农杆菌介导的无籽基因转化诺丽根段
|
摘要
[目的]为获得无籽诺丽植株。[方法]实验利用含有无籽基因的农杆菌对诺丽根段进行遗传转化,诺丽根段经预培养3 d、农杆菌液侵染20 min、共培养基暗培养3 d、筛选培养20 d后得到抗性芽,继代筛选后得到转化子;通过提取诺丽转化子叶片的DNA,以NptⅡ为引物进行PCR检测;将阳性植株进行生根培养后,炼苗并移栽至营养钵,观察其生长状况。[结果]表明:诺丽根段经过筛选培养后,获得225株抗性芽,抗性芽平均分化率为56. 25%;抗性芽继代筛选培养后得到94株转化子,对其进行PCR检测,获得18株转基因阳性植株;移栽后,能正常生根且于营养钵中正常生长。[结论]经PCR初步鉴定,无籽基因成功导入诺丽植株中,转化率为8%。
[Objective] To obtain seedless noni plants.[Methods]An Agrobacterium containing seedless genes is used in the experiment to carry out genetic transformation on the noni root segment. Noni root segment is precultured for 3 d,infected with Agrobacterium solution for 20 min,co-culture of dark culture for 3 d,and screened for 20 d to obtain resistant bud,and then get transformed seeds after secondary screening. By extracting DNA of blades of noni transformed seeds' leaves,NptⅡas primers for PCR detection; after the positive plants were taken root culture,the seedlings were refined and transplanted to the nutrition bowl to observe their growth status.[Results]show that after the noni root segment is screened and cultured,225 resistant buds are obtained,and the average differentiation rate of resistant buds is 56. 25%. 94 transformants are obtained after the secondary culture of resistant buds,which are detected by PCR and 18 transgenic positive plants are obtained. After transplantation,it can take root and grow normally in the nutrition bowl.[Conclusion] the seedless gene is successfully introduced into noni plants,according to primary evaluation of PCR,and the conversion rate is 8%.
[Objective] To obtain seedless noni plants.[Methods]An Agrobacterium containing seedless genes is used in the experiment to carry out genetic transformation on the noni root segment. Noni root segment is precultured for 3 d,infected with Agrobacterium solution for 20 min,co-culture of dark culture for 3 d,and screened for 20 d to obtain resistant bud,and then get transformed seeds after secondary screening. By extracting DNA of blades of noni transformed seeds' leaves,NptⅡas primers for PCR detection; after the positive plants were taken root culture,the seedlings were refined and transplanted to the nutrition bowl to observe their growth status.[Results]show that after the noni root segment is screened and cultured,225 resistant buds are obtained,and the average differentiation rate of resistant buds is 56. 25%. 94 transformants are obtained after the secondary culture of resistant buds,which are detected by PCR and 18 transgenic positive plants are obtained. After transplantation,it can take root and grow normally in the nutrition bowl.[Conclusion] the seedless gene is successfully introduced into noni plants,according to primary evaluation of PCR,and the conversion rate is 8%.
引文
[1]徐通,杨朴丽,杨焱.疏果对诺丽幼树生长及产量的影响[J].热带农业科技,2018,41(1):34-42.
[2]周偏,蔡坤,梁丛颖,等.诺丽酵素在自然发酵过程中体外抗氧化活性变化及品质研究[J].中国酿造,2018,37(1):92-96.
[3]晏永球,童应鹏,陆雨,等.诺丽的化学成分及药理活性研究进展[J].中草药,2017,48(9):1888-1905.
[4] Bussmann R W,Hennig L,Giannis A,et al. Anthraquinone content in noni(Morinda citrifolia L.)[J]. Evidence Based Complementary and Alternative Medicine,2013,12(4):5-10.
[5]李法营,蓝增全,刘昌芬,等.诺丽研究进展(一)—国内外研究进展[J].安徽农业科学,2009,37(32):15819-15821.
[6]乌云塔娜,吕祎馨,片山款则.无籽梨品种无籽果实形成原因的初步研究[J].经济林研究,2016,34(1):117-122.
[7]陈俊伟,谢鸣,吴江.遗传工程无籽果实原理及其研究进展[J].浙江农业学报,2003,15(6):365-371.
[8]杜浩,庄义庆,黄萍,等.农杆菌介导的甜高粱遗传转化研究进展[J].江苏农业科学,2018,46(4):29-32.
[9]丁霄,隋炯明,王晶珊,等.以木糖异构酶基因作为选择标记的花生遗传转化[J].核农学报,2012,26(3):444-448.
[10]李晓瑞,胡尚连,曹颖,等.农杆菌介导慈竹4CL基因遗传转化梁山慈竹[J].林业科学,2012,48(3):38-44.
[11] Maleki Samaneh Sadat,Mohammadi Kourosh,季孔庶.根癌农杆菌介导遗传转化烟草‘NC89’效率的影响因素[J].南京林业大学学报:自然科学版,2018,42(1):27-34.
[12]吴转娣,吴才文,曾千春,等. Cry1Ac和sck双价抗虫基因遗传转化甘蔗的研究[J].热带作物学报,2014,35(11):2236-2242.
[13]杜翔斐,彭庆务,王永飞.节瓜CqWRKY1基因的遗传转化[J].核农学报,2018,32(6):1060-1069.
[14]郭春艳,刘宏魁,吴颖,等.玉米冷响应基因Zm Cyp40克隆及其遗传转化[J].分子植物育种,2018,16(2):112-116.
[15]邓莉,张霞,曲延英,等.农杆菌介导转Bt基因海岛棉的获得及抗棉铃虫效果分析[J].分子植物育种,2018,16(2):440-444.
[16]王晓敏,王耀辉,耿思宇,等.利用花粉介导法获得油菜RNAi转基因植株[J].山西农业科学,2018,46(1):5-8.
[17]吕品,李朋伟,马双武,等.西瓜GGPS基因果实特异性表达载体构建与遗传转化[J].生物技术,2016,26(5):432-437.
[18]张璐,刘帅,魏萌涵,等.苹果s MdCAX1基因超表达载体的构建及遗传转化[J].核农学报,2016,30(8):1460-1469.
[19]王栋鑫,彭棣,张爽.农杆菌介导木本植物遗传转化的研究进展[J].北方园艺,2018,1(2):181-185.
[20]管岩岩,任国栋,郭新勇,等.天山雪莲遗传转化体系建立[J].西北农业学报,2010,19(7):84-88.
[21]朱军,韩锁义,袁美,等.农杆菌介导的花生遗传转化条件优化[J.]中国油料作物学报,2018,40(2):192-198.
[22]贾红梅,王碧玉,刘迪,等.农杆菌介导CBL基因对菊花品种‘C008’的转化[J].西北林学院学报,2017,32(1):184-189.
[23]张磊,李国领,张建周,等.农杆菌介导小麦遗传转化方法的优化与应用[J].河南农业大学学报,2017,51(6):781-785.
[24]张海芹,刘雅莉,张锋,等.根癌农杆菌介导的葡萄风信子遗传转化体系[J].西北农林科技大学学报:自然科学版,2017,45(2):135-142.
[25]陈长明,赵祥明,雷建军,等.基因枪法和农杆菌介导的Bt抗虫基因转化芥蓝[J].中国蔬菜,2016,1(8):21-28.
[26]赵辉,郭静远,孔华,等.金刚砂辅助农狗尾草种子高效遗传转化体系的建立[J].分子植物育种,2017,15(12):4955-4960.
[2]周偏,蔡坤,梁丛颖,等.诺丽酵素在自然发酵过程中体外抗氧化活性变化及品质研究[J].中国酿造,2018,37(1):92-96.
[3]晏永球,童应鹏,陆雨,等.诺丽的化学成分及药理活性研究进展[J].中草药,2017,48(9):1888-1905.
[4] Bussmann R W,Hennig L,Giannis A,et al. Anthraquinone content in noni(Morinda citrifolia L.)[J]. Evidence Based Complementary and Alternative Medicine,2013,12(4):5-10.
[5]李法营,蓝增全,刘昌芬,等.诺丽研究进展(一)—国内外研究进展[J].安徽农业科学,2009,37(32):15819-15821.
[6]乌云塔娜,吕祎馨,片山款则.无籽梨品种无籽果实形成原因的初步研究[J].经济林研究,2016,34(1):117-122.
[7]陈俊伟,谢鸣,吴江.遗传工程无籽果实原理及其研究进展[J].浙江农业学报,2003,15(6):365-371.
[8]杜浩,庄义庆,黄萍,等.农杆菌介导的甜高粱遗传转化研究进展[J].江苏农业科学,2018,46(4):29-32.
[9]丁霄,隋炯明,王晶珊,等.以木糖异构酶基因作为选择标记的花生遗传转化[J].核农学报,2012,26(3):444-448.
[10]李晓瑞,胡尚连,曹颖,等.农杆菌介导慈竹4CL基因遗传转化梁山慈竹[J].林业科学,2012,48(3):38-44.
[11] Maleki Samaneh Sadat,Mohammadi Kourosh,季孔庶.根癌农杆菌介导遗传转化烟草‘NC89’效率的影响因素[J].南京林业大学学报:自然科学版,2018,42(1):27-34.
[12]吴转娣,吴才文,曾千春,等. Cry1Ac和sck双价抗虫基因遗传转化甘蔗的研究[J].热带作物学报,2014,35(11):2236-2242.
[13]杜翔斐,彭庆务,王永飞.节瓜CqWRKY1基因的遗传转化[J].核农学报,2018,32(6):1060-1069.
[14]郭春艳,刘宏魁,吴颖,等.玉米冷响应基因Zm Cyp40克隆及其遗传转化[J].分子植物育种,2018,16(2):112-116.
[15]邓莉,张霞,曲延英,等.农杆菌介导转Bt基因海岛棉的获得及抗棉铃虫效果分析[J].分子植物育种,2018,16(2):440-444.
[16]王晓敏,王耀辉,耿思宇,等.利用花粉介导法获得油菜RNAi转基因植株[J].山西农业科学,2018,46(1):5-8.
[17]吕品,李朋伟,马双武,等.西瓜GGPS基因果实特异性表达载体构建与遗传转化[J].生物技术,2016,26(5):432-437.
[18]张璐,刘帅,魏萌涵,等.苹果s MdCAX1基因超表达载体的构建及遗传转化[J].核农学报,2016,30(8):1460-1469.
[19]王栋鑫,彭棣,张爽.农杆菌介导木本植物遗传转化的研究进展[J].北方园艺,2018,1(2):181-185.
[20]管岩岩,任国栋,郭新勇,等.天山雪莲遗传转化体系建立[J].西北农业学报,2010,19(7):84-88.
[21]朱军,韩锁义,袁美,等.农杆菌介导的花生遗传转化条件优化[J.]中国油料作物学报,2018,40(2):192-198.
[22]贾红梅,王碧玉,刘迪,等.农杆菌介导CBL基因对菊花品种‘C008’的转化[J].西北林学院学报,2017,32(1):184-189.
[23]张磊,李国领,张建周,等.农杆菌介导小麦遗传转化方法的优化与应用[J].河南农业大学学报,2017,51(6):781-785.
[24]张海芹,刘雅莉,张锋,等.根癌农杆菌介导的葡萄风信子遗传转化体系[J].西北农林科技大学学报:自然科学版,2017,45(2):135-142.
[25]陈长明,赵祥明,雷建军,等.基因枪法和农杆菌介导的Bt抗虫基因转化芥蓝[J].中国蔬菜,2016,1(8):21-28.
[26]赵辉,郭静远,孔华,等.金刚砂辅助农狗尾草种子高效遗传转化体系的建立[J].分子植物育种,2017,15(12):4955-4960.