应用PCR技术检测甘蔗线虫病抗性基因的研究
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摘要
本研究以43份未经线虫病常规鉴定的甘蔗质资源为供试材料，首先提取供试甘蔗基因组DNA，然后根据番茄抗根结线虫病基因和甜菜抗胞囊线虫病基因的保守序列区域，分别设计了两条上游引物、两条下游引物，对引物组合后，进行了PCR特异扩增，从中分别筛选出一对特异引物，并优化了PCR扩增反应体系中的各个影响因素。用抗根结线虫基因设计的引物进行扩增最终获得了一条大约460bp大小的特异片段；用抗胞囊线虫基因设计的引物进行扩增最终获得了一条大约690bp大小的特异片段，并进一步做了PCR-Southern杂交，以确认这两条特异片段的同源性程度和真实性，本实验所得结果如下：
1．建立了适合于甘蔗PCR特异扩增的反应体系：反应总体积为20μl，含10mmol／L Tris-HCl(pH8.3)，50mmol／L KCl，2.0mmol／L MgCl_2，100μmol／L dNTP，0.2μmol／L引物，40ng模板DNA，1.0U Taq酶；反应程序为：94℃，5min，1 cycle；94℃，40s，60℃，50s，72℃，90s，35 cycles；72℃延伸10min。
2．对43份未经线虫病常规鉴定的甘蔗种质资源根据不同的引物进行PCR检测。其中，用抗根结线虫病基因设计的引物进行扩增，结果有37份甘蔗出现特异条带，6份未出现任何条带；用抗胞囊线虫病基因设计的引物进行扩增，结果有39份甘蔗出现特异条带，4份未出现任何条带。
3．对两种PCR特异产物进行了PCR-Southern杂交，结果显示它们有杂交信号，说明这两个片段分别与两个抗线虫病基因有较高的同源性，为克隆甘蔗抗线虫病基因奠定了基础。
The nematode resistance of forty-three sugarcane cultivars (without conventional identification of nematode resistance) was tested in the experiment. Firstly, the sugarcane genomic DNA was extracted. Secondly, according to the sequence of root-knot nematode resistant gene in tomato and the sequence of nematode resistant gene in sugar beet, two forward primers and two reverse primers were produced separately by synthetic, 4 pairs of primers that were in .each kind were composed, and a pair of primers in each was selected among them by PCR. Thirdly, conditions for PCR in sugarcane were optimized, the results showed that a special fragment of about 460bp and a special fragment of about 690bp were appeared, and PCR-Southern blotting about them was made further in order to affirm the homogeneous sequences of nematode resistant gene. Finally , 43 sugarcane cultivars were primarily detected by PCR technique. The results were summarized as following :
1. An optimal reaction system for PCR in sugarcane was established : in a 20ul reaction solution, the optimal composition included 10 mmol/L Tris-HCl(pH8.3), 50 mmol/L KC1,1.5 mmol/L MgCl2,100umol/L dNTP ,0.2umol/L primers ,40ng of template DNA and 1.0 U Tag DNA Polymerase.The reaction program was devised in to 3 processes: The first one is denaturation at 94℃ for 5 min. The second one is 35 cycles of denaturation at 94 ℃ for 40s, annealing at 60℃ for 50s and extension at 72 ℃ for 90 s. The last one is extension at 72℃ for 10 min.
2. Among 43 sugarcane cultivars, 37 cultivars appeared a special fragment of about 630bp, and 6 cultivars had nothing by using root-knot nematode resistant primers. 39 cultivars appeared a special fragment of about 630bp, and 4 cultivars had nothing by using cytocyst nematode resistant primers.
3. The two kinds of special PCR products were hybridized by PCR-Southern blotting. The blotting signals appeared, which proved that there was a great homogeneous sequence between 460bp special fragment and root-knot nematode resistant gene in tomato and there was a great homogeneous sequence between 690bp special fragment and nematode resistant gene in sweet potato. They were the basis for cloning nematode resistant gene in sugarcane. 4.The results will be tested by field identification in the further.
1．建立了适合于甘蔗PCR特异扩增的反应体系：反应总体积为20μl，含10mmol／L Tris-HCl(pH8.3)，50mmol／L KCl，2.0mmol／L MgCl_2，100μmol／L dNTP，0.2μmol／L引物，40ng模板DNA，1.0U Taq酶；反应程序为：94℃，5min，1 cycle；94℃，40s，60℃，50s，72℃，90s，35 cycles；72℃延伸10min。
2．对43份未经线虫病常规鉴定的甘蔗种质资源根据不同的引物进行PCR检测。其中，用抗根结线虫病基因设计的引物进行扩增，结果有37份甘蔗出现特异条带，6份未出现任何条带；用抗胞囊线虫病基因设计的引物进行扩增，结果有39份甘蔗出现特异条带，4份未出现任何条带。
3．对两种PCR特异产物进行了PCR-Southern杂交，结果显示它们有杂交信号，说明这两个片段分别与两个抗线虫病基因有较高的同源性，为克隆甘蔗抗线虫病基因奠定了基础。
The nematode resistance of forty-three sugarcane cultivars (without conventional identification of nematode resistance) was tested in the experiment. Firstly, the sugarcane genomic DNA was extracted. Secondly, according to the sequence of root-knot nematode resistant gene in tomato and the sequence of nematode resistant gene in sugar beet, two forward primers and two reverse primers were produced separately by synthetic, 4 pairs of primers that were in .each kind were composed, and a pair of primers in each was selected among them by PCR. Thirdly, conditions for PCR in sugarcane were optimized, the results showed that a special fragment of about 460bp and a special fragment of about 690bp were appeared, and PCR-Southern blotting about them was made further in order to affirm the homogeneous sequences of nematode resistant gene. Finally , 43 sugarcane cultivars were primarily detected by PCR technique. The results were summarized as following :
1. An optimal reaction system for PCR in sugarcane was established : in a 20ul reaction solution, the optimal composition included 10 mmol/L Tris-HCl(pH8.3), 50 mmol/L KC1,1.5 mmol/L MgCl2,100umol/L dNTP ,0.2umol/L primers ,40ng of template DNA and 1.0 U Tag DNA Polymerase.The reaction program was devised in to 3 processes: The first one is denaturation at 94℃ for 5 min. The second one is 35 cycles of denaturation at 94 ℃ for 40s, annealing at 60℃ for 50s and extension at 72 ℃ for 90 s. The last one is extension at 72℃ for 10 min.
2. Among 43 sugarcane cultivars, 37 cultivars appeared a special fragment of about 630bp, and 6 cultivars had nothing by using root-knot nematode resistant primers. 39 cultivars appeared a special fragment of about 630bp, and 4 cultivars had nothing by using cytocyst nematode resistant primers.
3. The two kinds of special PCR products were hybridized by PCR-Southern blotting. The blotting signals appeared, which proved that there was a great homogeneous sequence between 460bp special fragment and root-knot nematode resistant gene in tomato and there was a great homogeneous sequence between 690bp special fragment and nematode resistant gene in sweet potato. They were the basis for cloning nematode resistant gene in sugarcane. 4.The results will be tested by field identification in the further.
引文
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[2] 张绍升编著．植物线虫病害诊断与治理．福建：福建科学技术出版社．1999
[3] Winstead N N and Sasser J N. Reaction of cucumber varieties to five root-knot nematodes (Meloidogyne spp.). Plant Diesase Reporter, 1856, 40(4): 272-275
[4] Karssen G. The Plant-parasitic Nematode Genus Meloidogyne Goldi, 1892 (Tylenchida) in Europe. Drukkerij Modern: Bennekom, 1999
[5] 杨宝君．十五种根结线虫病害的病原鉴定．植物病理学报，1984，14(2)：107-112
[6] 刘维志，段玉玺．植物病原线虫学．北京：中国农业出版社，2000，213-281
[7] 彭德良．蔬菜线虫病害的发生和防治．中国蔬菜，1998，4：57-58
[8] Jones M G K. Host cell response to endoparastic nematode attack: structure and function of giant cells and syncytia. Ann Appl Biol, 1981, 97:353-372
[9] Sijmons P C. Plant-nematode ineractions. Plant Mol Biol, 1993, 23:917-931
[10] Cai D, Kleine M, Kifle S, Harloff H J, et al. Positional cloning of a gene for nematode resistance in sugar beet. Science, 1997, 275:832-834
[11] Hallden C et al. The use of bulked segregant analysis to accumalate RAPD markers near a locus for beet cyst nematode resistance in Beta vulgaris. Plant Breed, 1996, 116:12-22
[12] Kaloshian I, Yaghoob J, Liharska T et al. Genetic and Physical localization of the root-knot nematode resistance locus Mi in tomato. Mol. Gen. Genet, 1998, 57:376-385
[13] Milligan S B, Bodeau J, Yaghoobi J et al. The rootknot nematode resistance gene Mi from tomato is a member of the leucine zipper, Nucleatide binding, Leucine-Rich repeat family of plant genes. The plant cell, 1998, 10:1307-1319
[14] Lagudah E S, Moulet O, Appels, R. Map-based cloning of a gene sequence encoding a nucleotide binding domain and a leucine-rich region at the Cre3 nematode resistance locus of wheat. Genome, 1997, 40(5): 659-665
[15] Ballvora A et al. Marker enrichment and high resolution map of the segment of potato chromosome Ⅶ. Harbouring the nematode resistance gene Grol. Mol. Gen. Genet, 1995, 249:82-90
[16] Jung C et al. Engineering nematode resistance in crop species. Trends in Plant Science, 1998, 3:266-271
[17] Niewoehner J. Development of PCR assays diagnostic for RFLP marker alleles closely linked to alleles Grol and Hl, conferring resistance to the root cyst nematode Globodera rostochiensis in potato. Mol. Breed, 1995, 1:65-78
[18] Ganal M W et al. Genetic mapping of a wide spectrum nematode resistance gene (Hera) against Globodera rostochiensis in tomato. Mol. Plant-Microbe Interact, 1995, 8:
886-891
[19] Vierling R A et al. Association of RFLP marker with loci conferring broad resistance to the soybean cyst nematode (Heterodera glycines). Theor. Appl. Genet, 1996, 92: 83-86
[20] Concibidio V C et al. Soybean cyst nematode resistance genes in 'Peking', PI90763, and PI88788 using DNA markers. Crop Sci, 1997, 37: 258-264
[21] Tamulocus J P et al. RFLP mapping of resistance to southern root-knot nematode in soybean. Crop Sci, 1997, 37: 1903-1909
[22] Kretschmer J M et al. RFLP mapping of the Ha2 cereal cyst nematode resistance gene in barley. Theor. Appl. Genet, 1997, 94: 1061-1064
[23] Williams K J et al. Development of a PCR-based allele-specific assay from an RFLP probe linked to resistance to cereal cyst nematode in wheat. Genome, 1996, 39: 798-801
[24] Garcia G M et al. Identification of RAPD, SCAR, and RFLP markers tightly linked to nematode resistance genes introgressed from Arachis cardenasii in to Arachis hypogaea.Genome, 1996, 39: 836-845
[25] Sijmons P C et al. Arabidopsis haliana as a new model hosts for plant-parasitic nematodes. Plant J, 1991, 1: 245-254
[26] Joan M H. Roy French. The polymerase chain reaction and plant disease diagnosis. Ann. Rev. Phytopathol, 1993, 31: 81-109
[27] Arnheim N, Erlich H. Polymerase chain reaction strategy. Ann. Rev. Biochem, 1992, 1:131
[28] Saeffan RJ, Atlas RM. Polymerase chain reaction: Applications in environmental microbiology. Ann. Rev. Microbiol, 1991, 45: 137
[29] 鲍晓明,黄百渠,李松涛等.用RAPD技术鉴定两个小冰麦易位系.遗传学报1993,20(1) : 81-87
[30] Myers TW, Gelfand DH. Reverse transcription and DNA amplification by a Thermus thermophillus DNA polymerase. Biochem, 1991, 30: 7661-7666
[31] Schafer C, Wostemeyer J. Random primer dependent PCR differentiates aggressive from non-aggressive isolates of the oilseed tape pathogen Phoma Ligain (Leptosphoeria maculans). Phytopathol, 1992, 136: 124-130
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