野生番茄LA2157中热稳定抗根结线虫基因Mi-9候选基因的分离与过表达载体构建
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摘要
根结线虫是番茄上的重要土传病害,选育抗根结线虫品种是最有效的防治方法,但是目前转育到栽培番茄中的抗根结线虫基因Mi-1在土温高于28℃时就丧失了抗性。本试验利用热稳定抗根结线虫野生番茄材料LA2157,根据Mi-1基因的序列信息,对其中热稳定抗根结线虫Mi-9基因进行同源克隆,在LA2157中共获得2个候选基因片段;通过In-Fusion克隆技术,将候选基因与过表达载体p BI121进行连接,经电泳检测和测序分析,最终构建Mi-9候选基因的过表达重组载体。
Root-knot nematode disease is one of the important tomato soil-borne diseases.Breeding varieties with root-knot nematode resistance is an effective control method to deal with this disease.But at present,Mi-1 the only gene transferred to tomato culture with resistance to root-knot nematode loses its resistance when the soil temperature is over 28 ℃.In this study,wild tomato accession LA2157 with heat-stable resistance to root-knot nematode was used.According to the sequence information of Mi-1,we carried out homologous cloning on Mi-9 gene with heat-stable resistance to root-knot nematode and gained 2 candidate gene segments from LA2157.The candidate gene was ligated with the overexpression vector p BI121 by In-Fusion cloning technique.Electrophoresis and sequencing analysis proved that the recombinant vectors of the candidate gene were constructed.
Root-knot nematode disease is one of the important tomato soil-borne diseases.Breeding varieties with root-knot nematode resistance is an effective control method to deal with this disease.But at present,Mi-1 the only gene transferred to tomato culture with resistance to root-knot nematode loses its resistance when the soil temperature is over 28 ℃.In this study,wild tomato accession LA2157 with heat-stable resistance to root-knot nematode was used.According to the sequence information of Mi-1,we carried out homologous cloning on Mi-9 gene with heat-stable resistance to root-knot nematode and gained 2 candidate gene segments from LA2157.The candidate gene was ligated with the overexpression vector p BI121 by In-Fusion cloning technique.Electrophoresis and sequencing analysis proved that the recombinant vectors of the candidate gene were constructed.
引文
陈菲帆,王琪琦,钟辉丽,付冰冰,任丹莉,李玉红.2015.基于In-Fusion技术的ERECTA基因植物表达载体的构建.北方园艺,(18):110-115.
刘维志.2000.植物病原线虫学.北京:中国农业出版社.
唐本安,唐敏,岳新民.2001.南方根结线虫生态地理居群对胡萝卜生产危害的研究.中国农学通报,17(1):28-32.
席先梅,白全江,张庆萍,李玉民,郭金涛,李敏.2017.设施黄瓜根结线虫化学防治技术研究.植物保护,43(2):235-240.
赵统敏,王银磊,杨玛丽,赵丽萍,余文贵.2012.番茄根结线虫病抗性基因的研究进展.江苏农业学报,28(6):1492-1497.
Aboul-Eid H Z,Noweer E M,Ashour N E,Ameen H H.2006.Evaluation of a nematode bio-product Dbx-20%against root-knot nematode Meloidogyne incognita affecting grapevine under field conditions.Communications in Agricultural&Applied Biological Sciences,71(3):659-663.
Ammiraju J,Veremis J,Huang X,Roberts P,Kaloshian I.2003.The heat-stable root-knot nematode resistance gene Mi-9from Lycopersicon peruvianum is localized on the short arm of chromosome 6.Theoretical&Applied Genetics,106(3):478-484.
Cap G B,Roberts P A,Thomason I J.1993.Inheritance of heat-stable resistance to Meloidogyne incognita in Lycopersicon peruvianum and its relationship to the Mi gene.Theoretical&Applied Genetics,85(6-7):777-783.
Dorhout R,Gommers F J,Koll?ffel C.1993.Phloem transport of carboxyfluorescein through tomato roots infected with Meloidogyne incognita.Physiological&Molecular Plant Pathology,43(1):1-10.
Filho H P M,Stevens M A.1980.Tomato breeding for nematode resistance:survey of resistant varieties for horticultural characteristics and genotype of acid phosphatases.Symposium on Production of Tomatoes for Processing,100:383-394.
Jablonska B,Ammiraju J S,Bhattarai K K,Mantelin S,Martinez de Ilarduya O,Roberts P A,Kaloshian I.2007.The Mi-9 gene from Solanum arcanum conferring heat-stable resistance to rootknot nematodes is a homolog of Mi-1.Plant Physiology,143(2):1044-1054.
Milligan S B,Bodeau J,Yaghoobi J,Kaloshian I,Zabel P,Williamson V M.1998.The root knot nematode resistance gene Mi from tomato is a member of the leucine zipper,nucleotide binding,leucine-rich repeat family of plant genes.Plant Cell,10(8):1307-1319.
Sasser J N.1980.Root-knot nematodes:a global menace to crop production.Plant Disease,64(1):36-41.
Smith P G.1944.Embryo culture of a tomato species hybrid.J Amer Soc Hort Sci,44:413-416.
Tzortzakakis E A,Adam M A M,Blok V C,Blok V C,Paraskevopoulos C,Bourtzis K.2005.Occurrence of resistance-breaking populations of root-knot nematodes on tomato in Greece.European Journal of Plant Pathology,113(1):101-105.
Veremis J C,Roberts P A.1996.Differentiation of Meloidogyne incognita and M.arenaria novel resistance phenotypes in Lycopersicon peruvianum and derived bridge-lines.Theoretical&Applied Genetics,93(5-6):960-967.
Wang Y,Yang W,Zhang W,Han Q,Feng M,Shen H.2013.Mapping of a heat-stable gene for resistance to southern root-knot nematode in Solanum lycopersicum.Plant Molecular Biology Reporter,31(2):352-362.
Yaghoobi J,Yates J L,Williamson V M.2005.Fine mapping of the nematode resistance gene Mi-3 in Solanum peruvianum and construction of a S.lycopersicum DNA contig spanning the locus.Molecular Genetics&Genomics,274(1):60-69.
刘维志.2000.植物病原线虫学.北京:中国农业出版社.
唐本安,唐敏,岳新民.2001.南方根结线虫生态地理居群对胡萝卜生产危害的研究.中国农学通报,17(1):28-32.
席先梅,白全江,张庆萍,李玉民,郭金涛,李敏.2017.设施黄瓜根结线虫化学防治技术研究.植物保护,43(2):235-240.
赵统敏,王银磊,杨玛丽,赵丽萍,余文贵.2012.番茄根结线虫病抗性基因的研究进展.江苏农业学报,28(6):1492-1497.
Aboul-Eid H Z,Noweer E M,Ashour N E,Ameen H H.2006.Evaluation of a nematode bio-product Dbx-20%against root-knot nematode Meloidogyne incognita affecting grapevine under field conditions.Communications in Agricultural&Applied Biological Sciences,71(3):659-663.
Ammiraju J,Veremis J,Huang X,Roberts P,Kaloshian I.2003.The heat-stable root-knot nematode resistance gene Mi-9from Lycopersicon peruvianum is localized on the short arm of chromosome 6.Theoretical&Applied Genetics,106(3):478-484.
Cap G B,Roberts P A,Thomason I J.1993.Inheritance of heat-stable resistance to Meloidogyne incognita in Lycopersicon peruvianum and its relationship to the Mi gene.Theoretical&Applied Genetics,85(6-7):777-783.
Dorhout R,Gommers F J,Koll?ffel C.1993.Phloem transport of carboxyfluorescein through tomato roots infected with Meloidogyne incognita.Physiological&Molecular Plant Pathology,43(1):1-10.
Filho H P M,Stevens M A.1980.Tomato breeding for nematode resistance:survey of resistant varieties for horticultural characteristics and genotype of acid phosphatases.Symposium on Production of Tomatoes for Processing,100:383-394.
Jablonska B,Ammiraju J S,Bhattarai K K,Mantelin S,Martinez de Ilarduya O,Roberts P A,Kaloshian I.2007.The Mi-9 gene from Solanum arcanum conferring heat-stable resistance to rootknot nematodes is a homolog of Mi-1.Plant Physiology,143(2):1044-1054.
Milligan S B,Bodeau J,Yaghoobi J,Kaloshian I,Zabel P,Williamson V M.1998.The root knot nematode resistance gene Mi from tomato is a member of the leucine zipper,nucleotide binding,leucine-rich repeat family of plant genes.Plant Cell,10(8):1307-1319.
Sasser J N.1980.Root-knot nematodes:a global menace to crop production.Plant Disease,64(1):36-41.
Smith P G.1944.Embryo culture of a tomato species hybrid.J Amer Soc Hort Sci,44:413-416.
Tzortzakakis E A,Adam M A M,Blok V C,Blok V C,Paraskevopoulos C,Bourtzis K.2005.Occurrence of resistance-breaking populations of root-knot nematodes on tomato in Greece.European Journal of Plant Pathology,113(1):101-105.
Veremis J C,Roberts P A.1996.Differentiation of Meloidogyne incognita and M.arenaria novel resistance phenotypes in Lycopersicon peruvianum and derived bridge-lines.Theoretical&Applied Genetics,93(5-6):960-967.
Wang Y,Yang W,Zhang W,Han Q,Feng M,Shen H.2013.Mapping of a heat-stable gene for resistance to southern root-knot nematode in Solanum lycopersicum.Plant Molecular Biology Reporter,31(2):352-362.
Yaghoobi J,Yates J L,Williamson V M.2005.Fine mapping of the nematode resistance gene Mi-3 in Solanum peruvianum and construction of a S.lycopersicum DNA contig spanning the locus.Molecular Genetics&Genomics,274(1):60-69.