外源野生胡麻总DNA遗传转化栽培胡麻及RAPD分子验证
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摘要
为了克服亚麻属不同种间存在的生殖隔离,从而利用亚麻野生种优异基因进行资源创制,采用花粉管通道法将亚麻野生种垂果亚麻的基因组DNA导入到栽培种坝选三号中,获得T0种子。T1播种后所获24个单株中有3株发生明显变异,转化后代的株高、工艺长度、分茎数、分枝数及单株蒴果数与受体坝选三号相比较,均发生明显增加。利用18条引物对供体、受体及24株T1导入材料的基因组DNA进行了RAPD扩增分析,从中筛选到6条多态性引物(OPH20、OPK14、S103、S118、OPJ4、OPT6)对8个样品的DNA扩增产物带型出现明显差异,占全部所用引物的33. 3%。RAPD分析表明,D14、D17、D22 3个植株的RAPD谱带中均有来自于供体的特异条带,结合考种数据,认为是供体DNA片段成功整合进了受体基因组中引起了表型上的变化。利用花粉管通道法向胡麻栽培种中导入野生种基因组DNA能够在当代引起多种变异,同时也可在分子水平上检测到基因组的差异,推测大片段外源DNA同源重组可能是变异的主要原因。
In order to overcome the reproductive isolation between different species of linseed,thus creat resources by using the excellent genes of flax wild species,we made exogenous DNA of wild flax directly introduce into cultivated specie Baxuan 3 by using pollen-tube pathway and obtained the T0 generation of seeds. After T1 generation seeds being planted,three of the twenty-four plants of after self-pollination showed obvious variation whose plant height,process length,stems per plant,branches per main stem and capsules per plant were significantly increased comparing with the recipients. The genomic DNA of donor,recipient and 24 T1 generation introduced materials were amplified by RAPD with 18 primers and six polymorphic primers(OPH20,OPK14,S103,S118,OPJ4,OPT6) were screened out. The banding patterns of DNA amplified products of 8 samples were significantly different,accounting for 33. 3% of the total primers used. The results showed that the RAPD bands of D14,D17 and D22 all had specific bands from donors,and speculated that large fragment exogenous DNA homologous recombination might be the main reason for variation combining phenotypic datas. Introducing genomic DNA of wild species into flax cultivars by pollen tube pathway method can cause multiple variations in the present generation,and can also detect the differences of genome at the molecular level. It is speculated that homologous recombination of large fragments of exogenous DNA may be the main cause of variation.
In order to overcome the reproductive isolation between different species of linseed,thus creat resources by using the excellent genes of flax wild species,we made exogenous DNA of wild flax directly introduce into cultivated specie Baxuan 3 by using pollen-tube pathway and obtained the T0 generation of seeds. After T1 generation seeds being planted,three of the twenty-four plants of after self-pollination showed obvious variation whose plant height,process length,stems per plant,branches per main stem and capsules per plant were significantly increased comparing with the recipients. The genomic DNA of donor,recipient and 24 T1 generation introduced materials were amplified by RAPD with 18 primers and six polymorphic primers(OPH20,OPK14,S103,S118,OPJ4,OPT6) were screened out. The banding patterns of DNA amplified products of 8 samples were significantly different,accounting for 33. 3% of the total primers used. The results showed that the RAPD bands of D14,D17 and D22 all had specific bands from donors,and speculated that large fragment exogenous DNA homologous recombination might be the main reason for variation combining phenotypic datas. Introducing genomic DNA of wild species into flax cultivars by pollen tube pathway method can cause multiple variations in the present generation,and can also detect the differences of genome at the molecular level. It is speculated that homologous recombination of large fragments of exogenous DNA may be the main cause of variation.
引文
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[12]贾影影,刘凤楼,王掌军,等.大豆DNA导入春小麦后的农艺性状和品质性状分析及RAPD标记验证[J].麦类作物学报,2016,36(7):833-840.
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[15]彭娜娜,王亚娟,王长有,等.普通小麦/山羊草属间杂交F1的形态学及细胞遗传学研究[J].麦类作物学报,2016,36(12):1563-1569.
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[19]王丽,马晓娣,米君,等.栽培亚麻×野生亚麻种间杂交胚胎发育的研究[J].河北农业大学学报,2007,30(5):19-22.
[20]张丽丽,白苇,米君,等.栽培亚麻×野生亚麻种间杂交种的真实性鉴定[J].华北农学报,2012,27(S):57-60.
[21]李闻娟,张建平,陈芳,等. bar基因的亚麻花粉管通道法转化[J].中国农学通报,2013,29(12):96-100.
[22]苏文杰.花粉管通道法转Bar基因胡麻后代的分子检测[J].种子科技,2017,35(3):117-120.
[2]刘传亮,田瑞平,孔德培,等.棉花规模化转基因技术体系构建及其应用[J].中国农业科学,2014,47(21):4183-4197.
[3]黄骏麒,钱思颍,刘桂铨,等.外源抗枯萎病棉DNA导入感病棉的抗性转移[J].中国农业科学,1986(3):32-36.
[4]郭彤,刘政,胡银岗.导入谷子基因组DNA对小麦光合生理特性的影响[J].麦类作物学报,2015,35(4):514-520.
[5]段晓岚,陈善葆.外源DNA导入水稻引起性状变异[J].中国农业科学,1985(3):6-11.
[6]雷勃钧,尹光初,王树林,等.外源野生大豆DNA导入栽培大豆引起的变异[J].中国油料作物学报,1989(3):11-14.
[7]雷勃钧,尹光初.外源DNA直接导入大豆的研究[J].大豆科学,1991,10(1):58-63.
[8]李艳超,赵青松,王凤敏,等.大豆遗传转化技术研究进展[J].大豆科学,2015,34(1):155-162.
[9]何欢,高嵩,韩丹,等.抗虫基因Cry1Iem转化大豆的研究[J].吉林农业大学学报,2017,39(6):665-669.
[10]王玉富,刘燕,康庆华,等.纤维亚麻新品种黑亚14号选育报告[J].中国麻业科学,2003,25(3):112-113.
[11]关凤芝,吴广文,宋宪友,等.纤维亚麻新品种黑亚17号的选育[J].黑龙江农业科学,2009(1):152-153.
[12]贾影影,刘凤楼,王掌军,等.大豆DNA导入春小麦后的农艺性状和品质性状分析及RAPD标记验证[J].麦类作物学报,2016,36(7):833-840.
[13]崔淑芳,钱玉源,王广恩,等.远缘杂交种冀棉25的选育及其育种利用[J].中国棉花,2018,45(3):21-25.
[14]王爱凡,康雷,李鹏飞,等.我国甘蓝型油菜远缘杂交和种质创新研究进展[J].中国油料作物学报,2016,38(5):691-698.
[15]彭娜娜,王亚娟,王长有,等.普通小麦/山羊草属间杂交F1的形态学及细胞遗传学研究[J].麦类作物学报,2016,36(12):1563-1569.
[16]兰海,苏鹏,周树峰,等.玉米与四倍体大刍草杂交结实性的遗传分析[J].核农学报,2014,28(3):393-397.
[17]米君.油纤兼用型亚麻种间杂交技术研究[J].河北农业科学,2004,8(3):6-7.
[18]米君,李爱荣,钱合顺,等.亚麻种间杂交技术研究初报[J].中国麻业科学,2008,30(3):136-140.
[19]王丽,马晓娣,米君,等.栽培亚麻×野生亚麻种间杂交胚胎发育的研究[J].河北农业大学学报,2007,30(5):19-22.
[20]张丽丽,白苇,米君,等.栽培亚麻×野生亚麻种间杂交种的真实性鉴定[J].华北农学报,2012,27(S):57-60.
[21]李闻娟,张建平,陈芳,等. bar基因的亚麻花粉管通道法转化[J].中国农学通报,2013,29(12):96-100.
[22]苏文杰.花粉管通道法转Bar基因胡麻后代的分子检测[J].种子科技,2017,35(3):117-120.