鸭梨几丁质PbChiIV的克隆与表达

doi:10.3864/j.issn.0578-1752.2015.11.019

摘要/Abstract

摘要： 【目的】从鸭梨果实中克隆PbChiIV的全长cDNA序列，检测PbChiIV在根、茎、叶、果实以及在水杨酸（SA）和梨轮纹病菌诱导下的表达特性，以探讨该基因与SA信号转导及抗梨轮纹病菌的相关性。【方法】设计特异引物，克隆PbChiIV的全长序列，将测序得到的核苷酸序列和推导的氨基酸序列在NCBI上用BLAST进行序列相似性分析，利用BiotEdit软件对氨基酸序列进行比对，利用MEGA6.0构建系统发育树，利用实时荧光定量PCR技术分析该基因在梨不同组织以及在SA和梨轮纹病菌诱导下的表达。【结果】克隆了PbChiIV的cDNA序列为819 bp，GenBank数据库登录号为KJ872676。生物信息学分析表明，PbChiIV编码272个氨基酸，与沙梨的同源性达100%，与毛果杨（XP_006376418.1）、葡萄（NP001268173.1）、拟南芥（CAA74930.1）、紫花苜蓿（ACL36992.1）、蒺藜苜蓿（AAR87869.1）、豇豆（CAA61281.1）、榛子（AEM97876.1）、东方山羊豆（AAP03085.1）、葡萄（NP_001268075.1）、华东葡萄（ABY66958.1）、葡萄（AAB65777.1）、烟草（BAF44533.1）和海岛棉（AER29902.1）的同源性分别为79%、73%、73%、72%、72% 、72%、69%、68%、67%、67%、65%、67%和62%，属于第IV类几丁质酶基因。表达分析表明，PbChiIV在根中的表达量最大，分别是茎和叶的4.32和2.96倍，其次是在果实中的表达量，分别是茎和叶的2.48和1.70倍，在叶片和茎中的表达相对较低。在鸭梨幼果和成熟期果实中，SA和梨轮纹病菌均可诱导该基因表达。SA处理后基因的最大表达量是对照的2.83和3.8倍，病原菌处理后基因的最大表达量是对照的1.82和1.66倍，SA、病原菌处理后基因的最大表达量是对照的2.49和3.43倍，表达量分别在72、24和72 h达到最大值。【结论】PbChiIV可能参与SA介导的植物抗病防卫反应的信号通路，推测其参与梨轮纹病菌引起的防卫反应，在鸭梨抗病过程中起作用。

关键词: 鸭梨')">

鸭梨, PbChiIV, 表达模式, SA, 梨轮纹病菌

Abstract: 【Objective】This study aimed to clone a full length cDNA of PbChiIV，fruit and the expression after treatment with the salicylic acid(SA) and Botryosphaeria berengeriana de. Not. f. sp. piricola (Nose) Koganecawa et. Sokwwa.【Method】The full lengthof PbChiIV was obtained from Pyrus bretschneideri ‘Yali’ using specific primers. The similarity of the nucleotide sequence and deduced amino acid sequence was analyzed by using BLAST on NCBI. BiotEdit software was used to compare the amino acid sequence. Phylogenetic tree was constructed by using MEGA6.0. Quantitative real-time PCR (qRT-PCR) was performed to determine the expression pattern of PbChiIV in different tissues and the expression of this gene after treatment with SA and B.berengeriana. f. sp. piricola.【Result】The cDNA lengths of PbChiIV genes was 819 bp (GenBank accession number: KJ872676), encoding 272 amino acids. The deduced amino acid sequence shares 100%, 79%, 73%, 73%, 72%, 72%, 72%, 69%, 68%, 67%, 67%, 65%, 67% and 62% identity with P. pyrifolia ACM45716.1, Populus trichocarpa XP_006376418.1, Vitis vinifera NP_001268173.1, Arabidopsis thaliana CAA74930.1, Medicago sativa ACL36992.1, Medicago truncatula AAR87869.1, Vigna unguiculata CAA61281.1, Corylus heterophylla AEM97876.1, Galega orientalis AAP03085.1, Vitis vinifera NP_001268075.1, Vitis pseudoreticulata ABY66958.1, Vitis vinifera AAB65777.1, Nicotiana tabacum BAF44533.1 and Gossypium barbadense AER29902.1. This gene belongs to IV chitinase gene. Expression analysis showed that the expression of PbChiIV in root was the highest, which was 4.32 and 2.96 times compared with stem and leaf, respectively. Followed by the expression in fruit, which was 2.48 and 1.70 times compared to that of stem and leaf, respectively. In the young fruit and mature fruit, the expression of the gene could be induced by SA and B.berengeriana. f. sp. piricola. The highest expression of the gene was 2.83 and 3.80 times compared with controls after treatment with SA, respectively. The highest expression of the gene was 1.82 and 1.66 times as compared with controls after treatment with pathogen, respectively. The highest expression of the gene was 2.49 and 3.43 times as compared with controls after treatment with SA and pathogen. In addition, the expression was up to the peak at 72, 24 and 72 h after treatment with SA and pathogen, respectively.【Conclusion】PbChiIV may be involved in SA-mediated basic signaling pathway of plant defense responses and disease resistance signal transduction network. It is speculated that PbChiIV is involved in defense responses mediated by B.berengeriana. f. sp. piricola. It might play an important role in disease resistance. from Pyrus bretschneideri ‘Yali’ and detect the expression characteristics of PbChiIV in root, stem, leaf

Key words: Pyrus bretschneideri ‘Yali’, PbChiIV de. Not. f. sp. piricola (Nose) Koganecawa et. Sokwwa), expression pattern, SA, pear ring rot pathogen (Botryosphaeria berengeriana

中图分类号:

鸭梨

李朋朋，刘宁，张玉星，彭建营，董金皋. 鸭梨几丁质PbChiIV的克隆与表达[J]. 中国农业科学, 2015, 48(11): 2279-2286.

LI Peng-peng, LIU Ning, ZHANG Yu-xing, PENG Jian-ying, DONG Jin-gao. Cloning and Expression of Chitinase PbChiIV Genes from Pyrus bretschneideri ‘Yali’[J]. Scientia Agricultura Sinica, 2015, 48(11): 2279-2286.

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