利用DDRT-PCR鉴定芸芥自交不亲和系与自交亲和系的差异表达cDNA（英文）
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摘要
芸芥与芸薹属植物具有亲缘关系,也是芸薹属植物的重要育种资源。自交亲和性是芸芥的一种重要变异性状。为了探明芸芥自交亲和基因的表达器官,并分离与自交亲和性有关的cDNA片段,以芸芥的一对近等基因系,自交亲和系(SC)与自交不亲和系(SI)为试材,利用差异显示RT-PCR技术分别对开花前和开花后的叶片、花药和柱头进行鉴定。结果表明,不论开花前还是开花后,芸芥自交亲和系与自交不亲和系的叶片或花药间的扩增带型是一样的,但在近等基因系的柱头间得到了差异表达条带。这证明芸芥自交亲和基因不是组成型表达,而是组织特异性表达,柱头是其唯一的表达器官。在开花前的自交亲和系中共得到了2条cDNA片段,一条小于100 bp,另一条为750~1 000 bp,在开花后的自交亲和系中得到了1条300 bp的cDNA片段。据分析这3条cDNA片段可能与芸芥的自交亲和性密切相关。在开花前的自交不亲和系中共得到了2条cDNA片段,一条为500 bp,另一条为750 bp,同时在开花后的自交不亲和系中得到了1条500~600 bp的cDNA片段。这些cDNA片段可以用来区分芸芥的自交亲和系与自交不亲和系,还可用于克隆自交亲和基因。
Yunjie is closely related to important vegetables and oil seed crops,and can be considered a genetic resource for all Brassiceae crops. Self compatibility is an important variant traits to Yunjie. This study aimed to probe the expression organ of self-compatible gene and isolate cDNA fragments related to self-compatibility of Yunjie.Leaf,anther and stigma from self-incompatible( SI) and self-compatible( SC) lines of Yunjie were examined by DDRT-PCR. The results showed that the amplification pattern of leaves and anthers were the same in SI and SC lines. However,different bands were obtained in stigmas of SI and SC lines before and after flowering. SC gene of E.sativa was not showing constitutive expression but showed tissue-specific expression in stigma. Only two fragments were amplified in SC lines before flowering,the one was less than 100 bp,the other was 750- 1 000 bp. A 300 bp fragment was found unique to self-compatible line after flowering. These three cDNA fragments might be closely related to the self-compatibility in Yunjie. In addition,two cDNA fragments were generated in SI line before flowering,one was 500 bp,the other was 750 bp. One band of 500- 600 bp was identified in SI line after flowering. These cDNA fragments could efficiently distinguish self-compatible and self-incompatible lines,and could also be usefulfor cloning self-compatible gene of Yunjie.
Yunjie is closely related to important vegetables and oil seed crops,and can be considered a genetic resource for all Brassiceae crops. Self compatibility is an important variant traits to Yunjie. This study aimed to probe the expression organ of self-compatible gene and isolate cDNA fragments related to self-compatibility of Yunjie.Leaf,anther and stigma from self-incompatible( SI) and self-compatible( SC) lines of Yunjie were examined by DDRT-PCR. The results showed that the amplification pattern of leaves and anthers were the same in SI and SC lines. However,different bands were obtained in stigmas of SI and SC lines before and after flowering. SC gene of E.sativa was not showing constitutive expression but showed tissue-specific expression in stigma. Only two fragments were amplified in SC lines before flowering,the one was less than 100 bp,the other was 750- 1 000 bp. A 300 bp fragment was found unique to self-compatible line after flowering. These three cDNA fragments might be closely related to the self-compatibility in Yunjie. In addition,two cDNA fragments were generated in SI line before flowering,one was 500 bp,the other was 750 bp. One band of 500- 600 bp was identified in SI line after flowering. These cDNA fragments could efficiently distinguish self-compatible and self-incompatible lines,and could also be usefulfor cloning self-compatible gene of Yunjie.
引文
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[27]Ni Z F,Sun Q X,Liu Z Y.Differential gene expression between heterotie and nonheterotic wheat hybrids and their parental inbreds in seedling leaves[J].Journal of Agricultural Biotechnology,1999,7(1):95-100.
[28]Sadia F,Amer J.Expression profiling of bioactive genes from a medicinal plant Nigella sativa L[J].Applied Biochemistry and Biotechnology,2013,170(6):1472-1481.
[2]Menéndez A I,Romero A M,Folcia A M.Aphid and episodic O3 injury in arugula plants grown in open-top field chambers[J].Agriculture,Ecosystems and Environment,2010(1/2):10-14.
[3]Michalis O,Chara P,Dimitra K.Relationships between nitrogen,dry matter accumulation and glucosinolates in Eruca sativa Mill[J].Plant and Soil,2012,1:347-358.
[4]Fan H L,Luo F F,Huang X L,et al.Preliminary studies of the variation in self-compatibility among 52 cultivars of Eruca sativa[J].Chinese Bulletin of Botany,2015,50(5):598-604.
[5]Sun W C,Pan Q Y,Liu Z G,et al.Overcoming self-incompatibility in Eruca sativa by chemical treatment of stigmas[J].Plant Genetic Resources,2005,1:13-18.
[6]Fan H L,Bai S W,Chang L G,et al.Genetic variation of self-compatibility in Eruca sativa Mill.[J].Chinese Journal of Oil Crop Sciences,2015,37(5):605-613.
[7]Coulibaly I,Noirot M,Loricux M,et al.Introgression of self-compatibility from Coffea heterocalyx to the cultivated species Coffea canephora[J].Theoretical and Applied Genetics,2002,105(617):994-999.
[8]Fang Z Y,Liu Y M,Yang L M,et al.An important change in seed production techniques of the cabbage[J].China Vegetables,2004,5:33.
[9]Yamane H,Ushijima K,Sassa H,et al.The use of the S haplotype-specific F-box protein gene,SFB,as a molecular marker for S-haplotypes and self-compatibility in Japanese apricot(Prunus mume)[J].Theoretical and Applied Genetics,2003,107(8):1357-1361.
[10]Goring D R,Glavin T L,Schafer U,et al.An S receptor kinase gene in self-compatible Brassica napus has a 1-bp deletion[J].The Plant Cell,1993,5(5):531-539.
[11]He Y T,Ma C Z,Ma Y,et al.Cloning of S-locus gene by PCR walking in Brassica campestris L.[J].Chinese Journal of Oil Crop Sciences,2004,26(1):1-5.
[12]Schopfer C R,Nasrallah M E,Nasrallah J B.The male determinant of self-incompatibility in Brassica[J].Sci-ence,1999,286(5445):1697-1700.
[13]Nasrallah J B,Rundle S J,Nasrallah M E.Genetic evidence for the requirement of the Brassica S_locus receptor kunase gene in the self-incompatibility response[J].Plant J,1994,5:373-384.
[14]Wang B C,Sun W C,Fan H L,et al.Studies on SOD,POD and CAT activity between self-compatible and selfincompatible lines in Eruca sativa Mill.[J].Chinese Journal of Oil Crop Sciences,2006,28(2):162-165.
[15]Chen J R,Fang Y,Sun W C,et al.Cloning and bioinformatics of xyloglucan endotrans glycosylase and hydrolase Es XTH1 gene in Eruca sativa[J].Chinese Journal of Oil Crop Sciences,2013,35(2):131-136.
[16]Fang Y,Yang G,Sun W C,et al.Cloning and expression of Dna J homolog gene from Eruca sativa[J].Acta Agriculturae Boreali-sinica,2015,30(6):61-65.
[17]Wang G L.Plant genetic engineering[M].Beijing:Science Press,1998:608-609.
[18]Golldack D,Su Hua,Quigley F,et al.Characterization of a HKT-type transporter in rice as a general alkali cation transporter[J].The Plant Journal,2002,31(4):529-542.
[19]Yan H F.mRNA differential expression from interaction of puccinia recondita with Lr38 based on DDRT-PCR[D].Baoding:Agricultural University of Hebei,2003.
[20]Yu J.Isolation of genes related to K562 cells differentiation suing a modified DDRT-PCR[D].Chongqing;Chongqing Medical University,2003.
[21]Yua H F,Zhao Z Q,Sheng X G,et al.Identification of S-haplotypes in DH-lines of broccoli(Brassica oleracea L.)[J].Journal Of Horticultural Science and Biotechnology.2014,89(4):430-434.
[22]Fan H L,Wang W Q,Li Y L,et al.Study on S alleles and their expression in self-incompatible lines of Eruca Sativa Mill.[J].Agricultural Research in the Arid Areas,2013,31(5):110-116.
[23]Ding C H,Du X W,Ying X,et al.Screening for differentially expressed genes in endophytic fungus strain 39during co-culture with herbal extract of its host dioscorea nipponica makino[J].Curr Microbiol,2014,69(4):517-524.
[24]Hannappel U,Balzer H J and Ganal N W.Direct isolationof cDNA sequences from specific chromosomal regions of the tomato genome by the differential display technique[J].Mol Gen Genet,1995,249:19-24.
[25]Mei J F,Tang C Q,Xu D L,et al.Study on diggerential gene expression of tea plant(Camellia sinensis)during cold acclimation in winter[J].Chinese Journal of Tropical Crops,2011,32(4):648-652.
[26]Liu H,Hu J,Pan L,et al.Robust ordered mRNA differential display:an improved method for global gene expression profiling[J].Biotechniques,2011,51(4):271-275.
[27]Ni Z F,Sun Q X,Liu Z Y.Differential gene expression between heterotie and nonheterotic wheat hybrids and their parental inbreds in seedling leaves[J].Journal of Agricultural Biotechnology,1999,7(1):95-100.
[28]Sadia F,Amer J.Expression profiling of bioactive genes from a medicinal plant Nigella sativa L[J].Applied Biochemistry and Biotechnology,2013,170(6):1472-1481.