苎麻转P_（SAG12）-IPT基因和B_t基因的分子检测及性状考察
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摘要
苎麻是我国传统的纤维作物之一,历史上在我国的农业生产中占有重要地位。随着社会的发展,市场需求的变化,苎麻生产的发展面临诸多挑战,如人口的增加,耕地面积的逐年减少等。因此,提高苎麻单产成为保持苎麻产业可持续发展的有效途径。目前,传统育种方法的局限性已逐步显现。植物基因工程为苎麻育种带来了新的途径。将苎麻导入各种目的基因后,经过选育,有望获得苎麻新品种。
P_(SAG12)-IPT基因能自我调节细胞分裂素的产生,延缓植物叶片的衰老。Bt基因为编码苏云金杆菌毒蛋白的基因,是当今农业上利用最广的一种杀虫基因。本实验室在建立了苎麻的遗传转化体系后,将P_(SAG12)-IPT基因和Bt基因分别导入苎麻品系“5041”和苎麻品种“芦竹青”中,并获得了大量的转基因株系。本研究对获得的转P_(SAG12)-IPT基因和Bt基因苎麻植株进行了分子检测,从分子水平上验证了目的基因已整合在苎麻基因组中,并能够稳定遗传;对转P_(SAG12)-IPT基因苎麻T_0代株系的光合特性等进行了测定,对转Bt基因苎麻T_0代株系的抗虫性进行了室内鉴定,并对两个转基因材料T_0代和T_1代株系的农艺性状和品质性状等进行了考察。主要研究结果如下:
1.利用PCR、Southern杂交等技术对转基因苎麻T_0代和T_1代株系进行检测,均检测到阳性植株。在T_0代转基因苎麻“5041”中,NptⅡ基因(选择标记基因)的PCR阳性率为80.1%,P_(SAG12)-IPT基因(目的基因)的PCR阳性率为69.8%,共转化率为66.1%;在T_0代转基因苎麻“芦竹青”中,NptⅡ基因(选择标记基因)的PCR阳性率为73.8%,Bt基因(目的基因)的PCR阳性率为33.4%,共转化率为33.4%。外源基因在不同材料中的遗传行为不同,对T_1代转基因苎麻进行Km选择培养和PCR检测发现,在转基因苎麻“5041”中,NptⅡ基因和P_(SAG12)-IPT基因的遗传符合3:1的比例,而在转基因苎麻“芦竹青”中,NptⅡ基因的遗传比符合1:1,Bt基因的遗传符合3:1的比例。Southern分子杂交结果进一步验证了上述结论。表明本实验室获得了转基因苎麻T_0代和T_1代植株,为进一步进行生理指标的测定和性状考察研究奠定了基础。
2.在大田环境下,转P_(SAG12)-IPT基因T_0代苎麻的光补偿点、表观量子效率和暗呼吸速率和对照间存在显著差异;在净光合速率(Pn)的日变化中,转基因苎麻部分株系表现为单峰曲线,其他株系表现为双峰曲线,而对照始终表现为单峰曲线;对转基因苎麻株系的叶绿素含量进行了测定,在头麻和二麻纤维成熟期,叶绿素b含量均低于对照,而叶绿素a/b值均高于对照。相关分析表明,在Pn和主要生理因子的关系中,转基因苎麻头、二麻呈显著性相关,三麻的相关性不显著。另外,T_0代转基因株系P513和P504的内源细胞分裂素含量显著高于对照,而且植株形态表现异常,T_0代其他转基因株系及T_1代各转基因株系内源细胞分裂素的含量和对照相比没有显著差异。P_(SAG12)-IPT基因的导入导致苎麻的生理特性发生了改变。
3.通过室内抗虫性生物检测,转Bt基因苎麻株系叶片对苎麻赤蛱蝶幼虫的生长具有抑制作用。转基因株系均表现出了一定的抗性,其中株系SBL2叶片喂养的苎麻赤蛱蝶幼虫死亡率达到38.7%,株系SBL3叶片喂养的苎麻赤蛱蝶蛹死亡率达到24.8%,株系SBL2叶片喂养的苎麻赤蛱蝶成虫畸形率达到40.0%。同时用转Bt基因植株和非转基因植株的叶片喂食苎麻赤蛱蝶幼虫,这些幼虫更趋向于取食非转基因叶片,转基因植株的叶片受到苎麻赤蛱蝶幼虫危害的程度要明显低于对照。在转Bt基因T_1代材料中,将阳性植株种植在常年苎麻赤蛱蝶发生严重的田块,在自然发虫状态下观察,非转基因植株的叶片被害虫取食,个别单株的叶片甚至被食光,而转基因植株没有受到苎麻赤蛱蝶幼虫的危害。
4.转P_(SAG12)-IPT基因苎麻T_0代株系和对照相比,植物学性状发生了一定程度的变异,如株系P504茎中上部发生很多分枝;株系P513在每季麻生育后期,整株叶片从下往上叶缘向上发生翻卷;部分株系发生了自交不育现象。转基因株系的农艺性状和品质性状也产生了显著变异:株系P502的主要性状指标优于受体品系5041;大部分株系部分性状指标优于对照,部分性状指标劣于对照;少数株系的某些性状甚至发生了特殊变异。转Bt基因苎麻和对照相比,T_0代株系性状并无大的变化,转Bt基因苎麻基本保持了原品种的特性。对两个转基因材料T_0代和T_1代的农艺性状和品质性状进行比较发现,外源基因没有改变转基因苎麻的遗传行为。
5.本研究获得了具有利用价值的转基因材料。如转P_(SAG12)-IPT基因株系P502,不仅单株原麻重高于对照,而且株高、分株力等农艺性状和纤维支数等品质性状显著优于对照,这将是一个好的育种材料;转P_(SAG12)-IPT基因株系P506和P512的单株原麻重极显著高于对照,转P_(SAG12)-IPT基因株系P513的纤维支数极显著高于对照,转Bt基因株系SBL2的抗虫性显著优于对照等,这些将是很好的育种中间材料。
Ramie(Boehmeria nivea L.Gaud),one of the traditional fiber crops,had plaid an important role in our agriculture production.With development of society and market demand,ramie production faces a challenge,such as increment of population,decrement of farmland year by year.Thus,it would be a effective way to enhance per unit yield for the continued advance of ramie production.For improving ramie yield and quality by traditional breeding difficultly at present,plant genetic engineering provide a new method for ramie breeding,it is hopeful to obtain novel cultivars by introducing goal gene into ramie plants.
P_(SAG12)-IPT(IPT gene with the SAG12 promoter) gene could autoregulate cytokinin production and delay leaf senescence.Bt(Bacillus thuringiensis) insecticidal gene encoded Bt toxin protein was applied on the most large scale in agriculture now.The two genes were introduced into ramie strain "5041" and cultivar "Luzhuqing" plants respectively,and an array of transgenic lines were obtained after we established ramie transformation system successfully.This paper carried out molecular detection on transgenic plants with P_(SAG12)-IPT and Bt gene,the results confirmed that the two gene had been integrated into ramie genome and could transcript normally.Photosynthetic characteristics of T_0 transgenic lines with P_(SAG12)-IPT gene were measured,the insect-resistance of the T_0 transgenic lines with Bt gene was bioassaied in the lab. Agronomic characters and quality character of all T_0,T_1 transgenic lines were tested.The main results in this study are as follows:
1.T_0,T_1 transgenic lines were detected by PCR and Southern blotting analysis,and each line had got positive plants.The PCR positive ratio of NptⅡgene was 80.1%、the PCR positive ratio of P_(SAG12)-IPT gene was 69.8%and co-transformation ratio was 66.1% among T_0 transgenic ramie strain"5041" plants,whereas the PCR positive ratio of NptⅡgene was 73.8%、the PCR positive ratio of Bt gene was 33.4%and co-transformation ratio was 33.4%among T_0 transgenic cultivar "Luzhuqing" plants.The behavior of exogenous genes in both strain and cultivar were different.Mendelian inheritance of PCR detection was showed single dominant locus and proved by Southern blotting analysis in T_1 of all transgenic plants.Finally,we got T_0,T_1 transgenic plants,which would provide material basis for further test of physiology and characters.
2.Light compensation point(LCP),apparent quantum yield(AQY) and dark respiration rate(Rd) of T_0 transgenic plants with P_(SAG12)-IPT gene were tested in the field in 2007,and they showed significance difference by compared with non-transgenic control.Part of the transgenic lines showed a single-peak curve,and the others showed a double-peak curve,but control always showed a single-peak curve in the photosynthetic rate(Pn) diurnal changes,the content of Chlorophyll(Chl) b of the control was higher than that of the transgenic lines,but the Chl a/b of the transgenic lines were higher than that of control in the first season and the second season in the fiber development period. Correlations between Pn and the major physiologic factors were analyzed and the results showed that different correlation coefficient existed in the first season、the second season and the third season.In addition,the endogenous cytokinins content of T_0 P513 and P504 line were significantly higher than that of control,but the major morphological traits were abnormal,there were no significant variation between the other lines(T_0 and T_1) and control.The integration of P_(SAG12)-IPT gene resulted in the changes of physiological traits.
3.Bt transgenic lines inhibited obviously the herbst larva growth in the lab.All transgenic lines exhibited insecticidal activity,the mortality of larva fed with SBL2 line reached 38.7%,the mortality of pupae fed with SBL3 line reached 24.8%,the misshapen rate of the adult fed with SBL2 line reached 40.0%.The herbst could distinguish non-Bt ramie leaves from Bt ramie leaves when two kinds of leaves were fed synchronously. Moreover,the herbst prefers to feed on Bt ramie leaves.T_1 Bt transgenic lines and control were planted in the field where herbst took place seriously average year,the results showed that non-Bt ramie leaves were fed on by larva,even very few had been eaten up, but Bt ramie grew normally.
4.The morphological traits of P_(SAG12)-IPT lines showed variation at some extent by compared with control,for example,there were many branches in middle section of P504 stem;the edge of the whole P513 leaves rolled up from bottom to top in late development period,part of lines exhibited inbred sterility.Agronomic characters and quality character of P_(SAG12)-IPT lines showed significantly variation:major character targets among P502 line were better than that of the cultivar 5041,Part of character targets were better than that of CK but any character targets were less than that of CK among major lines,There were even specific variance among the very few lines.T_0 Bt transgenic lines maintained the main traits compared to non-Bt ramie,two exogenous gene did not affect ramie heredity.
5.The favorable transgenic lines were obtained in this study.P502 line was significantly superior to the control,such as plant height,rate of effective tiller,raw fiber weight,fiber fineness,it would be a excellent breeding material,raw fiber weight of P506 line and P512 line,fiber fineness of P513 line and insect resistance of SBL2 line were better than that of control respectively,these lines would become breeding middle materials.
P_(SAG12)-IPT基因能自我调节细胞分裂素的产生,延缓植物叶片的衰老。Bt基因为编码苏云金杆菌毒蛋白的基因,是当今农业上利用最广的一种杀虫基因。本实验室在建立了苎麻的遗传转化体系后,将P_(SAG12)-IPT基因和Bt基因分别导入苎麻品系“5041”和苎麻品种“芦竹青”中,并获得了大量的转基因株系。本研究对获得的转P_(SAG12)-IPT基因和Bt基因苎麻植株进行了分子检测,从分子水平上验证了目的基因已整合在苎麻基因组中,并能够稳定遗传;对转P_(SAG12)-IPT基因苎麻T_0代株系的光合特性等进行了测定,对转Bt基因苎麻T_0代株系的抗虫性进行了室内鉴定,并对两个转基因材料T_0代和T_1代株系的农艺性状和品质性状等进行了考察。主要研究结果如下:
1.利用PCR、Southern杂交等技术对转基因苎麻T_0代和T_1代株系进行检测,均检测到阳性植株。在T_0代转基因苎麻“5041”中,NptⅡ基因(选择标记基因)的PCR阳性率为80.1%,P_(SAG12)-IPT基因(目的基因)的PCR阳性率为69.8%,共转化率为66.1%;在T_0代转基因苎麻“芦竹青”中,NptⅡ基因(选择标记基因)的PCR阳性率为73.8%,Bt基因(目的基因)的PCR阳性率为33.4%,共转化率为33.4%。外源基因在不同材料中的遗传行为不同,对T_1代转基因苎麻进行Km选择培养和PCR检测发现,在转基因苎麻“5041”中,NptⅡ基因和P_(SAG12)-IPT基因的遗传符合3:1的比例,而在转基因苎麻“芦竹青”中,NptⅡ基因的遗传比符合1:1,Bt基因的遗传符合3:1的比例。Southern分子杂交结果进一步验证了上述结论。表明本实验室获得了转基因苎麻T_0代和T_1代植株,为进一步进行生理指标的测定和性状考察研究奠定了基础。
2.在大田环境下,转P_(SAG12)-IPT基因T_0代苎麻的光补偿点、表观量子效率和暗呼吸速率和对照间存在显著差异;在净光合速率(Pn)的日变化中,转基因苎麻部分株系表现为单峰曲线,其他株系表现为双峰曲线,而对照始终表现为单峰曲线;对转基因苎麻株系的叶绿素含量进行了测定,在头麻和二麻纤维成熟期,叶绿素b含量均低于对照,而叶绿素a/b值均高于对照。相关分析表明,在Pn和主要生理因子的关系中,转基因苎麻头、二麻呈显著性相关,三麻的相关性不显著。另外,T_0代转基因株系P513和P504的内源细胞分裂素含量显著高于对照,而且植株形态表现异常,T_0代其他转基因株系及T_1代各转基因株系内源细胞分裂素的含量和对照相比没有显著差异。P_(SAG12)-IPT基因的导入导致苎麻的生理特性发生了改变。
3.通过室内抗虫性生物检测,转Bt基因苎麻株系叶片对苎麻赤蛱蝶幼虫的生长具有抑制作用。转基因株系均表现出了一定的抗性,其中株系SBL2叶片喂养的苎麻赤蛱蝶幼虫死亡率达到38.7%,株系SBL3叶片喂养的苎麻赤蛱蝶蛹死亡率达到24.8%,株系SBL2叶片喂养的苎麻赤蛱蝶成虫畸形率达到40.0%。同时用转Bt基因植株和非转基因植株的叶片喂食苎麻赤蛱蝶幼虫,这些幼虫更趋向于取食非转基因叶片,转基因植株的叶片受到苎麻赤蛱蝶幼虫危害的程度要明显低于对照。在转Bt基因T_1代材料中,将阳性植株种植在常年苎麻赤蛱蝶发生严重的田块,在自然发虫状态下观察,非转基因植株的叶片被害虫取食,个别单株的叶片甚至被食光,而转基因植株没有受到苎麻赤蛱蝶幼虫的危害。
4.转P_(SAG12)-IPT基因苎麻T_0代株系和对照相比,植物学性状发生了一定程度的变异,如株系P504茎中上部发生很多分枝;株系P513在每季麻生育后期,整株叶片从下往上叶缘向上发生翻卷;部分株系发生了自交不育现象。转基因株系的农艺性状和品质性状也产生了显著变异:株系P502的主要性状指标优于受体品系5041;大部分株系部分性状指标优于对照,部分性状指标劣于对照;少数株系的某些性状甚至发生了特殊变异。转Bt基因苎麻和对照相比,T_0代株系性状并无大的变化,转Bt基因苎麻基本保持了原品种的特性。对两个转基因材料T_0代和T_1代的农艺性状和品质性状进行比较发现,外源基因没有改变转基因苎麻的遗传行为。
5.本研究获得了具有利用价值的转基因材料。如转P_(SAG12)-IPT基因株系P502,不仅单株原麻重高于对照,而且株高、分株力等农艺性状和纤维支数等品质性状显著优于对照,这将是一个好的育种材料;转P_(SAG12)-IPT基因株系P506和P512的单株原麻重极显著高于对照,转P_(SAG12)-IPT基因株系P513的纤维支数极显著高于对照,转Bt基因株系SBL2的抗虫性显著优于对照等,这些将是很好的育种中间材料。
Ramie(Boehmeria nivea L.Gaud),one of the traditional fiber crops,had plaid an important role in our agriculture production.With development of society and market demand,ramie production faces a challenge,such as increment of population,decrement of farmland year by year.Thus,it would be a effective way to enhance per unit yield for the continued advance of ramie production.For improving ramie yield and quality by traditional breeding difficultly at present,plant genetic engineering provide a new method for ramie breeding,it is hopeful to obtain novel cultivars by introducing goal gene into ramie plants.
P_(SAG12)-IPT(IPT gene with the SAG12 promoter) gene could autoregulate cytokinin production and delay leaf senescence.Bt(Bacillus thuringiensis) insecticidal gene encoded Bt toxin protein was applied on the most large scale in agriculture now.The two genes were introduced into ramie strain "5041" and cultivar "Luzhuqing" plants respectively,and an array of transgenic lines were obtained after we established ramie transformation system successfully.This paper carried out molecular detection on transgenic plants with P_(SAG12)-IPT and Bt gene,the results confirmed that the two gene had been integrated into ramie genome and could transcript normally.Photosynthetic characteristics of T_0 transgenic lines with P_(SAG12)-IPT gene were measured,the insect-resistance of the T_0 transgenic lines with Bt gene was bioassaied in the lab. Agronomic characters and quality character of all T_0,T_1 transgenic lines were tested.The main results in this study are as follows:
1.T_0,T_1 transgenic lines were detected by PCR and Southern blotting analysis,and each line had got positive plants.The PCR positive ratio of NptⅡgene was 80.1%、the PCR positive ratio of P_(SAG12)-IPT gene was 69.8%and co-transformation ratio was 66.1% among T_0 transgenic ramie strain"5041" plants,whereas the PCR positive ratio of NptⅡgene was 73.8%、the PCR positive ratio of Bt gene was 33.4%and co-transformation ratio was 33.4%among T_0 transgenic cultivar "Luzhuqing" plants.The behavior of exogenous genes in both strain and cultivar were different.Mendelian inheritance of PCR detection was showed single dominant locus and proved by Southern blotting analysis in T_1 of all transgenic plants.Finally,we got T_0,T_1 transgenic plants,which would provide material basis for further test of physiology and characters.
2.Light compensation point(LCP),apparent quantum yield(AQY) and dark respiration rate(Rd) of T_0 transgenic plants with P_(SAG12)-IPT gene were tested in the field in 2007,and they showed significance difference by compared with non-transgenic control.Part of the transgenic lines showed a single-peak curve,and the others showed a double-peak curve,but control always showed a single-peak curve in the photosynthetic rate(Pn) diurnal changes,the content of Chlorophyll(Chl) b of the control was higher than that of the transgenic lines,but the Chl a/b of the transgenic lines were higher than that of control in the first season and the second season in the fiber development period. Correlations between Pn and the major physiologic factors were analyzed and the results showed that different correlation coefficient existed in the first season、the second season and the third season.In addition,the endogenous cytokinins content of T_0 P513 and P504 line were significantly higher than that of control,but the major morphological traits were abnormal,there were no significant variation between the other lines(T_0 and T_1) and control.The integration of P_(SAG12)-IPT gene resulted in the changes of physiological traits.
3.Bt transgenic lines inhibited obviously the herbst larva growth in the lab.All transgenic lines exhibited insecticidal activity,the mortality of larva fed with SBL2 line reached 38.7%,the mortality of pupae fed with SBL3 line reached 24.8%,the misshapen rate of the adult fed with SBL2 line reached 40.0%.The herbst could distinguish non-Bt ramie leaves from Bt ramie leaves when two kinds of leaves were fed synchronously. Moreover,the herbst prefers to feed on Bt ramie leaves.T_1 Bt transgenic lines and control were planted in the field where herbst took place seriously average year,the results showed that non-Bt ramie leaves were fed on by larva,even very few had been eaten up, but Bt ramie grew normally.
4.The morphological traits of P_(SAG12)-IPT lines showed variation at some extent by compared with control,for example,there were many branches in middle section of P504 stem;the edge of the whole P513 leaves rolled up from bottom to top in late development period,part of lines exhibited inbred sterility.Agronomic characters and quality character of P_(SAG12)-IPT lines showed significantly variation:major character targets among P502 line were better than that of the cultivar 5041,Part of character targets were better than that of CK but any character targets were less than that of CK among major lines,There were even specific variance among the very few lines.T_0 Bt transgenic lines maintained the main traits compared to non-Bt ramie,two exogenous gene did not affect ramie heredity.
5.The favorable transgenic lines were obtained in this study.P502 line was significantly superior to the control,such as plant height,rate of effective tiller,raw fiber weight,fiber fineness,it would be a excellent breeding material,raw fiber weight of P506 line and P512 line,fiber fineness of P513 line and insect resistance of SBL2 line were better than that of control respectively,these lines would become breeding middle materials.
引文
1.布都会,奚亚军,刘曙东,任鹏.转叶片衰老延缓基因P_(SAG12)-IPT基因小麦株系光合性能的研究.西北植物学报,2007,27(10):2104-2107
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