茶树几丁质酶基因克隆及响应坏损外担菌表达分析

Cloning of Chitinase Gene from Camellia sinensis and Analysis of Its Expression in Response to Exobasidium vexans Massee

茶饼病是由坏损外担菌(Exobasidium vexans Massee)引起的茶树(Camellia sinensis)芽叶病害。在植物中,几丁质酶是一种重要的病程相关蛋白。为探究几丁质酶在茶树抗坏损外担菌过程中的作用,本研究从茶树‘云抗10号’基因组中筛选出28个几丁质酶基因,以坏损外担菌高抗茶树‘梅占’和高感茶树‘黔湄601’为材料同源克隆几丁质酶基因,对克隆所获得的基因进行生物信息学分析。同时,对不同抗性的2种茶树进行坏损外担菌接种,在接种后的不同时期,利用实时定量PCR技术对克隆获得的基因的表达量进行分析。本研究在‘梅占’和‘黔湄601’两种茶树中都克隆获得4个几丁质酶基因,分别命名为CsC8、CsC13、CsC21和CsC23。进化树和保守结构域分析的结果发现,CsC8和CsC23属于肽酶家族,CsC13属于植物几丁质酶,CsC21虽然是几丁质酶但有可能不属于植物几丁质酶。高抗茶树‘梅占’接种坏损外担菌后,CsC8、CsC13和CsC23基因表达量均升高。高感茶树‘黔湄601’接种坏损外担菌后,CsC8和CsC13基因表达量升高,而CsC23基因在接种后几乎没有变化,且CsC13基因在高抗茶树‘梅占’中的表达量高于高感茶树‘黔湄601’的。因此,推测几丁质酶CsC13可能在茶树抗坏损外担菌的过程中起着重要作用。本研究为深入研究几丁质酶基因参与茶树抗坏损外担菌的机制奠定基础,同时为进一步探究植物肽酶功能提供理论依据。

Blister blight disease is a disease of Camellia sinensis buds and leaves caused by Exobasidium vexans Massee.Chitinase is an important pathogenesis-related protein in plants.In order to explore the role of chitinase in tea plant resistance to Exobasidium vexans Massee,28 chitinase genes were screened from the genome of tea plant‘Yunkang 10’in this study,and homologous cloning of chitinase gene sequences from Camellia sinensis was carried out using highly resistant Camellia sinensis‘Meizhan’and highly sensitive Camellia sinensis‘Qianmei 601’as materials,and the obtained genes from cloning were analyzed by bioinformatics.At the same time,two Camellia sinensises with different resistance were inoculated with Exobasidium vexans Massee,and the expression level of cloned genes were analyzed by real time quantitative PCR at different periods after inoculation.In this study,four chitinase genes were simultaneously cloned from two Camellia sinensises,named CsC8,CsC13,CsC21 and CsC23 respectively.The results of evolutionary tree and conservative domain analysis showed that CsC8 and CsC23 belong to peptidase family,CsC13 belonged to plant chitinase,CsC21 was a chitinase but may not belong to the plant chitinase.The expression levels of CsC8,CsC13 and CsC23 gene in highly resistant Camellia sinensis‘Meizhan’were all increased after inoculated with Exobasidium vexans Massee.The expression of CsC8 and CsC13 gene in highly sensitive Camellia sinensis‘Qianmei 601’was increased after inoculating with Exobasidium vexans Massee,but CsC23 gene had almost no change after inoculation,and the expression of and CsC13 gene in highly resistant Camellia sinensis‘Meizhan’was higher than that in highly sensitive Camellia sinensis‘Qianmei 601’.Therefore,it was speculated that chitinase CsC13 played an important role in the process of Camellia sinensis resistance to Exobasidium vexans Massee.This study lays a foundation for further research on the mechanism of chitinase gene involved in Camellia sinensis resistance to Exobasidium vexans Massee,and provides a theoretical basis for further research on the function of plant peptidase.