copy.Tissue-specific expression indicates that expression levels of rolB gene were root＞stem＞leaf,and pttGA20ox gene was expressed similarly in different parts of transgenic plantlets.
3)Demonstration of the transgene expression in transgenic poplar.
The transgenic plantlets exhibited a high sensitivity to auxin and possessed remarkably higher rooting ability compared to control at low auxin levels,rolB gene could be regulated by a short-period induction of exogenous auxin,whereas pttGA20ox gene was not regulated by exogenous auxin.The content of endogenous hormones(GA_3,IAA and ZR)in the transgenic plantlets were significantly different from control.
4)Characterization of the function of rolB-pttGA20ox double genes in transgenic poplar.
Phenotypic measurement shows that the amount and the length of roots were remarkably higher than those of the control.The average taproot length of transgenic plantlets was 43.3 cm,which was 12.7 cm longer than control.The average height of transgenic plantlets was 2.18 times as large as that of the control.
5)Validation of the stable expression of rolB-pttGA20ox double genes in transgenic tobacco.
The physiological detection shows that the target genes could steadily express in transgenic tobacco.The environment variations,such as transplantation,did not induce the silencing of transformed genes.
6)Demonstration of the genetic stability of rolB-pttGA20ox double genes in transgenic tobacco.
PCR screening of T_1 plantlets shows the stable inheritance of genes in the transgenic plantlets.The segregation ratio between transgenic versus non-transgenic plants was observed to be 3:1,which conformed to the Mendelian Law of single gene segregation.This also suggests that there was one insertion locus of transgene in the genome of transgenic tobacco.Physiological detection indicates that rolB-pttGA20ox double genes were expressed in T_1 transgenic tobacco.
引文
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