柑橘溃疡病相关基因CsPGIP的克隆与表达
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摘要
【目的】克隆CsPGIP并分析其表达特性,转化柑橘得到超表达转基因株系,并进行柑橘溃疡病抗性评价,为柑橘溃疡病分子育种提供理论依据。【方法】从晚锦橙和四季橘中克隆柑橘CsPGIP,使用MEGA6进行多序列比对并构建系统发育树;采用在线软件BaCelLo和SignalP 4.0进行亚细胞定位和信号肽预测并用GFP瞬时表达确定CsPGIP在细胞内的定位;利用实时荧光定量PCR(qRT-PCR)比较接种溃疡病菌前后高感品种和高抗品种中柑橘CsPGIP的表达特性,分析溃疡病菌侵染与CsPGIP表达的相关性;农杆菌介导遗传转化晚锦橙,采用GUS染色初筛、PCR鉴定和qRT-PCR相结合的方法鉴定超表达转基因株系;观察转基因和野生型株系表型变化,分析其株高、叶片表型;离体针刺法对超表达转基因株系和野生型株系进行柑橘溃疡病抗性评价,统计病斑面积和病情指数,分析CsPGIP表达对柑橘抗、感溃疡病的影响。【结果】晚锦橙和四季橘CsPGIP均编码328个氨基酸,与已报道的柑橘中的PGIP同源性高达99.39%,都包含2个PGIP基因典型的LRR结构域(LRR_1和LRR_2);构建系统进化树发现甜橙中的CsPGIP与葡萄中的PGIP(GSVIVT01033370001)遗传距离最近,相似度达到62.97%,推测CsPGIP与葡萄中的PGIP具有类似的抗病效果。亚细胞定位和信号肽预测结果表明CsPGIP属于分泌蛋白,GFP洋葱瞬时表达证明柑橘CsPGIP定位在细胞膜和细胞壁,与预测结果一致。高感品种晚锦橙和高抗品种四季橘接种溃疡病菌后CsPGIP的表达特性不同,在高感品种中表达显著下调,而高抗品种中表达显著上调且维持在较高水平,推测CsPGIP与柑橘溃疡病的抗性相关。构建CsPGIP超表达载体并转化晚锦橙,通过PCR鉴定和qRT-PCR确定其中9个(OE1、OE3、OE4、OE5、OE6、OE9、OE10、OE12和OE14)为CsPGIP超表达阳性株系。通过对转基因株系的表型观察发现OE3、OE14株系表型与野生型株系相比差异明显,植株表现为较矮小,其中OE14出现叶片卷曲、增厚的表型变化。对CsPGIP超表达转基因株系(8个株系)进行离体抗溃疡病评价,结果显示超表达转基因株系可以使柑橘溃疡病病斑面积降至野生型的24.11%—83.88%,其中OE1株系的病斑面积最小;从病情指数来看,除OE3株系外,其余株系的病情指数均比野生型显著下降(为野生型的23.12%—75.49%),其中OE1下降最显著,综上结果可知超表达CsPGIP可以有效抑制柑橘溃疡病菌的生长。【结论】CsPGIP是柑橘响应溃疡病菌侵染的重要基因,可抑制或减轻柑橘溃疡病的发病程度,在柑橘抗溃疡病机理研究方面具有较大的应用价值,也可作为柑橘抗溃疡病分子育种的一个候选基因。
【Objective】The objective of this study is to clone Cs PGIP and analyze its expression characteristics, construct Cs PGIP transgenic citrus and evaluate the resistance to citrus bacterial canker(CBC), and to provide a theoretical basis for molecular breeding of citrus bacterial canker.【Method】Cs PGIP was annotated from the genomic databases and cloned from Wanjincheng and Calamondin. MEGA6 was used for multiple sequence alignment and phylogenetic tree was constructed. Two online softwares BaCelLo and SignalP 4.0 were used for the prediction of subcellular localization and signal peptide. The predicted result was then demonstrated by GFP transient expression. The expression profile of Cs PGIP induced by Xanthomonas citri subsp. citri(Xcc) was also analyzed in Wanjincheng and Calamondin by using qRT-PCR method. The correlation between Xcc infection and Cs PGIP expression was analyzed. Genetic transformation of Wanjincheng was conducted by Agrobacterium-mediated method. The over-expressed lines were identified by GUS staining, PCR and qRT-PCR. The phenotypic changes of transgenic and wild-type lines were observed, plant height and leaf phenotype were analyzed. In vitro acupuncture was used to evaluate the resistance of transgenic lines and wild-type lines to citrus bacterial canker. The effect of CsPGIP expression on resistance and susceptibility to citrus bacterial canker was analyzed by statistical analysis of lesion area(LA) and disease index(DI). 【Result】The PGIP cloned from Wanjincheng and Calamondin encodes 328 amino acids, which is 99.39% homology with the reported Cs PGIP from Clementina, and contains two typical LRR domains(LRR_1 and LRR_2). In the phylogenetic tree, the genetic distance between CsPGIP and grape PGIP(GSVIVT01033370001) was the closest, and the similarity was 62.97%. It is inferred that Cs PGIP and grape PGIP have similar resistance to disease. The prediction of subcellular localization and signal peptide indicated that CsPGIP was a secretory protein, and GFP transient expression proved that CsPGIP located on cell membrane and cell wall, which was consistent with the predicted results.The expression of Cs PGIP in canker sensitive plant Wanjincheng and canker resistant plant Calamondin was different after inoculated with Xcc. The expression of Cs PGIP was significantly down-regulated in Wanjincheng, but significantly up-regulated and maintained at a high level in Calamondin. It is speculated that Cs PGIP was related to resistance to citrus bacterial canker. Cs PGIP over-expression vector was constructed and transformed into Wanjincheng, and nine Cs PGIP over-expression lines(OE1, OE3, OE4,OE5, OE6, OE9, OE10, OE12 and OE14) were identified as Cs PGIP over-expression positive lines by PCR identification and q RT-PCR. Through the phenotypic observation of transgenic lines, it was found that the phenotypes of OE3 and OE14 lines were significantly different from those of wild-type lines. The plant was short, in which OE14 was also abnormal with curly property and greater thickness. The in vitro canker resistance of eight Cs PGIP over-expression lines was evaluated. The results showed that the lesion area on the eight Cs PGIP over-expression lines was smaller compared to that on the wild-type(24.11%-83.88%), and the lesion area of OE1 was the smallest. In terms of disease index, the disease index of Cs PGIP over-expression lines(except OE3) was significantly lower than that of wild-type(23.12%-75.49%), and the decrease of OE1 was the most significant. The above results showed that over-expression of Cs PGIP could effectively inhibit the growth of citrus bacterial canker.【Conclusion】Cs PGIP is an important gene which can inhibit or reduce the incidence of citrus bacterial canker, and has a great application value in the mechanism study of citrus resistance to bacterial canker. In the same time, it can be used as a candidate gene for molecular breeding of citrus bacterial canker resistance.
【Objective】The objective of this study is to clone Cs PGIP and analyze its expression characteristics, construct Cs PGIP transgenic citrus and evaluate the resistance to citrus bacterial canker(CBC), and to provide a theoretical basis for molecular breeding of citrus bacterial canker.【Method】Cs PGIP was annotated from the genomic databases and cloned from Wanjincheng and Calamondin. MEGA6 was used for multiple sequence alignment and phylogenetic tree was constructed. Two online softwares BaCelLo and SignalP 4.0 were used for the prediction of subcellular localization and signal peptide. The predicted result was then demonstrated by GFP transient expression. The expression profile of Cs PGIP induced by Xanthomonas citri subsp. citri(Xcc) was also analyzed in Wanjincheng and Calamondin by using qRT-PCR method. The correlation between Xcc infection and Cs PGIP expression was analyzed. Genetic transformation of Wanjincheng was conducted by Agrobacterium-mediated method. The over-expressed lines were identified by GUS staining, PCR and qRT-PCR. The phenotypic changes of transgenic and wild-type lines were observed, plant height and leaf phenotype were analyzed. In vitro acupuncture was used to evaluate the resistance of transgenic lines and wild-type lines to citrus bacterial canker. The effect of CsPGIP expression on resistance and susceptibility to citrus bacterial canker was analyzed by statistical analysis of lesion area(LA) and disease index(DI). 【Result】The PGIP cloned from Wanjincheng and Calamondin encodes 328 amino acids, which is 99.39% homology with the reported Cs PGIP from Clementina, and contains two typical LRR domains(LRR_1 and LRR_2). In the phylogenetic tree, the genetic distance between CsPGIP and grape PGIP(GSVIVT01033370001) was the closest, and the similarity was 62.97%. It is inferred that Cs PGIP and grape PGIP have similar resistance to disease. The prediction of subcellular localization and signal peptide indicated that CsPGIP was a secretory protein, and GFP transient expression proved that CsPGIP located on cell membrane and cell wall, which was consistent with the predicted results.The expression of Cs PGIP in canker sensitive plant Wanjincheng and canker resistant plant Calamondin was different after inoculated with Xcc. The expression of Cs PGIP was significantly down-regulated in Wanjincheng, but significantly up-regulated and maintained at a high level in Calamondin. It is speculated that Cs PGIP was related to resistance to citrus bacterial canker. Cs PGIP over-expression vector was constructed and transformed into Wanjincheng, and nine Cs PGIP over-expression lines(OE1, OE3, OE4,OE5, OE6, OE9, OE10, OE12 and OE14) were identified as Cs PGIP over-expression positive lines by PCR identification and q RT-PCR. Through the phenotypic observation of transgenic lines, it was found that the phenotypes of OE3 and OE14 lines were significantly different from those of wild-type lines. The plant was short, in which OE14 was also abnormal with curly property and greater thickness. The in vitro canker resistance of eight Cs PGIP over-expression lines was evaluated. The results showed that the lesion area on the eight Cs PGIP over-expression lines was smaller compared to that on the wild-type(24.11%-83.88%), and the lesion area of OE1 was the smallest. In terms of disease index, the disease index of Cs PGIP over-expression lines(except OE3) was significantly lower than that of wild-type(23.12%-75.49%), and the decrease of OE1 was the most significant. The above results showed that over-expression of Cs PGIP could effectively inhibit the growth of citrus bacterial canker.【Conclusion】Cs PGIP is an important gene which can inhibit or reduce the incidence of citrus bacterial canker, and has a great application value in the mechanism study of citrus resistance to bacterial canker. In the same time, it can be used as a candidate gene for molecular breeding of citrus bacterial canker resistance.
引文
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[2]贾瑞瑞,周鹏飞,白晓晶,陈善春,许兰珍,彭爱红,雷天刚,姚利晓,陈敏,何永睿,李强.柑橘响应溃疡病菌转录因子CsBZIP40的克隆及功能分析.中国农业科学,2017,50(13):2488-2497.JIA R R,ZHOU P F,BAI X J,CHEN S C,XU L Z,PENG A H,LEI TG,YAO L X,CHEN M,HE Y R,LI Q.Gene cloning and expression analysis of canker-related transcription factor CsBZIP40 in citrus.Scientia Agricultura Sinica,2017,50(13):2488-2497.(in Chinese)
[3]杨枫,陈传武,范七君,石春梅,谢宗周,郭大勇,刘继红.温度和多胺对柑橘溃疡病发生的影响及作用机制.中国农业科学,2018,51(10):1899-1907.YANG F,CHEN C W,FAN Q J,SHI C M,XIE Z Z,GUO D Y,LIU JH.Influence of temperature and polyamines on occurrence of citrus canker disease and underlying mechanisms.Scientia Agricultura Sinica,2018,51(10):1899-1907.(in Chinese)
[4]陈力,王中康,黄冠军,曹月青,夏玉先,殷幼平.柑橘溃疡病生防菌株CQBS03的鉴定及其培养特性研究.中国农业科学,2008,41(8):2537-2545.CHEN L,WANG Z K,HUANG G J,CAO Y Q,XIA Y X,YIN Y P.Evaluation of Bacillus subtilis strain CQBS03 against Xanthomonas axonopodis pv.citri.Scientia Agricultura Sinica,2008,41(8):2537-2545.(in Chinese)
[5]陈波,罗庆华,谭雅芹,闫慧清.柑橘PGIP的B细胞抗原表位分析和原核表达.现代食品科技,2018,34(4):18-22.CHEN B,LUO Q H,TAN Y Q,YAN H Q.B cell epitopes analysis and prokaryotic expression of PGIP in citrus.Modern Food Science and Technology,2018,34(4):18-22.(in Chinese)
[6]FREIBERG A,MACHNER M P,PFELI W,SCHUBERT W D,HEINZ D W,SECKLER R.Folding and stability of the leucine-rich repeat domain of internal in B from Listeri monocytogenes.Journal of Molecular Biology,2004,337(2):453-461.
[7]LEHMANN P.Structure and evolution of plant disease resistance genes.Journal of Applied Genetics,2002,43(4):403-414.
[8]FERRARI S,GALLETTI R,VAIRO D,GERVONE F,DE LORENZOG.Antisense expression of the Arabidopsis thaliana At PGIP1 gene reduces polygalacturonase-inhibiting protein accumulation and enhances susceptibility to Botrytis cinerea.Molecular Plant-Microbe Interactions,2006,19(8):931-936.
[9]JOUBERT D A,KARS I,WAGEMAKERS L,BERGMANN C,KEMP G,VIVIER M A,VAN KAN J A.A polygalacturonaseinhibiting protein from grapevine reduces the symptoms of the endopolygalacturonase BcPG2 from Botrytis cinerea in Nicotiana benthamiana leaves without any evidence for in vitro interaction.Molecular Plant-Microbe Interactions,2007,20(4):392-402.
[10]CHENG Q,CAO Y Z,PAN H X,WANG M X,HUANG M R.Isolation and characterization of two genes encoding polygalacturonaseinhibiting protein from Populus deltoides.Journal of Genetics and Genomics,2008,35(10):631-638.
[11]HEGEDUS D D,LI R,BUCHWALDT L,PARKIN I,WHITWILL S,COUTU C,BEKKAOUI D,RIMMER S R.Brassica napus possesses an expanded set of polygalacturonase inhibitor protein genes that are differentially regulated in response to Sclerotinia sclerotiorum infection,wounding and defense hormone treatment.Planta,2008,228(2):241-253.
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